阅读说明：本技术 宿主细胞中风味化合物的产生 (Production of flavor compounds in host cells ) 是由 凯塔琳娜·灿卡尔 马蒂纳斯·朱利叶斯·贝克韦德 亨德里克·简·博施 彼得·汉斯·范·德·沙夫特 于 2018-11-19 设计创作，主要内容包括：本发明涉及生物技术领域；具体地涉及宿主细胞中风味化合物(覆盆子酮)的产生。(The invention relates to the field of biotechnology; in particular to the production of flavour compounds (raspberry ketones) in host cells.)

1. A prokaryotic microbial cell capable of expressing, preferably expressing, a functional heterologous enzyme having Tyrosine Ammonia Lyase (TAL) activity and further capable of expressing, preferably expressing, at least one functional enzyme selected from the group consisting of: 4-coumarate-coa ligase (4CL) and Benzylidene Acetone Synthase (BAS), and optionally also capable of expressing, preferably expressing, heterologous Benzylidene Acetone Reductase (BAR).

2. The cell of claim 1, wherein:

functional heterologous enzymes with TAL activity are derived from Pseudomonas capsulata (Rhodobacter capsulatus), Spanish saccharomycete (Saccharothrix espanaensis) or Flavobacterium johnsonii (Flavobacterium johnsoniae);

-the at least one functional enzyme selected from 4CL and BAS is 4CL from tobacco (Nicotiana tabacum), Arabidopsis thaliana (Arabidopsis thaliana), Physcomitrella patens (Physcomitrella patens) or Streptomyces coelicolor, or BAS from Rubus palmiformis (Rubus idaeus) or Rheum palmatum (Rheum palmatum); and

-the optional BAR is from Rubus palmatus (rubius idaeus).

3. The cell of claim 1 or 2, wherein:

-the functional heterologous enzyme with TAL activity is from rhodopseudomonas capsulata (Rhodobacter capsulatus);

-the at least one functional enzyme selected from 4CL and BAS is selected from 4CL from Physcomitrella patens (Physcomitrella patents), and BAS from Rheum palmatum (Rheum palmatum); and

-the optional BAR is from Rubus palmatus (rubius idaeus).

4. The cell of any one of claims 1 to 3, wherein:

-a functional heterologous enzyme having TAL activity has at least 60%, preferably at least 80%, more preferably at least 90%, most preferably 100% sequence identity to SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 5, preferably to SEQ ID No. 1, or is encoded by a polynucleotide having at least 60%, preferably at least 80%, more preferably at least 90%, most preferably 100% sequence identity to SEQ ID No. 2, SEQ ID No. 4, or SEQ ID No. 6, preferably to SEQ ID No. 2;

-4CL has at least 60%, preferably at least 80%, more preferably at least 90%, most preferably 100% sequence identity to SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, or SEQ ID No. 13, preferably to SEQ ID No. 11, or is encoded by a polynucleotide having at least 60%, preferably at least 80%, more preferably at least 90%, most preferably 100% sequence identity to SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, or SEQ ID No. 14, preferably to SEQ ID No. 12;

-BAL has at least 60%, preferably at least 80%, more preferably at least 90%, most preferably 100% sequence identity to SEQ ID No. 15, or SEQ ID No. 17, preferably to SEQ ID No. 17, or is encoded by a polynucleotide having at least 60%, preferably at least 80%, more preferably at least 90%, most preferably 100% sequence identity to SEQ ID No. 16 or SEQ ID No. 18, preferably to SEQ ID No. 18;

BAR has at least 60%, preferably at least 80%, more preferably at least 90%, most preferably 100% sequence identity to SEQ ID NO. 19 or is encoded by a polynucleotide having at least 60%, preferably at least 80%, more preferably at least 90%, most preferably 100% sequence identity to SEQ ID NO. 20.

5. The cell of any one of claims 1 to 4, wherein the polynucleotide sequence encoding at least one of the enzymes is codon optimized.

6. The cell according to any one of claims 1 to 5, wherein the cell is a gram-positive prokaryotic microbial cell, preferably a Corynebacterium (Corynebacterium), more preferably a Corynebacterium glutamicum (Corynebacterium glutamicum), even more preferably a Corynebacterium glutamicum ATCC13032, even more preferably a Corynebacterium (Corynebacterium) capable of producing at least twice as much L-tyrosine as Corynebacterium glutamicum ATCC 13032.

7. The cell of any one of claims 1-6, wherein at least two of the enzymes are encoded by a single recombinant polynucleotide construct.

8. The cell of any one of claims 1 to 7, which is capable of producing at least 5mg/L of raspberry ketone.

9. The cell of any one of claims 1-8, wherein the cell is Corynebacterium glutamicum (Corynebacterium glutamicum).

10. The cell of any one of claims 1 to 9, wherein the cell is Corynebacterium glutamicum (Corynebacterium glutamicum) and is capable of producing at least 5mg/L of raspberry ketone.

11. A method for producing a cell according to any one of claims 1 to 10, comprising

-contacting a prokaryotic cell with an expression construct encoding a functional heterologous enzyme having Tyrosine Ammonia Lyase (TAL) activity, and

-contacting the prokaryotic cell with an expression construct encoding at least one functional enzyme selected from the group consisting of 4-coumarate-coa ligase (4CL) and Benzylidene Acetone Synthase (BAS), and

-optionally contacting the prokaryotic cell with an expression construct encoding a heterologous Benzylidene Acetone Reductase (BAR).

12. A method for producing raspberry ketone comprises:

-culturing the cell according to any one of claims 1 to 10 under conditions conducive to the production of raspberry ketone, and optionally,

-isolating and/or purifying raspberry ketone from said cells and/or said culture medium.

13. Use of a functional heterologous enzyme having Tyrosine Ammonia Lyase (TAL) activity as defined in any of claims 1 to 4 for the production of raspberry ketone in a prokaryotic host cell, preferably a gram-positive prokaryotic host cell.

14. An expression vector comprising a first polynucleotide having at least 60%, 65%, 70%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68 or 70, more preferably to SEQ ID No. 52, or to SEQ ID No. 70, most preferably to SEQ ID No. 70, or wherein said expression vector consists of a polynucleotide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67 or 69, more preferably to SEQ ID No. 51, Or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 69, most preferably to SEQ ID NO. 69.

15. A polypeptide product expressed from the expression vector of claim 14.

Technical Field

The present invention relates to the field of biotechnology, in particular to the production of flavor compounds (raspberry ketones) in host cells.

Background

Raspberry ketone is one of the key flavor compounds with typical raspberry characteristics and low olfactory limits. It is an expensive flavour compound and is widely used in the food industry. Of course, raspberry ketones occur naturally in palmleaf raspberries (Rubusidaeus), but also in other fruits, such as peaches, grapes, apples, several kinds of berries, vegetables (e.g., rhubarb), and the bark of certain trees (e.g., yew, maple, and pine).

Raspberry ketones can be used in a variety of applications, for example, in formulating a variety of aromas, such as strawberry, kiwi and cherry aromas; and in cosmetics and as antiobesity agents.

Obtaining raspberry ketone from its natural source in a commercially relevant manner is not trivial, as the low content of fruits and other sources containing raspberry ketone makes the extraction and purification process unprofitable (less than 4mg of raspberry ketone per kg of raspberry can be obtained). Raspberry ketones have hitherto been produced chemically by condensation of p-hydroxybenzaldehyde with acetone, which is an environmentally unfriendly process. In addition, chemical synthesis of flavour compounds often leads to undesired racemic variants and mixtures (Vandamme and Soetaert; J Chem Techno Biotechnol 77: 1323-.

