Truncated promoters for recombinant gene expression

文档序号：1948437 发布日期：2021-12-10 浏览：8次中文

阅读说明：本技术 用于重组基因表达的截短的启动子 (Truncated promoters for recombinant gene expression ) 是由 F·克莱纳 L·斯特姆伯格 P·曼斯克于 2021-06-10 设计创作，主要内容包括：本发明涉及具有增加的相对表达效率的启动子,特别是具有增加的相对表达效率的甲酸脱氢酶启动子的启动子变体,用于重组蛋白产生和用于增加具有启动子活性的核苷酸序列的相对表达效率的方法。(The present invention relates to promoters with increased relative expression efficiency, in particular promoter variants of formate dehydrogenase promoters with increased relative expression efficiency, methods for recombinant protein production and for increasing the relative expression efficiency of nucleotide sequences with promoter activity.)

1. A promoter or promoter variant, characterized in that

The promoter is selected from SEQ ID NO: 1 is truncated to a length of 140-610 base pairs, preferably to a length of 140-560 base pairs, more preferably to a length of 160-560 base pairs, most preferably to a length of 160-500 base pairs;

wherein said promoter variant has at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 97% identity, most preferably at least 99% identity to said promoter.

2. The promoter or promoter variant of claim 1, wherein the relative expression efficiency of the promoter or promoter variant is greater than the relative expression efficiency of a polypeptide having the amino acid sequence of SEQ ID NO: 1 is at least 5%, preferably at least 10% higher.

3. The promoter or promoter variant of any of claims 1 and 2, wherein the promoter or promoter variant is comprised in an expression cassette, wherein the expression cassette further comprises a nucleotide sequence encoding a polypeptide and optionally a transcription terminator sequence, and wherein the promoter or promoter variant is operably linked to the nucleotide sequence encoding the polypeptide.

4. The promoter or promoter variant of any one of claims 1 and 2, wherein the promoter or promoter variant is comprised in a plasmid, and wherein the promoter or promoter variant is operably linked to a nucleotide sequence encoding a polypeptide.

5. The promoter or promoter variant of any one of claims 1 and 2, wherein the promoter or promoter variant is comprised in a yeast cell, and wherein the promoter or promoter variant is operably linked to a nucleotide sequence encoding a polypeptide.

6. The promoter or promoter variant of claim 5, wherein the yeast cell is a cell selected from the genera: pichia (Pichia), Komagataella, Ogataea, Candida (Candida), Hansenula (Hansenula), Saccharomyces (Saccharomyces), Schizosaccharomyces (Schizosaccharomyces), Kluyveromyces (Kluyveromyces), Zygosaccharomyces (Zygosaccharomyces), and Yarrowia, preferably Pichia pastoris (Pichia pastoris) cells or Yarrowia lipolytica (Yarrowia lipolytica) cells, most preferably Pichia pastoris cells.

7. A method for recombinantly producing a target polypeptide comprising the steps of:

a) introducing an expression cassette comprising a nucleotide sequence encoding the target polypeptide into at least one yeast cell;

b) producing a yeast cell culture by propagating the at least one yeast cell;

c) subjecting the yeast cell culture to conditions suitable for expression of a nucleotide sequence encoding the target polypeptide; and

d) isolating the produced target protein from the yeast cell culture,

it is characterized in that

The expression cassette further comprises the promoter or promoter variant of any one of claims 1 or 2 operably linked to a nucleotide sequence encoding the target polypeptide, and

wherein the yeast cell is a Pichia pastoris cell or a yarrowia lipolytica cell, preferably a Pichia pastoris cell.

8. The method of claim 7, wherein the conditions suitable for expression of the nucleotide sequence encoding the target polypeptide are fed-batch processes.

9. The process of claim 8, wherein the fed-batch process is operated at a glucose feed rate of at least 0.04mmol glucose per gram dry cell weight per hour (mmolGlu/g CDW/h) or at a glycerol feed rate of at least 0.07mmol glycerol per gram dry cell weight per hour (mmol Gly/g CDW/h), preferably at a glucose feed rate of at least 0.11mmol Glu/g CDW/h or at a glycerol feed rate of at least 0.21mmol Gly/g CDW/h, more preferably at a glucose feed rate of at least 0.18mmol Glu/g CDW/h or at a glycerol feed rate of at least 0.35mmol Gly/g CDW/h.

10. A method for increasing the relative expression efficiency of a nucleotide sequence having promoter activity,

the method comprises the following steps: by comparing the sequence of SEQ ID NO: 1, more preferably at least 95%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% identity, from the 5' -end to a length of 140-.

11. The method of claim 10, wherein the relative expression efficiency of the truncated promoter is greater than the relative expression efficiency of a polypeptide having the amino acid sequence of SEQ ID NO: 1, preferably at least 10%, more preferably at least 20%, even more preferably at least 50%, most preferably at least 100%.

Technical Field

The present invention relates to truncated promoters or variants thereof, methods for recombinantly producing a target polypeptide, and methods for increasing the relative efficiency of expression of a nucleotide sequence having promoter activity.

Background

The use of specific nucleotide sequences as promoters to facilitate recombinant gene expression and achieve target protein production has been studied in the biotechnology field for decades. Promoters used for gene expression in prokaryotic hosts are typically relatively short in length, typically consisting of less than 100 base pairs (bp), such as the T7 RNA polymerase promoter or the promoter of the e.coli lac operon. On the other hand, promoters for gene expression in eukaryotic hosts are typically 500 to 1000bp long, such as the FLD1 promoter or AOX1 promoter of Pichia pastoris (Pichia pastoris), and even promoters over 1kb (kilobase pair) are common.

Typical prokaryotic host organisms for recombinant gene expression are E.coli or Bacillus subtilis. Examples of eukaryotic host organisms are Saccharomyces cerevisiae (Saccharomyces cerevisiae), Trichoderma reesei (Trichoderma reesei) or Pichia pastoris. In recent years, strains of pichia pastoris have been established as a platform for recombinant production of industrially important proteins.

Well known promoters for regulating recombinant gene expression in Pichia pastoris are the methanol inducible promoter of the alcohol oxidase 1 gene (AOX1) and the constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase Gene (GAP). Hartner et al performed promoter engineering studies on the Pichia pastoris alcohol oxidase 1 promoter to identify transcription factor binding sites or to validate putative transcription factor binding sites by targeted short internal deletions in the Pichia pastoris alcohol oxidase 1 promoter nucleotide sequence (Hartner et al, nucleic Acids Res.2008.36(12: e 76)). Furthermore, in recent years, several novel promoter sequences with different regulation and expression strengths have been described (e.g.Vogl et al, ACS Synth biol.2016.5(2): 172-. Despite these scientific advances, the rational prediction of essential elements in eukaryotic promoters remains unreliable. The essential regulatory elements are typically separated from each other by hundreds or even thousands of base pairs in the primary nucleotide sequence and may interact only due to secondary or tertiary nucleotide structures. Therefore, the influence of insertion or deletion of a nucleotide sequence segment cannot be predicted. Thus, eukaryotic promoter engineering efforts still rely heavily on in-depth experiments with results that have heretofore been unpredictable.

In EP 0299108A 1, a nucleotide sequence comprising a control region and a structural gene encoding formate dehydrogenase is disclosed.

In WO 03/095653 a1, the nucleotide sequence of an engineered variant of the promoter from the formate dehydrogenase gene (FMD) of Ogataea angusta is disclosed. The disclosed invention relates to promoter variants comprising palindromic nucleotide sequences to achieve higher transcription rates.

In WO2017/109082a1, a variant of the formate dehydrogenase promoter for recombinant gene expression in yeast cells of Komagataella species is disclosed, which is about 620bp in length and at least 90% identical to the non-mutated wild-type FMD promoter of Ogataea angusta (also known as Ogataea polymorpha or Hansenula polymorpha). These variants illustrate the extent of single point mutations or deletions or insertions that can be introduced into the nucleotide sequence without adversely affecting its promoter function. It is emphasized that the indicated promoters can be induced not only by methanol but also advantageously by the so-called derepression action. Induction by derepression is described as being achieved by depletion of inhibitory carbon sources (e.g. glucose or glycerol) or by alternative supply of non-inhibitory carbon sources (e.g. sorbitol). Notably, when glucose feeding was completely stopped, a particularly high induction of the wild-type FMD promoter was shown.

