阅读说明：本技术 可调控启动子 (Regulatable promoter ) 是由 D.玛塔诺维克 B.加瑟 M.茂勒 R.普里尔霍佛 J.克雷恩 J.温格 于 2012-10-05 设计创作，主要内容包括：一种通过培养包含表达构建体的重组真核细胞系来产生感兴趣蛋白质(POI)的方法,所述表达载体包含可调控的启动子和在所述启动子的转录调控下的编码POI的核酸分子,所述方法包括以下步骤：a)用阻遏所述启动子的基础碳源培养所述细胞系,b)用使所述启动子去阻遏的有限量的补充碳源培养所述细胞系,从而诱导POI以相比于天然pGAP启动子至少15％的转录速率产生,和c)产生并回收所述POI；以及另外地,分离的可调控的启动子和相应的表达系统。(A method for producing a protein of interest (POI) by culturing a recombinant eukaryotic cell line comprising an expression construct, said expression vector comprising a regulatable promoter and a nucleic acid molecule encoding the POI under the transcriptional control of said promoter, said method comprising the steps of: a) culturing the cell line with a basic carbon source that represses the promoter, b) culturing the cell line with a limited amount of a supplemental carbon source that derepresses the promoter, thereby inducing the production of the POI at a transcription rate of at least 15% compared to the native pGAP promoter, and c) producing and recovering the POI; and, additionally, isolated regulatable promoters and corresponding expression systems.)

1. A method for producing a protein of interest by culturing a recombinant methylotrophic yeast comprising an expression construct comprising a regulatable promoter and a nucleic acid molecule encoding the protein of interest under transcriptional control of the promoter, the method comprising the steps of:

a) culturing the cell line with a basic carbon source that represses the promoter, wherein the basic carbon source is glucose, glycerol, or a mixture thereof,

b) culturing said cell line under glucose-free or limited glucose-containing conditions in which said promoter is derepressed and fully induced, and

c) producing and recovering the protein of interest,

wherein the promoter comprises a nucleic acid sequence selected from the group consisting of:

i) pG1 consisting of the nucleotide sequence of SEQ ID 1, pG3 consisting of the nucleotide sequence of SEQ ID2, pG4 consisting of the nucleotide sequence of SEQ ID 4, pG6 consisting of the nucleotide sequence of SEQ ID 3, pG7 consisting of the nucleotide sequence of SEQ ID 5, or pG8 consisting of the nucleotide sequence of SEQ ID 6; and

ii) pG1a consisting of the nucleotide sequence of SEQ ID 41, pG1b consisting of the nucleotide sequence of SEQ ID 42, pG1c consisting of the nucleotide sequence of SEQ ID 43, pG1d consisting of the nucleotide sequence of SEQ ID 44, pG1e consisting of the nucleotide sequence of SEQ ID 45 and pG1f consisting of the nucleotide sequence of SEQ ID 46.

2. The method of claim 1, wherein the promoter is a functionally active promoter that is a carbon source controllable promoter capable of expressing a protein of interest in a recombinant eukaryotic cell at a transcription rate of at least 20% compared to the cell's native pGAP promoter in a fully induced state and in a chemically defined and methanol-free feed medium.

3. The method according to claim 1, wherein said step b) employs a feed medium that provides no glucose or a limited amount of glucose.

4. The method according to any one of claims 1 to 2, wherein the limited amount is 0-1g/L in the culture medium.

5. The method according to claim 1, wherein the glucose is growth limiting to maintain a specific growth rate at 0.02h^-1To 0.2h^-1Within the range of (1).

6. The method according to claim 1, wherein the glucose is growth limiting to maintain a specific growth rate at 0.02h^-1To 0.15h^-1Within the range of (1).

7. The method of claim 1, wherein the promoter is pG1 consisting of the nucleotide sequence of SEQ ID 1 or a functionally active variant thereof.

8. The method according to claim 1, wherein the promoter is selected from the group consisting of: pG1 consisting of the nucleotide sequence of SEQ ID 1, pG1a consisting of the nucleotide sequence of SEQ ID 41, pG1b consisting of the nucleotide sequence of SEQ ID 42, pG1c consisting of the nucleotide sequence of SEQ ID 43, pG1d consisting of the nucleotide sequence of SEQ ID 44, pG1e consisting of the nucleotide sequence of SEQ ID 45 and pG1f consisting of the nucleotide sequence of SEQ ID 46.

9. The method according to claim 1, wherein the protein of interest is a heterologous protein.

10. The method according to claim 9, wherein the heterologous protein is selected from the group consisting of therapeutic proteins including antibodies or fragments thereof, enzymes and peptides, protein antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccines and vaccine-like proteins or particles, growth factors, hormones and cytokines.

11. An isolated nucleic acid comprising a nucleic acid sequence selected from the group consisting of:

i) pG1 consisting of the nucleotide sequence of SEQ ID 1, pG3 consisting of the nucleotide sequence of SEQ ID2, pG6 consisting of the nucleotide sequence of SEQ ID 3, pG7 consisting of the nucleotide sequence of SEQ ID 5, or pG8 consisting of the nucleotide sequence of SEQ ID 6; and

ii) pG1a consisting of the nucleotide sequence of SEQ ID 41, pG1b consisting of the nucleotide sequence of SEQ ID 42, pG1c consisting of the nucleotide sequence of SEQ ID 43, pG1d consisting of the nucleotide sequence of SEQ ID 44, pG1e consisting of the nucleotide sequence of SEQ ID 45 and pG1f consisting of the nucleotide sequence of SEQ ID 46.

12. The nucleic acid of claim 11, comprising a functionally active promoter, which is a carbon source-controllable promoter, which is capable of expressing a protein of interest in a recombinant eukaryotic cell at a transcription rate of at least 20% compared to the cell's native pGAP promoter in a fully induced state and in a chemically defined and methanol-free feed medium.

13. An expression construct comprising a nucleic acid according to claim 12, wherein said promoter is operably linked to a nucleotide sequence encoding a protein of interest under the transcriptional control of said promoter, said nucleic acid not being naturally associated with the nucleotide sequence encoding said protein of interest.

14. A recombinant methylotrophic yeast comprising the expression construct of claim 13.

Technical Field

The present invention relates to regulatable promoters and methods for producing a protein of interest under the control of a regulatable promoter in eukaryotic cell culture.

Background

The successful production of recombinant proteins has been achieved using eukaryotic hosts. The most prominent examples are yeasts such as Saccharomyces cerevisiae (Saccharomyces cerevisiae), Pichia pastoris (Pichia pastoris) or Hansenula polymorpha (Hansenula polymorpha), filamentous fungi such as Aspergillus awamori or Trichoderma reesei (Trichoderma reesei), or mammalian cells such as for example CHO cells. Although it is easy to achieve the production of some proteins at high rates, there are many other proteins that are only available at rather low levels.

Heterologous expression of a gene in a host organism generally requires a vector that allows stable transformation of the host organism. The vector will provide the gene and a functional promoter adjacent to the 5' end of the coding sequence. Thus, transcription is regulated and initiated by the promoter sequence. Most promoters used to date originate from genes encoding proteins that are usually present in high concentrations in cells.

EP0103409a2 discloses the use of yeast promoters associated with the expression of specific enzymes in the glycolytic pathway, i.e. promoters involved in the expression of pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, phosphoglycerate mutase, hexokinase 1 and 2, glucokinase, phosphofructokinase, aldolase and glycolytic regulatory genes.

WO 97/44470 describes yeast promoters from Yarrowia lipolytica for the translation of the elongation factor 1(TEF1) protein and for the ribosomal protein S7, which are suitable for heterologous expression of proteins in yeast, while EP1951877A1 describes the use of the Pichia pastoris TEF1 promoter for the production of heterologous proteins.

WO2005003310 provides methods for expressing a coding sequence of interest in yeast using a promoter for glyceraldehyde-3-phosphate dehydrogenase or phosphoglycerate mutase from the oleaginous yeast yarrowia lipolytica.

Promoter sequences derived from genes involved in the methanol metabolic pathway of pichia pastoris are disclosed in US4808537 and US4855231 (alcohol oxidase AOX1, AOX2) and US6730499B1 (formaldehyde dehydrogenase FLD 1). US20080153126a1 includes a mutant promoter sequence based on the AOX1 promoter.

The AOX1 promoter is induced only in response to methanol and repressed by other carbon sources such as glucose or ethanol. Methanol has the disadvantage that it is not suitable for producing certain products because of its potential hazard due to toxicity and flammability. Therefore, alternatives to the AOX1 promoter were sought.

US2008299616a1 introduces regulatory sequences for the malate synthase (MLS1) gene for heterologous gene expression in pichia pastoris, which is repressed in glucose-containing medium but derepressed under glucose starvation conditions or in the presence of acetate. However, this system is not considered suitable for an efficient production method, since the MLS1 promoter is weaker and less active under derepressed conditions.

And Schmuller (mol.cell biol.199414 (6):3613-22) describe the regulatory region of the isocitrate lyase gene ICL1, which is derepressed after transfer of the cells from fermentative growth conditions to non-fermentative growth conditions.

WO2008063302a2 describes the use of novel inducible promoters derived from the genes ADH1 (alcohol dehydrogenase), ENO1 (enolase) and GUT1 of pichia pastoris for the expression of heterologous proteins, the pichia pastoris omega 3-fatty acid dehydrogenase promoter sequence in CN1966688A, and the pichia pastoris derived auto-inducible (auto-inducible) NPS promoter in WO002007117062a1, which is induced by phosphorus restriction.

WO2008128701a2 describes the use of a novel promoter in which the promoter of the THI3 (thiamine metabolism) gene from pichia pastoris is repressed in a thiamine-containing medium and derepressed when thiamine is depleted.

US2009325241a1 describes a method for producing ethanol in yeast cells using a xylose-inducible promoter (FAS2 promoter).

It would be desirable to provide improved recombinant eukaryotic cell lines for producing fermentation products that can be isolated in high yield. It is therefore an object of the present invention to provide alternative regulatory elements suitable for recombinant production methods, which are simple and efficient.

Disclosure of Invention

The object is solved by the claimed subject matter.

According to the present invention there is provided a method of producing a protein of interest (POI) by culturing a recombinant eukaryotic cell line comprising an expression vector comprising a regulatable promoter and a nucleic acid molecule encoding the POI under the transcriptional control of the promoter, the method comprising the steps of:

a) culturing said cell line with a basal carbon source repressing said promoter,

b) culturing said cell line with no or limited amount of supplemental carbon source that derepresses said promoter, thereby inducing production of said POI at a transcription rate of at least 15% compared to the native pGAP promoter of said cell, and

c) generating and recovering the POI.

The culturing step specifically comprises culturing the cell line in the presence of the carbon source, whereby in a medium comprising the carbon source, or in step b) also in the absence of a supplemental carbon source.

Induction of production of said POI refers in particular to induction of transcription, including in particular further translation and optionally expression of said POI.

The transcription rate refers in particular to the amount of transcripts obtained when the promoter is fully induced. The promoter is considered to be derepressed and fully induced if the culture conditions provide for a presumably maximal induction, for example at a glucose concentration of less than 0.4g/L, preferably less than 0.04g/L, in particular less than 0.02 g/L. Preferably, the fully induced promoter shows a transcription rate of at least 150% or at least 200% compared to the native pGAP promoter of at least 15%, preferably at least 20%, more preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90% and at least 100% or even higher. The transcription rate can be measured, for example, by the amount of transcripts of a reporter gene, such as eGFP, which, as described in the examples section below, shows a relatively high transcription rate of at least 50% of the pG1 promoter compared to the native pGAP promoter when clones are cultured in solution. Alternatively, the transcription rate can be determined by the transcription intensity on a microarray, where the microarray data show a difference in expression level between repressed and derepressed states and a high signal intensity in the fully induced state compared to the native pGAP promoter. Such microarray data specifically showed transcription rates of over 200% for pG1, over 30% for pG3 and pG4, over 60% for pG6, over 30% for pG7, and over 20% for pG8 (each compared to native pGAP). The promoters of the prior art were found to be too weak to be suitable for the purposes of the present invention.

In particular, the native pGAP promoter is active in the recombinant eukaryotic cell (including unmodified (non-recombinant) or recombinant eukaryotic cells) in a manner similar to that found in native eukaryotic cells of the same species or strain. Such native pGAP promoters are generally understood to be endogenous promoters, and as such, are eukaryotic homologous, and serve as standard or reference promoters for comparison purposes.

For example, the native pGAP promoter of pichia pastoris is an unmodified endogenous promoter sequence in pichia pastoris, as used to control GAPDH expression in pichia pastoris, e.g., having the sequence shown in fig. 13: the native pGAP promoter sequence (SEQ ID 13) of Pichia pastoris (GS 115). If Pichia pastoris is used as host for the production of POIs according to the invention, the transcription strength or rate of the promoter according to the invention is compared to such native pGAP promoters of Pichia pastoris.

As a further example, the natural pGAP promoter of Saccharomyces cerevisiae is the unmodified endogenous promoter sequence of Saccharomyces cerevisiae, as used to control GAPDH expression in Saccharomyces cerevisiae. If Saccharomyces cerevisiae is used as host for the production of POIs according to the invention, the transcription strength or rate of the promoter according to the invention is compared with such native pGAP promoters of Saccharomyces cerevisiae.

Thus, the relative transcriptional strength or rate of a promoter according to the invention is typically compared to the native pGAP promoter of a cell of the same species or strain used as the host for the production of the POI.

According to a specific embodiment, the base carbon source is different, e.g. quantitatively and/or qualitatively, from the supplemental carbon source. The quantitative differences may provide different conditions to repress or derepress promoter activity.

According to a further specific embodiment, the base and supplemental carbon sources comprise the same type of molecule or carbohydrate, preferably at different concentrations. According to yet another specific embodiment, the carbon source is a mixture of two or more different carbon sources.

Any organic carbon suitable for eukaryotic cell culture can be used. According to a specific embodiment, the carbon source is a hexose such as glucose, fructose, galactose or mannose, a disaccharide such as sucrose, an alcohol such as glycerol or ethanol, or a mixture thereof.

According to a particularly preferred embodiment, the basic carbon source is selected from the group consisting of: glucose, glycerol, ethanol, or mixtures thereof, and complex nutritional materials. According to a preferred embodiment, the base carbon source is glycerol.

According to yet another specific embodiment, the supplemental carbon source is a hexose such as glucose, fructose, galactose and mannose, a disaccharide such as sucrose, an alcohol such as glycerol or ethanol, or a mixture thereof. According to a preferred embodiment, the supplemental carbon source is glucose.

In particular, the method may employ glycerol as the basic carbon source and glucose as the supplemental carbon source.

The derepression conditions may be suitably achieved by specific means. Optionally, step b) employs a feed medium that provides no or limited amounts of supplemental carbon sources.

Specifically, the feed medium is chemically defined and methanol-free.

The feed medium may be added to the medium in liquid form or in other alternative forms such as a solid (e.g., tablets or other sustained release means) or a gas (e.g., carbon dioxide). However, according to a preferred embodiment, the limited amount of supplemental carbon source added to the cell culture medium may even be 0. Preferably, the concentration of the supplemental carbon source in the culture medium is 0-1g/L, preferably less than 0.6g/L, more preferably less than 0.3g/L, more preferably less than 0.1g/L, preferably 1-50mg/L, more preferably 1-10mg/L, particularly preferably 1mg/L or even lower, such as below the limit of detection as measured by a suitable standard assay, e.g.as the residual concentration in the culture medium as determined at the time of consumption by the growing cell culture.

In a preferred method, the limited amount of supplemental carbon source provides a residual amount in the cell culture below the detection limit, as determined at the end of the production phase or in the output of the fermentation process, preferably in the fermentation broth at the time of harvesting the fermentation product.

Preferably, the limited amount of supplemental carbon source is growth-limiting to maintain a range of 0.02h^-1To 0.2h^-1Preferably 0.02h^-1To 0.15h^-1Specific growth rate of the inner layer.

According to a particular aspect of the invention, the promoter is the pichia pastoris promoter or a functionally active variant thereof.

In this context, a promoter according to the invention shall always refer to the sequences described herein and functionally active variants thereof. As explained in detail below, such variants include homologues and analogues derived from species other than pichia pastoris.

The method according to the invention may employ a promoter which is a wild-type promoter of pichia pastoris or a functionally active variant thereof, for example a wild-type promoter capable of controlling the transcription of a particular gene in a wild-type or recombinant eukaryotic cell, for example a selected gene selected from the group consisting of: g1(SEQ ID 7), such as encoding a (high affinity) glucose transporter, G3(SEQ ID 8), G4(SEQ ID 9), such as encoding a mitochondrial aldehyde dehydrogenase, G6(SEQ ID 10), G7(SEQ ID 11), such as encoding a member of the major facilitator sugar transporter family (major facilitator transporter family), or G8(SEQ ID 12), such as encoding a member of the Gti1_ Pac2 superfamily, or a functionally active variant thereof.

According to the present invention, there is specifically provided a promoter or functionally active variant thereof which will be naturally associated with one of such genes in a wild-type yeast cell.

According to a particular embodiment, the cell line is selected from the group consisting of: mammalian, insect, yeast, filamentous fungi and plant cell lines, preferably yeast.

Specifically, the yeast is selected from the group consisting of: pichia (Pichia), Candida (Candida), Torulopsis (Torulopsis), Arxula, Hansenula (Hensenula), Yarrowia (Yarrowia), Kluyveromyces (Kluyveromyces), Saccharomyces (Saccharomyces), Komagataella, preferably methylotrophic yeasts.

One particularly preferred yeast is pichia pastoris, Komagataella pastoris, k.phaffii, or k.pseudopsathoris.

According to yet another specific embodiment, the promoter is not naturally associated with the nucleotide sequence encoding the POI.

In particular, the POI is a eukaryotic, preferably mammalian, protein.

The POI produced according to the present invention may be a multimeric protein, preferably a dimer or tetramer.

According to one aspect of the invention, the POI is a recombinant or heterologous protein, preferably selected from the group consisting of therapeutic proteins including antibodies or fragments thereof, enzymes and peptides, protein antibiotics, toxin fusion proteins, sugar-protein conjugates, structural proteins, regulatory proteins, vaccines and vaccine-like proteins or particles, processing enzymes, growth factors, hormones and cytokines, or metabolites of the POI.

One particular POI is an antigen binding molecule such as an antibody or fragment thereof. Specific POIs are antibodies such as monoclonal antibodies (mAbs), immunoglobulins (Ig) or immunoglobulin classes G (IgG), heavy chain antibodies (HcAb), or fragments thereof such as fragment-antigen binding (Fab), Fd, single chain variable fragments (scFv), or engineered variants thereof such as, for example, Fv dimers (diabodies), Fv trimers (triabodies), Fv tetramers or minibodies (minibodies), and single domain antibodies such as VH or VHH or V-NAR.

According to a particular embodiment, the fermentation product is prepared using a POI, a metabolite or derivative thereof.

In accordance with another aspect of the invention, there is provided a method for regulating expression of a POI in a recombinant eukaryotic cell under the transcriptional control of a carbon source controllable promoter having a transcriptional strength of at least 15% compared to the native pGAP promoter of the cell, wherein the expression is induced under carbon source limiting conditions. The carbon source-controllable promoter preferably has a transcription strength of at least 20% compared to the reference pGAP promoter, in particular compared to the native pGAP promoter as described above in terms of transcription rate. Thus, a fully induced promoter has an even higher transcription intensity of preferably at least 15%, preferably at least 20%, more preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90% and at least 100% or at least 150% or at least 200% compared to the native pGAP promoter of the cell, as determined in the eukaryotic cell selected for POI production.

In a preferred embodiment, such promoters are used which have a transcriptional activity or transcriptional strength in a derepressed state which is at least 2-fold, more preferably at least 5-fold, even more preferably at least 10-fold, more preferably at least 20-fold, more preferably at least 30, 40, 50, or 100-fold in the derepressed state compared to the repressed state.

In accordance with another aspect of the present invention, there is provided a method of producing a POI in a recombinant eukaryotic cell under the transcriptional control of a carbon source-controllable promoter, wherein the promoter has a transcriptional strength as described above, i.e., at least 15% compared to the cell's native pGAP promoter. The carbon source-controllable promoter preferably has an even higher transcription strength of at least 20%, more preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90% and at least 100% or at least 150% or at least 200% compared to the reference pGAP promoter. In a preferred embodiment, such promoters are used which have transcriptional activity in a derepressed state which is at least 2-fold, more preferably at least 5-fold, even more preferably at least 10-fold, more preferably at least 20-fold, more preferably at least 30, 40, 50, or 100-fold in the derepressed state compared to the repressed state. Suitably, a specific promoter according to the invention is used in such a method.

In a particular preferred method according to the invention, the promoter is a regulatable promoter comprising a nucleic acid sequence selected from the group consisting of:

a) pG1(SEQ ID 1), pG3(SEQ ID2), pG4(SEQ ID 4), pG6(SEQ ID 3), pG7(SEQ ID 5), or pG8(SEQ ID 6);

b) a sequence having at least 60% homology to: pG1(SEQ ID 1), pG3(SEQ ID2), pG4(SEQ ID 4), pG6(SEQ ID 3), pG7(SEQ ID 5), or pG8(SEQ ID 6);

c) a sequence that hybridizes under stringent conditions to: pG1(SEQ ID 1), pG3(SEQ ID2), pG4(SEQ ID 4), pG6(SEQ ID 3), pG7(SEQ ID 5), or pG8(SEQ ID 6); and

d) fragments or variants derived from a), b) or c),

wherein the promoter is a functionally active promoter which is a carbon source regulatable promoter capable of expressing the POI in a recombinant eukaryotic cell at a transcription rate of at least 15% compared to the native pGAP promoter of the cell.

Specifically, variants of pG1(SEQ ID 1), pG3(SEQ ID2), pG4(SEQ ID 4), pG6(SEQ ID 3), pG7(SEQ ID 5) or pG8(SEQ ID 6) are functionally active variants selected from the group consisting of: homologues having at least about 60% nucleotide sequence identity, homologues obtainable by modifying a parent nucleotide sequence by insertion, deletion or substitution of one or more nucleotides within the sequence or at one or both distal ends of the sequence (preferably having a nucleotide sequence of at least 200bp, preferably at least 250bp, preferably at least 300bp, more preferably at least 400bp, at least 500bp, at least 600bp, at least 700bp, at least 800bp, at least 900bp, or at least 1000bp), and analogues derived from species other than pichia pastoris.