In raspberry, raspberry ketone synthesis is the bi-enzymatic part of the phenylpropanoid pathway. This pathway has been described by Borejsza-Wysockiki and Hrazdina (1994). In the first step, coumaroyl-coa, which is present in many plant tissues, is condensed with malonyl-coa to benzylidene acetone (p-hydroxyphenylbut-3-en-2-one). The enzyme that catalyzes this step is called Benzylidene Acetone Synthase (BAS). In the second step, the double bond in benzylidene acetone is reduced to obtain raspberry ketone (p-hydroxyphenyl-2-butanone). The enzyme that catalyzes this step is called Benzylidene Acetone Reductase (BAR), which requires the presence of NADPH.

Benzylidene Acetone Synthase (BAS), EC 2.3.1. Benzylidene acetone synthase condenses an acetone unit of malonyl-coa with p-coumaric acid to form benzylidene acetone. The polyketide synthase family is described in detail in Schroder (1999). Abe et al (2001) teach the cloning of BAS genes from rhubarb. Koeduka et al (2011) teach the characterization of raspberry BAS.

Soluble enzymes that use NADPH to catalyze the reduction of double bonds, such as Benzylidene Acetone Reductase (BAR), are classified as the enzyme class ec1.3.1.x by the international union of biochemistry and molecular biology. For example, an enzyme from Arthrobacter sp (Arthrobacter sp.) annotated as EC 1.3.1.11 was reported to remove double bonds from coumaric acid (Levi and Weinstein, 1964), but no gene associated with this enzyme activity was found. Other enzymes in enzyme class EC1.3.1.X are orotate reductase, 2-hexadecenal reductase, cholestenone 5. alpha. -reductase, etc., the genes of which are known. However, none of these enzymes have been reported to have Benzylidene Acetone Reductase (BAR) activity. It is known from the literature that 4-hydroxybenzylideneacetone can be converted into raspberry ketones by fungi or yeasts such as Pichia (Pichia), Saccharomyces (Saccharomyces), Beauveria (Beauveria), Kloeckera (Kloeeckera), Aureobasidium (Aureobasidium), Cladosporium (Cladosporium), Geotrichum (Geotrichum), Mucor (Mucor) and Candida (Candida spp.) (Fuganti and Zucchi, 1998). Beekwilder et al, (2007) reported that BAR activity was present in e. However, no gene has been found which is involved in this enzyme activity.

Attempts to biosynthesize raspberry ketone have been described. Hugueny et al (1995, Bioflavour 95pp 269-273) teach biotechnological methods for producing raspberry ketones. The method comprises culturing a microorganism having a secondary Alcohol Dehydrogenase (ADH), such as Candida boidinii (Candida boidinii), and adding a precursor betulin to the culture medium. In this cellular environment, secondary ADH dehydrogenates rhododendrol to raspberry ketone. Abe et al (2001) teach the cloning of rheum BAS, gene expression in e.coli (e.coli), purification of recombinant BAS protein, and in vitro synthesis of benzylidene acetone. However, this study does not teach in vivo synthesis of benzylidene acetone or raspberry ketone.

EP1226265 teaches the biological production of p-hydroxycinnamic acid (also known as p-coumaric acid) which can be used as a precursor for raspberry ketones. EP1226265 describes a phenyl-ammonia-lyase (PAL) (ec 4.3.1.5); it does not teach the in vivo synthesis of raspberry ketones. GB2416769 and GB2416770 describe the biological production of raspberry ketones in escherichia coli (e.coli) from coumaric acid by precursors added to the fermentation. Likewise, in Beekwilder et al (2007), raspberry ketones were produced in escherichia coli (e.coli) from p-coumaric acid, a precursor added to the fermentation. In 2016, Lee et al published their results, namely the production of raspberry ketone in Saccharomyces cerevisiae (Saccharomyces cerevisiae) from p-coumaric acid, a precursor added to the fermentation. In addition, they produced small amounts of raspberry ketone (0.49mg/L) by Saccharomyces cerevisiae (Saccharomyces cerevisiae) using anaerobic fermentation. The addition of a precursor capable of producing raspberry ketone in a microbial host cell is not efficient. In addition, the use of eukaryotic cells in anaerobic fermentation is laborious and inefficient, certainly on an industrial scale. Thus, there is a need for de novo production of raspberry ketones in prokaryotic microbial host cells using simple fermentation conditions. To date, the de novo production of raspberry ketones from such prokaryotic host cells using such simple aerobic fermentation has not been proposed.

Disclosure of Invention

The present invention relates to a cell capable of producing raspberry ketone, and to methods of using such a cell. In a first aspect, the present invention provides a prokaryotic microbial cell capable of expressing, preferably expressing, a functional heterologous enzyme having Tyrosine Ammonia Lyase (TAL) activity and further capable of expressing, preferably expressing, at least one functional enzyme selected from the group consisting of: 4-coumarate-coa ligase (4CL) and Benzylidene Acetone Synthase (BAS), and optionally also capable of expressing, preferably expressing, heterologous Benzylidene Acetone Reductase (BAR). In an embodiment of this aspect, there is provided a cell wherein the functional heterologous enzyme having TAL activity is from rhodopseudomonas capsulata (Rhodobacter capsulatus), saccharothrix Spanisi (Saccharothricinaeensis) or Flavobacterium johnsonii (Flavobacterium johnsoniae); wherein the 4CL is from tobacco (Nicotiana tabacum), Arabidopsis (Arabidopsis thaliana), Physcomitrella patens (Physcomitrella patents) or Streptomyces coelicolor; wherein the BAS is from Rubus palmatus (Rubus idaeus) or Rheum palmatum (Rheum palmatum), and wherein the optional BAR is from Rubus palmatus (Rubus idaeus). In a particular embodiment, this aspect provides a cell wherein the polynucleotide sequence of at least one of the enzymes is codon optimized. In a further embodiment, this aspect provides a cell, wherein the cell is a gram-positive prokaryotic microbial cell, preferably a Corynebacterium (Corynebacterium), more preferably a Corynebacterium glutamicum (Corynebacterium glutamicum), even more preferably a Corynebacterium glutamicum ATCC13032, even more preferably a Corynebacterium glutamicum (Corynebacterium) capable of producing at least twice as much L-tyrosine as compared to Corynebacterium glutamicum ATCC 13032. In a preferred embodiment of this aspect, a cell is provided wherein at least two enzymes are encoded by a single recombinant polynucleotide construct. In a further preferred embodiment of this aspect, there is provided a cell capable of producing at least 5mg/l of raspberry ketone.

In a second aspect, the invention provides a method for producing a cell according to the first aspect, the method comprising contacting a prokaryotic cell with an expression construct encoding a functional heterologous enzyme having Tyrosine Ammonia Lyase (TAL) activity, and contacting the prokaryotic cell with an expression construct encoding at least one functional enzyme selected from the group consisting of 4-coumarate-coa ligase (4CL) and Benzylidene Acetone Synthase (BAS), and optionally contacting the prokaryotic cell with an expression construct encoding a heterologous Benzylidene Acetone Reductase (BAR).