In Song et al (Bioechnol Lett.2003.25(23):1999-2006), expression vectors comprising a 645bp variant of the FMD promoter or the methanol oxidase promoter of Hansenula polymorpha were described for the production of green fluorescent protein as a reporter protein in Hansenula polymorpha.

In Vogl et al (AMB express.2020.10(1):38), it was described that the 623bp wild-type FMD promoter from Hansenula polymorpha is capable of strong derepression expression in Pichia pastoris. It is particularly emphasized that derepression leads to strong activation of the HpFMD promoter in pichia pastoris when the inhibitory carbon source (e.g. glucose or glycerol) is depleted.

In Gellisen G et al: heterologus gene expression in Hansnula polymorpha: effective mutation of glucoamyylase ", Biotechnology, the international simple for induced biological, natural publishing Group, US, vo.9, March 1,1991(1991-03-01), p.291 295, the glucoamylase gene from Schwanniomyces occidentalis (GAM1) was introduced into the genome of the methylotrophic yeast Hansenula polymorpha ahs and the potential of this organism as a host for high level expression of Heterologous genes encoding secreted proteins was investigated. Colonies secreting active glucoamylase were found to be isolated after insertion or transformation, and some cycles were shown to be performed to produce mitotically stable strains carrying integrated foreign DNA.

In the work flow required to achieve recombinant gene expression, one essential step is the amplification of the expression plasmid and in particular the expression cassette. Plasmid amplification is usually carried out in bacterial cells (e.g.E.coli). After isolation of the amplified plasmid from the cells, the expression cassette can be excised from the circular plasmid by restriction enzyme digestion. Alternatively, the expression cassette may be obtained by DNA amplification by Polymerase Chain Reaction (PCR). Notably, both strategies rely on DNA replication by DNA polymerases, which tend to introduce unwanted mutations into the expression cassette. Although improvements were made to the E.coli strains used for plasmid amplification and the polymerase used for PCR, a certain number of mutant expression cassette molecules must be taken into account in any amplification step and cannot therefore be excluded. Statistically, DNA polymerases produce erroneous nucleotide sequences after processing a certain number of nucleotides. Thus, the longer the expression cassette, the higher the risk of introducing unwanted mutations during replication of the DNA by the polymerase. Furthermore, the yield of host cells successfully transformed with the expression cassette benefits from a higher number of molecules. However, it is known that PCR produces fewer products, i.e., fewer molecules, when the nucleotide sequence to be amplified is longer. Thus, an increase in the yield of PCR products can be achieved by reducing the length of the nucleotide sequence to be amplified. It is therefore desirable to use expression cassettes which are as short as possible, while still achieving their purpose. In general, it is known that molecular assembly of gene expression cassettes or plasmids is achieved with a high success rate when the length of the individual polynucleotide fragments to be assembled is short. Thus, also in this regard, shorter segments are expected to function rather than longer segments. However, shortening a fragment by truncating a functional polynucleotide sequence (e.g., a promoter) would not be expected to negatively impact the activity of such a functional sequence. In particular, the effect on eukaryotic promoter function after omitting the nucleotide stretch of the promoter sequence has been unpredictable to date (Hartner et al, nucleic Acids Res.2008.36(12: e 76)).

It is therefore an object of the present invention to provide more efficient eukaryotic promoters with high expression strength at shorter lengths, i.e. eukaryotic promoters with increased relative expression efficiency.

This object is achieved by providing a promoter, wherein said promoter is selected from the group consisting of SEQ ID NO: the 5' -end of the nucleotide sequence of 1 is truncated to a length of 140-610 base pairs, preferably to a length of 140-560 base pairs, more preferably to a length of 160-560 base pairs, most preferably to a length of 160-500 base pairs; or by providing a variant thereof, i.e. a promoter variant, wherein said promoter variant has at least 80% identity, preferably at least 90% identity, preferably at least 95% identity, more preferably at least 97% identity, most preferably at least 99% identity to said promoter. Those skilled in the art will appreciate the following unexpected effects: although having the sequence of SEQ ID NO: 1 or a variant thereof, but the activity of the truncated promoter or promoter variant as described herein has not been eliminated. By providing such truncated or shortened promoters or promoter variants, even short synthetic oligonucleotides, e.g. primer oligonucleotides, can be used, which can be added to other recombinant nucleotide fragments in a simple PCR reaction, thus greatly accelerating the steps required for assembly of the recombinant gene expression cassette, while reducing the risk of introducing point mutations (as in the case when the assembly of larger polynucleotide fragments has to be relied upon).

It is common practice in the art to write deoxyribonucleic acid (DNA) sequences, particularly promoter sequences in the 5 '-3' direction. The 5 '-end refers to the 5' -carbon atom of the ribose moiety of the nucleotide, and the 3 '-end refers to the 3' -carbon atom. For the avoidance of any doubt, the 5' -end of the nucleotide sequence of the promoter is distal to the nucleotide sequence of the promoter. In other words, the 5' -end is determined by the nucleotide farthest from the start codon of the open reading frame, the transcription of which is regulated by the promoter. The 5' -end of the nucleotide sequence of the promoter is the most upstream nucleotide of the start codon of the open reading frame whose transcription is regulated by the promoter. In contrast, the 3' -end of the nucleotide sequence of a promoter is the closest nucleotide upstream of the start codon of the open reading frame whose transcription is regulated by the promoter. In other words, the 3' -end of the nucleotide sequence of the promoter is the last nucleotide of the promoter upstream of the start codon of the open reading frame whose transcription is regulated by the promoter.

The skilled person is aware of the fact that small amounts of single point mutations, single nucleotide insertions or single nucleotide deletions can be introduced into a promoter nucleotide sequence without substantially reducing the activity or function of the promoter nucleotide sequence. Promoter activity or function will be considered significantly reduced when one or more single point mutations, insertions or deletions will result in a functional change of at least 60% compared to the function of the unaltered promoter. Thus, the invention also includes mutant promoter variants having at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 97% identity, most preferably at least 99% identity to a truncated promoter as described herein. For the sake of clarity only, it is noted that promoters or promoter variants are not included in the present invention, which are derived from SEQ ID NO: 1 to a length of more than 610 base pairs (e.g., with a promoter or promoter variant of 611bp, 612bp, 620bp, or even longer). Even if such longer promoters or promoter variants comprise promoter sequences of less than 610bp, these promoters or promoter variants with a length of more than 610 base pairs are not included in the present invention, since their relative expression efficiency is not considered to be increased sufficiently. In line with this, promoter variants having at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 97% identity, most preferably at least 99% identity to a truncated promoter as described herein and a length of more than 610 base pairs are also not included in the present invention. Also correspondingly, the peptide having SEQ ID NO: 1 is not included in the present invention even if it comprises a promoter having less than 610 bp. In other words, the promoter or promoter variant according to the invention does not occur naturally, but requires at least one truncation step by the experimenter. Thus, the promoter or promoter variant according to the invention relates only to non-natural or recombinant artificial molecules.

One skilled in the art is aware of methods for determining the degree of sequence identity, expressed as a percentage (%), between two or more nucleotide sequences. As an example, the Needleman-Wunsch algorithm (Needleman, Wunsch. J Mol biol.1970.48(3): 443-. For example, the default algorithm parameters (match/mismatch score: 2, -3. gap cost: presence: 5 extensions: 2) are used from NCBI (national center for Biotechnology information;https://blast.ncbi.nlm.nih.gov/ Blast.cgiPAGE_TYPE＝BlastSearch&PR OG_DEF＝blastn&BLAST_PROG_DEF＝blastn& BLAST_SPEC＝Global Aln&LINK_LOC＝BlastHomeLink) The appropriate tools are available online. It is believed that using the default settings for the DNA input sequence, a global alignment algorithm, such as the Clustal Omega tool from the European bioinformatics institute (https:// www.ebi.ac.uk/Tools/msa/clustalo /), will be used. When the degree of sequence identity between a promoter and a promoter variant comprising a point mutation and/or a point insertion and/or a point deletion is determined using the Needleman-Wunsch algorithm or a comparable global alignment algorithm, only the corresponding nucleotide sequences, i.e. nucleotide sequences of the same length, are compared. For example, if the promoter variant is derived from SEQ ID NO: 1 to a length of 160bp and the promoter variant further comprises three point mutations, the corresponding promoter sequence to which the promoter variant is to be aligned will also have a length of 160 bp. In other words, to allow correct determination of the sequence contained in SEQ ID NO: 1 and one or more promoter variants, the sequence identity between the sequence segment in SEQ ID NO: 1 should be the same length as the promoter variant. Only such SEQ ID NO: 1 would be the nucleotide sequence segment of SEQ ID NO: 1, so that it will only compare with the promoter variant in a global alignment if it does not contain any point mutations or insertions or deletionsSEQ ID NO: 1 is 100% identical.