Some preferred functionally active variants of the promoter according to the invention are fragments of any of the pG1, pG3, pG4, pG6, pG7 or pG8 promoter nucleotide sequences, preferably fragments comprising the 3 ' end of the promoter nucleotide sequence, such as a nucleotide sequence derived from one of said promoter nucleotide sequences, having a specific length and a deletion in the 5 ' end region, such as a cleavage of the nucleotide sequence at the 5 ' end, such that a specific length ranging from the 3 ' end to the altered 5 ' end is obtained, such as a nucleotide sequence length of at least 200bp, preferably at least 250bp, preferably at least 300bp, more preferably at least 400bp, at least 500bp, at least 600bp, at least 700bp, at least 800bp, at least 900bp, or at least 1000 bp.

Exemplary variants comprising or consisting of such fragments, e.g., fragments having a specific length in the range of 200 to 1000bp, preferably in the range of 250 to 1000bp, more preferably in the range of 300 to 1000bp, e.g., fragments comprising a 3' terminal sequence, have been shown to be functionally active. For example, functionally active variants of pG1 are selected from the group consisting of: pG1a (SEQ ID 41), pG1b (SEQ ID 42), pG1c (SEQ ID 43), pG1d (SEQ ID 44), pG1e (SEQ ID 45) and pG1f (SEQ ID 46), such that a nucleotide sequence ranging from 300 and 1000bp comprises the 3' terminal sequence up to nucleotide 1001.

According to another aspect of the invention, there is provided an isolated nucleic acid comprising a nucleic acid sequence selected from the group consisting of:

a) pG1(SEQ ID 1), pG3(SEQ ID2), pG6(SEQ ID 3), pG7(SEQ ID 5) or pG8(SEQ ID 6),

b) a sequence having at least 60% homology to: pG1(SEQ ID 1), pG3(SEQ ID2), pG6(SEQ ID 3), pG7(SEQ ID 5) or pG8(SEQ ID 6),

c) a sequence that hybridizes under stringent conditions to: pG1(SEQ ID 1), pG3(SEQ ID2), pG6(SEQ ID 3), pG7(SEQ ID 5) or pG8(SEQ ID 6), and

d) fragments or variants derived from a), b) or c),

wherein the nucleic acid comprises a functionally active promoter which is a carbon source regulatable promoter capable of expressing the POI in a recombinant eukaryotic cell at a transcription rate of at least 15% compared to the native pGAP promoter of the cell.

Specifically, the variant of pG1(SEQ ID 1), pG3(SEQ ID2), pG6(SEQ ID 3), pG7(SEQ ID 5) or pG8(SEQ ID 6) is a functionally active variant selected from the group consisting of: homologues having at least about 60% nucleotide sequence identity, homologues obtainable by modifying a parent nucleotide sequence by insertion, deletion or substitution of one or more nucleotides within the sequence or at one or both distal ends of the sequence (preferably having a nucleotide sequence of at least 200bp, preferably at least 250bp, preferably at least 300bp, more preferably at least 400bp, at least 500bp, at least 600bp, at least 700bp, at least 800bp, at least 900bp, or at least 1000bp), and analogues derived from species other than pichia pastoris.

Some preferred functionally active variants of the promoter according to the invention are fragments of any of the pG1, pG3, pG6, pG7 or pG8 promoter nucleotide sequences, preferably fragments comprising the 3 ' end of the promoter nucleotide sequence, for example a nucleotide sequence derived from one of the promoter nucleotide sequences, having a specific length and a deletion in the 5 ' end region, for example a cleavage of the nucleotide sequence at the 5 ' end, such that a specific length ranging from the 3 ' end to the altered 5 ' end is obtained, for example a nucleotide sequence length of at least 200bp, preferably at least 250bp, preferably at least 300bp, more preferably at least 400bp, at least 500bp, at least 600bp, at least 700bp, at least 800bp, at least 900bp, or at least 1000 bp.

Exemplary variants comprising or consisting of such fragments, e.g., fragments having a specific length in the range of 200 to 1000bp, preferably in the range of 250 to 1000bp, more preferably in the range of 300 to 1000bp, e.g., fragments comprising a 3' terminal sequence, have been shown to be functionally active. For example, functionally active variants of pG1 are selected from the group consisting of: pG1a (SEQ ID 41), pG1b (SEQ ID 42), pG1c (SEQ ID 43), pG1d (SEQ ID 44), pG1e (SEQ ID 45) and pG1f (SEQ ID 46), such that one nucleotide sequence ranges from 300 and 1000bp, which comprises the 3' terminal sequence up to nucleotide 1001.

The carbon source-controllable promoter preferably has a transcription strength as described above, preferably at least 20% compared to the reference pGAP promoter, more preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90% and at least 100% or even more higher at least 150% or at least 200% compared to the native pGAP promoter. In a preferred embodiment, such promoters are used which have transcriptional activity in a derepressed state which is at least 2-fold, more preferably at least 5-fold, even more preferably at least 10-fold, more preferably at least 20-fold, more preferably at least 30, 40, 50, or 100-fold in the derepressed state compared to the repressed state. Suitably, a specific promoter according to the invention is used in such a method.

According to a further aspect of the invention there is provided an expression construct comprising a promoter according to the invention operably linked to a nucleotide sequence encoding a POI under the transcriptional control of said promoter, which promoter is not naturally associated with the coding sequence of the POI.

Yet another aspect of the invention refers to a vector comprising a construct according to the invention.

Yet another aspect of the invention refers to a recombinant eukaryotic cell comprising a construct or vector according to the invention.

In particular, the cell is selected from the group consisting of: mammalian, insect, yeast, filamentous fungi and plant cell lines, preferably yeast.

The yeast may suitably be selected from the group consisting of: pichia (Pichia), Candida (Candida), Torulopsis (Torulopsis), Arxula, Hansenula (Hensenula), Yarrowia (Yarrowia), Kluyveromyces (Kluyveromyces), Saccharomyces (Saccharomyces), Komagataella, preferably methylotrophic yeasts.

Preferably, the yeast is pichia pastoris, Komagataella pastoris, k.

According to a specific embodiment, a cell is used which has a higher specific growth rate in the presence of excess carbon source relative to the conditions of a limited carbon source.

Yet another aspect of the invention is directed to the use of a recombinant eukaryotic cell of the invention for the production of a POI.

According to a further aspect of the invention, there is provided a method for screening or identifying a carbon source-regulatable promoter from a eukaryotic cell, comprising the steps of:

a) culturing eukaryotic cells in batch culture in the presence of a carbon source under cell growth conditions,

b) further culturing the cells in fed-batch culture in the presence of a limited amount of a supplemental carbon source,

c) providing a sample of the cell culture of steps a) and b), and

d) performing a transcriptional analysis in said sample to identify a regulatable promoter that exhibits a higher transcriptional intensity in the cells of step b) than in the cells of step a).

The higher transcription intensity can be determined by the transcription intensity in the fully induced state, which is for example obtained under glucose-limited chemostat culture conditions, which is at least 2-fold, more preferably at least 5-fold, even more preferably at least 10-fold, more preferably at least 20-fold, more preferably at least 30, 40, 50, or 100-fold in the derepressed state compared to the repressed state.

Preferably, the transcriptional analysis is quantitative or semi-quantitative, preferably using DNA microarrays, RNA sequencing, and transcriptome analysis.

Drawings

FIG. 1: the promoter sequence pG1(SEQ ID 1) of Pichia pastoris.

FIG. 2: the promoter sequence pG3(SEQ ID2) of Pichia pastoris.

FIG. 3: the promoter sequence pG4(SEQ ID 4) of Pichia pastoris.

FIG. 4: the promoter sequence pG6(SEQ ID 3) of Pichia pastoris.

FIG. 5: the promoter sequence pG7 of Pichia pastoris (SEQ ID 5).

FIG. 6: the promoter sequence of Pichia pastoris pG8(SEQ ID 6).

FIG. 7: the coding sequence of the G1 gene of the Pichia pastoris GS115 genome (SEQ ID 7).

FIG. 8: the coding sequence of the G3 gene of the Pichia pastoris GS115 genome (SEQ ID 8).

FIG. 9: the coding sequence of the G4 gene of the Pichia pastoris GS115 genome (SEQ ID 9).

FIG. 10: the coding sequence of the G6 gene of the Pichia pastoris GS115 genome (SEQ ID 10).

FIG. 11: the coding sequence of the G7 gene of the Pichia pastoris GS115 genome (SEQ ID 11).

FIG. 12: the coding sequence of the G8 gene of the Pichia pastoris GS115 genome (SEQ ID 12).

FIG. 13: native pGAP promoter sequence (SEQ ID 13) of Pichia pastoris (GS115)

#	Name (R)	PAS*	PIPA*	GS115 description
					pGAP	TDH3	PAS_chr2-1_0437	PIPA02510	Glyceraldehyde-3-phosphate dehydrogenase

PAS: ORF name in pichia pastoris GS 115; PIPA: ORF name in Pichia pastoris strain type DSMZ70382

FIG. 14: derepression properties of pG1 (round), pG3 (triangle), pG4 (diamond) and pG6 (square) promoters: for pG1, the maximum transcriptional activity was reached at about 0.04g glucose/L or less, while all other pG promoters reached the maximum transcriptional activity at about 4g/L or less. To compare the relative induction behavior of different promoters, the data were normalized by removing each value from the D20 value of the corresponding promoter construct. Thus, the data are relative fluorescence values, while the data point at D20 is 1.0.

FIG. 15: a functionally active variant of the promoter sequence pG 1; pG1a-f of Pichia pastoris (SEQ ID 41-46).

Detailed Description

Specific terms as used throughout this specification have the following meanings.

As used herein, the term "carbon source" means a fermentable carbon substrate suitable as a source of energy for a microorganism, typically a carbohydrate source, such as those capable of being metabolized by a host organism or a production cell line, specifically a source selected from the group consisting of: monosaccharides, oligosaccharides, polysaccharides, alcohols including glycerol, are provided in purified form or as raw materials such as complex nutritional materials. According to the invention, the carbon source can be used as a single carbon source or as a mixture of different carbon sources.

As used in accordance with the present invention, a "basic carbon source" is generally a carbon source suitable for cell growth, such as a nutrient for eukaryotic cells. The basic carbon source may be provided in a medium, such as a basal medium or a complex medium, but may also be provided in a chemically defined medium containing a purified carbon source. The base carbon source is typically provided in an amount that provides for cell growth, particularly during a growth phase in a culture process, e.g. to obtain a cell density of at least 5g/L cell dry mass, preferably at least 10g/L cell dry mass or at least 15g/L cell dry mass, e.g. exhibiting a survival of more than 90%, preferably more than 95%, during a standard subculture step.

According to the present invention, the basic carbon source is usually used in excess or surplus, which is understood to be an excess of energy source to increase the biomass, for example during the growth phase of the cell line in a fed-batch culture process. The excess is in particular over a limited amount of supplemental carbon source to achieve a residual concentration in the fermentation broth that is measurable and typically at least 10-fold, preferably at least 50-fold or at least 100-fold higher than during the feeding of the limited amount of supplemental carbon source.

The term "chemically defined" with respect to a cell culture medium, such as a feed medium in a fed-batch process, means a growth medium suitable for use in vitro cell culture of a production cell line, wherein all chemical components and peptides are known. In general, chemically defined media are completely free of animal-derived components and represent a pure and constant cell culture environment.

"supplemental carbon source" as used according to the invention is typically a supplemental substrate that promotes the production of the fermentation product of the producer cell line, particularly during the production phase of the cultivation process. The production phase is in particular after the growth phase, for example in batch, fed-batch and continuous culture processes. The supplemental carbon source may specifically be comprised in the feed of a fed-batch process.

By "limited amount" of a carbon source or "limited carbon source" is herein understood the amount of carbon source required to maintain the production cell line in the production phase or mode. Such limited amounts can be employed in fed-batch processes where a carbon source is contained in a feed medium and supplied to the culture at a low feed rate for sustained energy delivery to produce the POI, while maintaining the biomass at a low growth rate. Feed medium is typically added to the fermentation broth during the production phase of the cell culture.

The limited amount of supplemental carbon source can be determined, for example, by the residual amount of supplemental carbon source in the cell culture broth, which is below a predetermined threshold or even below the detection limit as measured in a standard (sugar) assay. The residual amount will typically be determined in the fermentation broth at the time the fermentation product is harvested.

The limited amount of the supplemental carbon source may also be determined by defining the average feed rate of the supplemental carbon source to the fermentor, e.g., as determined by the amount added over the entire cultivation process, e.g., fed-batch phase, by the cultivation time, to determine the average amount over time. This average feed rate is kept low to ensure complete use of the supplemental carbon source by the cell culture, e.g., 0.6g L^-1h^-1(g carbon source per L initial fermentation volume and h time) to 25g L^-1h^-1Preferably 1.6g L^-1h^-1To 20g L^-1h^-1。

The limited amount of supplemental carbon source may also be determined by measuring the specific growth rate before and during the production process, which specific growth rate is kept low during the production phase, e.g. within a predetermined range, such as at 0.02h^-1To 0.20h^-1In the range of (1), preferably 0.02h^-1To 0.15h^-1。

As used herein, the term "cell line" refers to an established clone of a particular cell type that has acquired the ability to proliferate over an extended period of time. The term "host cell line" refers to a cell line as used for expressing endogenous or recombinant genes or products of metabolic pathways to produce polypeptides or cellular metabolites mediated by such polypeptides. By "production host cell line" or "production cell line" is generally understood a cell line which is ready for cultivation in a bioreactor to obtain a production process product, such as a POI. The term "eukaryotic host" or "eukaryotic cell line" means any eukaryotic cell or organism that can be cultured to produce a POI or host cell metabolite. It is fully understood that the term does not include humans.

The term "expression" or "expression system" or "expression cassette" refers to a nucleic acid molecule containing a desired coding sequence and control sequences in operable linkage such that a host transformed or transfected with these sequences can produce the encoded protein or host cell metabolite. To effect transformation, the expression system may be incorporated into a vector; however, the relevant DNA may also be integrated into the host chromosome. Expression may refer to secreted or non-secreted expression products, including polypeptides or metabolites.

An "expression construct" or "vector" is used herein to define a recombinant nucleotide sequence, i.e., a DNA sequence required for transcription of a recombinant gene and translation of its mRNA, cloned in a suitable host organism. Expression vectors typically comprise an origin for autonomous replication in a host cell, a selectable marker (e.g., an amino acid synthesis gene or a gene that confers resistance to an antibiotic such as neomycin, kanamycin, G418, or hygromycin), a number of restriction enzyme cleavage sites, a suitable promoter sequence, and a transcription terminator, the components of which are operably linked together. As used herein, the terms "plasmid" and "vector" include autonomously replicating nucleotide sequences as well as genomically integrated nucleotide sequences.

As used herein, the term "variant" in the context of the present invention refers to any sequence having a particular homology or similarity. Variant promoters may, for example, be derived from the promoter sequences pG1(SEQ ID 1), pG3(SEQ ID2), pG4(SEQ ID 4), pG6(SEQ ID 3), pG7(SEQ ID 5) or pG8(SEQ ID 6), which are mutagenized to generate sequences suitable for use as promoters in recombinant cell lines. Such variant promoters may be obtained from libraries of mutant sequences by selecting those library members having predetermined characteristics. Variant promoters may have the same or even improved properties, for example improved in inducing POI production, with increased differential effect under repressing and derepressing conditions. The variant promoter may also be derived from similar sequences, for example from eukaryotic species other than pichia pastoris or from genera other than pichia, such as kluyveromyces lactis (k.lactis), z.rouxii, p.stipitis, hansenula polymorpha. In particular, similar promoter sequences naturally associated with genes similar to the corresponding pichia pastoris gene can be used as such or as parent sequences to produce functionally active variants thereof. In particular, the amount of the solvent to be used,

a promoter similar to pG1, characterized in that it is naturally associated with a gene similar to G1 (high affinity glucose transporter; Pichia pastoris GS115 describes: a putative transporter, a member of the sugar handling family; coding sequence SEQ ID 7);

-a promoter similar to pG3 characterized in that it is naturally associated with a gene similar to G3 (coding sequence SEQ ID 8);

a promoter similar to pG4, characterized in that it is naturally associated with a gene similar to G4 (Pichia pastoris GS 115: predicted mitochondrial aldehyde dehydrogenase; coding sequence SEQ ID 9);

-a promoter similar to pG6 characterized in that it is naturally associated with a gene similar to G6 (coding sequence SEQ ID 10);

a promoter similar to pG7, characterized in that it is naturally associated with a gene similar to G7 (Pichia pastoris GS 115: a member of the main facilitator sugar transporter family; coding sequence SEQ ID 11);

a promoter similar to pG8 is characterized in that it is naturally associated with a gene similar to G8 (Pichia pastoris GS 115: Gti1_ Pac2 superfamily member; coding sequence SEQ ID 12).

The properties of such similar promoter sequences or functionally active variants thereof can be determined using standard techniques.

"functionally active" variants of a nucleotide or promoter sequence as used herein means a sequence which results from modification of the parent sequence by insertion, deletion or substitution of one or more nucleotides within the sequence or at one or both of the distal ends of the sequence, and which modification does not affect (in particular does not impair) the activity of the sequence.

In particular, functionally active variants of the promoter sequence according to the invention are selected from the group consisting of:

-homologues having at least 60% nucleotide sequence identity,

homologues obtainable by modification of a parent nucleotide sequence by insertion, deletion or substitution of one or more nucleotides within the sequence or at one or both of the distal ends of the sequence, preferably having (i.e. comprising or consisting of) a nucleotide sequence of at least 200bp, preferably at least 300bp, more preferably at least 400bp, at least 500bp, at least 600bp, at least 700bp, at least 800bp, at least 900bp or at least 1000bp, and

-analogues derived from species other than pichia pastoris.

Particularly preferred functionally active variants are those fragments which are derived from the promoter and/or promoter sequence according to the invention by modification and which have (i.e. comprise or consist of) a nucleotide sequence of at least 200bp, preferably at least 250bp, preferably at least 300bp, more preferably at least 400bp, at least 500bp, at least 600bp, at least 700bp, at least 800bp, at least 900bp or at least 1000 bp.

Some preferred functionally active variants of the promoter according to the invention are fragments of any of the promoter nucleotide sequences pG1, pG3, pG4, pG6, pG7 or pG8, preferably a fragment comprising the 3 ' end of a promoter nucleotide sequence, such as a nucleotide sequence derived from one of said promoter nucleotide sequences, having a specific length and a deletion in the 5 ' end region, such as a cleavage of the nucleotide sequence at the 5 ' end, such that a specific length ranging from the 3 ' end to the altered 5 ' end is obtained, such as a nucleotide sequence length of at least 200bp, preferably at least 250bp, preferably at least 300bp, more preferably at least 400bp, at least 500bp, at least 600bp, at least 700bp, at least 800bp, at least 900bp or at least 1000 bp.

Exemplary variants comprising or consisting of such fragments, e.g., fragments having a specified length in the range of 200 to 1000bp, preferably in the range of 250 to 1000bp, more preferably in the range of 300 to 1000bp, e.g., fragments comprising a 3' terminal sequence, have been shown to be functionally active. For example, functionally active variants of pG1 are selected from the group consisting of: pG1a (SEQ ID 41), pG1b (SEQ ID 42), pG1c (SEQ ID 43), pG1d (SEQ ID 44), pG1e (SEQ ID 45) and pG1f (SEQ ID 46), such that one nucleotide sequence ranges from 300 and 1000bp, which comprises the 3' terminal sequence up to nucleotide 1001.

As used herein, the term "regulatable" with respect to a promoter refers to a promoter that is repressed in a eukaryotic cell in the presence of an excess of a carbon source (nutrient substrate) during the growth phase of a batch culture, and that is derepressed to exert strong promoter activity during the production phase of a producer cell line, e.g., when the amount of carbon is reduced, such as when a growth-limiting carbon source (nutrient substrate) is supplied to the culture in accordance with a fed-batch strategy. In this respect, the term "controllable" is understood as "controllable for carbon source limitation" or "controllable for glucose limitation", and refers to the derepression of a promoter by carbon consumption, reduction, shortage or exhaustion, or by limited addition of a carbon source so that it is readily consumed by the cell.

A functionally active promoter according to the invention is a relatively strong regulatable promoter which is silenced or repressed under cell growth conditions (growth phase) and activated or derepressed under production conditions (production phase) and is therefore suitable for inducing POI production in a producer cell line by carbon source limitation. Thus, functionally active variants of the promoter have at least such controllable properties.

The strength of a regulatable promoter according to the invention refers to its transcription strength, represented by the efficiency of initiation of transcription that occurs at the promoter at a high or low frequency. The higher the intensity of transcription, the more frequently transcription will occur at that promoter. Promoter strength is important because it determines how frequently a given mRNA sequence is transcribed, effectively giving some genes a higher priority for transcription than others, resulting in a higher concentration of transcripts. For example, genes encoding large amounts of a desired protein often have relatively strong promoters. RNA polymerases can only perform one transcription task at a time and therefore must prioritize their work to be efficient. The difference in promoter strength was chosen to allow for this preference. According to the present invention, the regulatable promoter is relatively strong in a fully induced state, which is generally understood to be a state of about maximal activity. The relative strength is usually determined against a standard promoter, such as the corresponding pGAP promoter of the cell used as host cell. The transcription frequency is generally understood as the rate of transcription, e.g. as determined by the amount of transcripts in a suitable assay, e.g. RT-PCR or Northern blot. For example, the transcriptional strength of the promoter according to the present invention was determined in a host cell that is pichia pastoris and compared to the natural pGAP promoter of pichia pastoris.

The pGAP promoter promotes the expression of the gap gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a constitutive promoter present in any microorganism capable of growing on glucose. GAPDH (EC 1\2\1\12), a key enzyme in glycolysis, plays a crucial role in catabolism and anabolic sugar metabolism.