In a third aspect, the present invention provides a method of producing raspberry ketone, the method comprising culturing a cell according to the first aspect of the invention under conditions conducive to the production of raspberry ketone, and optionally isolating and/or purifying raspberry ketone from the cell and/or the culture medium.

In a fourth aspect, the present invention provides the use of a functional heterologous enzyme having Tyrosine Ammonia Lyase (TAL) activity according to the first aspect of the invention for the production of raspberry ketones in prokaryotic host cells, preferably gram-positive prokaryotic cells.

In a fifth aspect, the present invention provides an expression vector comprising a specific operator sequence. In a sixth aspect, the invention provides a polypeptide product expressed by an expression vector according to the fifth aspect.

Drawings

FIG. 1 shows a schematic view of a-a biosynthetic pathway to produce raspberry ketone from L-tyrosine; TAL ═ tyrosine ammonia lyase; 4CL ═ 4-coumarate-coa ligase; BAS ═ benzylidene acetone synthase; BAR ═ benzylidene acetone reductase.

FIG. 2-a plasmid map of plasmid pECXK _ P; RpBAS ═ rheum palmatum benzylidene acetone synthase, Pp4CL ═ physcomitrella patens 4-coumarate coenzyme a ligase, RcTAL ═ rhodopseudomonas capsulata tyrosine ammonia lyase, Laclq ═ repressor gene, Ptrc ═ IPTG-induced Ptrc promoter; t1 and T2 ═ rrnB transcription terminators T1 and T2; KanR ═ kanamycin resistance; repA — origin of replication pGA 1; peri is a positive effector of plasmid replication.

FIG. 3-a plasmid map of plasmid pECXK _ PB; RpBAS ═ rheum palmatum benzylidene acetone synthase; pp4CL ═ physcomitrella patens 4-coumarate-coa ligase; RcTAL ═ capsular rhodopseudomonas sp tyrosine ammonia lyase; RiBAR ═ rubus chingii benzylidene acetone reductase; laclq ═ repressor genes; ptrc ═ IPTG-induced Ptrc promoter; t1 and T2 ═ rrnB transcription terminators T1 and T2; KanR ═ kanamycin resistance; repA — origin of replication pGA 1; peri is a positive effector of plasmid replication.

FIG. 4De novo production of 4-coumaric acid in E.coli

HPLC chromatograms of the strain A.) Ec _ RK _ EV, B.) Ec _ RK _ P and C.) Ec _ RK _ PB and D.) 4-coumaric acid standards are shown. A peak of 4-coumaric acid was observed at retention time 17.9 minutes.

FIG. 5De novo production of hydroxyphenyl butenones in E.coli

HPLC chromatograms of a) Ec _ RK _ EV, B.) Ec _ RK _ P and C.) Ec _ RK _ PB and D.) hydroxyphenyl butenone standards are shown. A peak of hydroxyphenyl butenone was observed at a retention time of 23.1 minutes and indicated by an arrow.

FIG. 6HPLC analysis of the de novo production of hydroxyphenylbutenone in corynebacterium glutamicum (c.

HPLC chromatograms of a) Cg _ RK _ EV, B.) Cg _ RK _ P and C.) Cg _ RK _ PB and D.) hydroxyphenyl butenone standards are shown. A peak of hydroxyphenyl butenone was observed at a retention time of 23.1 minutes and indicated by an arrow.

FIG. 7GC-MS analysis of raspberry ketone production in Corynebacterium glutamicum.

GC-MS chromatograms for strains Cg _ RK _ EV, Cg _ RK _ P and Cg _ RK _ PB are shown in Panel A. A raspberry ketone peak was observed at retention time 14.2 minutes. In panel B, the mass spectrum of raspberry ketone produced by strain Cg _ RK _ PB is shown. In fig. C, the mass spectrum of the authentic raspberry ketone standard is shown.

Detailed Description

The inventors have determined that, surprisingly, raspberry ketones can be synthesized de novo from glucose by recombinant prokaryotic microbial host cells using simple aerobic fermentation conditions.

Thus, in a first aspect, the present invention provides a prokaryotic microbial cell capable of expressing, preferably expressing, a functional heterologous enzyme having Tyrosine Ammonia Lyase (TAL) activity and further capable of expressing, preferably expressing, at least one functional enzyme selected from the group consisting of: 4-coumarate-coa ligase (4CL) and Benzylidene Acetone Synthase (BAS), and optionally also capable of expressing, preferably expressing, heterologous Benzylidene Acetone Reductase (BAR). Such prokaryotic microbial cells are hereinafter referred to as cells according to the invention.

In a preferred embodiment, this aspect provides a prokaryotic microbial cell that expresses a functional heterologous enzyme having Tyrosine Ammonia Lyase (TAL) activity and further expresses at least one functional enzyme selected from the group consisting of: 4-coumarate-coa ligase (4CL) and Benzylidene Acetone Synthase (BAS), and optionally also expressing heterologous Benzylidene Acetone Reductase (BAR).

In a particular embodiment, this aspect provides a prokaryotic microbial cell that expresses a functional heterologous enzyme having Tyrosine Ammonia Lyase (TAL) activity, and further expresses a functional 4-coumarate-coa ligase (4CL) enzyme, and optionally further expresses a heterologous Benzylidene Acetone Reductase (BAR).

In a particular embodiment, this aspect provides a prokaryotic microbial cell that expresses a functional heterologous enzyme having Tyrosine Ammonia Lyase (TAL) activity, and also expresses a functional Benzylidene Acetone Synthase (BAS) enzyme, and optionally also expresses a heterologous Benzylidene Acetone Reductase (BAR).

In a particular embodiment, this aspect provides a prokaryotic microbial cell that expresses a functional heterologous enzyme having Tyrosine Ammonia Lyase (TAL) activity, and further expresses a functional 4-coumarate-coa ligase (4CL) enzyme and a functional Benzylidene Acetone Synthase (BAS) enzyme, and optionally further expresses a heterologous Benzylidene Acetone Reductase (BAR).

In a particular embodiment, this aspect provides a prokaryotic microbial cell that expresses a functional heterologous enzyme having Tyrosine Ammonia Lyase (TAL) activity, and further expresses a functional 4-coumarate-coa ligase (4CL) enzyme and a functional Benzylidene Acetone Synthase (BAS) enzyme, and further expresses a heterologous Benzylidene Acetone Reductase (BAR).

Prokaryotic microbial cells are well known in the art. Prokaryotes are single-celled organisms that lack membrane-bound nuclei, mitochondria, and other membrane-bound organelles. Examples of prokaryotes are bacteria and archaea. Preferred prokaryotic cells are bacteria, such as E.coli, or preferably gram-positive bacteria, such as Corynebacterium (Corynebacterium), more preferably, for example, Corynebacterium glutamicum (Corynebacterium glutamicum), since their cultivation is well-established. In a preferred embodiment, this aspect provides a cell according to the invention, wherein the cell is a gram-positive prokaryotic microbial cell, preferably a Corynebacterium (Corynebacterium), more preferably a Corynebacterium glutamicum (Corynebacterium glutamicum), even more preferably a Corynebacterium glutamicum ATCC13032, even more preferably a Corynebacterium glutamicum (Corynebacterium) capable of producing at least twice as much L-tyrosine as compared to Corynebacterium glutamicum ATCC 13032. Strain ATCC13032 is known to those skilled in the art; this strain is also known as DSM 20300, JCM 1318, LMG3730 and NCIMB 10025.