In a preferred embodiment of the invention, the relative expression efficiency of the promoter or promoter variant as described herein is greater than the relative expression efficiency of a polypeptide having the sequence of SEQ ID NO: 1 is at least 5%, preferably at least 10% higher. Thus, it is possible to achieve an expression strength of a truncated promoter or promoter variant which is at least as high as that of a non-truncated promoter in terms of its length.

The term "relative expression efficiency" as used herein refers to the amount of target protein produced per base pair of the promoter nucleotide sequence. In the case where the target protein is an enzyme, "relative expression efficiency" (REE) may refer to the amount of enzyme activity per base pair of the promoter nucleotide sequence. For example, if expression of a non-truncated promoter with 623bp produces 80 units of enzyme activity per liter (U/L), its relative expression efficiency will be 80/623 ═ 0.128U/L/bp. If 83U/L is produced by expression from a promoter with a truncation of 500bp, its relative expression efficiency will be 83/500 ═ 0.166U/L/bp. The increase in "relative expression efficiency" of a truncated promoter relative to a non-truncated promoter, expressed as a percentage, can be calculated by multiplying the relative expression efficiency of the truncated promoter variant by 100 and dividing by the relative expression efficiency of the non-truncated promoter: 0.166 × 100/0.128 ═ 129.3%. Thus, the relative expression efficiency of the promoter with the truncation of 500bp was 29.3% higher than that of the promoter with 623 bp. In other words, the relative expression efficiency of a promoter with a truncation of 500bp will be 1.293 times higher than that of a promoter with 623 bp.

For the avoidance of doubt, a truncated promoter is considered to have a higher REE than a non-truncated promoter when the higher REE is determined by small scale culture (e.g. culture in a deep well plate) or medium scale culture (e.g. culture in a shake flask) or large scale culture (e.g. culture in a bioreactor). It is well known in the art that transformation of yeast, such as Pichia pastoris, typically results in a large number of transformed strains that behave inconsistently, for example Vogl et al (Appl Environ Microbiol.2018.84(6): e 02712-17). Thus, when comparing different expression strategies, e.g. different promoter variants, a preliminary screening of multiple transformants has to be performed to exclude clearly poor strains or clearly poorly performing strains. Without wishing to be bound by theory, it is believed that such poor or poorly performing strains are the result of destructive genomic integration, wherein the expression cassette is integrated into a genomic locus that disrupts or at least reduces the expression capacity of the expression cassette. Therefore, it is necessary to select only strains for comparison with each other, which represent the respective expression strategies. In other words, it is noted that only strains carrying the same amount of integrated expression cassette are compared, as well as at least 5, preferably at least 6, more preferably at least 7, even more preferably at least 8, most preferably at least 9 of the 90 transformants from each expression strategy.

The present invention also relates to a promoter or promoter variant as described herein, wherein the promoter or promoter variant is comprised in an expression cassette, wherein the expression cassette further comprises a nucleotide sequence encoding a polypeptide and optionally a transcription terminator sequence, and wherein the promoter or promoter variant is operably linked to the nucleotide sequence encoding the polypeptide. For clarity only, an optional transcription terminator sequence is also operably linked to the nucleotide sequence encoding the polypeptide. Surprisingly, by providing such an expression cassette, the same level of recombinant gene expression as a non-truncated promoter can be achieved, while having a smaller molecular size due to the reduced length of the promoter. Thus, when a cell of a production host organism is transformed with a certain amount of DNA of such an expression cassette, more expression cassette molecules can be introduced into the cell than when the production host organism is transformed with the same amount of DNA of an expression cassette comprising a non-truncated and thus longer promoter. In a preferred embodiment of the invention, the promoter or promoter variant as described herein is operably linked to a recombinant nucleotide sequence encoding a heterologous polypeptide. Thus, proteins may be produced that are not normally produced by the native host cell. The present invention also relates to a promoter or promoter variant as described herein, wherein said promoter or promoter variant is comprised in a plasmid, and wherein said promoter or promoter variant is operably linked to a nucleotide sequence encoding a polypeptide.

The term "operably linked" as used herein is to be understood as meaning that the promoter or promoter variant is linked to the nucleotide sequence encoding the polypeptide in an orientation which allows for promoting gene expression, i.e. transcription, of the nucleotide sequence encoding the polypeptide by the promoter or promoter variant. In other words, the 3 '-end of the promoter or promoter variant is linked to the 5' -end of the nucleotide sequence comprising the open reading frame encoding the polypeptide. The 5' -end of the open reading frame encoding a polypeptide is typically the start codon for the first amino acid of the encoding polypeptide. Typically, although not always necessary, the initiation codon has the sequence 5' -A-T-G-3 ', wherein the 5' -terminus of the open reading frame encoding the polypeptide is "A", i.e., the nucleotide deoxyadenosine monophosphate, and the initiation codon typically encodes the amino acid methionine. Typically, the 5 '-end of the open reading frame encoding the polypeptide is located near the 3' -end of the promoter or promoter variant. The 5 '-end of the open reading frame encoding the polypeptide may be linked directly to the 3' -end of the promoter or promoter variant. Alternatively, the 5 '-end of the open reading frame encoding the polypeptide may be separated from the 3' -end of the promoter or promoter variant by one or more nucleotides without substantially altering the activity of the promoter or promoter variant on expression of the open reading frame encoding the polypeptide operably linked to the promoter or promoter variant. It is contemplated that the one or more nucleotides separating the 3 '-end of the promoter or promoter variant from the 5' -end of the open reading frame encoding the polypeptide can be, for example, a nucleotide sequence recognized by one or more restriction enzymes. Preferably, the one or more nucleotides separating the 3 '-end of the promoter or promoter variant from the 5' -end of the open reading frame encoding the polypeptide is shorter than 100 nucleotides, more preferably shorter than 50 nucleotides, even more preferably shorter than 20 nucleotides, most preferably shorter than 10 nucleotides. The terms "protein" and "polypeptide" are used interchangeably.

The invention also relates to a promoter or promoter variant as described herein, wherein said promoter or promoter variant is comprised in a yeast cell, and wherein said promoter or promoter variant is comprised in a yeast cellThe promoter or promoter variant is operably linked to a nucleotide sequence encoding a polypeptide. Thus, biotechnological protein production can be achieved in suitable host organisms. In a preferred embodiment, the yeast cell is a cell selected from the genera: pichia (Pichia), Komagataella, Ogataea, Candida (Candida), Hansenula (Hansenula), Saccharomyces (Saccharomyces), Schizosaccharomyces (Schizosaccharomyces), Kluyveromyces (Kluyveromyces), Zygosaccharomyces (Zygosaccharomyces), and Yarrowia, preferably Pichia pastoris cells or Yarrowia lipolytica (Yarrowia lipolytica) cells, most preferably Pichia pastoris cells. By selecting yeast cells from the genera: pichia, Komagataella, Ogataea, Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Kluyveromyces, Zygosaccharomyces, and Yarrowia, and in particular by selecting yeast cells from the organisms Pichia pastoris or Yarrowia lipolytica, preferably Pichia pastoris, protein production can be achieved in a particularly efficient manner. The skilled person is aware of the fact that the designation "Pichia pastoris" as used herein refers to several yeast strains which are also classified into the Komagataella pastoris and Komagataella phaffii taxonomic groups (Yamada et al, Biosci Biotechnol biochem.1995.59(3): 439. cndot. 444; Kurtzman.J. Ind Microbiol Biotechnol.2009.35(11): 1435. cndot. 1438). Other synonymous, albeit less common, names for Pichia pastoris strains are Zygosaccharomyces pastoris Guillierend, Endomyces pastoris (Guillierend), Saccharomyces pastori (Guillierend), Petasospora pastori (Guillierend), Zymopicia pastori (Guillierend) or Zygowia pastori (Guillierend) (Wolf et al, Sprin-Verlag Beilinger Heelsberg 2003. minor-yeast strain 448. Biotechn. 978. and 3. 488;http://www.mycobank.org/BioloMICS.aspxTableKey＝ 14682616000000067&Rec＝107579&Fields＝All). Of particular industrial relevanceThe Pichia pastoris platform strain of (A) is especially Komagataella phaffii CBS 7435 (also deposited for example as ATCC 76273 or NRRL Y-11430), or Komagataella phaffii GS115 (also deposited for example as ATCC 20864), or Komagataella pastoris CBS 704 (also deposited for example as ATCC 28485 or NRRL Y-1603).