The regulatable promoters according to the present invention exert a relatively high transcription intensity, which is reflected by a transcription rate or transcription intensity of at least 15% compared to the native pGAP promoter (sometimes referred to as the "homologous pGAP promoter") in the host cell. Preferably, the transcription rate or intensity is at least 20%, in particularly preferred cases at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and at least 100% or even higher, such as at least 150% or at least 200%, compared to the native pGAP promoter, e.g. as determined in a eukaryotic cell selected as the host cell for the production of the POI.

Particularly preferred is a regulatable promoter which, in the induced state, has the transcription strength of at least one of the promoters pG1, pG3, PG4, pG6, pG7 or pG 8. Comparative transcription intensities using the pGAP promoter as reference can be determined by standard means, e.g.by measuring the amount of transcript, for example using microarrays, or in cell culture, e.g.by measuring the amount of the corresponding gene expression product in recombinant cells. Exemplary tests are exemplified in the examples section.

In particular, the promoter according to the invention is a carbon source controllable promoter with a differential promoter strength, as determined in a test comparing the strength in the presence of glucose and glucose limitation, showing that it is still repressed at relatively high glucose concentrations, preferably at a concentration of at least 10g/L, preferably at least 20 g/L. In particular, the promoter according to the invention is fully induced at a limited glucose concentration and a glucose threshold concentration for fully inducible promoters, which threshold is below 20g/L, preferably below 10g/L, below 1g/L, even below 0.1g/L or below 50mg/L, preferably at a glucose concentration below 40mg/L with a full transcription strength of e.g.at least 50% of the native, homologous pGAP promoter.

Preferably, differential promoter strength is determined by initiating POI production when switching to inducing conditions below a predetermined carbon source threshold and comparing to the strength in repressive state. The transcription intensity is generally understood to be the intensity in the fully induced state, i.e.exhibiting about the maximum activity under derepression conditions. Differential promoter strength is, for example, measured in terms of efficiency or yield of POI production in the recombinant host cell line under derepressed conditions as compared to repressing conditions, or by the amount of transcript. The regulatable promoter according to the invention has a preferred differential promoter strength which is at least 2-fold, more preferably at least 5-fold, even more preferably at least 10-fold, more preferably at least 20-fold, more preferably at least 30, 40, 50 or 100-fold in the derepressed state compared to the repressed state, also understood as fold induction. Such differential promoter strengths can be determined by tests as exemplified by the accompanying examples.

The promoters of the prior art (MLS1 promoter or ICL1 promoter) appeared to have a differential promoter strength significantly lower than 2-fold induction. Such prior art promoters are also not useful for industrial POI production, with promoter strengths of about 5% compared to the pGAP promoter standard. This has been demonstrated in direct comparison with the promoters according to the invention.

The term "homology" refers to two or more nucleotide sequences having identical or conserved base pairs at corresponding positions to a particular degree, up to a degree approaching 100%. Homologous sequences typically have at least about 50% nucleotide sequence identity, preferably at least about 60% identity, more preferably at least about 70% identity, more preferably at least about 80% identity, more preferably at least about 90% identity, more preferably at least about 95% identity.

The homologous promoter sequence according to the present invention preferably has a specific homology with any of the pichia pastoris pG1, pG3, pG4, pG6, pG7 or pG8 promoter nucleotide sequences at least a specific part of the nucleotide sequence, such as a part comprising the 3 'region of the respective promoter nucleotide sequence, preferably a part having a specific length up to the 3' end of the respective promoter nucleotide sequence, such as a part having a length of at least 200bp, preferably at least 250bp, preferably at least 300bp, more preferably at least 400bp, at least 500bp, at least 600bp, at least 700bp, at least 800bp, at least 900bp or at least 1000bp, and analogues derived from species other than pichia pastoris. In particular, at least those portions are preferably homologous within the range of 300-1000bp (comprising the 3' -terminal sequence of the nucleotide sequence of the corresponding promoter).

Similar sequences are typically derived from other species or strains. It is expressly understood that any similar promoter sequence of the present invention derived from a species other than pichia pastoris can comprise homologous sequences, i.e., sequences having particular homology as described herein. Thus, the term "homologous" may also encompass similar sequences. In another aspect, it is understood that the invention also refers to analogous sequences and homologues thereof comprising a specific homology.

"percent (%) identity" is defined in terms of the nucleotide sequence of a gene as the percentage of nucleotides in a candidate DNA sequence that are identical to the nucleotides in the DNA sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent nucleotide sequence identity can be performed in a variety of ways within the skill of the art, for example using publicly available computer software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to obtain maximum alignment over the full length of the sequences being compared.

As used in the context of the present invention, the term "mutagenesis" refers to a method of providing a mutant of a nucleotide sequence, e.g. via insertion, deletion and/or substitution of one or more nucleotides, thereby obtaining a variant thereof having at least one change in a non-coding or coding region. Mutagenesis may be achieved via random, semi-random or site-directed mutagenesis. Large randomized gene libraries with high gene diversity are often generated, which can be selected according to a particular desired genotype or phenotype.

As used herein, the term "protein of interest (POI)" refers to a polypeptide or protein produced in a host cell by recombinant techniques. More specifically, the protein may be a polypeptide that does not naturally occur in the host cell, i.e., a heterologous protein, or may be native to the host cell, i.e., a protein that is homologous to the host cell, but is produced, for example, by transformation with a self-replicating vector containing the nucleic acid sequence encoding the POI, or by integration of one or more copies of the nucleic acid sequence encoding the POI into the genome of the host cell by recombinant techniques, or by recombinant modification of one or more regulatory sequences (e.g., promoter sequences) that control expression of the gene encoding the POI. In some cases, as used herein, the term POI also refers to any metabolite of the host cell, as mediated by a recombinantly expressed protein.

As used herein, the term "recombinant" means "made by or the result of genetic engineering. Thus, a "recombinant microorganism" comprises at least one "recombinant nucleic acid". The recombinant microorganism comprises in particular an expression vector or a cloning vector, or it has been genetically engineered to contain a recombinant nucleic acid sequence. A "recombinant protein" is produced by expressing the corresponding recombinant nucleic acid in a host. A "recombinant promoter" is a genetically engineered non-coding nucleotide sequence that is suitable for use as a functionally active promoter as described herein.

Surprisingly, it was found that eukaryotic cells can induce POI production by limiting the availability of carbon sources. It was found that carbon starvation conditions trigger induction of strong promoter activity, which was previously unknown in the art. The Pichia pastoris MLS1 promoter repressed under sugar restriction as described in US2008299616A1 is in fact a comparably weaker regulatable promoter for POI production. Thus, surprisingly, such strongly regulatable promoters of pichia pastoris can be identified and can be used in eukaryotic production cell lines, in particular for recombinant POI production.

Although the 9.43Mbp genomic sequence of the Pichia pastoris GS115 strain has been determined and disclosed in US20110021378A1, the properties of the individual sequences, such as the promoter sequence, have not been investigated in detail. For example, the pG4 sequence (SEQ ID 4) as described herein was identified as a promoter sequence in US20110021378a1, however, its regulatory properties or its use under carbon starvation conditions are unknown. Even surprisingly, such promoters can be effectively used in the method according to the invention. The regulated promoters in the prior art, as used in commercial scale POI production, are derived primarily from methanol metabolic pathways and require the addition of methanol to induce POI production, which is often undesirable. The advantage of the method according to the invention is that it can provide increased production by enhanced expression and has a reduced risk of contamination due to specific promoter regulation, especially when using chemically defined methanol-free media.

It was found that the regulatable promoter according to the invention exerts its regulatable activity only when very specific media suitable for establishing repressing and derepressing conditions of the promoter are used. For example, Pichia pastoris can be successfully cultured under industrial process conditions. First a batch culture on a basic carbon source such as glycerol is used, followed by a fed-batch with limited feed of a supplemental carbon source such as glucose. Samples were taken near the end of the first batch phase and in limited growth conditions (e.g., using a limited amount of supplemental carbon source). Transcriptome analysis using DNA microarrays revealed specific genes that were strongly active on supplemental carbon sources and were weaker or inactive in the presence of excess carbon (i.e., excess base carbon source). At least 6 promoter sequences were identified as regulatable promoters according to the invention, namely pG1(SEQ ID 1), pG3(SEQ ID2), pG4(SEQ ID 4), pG6(SEQ ID 3), pG7(SEQ ID 5) and pG8(SEQ ID 6). The comparable MLS1 or ICL1 promoters in the prior art are only weak, have a lower strength than the pG1 promoter 1/10, and have no detectable regulation.

The characteristics of repressible recombinant gene expression on basal carbon sources, and strong expression (i.e., induced by substrate changes) on limited supplemental carbon sources can be verified in fermentation processes.

Nucleotide sequences that can be used as control sequences according to the present invention, which will provide improved recombinant protein production, are available from a variety of sources. The promoter according to the invention is preferably derived from a yeast cell, most preferably from a methylotrophic yeast such as a species from pichia or from pichia pastoris, which promoter can then be used as a parent sequence to generate suitable variants, e.g. mutants or the like.

A yeast cell covering a range of yeast cells, in particular pichia strains, may be suitable for obtaining the corresponding promoter sequence responsible for protein production under carbon starvation conditions, or the corresponding analogue in different species.

Variants of the identified pichia pastoris promoter, including functionally active variants such as homologues and analogues, can be produced using standard techniques. Promoters can be modified, for example, to generate promoter variants with altered expression levels and regulatory properties.

For example, a promoter library can be prepared by mutagenesis of a promoter sequence according to the invention, which can be used as a parent molecule, for example, to fine-tune gene expression in eukaryotic cells by analyzing the expression of the variants under different fermentation strategies and selecting suitable variants. Synthetic libraries of variants can be used, for example, to select promoters that meet the requirements for producing a selected POI. Such variants may have increased expression efficiency in eukaryotic host cells and high expression upon depletion of a carbon source.

The differential fermentation strategy will distinguish between a growth phase, such as step a) according to the invention, and a production phase, such as step b).

Growth and/or production can suitably take place in batch mode, fed-batch mode or continuous mode. Any suitable bioreactor may be used, including batch, fed-batch, continuous, stirred-cell reactors or airlift reactors.

It would be advantageous to provide a fermentation process on a pilot plant (pilot) or industrial scale. The industrial process scale will preferably employ a volume of at least 10L, in particular at least 50L, preferably at least 1m³Preferably at least 10m³Most preferably at least 100m³。

Preference is given to production conditions on an industrial scale, which means, for example, in the range from 100L to 10m³Fed-batch culture in reactor volumes of greater or typical process times of several days, or continuous processes in fermenter volumes of about 50-1000L or greater, for about 0.02-0.15 h^-1The dilution rate of (c).

Suitable culturing techniques may encompass culturing in a bioreactor starting with a batch phase, followed by a shorter exponential fed-batch phase at a particular high growth rate, followed by a fed-batch phase at a particular low growth rate. Another suitable culture technique may encompass a batch phase followed by a continuous culture phase at a low dilution rate.

A preferred embodiment of the invention comprises batch culture to provide biomass followed by fed-batch culture to obtain high yield POI production.

For example, the cell line may be grown on glycerol or glucose in step a) according to the invention to obtain biomass.

The host cell line according to the invention is preferably cultured in a bioreactor under growth conditions to obtain a cell density of at least 1g/L dry cell weight, more preferably at least 10g/L dry cell weight, preferably at least 20g/L dry cell weight. It would be advantageous to provide yields of such biomass production on a pilot plant or industrial scale.

Growth media, particularly basal growth media, that allow biomass to accumulate typically contain a carbon source, a nitrogen source, a sulfur source, and a phosphorus source. Typically, such media also comprise trace elements and vitamins, and may also comprise amino acids, peptones or yeast extract.

Preferred nitrogen sources include NH₄H₂PO₄Or NH₃Or (NH4)₂SO₄；

Preferred sulfur sources include MgSO₄Or (NH4)₂SO₄Or K₂SO₄；

Preferred phosphorus sources include NH₄H₂PO₄Or H₃PO₄Or NaH₂PO₄、KH₂PO₄、Na₂HPO₄Or K₂HPO₄；

Other typical media components include KCl, CaCl₂And trace elements such as: fe. Co, Cu, Ni, Zn, Mo, Mn, I, B;

preferably the medium is vitamin B₇Supplementing;

typical growth media for Pichia pastoris contain glycerol or glucose, NH₄H₂PO₄、MgSO₄、KCl、CaCl₂Biotin and trace elements.

In the production phase, in particular production media are used which have only a limited amount of supplemental carbon sources.

Preferably, the host cell line is cultured in mineral medium with a suitable carbon source, thereby further simplifying the isolation process significantly. An example of a preferred mineral medium is a medium containing: carbon sources (e.g. glucose, glycerol or methanol), salts containing macro-elements (potassium, magnesium, calcium, ammonium, chloride, sulphur, phosphorus) and trace elements (ketones, iodides, manganese, molybdates, cobalt, zinc, and iron salts, and boric acid), and optionally vitamins or amino acids, for example to compensate for auxotrophy.

The cells are cultured under conditions suitable to effect expression of the desired POI, which can be purified from the cells or the culture medium, depending on the expression system and the nature of the protein expressed, e.g., whether the protein is fused to a signal peptide and whether the protein is soluble or membrane bound. As will be appreciated by the skilled artisan, the culture conditions will vary depending upon factors including the type of host cell and the particular expression vector employed.

Preferably, induction of POI production by a promoter according to the invention is controlled by culturing the cells on a limited amount of a supplemental carbon source, which is the sole source of carbon and energy. Cells grow very slowly under carbon-limited conditions, but produce high yields of POI under the control of a regulatable promoter.

In particular, the difference in promoter activity in the derepressed state compared to the repressed state is at least 2-fold, preferably at least 5-fold, more preferably at least 10-fold, more preferably at least 20-fold, more preferably at least 30, 40, 50 or 100-fold.

By selecting suitable promoter sequences according to the invention, optionally in combination with other preferred regulatory sequences, it is possible to provide at least about the same, or at least about 1.5-fold, or at least about 2-fold, or at least about 5-fold, 10-fold, or at least up to about 15-fold activity (as represented by promoter activity or transcription strength, or as regulated by promoter strength) under comparable conditions relative to the GAP promoter homologous to the producer cell, the native pGAP, or the GAP promoter isolated from pichia pastoris.

A typical production medium contains a supplemental carbon source, and additional NH₄H₂PO₄、MgSO₄、KCl、CaCl₂Biotin and trace elements.

For example, a feed of supplemental carbon source added to the fermentation may comprise a carbon source having up to 50 wt% fermentable sugars. The low feed rate of the supplemented medium will limit the effect of product inhibition on cell growth, so that a high product yield based on substrate provision will be possible.

Preferably, the fermentation is carried out at a pH of from 3 to 7.5.

Typical fermentation times are about 24 to 120 hours, and temperatures in the range of 20 ℃ to 35 ℃, preferably 22-30 ℃.

In general, recombinant nucleic acids or organisms as referred to herein can be produced by recombinant techniques well known to those skilled in the art. In accordance with the present invention, conventional molecular biology, microbiology and recombinant DNA techniques within the skill of the art may be employed. Such techniques are fully explained in the literature. See, e.g., Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual" (1982).

According to a preferred embodiment of the invention, the recombinant construct is obtained by ligating the promoter and the gene concerned into a vector. These genes can be stably integrated into the host cell genome by transforming the host cell with such vectors.

Expression vectors can include, but are not limited to, cloning vectors, modified cloning vectors, and specifically designed plasmids. The preferred expression vector as used in the present invention may be any expression vector suitable for expressing a recombinant gene in a host cell and is selected according to the host organism. A recombinant expression vector may be any vector which is capable of replication or integration into the genome of a host organism, also referred to as host vector.

In the present invention, a plasmid derived from pPUZZLE is preferably used as the vector.

Suitable expression vectors typically comprise additional regulatory sequences suitable for expression of the DNA encoding the POI in a eukaryotic host cell. Examples of regulatory sequences include operators, enhancers, ribosome binding sites, and sequences that control the initiation and termination of transcription and translation. The control sequences may be operably linked to the DNA sequence to be expressed.

To allow expression of the recombinant nucleotide sequence in a host cell, the expression vector may provide a promoter according to the invention adjacent to the 5' end of the coding sequence, e.g. upstream of the signal peptide gene. Thus, transcription is regulated and initiated by the promoter sequence.

The signal peptide may be a heterologous signal peptide or a hybrid of native and heterologous signal peptides, and may in particular be heterologous or homologous to the host organism producing the protein. The function of the signal peptide is to allow the POI to be secreted into the endoplasmic reticulum. It is generally a short (3-60 amino acid long) peptide chain that directs protein transport outside the plasma membrane, thereby making it easy to isolate and purify the heterologous protein. Some signal peptides are cleaved from the protein by signal peptidases after transport of the protein.

Exemplary signal peptides are the signal sequence from the Saccharomyces cerevisiae alpha-mating factor prepropeptide and the signal peptide from the Pichia pastoris acid phosphatase gene (PHO 1).

A promoter sequence is understood to be operably linked to a coding sequence if the promoter controls the transcription of the coding sequence. If the promoter sequence is not naturally associated with the coding sequence, its transcription is not under the control of the promoter in the native (wild-type) cell, or the sequence is recombined with a different contiguous sequence.

To confirm the function of the relevant sequences, expression vectors containing one or more regulatory elements can be constructed to drive expression of the POI and to compare the yield of expression to constructs with conventional regulatory elements. A detailed description of the experimental protocol can be found in the examples below. The identified genes can be amplified by PCR from pichia pastoris using specific nucleotide primers, cloned into expression vectors and transformed into eukaryotic cell lines, e.g., using yeast vectors and strains of pichia pastoris, for high level production of a variety of different POIs. To assess the influence of the promoters according to the invention on the amount of recombinant POI thus produced, the eukaryotic cell lines can be cultured in shake flask experiments and fed-batch or chemostat fermentations and compared with strains comprising conventional non-carbon-source-regulatable promoters, such as for example the standard pGAP promoter in the corresponding cell. In particular, the choice of promoter has a great influence on recombinant protein production.

Preferred transformation methods for microbial uptake of recombinant DNA fragments include chemical transformation, electroporation or transformation by protoplast formation (proplastation). The transformant according to the present invention can be obtained by introducing such vector DNA, for example, plasmid DNA, into a host and selecting a transformant that expresses the relevant protein or a metabolite of the host cell at a high yield.

The POI can be produced using a recombinant host cell line, by culturing the transformant, thus obtained in a suitable medium, isolating the expressed product or metabolite from the culture, and optionally purifying it by a suitable method.

The transformant according to the present invention can be obtained by introducing such vector DNA, for example, plasmid DNA, into a host and selecting a transformant that expresses the POI or the metabolite of the host cell at a high yield. The host cell is treated to be able to incorporate foreign DNA by methods conventionally used for transforming eukaryotic cells, such as the electric pulse method, the protoplast method, the lithium acetate method and methods modified therefrom. Preferably, pichia pastoris is transformed by electroporation.

Preferred host cell lines according to the invention maintain the genetic profile employed according to the invention and the production level remains high, e.g. at least at the μ g level, even after culture for about 20 generations, preferably at least 30 generations, more preferably at least 40 generations, most preferably at least 50 generations. Stable recombinant host cells are considered to be a great advantage when used for industrial scale production.

Several different approaches for producing POIs according to the method of the invention are preferred. By transforming eukaryotic host cells with an expression vector carrying a recombinant DNA encoding a protein of interest and at least one regulatory element as described above, preparing a culture of transformed cells, growing the culture, inducing transcription and POI production, and recovering the products of the fermentation process, the substance can be expressed, processed and optionally secreted.

Preferably, the POI is expressed under conditions that result in a yield of at least 1mg/L, preferably at least 10mg/L, preferably at least 100mg/L, and most preferably at least 1 g/L.

Preferably, the host cell according to the invention is tested for expression capacity or yield by: ELISA, activity assay, HPLC, or other suitable test.

It is understood that the methods disclosed herein may further comprise culturing the recombinant host cell, preferably in secreted form or as an intracellular product, under conditions that allow expression of the POI. The recombinantly produced POI or host cell metabolite can then be isolated from the cell culture medium and further purified by techniques well known to those skilled in the art.

The POI produced according to the present invention can generally be isolated and purified using techniques known in the art, including increasing the concentration of the desired POI and/or decreasing the concentration of at least one impurity.

If the POI is secreted from the cell, it can be isolated and purified from the culture medium using techniques known in the art. Secretion of the recombinant expression product from the host cell is generally advantageous for reasons including facilitating the purification process, since the product is recovered from the culture supernatant rather than from a complex protein mixture, which is produced upon disruption of the yeast cell to release intracellular proteins.

The cultured transformant cells may also be sonicated or mechanically, enzymatically or chemically disrupted to obtain a cell extract containing the desired POI, from which the POI is isolated and purified.

Separation and purification methods for obtaining a recombinant polypeptide or protein product, such as methods utilizing difference in solubility such as salting out and solvent precipitation, methods utilizing difference in molecular weight such as ultrafiltration and gel electrophoresis, methods utilizing difference in charge such as ion exchange chromatography, methods utilizing specific affinity such as affinity chromatography, methods utilizing difference in hydrophobicity such as reversed-phase high-performance liquid chromatography, and methods utilizing difference in isoelectric point such as isoelectric focusing, can be used.

The highly purified product is substantially free of contaminating proteins and preferably has a purity of at least 90%, more preferably at least 95%, or even at least 98%, up to 100%. The purified product may be obtained by purifying the cell culture supernatant or from cell debris.

As the separation and purification method, the following standard methods are preferred: cell disruption if the POI is obtained intracellularly, cell (debris) separation and washing by microfiltration or Tangential Flow Filter (TFF) or centrifugation, purification of the POI by precipitation or heat treatment, POI activation by enzymatic digestion, purification of the POI by chromatography such as Ion Exchange (IEX), Hydrophobic Interaction Chromatography (HIC), affinity chromatography, Size Exclusion (SEC) or HPLC chromatography, POI concentration precipitation and washing by ultrafiltration steps (POI concentration of concentration and washing by ultrafiltration steps).

The isolated and purified POI can be identified by conventional methods such as Western blot, HPLC, activity assay or ELISA.