Throughout this application, expression is considered to be the transcription of a gene into a functional mRNA, resulting in a functional polypeptide, e.g., an enzyme. Enzymes are polypeptides that can catalyze reactions. In this context, an increase in expression of an enzyme may be considered to be an increase in the level of mRNA encoding the enzyme, an increase in the level of an enzyme polypeptide or an increase in the overall activity of the enzyme. Preferably, an increase in expression of the enzyme results in an increase in activity of the enzyme, which may be caused by an increase in the level of enzyme polypeptide. Enzymes that exhibit activity in catalyzing their associated reactions are functional enzymes. The relevant reactions for TAL, 4CL, BAS, and BAR are described elsewhere herein. The activity can be determined by monitoring the increase in product concentration using chromatographic techniques such as Gas Chromatography (GC), High Performance Liquid Chromatography (HPLC) or GC advantageously coupled with mass spectrometric methods or liquid chromatography such as LCMS or GCMS. The preferred method for detecting intermediates is GCMS. The preferred method for detecting raspberry ketone is GCMS.

In the context of the present application, a cell capable of expressing an enzyme is a cell comprising the genetic information required for expression of the enzyme, preferably a nucleic acid encoding the enzyme. This does not mean that the enzyme must be expressed. By way of non-limiting example, a cell comprising a nucleic acid encoding an enzyme, wherein the nucleic acid is under the control of a promoter that is responsive to a particular induction, which promoter is not necessarily induced and thus does not necessarily express the enzyme, and in fact, the cell is capable of such expression. In a preferred embodiment of the invention, a preferred cell capable of expressing a functional enzyme is a cell expressing said functional enzyme.

Heterologous enzymes are enzymes derived from different organisms. Heterologous polynucleotides are polynucleotides derived from different organisms. The heterologous polynucleotide may be a synthetic polynucleotide. In a preferred embodiment, a cell according to the invention is provided, wherein the polynucleotide sequence encoding at least one of the enzymes is codon-optimized. A codon-optimized polynucleotide sequence is one in which codon usage bias has been mitigated by selecting alternative codons without altering the encoded polypeptide. For example, a rare codon can be replaced by a more common codon, or a region containing many identical codons can be interrupted by a substitution of a synonymous codon to reduce the need for many identical codons. Codon optimization is known in the art, and bioinformatic tools for codon optimization are freely available from different suppliers over the internet.

Tyrosine ammonia lyase, hereinafter referred to as TAL, also known as tyrosine ammonia lyase, L-tyrosine ammonia lyase and tyrosinase. It is an enzyme (EC4.3.1.23) in the biosynthetic pathway of natural phenols, which converts L-tyrosine to p-coumaric acid, releasing ammonia. P-coumaric acid is also known as p-coumaric acid, 4-hydroxycinnamic acid and (E) -3- (4-hydroxyphenyl) -2-propenoic acid. Preferred TALs or preferred enzymes having TAL activity are heterologous TALs or heterologous enzymes having TAL activity, which are TALs or enzymes having TAL activity derived from an organism different from the cell according to the invention. Preferred organisms from which TAL or enzymes having TAL activity can be derived are Pseudomonas rhodochrous (Rhodobacter Cystus) (e.g., RcTAL, represented by SEQ ID NO:1 and encoded by SEQ ID NO:2, see Kyndt et al, 2002FEBS Lett.512:240-244), Spanish (Saccharothrix espanaensis) (e.g., SeSam8, represented by SEQ ID NO:3 and encoded by SEQ ID NO:4, see Berner et al, 2006J Baciol 188:2666-2673), and Flavobacterium johnsonii (e.g., FjTAL, represented by SEQ ID NO:5 and encoded by SEQ ID NO:6, see Jendersen et al, 2015Appl Environ Microbiol81: 4458-4476). Preferred TALs or enzymes having TAL activity have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, preferably at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100%, preferably at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, more preferably at least 90%, most preferably 100% sequence identity to SEQ ID NO 1, SEQ ID NO 3 and SEQ ID NO 5, preferably to SEQ ID NO 1, or are encoded by polynucleotides having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, (97%,) sequence identity to SEQ ID NO 2, SEQ ID NO 4 or SEQ ID NO 6, preferably to SEQ ID NO 2, 98%, 99% or 100%, preferably at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100%, more preferably at least 90%, most preferably 100%. Preferred polynucleotides encoding TAL or encoding enzymes having TAL activity are codon optimized. When an enzyme to be tested for TAL activity is contacted with L-tyrosine in a suitable buffer, the TAL activity of the enzyme can be determined by monitoring the production of coumaric acid or ammonia evolution by chromatography.

4-Coumaric acid-CoA ligase, hereinafter referred to as 4CL, also known as 4CL, 4-coumaroyl-CoA synthetase, p-coumaroyl-CoA ligase, p-coumaroyl-CoA synthetase, p-coumaroyl-CoA ligase, feruloyl-CoA ligase, hydroxycinnamoyl-CoA synthetase and other various names. It is an enzyme (EC 6.2.1.12) that catalyzes the conjugation of coenzyme a (coa) to 4-coumaric acid to form 4-coumaroyl-coenzyme a at the expense of ATP. 4-Coumaroyl-CoA is also known as coumaroyl-CoA. Preferred 4CL is heterologous 4CL, which is a 4CL derived from an organism different from the cell according to the invention. Preferred organisms from which the 4CL may be derived are tobacco (Nicotiana tabacum) (e.g., Nt4CL, represented by SEQ ID NO:7, and encoded by SEQ ID NO:8, see Lee & Douglas.1996plant Physiol.112: 193) -205), Arabidopsis (Arabidopsis thaliana) (e.g., At4CL, represented by SEQ ID NO:9, and encoded by SEQ ID NO:10, see Ehlting et al, 1999plant.J.19:9-20), Physcomitrella patens (e.g., Pp4CL, represented by SEQ ID NO:11, and encoded by SEQ ID NO:12, see Silber et al, Phytocmom.69: 2449-2456), and Streptomyces coelicolor (Streptomyces coelicolor) (e.g., Sc4CL, represented by SEQ ID NO:13, and encoded by SEQ ID NO: 14). Preferred 4CL has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, preferably at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100%, preferably at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, more preferably at least 90%, most preferably 100% sequence identity to SEQ ID NO 7, 9, 11, or 13, or is encoded by a polynucleotide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% sequence identity to SEQ ID NO 8, 10, 12, or 14, preferably to SEQ ID NO 12, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, preferably at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, more preferably at least 90%, most preferably 100%. Preferred polynucleotides encoding 4CL are codon optimized.