It is another object of the invention to provide a method that allows high product titers through recombinant protein production. Thus, another aspect of the invention relates to a method for recombinantly producing a target polypeptide comprising the steps of: introducing an expression cassette comprising a nucleotide sequence encoding the target polypeptide into at least one yeast cell; producing a yeast cell culture by propagating the at least one yeast cell; subjecting the yeast cell culture to conditions suitable for expression of a nucleotide sequence encoding the target polypeptide; and isolating the produced target protein from the yeast cell culture; wherein the expression cassette further comprises a promoter or promoter variant as described herein operably linked to a nucleotide sequence encoding the target polypeptide, and wherein the yeast cell is a pichia pastoris cell or a yarrowia lipolytica cell, preferably a pichia pastoris cell. By applying this method, it has surprisingly been found that particularly large amounts of target polypeptide can be produced. It is noted that the method for recombinantly producing a target polypeptide according to the invention may also be performed in such a way that the expression cassette comprises a nucleic acid sequence comprising SEQ ID NO: 1.

The expression cassette can be introduced into the yeast cells by applying common yeast transformation protocols known to the person skilled in the art, for example by electroporation (Lin-Cereghino et al, biotechniques.2005.38(1): 44-48). The expression cassette may be provided alone or as part of a larger polynucleotide construct (e.g., a linear or circular plasmid). Yeast Cell cultures are produced by propagating one or more yeast cells by applying culture protocols known in the art to be suitable for culturing yeast, such as Krainer et al (Microb Cell fact.2012.11:22) or Cregg (Humana Press Inc.2007.Pichia protocols. ISBN 978-1-59745-456-8). For the purpose of isolating the produced target protein from the yeast cell culture, several options are available, including but not limited to separating the cells from the liquid by centrifugation and collecting the supernatant comprising the secreted target protein, or lysing the yeast cells and collecting the lysate comprising the produced target protein. In addition, further purification steps (e.g., fractional precipitation or chromatographic methods) may be applied to isolate the target protein produced.

In a preferred embodiment of the invention, the method as described herein is performed in a manner wherein the conditions suitable for expression of the target polypeptide are a fed-batch process. Preferably, the fed-batch process is operated at a glucose feed rate of at least 0.04mmol glucose per gram dry cell weight per hour (mmol Glu/g CDW/h) or at a glycerol feed rate of at least 0.07mmol glycerol per gram dry cell weight per hour (mmol Gly/g CDW/h), preferably at a glucose feed rate of at least 0.11mmol Glu/g CDW/h or at a glycerol feed rate of at least 0.21mmol Gly/g CDW/h, more preferably at a glucose feed rate of at least 0.18mmol Glu/g CDW/h or at a glycerol feed rate of at least 0.35mmol Gly/g CDW/h. Contrary to the current teaching in the prior art, it was found that not derepression by consumption of glucose or glycerol, but active feeding with glucose or glycerol as described herein increases the production of the target polypeptide under the control of an HpFMD based promoter, in particular when a glucose feeding rate of at least 0.04mmol Glu/g CDW/h or a glycerol feeding rate of at least 0.07mmol Gly/g CDW/h is applied, preferably at least 0.11mmol Glu/g CDW/h or at least 0.21mmol Gly/g CDW/h, more preferably at least 0.18mmol Glu/g CDW/h or at least 0.35mmol Gly/g CDW/h. In a preferred embodiment of the invention, the yeast cell is a pichia pastoris cell or a yarrowia lipolytica cell, preferably a pichia pastoris cell. Thus, recombinant production can be performed by methods as described herein to produce particularly high product titers.

Another aspect of the present invention relates to a method for increasing the relative expression efficiency of a nucleotide sequence having promoter activity, the method comprising the steps of: by comparing the sequence of SEQ ID NO: 1, more preferably at least 95%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% identity, from the 5' -end to a length of 140-. For the avoidance of doubt, truncation is only performed from the 5 '-end, while the 3' -end of the nucleotide sequence remains non-truncated. Preferably, the relative expression efficiency of the truncated promoter is greater than the relative expression efficiency of a polypeptide having the sequence of SEQ ID NO: 1, preferably at least 10%, more preferably at least 20%, even more preferably at least 50%, most preferably at least 100%.

The invention is also characterized by the following items:

1. a promoter, wherein the promoter is selected from the group consisting of SEQ ID NO: 1 to a length of 160-500 base pairs at the 5' -end; or a variant thereof (promoter variant), wherein said promoter variant has at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98%, most preferably at least 99% identity to said promoter.

2. The promoter of item 1, wherein the promoter or promoter variant comprises at least one amino acid sequence relative to SEQ ID NO: 1 non-naturally occurring replacement modifications.

3. A promoter, wherein the promoter is selected from the group consisting of SEQ ID NO: 1. the 5' -end of any one of the nucleotide sequences of 6-24 is truncated to a length of 560 base pairs, preferably 500 base pairs, more preferably 450 base pairs, more preferably 380 base pairs, more preferably 300 base pairs, more preferably 280 base pairs, more preferably 170 base pairs, most preferably 160 base pairs.

4. A promoter, wherein the promoter is selected from the group consisting of SEQ ID NO: 1 to a length of 560 base pairs, preferably 500 base pairs, more preferably 450 base pairs, more preferably 380 base pairs, more preferably 300 base pairs, more preferably 280 base pairs, more preferably 170 base pairs, most preferably 160 base pairs.

5. The promoter of item 4, wherein the promoter is selected from the group consisting of SEQ ID NO: 1 to a length of 160 base pairs, and wherein the promoter has the sequence of SEQ ID NO: 5.

6. The promoter or promoter variant according to any one of items 1 to 5, wherein the relative expression efficiency of the promoter or promoter variant is higher than that of a polypeptide having the sequence of SEQ ID NO: 1, preferably at least 10%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, most preferably at least 100%.

7. An expression cassette comprising a promoter or promoter variant according to any of items 1 to 6, and a nucleotide sequence encoding a polypeptide and optionally a transcription terminator sequence, wherein the promoter or promoter variant is operably linked to the nucleotide sequence encoding the polypeptide.

8. The expression cassette of clause 7, wherein the nucleotide sequence encoding the polypeptide is a heterologous nucleic acid sequence.

9. The expression cassette of any one of items 7 and 8, wherein the nucleotide sequence encoding a polypeptide encodes a heterologous polypeptide.

10. A plasmid comprising a promoter or promoter variant according to any one of items 1 to 6, or an expression cassette according to any one of items 7 to 9.

11. A yeast cell comprising a promoter or promoter variant according to any of items 1 to 6, or an expression cassette according to any of items 7 to 9, or a plasmid according to item 10, wherein the yeast cell is a cell selected from the genera: pichia, Komagataella, Ogataea, Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Kluyveromyces, Zygosaccharomyces, and Yarrowia, preferably Pichia pastoris cells or Yarrowia lipolytica cells, most preferably Pichia pastoris cells.