The POI can be any eukaryotic, prokaryotic, or synthetic polypeptide. It may be a secreted protein or an intracellular protein. The invention also provides for the recombinant production of functional homologues, functionally equivalent variants, derivatives and biologically active fragments of naturally occurring proteins. Preferably, the functional homologue is identical or corresponds to the sequence and has a functional feature of the sequence.

The POI referred to herein may be a product homologous or heterologous to the eukaryotic host cell, which is preferably used for therapeutic, prophylactic, diagnostic, analytical or industrial use.

Preferably, the POI is a heterologous recombinant polypeptide or protein, which is produced in a eukaryotic cell (preferably a yeast cell), preferably as a secreted protein. Examples of proteins which are preferably produced are immunoglobulins, immunoglobulin fragments, aprotinin (aprotinin), tissue Factor pathway inhibitors or other protease inhibitors, and insulin or insulin precursors, insulin analogues, growth hormones, interleukins, tissue plasminogen activator, transforming growth factors a or b, glucagon-like peptide 1(GLP-1), glucagon-like peptide 2(GLP-2), GRPP, Factor VII, Factor VIIl, Factor XIII, platelet-derived growth Factor 1, serum albumin, enzymes such as lipases or proteases, or functional homologues, functionally equivalent variants, derivatives or biologically active fragments having a similar function to the native protein. The POI may be structurally similar to the native protein and may be derived from the native protein by the addition of one or more amino acids at one or both or side chains at the C and N terminal ends of the native protein, substitution of one or more amino acids at one or many different positions in the native amino acid sequence, deletion of one or more amino acids at either or both ends of the native protein or at one or several positions in the amino acid sequence, or insertion of one or more amino acids at one or more positions in the native amino acid sequence. Such modifications are well known for several of the proteins mentioned above.

The POI may be selected from the group consisting of substrates, enzymes, inhibitors or cofactors providing a biochemical reaction in the host cell with the purpose of obtaining products of said biochemical reaction or of several reaction cascades, e.g. obtaining metabolites of the host cell. Exemplary products may be vitamins, such as riboflavin, organic acids and alcohols, which can be obtained in increased yield after expression of a recombinant protein or POI according to the invention.

In general, the host cell expressing the recombinant product may be any eukaryotic cell suitable for recombinant expression of the POI.

Examples of preferred mammalian cells are BHK, CHO (CHO-DG44, CHO-DUXB11, CHO-DUKX, CHO-K1, CHOK1SV, CHO-S), HeLa, HEK293, MDCK, NIH3T3, NS0, PER. C6, SP2/0 and VERO cells.

Examples of preferred yeast cells for use as host cells according to the invention include, but are not limited to, Saccharomyces (e.g., Saccharomyces cerevisiae), Pichia (e.g., Pichia pastoris or Pichia methanolica), Komagataella (K.pastoris, K.pseudoopsis or K.phaffii), Hansenula polymorpha or Kluyveromyces lactis.

More recent literature has divided and renamed Pichia pastoris, Komagataella phaffii and Komagataella pseudophosphatris. Pichia pastoris is used synonymously herein for all Komagataella pastoris, Komagataella phaffii and Komagataella pseudophora.

Preferred yeast host cells are derived from methylotrophic yeasts such as those from pichia pastoris or Komagataella, e.g. pichia pastoris or Komagataella pastoris or k. Examples of hosts include yeasts such as Pichia pastoris. Examples of Pichia pastoris strains include CBS 704 (NRRL Y-1603-DSMZ 70382), CBS2612 (NRRL Y-7556), CBS 7435 (NRRL Y-11430), CBS 9173-9189(CBS strain: CBS-KNAW Fungal Biodiversity Centre, Central nerve voor Schimmelcultures, Ultrecht, The Netherlands), and DSMZ 70877(German Collection of Micro-organisms and cells), as well as strains from Invitron, such as X-33, GS115, KM71 and SMD 1168. Examples of Saccharomyces cerevisiae strains include W303, CEN.PK and the BY series (collection EUROSCARF). All of the above-described strains have been successfully used to produce transformants and express heterologous genes.

In accordance with the present invention, preferred yeast host cells, such as Pichia pastoris or Saccharomyces cerevisiae host cells, contain heterologous or recombinant promoter sequences, which may be derived from a Pichia pastoris or Saccharomyces cerevisiae strain different from the production host. In another particular embodiment, the host cell according to the invention comprises a recombinant expression construct according to the invention comprising a promoter derived from the same genus, species or strain as the host cell.

The promoter may be a promoter according to the present invention or any other DNA sequence showing transcriptional activity in the host cell, and may be derived from genes encoding proteins either homologous or heterologous to the host. Preferably, the promoter is derived from a gene encoding a protein homologous to the host cell.

For example, a promoter according to the present invention may be derived from a yeast, such as a strain of saccharomyces cerevisiae, and may be used to express a POI in a yeast. A particularly preferred embodiment relates to a method of using a promoter according to the invention derived from pichia pastoris for the production of a recombinant POI in a pichia pastoris producer host cell line. The homologous origin of the nucleotide sequence facilitates its incorporation into a host cell of the same genus or species, thus enabling stable production of the POI, possibly in increased yield in an industrial manufacturing process. Also, functionally active variants of promoters from other suitable yeasts or other fungi or other organisms such as vertebrates or plants may be used.

If the POI is a protein that is homologous to the host cell, i.e.a protein that is naturally present in the host cell, then expression of the POI in the host cell can be regulated by exchanging its native promoter sequence for a promoter sequence according to the present invention.

This object can be achieved, for example, by transforming a host cell with a recombinant DNA molecule comprising a homologous sequence of a target gene to allow site-specific recombination, a promoter sequence and a selectable marker suitable for use in a host cell. This site-specific recombination should occur so that the promoter sequence is operably linked to the nucleotide sequence encoding the POI. This results in expression of the POI from the promoter sequence according to the invention rather than from the native promoter sequence.

In a particularly preferred embodiment of the invention, the promoter sequence has increased promoter activity relative to the native promoter sequence of the POI.

According to the present invention, preferably a pichia pastoris host cell line is provided comprising a promoter sequence according to the present invention operably linked to a nucleotide sequence encoding a POI.

According to the present invention there may also be provided a wildcard vector or host cell according to the invention which comprises a promoter according to the invention and which is ready for incorporation of a gene of interest encoding a POI. Thus, the wildcard cell line is a preformed host cell line characterized by its expression capacity. This is in accordance with an innovative "wildcard" platform strategy for generating producer cell lines to generate POIs, for example using site-specific recombinase mediated cassette exchange. Such new host cells facilitate cloning of a gene of interest (GOI), for example, into a predetermined genomic expression hotspot within days to obtain a reproducible, efficient production cell line.

According to a preferred embodiment, the method according to the invention employs recombinant nucleotide sequences encoding a POI, provided as single or multiple copies per cell on a plasmid suitable for integration into the genome of a host cell. The recombinant nucleotide sequence encoding the POI may also be provided in single or multiple copies per cell on autonomously replicating plasmids.

Preferred methods according to the invention employ plasmids which are eukaryotic expression vectors, preferably yeast expression vectors. Expression vectors can include, but are not limited to, cloning vectors, modified cloning vectors, and specifically designed plasmids. Preferred expression vectors for use in the present invention may be any expression vector suitable for expression of a recombinant gene in a host cell and selected according to the host organism. The recombinant expression vector may be any vector (which is also referred to as host vector) capable of replication or integration into the genome of the host organism, such as a yeast vector, carrying the DNA construct according to the invention. Preferred yeast expression vectors are for expression in yeast selected from the group consisting of: methylotrophic yeasts represented by Hansenula, Pichia, Candida and Torulopsis.

In the present invention, plasmids derived from pPICZ, pGAPZ, pPIC9, pPICZalfa, pGAPZalfalfa, pPIC9K, pGAPHis or pPUZZLE are preferably used as the vector.

According to a preferred embodiment of the invention, the recombinant construct is obtained by ligating the relevant gene into a vector. These genes can be stably integrated into the host cell genome by transforming the host cell with such vectors. The polypeptides encoded by the genes can be produced using recombinant host cell lines, by culturing the transformants, thus obtained in a suitable medium, isolating the expressed POI from the culture and purifying it by methods suitable for expressing the product, in particular separating the POI from contaminating proteins.

The expression vector may comprise one or more phenotypic selectable markers, such as genes encoding proteins that confer antibiotic resistance or supply autotrophic requirements. Yeast vectors commonly contain an origin of replication, an Autonomously Replicating Sequence (ARS) or sequences for integration into the host genome, a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker from a yeast plasmid.

Protocols for ligating DNA sequences, e.g., the DNA sequence encoding the precursor sequence and/or POI, promoter and terminator, respectively, and inserting them into suitable vectors containing the information required for integration or host replication are well known to those skilled in the art, e.g., as described by J.Sambrook et al, "Molecular Cloning 2nd ed.", Cold Spring Harbor Laboratory Press (1989).

It will be appreciated that vectors using the regulatory elements and/or POIs according to the invention as integration targets may be constructed by first preparing a DNA construct containing the entire DNA sequence encoding the regulatory element and/or POI, and then inserting this fragment into a suitable expression vector, or by successively inserting DNA fragments containing the genetic information of the respective element (e.g.signal, leader or heterologous protein) followed by ligation.

A polyclonal vector, which is a vector having multiple cloning sites at which desired heterologous genes can be incorporated to provide an expression vector, can also be used in accordance with the present invention. In the expression vector, a promoter is placed upstream of the gene of POI and regulates the expression of the gene. In the case of a polyclonal vector, since the gene of POI is introduced at the multiple cloning site, a promoter is placed upstream of the multiple cloning site.

The DNA construct as provided to obtain a recombinant host cell according to the invention may be prepared synthetically by established standard methods, for example the phosphoramidite (phosphoramidite) method. The DNA construct may also be of genomic or cDNA origin, for example, obtained by preparing a genomic or cDNA library and screening for DNA sequences encoding all or part of a polypeptide of the invention by hybridization using synthetic oligonucleotide probes according to standard techniques (Sambrook et al, Molecular Cloning: A laboratory Manual, Cold Spring Harbor, 1989). Finally, the DNA construct may be of mixed synthetic and genomic, mixed synthetic and cDNA, or mixed genomic and cDNA origin, prepared by appropriately annealing fragments of synthetic, genomic or cDNA origin (which correspond to various parts of the complete DNA construct) in accordance with standard techniques.

In another preferred embodiment, the yeast expression vector is capable of stable integration into the yeast genome, e.g., by homologous recombination.

Preferably, a transformant host cell according to the present invention obtained by transforming a cell with a regulatory element and/or POI gene according to the present invention can be first cultured under conditions effective to grow to a large number of cells without the burden of expressing a heterologous protein. When the cell line is ready for POI expression, culture techniques are selected to produce the expression product.

The following defined subjects are to be considered as embodiments of the invention:

1. a method for producing a protein of interest (POI) by culturing a recombinant eukaryotic cell line comprising an expression construct, said expression vector comprising a regulatable promoter and a nucleic acid molecule encoding the POI under the transcriptional control of said promoter, said method comprising the steps of:

a) culturing said cell line with a basal carbon source repressing said promoter,

b) culturing the cell line under conditions of no supplemental carbon source or limited supplemental carbon source that derepress the promoter, thereby inducing the POI to be produced at a transcription rate of at least 15% compared to the cell's native pGAP promoter, and

c) generating and recovering the POI.

2. The method according to item 1, wherein the base carbon source is selected from the group consisting of: glucose, glycerol, ethanol, and complex nutritional materials.

3. The method according to item 1 or 2, wherein the supplemental carbon source is a hexose such as glucose, fructose, galactose or mannose, a disaccharide such as sucrose, an alcohol such as glycerol or ethanol, or a mixture thereof.

4. The method according to any one of items 1 to 3, wherein the base carbon source is glycerol and the supplemental carbon source is glucose.

5. The method according to any one of items 1 to 4, wherein step b) employs a feed medium that does not provide or in limited amounts said supplemental carbon source, preferably 0-1g/L in said medium.

6. The method according to clause 5, wherein the feed medium is chemically defined and methanol-free.

7. The method according to any one of items 1 to 6, wherein the limited amount of supplemental carbon source is growth-limiting to maintain a range of 0.02h^-1To 0.2h^-1Preferably 0.02h^-1To 0.15h^-1Specific growth rate of the inner layer.

8. The method according to clause 7, wherein the limited amount of supplemental carbon source provides a residual amount in the cell culture that is below the detection limit.

9. The method according to any one of items 1 to 8, wherein the promoter is capable of controlling transcription of a gene selected from the group consisting of: g1(SEQ ID 7), G3(SEQ ID 8), G4(SEQ ID 9), G6(SEQ ID 10), G7(SEQ ID 11) or G8(SEQ ID 12), or a functionally active variant thereof.

10. The method according to item 9, wherein the functionally active variant is selected from the group consisting of: homologues having at least about 60% nucleotide sequence identity, homologues modified by insertion, deletion or substitution of one or more nucleotides within the parent nucleotide sequence or at one or both of the distal ends of the sequence, preferably nucleotide sequences comprising or consisting of at least 200bp, and analogues derived from species other than Pichia pastoris.

11. A method according to item 9 or 10, wherein the functionally active variant of pG1 is selected from the group consisting of: pG1a (SEQ ID 41), pG1b (SEQ ID 42), pG1c (SEQ ID 43), pG1d (SEQ ID 44), pG1e (SEQ ID 45) and pG1f (SEQ ID 46).

12. The method according to any one of items 1 to 11, wherein the promoter is a pichia pastoris promoter or a functionally active variant thereof.

13. The method according to any one of items 1 to 12, wherein the cell line is selected from the group consisting of: mammalian, insect, yeast, filamentous fungi and plant cell lines, preferably yeast.

14. The method according to item 13, wherein the yeast is selected from the group consisting of: pichia (Pichia), Candida (Candida), Torulopsis (Torulopsis), Arxula, Hansenula (Hensenula), Yarrowia (Yarrowia), Kluyveromyces (Kluyveromyces), Saccharomyces (Saccharomyces), Komagataella, preferably methylotrophic yeasts.

15. The process according to item 14, wherein the yeast is pichia pastoris, Komagataella pastoris, k.

16. A method according to any one of claims 1 to 15 wherein the promoter is not naturally associated with the nucleotide sequence encoding the POI.

17. The method according to any one of items 1 to 16, wherein the POI is a heterologous protein, preferably selected from the group consisting of therapeutic proteins including antibodies or fragments thereof, enzymes and peptides, protein antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccines and vaccine-like proteins or particles, processing enzymes, growth factors, hormones and cytokines, or metabolites of POI.

18. The method according to any one of claims 1 to 17, wherein the POI is a eukaryotic protein, preferably a mammalian protein.

19. The method according to any one of claims 1 to 18, wherein the POI is a multimeric protein, preferably a dimer or tetramer.

20. A method according to any one of claims 1 to 19 wherein the POI is an antigen binding molecule such as an antibody or fragment thereof.

21. The method according to any one of items 1 to 20, wherein the POI, metabolite or derivative thereof is used to produce a fermentation product.

22. A method for regulating expression of a POI in a recombinant eukaryotic cell under the transcriptional control of a carbon source-controllable promoter having a transcriptional strength of at least 15% compared to the cell's native pGAP promoter, wherein said expression is induced under conditions that restrict the carbon source.

23. A method for producing a POI under the transcriptional control of a carbon source-controllable promoter in a recombinant eukaryotic cell, wherein the promoter has a transcriptional strength of at least 15% compared to the cell's native pGAP promoter.

24. The method according to any one of items 1 to 23, wherein the regulatable promoter comprises a nucleic acid sequence selected from the group consisting of:

a) pG1(SEQ ID 1), pG3(SEQ ID2), pG4(SEQ ID 4), pG6(SEQ ID 3), pG7(SEQ ID 5), or pG8(SEQ ID 6);

b) a sequence having at least 60% homology to: pG1(SEQ ID 1), pG3(SEQ ID2), pG4(SEQ ID 4), pG6(SEQ ID 3), pG7(SEQ ID 5), or pG8(SEQ ID 6);

c) a sequence that hybridizes under stringent conditions to: pG1(SEQ ID 1), pG3(SEQ ID2), pG4(SEQ ID 4), pG6(SEQ ID 3), pG7(SEQ ID 5), or pG8(SEQ ID 6); and

d) fragments or variants derived from a), b) or c),

wherein the promoter is a functionally active promoter which is a carbon source regulatable promoter capable of expressing the POI in a recombinant eukaryotic cell at a transcription rate of at least 15% compared to the native pGAP promoter of the cell.

25. The method according to item 24, wherein the variant of pG1(SEQ ID 1), pG3(SEQ ID2), pG4(SEQ ID 4), pG6(SEQ ID 3), pG7(SEQ ID 5), or pG8(SEQ ID 6) is a functionally active variant selected from the group consisting of: homologues having at least about 60% nucleotide sequence identity, homologues modified by insertion, deletion or substitution of one or more nucleotides within the parent nucleotide sequence or at one or both of the distal ends of the sequence, preferably nucleotide sequences comprising or consisting of at least 200bp, and analogues derived from species other than pichia pastoris.

26. A method according to item 24 or 25, wherein the functionally active variant of pG1 is selected from the group consisting of: pG1a (SEQ ID 41), pG1b (SEQ ID 42), pG1c (SEQ ID 43), pG1d (SEQ ID 44), pG1e (SEQ ID 45) and pG1f (SEQ ID 46).

27. An isolated nucleic acid comprising a nucleic acid sequence selected from the group consisting of:

a) pG1(SEQ ID 1), pG3(SEQ ID2), pG6(SEQ ID 3), pG7(SEQ ID 5), or pG8(SEQ ID 6);

b) a sequence having at least 60% homology to: pG1(SEQ ID 1), pG3(SEQ ID2), pG6(SEQ ID 3), pG7(SEQ ID 5), or pG8(SEQ ID 6);

c) a sequence that hybridizes under stringent conditions to: pG1(SEQ ID 1), pG3(SEQ ID2), pG6(SEQ ID 3), pG7(SEQ ID 5), or pG8(SEQ ID 6); and

d) fragments or variants derived from a), b) or c),

wherein the nucleic acid comprises a functionally active promoter which is a carbon source regulatable promoter capable of expressing the POI in a recombinant eukaryotic cell at a transcription rate of at least 15% compared to the native pGAP promoter of the cell.

28. The nucleic acid according to item 27, wherein the variant of pG1(SEQ ID 1), pG3(SEQ ID2), pG6(SEQ ID 3), pG7(SEQ ID 5) or pG8(SEQ ID 6) is a functionally active variant selected from the group consisting of: homologues having at least about 60% nucleotide sequence identity, homologues modified by insertion, deletion or substitution of one or more nucleotides within the parent nucleotide sequence or at one or both of the distal ends of the sequence, preferably having a nucleotide sequence of at least 200bp, and analogues derived from species other than pichia pastoris.

29. A nucleic acid according to item 27 or 28, wherein the functionally active variant of pG1 is selected from the group consisting of: pG1a (SEQ ID 41), pG1b (SEQ ID 42), pG1c (SEQ ID 43), pG1d (SEQ ID 44), pG1e (SEQ ID 45) and pG1f (SEQ ID 46).

30. An expression construct comprising a nucleic acid according to any one of items 27 to 29 operably linked to a nucleotide sequence encoding a POI that is not naturally associated with the nucleotide sequence encoding the POI, under the transcriptional control of the promoter.

31. A vector comprising a construct according to item 30.

32. A recombinant eukaryotic cell comprising the construct of item 30, or the vector of item 31.

33. A cell according to item 31, selected from the group consisting of: mammalian, insect, yeast, filamentous fungi and plant cell lines, preferably yeast.

34. A cell according to item 32, wherein the yeast is selected from the group consisting of: pichia (Pichia), Candida (Candida), Torulopsis (Torulopsis), Arxula, Hansenula (Hensenula), Yarrowia (Yarrowia), Kluyveromyces (Kluyveromyces), Saccharomyces (Saccharomyces), Komagataella, preferably methylotrophic yeasts.

35. The cell according to item 34, wherein the yeast is pichia pastoris, Komagataella pastoris, k.

36. A cell according to any one of items 32 to 35 having a higher specific growth rate relative to a limited carbon source condition in the presence of excess carbon source.

37. A method for identifying a carbon source-controllable promoter from a eukaryotic cell, comprising the steps of:

a) culturing eukaryotic cells in batch culture in the presence of a carbon source under cell growth conditions,

b) further culturing the cells in fed-batch culture in the presence of a limited amount of a supplemental carbon source,

c) providing a sample of the cell culture of steps a) and b), and

d) performing a transcriptional analysis in said sample to identify a regulatable promoter that exhibits a higher transcriptional intensity in the cells of step b) than in the cells of step a).

38. The method according to item 37, wherein the transcriptional analysis is quantitative or semi-quantitative, preferably using DNA microarray, RNA sequencing and transcriptome analysis.

Specific examples relate to fed-batch fermentation of reporter protein-producing recombinant pichia pastoris cell lines using glycerol batch medium and glucose fed-batch medium. Comparative promoter activity studies have demonstrated that the promoter according to the invention can be successfully activated to induce recombinant protein production.

According to a separate example, Human Serum Albumin (HSA) was produced as POI under the control of a glucose-restricted inducible promoter, and HSA yield and gene copy number were determined.

According to another example, fed-batch culture of a Pichia pastoris strain expressing HSA under control of a promoter according to the invention is performed. It was found that the induction of promoter activity under glucose-limiting conditions was even more than 120-fold (for pG1) and more than 20-fold (for pG6) compared to the repressed state.

Another example relates to the expression of porcine carboxypeptidase B as a model protein under the transcriptional control of pG1 and pG6 promoters.

Yet another example relates to the expression of antibody fragments under the transcriptional control of pG 1.

A further example demonstrates the functional activity of the promoter variants according to the invention, such as pG1 fragments ranging from 300 to 1000bp in length. Additional experiments have shown that even shorter pG1 fragments are functionally active in a similar context, such as fragments ranging from 200 to 1000bp, or fragments ranging from 250 to 1000.

The foregoing description will be more fully understood with reference to the following examples. However, such examples are merely representative of methods of practicing one or more embodiments of the present invention and should not be construed as limiting the scope of the invention.

Examples

The following examples illustrate materials and methods for identifying novel regulatable promoters and analyzing their expression characteristics in pichia pastoris.