Benzylidene acetone synthase, hereinafter BAS, is a plant-specific type III polyketide synthase (PKS). It is an enzyme (EC 2.3.1.212) that catalyzes the conversion of 4-coumaroyl-coenzyme A to 4-hydroxybenzylideneacetone. 4-hydroxybenzylideneacetone is also known as hydroxyphenylbutenone, p-hydroxybenzylideneacetone, 1- (4-hydroxybenzylidene) acetone and (3E) -4- (4-hydroxyphenyl) -3-buten-2-one. Preferred BAS are heterologous BAS, which are BAS derived from an organism different from the cell according to the invention. Preferred organisms from which BAS can be derived are Rubus palmatus (Rubus idaeus), (e.g., RiPKS, represented by SEQ ID NO:15 and encoded by SEQ ID NO:16, see Zheng & Hrazdina 2008Arch Biochem Biophys 470:139-145) and Rheum palmatum (e.g., represented by SEQ ID NO:17 and encoded by SEQ ID NO:18, see Abe et al, 2001Eur J Biochem 268: 3354-3359). Preferred BASs have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, preferably at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, more preferably at least 90%, most preferably 100% sequence identity to SEQ ID NO 15, or to SEQ ID NO 17, preferably to SEQ ID NO 17, or are encoded by polynucleotides having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, preferably at least 80%, 85%, 90% sequence identity to SEQ ID NO 18, or by polynucleotides encoding a polynucleotide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or to SEQ ID NO 18, preferably to SEQ ID NO 18, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, more preferably at least 90%, most preferably 100%. Preferred polynucleotides encoding BAS are codon optimized.

Benzylidene acetone reductase, hereinafter referred to as BAR. It is an enzyme (EC 1.3.1.-) that catalyzes the conversion of 4-hydroxybenzylideneacetone to raspberry ketone. Raspberry ketones are also known as 4- (4-hydroxyphenyl) butan-2-one, p-hydroxybenzylacetone, 4- (p-hydroxyphenyl) -2-butanone, hydroxyphenylbutanone, oxyphenyl ketone, rhegmine (rheosmin) and raspberry ketone. Preferred BARs are heterologous BARs, which are BARs derived from an organism different from the cell according to the invention. A preferred organism from which BAR may be derived is Rubus palmatus (Rubus idaeus) (e.g., RiBAR, represented by SEQ ID NO:19 and encoded by SEQ ID NO:20, see Koeduka et al, 2011Biochem Biophys Res Commun 412: 104-108). Preferred BARs have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, preferably at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, more preferably at least 90%, most preferably 100% sequence identity to SEQ ID NO 19 or are encoded by a polynucleotide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, preferably at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, more preferably at least 90%, (preferably at least 90%,), Most preferably 100% sequence identity. Preferred polynucleotides encoding BARs are codon optimized.

In this aspect, preferred cells according to the invention are provided, wherein

Functional heterologous enzymes with TAL activity are derived from Pseudomonas capsulata (Rhodobacter capsulatus), Spanish saccharomycete (Saccharothrix espanaensis) or Flavobacterium johnsonii (Flavobacterium johnsoniae); preferably, it is derived from rhodopseudomonas capsulata (Rhodobacter capsulatus);

-the at least one functional enzyme selected from 4CL and BAS is 4CL from tobacco (Nicotiana tabacum), Arabidopsis thaliana (Arabidopsis thaliana), Physcomitrella patens (Physcomitrella patens) or Streptomyces coelicolor, or BAS from Rubus palmiformis (Rubus idaeus) or Rheum palmatum (Rheum palmatum); preferably, BAS is from rheum palmatum; preferably, the 4CL is from physcomitrella patens; and

-the optional BAR is from Rubus palmatus (rubius idaeus).

In a more preferred embodiment, a cell according to the invention is provided, wherein

Functional heterologous enzymes with TAL activity are derived from Pseudomonas capsulata (Rhodobacter capsulatus), Spanish saccharomycete (Saccharothrix espanaensis) or Flavobacterium johnsonii (Flavobacterium johnsoniae); preferably, it is derived from rhodopseudomonas capsulata (Rhodobacter capsulatus);

-the at least one functional enzyme selected from 4CL and BAS is 4CL from tobacco (Nicotiana tabacum), Arabidopsis thaliana (Arabidopsis thaliana), Physcomitrella patens (Physcomitrella patens) or Streptomyces coelicolor, or BAS from Rubus palmiformis (Rubus idaeus) or Rheum palmatum (Rheum palmatum); preferably, BAS is from rheum palmatum; preferably, the 4CL is from physcomitrella patens; and

-the optional BAR is from Rubus palmatus (rubius idaeus).

In an even more preferred embodiment, there is provided a cell according to the invention, wherein:

-the functional heterologous enzyme with TAL activity is from rhodopseudomonas capsulata (Rhodobacter capsulatus);

-the at least one functional enzyme selected from 4CL and BAS is selected from 4CL from Physcomitrella patens (Physcomitrella patents), and BAS from Rheum palmatum (Rheum palmatum); and

-the optional BAR is from Rubus palmatus (rubius idaeus).

In a particular embodiment, both BAS and 4CL are expressed in a cell according to the invention. In such embodiments, there is provided a cell according to the invention, wherein:

functional heterologous enzymes with TAL activity are derived from Pseudomonas capsulata (Rhodobacter capsulatus), Spanish saccharomycete (Saccharothrix espanaensis) or Flavobacterium johnsonii (Flavobacterium johnsoniae);

-4Cl is from tobacco (Nicotiana tabacum), Arabidopsis (Arabidopsis thaliana), Physcomitrella patens (Physcomitrella patents) or Streptomyces coelicolor;

BAS is from Rubus palmatus (Rubus idaeus) or Rheum palmatum (Rheum palmatum); and

-the optional BAR is from Rubus palmatus (rubius idaeus).

In a more preferred such embodiment, there is provided a cell according to the invention, wherein:

-the functional heterologous enzyme with TAL activity is from rhodopseudomonas capsulata (Rhodobacter capsulatus);

-4CL from Physcomitrella patens (Physcomitrella patents);

BAS is from Rheum palmatum (Rheum palmatum); and

-the optional BAR is from Rubus palmatus (rubius idaeus).

In a preferred embodiment of this aspect, the invention provides a cell according to the invention, wherein:

-a functional heterologous enzyme having TAL activity has at least 60%, preferably at least 80%, more preferably at least 90%, most preferably 100% sequence identity to SEQ ID No. 1, SEQ ID No. 3, or SEQ ID No. 5, preferably to SEQ ID No. 1, or is encoded by a polynucleotide having at least 60%, preferably at least 80%, more preferably at least 90%, most preferably 100% sequence identity to SEQ ID No. 2, SEQ ID No. 4, or SEQ ID No. 6, preferably to SEQ ID No. 2;

-4CL has at least 60%, preferably at least 80%, more preferably at least 90%, most preferably 100% sequence identity to SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, or SEQ ID No. 13, preferably to SEQ ID No. 11, or is encoded by a polynucleotide having at least 60%, preferably at least 80%, more preferably at least 90%, most preferably 100% sequence identity to SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, or SEQ ID No. 14, preferably to SEQ ID No. 12;

-the BAS has at least 60%, preferably at least 80%, more preferably at least 90%, most preferably 100% sequence identity to SEQ ID No. 15, or SEQ ID No. 17, preferably to SEQ ID No. 17, or is encoded by a polynucleotide having at least 60%, preferably at least 80%, more preferably at least 90%, most preferably 100% sequence identity to SEQ ID No. 16 or SEQ ID No. 18, preferably to SEQ ID No. 18;

BAR has at least 60%, preferably at least 80%, more preferably at least 90%, most preferably 100% sequence identity to SEQ ID NO. 19 or is encoded by a polynucleotide having at least 60%, preferably at least 80%, more preferably at least 90%, most preferably 100% sequence identity to SEQ ID NO. 20.

In this regard, it may be advantageous to use polynucleotide constructs encoding more than one enzyme. For example, polycistronic constructs, or operons, in which more than one enzyme is under the control of a single promoter may be used. Such constructs can be produced using recombinant DNA techniques well known in the art.