12. A method for the recombinant production of a target polypeptide comprising the steps of a) introducing an expression cassette comprising a nucleotide sequence encoding said target polypeptide into at least one yeast cell; b) producing a yeast cell culture by propagating the at least one yeast cell; c) subjecting the yeast cell culture to conditions suitable for expression of a nucleotide sequence encoding the target polypeptide; and d) isolating the produced target protein from the yeast cell culture, wherein the expression cassette comprises the promoter or promoter variant according to any one of items 1 to 6 operably linked to a nucleotide sequence encoding the target polypeptide.

13. A method for the recombinant production of a target polypeptide comprising the steps of a) introducing an expression cassette comprising a nucleotide sequence encoding said target polypeptide into at least one yeast cell; b) producing a yeast cell culture by propagating the at least one yeast cell; c) subjecting the yeast cell culture to conditions suitable for expression of a nucleotide sequence encoding the target polypeptide; and d) isolating the produced target protein from the yeast cell culture, wherein the expression cassette comprises a promoter comprising the nucleotide sequence of SEQ ID NO: 1.

14. The method of any of clauses 12 and 13, wherein the expression cassette further comprises a transcription terminator sequence.

15. The method of any one of clauses 12 to 14, wherein the conditions suitable for expression of the nucleotide sequence encoding the target polypeptide are a fed-batch process.

16. The method of clause 15, wherein the fed-batch process is operated at a glucose feed rate of at least 0.04mmol glucose per gram dry cell weight per hour (mmol Glu/g CDW/h) or at a glycerol feed rate of at least 0.07mmol glycerol per gram dry cell weight per hour (mmol Gly/g CDW/h), preferably at a glucose feed rate of at least 0.11mmol Glu/g CDW/h or at a glycerol feed rate of at least 0.21mmol Gly/g CDW/h, more preferably at a glucose feed rate of at least 0.18mmol Glu/g CDW/h or at a glycerol feed rate of at least 0.35mmol Gly/g CDW/h.

17. The method of any one of items 12 to 16, wherein the nucleotide sequence encoding the target polypeptide is a heterologous nucleic acid sequence.

18. The method of clause 17, wherein the nucleotide sequence encoding the target polypeptide encodes a heterologous polypeptide.

19. The method of any of items 12 to 18, wherein the yeast cell is a pichia pastoris cell or a yarrowia lipolytica cell, preferably a pichia pastoris cell.

20. A method for increasing the relative efficiency of expression of a nucleotide sequence having promoter activity, the method comprising the steps of: by comparing the sequence of SEQ ID NO: 1, more preferably at least 95%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% identity, from the 5' -end to a length of 140-.

21. The method of clause 20, wherein the promoter or promoter variant contains at least one amino acid sequence that is complementary to the amino acid sequence of SEQ ID NO: 1 non-naturally occurring replacement modifications.

22. The method of any one of clauses 20 and 21, wherein the relative expression efficiency of the truncated promoter is greater than the relative expression efficiency of a polypeptide having the amino acid sequence of SEQ ID NO: 1, preferably at least 10%, more preferably at least 20%, even more preferably at least 50%, most preferably at least 100%.

23. The method of any of clauses 20 to 22, wherein the relative expression efficiency of the truncated promoter is increased in a yeast cell, wherein the yeast cell is a pichia pastoris cell or a yarrowia lipolytica cell, preferably a pichia pastoris cell.

The solution according to the invention is further described below by means of non-limiting figures and examples.

Drawings

Fig. 1 is a bar graph showing the sequence of a sequence encoded from a sequence having SEQ ID NO: 1 and truncated promoter variants thereof under the control of a promoter of the nucleotide sequence of 1.

Fig. 2 is a bar graph showing the sequence of a sequence encoded from a sequence having SEQ ID NO: 2 and truncated promoter variants thereof as a reporter protein produced under the control of a promoter having the nucleotide sequence of SEQ ID NO: 1 and truncated promoter variants thereof under control of a reporter protein.

Fig. 3 is a bar graph showing the sequence of a sequence encoded from a sequence having SEQ ID NO: 3 and truncated promoter variants thereof, as a reporter protein produced under the control of a promoter having the nucleotide sequence of SEQ ID NO: 1 and truncated promoter variants thereof under control of a reporter protein.

Fig. 4 is a bar graph showing the sequence of a sequence encoded from a sequence having SEQ ID NO: 4 and truncated promoter variants thereof, as a reporter protein produced under the control of a promoter having the nucleotide sequence of SEQ ID NO: 1 and truncated promoter variants thereof under control of a reporter protein.

Fig. 5 is a bar graph showing the sequence as a result of the sequence set forth in the sequence having SEQ ID NO: 1 and truncated promoter variants thereof, and a phosphatase recombinantly produced under the control of the promoter of the nucleotide sequence of 1 (UniProtKB entry: P11491).

Fig. 6 is a bar graph showing the sequence as a result of the sequence set forth in the sequence having SEQ ID NO: 1 and truncated promoter variants thereof under the control of the promoter of the nucleotide sequence of 1 and the truncated promoter variants thereof.

Fig. 7 is a bar graph showing the sequence as a result of the sequence set forth in the sequence having SEQ ID NO: 1 and truncated promoter variants thereof under the control of a peroxidase (UniProtKB entry: P00433) produced recombinantly (UniProtKB).

FIG. 8 is a graph showing the expression of a nucleotide sequence in a medium having SEQ ID NO: 1 as a reporter signal under the control of a promoter for the nucleotide sequence of (1).

Fig. 9 is a graph showing the expression of a nucleotide sequence in a medium having SEQ ID NO: 1 as a reporter signal under the control of a truncated variant of the promoter of the nucleotide sequence of 1.

Fig. 10 is a graph showing the amount of bulk enzyme activity as a reporter signal produced by several strains, wherein the reporter is expressed throughout the culture in a nucleic acid having the sequence of SEQ ID NO: 1 under the control of a truncated variant of a promoter of the nucleotide sequence of seq id no.

Detailed Description

Referring to fig. 1-4, a sequence derived from a nucleotide sequence having SEQ ID NO: 1-4 and truncated variants thereof under the control of a promoter of the nucleotide sequence of any one of claims 1-4. Shown is the median of the standard deviations obtained from at least nine different transformed strains for each promoter construct. Although surprisingly found to have SEQ ID NO: 1 (fig. 1), but neither the promoter of the pichia pastoris ADH2 gene encoding pichia pastoris alcohol dehydrogenase 2 (SEQ ID NO: 2; fig. 2) nor the promoter of the pichia pastoris AOX1 gene encoding pichia pastoris alcohol oxidase 1 (SEQ ID NO: 3; fig. 3) nor the promoter of the pichia pastoris FDH1 gene encoding pichia pastoris formate dehydrogenase 1 (SEQ ID NO: 4; fig. 4) can achieve this approach without significant loss of promoter activity. Thus, for a polypeptide having SEQ ID NO: 1 appears to be an exception rather than a rule.

Referring to fig. 5-7, a polypeptide is shown as a polypeptide from SEQ ID NO: 1 and truncated variants thereof under the control of the promoter of the nucleotide sequence of 1, and a bar graph of the amount of volumetric enzyme activity of reporter signal for three different reporter enzymes. Shown are bar graphs representing the mean of the standard deviations obtained from at least nine different transformed strains when cultured in 96-deep well plates. Substantially the same effect as that of esterase (FIG. 1) was observed for phosphatase (FIG. 5), alcohol dehydrogenase (FIG. 6) and peroxidase (FIG. 7). Based on these results, it can be concluded that the polypeptide having SEQ ID NO: 1 occurs independently of the protein encoded in the open reading frame whose expression is regulated by the promoter.

Referring to fig. 8-10, a sequence as set forth in SEQ ID NO: 1 (figure 8) or a truncated variant thereof (figures 9 and 10) throughout the bioreactor culture process. Carries a polypeptide comprising a sequence having SEQ ID NO: 1 (fig. 9) and a strain carrying an expression cassette comprising a truncated promoter of 280bp length having the nucleotide sequence of SEQ ID NO: 1 (fig. 10) was found to produce an expression cassette comprising a truncated promoter of 160bp length (fig. 10) that is at least as long as a polynucleotide sequence carrying a promoter having the nucleotide sequence of SEQ ID NO: 1 nucleotide sequence of 623bp long non-truncated promoter and the same amount of target enzyme.