Example 1: identification of multiple potent regulated genes in Pichia pastoris under glucose-limited conditions

In order to identify a number of potent regulated genes and their corresponding promoters in pichia pastoris under glucose-limited conditions, analysis of gene expression patterns was performed using microarrays. Pichia pastoris cells grown in glycerol batch (excess carbon source) were compared to cells cultured in conditions with glucose as growth limitation (chemostat), thereby mimicking the course of a protein production process that is typically carried out in fed-batch mode.

a) Bacterial strains

A wild-type Pichia pastoris strain (CBS2612, CBS-KNAW Fungal Biodiversity Centre, Central bureau voor Schimmelcultures, Utrecht, The Netherlands) was used, which was able to grow on minimal medium without supplements.

b) Culture of Pichia pastoris

The fermentation was carried out in a Minifors reactor (Infors-HT, Switzerland) with a final working volume of 2.5L.

The following media were used:

PTM₁stock solutions of trace salts, each liter containing

6.0g CuSO₄·5H₂O，0.08g NaI，3.36g MnSO₄·H₂O，0.2g Na₂MoO₄·2H₂O，0.02g H₃BO₃，0.82g CoCl₂，20.0g ZnCl₂，65.0g FeSO₄·7H₂O, 0.2g biotin and 5.0ml H₂SO₄(95％-98％)。

Glycerol batch medium, per liter

2g citric acid monohydrate (C)₆H₈O₇·H₂O), 39.2g of glycerol, 20.8g of NH₄H₂PO₄，0.5g MgSO₄·7H₂O，1.6g KCl，0.022g CaCl₂·2H₂O, 0.8mg biotin and 4.6ml of PTM1 trace salt stock solution. HCl was added to set the pH to 5.

Glycerol fed-batch medium, per liter

632g of glycerol, 8g of MgSO₄·7H₂O, 22g KCl, and 0.058g CaCl₂·2H₂O。

Chemostat medium containing per liter

2g citric acid monohydrate (C)₆H₈O₇·H₂O), 99.42g of glucose monohydrate, 22g of NH₄H₂PO₄，1.3g MgSO₄·7H₂O，3.4g KCl，0.02g CaCl₂·2H₂O, 0.4mg biotin and 3.2ml of PTM1 trace salt stock solution. HCl was added to set the pH to 5.

The dissolved oxygen was controlled at a stirrer speed (500-. The ventilation rate was 60L h^-1Air, temperature controlled at 25 ℃ and NH added₄OH (25%) to control pH set point to 5.

To start the fermentation, 1.5L of batch medium was sterile filtered into the fermentor and Pichia pastoris was inoculated at starting optical density (OD600)1 (from YPG, 180rpm, overnight preculture at 28 ℃). The dry biomass concentration at the batch phase reached about 25h was about 20g/L, followed by 10h of exponential fed-batch culture using glucose medium, yielding a dry biomass concentration of about 50 g/L. Then, the volume was reduced to 1.5L and 0.15L h at 0.15L^-1The chemostat culture was started, resulting in a constant growth rate of μ ═ 0.1. The fermentation was terminated 50h after the start of the chemostat.

The fermentation was performed 3 times to obtain the biological replicate fractions required for reliable microarray analysis.

Carbon-limited conditions (no detectable residual glucose) during the chemostat were verified by HPLC analysis of the culture supernatant.

c) Sampling

Samples were taken at the end of the glycerol batch phase and under steady state conditions of the glucose chemostat. Routine sampling was done together during each fermentation period for determination of optical density or yeast dry weight, qualitative microscopy and cell viability analysis. For microarray analysis, samples were taken and processed as follows: for optimal quenching, 9mL of cell culture broth was immediately mixed with 4.5mL of ice-cold 5% phenol (Sigma) solution (in absolute ethanol) and aliquoted. Every 2mL was centrifuged (13200rpm for 1 min) in a pre-cooled collection tube (GE healthcare, NJ), the supernatant was removed completely and the tube was stored at-80 ℃ until RNA purification.

d) RNA purification and sample preparation for microarray hybridization

RNA was isolated using TRI reagent according to the supplier's instructions (Ambion, US). Cell pellet was resuspended in TRI reagent and homogenized with glass beads at 5ms^-1FastPrep 24(m.p. biomedicals, CA) was used for 40 seconds. After addition of chloroform, the samples were centrifuged and total RNA was precipitated from the aqueous phase by addition of isopropanol. The pellet was washed with 70% ethanol, dried and resuspended in RNAse-free water. The RNA concentration was determined by measuring OD260 using a Nanodrop 1000 spectrophotometer (Nanodrop products, DE). Residual DNA was removed from the sample using DNAfree Kit (Ambion, CA). A sample volume equivalent to 10. mu.g of RNA was diluted to 50. mu.L in RNAse-free water, and DNAse buffer I and rDNAse I were added and incubated at 37 ℃ for 30 minutes. After addition of DNAse inactivation reagent, the sample was centrifuged and the supernatant was transferred to a new tube. RNA concentration was again determined as described above. In addition, RNA integrity was analyzed using RNA nano-chips (Agilent). To monitor the microarray flow from amplification and labeling of samples to hybridization, a Spike In Kit (Agilent, product No.: 5188-. It contains 10 different polyadenylated transcripts from adenovirus which are amplified, labeled and co-hybridized with the RNA sample itself. The samples were labeled with Cy3 and Cy5 using the Quick Amp labeling Kit (Agilent, prod. No.: 5190-. Thus, 500ng of purified sample RNA was diluted in 8.3. mu.L of RNAse-free water, 2. mu.L of Spike A or B, and 1.2. mu. L T7 promoter primer were added. The mixture was denatured at 65 ℃ for 10 minutes and kept on ice for 5 minutes. Then, 8.5. mu.L of cDNA master mix (each sample: 4. mu.L of 5 Xfirst strand buffer, 2. mu.L of 0.1M DTT, 1. mu.L of 10mM dNTP mix, 1. mu.L of MMLV-RT, 0.5. mu.L of RNAse out) was added, incubated at 40 ℃ for 2 hours, then transferred to 65 ℃ for 15 minutes and placed on ice for 5 minutes. A master mix of transcripts (15.3. mu.L nuclease-free water, 20. mu.L transcription buffer, 6. mu.L 0.1M DTT, 6.4. mu.L 50% PEG, 0.5. mu.L RNAse inhibitor, 0.6. mu.L inorganic phosphatase, 0.8. mu. L T7 RNA polymerase, 2.4. mu.L Cyanin 3 or Cyanin 5 per sample) was prepared and added to each tube and incubated at 40 ℃ for 2 hours. To purify the obtained labeled cRNA, RNeasy Mini Kit (Qiagen, catalog No. 74104) was used. Samples were stored at-80 deg.C. Quantification of cRNA concentration and labeling efficiency was done at the Nanodrop spectrophotometer.

e) Microarray analysis

To identify strong genes that are effectively regulated in glucose-limited chemostat cultures, 3 replicates of their biological samples were compared to the same reference and each in one staining exchange (dyswap). The reference samples were generated by combining samples of glycerol batch cultures in equal amounts.

A Gene Expression hybridization Kit (Agilent, Cat. No.5188-5242) was used for hybridization of labeled sample cRNA. For the preparation of the hybridized samples, each 300ng of cRNA (Cy3 and Cy 5) and 6. mu.L of 10-fold blocking reagent were diluted with nuclease-free water to a final volume of 24. mu.L. After addition of 1. mu.L of 25-fold fragmentation buffer, the mixture was incubated at 60 ℃ for 30 minutes. Then, 25. mu.L of GEx hybridization Buffer HI-RPM was added to stop the reaction. After centrifugation at 13,200rpm for 1 minute, the samples were chilled on ice and immediately used for hybridization. Pichia pastoris-specific oligonucleotide arrays (AMAD-ID: 026594, 8X15K custom array, Agilent) were used, with their own design. Microarray hybridization was performed according to Microarray hybridization Chamber User Guide (Agilent G2534A). First, a gasketslide (gasketslide) was removed of the lid and placed on a chamber (chamber) base with the Agilent label facing up. Samples (40 μ Ι per array) were loaded into the center of each of the 8 squares. The microarray slide was then carefully placed on a backing slide (Agilent label down) and the chamber lid was placed on top and held in place with a clamp (clamp). Hybridization was carried out in a hybridization oven at 65 ℃ for 17 hours. The microarray chip is washed prior to scanning. Thus, while submerged in wash buffer 1, the chamber is removed, and the sandwich slides are detached from each other. The microarray was transferred directly to another dish with wash buffer 1, washed for 1 minute, transferred to wash buffer 2 (temperature at least 30 ℃) and washed for another 1 minute. After drying the microarray slide by contacting the slide side with a tissue, it was placed in the slide support (Agilent label up). The slide holder was placed in a conveyor belt (carousel) and scanning was started.

f) Data acquisition and statistical evaluation of microarray data

The images were scanned with a G2565AA microarray scanner (Agilent) at 50nm resolution and input into the Agilent Feature Extraction 9.5 software. Agilent Feature Extraction 9.5 was used for quantification of spot intensities. The raw mean point intensity data is then input into open source software R for further normalization and data analysis.

For data pre-processing and normalization, R-packages limma, vsn and marray were used. The intensity data were normalized with VSN without background correction and after normalization were converted to log2 ratios of Cy5 channels versus Cy3 channels. Differential expression was calculated using the lmfit and eBayes functions of the limma package.

The microarray data were reviewed for entries with high differences in expression levels between repressed to induced states (fold change) and high signal intensity in induced states to identify strongly expressed, effectively regulated genes. A list of selected genes is shown in table 1, fold change means signal intensity in the induced state divided by signal intensity in the repressed state. The data for pGAP and pMLS1, pICL1 were added as references.

Table 1: microarray data for promoters selected for further characterization and pGAP, ICL1 and MLS1 as controls

Induced states in green channels

Example 2:comparative promoter Activity Studies of newly identified promoters in Pichia pastoris, using eGFP as a reporter Gene for intracellular expression

To analyze the newly identified promoters for their properties under glucose-limiting conditions, shake flask screening was performed as follows: a 24 hour preculture-simulated batch phase (repressive state of the promoter) was done with rich medium containing glycerol as carbon source, followed by a main culture-simulated glucose limited fed-batch phase (inducible state of the promoter) using minimal medium and glucose feeding beads.

a) Strains and expression vectors

A Pichia pastoris wild type strain (CBS2612, CBS-KNAW Fungal Biodiversity Centre, Central bureau voor Schimmelcultures, Utrecht, The Netherlands) was used as The host strain. Transformation of the strain was performed using an own vector called pPUZZLE (Stadlmayr et al J. Biotechnol 2010 Dec; 150(4):519-29) containing the origin of replication of E.coli (pUC19), an antibiotic resistance cassette (Sh ble gene conferring resistance to neomycin) for selection in E.coli and yeast, an expression cassette for the gene of interest (GOI) (consisting of a multiple cloning site and the Saccharomyces cerevisiae CYC1 transcription terminator), and a locus for integration into the P.pastoris genome (3' AOX1 region).

b) The newly identified promoters pG1, pG3, pG4 and pG6 were amplified and cloned into pPUZZLE expression vector containing eGFP as GOI

A list of the newly identified promoter sequences and their corresponding genes (see example 1) is shown in Table 2. The 5' non-coding region of the corresponding gene up to 1000bp of the start codon ATG was amplified as promoter sequence by PCR (Phusion Polymerase, New England Biolabs) using the primers shown in Table 2. These sequences were cloned into the pPUZZLE expression vector pPM1aZ10_ eGFP to yield pPM1aZ10_ pG1_ eGFP, pPM1aZ10_ pG3_ eGFP, pPM1aZ10_ pG4_ eGFP and pPM1aZ10_ pG6_ eGFP. In addition, the vector pPM1aZ10_ pGAP _ eGFP containing the commonly used glyceraldehyde 3-phosphate dehydrogenase promoter (pGAP of Pichia pastoris, SEQ ID 25 herein) was used as a reference. Using ApaI and SbfI restriction sites (see tables 2 and 3), the promoter was inserted upstream of the start codon of the eGFP gene. The correctness of the promoter sequence was verified by Sanger sequencing.

Table 2: primers for PCR amplification of promoters

Table 3: amplification primers, cloning enzymes and length of cloned promoter

c) expression of eGFP in Pichia pastoris for analysis of promoter activity

All plasmids were linearized with AscI within the 3' AOX genomic integration region followed by electroporation (2kV,4ms, GenePulser, BioRad) into electrocompetent Pichia pastoris.

Selection of positive transformants was performed on YPD plates (10 g yeast extract, 20g peptone, 20g glucose, 20g agar-agar per liter) containing 25. mu.g/mL neomycin (Invivogen, CA). Colony PCR was used to ensure the presence of the transformation plasmid. Thus, genomic DNA was obtained by hot-boiling and freezing Pichia pastoris colonies for 5 minutes each, and PCR was directly performed with appropriate primers. For expression screening, a single colony was inoculated into liquid YPG-Zeo medium (per liter: 20g peptone, 10g yeast extract, 12.6g glycerol and 25mg neomycin) as a preculture. After about 24h, a preculture with an OD600 of 0.1 was inoculated with 10ml of YP medium (per liter: 20g peptone, 10g yeast extract) and 2 parts main culture in glucose-fed beads (Kuhner, CH). Glucose-limiting growth conditions are achieved due to the slow glucose release kinetics of these feeding beads, which are described by the following equation: (glucose) ═ 1.63 × t^0.74[mg/Disc]. Samples were taken at the end of preculture and 24 and 48 hours after inoculation of the main culture. Cell density was determined by measuring OD600 and eGFP expression was analyzed by flow cytometry as described in Stadlmayr et al (J.Biotechnology 2010 Dec; 150(4): 519-29). For each sample, 10,000 cells were analyzed. The autofluorescence of pichia pastoris was measured using untransformed pichia pastoris wild type cells and subtracted from the signal. Relative eGFP expression level: (Fluorescence intensity versus cell size) is shown as a percentage of the eGFP expression level of clones expressing eGFP under the control of the constitutive pGAP promoter.

Other similar studies were done using the promoters pG7 and pG 8. Cloning was done as described in example 2b, but wild-type Pichia pastoris strain X-33(Invitrogen) was used to transform pPM1aZ10_ pG7_ eGFP and pPM1aZ10_ pG8_ eGFP. The primers and cloned fragments used are listed in tables 2 and 3. The results are shown in Table 4.

Table 4: screening results for pichia pastoris clones expressing eGFP under the control of the new promoter; data shown (fluorescence/cell size) are related to pGAP;

d) determination of eGFP Gene Copy Number (GCN) in selected eGFP-expressing clones

Expression intensity is often correlated with the number of expression cassettes integrated into the pichia pastoris genome. Thus, the copy number of the gene of eGFP was determined. Genomic DNA was isolated using the DNeasy Blood & Tissue Kit (Quiagen, Cat. 69504). Quantitative PCR was used to determine gene copy number. Thus, the SenseMix SYBR Kit (Bioline, QT605-05) was used. Sensi Mix SYBR was mixed with primers and samples and used for real-time analysis in a real-time PCR cycler (Rotor Gene, Qiagen). The list of primers is shown in table 5. All samples were analyzed in triplicate or quadruplicate. Rotator Gene software was used for data analysis. The actin gene ACT1 was used as a calibrator. The results are shown in Table 6.

Table 5: primers for copy number determination by real-time PCR

Table 6: screening results (fluorescence/cell size associated with pGAP) and gene copy number for selected pichia pastoris clones expressing eGFP under control of pG1 and pG 6;

e) analysis of pG1 promoter strength in fed-batch fermentation of an eGFP clone

Fed-batch fermentation was performed in a DASGIP reactor with a final working volume of 0.7L.

The following media were used:

PTM1 Trace salt stock solution containing per liter

6.0g CuSO₄·5H₂O，0.08g NaI，3.36g MnSO₄·H₂O，0.2g Na₂MoO₄·2H₂O，0.02g H₃BO₃，0.82g CoCl₂，20.0g ZnCl₂，65.0g FeSO_4·7H₂O, 0.2g biotin and 5.0ml H₂SO₄(95％-98％)。

Glycerol batch medium, per liter

2g citric acid monohydrate (C)₆H₈O₇·H₂O), 39.2g of glycerol, 12.6g of NH₄H₂PO₄，0.5g MgSO₄·7H₂O，0.9g KCl，0.022g CaCl₂·2H₂O, 0.4mg biotin and 4.6ml of PTM1 trace salt stock solution. HCl was added to set the pH to 5.

Glucose fed-batch Medium, containing per liter

464g of glucose monohydrate, 5.2g of MgSO₄·7H2O，8.4g KCl，0.28g CaCl₂·2H₂O, 0.34mg biotin and 10.1mL of PTM1 trace salt stock solution.

The dissolved oxygen was controlled to DO of 20% at the stirrer speed (400-. The ventilation rate was 24L h^-1Air, temperature controlled at 25 ℃ and NH added₄OH (25%) to control pH set point to 5.

To start the fermentation, 400mL of batch medium was sterile filtered into the fermentor and the pichia pastoris clone pG1_ eGFP #8 was inoculated at starting optical density (OD600)1 (from the preculture). About 25h of batch phase (to reach a dry biomass concentration of about 20 g/L) was followed by a glucose limited fed-batch starting at an exponential feed of 7h and a constant feed of 15g/L for 13h, resulting in a final dry biomass concentration of about 100 g/L. Samples were taken during the batch and fed-batch phases and analyzed for eGFP expression using a plate reader (Infinite 200, Tecan, CH). Thus, the sample was diluted to an optical density (OD600) of 5. The results are shown in Table 7 as relative fluorescence values per bioreactor (FL/r).

Table 7: relative fluorescence/bioreactor of two different pichia pastoris clones expressing eGFP under control of pGAP or pG1 in optimized fed-batch fermentations.

Example 3: comparative promoter activity study of newly identified promoters in Pichia pastoris, using Human Serum Albumin (HSA) as the reporter gene for extracellular expression

To analyze the characteristics of the newly identified promoters under glucose limiting conditions, shake flask screening was performed as follows: a 24 hour pre-culture-simulated batch phase (repression of promoter) was done with rich medium containing glycerol as carbon source, followed by a main culture-simulated glucose-limited fed-batch phase (inducible of promoter) using minimal medium and glucose-fed beads.

a) Strains and expression vectors

A Pichia pastoris wild type strain (CBS2612, CBS-KNAW Fungal Biodiversity Centre, Central bureau voor Schimmelcultures, Utrecht, The Netherlands) was used as The host strain. Transformation of the strain was performed using an own vector called pPUZZLE (Stadlmayr et al J. Biotechnol 2010 Dec; 150(4):519-29), and positive transformants were selected on the basis of neomycin resistance. For secretory expression of Human Serum Albumin (HSA), its native secretion leader is used.

b) Amplification and cloning of newly identified promoters pG1, pG3, pG4 and pG6 into self-contained expression vectors

The 4 promoters amplified in example 2b were cloned into the pPUZZLE expression vector pPM1aZ10_ HSA to yield pPM1aZ10_ pG1_ HSA, pPM1aZ10_ pG3_ HSA, pPM1aZ10_ pG4_ HSA, and pPM1aZ10_ pG6_ HSA. In addition, the vector pPM1aZ10_ pGAP _ HSA containing the commonly used glyceraldehyde 3-phosphate dehydrogenase promoter (pGAP) was used as a reference. The promoter was inserted upstream of the initiation codon of the HSA gene using ApaI and SbfI restriction sites (see table 3). The correctness of the promoter sequence was verified by Sanger sequencing.

c) Expression of HSA in Pichia pastoris under control of a newly identified glucose-restricted inducible promoter

All plasmids were linearized using AscI restriction enzymes, followed by electroporation (using standard transformation protocols for pichia pastoris) into pichia pastoris. Selection of positive transformants was carried out on YPD plates (per liter: 10g yeast extract, 20g peptone, 20g glucose, 20g agar-agar) containing 25. mu.g/mL neomycin. Colony PCR was used to ensure the presence of the transformation plasmid as described in example 2 c.

For HSA expression screening, a single colony was inoculated into liquid YPG-Zeo medium (per liter: 20g peptone, 10g yeast extract, 12.6g glycerol and 25mg neomycin) as a preculture. After about 24h, the main culture in YP medium (per liter: 20g peptone, 10g yeast extract) and glucose feed beads (Kuhner, CH) was inoculated with a preculture with an OD600 of 1. Glucose-limiting growth conditions are achieved due to the slow glucose release kinetics of these feeding beads, which are described by the following equation: (glucose) ═ 1.63 × t^0.74[mg/Disc]. Samples were taken at the end of preculture and 24 and 48 hours after inoculation of the main culture. Biomass concentration was determined by measuring OD600 or cell wet weight. HSA concentration in culture supernatantsQuantification was performed by the human albumin ELISA quantification kit (catalog No. E80-129, Bethy Laboratories, TX, USA) according to the supplier's instruction manual. HSA standard substance 400ng mL^-1The starting concentration of (2) was used. Samples were diluted accordingly in sample diluent (50mM Tris-HCl, 140mM NaCl, 1% (w/v) BSA, 0.05% (v/v) Tween20, pH 8.0). The HSA titers obtained from the screening of several clones for each construct are presented in table 8.

Table 8: screening results for Pichia pastoris clones expressing HSA under the control of pGAP, pG1 and pG6

d) Determination of the copy number of the HSA Gene

Genomic DNA isolation and qPCR measurements were performed as in example 2d using the primers given in table 9. The results are shown in Table 10.

Table 9: primers for determining gene copy number by real-time PCR

Table 10: screening and gene copy number results for selected pichia pastoris clones expressing HSA under the control of pGAP, pG1 and pG 6;

e) fed-batch culture of Pichia pastoris strains expressing HSA under control of pG1 and pG6 promoters

Fermentation was carried out in a DASGIP bioreactor with a final working volume of 0.7L. Strain pG1_ HSA #23 has two copies of the HSA gene, and strain pG6_ HSA #36 carries only one copy of the HSA gene. Thus, two different pichia pastoris strains expressing HSA under pGAP control (pGAP _ HSA #3 has one copy of the HSA gene and pGAP _ HSA #4 has two copies of the HSA gene) were cultured as references. All fermentations were performed in duplicate.