Thus, in a preferred embodiment, this aspect provides a cell according to the invention, wherein at least two enzymes are encoded by a single recombinant polynucleotide construct. Such a construct may be contained in an expression vector, which may be a plasmid. The two enzymes may also be a single fusion polypeptide.

Raspberry ketone is 4- (4-hydroxyphenyl) butan-2-one and is also known as p-hydroxybenzylacetone, 4- (p-hydroxyphenyl) -2-butanone, hydroxyphenylbutanone, oxyphenyl ketone, rheamin (rheosmin) and raspberry ketone. It is a natural phenolic compound, which is the main aroma compound of red raspberry. It is present in a variety of fruits, including cranberries and blackberries. The cells according to the invention are capable of producing raspberry ketone and produce raspberry ketone when cultured. Preferred cells according to the invention are capable of producing, preferably at least 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50mg/L of raspberry ketone, preferably at least 5 mg/L. More preferably, the cell according to the invention is capable of producing, preferably, at least 100mg/L, 200mg/L, 300mg/L, 400mg/L, 500mg/L, 600mg/L, 700mg/L, 800mg/L, 900mg/L, 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L, 11g/L, 12g/L, 13g/L, 14g/L, 15g/L, 16g/L, 17g/L, 18g/L, 19g/L, 20g/L, 21g/L, 22g/L, 23g/L, 24g/L, or 25g/L of raspberry ketone is produced. Preferably, the raspberry ketone is detected in the cell according to the invention and/or in its culture broth or in the headspace in which the cell is cultured.

Accordingly, this aspect provides a cell according to the invention, which is capable of producing at least 5mg/L of raspberry ketone. A preferred embodiment provides a cell according to the invention, which is capable of producing at least 1g/L of raspberry ketone. A more preferred embodiment provides a cell according to the invention, which is capable of producing at least 5g/L of raspberry ketone. Still more preferred embodiments provide a cell according to the invention, which is capable of producing at least 10g/L of raspberry ketone, most preferably at least 20g/L of raspberry ketone. The yield of raspberry ketone can be monitored using chromatographic techniques on a sample obtained from the production medium. Such amounts are preferably obtained at up to 2 weeks, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 15 hours, 10 hours, 5 hours, 4 hours, 3 hours, 2 hours or 1 hour of culture, preferably after 24 hours of culture.

Production of cells

In a second aspect, the invention provides a method for producing a cell according to the invention. The features of this aspect are preferably those of the first aspect of the invention. In an embodiment of this aspect, there is provided a method for producing a cell according to the invention, comprising

-contacting a prokaryotic cell with an expression construct encoding a functional heterologous enzyme having Tyrosine Ammonia Lyase (TAL) activity, and

-contacting the prokaryotic cell with an expression construct encoding at least one functional enzyme selected from the group consisting of 4-coumarate-coa ligase (4CL) and Benzylidene Acetone Synthase (BAS), and

-optionally contacting the prokaryotic cell with an expression construct encoding a heterologous Benzylidene Acetone Reductase (BAR).

In a preferred embodiment of this aspect, the invention provides a method for producing a cell according to the invention, comprising

-contacting a prokaryotic cell with an expression construct encoding a functional heterologous enzyme having Tyrosine Ammonia Lyase (TAL) activity, and

-contacting the prokaryotic cell with an expression construct encoding a functional 4-coumarate-coa ligase (4CL) and a functional Benzylidene Acetone Synthase (BAS), and

-optionally contacting the prokaryotic cell with an expression construct encoding a heterologous Benzylidene Acetone Reductase (BAR).

In a preferred embodiment of this aspect, the invention provides a method for producing a cell according to the invention, comprising

-contacting a prokaryotic cell with an expression construct encoding a functional heterologous enzyme having Tyrosine Ammonia Lyase (TAL) activity, and

-contacting the prokaryotic cell with an expression construct encoding a functional 4-coumarate-coa ligase (4CL), and

-optionally contacting the prokaryotic cell with an expression construct encoding a heterologous Benzylidene Acetone Reductase (BAR).

In a preferred embodiment of this aspect, the invention provides a method for producing a cell according to the invention, comprising

-contacting a prokaryotic cell with an expression construct encoding a functional heterologous enzyme having Tyrosine Ammonia Lyase (TAL) activity, and

-contacting the prokaryotic cell with an expression construct encoding a functional Benzylidene Acetone Synthase (BAS), and

-optionally contacting the prokaryotic cell with an expression construct encoding a heterologous Benzylidene Acetone Reductase (BAR).

Corynebacterium (Corynebacterium), more preferably Corynebacterium glutamicum (Corynebacterium glutamicum)

Suitable cell types are defined herein before. Preferred prokaryotic cells are gram-positive cells, more preferably Corynebacterium, even more preferably Corynebacterium glutamicum. Thus, in a preferred embodiment of this aspect, the invention provides a method for producing a cell according to the invention, comprising

-contacting a Corynebacterium (Corynebacterium) cell, preferably a Corynebacterium glutamicum (Corynebacterium glutamicum) cell, with an expression construct encoding a functional heterologous enzyme having Tyrosine Ammonia Lyase (TAL) activity, and

-contacting a Corynebacterium (Corynebacterium) cell with an expression construct encoding at least one functional enzyme selected from the group consisting of 4-coumarate-coa ligase (4CL) and Benzylidene Acetone Synthase (BAS), and

-optionally contacting a Corynebacterium (Corynebacterium) cell with an expression construct encoding a heterologous Benzylidene Acetone Reductase (BAR).

For the expression of the enzyme in the prokaryotic cell according to the invention and for the further genetic modification of the prokaryotic cell according to the invention, the cell may be transformed with the nucleic acid or the nucleic acid construct described herein by any method known to the person skilled in the art. Such methods are known, for example, from standard manuals, such as Sambrook and Russel (2001) "molecular cloning Laboratory Manual (3 rd edition), Cold Spring Harbor Laboratory Press, or F.Ausubel et al, editors" Current protocols in molecular biology ", Green publishing and Wiley Interscience, New York (1987). Methods for transformation and genetic modification of bacterial cells are known, for example, from U.S. Pat. No. 6,699,696 or U.S. Pat. No. 4,778,759. For Corynebacterium (Corynebacterium), reference is made to the Eggeling & Reyes2005 experiment. In Eggeling, L., Bott, M. (eds.), the Handbook of Corynebacterium glutamicum (Handbook of Corynebacterium glutamicum) CRC Press, Boca Raton, FL, pp.3535-3566. Examples are transformation using competent or super-competent cells, electroporation, use of transfected lipids, use of transfected polymers or stereogenic transformation. A preferred method is electroporation.

When the nucleic acid construct is used for expressing an enzyme in a prokaryotic cell according to the invention, a selectable marker may be present in the nucleic acid construct comprising the polynucleotide encoding the enzyme. The term "marker" as used herein refers to a gene encoding a trait or phenotype that allows for the selection or screening of cells containing the marker. The marker gene may be an antibiotic resistance gene whereby the appropriate antibiotic can be used to select for transformed cells from untransformed cells. Preferred selectable markers are kanamycin and its corresponding resistance gene. Preferably, however, non-antibiotic resistance markers are used, such as auxotrophic markers (URA3, TRP1, LEU 2). For example, preferred cells according to the invention transformed with the nucleic acid construct are marker-free genes. (Cheah et al, 2013) describes a method for constructing a microbial host cell free of recombinant marker genes, which is based on the use of bidirectional markers. Alternatively, screenable markers such as green fluorescent protein, lacZ, luciferase, chloramphenicol acetyltransferase, β -glucuronidase may be incorporated into the nucleic acid construct according to the invention to screen for transformed cells.