Examples

Example 1: expression studies using truncated eukaryotic promoters in pichia pastoris

To assess whether eukaryotic promoters can be truncated without substantial loss of promoter activity, full-length polynucleotide fragments of the promoters were selected either as synthetically produced fragments or directly amplified from the respective eukaryotes. In particular, the Ogataea polymorpha FMD promoter (SEQ ID NO: 1), the Pichia pastoris ADH2 promoter (SEQ ID NO: 2), the Pichia pastoris AOX1 promoter (SEQ ID NO: 3), and the Pichia pastoris FDH1 promoter (SEQ ID NO: 4) were studied. To obtain truncated variants of these promoters, oligonucleotide primers were designed for amplification by Polymerase Chain Reaction (PCR), where a reverse primer was designed to bind to the 3' -end of the promoter and a forward primer was designed to bind within the full-length promoter sequence, depending on the desired final length of the PCR product. For the Ogataea polymorpha FMD promoter, a truncated promoter with 612, 610, 600, 590, 580, 570, 560, 550, 540, 530, 520, 510, 500, 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 158, 156, 154, 152, 150, 130, 120, 110, 100bp was tested. Variants with 1000, 500, 350, 280, 160, 140bp were tested for the pichia pastoris ADH2 promoter, the pichia pastoris AOX1 promoter and the pichia pastoris FDH1 promoter. The full-length promoter or truncated variants thereof are cloned into expression cassettes for recombinant gene expression in Pichia pastoris using standard Cloning methods of the prior art (e.g.J.M.Cregg, Pichia Protocols, 2 nd Edition, ISBN-10:1588294293,2007; J.Sambrook et al, 2012.Molecular Cloning, A Laboratory Manual 4th Edition, Cold Spring Harbor). These expression cassettes comprise a full-length promoter or a truncated variant thereof, an open reading frame encoding a secretory reporter protein, a transcription terminator (pichia pastoris AOX1 transcription terminator) and an antibiotic selection marker. In order to quantify the amount of target protein produced by the corresponding full-length promoter or truncated variants thereof, esterase (UniProtKB entry: D2D3B6) was used as reporter protein. Esterase secretion from transformed cells was achieved using a s.cerevisiae mating factor alpha prepropeptide (prepro peptide) fused to the N-terminus of the esterase replacing the first 47 amino acids of the full-length esterase polypeptide.

Any of the resulting expression cassettes were used to transform Pichia pastoris wild-type strains by electroporation, essentially following the protocol of Lin-Cereghino et al (biotechniques.2005.38(1): 44-48). However, any alternative for transforming Pichia pastoris is also suitable. After transformation, the cells were incubated on agar plates containing appropriate antibiotics to select for transformed strains.

Microculturing of at least 9 transformed strains per transformation cassette was performed in 96-well plates (Hamilton 2.2mL PP-well plates) in a protocol similar to that described by Weis et al (FEMS Yeast Res.2004.5(2): 179-189). Cells were grown in 450. mu.L of YPD (1% w/v yeast extract, 2% w/v peptone, 2% w/v glucose) per well at 28 ℃ at 320rpm at 90% humidity in a Kuhner ISF1-X Climo-Shaker (throw 50mm) for about 60 hours. In the case of strains transformed with expression cassettes containing the AOX1 promoter or the FDH1 promoter, expression was induced 60 hours after inoculation by adding 100. mu.L of YPM 0.5% (1% w/v yeast extract, 2% w/v peptone, 0.5% w/v methanol) per well. In the case of strains transformed with expression cassettes containing the FMD promoter or ADH1 promoter, 100 μ L YPD was added per well 60 hours after inoculation. After about 120 hours of incubation, the 96-deep well plate was centrifuged to separate the biomass from the supernatant. The supernatant was used to analyze the secreted target reporter protein produced. To allow plate-to-plate comparability, stable esterase producing strains were also cultured in at least four wells of each plate as positive control and calibration strains.

The amount of target reporter protein secreted into the supernatant was determined by quantifying the amount of bulk esterase activity in the supernatant: the enzyme activity was quantified by measuring the hydrolysis of the esterase substrate 4-nitrophenyl 2- (trimethylsilyl) ethyl carbonate, which was measured photometrically by tracking the increase in absorbance at 405nm as described by Ruth et al (Microb Cell fact.2014.13: 120). The amount of signal of the reporter protein produced by the tested expression cassettes for the non-truncated promoters of FMD, ADH2, AOX1 and FDH1 and truncated promoter variants thereof are shown in figures 1, 2, 3 and 4, respectively. It is well known in the art that Pichia pastoris transformants tend to show clonal variation (e.g., Vogl et al, Appl Environ Microbiol.2018.84(6): e 02712-17). In contrast to other expression systems (e.g.E.coli), it is therefore necessary to test not only one transformed strain but also a plurality of strains, as described herein. Notably, clonal variation was found to be more pronounced when the promoter was shorter, as reflected by a higher standard deviation, and as seen, for example, for truncated variants of the FMD promoter having 160bp or 150bp compared to the longer variant having 600 bp. Without wishing to be bound by theory, it is believed that this effect is due to the greater influence of the integration site of the shorter promoter.

The median of the enzyme activities produced by at least nine transformed strains for each expression cassette was used to calculate the relative expression efficiency of the corresponding promoter or promoter variant. The relative expression efficiency was calculated by dividing the median value of the enzyme activities produced by the length of the promoter used to control the expression of the target reporter gene. In the case of the FMD promoter, truncated promoter variants having a length of 140 to 610bp were found to have at least a 5% increase in relative expression efficiency compared to the full-length promoter, see table 1 below. Essentially the same results were obtained regardless of which pichia pastoris strain (k.phaffii CBS 7435, k.phaffii GS115 or k.pastoris CBS 704) was transformed.

Table 1: relative Expression Efficiency (REE) of the full-length FMD promoter (623bp) and its truncated variants.

To demonstrate the mutational stability of the truncated promoter variants, the promoter sequences were tested against the sequences from SEQ ID NO: 1 to a length of 160bp, has at least 80% sequence identity. Mutant variants are represented by SEQ ID NO: 6-24 to a length of 160bp, whereby these truncated mutant variants have a sequence identical to the sequence given in SEQ ID NO: 1 to a length of 160bp has a sequence identity of 80.74% to 99.38%. For all mutant variants, a mutation that is identical to that observed for SEQ ID NO: 1, or a truncated variant of the promoter of the nucleotide sequence of 1. Despite approximately one quarter of the length, the reporter signal obtained is comparable to that of the non-truncated promoter, so that the novel truncated promoter achieves an increase in relative expression efficiency of at least 5%.

In contrast, truncated variants of any of the promoters of ADH2, AOX1, or FDH1 did not result in increased relative expression efficiency compared to the respective full-length promoters (each 1000bp in length), see table 2 below.

Table 2: relative Expression Efficiency (REE) of full-length promoters (each 1000bp) of ADH2, AOX1, or FDH1, and truncated variants thereof.

In summary, it was surprisingly found that only certain truncated variants of the o.polymorpha FMD promoter, but not any other eukaryotic promoter tested, resulted in increased relative expression efficiency of pichia pastoris, compared to the non-truncated full-length promoter.

Example 2: recombinant production of different reporter proteins in small-scale culture using promoters with increased relative expression efficiency

The regulation of expression by the promoter takes place at the DNA level, in particular at the level of expression of the DNA upstream of the open reading frame regulated by the promoter. Thus, one skilled in the art would not expect substantial differences in promoter activity when expression of different open reading frames is modulated. In other words, it would not be expected that the production of a protein different from the esterase described in example 1 would produce substantially different results in terms of promoter activity or functionality compared to the production of the esterase described in example 1. However, to experimentally verify the general applicability of truncated promoter variants with increased relative expression efficiency for expression of any recombinant target gene, other expression cassettes carrying open reading frames encoding phosphatases, alcohol dehydrogenases or peroxidases were generated.