The following media were used:

PTM1 Trace salt stock solution containing per liter

6.0g CuSO₄·5H₂O，0.08g NaI，3.36g MnSO₄·H₂O，0.2g Na₂MoO₄·2H₂O，0.02g H₃BO₃，0.82g CoCl₂，20.0g ZnCl₂，65.0g FeSO_4·7H₂O, 0.2g biotin and 5.0ml H₂SO₄(95％-98％)。

Glycerol batch medium, per liter

39.2g of glycerol, 27.9g H₃PO₄(85％)，7.8g MgSO₄·7H₂O，2.6g KOH，9.5g K₂SO₄，0.6g CaSO₄2H2O, 0.4mg biotin and 4.6ml of a PTM1 trace salt stock. After sterile filtration into the fermentor, the pH was adjusted to 5.85.

Glucose fed-batch Medium, containing per liter

550g glucose monohydrate, 6.5g MgSO₄·7H₂O，10g KCl，0.35g CaCl₂·2H₂O, 0.4mg biotin and 12ml of PTM1 trace salt stock solution.

The dissolved oxygen was controlled to DO of 20% at the stirrer speed (400-. The ventilation rate was 24L h^-1Air, temperature controlled at 25 ℃ and NH added₄OH (25%) to control pH set point at 5.85.

To start the fermentation, 400mL of batch medium was sterile filtered into the fermentor and the pichia pastoris clone was inoculated (from the preculture) at the starting optical density (OD600) 1. A batch phase of about 25h reached a dry biomass concentration of about 20g/L, followed by a constant fed-batch (up to 100 hours) using a glucose medium, resulting in a final dry biomass concentration of about 100 g/L. The pH was 5.85 during the batch phase and was maintained at 5.85 throughout the fermentation. Samples were taken during the batch and fed-batch phases. HSA concentrations were quantified using the human albumin ELISA quantification kit (Bethyyl, cat. No. E80-129), as described in example 3 c. The biomass concentrations and HSA titers are shown in table 11, and the product yields (amount of HSA secreted/biomass, HSA/YDM) at the end of the batch (repressing conditions for pG1 and pG6) and at the end of the fed-batch (inducing conditions for pG1 and pG6) are given in table 12. Thereby enabling verification of the induction strategy. pG1 and pG6 were repressed by carbon source excess (in glycerol batch), showing little detectable HAS, in contrast to pGAP driven clones. Induction of pG1 and pG6 occurred when switching to C-limiting conditions at the beginning of the fed-batch phase. The induction of pG1 (HSA/YDM) was over 120-fold compared to the repressed state, the induction of pG6 was over 20-fold compared to the repressed state, and little change was observed for pGAP (3-fold increase in HSA/YDM compared to batch phase).

Table 11: yeast dry weights and HSA titers at batch and fed-batch Ends for 7 fermentations of Pichia pastoris clones expressing HSA under control of pGAP, pG1 or pG6

Table 12: HSA titers at batch and fed-batch end of 7 fermentations of Pichia pastoris clones expressing HSA under control of pGAP, pG1 or pG6 versus yeast dry weight

Example 4: comparative promoter Activity Studies of newly identified promoters in Pichia pastoris at various glucose concentrations, using eGFP as a reporter for intracellular expression

To analyze the characteristics of the newly identified promoters at various glucose concentrations, shake flask screens were performed as follows: preculture was completed for 24 hours with rich medium containing glycerol as carbon source (repression of promoter), followed by main culture with minimal medium and glucose as carbon source (inducible state of promoter);

a) strains and expression vectors

A Pichia pastoris wild type strain (CBS2612, CBS-KNAW Fungal Biodiversity Centre, Central bureau voor Schimmelcultures, Utrecht, The Netherlands) was used as The host strain. Transformation of the strain was performed using an own vector called pPUZZLE (Stadlmayr et al J. Biotechnol 2010 Dec; 150(4):519-29), and positive transformants were selected on the basis of neomycin resistance.

b) The newly identified promoters pG1, pG3, pG4 and pG6 were amplified and cloned into pPUZZLE expression vector containing eGFP as GOI

Amplification and cloning was done as described in example 2.

c) expression of eGFP in Pichia pastoris for analysis of promoter activity

Transformation and clonal selection was accomplished as described in example 2.

For expression screening, a single colony was inoculated into liquid YPG-Zeo medium (per liter: 20g peptone, 10g yeast extract, 12.6g glycerol and 25mg neomycin) as a preculture. After about 24h, 10ml of the main culture in YP medium (per liter: 20g of peptone, 10g of yeast extract) were inoculated with a preculture with an OD600 of 0.01 and glucose as carbon source. From 20 to 0.001g L^-1Glucose was used at various concentrations.

Samples were taken 1-8 hours after inoculation of the main culture. eGFP expression Positive transformants were selected based on neomycin resistance by flow cytometry as described in Stadlmayr et al (J.Biotechnology 2010 Dec; 150(4): 519-29). For each sample, 10,000 cells were analyzed. The autofluorescence of pichia pastoris was measured using untransformed pichia pastoris wild-type cells.

Example 5: comparative promoter activity study of newly identified promoters in Pichia pastoris, using porcine carboxypeptidase B (CpB) as intracellular expression reporter gene

To analyze the characteristics of the newly identified promoters under glucose limiting conditions, shake flask screening was performed as follows: a 24 hour pre-culture-simulated batch phase (repression of promoter) was done with rich medium containing glycerol as carbon source, followed by main culture using minimal medium and glucose feeding beads-simulated glucose limited fed-batch phase (inducible of promoter);

a) strains and expression vectors

A Pichia pastoris wild type strain (CBS2612, CBS-KNAW Fungal Biodiversity Centre, Central bureau voor Schimmelcultures, Utrecht, The Netherlands) was used as The host strain. Transformation of the strain was performed using an own vector called pPUZZLE (Stadlmayr et al J. Biotechnol 2010 Dec; 150(4):519-29), and positive transformants were selected on the basis of neomycin resistance. For secretory expression of porcine carboxypeptidase B (CpB), the yeast alpha mating factor leader was used.

b) The newly identified promoters pG1, pG3, pG4 and pG6 were amplified and cloned into the own expression vector

The two promoters amplified in example 2b were cloned into the pPUZZLE expression vector pPM1aZ30_ aMF _ CpB, yielding pPM1aZ30_ pG1_ aMF _ CpB and pPM1aZ30_ pG6_ aMF _ CpB. In addition, the vector pPM1dZ30_ pGAP _ CPB containing the commonly used glyceraldehyde 3-phosphate dehydrogenase promoter (pGAP) was used as a reference. The promoter was inserted upstream of the start codon of the CpB gene using ApaI and SbfI restriction sites. The correctness of the promoter sequence was verified by Sanger sequencing.

c) Cpb expression in pichia pastoris under control of a newly identified glucose-restricted inducible promoter

The plasmid was linearized using either SpeI or SapI restriction enzymes, followed by electroporation (using standard transformation protocols for pichia pastoris) into pichia pastoris. Selection of positive transformants was carried out on YPD plates (per liter: 10g yeast extract, 20g peptone, 20g glucose, 20g agar-agar) containing 25. mu.g/mL neomycin. Colony PCR was used to ensure the presence of the transformation plasmid, as described in example 2 c.

For CpB expression screening, single colonies were inoculated into liquid YPG-Zeo medium (per liter: 20g peptone, 10g yeast extract, 12.6g glycerol and 25mg neomycin) as a preculture. After about 24h, the main culture in YP medium (per liter: 20g peptone, 10g yeast extract) and glucose feed beads (Kuhner, CH) was inoculated with a preculture with an OD600 of 1. Glucose-limiting growth conditions are achieved due to the slow glucose release kinetics of these feeding beads, which are described by the following equation: (glucose) ═ 1.63 × t^0.74[mg/Disc]. Samples were taken at the end of preculture and 24 and 48 hours after inoculation of the main culture. Biomass concentration was determined by measuring OD600 or cell wet weight. CpB concentration in the culture supernatant was quantified by enzyme assay based on conversion of CpB to hippuric acid by hippuric-L-arginine. The reaction kinetics were measured by monitoring the absorption at 254nm at 25 ℃ using a Hitachi U-2910 spectrophotometer at the beginning of the reaction. Samples and standards were buffered with assay buffer (25mM Tris, 100mM HCl, pH 7.65) and activation buffer (0.01mgL-1 trypsin, 300mM Tris, 1. mu.M ZnCl) was used₂pH 7.65). Activation buffer without trypsin was used instead of the sample as a negative control. The reaction was started by adding substrate solution (1 mM malonyl-L-arginine in assay buffer).

d) Fed-batch culture of Pichia pastoris strains expressing CpB under control of pG6 promoter

The fed-batch fermentation was completed as described in example 3 e. Clone pPM1aZ10_ pG6_ CpB #4 produced no detectable CpB in the batch, while producing over 210mg/L of CpB at the end of the fed-batch.

Example 6: comparative promoter Activity Studies of newly identified promoters pG1 and pG6 in Pichia pastoris multicopy clones, using Human Serum Albumin (HSA) as an extracellularly expressed reporter gene

To analyze the characteristics of the newly identified promoters under glucose limiting conditions, shake flask screening was performed as follows: a 24 hour pre-culture-simulated batch phase (repression of promoter) was done with rich medium containing glycerol as carbon source, followed by main culture using minimal medium and glucose feeding beads-simulated glucose limited fed-batch phase (inducible of promoter);

a) strains and expression vectors

A Pichia pastoris wild type strain (CBS2612, CBS-KNAW Fungal Biodiversity Centre, Central bureau voor Schimmelcultures, Utrecht, The Netherlands) was used as The host strain. Transformation of the strain was performed using an own vector called pPUZZLE (Stadlmayr et al J. Biotechnol 2010 Dec; 150(4):519-29), and positive transformants were selected on the basis of neomycin resistance. For secretory expression of Human Serum Albumin (HSA), its native secretion leader is used.

b) The newly identified promoters pG1 and pG6 were amplified and cloned into the own expression vector

The two promoters amplified in example 2b were cloned into the pPUZZLE expression vector pPM1nZ30_ HSA, yielding pPM1nZ30_ pG1_ HSA and pPM1nZ30_ pG6_ HSA.

The promoter was inserted upstream of the initiation codon of the HSA gene using ApaI and SbfI restriction sites. The correctness of the promoter sequence was verified by Sanger sequencing.

c) Expression of HSA in Pichia pastoris under control of a newly identified glucose-restricted inducible promoter

All plasmids were linearized using AscI restriction enzymes, followed by electroporation (using standard transformation protocols for pichia pastoris) into pichia pastoris. Selection of positive transformants was carried out on YPD plates (per liter: 10g yeast extract, 20g peptone, 20g glucose, 20g agar-agar) containing 25. mu.g/mL neomycin. Gene copy number amplification was performed as described in Marx et al (FEMS Yeast Res.2009Dec; 9(8): 1260-70). Colony PCR was used to ensure the presence of the transformation plasmid, as described in example 2 c.

For HSA expression screening, a single colony was inoculated into the fluidAs preculture, somatic YPG-Zeo medium (per liter: 20g peptone, 10g yeast extract, 12.6g glycerol and 25mg neomycin) was used. After about 24h, the main culture in YP medium (per liter: 20g peptone, 10g yeast extract) and glucose feed beads (Kuhner, CH) was inoculated with a preculture with an OD600 of 1. Glucose-limiting growth conditions are achieved due to the slow glucose release kinetics of these feeding beads, which are described by the following equation: (glucose) ═ 1.63 × t^0.74[mg/Disc]. Samples were taken at the end of preculture and 24 and 48 hours after inoculation of the main culture. Biomass concentration was determined by measuring OD600 or cell wet weight. HSA concentration in the culture supernatant was quantified by the human albumin ELISA quantification kit (catalog No. E80-129, Bethy Laboratories, TX, USA) according to the supplier's instruction manual. HSA standard substance 400ng mL^-1The starting concentration of (2) was used. Samples were diluted accordingly in sample diluent (50mM Tris-HCl, 140mM NaCl, 1% (w/v) BSA, 0.05% (v/v) Tween20, pH 8.0). The HSA titers obtained from screening several multicopy clones and single copy clones from example 3c are presented in table 13.

Cloning	HSA titer (mg/L)
		pPM1aZ10_pG1_HSA#23	8.20
pPM1nZ30_pG1_HSA#C2	19.55
		pPM1nZ30_pG1_HSA#4*1000	21.59
pPM1nZ30_pG1_HSA#5*1000	21.33
		pPM1nZ30_pG1_HSA#X4	27.22
pPM1nZ30_pG1_HSA#X5	6.90
		pPM1aZ10_pG6_HSA#36	1.55
pPM1nZ30_pG6_HSA#C6	14.12
		pPM1nZ30_pG6_HSA#2*1000	15.85
pPM1nZ30_pG6_HSA#X5	11.52
		pPM1nZ30_pG6_HSA#X8	7.87

Table 13: screening results for Pichia pastoris multicopy clones expressing HSA under the control of pGAP, pG1 and pG6

d) Determination of the copy number of the HSA Gene

Genomic DNA isolation and qPCR measurements were performed as in example 2d using the primers given in table 9. The results are shown in Table 14.

Table 14: screening and Gene copy number results for selected Pichia pastoris multicopy clones expressing HSA under the control of pGAP, pG1 and pG6

e) Fed-batch culture of multicopy pichia pastoris strains expressing HSA under control of pG1 and pG6 promoters

The fed-batch fermentation was completed as described in example 3 e. Clones pPM1nZ30_ pG1_ HSA #4 x 1000 and pPM1nZ30_ pG6_ HSA # C6 reached 1060 and 728mg/L HSA, respectively, at the end of the fed-batch.

Example 7: comparative promoter Activity Studies of the newly identified promoter pG1 in Pichia pastoris, using antibody fragment (Fab) as the reporter gene for extracellular expression

To analyze the characteristics of the newly identified promoters under glucose limiting conditions, shake flask screening was performed as follows: a 24 hour pre-culture-simulated batch phase (repression of promoter) was done with rich medium containing glycerol as carbon source, followed by main culture using minimal medium and glucose feeding beads-simulated glucose limited fed-batch phase (inducible of promoter);

a) strains and expression vectors

A Pichia pastoris wild type strain (CBS2612, CBS-KNAW Fungal Biodiversity Centre, Central bureau voor Schimmelcultures, Utrecht, The Netherlands) was used as The host strain. Transformation of the strain was performed using an own vector called pPUZZLE (Stadlmayr et al J. Biotechnol 2010 Dec; 150(4):519-29), and positive transformants were selected on the basis of neomycin resistance. For secretory expression of antibody Fab fragments, yeast alpha mating factor leader was used.

b) Amplification and cloning of the newly identified promoter pG1 into the own expression vector

The pG1 promoter amplified in example 2b was cloned into the pPUZZLE expression vector containing Fab as GOI as described in example 5 b. The promoter was inserted upstream of the start codon of the Fab gene using ApaI and SbfI restriction sites. The correctness of the promoter sequence was verified by Sanger sequencing.

c) Fab expression in Pichia pastoris under the control of a newly identified glucose-limiting inducible promoter pG1

The plasmid was linearized using either SpeI or SapI restriction enzymes, followed by electroporation (using standard transformation protocols for pichia pastoris) into pichia pastoris. Selection of positive transformants was carried out on YPD plates (per liter: 10g yeast extract, 20g peptone, 20g glucose, 20g agar-agar) containing 25. mu.g/mL neomycin. Colony PCR was used to ensure the presence of the transformation plasmid, as described in example 2 c.

For Fab expression screening, single colonies were inoculated into liquid YPG-Zeo medium (per liter: 20g peptone, 10g yeast extract, 12.6g glycerol and 25mg neomycin) as a preculture. After about 24h, the main culture in YP medium (per liter: 20g peptone, 10g yeast extract) and glucose feed beads (Kuhner, CH) was inoculated with a preculture with an OD600 of 1. Glucose-limiting growth conditions are achieved due to the slow glucose release kinetics of these feeding beads, which are described by the following equation: (glucose) ═ 1.63 × t^0.74[mg/Disc]. Samples were taken at the end of preculture and 24 and 48 hours after inoculation of the main culture. Biomass concentration was determined by measuring OD600 or cell wet weight. Fab expression levels were quantified by ELISA using anti-human Kappa light chain (bound and free) -alkaline phosphatase antibodies generated in goats. The Fab titers from screening several Fab expressing clones under the control of pGAP and pG1 are presented in table 15.

Table 15: screening results for Pichia pastoris clones expressing Fab under the control of pGAP and pG1

d) Fed-batch culture of Pichia pastoris strains expressing Fab under control of pG1 promoter

Fed-batch fermentation was done similarly as described in example 3e, but using glucose fed-batch as described in example 2 e. At the end of the fed-batch, clones pPM1aZ30_ pG1_ Fab # C4 and pPM1aZ30_ pG1_ Fab # C7 reached 165 and 131mg/L Fab, respectively.

Example 8: exponential fed-batch fermentation to control specific growth rate of newly identified promoters at maximum volumetric productivity

Chemostat cultures of pichia pastoris clones expressing reporter genes under control of newly identified promoters were used to determine specific and volumetric productivity at different growth rates. Exponential fed-batch fermentation, as described by Maurer et al (Microb Cell fact.2006Dec 11; 5:37), can be used to grow Pichia pastoris clones at specific growth rates for improved production throughout the feeding phase. Thereby, the space-time yield can be optimized. With optimized feed, the space-time yield of the fed-batch phase improved by more than 35%.

Example 9: determination of promoter/transcription intensity: identification of comparative promoter Activity Studies for promoter Regulation at different glucose concentrations, Using eGFP as a reporter Gene for intracellular expression

The regulatory properties of the promoter were analyzed by screening clones expressing eGFP under the control of the promoter. Thus, a single colony was inoculated into liquid YPG-Zeo medium (per liter: 20g of peptone, 10g of yeast extract, 12.6g of glycerol and 25mg of neomycin) as a preculture. After about 24h, 10ml of a main culture in YP medium (20g peptone, 10g yeast extract per liter) and different concentrations of glucose (20,10,5,2.5,1.25,0.625,0.313,0.156,0.078,0.039,0.020,0.010,0.005 and 0.002g/L) were inoculated with a preculture with an OD600 of 0.01. Samples were taken after 6 hours and analyzed by flow cytometry as described by Stadlmayr et al (J Biotechnol.2010Dec; 150(4): 519-29). Fluorescence (forward scatter to the power of 1.5) was calculated for each cell/data point as a function of cell size and its geometric mean was used to compare eGFP expression levels generated at different glucose concentrations. Clones expressing eGFP under pGAP control were used as reference (pichia pastoris pGAP, SEQ ID 25 herein). The autofluorescence of pichia pastoris was measured using untransformed pichia pastoris wild type cells and subtracted from the signal. Table 16 shows complete induction of pG1 promoter at about 40mg/L glucose or less and comparison of the transcription strength with the native pGAP promoter.

％pGAP	Glucose (g/L)
		14.7	20
17.4	10
		23.7	5
25.4	2.5
		28.2	1.25
30.6	0.625
		36.9	0.3125
44.5	0.15625
		50.9	0.078125
56.2	0.0390625
		55.0	0.0195313
57.5	0.0097656
		59.2	0.0048828
59.6	0.0024414

Table 16: relative eGFP expression (relative to pGAP) of Pichia pastoris clones expressing eGFP under the control of pG1 promoter at different glucose concentrations (20-0.002 g/L)

Other similar studies were performed to compare the relative transcriptional strengths of the derepressed promoters pG1, pG3, pG4, pG6, and pG 7. Clones expressing eGFP under the control of one of the promoters were cultured in YPG (20g/L glycerol, repressed) and then inoculated into YP medium containing different amounts of glucose (20 to 0.002g/L (D20, D10, … D0.002), induced) and cultured for 5-6 hours. Cells were analyzed by flow cytometry and the results were evaluated as follows: the fluorescence of each cell was correlated to cell size (forward scatter multiplied by power 1.5) and its geometric mean was used to compare different glucose concentrations. The conclusive results of these screens are shown in FIG. 14, which shows that logarithmic glucose concentration versus relative fluorescence better plots the induction behavior of glucose-restricted regulatable promoters. FIG. 14 shows the complete induction of pG1 promoter at about 40mg/L glucose or less, and the complete induction of pG3, pG4, and pG6 promoters at about 4g/L or less, as well as the transcriptional strength, as compared to the native pGAP promoter. The induction behavior of pG7 was similar to that of pG1 (data not shown). Based on previous results for pG8, its inducible behavior was assumed to be within the scope of other promoters.

Example 10: comparison of the prior art pICL1 and pMLS1 promoters with pG1 in a glucose concentration screening assay

Comparative promoter activity studies were performed according to example 9, using the pICL1 and pMLS1 promoters as references to compare with the pG1 promoter according to the invention.

Both the pICL1 and pMLS1 promoters were found to be very weak in activity, with no significant difference in high (D20: 20 g/L/repressed) or low (D0.04: 0.04g/L induction ═ derepression) glucose concentrations. In either case, the activity is much lower than that of the pG1 promoter repressed in the same background. The results are shown in table 17, which is the% promoter activity relative to the pGAP promoter.

Table 17: relative fluorescence of strains expressing eGFP under the control of pG1, pICL1 and pMLS1, respectively, grown in media containing 20g/L (D20) or 0.04g/L (D0.04) glucose.

Example 11: comparison of pG1 variants

Shorter variants of the pG1 promoter were cloned as described in example 2a and screened similarly to that described in example 2c, but in a scale-down apparatus using 24-well plates (Whatman, UK, art. Nr.7701-5110) and feeding beads 1/4(12mm, Kuhner, CH) instead of the whole. Clones expressed under the control of pG1 and pGAP were used as controls. The forward primers and lengths of pG1 and its variants are listed in Table 18. There was no significant difference in the relative fluorescence of cells expressing eGFP under the control of pG1 and pG1 variants a-f.

Table 18: pG1 and variants thereof: the forward primer and the 5 'start and 3' end positions in the pG1 sequence (SEQ ID 1). The sequence of pG1a-f is shown in FIG. 15(SEQ ID 41-46).