Method for producing raspberry ketone

The prokaryotic cells according to the invention can be used for the de novo production of raspberry ketones using a simple aerobic fermentation process. Accordingly, in a third aspect, the present invention provides a method of producing raspberry ketone, comprising:

-culturing a cell according to the invention under conditions conducive to the production of raspberry ketone, and optionally,

-isolating and/or purifying raspberry ketone from the cells and/or the culture medium.

In a preferred embodiment, this aspect provides a method of producing raspberry ketone, comprising:

-culturing a cell according to the invention under conditions conducive to the production of raspberry ketone, and

-isolating and/or purifying raspberry ketone from the cells and/or the culture medium. The features of this aspect are preferably those of the first and second aspects of the invention.

This method will be referred to as the method according to the invention below. Preferably, the method according to the invention for producing raspberry ketone comprises culturing a cell according to the invention, preferably a gram-positive cell as defined in the first aspect of the invention, and more preferably a Corynebacterium (Corynebacterium) cell as defined in the first aspect, wherein the culture conditions comprise culturing the cell in LB medium or YT medium or CgXII medium, preferably CgXII medium, at about 30 ℃, wherein the medium is preferably supplemented with about 20g/L D-glucose, optionally supplemented with kanamycin (preferably about 50 μ g/mL). The culture is preferably shaken or agitated at about 250 rpm. The culturing may include induction with isopropyl beta-D-1-thiogalactoside (IPTG) or arabinose or another suitable inducer, as will be apparent to those skilled in the art. The culture may require the use of a starter culture. Other examples of advantageous conditions are provided in the examples.

Typically, a single colony was inoculated into 5mL of LB medium supplemented with 50. mu.g/mL kanamycin and 1% glucose. The starter culture can be grown overnight at 37 ℃ and 230 rpm. In a 100mL Erlenmeyer flask, about 200. mu.l of the starting culture can be used to inoculate about 20mL of 2XYT medium (16g/L tryptone, 10g/L yeast extract, 10g/L NaCl) supplemented with about 50. mu.g/mL kanamycin and incubated at 37 ℃ and 230rpm until an optical density at 600nm (OD600 or A600) of 0.4-0.6. Subsequently, 1mM IPTG may be added to the medium and the culture incubated at 30 ℃ at 250 rpm. Optionally, the culture may be supplemented with 4-coumaric acid, preferably with about 3mM 4-coumaric acid. The total bacterial culture is preferably collected 24 hours after induction by IPTG and may be stored at-20 ℃. This method is particularly applicable to e.

Alternatively, the starter culture can be grown in 25mL LB medium supplemented with 50. mu.g/mL kanamycin and 1% glucose at 250rpm and 30 ℃ for 48 h. The starter culture can then be centrifuged at about 5000rpm for about 10 minutes and the bacterial pellet resuspended in about 1.5ml of CgXII minimal medium. Subsequently, the culture can be transferred to an about 100mL Erlenmeyer flask containing about 25mL CgXII minimal medium supplemented with about 50. mu.g/mL kanamycin and about 20g/L D-glucose, which can then be directly induced with about 1mM IPTG. The bacterial culture may be incubated at about 30 ℃ and about 250rpm for about 30 hours, up to about 4 days. Optionally, the culture may be supplemented with 4-coumaric acid, preferably with about 3mM 4-coumaric acid. After fermentation, the entire bacterial culture can be collected and stored at-20 ℃. The method is particularly applicable to Corynebacterium glutamicum (C.glutamicum).

In a preferred method, raspberry ketone is separated from the culture broth. This may be done continuously during the production process or after the production process. The separation may be based on any separation method known to the person skilled in the art. Chromatography or liquid/liquid extraction are attractive techniques.

The raspberry ketone produced by the cells according to the invention and the method according to the invention has specific properties, for example is free of trace impurities remaining after chemical synthesis. Thus, there is provided raspberry ketone obtainable by the method according to the invention.

The raspberry ketone obtained using the process according to the invention can be conveniently used in products. Thus, there is provided a pharmaceutical, flavour, fragrance, cosmetic or food composition comprising raspberry ketone obtainable by the method according to the invention.

The process according to the invention is preferably a de novo process, i.e. a process for producing raspberry ketone from the head. In the de novo method, no proprietary precursors are added to the culture, which are then transformed by fermentation. In other words, in the de novo process, the intended product is formed from metabolites that are conventionally present in the culture broth. As a non-limiting example, the addition of 4-coumaric acid to the culture broth in a process to obtain raspberry ketone can be considered not a head-on process, as raspberry ketone would be formed at least in part from exogenously added 4-coumaric acid, and 4-coumaric acid is not a conventional ingredient of the culture broth. As a non-limiting example, since L-tyrosine is typically present in culture broth, formation of raspberry ketone from L-tyrosine can be considered de novo.

Use of TALs

The function of TAL is an important feature of the present invention. Thus, in a fourth aspect, the present invention provides the use of a functional enzyme having Tyrosine Ammonia Lyase (TAL) activity as defined herein before for the production of raspberry ketones in prokaryotic host cells, preferably gram-positive prokaryotic host cells. The features of this aspect are preferably those of the first, second and third aspects of the invention. In a preferred embodiment of this aspect, the present invention provides the use of a functional heterologous enzyme having Tyrosine Ammonia Lyase (TAL) activity as defined herein before for the production of raspberry ketones in prokaryotic host cells, preferably gram-positive prokaryotic host cells. In a preferred embodiment of this aspect, the present invention provides the use of a functional heterologous enzyme having Tyrosine Ammonia Lyase (TAL) activity as defined herein before for the production of raspberry ketones in gram-positive prokaryotic host cells, preferably corynebacterium host cells, more preferably corynebacterium glutamicum host cells. Features and definitions are provided elsewhere herein.

Expression vector

In a fifth aspect of the invention, there is provided an operon and an expression vector as defined herein before. The features of this aspect are preferably those of the first, second, third and fourth aspects of the invention. Such expression vectors are referred to herein as expression vectors according to the present invention. Such an operon is herein referred to as an operon according to the present invention. Thus, a preferred embodiment of this fifth aspect provides an expression vector comprising a first polynucleotide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68 or 70, more preferably with SEQ ID NO 52, or with SEQ ID NO 70, most preferably with SEQ ID NO 70, or wherein the expression vector consists of a polynucleotide which is identical to SEQ ID NO 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67 or 69, more preferably at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 51, or to SEQ ID NO 69, most preferably to SEQ ID NO 69.

A more preferred embodiment of this fifth aspect provides an expression vector comprising a first polynucleotide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, preferably at least 90% sequence identity to SEQ ID No. 52, or to SEQ ID No. 70, most preferably to SEQ ID No. 70, or wherein the expression vector consists of a polynucleotide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, preferably at least 90% sequence identity to SEQ ID No. 51, or to SEQ ID No. 69, most preferably to SEQ ID No. 69.