The expression of genes comprising an open reading frame encoding phosphatase, alcohol dehydrogenase or peroxidase is designed to be regulated by the full-length o.polymorpha FMD promoter (623bp) or truncated variants thereof of 500bp, 350bp, 280bp, 160bp or 140bp in length. Transformation and culture of pichia pastoris in 96 deep well plates was essentially performed as described for the promoter in example 1.

To determine the amount of phosphatase produced, a p-nitrophenol (pNPP) assay was performed essentially as described in Takai and Mieskes (Biochem J.1991.275(1):233-₂、0.1mM ZnCl₂40mM Tris-HCl, pH 8.1. To determine the amount of alcohol dehydrogenase produced, the enzyme activity is determined essentially as described in WO2016154640A 1. Pichia pastoris strains producing alcohol dehydrogenase were cultured in YPD containing additional 1mM pyrroloquinoline quinone and 1mM calcium chloride. To determine the amount of alcohol dehydrogenase produced, the enzyme activity was determined by mixing 5. mu.L of the clear culture supernatant containing the enzyme produced with 280. mu.L of assay buffer (20mM potassium ferricyanide (III), 50mM Tris-HCl, pH7.5, 50ppm deoxynivalenol). The assay reaction was incubated at 30 ℃ for 60 minutes. Samples of 50. mu.L were periodically withdrawn from the reaction and analyzed by HPLC. To determine the amount of peroxidase produced, the enzyme activity was measured analogously to Krainer et al (J Biotechnol.201.233:181-189) by exchanging the substrate ABTS (CAS No: 30931-67-0) with TMB (3,3',5,5' -tetramethylbenzidine; CAS No: 54827-17-7); the product formation was followed at 653 nm. The amount of reporter enzyme signal generated by the tested promoter variants for phosphatase, alcohol dehydrogenase and peroxidase are shown in figures 5, 6 and 7, respectively.

Relative expression efficiencies were determined in analogy to the protocol described in example 1 and are shown in table 3.

Table 3: relative Efficiency of Expression (REE) of phosphatase, alcohol dehydrogenase or peroxidase produced under the control of FMD full-length promoter (623bp) or truncated variants thereof (500bp, 350bp, 280bp, 160bp, 140 bp).

In summary, it was found that the functionality and applicability of truncated promoter variants of the FMD wild-type promoter is not limited to a single open reading frame, but is well suited for regulating the expression of any target gene.

Example 3: recombinant protein production using promoters with increased relative expression efficiency in bioreactor culture

It is known that small-scale cultivation of microorganisms, for example in 24-well or 96-well plates or in shake flasks, tends to yield different data compared to cultivation in bioreactors. Without wishing to be bound by theory, it is believed that these differences stem from different conditions between culture types with respect to nutrient supply and pH and temperature control, oxygen supply and agitation. Although small-scale culture is well suited for general concepts such as testing scientific hypotheses and screening large numbers of microbial transformants for strain production, bioreactor culture is the first choice for evaluating production performance data.

To demonstrate the applicability of truncated variants of the o polymorpha FMD promoter not only in small-scale culture but also in large-scale culture, pichia pastoris strains harboring expression cassettes comprising truncated FMD promoter variants were cultured in bioreactors and compared to strains harboring expression cassettes comprising non-truncated FMD promoter as benchmark. As the reporter enzyme, the esterase of example 1 was used.

The preculture was incubated in YPG medium containing 10g/kg yeast extract, 20g/kg soy peptone, 0.3mL/kg antifoam Struktol J650 and 40g/kg glycerol in 500mL baffled shake flasks at 180rpm and 28 ℃ for 24 h. 500mL batches of media as reported by Krainer et al (Microb Cell fact.2015.14:4) were sterilized in 1L glass bioreactor vessels (DASGIP, Switzerland) prior to inoculation. The batch phase was carried out at 28 ℃ for 12 hours, the minimum saturation of 30% of dissolved oxygen throughout the cultivation being controlled by a cascade arrangement of stirrer speed, air flow and oxygen flow. 13 hours after inoculation, a linear increase in glycerol feed rate starting at 6.85g/L/h was initiated for 17 hours to a final maximum glycerol feed rate of 8.72 g/L/h. The feed rate was then set to promote a constant growth rate of 0.014/h and the temperature was set to 24 ℃. This feed rate was continued until harvest 120 hours after inoculation. The feed medium contained 696g/L glycerol, 4.4g/L of a trace element solution (Krainer et al, Microb Cell fact.2015.14:4), 200ppm biotin and 0.3mL/kg of an antifoam Struktol J650. The supernatant was periodically sampled by centrifuging the culture broth in a bench top Centrifuge (Eppendorf Centrifuge 5810R, Eppendorf, Germany) at 4000rpm and was further used for the enzyme activity assay.

The amount of reporter signal produced by the strain carrying the expression cassette comprising the non-truncated FMD promoter (i.e.the promoter having the nucleotide sequence of SEQ ID NO: 1) throughout the culture is shown in FIG. 8. After about 119h of incubation, the obtained volumetric enzyme activity was set to 100% as reporter signal. Carries a polypeptide comprising a sequence selected from SEQ ID NO: 1 to a promoter of 280bp length, the strain produced 115% of the reporter signal after about 119 hours of culture (figure 9). Culturing a cell harboring a nucleic acid comprising a sequence selected from SEQ ID NO: 1 to 160bp in length and produces reporter signals of 112.6%, 160.4%, 139.1%, 149.2%, 107.0%, 110.1% and 134.5% after about 119h of culture (fig. 10). The conclusion is that the Relative Expression Efficiency (REE) of the 623bp promoter is 0.161%/bp, the REE of the 280bp promoter is 0.411%/bp, and the REE of the 160bp promoter is 0.699 to 1.003%/bp. Thus, the REE of the 280bp promoter was found to be 2.56 times that of the non-truncated 623bp promoter, and the REE of the 160bp promoter was found to be 4.16-6.25 times that of the 623bp promoter. Surprisingly, it was found that all strains carrying a truncated promoter produce more target enzyme than a non-truncated promoter.

Example 4: induction of promoters by glycerol or glucose feeding

In WO2017/109082A1 and Vogl et al (AMB express.2020.10(1):38), it is taught that in Pichia pastoris expression from the 623bp wild-type HpFMD promoter is activated, i.e.induced, when glucose or glycerol is depleted. To investigate whether the truncated promoters described herein would follow this dogma, the actual consumption of glucose/glycerol without feed was tested 17 hours after the linearly increasing glycerol feed rate starting at 6.85g/L/h as described in example 3, i.e.30 hours after the start of the culture, and different constant glucose or glycerol feed rates of 0.07mmol glycerol/g CDW/h or 0.04mmol glucose/g CDW/h to 0.48mmol glycerol/g CDW/h or 0.25mmol glucose/g CDW/h. Otherwise, the culture was performed essentially as described in example 3. In addition, these conditions were also tested against the non-truncated wild-type FMD promoter. As the reporter enzyme, the esterase of example 1 was used.

Reporter signals from the generated reporter enzymes obtained from different glucose or glycerol feed rates as described above are shown as examples in table 4, where expression is regulated by a truncated FMD promoter with 280 bp.

Table 4: volumetric enzyme activity was obtained after approximately 60 hours of culture, where promoter induction was promoted by depletion of glucose/glycerol (i.e., 0.00mmol/g CDW/h) or feeding at different rates of glucose or glycerol. The reporter signal obtained at glucose depletion was set at 100%.

Unexpectedly, even a positive correlation between the feed rate and the specific production rate can be observed: higher feed rates result in higher volumes and specific activities. The same trend was found when expression was regulated by a 623bp promoter, and when expression was regulated by a promoter variant with a truncation of 500bp, 350bp, 280bp or 160 bp. Based on this, it was concluded that, surprisingly and differently from the previous publications, the best mode of operation of the wild-type FMD promoter or truncated variant thereof in pichia pastoris is not activated by depletion of glucose or glycerol, but by feeding at least 0.07mmol Gly/g CDW/h or 0.04mmol Glu/g CDW/h.