The present invention provides the following:

1. a method for producing a protein of interest (POI) by culturing a recombinant eukaryotic cell line comprising an expression construct, said expression vector comprising a regulatable promoter and a nucleic acid molecule encoding the POI under the transcriptional control of said promoter, said method comprising the steps of:

a) culturing said cell line with a basal carbon source repressing said promoter,

b) culturing said cell line under carbon source-supplementation-free conditions or limited carbon source supplementation conditions that derepress said promoter, thereby inducing production of said POI at a transcription rate of at least 15% compared to the native pGAP promoter of said cell, and

c) generating and recovering the POI.

2. The method according to item 1, wherein the base carbon source is selected from the group consisting of: glucose, glycerol, ethanol, mixtures thereof, and complex nutritional materials.

3. The method according to item 1 or 2, wherein the supplemental carbon source is a hexose such as glucose, fructose, galactose or mannose, a disaccharide such as sucrose, an alcohol such as glycerol or ethanol, or a mixture thereof.

4. The method according to any one of items 1 to 3, wherein said step b) employs a feed medium that does not provide or provides a limited amount of said supplemental carbon source, preferably said limited amount is 0-1g/L in said medium.

5. The method according to any one of items 1 to 4, wherein the limited amount of supplemental carbon source is growth-limiting to maintain a specific growth rate of 0.02h^-1To 0.2h^-1Preferably 0.02h^-1To 0.15h^-1And (4) the following steps.

6. The method according to any one of items 1 to 5, wherein the promoter is capable of controlling transcription of a gene selected from the group consisting of: g1(SEQ ID 7), G3(SEQ ID 8), G4(SEQ ID 9), G6(SEQ ID 10), G7(SEQ ID 11) and G8(SEQ ID 12), or a functionally active variant thereof.

7. The method according to item 6, wherein the functionally active variant is selected from the group consisting of: homologues having at least about 60% nucleotide sequence identity, homologues modified by insertion, deletion or substitution of one or more nucleotides within the parent nucleotide sequence or at one or both of the distal ends of the sequence, preferably having a nucleotide sequence of at least 200bp, and analogues derived from species other than Pichia pastoris (Pichia pastoris).

8. The method according to item 6 or 7, wherein the functionally active variant of pG1 is selected from the group consisting of: pG1a (SEQ ID 41), pG1b (SEQ ID 42), pG1c (SEQ ID 43), pG1d (SEQ ID 44), pG1e (SEQ ID 45) and pG1f (SEQ ID 46).

9. The method according to any one of items 1 to 8, wherein the cell line is selected from the group consisting of: mammalian, insect, yeast, filamentous fungi and plant cell lines, preferably yeast.

10. The method according to any one of items 1 to 9, wherein the POI is a heterologous protein, preferably selected from the group consisting of therapeutic proteins including antibodies or fragments thereof, enzymes and peptides, protein antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccines and vaccine-like proteins or particles, processing enzymes, growth factors, hormones and cytokines, or metabolites of POI.

11. A method for regulating expression of a POI in a recombinant eukaryotic cell under the transcriptional control of a carbon source-controllable promoter having a transcriptional strength of at least 15% compared to the cell's native pGAP promoter, wherein said expression is induced under conditions that restrict the carbon source.

12. A method for producing a POI in a recombinant eukaryotic cell under the transcriptional control of a carbon source-controllable promoter, wherein the promoter has a transcriptional strength of at least 15% compared to the native pGAP promoter of the cell.

13. The method according to any one of items 1 to 12, wherein the regulatable promoter comprises a nucleic acid sequence selected from the group consisting of:

a) pG1(SEQ ID 1), pG3(SEQ ID2), pG4(SEQ ID 4), pG6(SEQ ID 3), pG7(SEQ ID 5), or pG8(SEQ ID 6);

b) a sequence having at least 60% homology to: pG1(SEQ ID 1), pG3(SEQ ID2), pG4(SEQ ID 4), pG6(SEQ ID 3), pG7(SEQ ID 5), or pG8(SEQ ID 6);

c) a sequence that hybridizes under stringent conditions to: pG1(SEQ ID 1), pG3(SEQ ID2), pG4(SEQ ID 4), pG6(SEQ ID 3), pG7(SEQ ID 5), or pG8(SEQ ID 6); and

d) fragments or variants derived from a), b) or c),

wherein the promoter is a functionally active promoter which is a carbon source regulatable promoter capable of expressing the POI in a recombinant eukaryotic cell at a transcription rate of at least 15% compared to the native pGAP promoter of the cell.

14. The method according to item 13, wherein the variant of pG1(SEQ ID 1), pG3(SEQ ID2), pG4(SEQ ID 4), pG6(SEQ ID 3), pG7(SEQ ID 5), or pG8(SEQ ID 6) is a functionally active variant selected from the group consisting of: homologues having at least about 60% nucleotide sequence identity, homologues modified by insertion, deletion or substitution of one or more nucleotides within the parent nucleotide sequence or at one or both of the distal ends of the sequence, preferably having a nucleotide sequence of at least 200bp, and analogues derived from species other than pichia pastoris.

15. A method according to item 13 or 14, wherein the functionally active variant of pG1 is selected from the group consisting of: pG1a (SEQ ID 41), pG1b (SEQ ID 42), pG1c (SEQ ID 43), pG1d (SEQ ID 44), pG1e (SEQ ID 45) and pG1f (SEQ ID 46).

16. An isolated nucleic acid comprising a nucleic acid sequence selected from the group consisting of:

a) pG1(SEQ ID 1), pG3(SEQ ID2), pG6(SEQ ID 3), pG7(SEQ ID 5), or pG8(SEQ ID 6);

b) a sequence having at least 60% homology to: pG1(SEQ ID 1), pG3(SEQ ID2), pG6(SEQ ID 3), pG7(SEQ ID 5), or pG8(SEQ ID 6);

c) a sequence that hybridizes under stringent conditions to: pG1(SEQ ID 1), pG3(SEQ ID2), pG6(SEQ ID 3), pG7(SEQ ID 5), or pG8(SEQ ID 6); and

d) fragments or variants derived from a), b) or c),

wherein the nucleic acid comprises a functionally active promoter which is a carbon source regulatable promoter capable of expressing the POI in a recombinant eukaryotic cell at a transcription rate of at least 15% compared to the native pGAP promoter of the cell.

17. The nucleic acid according to item 16, wherein the variant of pG1(SEQ ID 1), pG3(SEQ ID2), pG6(SEQ ID 3), pG7(SEQ ID 5) or pG8(SEQ ID 6) is a functionally active variant selected from the group consisting of: homologues having at least about 60% nucleotide sequence identity, homologues modified by insertion, deletion or substitution of one or more nucleotides within the parent nucleotide sequence or at one or both of the distal ends of the sequence, preferably having a nucleotide sequence of at least 200bp, and analogues derived from species other than pichia pastoris.

18. The nucleic acid according to item 16 or 17, wherein the functionally active variant of pG1 is selected from the group consisting of: pG1a (SEQ ID 41), pG1b (SEQ ID 42), pG1c (SEQ ID 43), pG1d (SEQ ID 44), pG1e (SEQ ID 45) and pG1f (SEQ ID 46).

19. An expression construct comprising a nucleic acid according to items 16 to 18 operably linked to a nucleotide sequence encoding a POI which is not naturally associated with the nucleotide sequence encoding the POI, under the transcriptional control of the promoter.

20. A recombinant eukaryotic cell comprising the construct of item 19.

21. A method for identifying a carbon source-controllable promoter from a eukaryotic cell, comprising the steps of:

a) culturing eukaryotic cells in batch culture in the presence of a carbon source under cell growth conditions,

b) further culturing the cells in fed-batch culture in the presence of a limited amount of a supplemental carbon source,

c) providing a sample of the cell culture of steps a) and b), and

d) performing a transcriptional analysis in said sample to identify a regulatable promoter that exhibits a higher transcriptional intensity in the cells of step b) than in the cells of step a).

Sequence listing

<110> Longzai Co., Ltd. (Lonza Ltd.)

<120> regulatable promoter

<130> LO001P

<160> 46

<170> PatentIn version 3.3

<210> 1

<211> 1001

<212> DNA

<213> Pichia pastoris (Pichia pastoris)

<400> 1

atttccaccc ccatcccagt agaatgtagg gtccccaaac atttgctccc cctagtctcc 60

agggaaatgt aaaatatact gctaatagaa aacagtaaga cgctcagttg tcaggataat 120

tacgttcgac tgtagtaaaa caggaatctg tattgttaga aagaacgaga gttttttacg 180

gcgccgccat attgggccgt gtgaaaacag cttgaaaccc cactactttc aaaggttctg 240

ttgctataca cgaaccatgt ttaaccaacc tcgcttttga cttgactgaa gtcatcggtt 300

aacaatcaag taccctagtc tgtctgaatg ctcctttcca tattcagtag gtgtttcttg 360

cacttttgca tgcactgcgg aagaattagc caatagcgcg tttcatatgc gcttttaccc 420

cctcttttgt caagcgcaaa atgcctgtaa gatttggtgg gggtgtgagc cgttagctga 480

agtacaacag gctaattccc tgaaaaaact gcagatagac ttcaagatct cagggattcc 540

cactatttgg tattctgata tgtttttcct gatatgcatc aaaactctaa tctaaaacct 600

gaatctccgc tatttttttt ttttttttga tgaccccgtt ttcgtgacaa attaatttcc 660

aacggggtct tgtccggata agagaatttt gtttgattat ccgttcggat aaatggacgc 720

ctgctccata tttttccggt tattacccca cctggaagtg cccagaattt tccggggatt 780

acggataata cggtggtctg gattaattaa tacgccaagt cttacatttt gttgcagtct 840

cgtgcgagta tgtgcaataa taaacaagat gagccaattt attggattag ttgcagcttg 900

accccgccat agctaggcat agccaagtgc tatgggtgtt agatgatgca cttggatgca 960

gtgagttttg gagtataaaa gatccttaaa attccaccct t 1001

<210> 2

<211> 1000

<212> DNA

<213> Pichia pastoris (Pichia pastoris)

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gtaaatagcg gcagcaatcc agtaaccttt tctgaatagc agagccttaa ctaaaataat 60

ggccagggta aaaaattcga aatttgacac caaaaataaa gacttgtcgt tataagtctt 120

aacaaagtcc gcaattttgg agctaacggt ggcggttgct gggatattca ataatggtag 180

aatgttgctg cgggtatatg acagagcgtg aaacacactg aacaaggtaa atggaacaac 240

agcaattgca atatggggga ggatagtcaa gaacaaagca gcaatggcaa agtactgaat 300

attctccaaa gccaaaaggt ccagtggttt caacgacaaa gtcttgttgg tatagctttg 360

gaacaaaagg acaccgaaag actcgacagc gcccacaaat acagcgttgt agaagaacga 420

attgattgct ccagagcttc taatagtcag aagatacccc aaacctccga gcaacgttag 480

cacatgacct aagaaccagg cgaagtgaag agtctggaat aacgacaccc agtcagtttt 540

tcctgagctc ctggtgggat tggtagaagc atttgatttg cttggagtgg ttttatttga 600

agatggtgtt gaagccattg ttgctaaaga gtcggagttt tgcttttagg gtttgttaag 660

caaaggagga aaaactgcgc cgtttgaagt cccaggtagt ttcgcgtgtg aggccagcca 720

gggaaagctt ccttcggtac ttttttttct tttgcaggtt ccggacggat taagcttcgg 780

gttatgaggg gggcggtagc caattccgga cacaatattg cgtcgcagct agtcaccccg 840

ccataaatat acgcaggatt gaggtaataa catcgatagt cttagtaatt aatacaattc 900

agtggcgaat ttggcaacat gacgtaaggc ccactgttgt ctataaaagg ggatgaattt 960

tcatgttttt gaggcctccc ggacaattta ttgaactcaa 1000

<210> 3

<211> 1001

<212> DNA

<213> Pichia pastoris (Pichia pastoris)

<400> 3

agaccagcag tttaactacg caaatccaca ggaatttcta catcacaata ccaatggtaa 60

taccacgacg tcaaggaatg gaaacgacga cttggaggaa gacttcgtca acctcttgcg 120

gagtacccga ggctaagaca ataagaagaa aaaaaaaaga aaagcggtgg gggagggatt 180

attaaataag gattatgtaa ccccagggta ccgttctata catatttaag gattatttag 240

gacaatcgat gaaatcggca tcaaactgga tgggagtata gtgtccggat aatcggataa 300

atcatcttgc gaggagccgc ttggttggtt ggtgagagga gtgaaatatg tgtctcctca 360

cccaagaatc gcgatatcag caccctgtgg gggacactat tggcctccct cccaaacctt 420

cgatgtggta gtgctttatt atattgatta cattgattac atagctaaac cctgcctggt 480

tgcaagttga gctccgaatt ccaatattag taaaatgcct gcaagataac ctcggtatgg 540

cgtccgaccc cgcttaatta ttttaactcc tttccaacga ggacttcgta atttttgatt 600

agggagttga gaaacggggg gtcttgatac ctcctcgatt tcagatccca ccccctctca 660

gtcccaagtg ggacccccct cggccgtgaa atgcgcgcac tttagttttt ttcgcatgta 720

aacgccggtg tccgtcaatt aaaagtcgca gactagggtg aactttacca tttttgtcgc 780

actccgtctc ctcggaatag gggtgtagta attctgcagt agtgcaattt ttaccccgcc 840

aagggggggc gaaaagagac gacctcatca cgcattctcc agtcgctctc tacgcctaca 900

gcaccgacgt agttaacttt ctcccatata taaagcaatt gccattcccc tgaaaacttt 960

aacctctgct ttttcttgat ttttccttgc ccaaagaaaa g 1001

<210> 4

<211> 1000

<212> DNA

<213> Pichia pastoris (Pichia pastoris)

<400> 4

tggactgttc aatttgaagt cgatgctgac gatgtcaaga gagatgctca attatatttg 60

tcatttgctg gttacactgg aaacgctact tttgttggcg gaaactctac cagtttggcc 120

gtccatgtaa acgatgtcgt tctgggccgt gaccgtttca acacgaacat aaccaatgac 180

aaatccactt acaggtctag ttcatatgga ggcaattggt accttacttc tttggatgtc 240

ccaagtgggg ctttaacgtc tggtactaac aatgtctcgt ttgtcactac aaactccgag 300

gtaaataaag gattcttgtg ggattctctc aagtttgttt ggaagttgta acaggtttat 360

aagcatatcg tgcgcttgtc cacaattgaa tcatttattg ttgcgagata catgaacaaa 420

gtgtgaactg ggacccatta ctacaattcc cacgcaaccg ttgtttcaaa gcccatattt 480

tttgacaatt gtttcgttac acccccagtt tgatgtacat cgcttgcaat gatgtgtgtc 540

ccggagtatt ttccatattc agcttgaatt cgtatactca accaatatct gggggtatac 600

ttttatgtaa cctatacaaa tcaactatac tatttcacct ttcgaccaat catctcccat 660

cttgttaagt tttgcttcct atatccctga ccctgacatc acccatgatt ccgctcaacg 720

gttctcctct acatcgtccc tcttttggag agggtgttca gtttgacatt caaattaccc 780

cccgccatca cgcgcaaccg agaccgcacc cccgaatttt cacaaattac cccacaccct 840

atactccacc actatgaggg ttattagaac tgatcacgta taaataccac cgcaagttcc 900

caagggatcg tgttcttctt ctccaattgc aatcatattt ctgactcttt ctagttcaga 960

ttaattcctt tacacttgct tttttccctt acctttatcc 1000

<210> 5

<211> 1000

<212> DNA

<213> Pichia pastoris (Pichia pastoris)

<400> 5

aattgattaa gttcagtgaa atttcaaacc gctatacaca acaggacaac tttgagttta 60

gaaaaatccg atgtagtgta acggctagca cggtccgctt tcaccgggca gacccgggtt 120

cgactcccgg catcggagtt tatttttcca tttcgttctt tagagtattc tcctcagcat 180

gcccccctga atttttcctt ttttccatgt gtcccatttt tccacttttt ttacagtttt 240

cctcgtgatg ttaattggct acacaaaagc tgccacacga aaccttaatc acgaaaaact 300

atacagcctt cactaatccg tagccccata atatgttgtc cacgtgctgt tgggtactac 360

ctgtagactc tcatacccca ctccgtcttt ctccaacaat taacgcagta ccgagattta 420

tcagcagact caaattgggc aaactctgta tttttccttg cccgcataat ttatgggtct 480

caggcctcca cgtttcctgt ttacttgaag aatattggct gcggaaaaag tggtaaggac 540

aacccccttt taattggatc cagtttttcc gaaatgttcc gatccgtacg tcatctccga 600

agccgtacat tttcactcaa tctacgtagc tttggactca gcgctcctgg aattgccagg 660

acagtttact tgagttgata ttcccttgta gattgtgtgc ttctttttcc aaaatttgag 720

gcttcgtttg aaaagtggaa tctggtcgct agatcacttc atgcctattt ttcacggaaa 780

aataagtggt actatgcacc ccttaaacct aaagaaaaac ggaaaaatta ccccaaaacc 840

tggtgatgtt tttcgcccct ttctttttat ccgagttttt cttttttctt gtctgccaaa 900

ttcctctcct gaccttagcg tccccggaaa aaattaacta cttaaggacc gaatgagccc 960

cagcttttcc ccttctcttc attattcccc ataatataat 1000

<210> 6

<211> 1000

<212> DNA

<213> Pichia pastoris (Pichia pastoris)

<400> 6

ctgcacaacc attgccagta aggacgaaga gaaggcccca ctacccaaaa ttcaggataa 60

cgtcttcata ccatgcagcg acgcctacaa gacgctgtca agacatgcca acttcaacga 120

agtgaacttt aacacattga tcgggaaatt gaccaccaag ggaatgctgg ttgaggctgg 180

aagcgttgcc agtgtcctga gggaactgga ccgaaagttt agtaatgcat aagaggatat 240

atataggaat gcagtaataa tattagtacc cattaagtgg gctaagccat tggaaggccg 300

tctgactgat ggtggtgttc ttctcattta gatagtgcat ttgcaactac cgtctgagat 360

tgagtttgat gtgaagctcc agcgccaaaa cagtataaga accttatctc cgcattattg 420

ttcttgcgta aaagtttgtg tgaagaaaca ggggtagttg cgcagattag ttgtaatatg 480

cgcataggat gggtcattga cttctttcct cgaaagagcc acaccgttag ctaaaaaagg 540

acgcgcatct accccaaaat agaatgtggg gaaataggac gcgcaacttc ctctcaatca 600

ctggacgtca gaaaaacaaa tgcgcaatcg agtcaccctc cgtgataccc tccgtgatac 660

cccctctccg tctattctga cagcgtctcc ccatgacgtt tcaatctact tagaaaagat 720

ttcgtttttt tttccttcaa ttacacgatc tcatcttctg caagggtctg gaggacatca 780

ccaatctgcg actccataac ttagtcctga gtttatattt acgcttcatc tgatgagtag 840

gaagaaaaag tttcacgaaa ttcccccgcc aacttgccct tcggaataag cagccactct 900

ccttctgccc atagtaagct tgcgcgaggc cccaacttgg ccagaaactt taaatatgcc 960

aaacaatctc ccccaatcta agttctccct cttctaaaaa 1000

<210> 7

<211> 1662

<212> DNA

<213> Pichia pastoris (Pichia pastoris)

<400> 7

atgtcctcgt tttttctaaa caaccaaaca gtaaagatga tgacgccttt gggaagagct 60

agagctctaa attttcaggg caaagtttat gataaatttc caaaaactta caatatctac 120

gctattgcaa taacagccac cgtttctgga ctgatgttcg gttttgatat ttcttctgtg 180

tcctcgttcg taagtcagga tcattacaga aactacttca accgtcccga cagtttgacg 240

caagggggta tcaccgcaag tatggctgga ggttctttct tgggttcgtt attttcttct 300

gacttccagg atatctttgg aagaagagtt gctctgcata tgtgcagtgt cctctggatt 360

atcggggcca ttcttcaatg cgctgcacaa aaccaaggta tgctgatcgc agggagattg 420

atttccggta tcggtgtcgg gtttggttca gcttcagctc cagtctattg ttctgaagtt 480

gctccagcaa agattagagg aatgattgga ggattatttc aattttctgt cactgtgggt 540

atcatgataa tgttttatat cggatatgga tgtcactaca ttgacggcgt tgcatcattt 600

agactggcct ggggtttgca aatggttcca ggtcttattc ttttggtcgg tgtattcttc 660

cttcctgagt ctccaagatg gctggctaac cacaaccgct gggaagacgc agttgaggtt 720

attgctaatg ttgttgcaaa aggtgacaga gaaaacgccg atgtgcgtct gcaattggat 780

gaagttcagg agcaactatt gattgacaaa gatgcttctg attttggtta ccttgatttg 840

tttaagaaag attgtatcaa acgtaccttc attggagtgt cagctcaagt gtggcaacaa 900

ctttgtggta ttaatgttgc aatgtactac gttgtgtatc tcttccaaat ggctggtttt 960

actggaaatg tggcgttggt atcgtcctca attcaatatg ttttgaatgt tgttatgact 1020

gttccagctt tgtttctaat ggaccgtata ggcagacgac ccctactaat tggtggtggt 1080

attttcatgt gtatttggct gtttggagtg gcaggattat taggcactta ctctgaacca 1140

attgaaaatt tcagcggtga tgatactgtc agaattacta ttcctgacca gcacaaggct 1200

gcagcaaggg gtgttattgc ctgttcctat ctattcgtgt gctcctttgc tccaacctgg 1260

ggtatctgca tttgggttta tgcctctgaa attttcaaca acagacaaag agcaaaggga 1320

gcagcatttg ctgcctccgc taactggatt ttcaactttg ccttggctat gttcgtgcca 1380

tcagccttta gaaacattac atggaagact tacatcattt ttggagtatt ttcgttctgc 1440

ttaacaatcc atgttttctt acaattccca gaaaccagag gtaagacttt ggaagaaatt 1500

gatcaaatgt ttaaggacaa tattccagct tggagaagtg cttcgtacgt tccagatatg 1560

ccaattttca acaaagagaa ggtagtatct actgagcatg cagaaaatgc ttccagctcg 1620

tccgaaaaag ccttgatggt tcaggaagag gaatctgtat aa 1662

<210> 8

<211> 987

<212> DNA

<213> Pichia pastoris (Pichia pastoris)