Other preferred embodiments of this fifth aspect provide an expression vector comprising a first polynucleotide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, preferably at least 90% sequence identity to SEQ ID NO 46, 48, 52 or 70, most preferably to SEQ ID NO 46, 48, 52 or 54, or wherein the expression vector consists of a polynucleotide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, preferably at least 90% sequence identity to SEQ ID NO 45, 47, 51, 53 or 69, most preferably to SEQ ID NO 45, 47, 51, 53.

The polynucleotide is represented by a nucleotide sequence. The polypeptide is represented by an amino acid sequence. A nucleic acid construct is defined as a polynucleotide that is isolated from a naturally occurring gene, or that has been modified to contain polynucleotide fragments that are combined or juxtaposed in a manner that would not otherwise exist in nature. Optionally, the polynucleotide present in the nucleic acid construct is operably linked to one or more control sequences that direct the production or expression of the peptide or polypeptide in a cell or subject.

The polynucleotides described herein may be native or may be codon optimized. Codon optimization adapts the codon usage of the encoded polypeptide to the codon bias of the organism in which the polypeptide is to be produced. Codon optimization generally contributes to an increase in the level of production of the polypeptide encoded by the host cell (e.g., in a host preferred herein). Corynebacterium (Corynebacterium) for the person skilled in the art many algorithms are available for codon optimization. One preferred method is the "monte carlo algorithm-based guided random method that can be used in the genome over the internet. us/OPTIMIZER/(P.Puigb, E.Guzm a, A.Romeu, and S.Garcia-Valley.nucleic acid Res.2007 July; 35(Web Server issue): W126-W131).

As used herein, the term "heterologous sequence" or "heterologous nucleic acid" is not naturally occurring, and is operably linked as a contiguous sequence to the first nucleotide sequence. As used herein, the term "heterologous" may mean "recombinant". "recombinant" refers to a genetic entity other than that which is ubiquitous in nature. When applied to a nucleotide sequence or nucleic acid molecule, this means that the nucleotide sequence or nucleic acid molecule is the product of various combinations of cloning, restriction and/or ligation steps, as well as other procedures that result in the production of constructs that differ from sequences or molecules found in nature. A recombinant oligonucleotide may be an oligonucleotide comprising a sequence from more than one single source.

"operably linked" is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to a nucleotide sequence encoding a polypeptide of the invention such that the control sequence directs the production/expression of a peptide or polypeptide of the invention in a cell and/or in a subject.

"operably linked" may also be used to define a configuration in which the sequences are appropriately placed at a position relative to another sequence encoding a functional domain such that a chimeric polypeptide is encoded in a cell and/or in a subject. Throughout this application, any nucleic acid sequence encoding an enzyme is preferably operably linked to another such sequence or promoter.

As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more nucleic acid molecules located upstream in the direction of transcription relative to the transcription initiation site of the nucleic acid molecule and structurally recognized by the presence of a binding site for a DNA-dependent RNA polymerase, the transcription initiation site, and any other DNA sequences, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to those of skill in the art to act directly or indirectly to regulate the amount of transcription from a promoter. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation.

Optional further elements that may be present in the nucleic acid construct according to the invention include, but are not limited to, one or more leader sequences, enhancers, integration factors and/or reporter genes, intron sequences, centromeres, telomeres and/or Matrix Attachment (MAR) sequences. The nucleic acid constructs according to the invention can be provided in a manner known per se, which generally involves techniques such as restriction and ligation of nucleic acids/nucleic acid sequences, for which reference is made to standard manuals, for example Sambrook and Russell (2001) "molecular cloning: a Laboratory Manual (3 rd edition), Cold Spring Harbor Laboratory Press.

Polypeptides

In a sixth aspect, the invention provides a polypeptide product expressed by an expression vector according to the invention. Features and definitions are provided elsewhere herein and are preferably features and definitions of the first, second, third, fourth and fifth aspects of the present invention.

Definition of

In the context of amino acid sequences or nucleic acid sequences, "sequence identity" or "identity" is defined herein as the relationship between two or more amino acid (peptide, polypeptide, or protein) sequences or two or more nucleic acid (nucleotide, polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleotide sequences as appropriate, as determined by the match between such sequence strings. In the present invention, sequence identity to a particular sequence preferably refers to sequence identity over the entire length of the particular polypeptide or polynucleotide sequence. The sequence information provided herein should not be construed narrowly as requiring inclusion of misidentified bases. The skilled person is able to identify such erroneously identified bases and knows how to correct these errors.

Preferred methods of determining identity are designed to give the maximum match between test sequences. Methods for determining consistency and similarity are compiled in publicly available computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, for example, the GCG program package (Devereux, J. et al, Nucleic Acids Research 12(1):387(1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S.F. et al, J.mol.biol.215,403-410 (1990)). BLAST X programs are publicly available from NCBI and other sources (BLAST handbook, Altschul, S. et al, NCBI NLM NIH Bethesda, MD 20894; Altschul, S. et al, J.mol.biol.215,403-410 (1990)). The well-known Smith Waterman (Smith Waterman) algorithm can also be used to determine identity.

Preferred parameters for polypeptide sequence comparison include the following: the algorithm is as follows: needleman and Wunsch, J.mol.biol.48: 443-; comparing the matrixes: BLOSUM62 from Hentikoff and Hentikoff, Proc.Natl.Acad.Sci.USA.89: 10915-; gap penalties: 12; and gap length penalties: 4. programs for these parameters are publicly available as "Ogap" programs from Genetics Computer Group at madison division, university of wisconsin, usa. The foregoing parameters are the default parameters for amino acid comparisons (with no penalty for end gaps).

Preferred parameters for nucleic acid comparison include the following: the algorithm is as follows: needleman and Wunsch, J.mol.biol.48: 443-; comparing the matrixes: match +10, mismatch-0; gap penalties: 50; gap length penalty: 3. available from the Gap program of Genetics Computer Group at middison division, university of wisconsin, usa. The default parameters for nucleic acid comparisons are given above.

Moderate conditions are defined herein as allowing a nucleic acid sequence of at least 50 nucleotides, preferably about 200 or more nucleotides to hybridize in a solution comprising about 1M salt, preferably 6 xssc, or any other solution having comparable ionic strength at a temperature of about 45 ℃ and to be washed in a solution comprising about 1M salt, preferably 6 xssc, or any other solution having comparable ionic strength at room temperature. Preferably, the hybridization is carried out overnight, i.e. at least 10 hours, and preferably the washing is carried out for at least one hour with at least two changes of the washing solution. These conditions generally allow for specific hybridization of sequences with up to 50% sequence identity. One skilled in the art will be able to modify these hybridization conditions to specifically recognize sequences that vary in identity between 50% and 90%.

Expression is understood to include any step involved in the production of a peptide or polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, the use of the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that one and only one of the elements is present. Thus, the indefinite article "a" or "an" usually means "at least one".

The word "about" or "approximately" when used in conjunction with a numerical value (e.g., about 10) preferably means that the value can be plus or minus 10% of the value (of 10) for the given value.

The sequence information provided herein should not be construed narrowly as requiring inclusion of misidentified bases. The skilled person is able to identify such erroneously identified bases and knows how to correct these errors. In the case of a sequence error, the sequence of the enzyme obtainable by expressing the genes represented by SEQ ID NOs 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 containing the enzyme encoding the polynucleotide sequence should win.

All patents and documents cited in this specification are incorporated herein by reference in their entirety.