Example 5: recombinant gene expression with truncated eukaryotic promoter in yarrowia lipolytica

To test whether expression by the truncated FMD promoter was limited to pichia pastoris strains (as found for k.phaffii CBS 7435, k.phaffii GS115 and k.pastoris CBS 704), yarrowia lipolytica was tested. Transformation of yarrowia lipolytica CBS7504 strain with the expression cassette as described in example 1 was carried out by electroporation according to the protocol of Lin-Cereghino et al (biotechniques.2005.38(1): 44-48). Nevertheless, an alternative to the transformation of yarrowia lipolytica can be employed to transform such yeast with an expression cassette. Subsequently, cells were cultured and selected on agar plates containing the appropriate antibiotic for selection of positive transformants. Culturing of at least 50 positive transformants was performed in 96-well plates essentially as described by Weis et al (FEMS Yeast Res.2004.5(2): 179-189): cell growth was achieved in 450. mu.L of YPD (1% w/v yeast extract, 2% w/v peptone, 2% w/v glucose) per well at 28 ℃ at 320rpm at 90% humidity in Kuhner ISF1-X Climo-Shaker for 60 hours, similar to example 1. After 60 hours of culture at 320rpm and 28 ℃, the cells were fed twice with 100 μ L per well of YPD, 60 hours and 85 hours after seeding, then cultured for another 24 hours, and then harvested by centrifugation. The level of reporter protein in the supernatant was determined by monitoring the volume esterase activity of the hydrolysis of 4-nitrophenyl 2- (trimethylsilyl) ethyl carbonate. After the hydrolysis reaction, the increase in absorbance at 405nm was photometrically measured essentially as described by Ruth et al (Microb Cell fact.2014.13: 120). Similar to recombinant production in pichia pastoris strains controlled by truncated FMD promoters, essentially the same increase in REE was found when these promoters were used to regulate recombinant protein production in yarrowia lipolytica. It was found that not only pichia pastoris, but also yarrowia lipolytica showed the ability to produce and secrete target reporter proteins, the production of which was controlled by a truncated FMD promoter.

Sequence listing

<110> Eleberg Ltd

<120> truncated promoter for recombinant gene expression

<130> P06005

<160> 24

<170> BiSSAP 1.3.6

<210> 1

<211> 623

<212> DNA

<213> Ogataea polymorpha

<400> 1

aatgtatcta aacgcaaact ccgagctgga aaaatgttac cggcgatgcg cggacaattt 60

agaggcggcg atcaagaaac acctgctggg cgagcagtct ggagcacagt cttcgatggg 120

cccgagatcc caccgcgttc ctgggtaccg ggacgtgagg cagcgcgaca tccatcaaat 180

ataccaggcg ccaaccgagt ctctcggaaa acagcttctg gatatcttcc gctggcggcg 240

caacgacgaa taatagtccc tggaggtgac ggaatatata tgtgtggagg gtaaatctga 300

cagggtgtag caaaggtaat attttcctaa aacatgcaat cggctgcccc gcaacgggaa 360

aaagaatgac tttggcactc ttcaccagag tggggtgtcc cgctcgtgtg tgcaaatagg 420

ctcccactgg tcaccccgga ttttgcagaa aaacagcaag ttccggggtg tctcactggt 480

gtccgccaat aagaggagcc ggcaggcacg gagtctacat caagctgtct ccgatacact 540

cgactaccat ccgggtctct cagagagggg aatggcacta taaataccgc ctccttgcgc 600

tctctgcctt catcaatcaa atc 623

<210> 2

<211> 1000

<212> DNA

<213> Komagataella pastoris

<400> 2

cgattgcccc tctacaggca taagggtgtg actttgtggg cttgaatttt acaccccctc 60

caacttttct cgcatcaatt gatcctgtta ccaatattgc atgcccggag gagacttgcc 120

ccctaatttc gcggcgtcgt cccggatcgc agggtgagac tgtagagacc ccacatagtg 180

acaatgatta tgtaagaaga ggggggtgat tcggccggct atcgaactct aacaactagg 240

ggggtgaaca atgcccagca gtcctcccca ctctttgaca aatcagtatc accgattaac 300

accccaaatc ttattctcaa cggtccctca tccttgcacc cctctttgga caaatggcag 360

ttagcattgg tgcactgact gactgcccaa ccttaaaccc aaatttctta gaaggggccc 420

atctagttag cgaggggtga aaaattcctc catcggagat gtattgaccg taagttgctg 480

cttaaaaaaa atcagttcag atagcgagac ttttttgatt tcgcaacggg agtgcctgtt 540

ccattcgatt gcaattctca ccccttctgc ccagtcctgc caattgccca tgaatctgct 600

aatttcgttg attcccaccc ccctttccaa ctccacaaat tgtccaatct cgttttccat 660

ttgggagaat ctgcatgtcg actacataaa gcgaccggtg tccgaaaaga tctgtgtagt 720

tttcaacatt ttgtgctccc cccgctgttt gaaaacgggg gtgagcgctc tccggggtgc 780

gaattcgtgc ccaattcctt tcaccctgcc tattgtagac gtcaacccgc atctggtgcg 840

aatatagcgc acccccaatg atcacaccaa caattggtcc acccctcccc aatctctaat 900

attcacaatt cacctcacta taaatacccc tgtcctgctc ccaaattctt ttttccttct 960

tccatcagct actagctttt atcttattta ctttacgaaa 1000

<210> 3

<211> 1000

<212> DNA

<213> Komagataella pastoris

<400> 3

atgttggtat tgtgaaatag acgcagatcg ggaacactga aaaataacag ttattattcg 60

agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 120

gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 180

tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 240

agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 300

acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 360

tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 420

agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 480

gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 540

ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 600

cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 660

ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 720

ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 780

gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 840

atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 900

actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 960

caacttgaga agatcaaaaa acaactaatt attcgaaacg 1000

<210> 4

<211> 1000

<212> DNA

<213> Komagataella pastoris

<400> 4

aaatggcaga aggatcagcc tggacgaagc aaccagttcc aactgctaag taaagaagat 60

gctagacgaa ggagacttca gaggtgaaaa gtttgcaaga agagagctgc gggaaataaa 120

ttttcaattt aaggacttga gtgcgtccat attcgtgtac gtgtccaact gttttccatt 180

acctaagaaa aacataaaga ttaaaaagat aaacccaatc gggaaacttt agcgtgccgt 240

ttcggattcc gaaaaacttt tggagcgcca gatgactatg gaaagaggag tgtaccaaaa 300

tggcaagtcg ggggctactc accggatagc caatacattc tctaggaacc agggatgaat 360

ccaggttttt gttgtcacgg taggtcaagc attcacttct taggaatatc tcgttgaaag 420

ctacttgaaa tcccattggg tgcggaacca gcttctaatt aaatagttcg atgatgttct 480

ctaagtggga ctctacggct caaacttcta cacagcatca tcttagtagt cccttcccaa 540

aacaccattc taggtttcgg aacgtaacga aacaatgttc ctctcttcac attgggccgt 600

tactctagcc ttccgaagaa ccaataaaag ggaccggctg aaacgggtgt ggaaactcct 660

gtccagttta tggcaaaggc tacagaaatc ccaatcttgt cgggatgttg ctcctcccaa 720

acgccatatt gtactgcagt tggtgcgcat tttagggaaa atttacccca gatgtcctga 780

ttttcgaggg ctacccccaa ctccctgtgc ttatacttag tctaattcta ttcagtgtgc 840

tgacctacac gtaatgatgt cgtaacccag ttaaatggcc gaaaaactat ttaagtaagt 900

ttatttctcc tccagatgag actctccttc ttttctccgc tagttatcaa actataaacc 960

tattttacct caaatacctc caacatcacc cacttaaaca 1000

<210> 5

<211> 160

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic promoter

<400> 5

cggggtgtct cactggtgtc cgccaataag aggagccggc aggcacggag tctacatcaa 60

gctgtctccg atacactcga ctaccatccg ggtctctcag agaggggaat ggcactataa 120

ataccgcctc cttgcgctct ctgccttcat caatcaaatc 160

<210> 6

<211> 623

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic promoter

<400> 6