<400> 8

atggtagttg caatcgaagg tggtacaggc ttaggcctta tgaatcttac ttggaaacca 60

actccaaccc caattgatga tgcaattgag acaattagat atgctgttga ggaagctggt 120

gtcagatact tgaacggagg agagttctac aactttcctc ttgattcaaa cctgaatttg 180

cagtacattc aggaatttgc aaaaaggtac cccgagctat ataaaaaggt gagtctgtcg 240

gtaaaaggtg ctgtcagttt ggtcgatgtg agccccgatt cttccccgga gaaccttgaa 300

aaatcgattt caaacataac caaacatttg ccgaacaact tcctgccaat ttttgagcct 360

gctagaatcg ataaacgtta ctccattgag gagacaataa agaatctctc taagttcgtc 420

gaagatggca gaattggagg tatttcactt agtgaagttg gtgctgacac tatcagaaga 480

gctgcgaaag tggctcccat cgcctgtgtg gaagtggagt tttctctatt gactagagat 540

attcttcata atggagttct tgctgcttgt gaggatttga acattcctat tattgcctac 600

agtcccttgg gaagaggatt tttgactgga acgataaaca gcaaagctga cattcctgaa 660

ggtgatatca ggttaagttt ggaaagattc aatgacgatg aagttattga acacaatttg 720

aaacttgttc acggtttgaa aaagatagcc gacaaaaaag gagtcacatt ggctcaattg 780

tctcttgcgt ggttacgaaa gtttggagat aaacacgtca aggtgcttcc tattccaagc 840

tgctcatctc ctcgtagagt tgcagaaaac acaaaagaga tttccttgac tgatagcgag 900

ttccaggaga ttactgactt tgcagagtcg gttccaatca aaggtggtcg ttacaacaaa 960

gcaagtgagg ctgttcttaa cggttag 987

<210> 9

<211> 1506

<212> DNA

<213> Pichia pastoris (Pichia pastoris)

<400> 9

atgacatttg ctcctccctt agaattcgag attgaccttc ctaacggatt gaagtacact 60

caaccattgg gactcttcat caacaatgag tttgttgaag gtgtagaggg aaagctctta 120

ccagtgatca atccttgtga tgagactaaa ataacccaag tttgggaagc ttctgcagcg 180

gatgttgacc gtgctgttga tgccgctgaa gatgctttca acaactccgt atgggctact 240

caggacccat tagagagggg aaagctgatg aacaaattgg cagaccttat cgatcgtgac 300

ttcaacatct tggctggtat cgaatccatc gacaatggta aggcctatac ctctgcccag 360

ggtgatgtta ctcttgctgt caactacatc agatcctgtg ctggatgggc cgacaagatt 420

ttgggaaacg ttgttgattc cggaaacacc caccttaact tggttaaaag agagccattg 480

ggtgttgtgg gacaaattat cccatggaac tttcctctcc tgatgttggc ttggaagttg 540

ggacctgcgc tggccacagg taacactgtt gttttgaaga ctgccgagtc tacccctctg 600

tcgggtttat acgttgccaa attgatcaag gaggccggtt tcccacctgg tgtggttaac 660

attctcagtg gtttcggtaa cccagctgga gctgccatcg ctgctcatcc cagaatcaag 720

aagattgctt tcaccggatc cactgcaaca ggccgtaaga tcatggaagc agccgctaaa 780

tctaacctga aaaaagtcac tttggaacta ggtggtaaat ctccaaacat tgtgtttgaa 840

gatgctgata tccagaagac tatccataac attattttgg gaatcttctt caattctggt 900

gaagtctgtt gtgcaggttc cagagtctac attcaagaca ctgtgtatga agaagtgctt 960

gaagccttca agaaggagac tgataacgtt aaggttggtg gaccattcga agaaggtgtc 1020

ttccaagggc ctcagacctc tgagttgcaa cttaacagaa tccttagtta catcaaacac 1080

ggtaaggatg aaggtgctcg tgtaattacc ggtggttcaa gataccgtaa ccgaggttac 1140

tacattaagc ccacaatttt tgctgacgtt actgaagaca tgaagattgt caaggaggag 1200

atttttggtc ctgtggttac tatcactaag ttctctaccg tggatgaggt tgttggatat 1260

gccaacaaca ccaactatgg tctagctgct ggtattcaca caaacaactt gaacaaagcc 1320

attgatgttg ccagtagaat caaggcgggt gtcgtttgga ttaacaccta caacgatttc 1380

caccacatgg ttcctttcgg aggttatgga gaatctggta ttggcagaga gcttggtgct 1440

gaggctttgg ataactacac tcaagccaag gctatcagaa ttgcttacac tcctgaacat 1500

aagtag 1506

<210> 10

<211> 1578

<212> DNA

<213> Pichia pastoris (Pichia pastoris)

<400> 10

atgcttagaa cttctccagc tactaagaaa gctctcaagt cgcagattaa cgccttcaac 60

gttgctgcct tgagattcta ctcctcattg cctttgcagg ttccaattac cttgccaaac 120

ggtaagacct acaatcagcc aacaggtttg tttatcaaca atgagttcgt tccttctaag 180

caaggtaaga cctttgctgt tttaaaccct tccactgagg aggagattac tcacgtctac 240

gagtccagag aggacgacgt tgagttagcc gttgcagccg ctcaaaaggc tttcgactca 300

acctggtcca cccaggaccc tgctgagaga ggtaaggtct tgaacaagtt ggctgacctg 360

atcgaggagc actctgagac ccttgccgcc atcgagtcct tggacaacgg taaggccatt 420

tcctccgcta gaggtgatgt tggtctggtt gtcgcctact tgaagtcctg tgccggttgg 480

gccgacaagg ttttcggtag agttgttgaa accggaagct cccacttcaa ctacgttaga 540

agagagccat tgggtgtttg tggtcagatt atcccatgga actttcctct tctgatgtgg 600

tcctggaaag ttggtccagc tttggccact ggtaacactg ttgtcctgaa gacagccgag 660

tctactcctc tgtccgccct gtacgtttcc caattggtca aggaggccgg tatcccagct 720

ggtgtccaca acattgtgtc cggtttcggt aagattactg gtgaagctat tgctactcat 780

cctaagatca agaaggttgc cttcactggt tctaccgcca ctggtcgtca catcatgaag 840

gctgctgccg aatccaactt gaagaaggtt actttggagt tgggtggtaa atctcctaac 900

atcgtgttca acgatgctaa cattaagcaa gctgtcgcca acatcatcct cggtatttac 960

tacaactctg gagaagtttg ttgtgctggt tccagagttt atgttcaatc cggtatttac 1020

gacgagcttt tggccgaatt caagactgct gctgagaatg tcaaggttgg taacccattc 1080

gacgaggaca ccttccaagg tgctcaaacc tctcagcaac aattggagaa gattttgggt 1140

ttcgttgagc gtggtaagaa ggacggtgct actttgatta ctggtggtgg cagattaggt 1200

gacaagggtt acttcgtcca gccaactatc ttcggtgatg ttacaccaga gatggagatt 1260

gtcaaggaag agatctttgg tcctgttgtc actatcagca agtttgacac cattgatgag 1320

gttgtcgacc ttgctaacga ctctcaatac ggtcttgctg ctggtatcca ctctgacgat 1380

atcaacaagg tcattgacgt tgctgctaga atcaagtccg gtaccgtgtg ggtcaacacc 1440

tacaacgatt tccaccaaat ggttccattc ggtggatttg gccaatccgg tattggtcgt 1500

gagatgggtg ttgaagcttt ggaaaactac acccaataca aggctatccg tgtcaagatc 1560

aaccacaaga acgagtaa 1578

<210> 11

<211> 1614

<212> DNA

<213> Pichia pastoris (Pichia pastoris)

<400> 11

atgagttcaa cagatatcca aggtgatcaa ggtgacaatg aaaagatata cgccattgag 60

agcagtccct ccaatgagca aataaaagat attcatgagg ctccggccga caacaaaagt 120

gaactagaca tcccagtcaa acccaagggt tcctatatct tggtgtctgt gttatgtctt 180

ctagtcgcat tcggtggttt cgtgttcggt tgggataccg gtaccatctc aggtttcgtt 240

aacatgtctg actttacgag acgtttcgga cagtttaacg gtgaaacgta ttacctttct 300

aaagtgagag ttggtttaat tgtttctatt ttcaacattg gttgtgctat cggaggtgtc 360

actctaggta aacttggtga catttggggt agaaagaagg ctttgatgtt cgtcatggtc 420

atctatatgg tcggtatttt gattcaaatt gcttccattg acaaatggta ccagtatttc 480

attggaagaa ttattgcagg tctggccgtc ggtgcagttt ccgttttatc ccccatgttc 540

atcagtgaga cttctcctaa acacatcaga ggttccttag tctcctgcta ccaattaatg 600

attacagccg gtattttctt gggttactgt accacttacg gaaccaagac ttacaccgac 660

tccacccaat ggagagttcc tttgggattg tgtttcgctt gggccattct gatgattgtt 720

ggtatgacct tcatgccaga gtccccacgt ttcttggttg aggttaacag agtcgacgag 780

gctatgaagt ccattgccag agttaacaag gtctctatcg acgatccatc tgtctacaat 840

gagatgagac ttatttctga cggtattgag aaggagaagg aggctggtag cgtttcttgg 900

ggtgaactgt tcactggtaa gccaaagatt ttctaccgtc tattgattgg tattttcatg 960

caatctttgc aacaattgac cggtaacaac tatttcttct actacggaac taccattttc 1020

aaggctgtcg gattggacga ttctttccaa acttctatca ttcttggtgt tgtcaatttt 1080

gcttccacat tcctaggtat ctacaccatg gataaatttg gtagaagaag aacactttta 1140

ggaggttctg gagccatggt tgtttgtttg gtcattttca gttccgttgg tgtcaagtct 1200

ctttatgaga acggtaagga tgatccatcc aaaccagcag gtaacgccat gattgtcttc 1260

acctgtctgt tcattttctt ctttgcatgt acctgggctc caggtgtttt cgtcgttgtg 1320

tctgaaacct acccacttag aattagatcc aagggtatgg ccatcgctca aggttccaat 1380

tggctttggg gtttcctcat tgccttcttc actccattta tctcaggtgc cattgatttc 1440

gcctacggtt acgtctttat gggatgtact ctgttcgcct tcttctttgt gtacttcttc 1500

gttcctgaaa ccaagggtct gtcgctggaa gacgttgatg aagtctatga gaaccttacc 1560

ttcggaagag catatgcata cagccacacg attaaagaca agggcgccct ataa 1614

<210> 12

<211> 1032

<212> DNA

<213> Pichia pastoris (Pichia pastoris)

<400> 12

atggccctat ctcctaccta tcagggctac atatctacca ctggcgacgc gttgatcgtg 60

atccaggcag ctctaaataa ccatttgaat cttcttcccc gaagaccaag agaaagagag 120

cgagatgggc taatacgatc aggtaacgta tttgtttttg tcgagcaacg gtctcatatc 180

aaacgatgga ccgatggtat cccctggtct ccatctagag tccttggaaa gttcttgttg 240

tatcgggaac tggacaagga tacccccaaa aactcgcaaa gtgacgaaga tactgaggag 300

gggagaaaga ggcgaaagac ttctgtggat gtaaccgatc caaataccag gcagttggtg 360

ggatcattgg tgacttccta tgacttcaaa gaggatggac ttattaaaaa aacactctcc 420

ttgactttcc agaccggtgc taatgaagaa agggaaacag tgcacttgat tagttattat 480

actccggaag atgtaacgaa ccatcgtttg aacaggccgt ctgacaatcc atatctggcc 540

aatatcactg tttcagagtc attattgact gccttgagag agagtaccct tggaggaaga 600

gcaacgtctg atgacgagct ttctttagtc agaagtaact cgttagagta ccaagaggta 660

ccaatgaaca tatctatgtc tttaccttta tcaactccac tttccttgaa cacaggagta 720

aactcaacta cccagctgca acagcaacaa ctacaacaac aacaacagca acagcaacag 780

cagcagcaac aacagcagca acaacagcaa ccggtagcat cccttccaaa atttgatgga 840

tcctttctat tacaacaggg tgtaattcca gttcctcatt tcatggacca aaaaatggga 900

agtagcaatt cgtggattaa caattggttt cgtccaaatt cgtcagaatc aaatgggcta 960

tcggttatcg gacctcacaa gggatatgac gaacaaagtc cagcaacgag ttatactttg 1020

aatgaacgtt ga 1032

<210> 13

<211> 491

<212> DNA

<213> Pichia pastoris (Pichia pastoris)

<400> 13

cttttttgta gaaatgtctt ggtgtcctcg tccaatcagg tagccatctc tgaaatatct 60

ggctccgttg caactccgaa cgacctgctg gcaacgtaaa attctccggg gtaaaactta 120

aatgtggagt aatggaacca gaaacgtctc ttcccttctc tctccttcca ccgcccgtta 180

ccgtccctag gaaattttac tctgctggag agcttcttct acggccccct tgcagcaatg 240

ctcttcccag cattacgttg cgggtaaaac ggaggtcgtg tacccgacct agcagcccag 300

ggatggaaaa gtcccggccg tcgctggcaa taatagcggg cggacgcatg tcatgagatt 360

attggaaacc accagaatcg aatataaaag gcgaacacct ttcccaattt tggtttctcc 420

tgacccaaag actttaaatt taatttattt gtccctattt caatcaattg aacaactatc 480

acctgcaggc c 491

<210> 14

<211> 34

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 14

gatagggccc caaacatttg ctccccctag tctc 34

<210> 15

<211> 39

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 15

gatacctgca ggaagggtgg aattttaagg atcttttat 39

<210> 16

<211> 36

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 16

gatagggccc cagcaatcca gtaacctttt ctgaat 36

<210> 17

<211> 36

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 17

gatacctgca ggttgagttc aataaattgt ccggga 36

<210> 18

<211> 35

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 18

gatagggccc tggactgttc aatttgaagt cgatg 35

<210> 19

<211> 37

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 19

gatacctgca ggggataaag gtaagggaaa aaagcaa 37

<210> 20

<211> 36

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 20

gatagggccc agaccagcag tttaactacg caaatc 36

<210> 21

<211> 36

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 21

gatacctgca ggcttttctt tgggcaagga aaaatc 36

<210> 22

<211> 39

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 22

gatagggccc aattgattaa gttcagtgaa atttcaaac 39

<210> 23

<211> 42

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 23

gatacctgca ggattatatt atggggaata atgaagagaa gg 42

<210> 24

<211> 33

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 24

gatagggccc ctgcacaacc attgccagta agg 33

<210> 25

<211> 40

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 25

gatacctgca ggtttttaga agagggagaa cttagattgg 40

<210> 26

<211> 24

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 26

cctgaggctt tgttccaccc atct 24

<210> 27

<211> 30

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 27

ggaacatagt agtaccaccg gacataacga 30

<210> 28

<211> 22

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 28

tcgccgacca ctaccagcag aa 22

<210> 29

<211> 23

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 29

accatgtgat cgcgcttctc gtt 23

<210> 30

<211> 24

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 30

cctgaggctt tgttccaccc atct 24

<210> 31

<211> 30

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 31

ggaacatagt agtaccaccg gacataacga 30

<210> 32

<211> 28

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 32

aaacctagga aaagtgggca gcaaatgt 28

<210> 33

<211> 29

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 33

actctgtcac ttactggcgt tttctcatg 29

<210> 34

<211> 34

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 34

gatagggccc caaacatttg ctccccctag tctc 34

<210> 35

<211> 39

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 35

gatagggccc ggaatctgta ttgttagaaa gaacgagag 39

<210> 36

<211> 36

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 36

gatagggccc ccatattcag taggtgtttc ttgcac 36

<210> 37

<211> 36

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 37

gatagggccc ctgcagatag acttcaagat ctcagg 36

<210> 38

<211> 32

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 38

gatagggccc gaccccgttt tcgtgacaaa tt 32

<210> 39

<211> 37

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 39

gatagggccc ccggataaga gaattttgtt tgattat 37

<210> 40

<211> 31

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 40

gatagggccc gcctgctcca tatttttccg g 31

<210> 41

<211> 859

<212> DNA

<213> Artificial

<220>

<223> promoter variants

<400> 41

ggaatctgta ttgttagaaa gaacgagagt tttttacggc gccgccatat tgggccgtgt 60

gaaaacagct tgaaacccca ctactttcaa aggttctgtt gctatacacg aaccatgttt 120

aaccaacctc gcttttgact tgactgaagt catcggttaa caatcaagta ccctagtctg 180

tctgaatgct cctttccata ttcagtaggt gtttcttgca cttttgcatg cactgcggaa 240

gaattagcca atagcgcgtt tcatatgcgc ttttaccccc tcttttgtca agcgcaaaat 300

gcctgtaaga tttggtgggg gtgtgagccg ttagctgaag tacaacaggc taattccctg 360

aaaaaactgc agatagactt caagatctca gggattccca ctatttggta ttctgatatg 420

tttttcctga tatgcatcaa aactctaatc taaaacctga atctccgcta tttttttttt 480

ttttttgatg accccgtttt cgtgacaaat taatttccaa cggggtcttg tccggataag 540

agaattttgt ttgattatcc gttcggataa atggacgcct gctccatatt tttccggtta 600

ttaccccacc tggaagtgcc cagaattttc cggggattac ggataatacg gtggtctgga 660

ttaattaata cgccaagtct tacattttgt tgcagtctcg tgcgagtatg tgcaataata 720

aacaagatga gccaatttat tggattagtt gcagcttgac cccgccatag ctaggcatag 780

ccaagtgcta tgggtgttag atgatgcact tggatgcagt gagttttgga gtataaaaga 840

tccttaaaat tccaccctt 859

<210> 42

<211> 664

<212> DNA

<213> Artificial

<220>

<223> promoter variants

<400> 42

ccatattcag taggtgtttc ttgcactttt gcatgcactg cggaagaatt agccaatagc 60

gcgtttcata tgcgctttta ccccctcttt tgtcaagcgc aaaatgcctg taagatttgg 120

tgggggtgtg agccgttagc tgaagtacaa caggctaatt ccctgaaaaa actgcagata 180

gacttcaaga tctcagggat tcccactatt tggtattctg atatgttttt cctgatatgc 240

atcaaaactc taatctaaaa cctgaatctc cgctattttt tttttttttt tgatgacccc 300

gttttcgtga caaattaatt tccaacgggg tcttgtccgg ataagagaat tttgtttgat 360

tatccgttcg gataaatgga cgcctgctcc atatttttcc ggttattacc ccacctggaa 420

gtgcccagaa ttttccgggg attacggata atacggtggt ctggattaat taatacgcca 480

agtcttacat tttgttgcag tctcgtgcga gtatgtgcaa taataaacaa gatgagccaa 540

tttattggat tagttgcagc ttgaccccgc catagctagg catagccaag tgctatgggt 600

gttagatgat gcacttggat gcagtgagtt ttggagtata aaagatcctt aaaattccac 660

cctt 664

<210> 43

<211> 493

<212> DNA

<213> Artificial

<220>

<223> promoter variants

<400> 43

ctgcagatag acttcaagat ctcagggatt cccactattt ggtattctga tatgtttttc 60

ctgatatgca tcaaaactct aatctaaaac ctgaatctcc gctatttttt tttttttttt 120

gatgaccccg ttttcgtgac aaattaattt ccaacggggt cttgtccgga taagagaatt 180

ttgtttgatt atccgttcgg ataaatggac gcctgctcca tatttttccg gttattaccc 240

cacctggaag tgcccagaat tttccgggga ttacggataa tacggtggtc tggattaatt 300

aatacgccaa gtcttacatt ttgttgcagt ctcgtgcgag tatgtgcaat aataaacaag 360

atgagccaat ttattggatt agttgcagct tgaccccgcc atagctaggc atagccaagt 420

gctatgggtg ttagatgatg cacttggatg cagtgagttt tggagtataa aagatcctta 480

aaattccacc ctt 493

<210> 44

<211> 370

<212> DNA

<213> Artificial

<220>

<223> promoter variants

<400> 44

gaccccgttt tcgtgacaaa ttaatttcca acggggtctt gtccggataa gagaattttg 60

tttgattatc cgttcggata aatggacgcc tgctccatat ttttccggtt attaccccac 120

ctggaagtgc ccagaatttt ccggggatta cggataatac ggtggtctgg attaattaat 180

acgccaagtc ttacattttg ttgcagtctc gtgcgagtat gtgcaataat aaacaagatg 240

agccaattta ttggattagt tgcagcttga ccccgccata gctaggcata gccaagtgct 300

atgggtgtta gatgatgcac ttggatgcag tgagttttgg agtataaaag atccttaaaa 360

ttccaccctt 370

<210> 45

<211> 328

<212> DNA

<213> Artificial

<220>

<223> promoter variants

<400> 45

ccggataaga gaattttgtt tgattatccg ttcggataaa tggacgcctg ctccatattt 60

ttccggttat taccccacct ggaagtgccc agaattttcc ggggattacg gataatacgg 120

tggtctggat taattaatac gccaagtctt acattttgtt gcagtctcgt gcgagtatgt 180

gcaataataa acaagatgag ccaatttatt ggattagttg cagcttgacc ccgccatagc 240

taggcatagc caagtgctat gggtgttaga tgatgcactt ggatgcagtg agttttggag 300

tataaaagat ccttaaaatt ccaccctt 328

<210> 46

<211> 283

<212> DNA

<213> Artificial

<220>

<223> promoter variants

<400> 46

gcctgctcca tatttttccg gttattaccc cacctggaag tgcccagaat tttccgggga 60

ttacggataa tacggtggtc tggattaatt aatacgccaa gtcttacatt ttgttgcagt 120

ctcgtgcgag tatgtgcaat aataaacaag atgagccaat ttattggatt agttgcagct 180

tgaccccgcc atagctaggc atagccaagt gctatgggtg ttagatgatg cacttggatg 240

cagtgagttt tggagtataa aagatcctta aaattccacc ctt 283