BACKGROUND

Vanilloids are defined as chemical compounds possessing a vanillyl group whose chemical structure is shown below:

wherein R is selected from the group consisting of an hydrogen H, a methyl - C¾, an ethyl -CH2CH ₃ , an aldehyde and a carboxylic acid. Optionally, the OH group can be substituted with an O-glycosyl group. In a non-limitative manner, vanilloids include vanillyl alcohol, vanillin, vanillic acid, vanillin β-D-glucoside, acetovanillon, vanillylmandclic acid, homovanillic acid and capsaicin.

By extension, vanilloids also preferably includes chemical compounds possessing a group whose chemical structure is shown below:

wherein R is selected from the group consisting of an hydrogen H, a methyl -

CH ₃ , an ethyl -CH:CH ₃ , an aldehyde and a carboxylic acid. Optionally, the OH group can be substituted with an O-glycosyl group. In a non-limitative manner, these chemical compunds include ethyl vanillyl alcohol, ethylvanillin, ethyl vanillic acid, ethylvanillin β- D-glucoside, acetoethyl vani 1 Ion, ethylvanillylmandelic acid and homoethylvanillic acid.

Among vanilloids, vanillin, of chemical name 4-hydroxy-3- methoxybenzaldehyde. is one of the most important aromatic flavor compound used in foods, beverages, fragrances, pharmaceuticals and polymers. Vanillin was historically extracted from Vanilla planifolia, Vanilla tahitiensis and Vanilla pompona pods. The demand getting higher today, less than 5% of worldwide vanillin production comes from natural vanilla pods. Currently, chemical synthesis is the most important process for producing vanillin. There is a growing interest in other sources of vanillin and in particular in byconversion processes. The use of microbial cells and their enzymes as biocatalysts for the synthesis of chemicals and flavor compounds have attracted much attention lately. Advantageously, under certain criteria, the products of such bioconversion may be considered 'natural ^' by legislations, such as the EU one.

Bioconversion processes are based on the following substrates: lignin, phenolic stilbenes, isoeugenol, eugenol, ferulic acid, sugars, phenolic stilbenes, waste residues and aromatic amino acids. The recent review from Kaur and Chakraborty (Kaur

B, Chakraborty D. "Biotechnological and molecular approaches for vanillin production: a review Appl Biochem Biotechnol. 2013 Feb; 169(4): 1353-72) list several biosynthetic pathways and appropriate cells used for bioconversion of vanilloids.

De novo synthesis from glucose and using metabolically engineered yeast strains was recently described (Hansen EH, M oiler BL, Kock GR. Biinner CM, Kristensen

C, Jensen OR, Okkels FT, Olsen CE, Motawia MS. Hansen J. "De novo biosynthesis of vanillin in fission yeast (Schizosaccharomyces pombe) and baker's yeast (Saccharomyces cerevisiae)" . Appl Environ Microbiol. 2009 May;75(9):2765-74). The engineered pathway involves a 3-dehydroshikimate dehydratase (3DSD), an aromatic carboxylic acid reductase (AC'AR) and an O-methyltransferase. as shown in figure 2, pathways 1 and 2. In S. cerevisiae, because the ACAR. enzyme requires an activation mediated by a phosphopantetheinyl transferase, the latter enzyme was also introduced in the yeast. Prevention of reduction of vanillin to vanill l alcohol was achieved by deleting the host alcohol dehydrogenase encoding gene adh6. Later, the same authors improved their engineered pathway by using mutants of human Catechol O-methyltransferase having better substrate specificity, thereby limiting the production of isovanillin (WO 2013/022881).

Another engineered pathway for producing a vanilloid in yeast is inspired from the natural biosynthesis pathway observed in the Vanilla planifolia orchid, such as described in patent application US 2003/0070188. This engineered pathway comprises the following enzymes: phenylalanine/tyrosine ammonia lyase (PAL/TAL). cinnamate-4- hydrolase (C4H), 4-coumarate coenzyme A ligase (4CL). enoyl-CoA hydratase/aldolase (EOT), a hydroxybenzoic acid hydroxylase (HBH) and a caffcic acid O-methyltransferase (COMT). as shown in figures 1 and 2, pathway 3. This pathway uses aromatic amino acids such as phenylalanine and tyrosine as primary substrates. Enzymes of this pathway have been successfully introduced into yeast, able to bio-synthetize vanilloids (patent application EP 13156021.1 , not published, herein incorporated by reference).

However, even if these engineered pathways are effective for producing vanilloids, they do not allow production of these compounds in amounts which are compatible with the economical and industrial requirements. Different propositions have been made to improve the production of vanilloids by fermentation in cells.

Among them, alcohol dehydrogenases ADH6 & ADH7 are known to convert vanillin into its corresponding vanillyl alcohol. Studies have suggested the deletion of the adhe gene in vanillin-producing yeasts (Hansen et al., 2009, previously cited).

The article from Brochado et al. (Brochado AR, Matos C, M oiler BL, Hansen

J, Mortensen UH, Patil KR. Improved vanillin production in baker's yeast through in silica design". Microb Cell Fact. 2010 Nov 8:9:84) suggested the deletion of genes encoding pyruvate decarboxylase (PDC1) and glutamate dehydrogenase (GDH l ), since the deletion of these genes increases the respiratory capacity of the cell and the availability of co-factors (ATP, NADPH... ) for the biosynthesis pathway of vanilloids.

Most of phenolic compounds such as vanillin show some toxicity for many living organisms with increased concentration of vanilloids. In case of Saccharomyces cerevisiae, growth defect is significant with concentrations as low as 0.5 g/l. To avoid the impaired growth of producing microorganisms, it has been proposed to isolate the produced vanilloids from the culture medium, in particular with resins. Another suggested solution is promoting conversion of vanillin into vanillin β-D-glucoside, this glycosylation inhibiting its toxic effect (Hansen et al., 2009; Brochado et al., 2010. both previously cited).

However, the highest amount of vanillin produced in byconversion process with these genetically modified microorganisms is still limited to about 1 μΜ; indeed several problems subsist and have to be solved to improve vanillin production. Specific disadvantages linked to byconversion processes are in particular the following:

• In living cells, intermediates of the biosynthesis pathway are subject to degradation by endogenous enzymes. In particular, phcnylacrylic acids such as cinnamic acid and coumaric acid can be decarboxylated by endogenous yeasts enzymes and converted into styrene and 4-vinylphenol. Also, aldehydes compounds in the pathway are subject to reduction in their corresponding primary alcohol by various alcohol dehydrogenases. Finally, the aromatic amino acids, first intermediates of the pathway, are also subject to consumption in concurrent metabolic pathways.

• Efficiency of byconversion is linked to the growth ability of the producing cells; therefore, it is essential to determine the best conditions of growth for said producing cells. Different parameters can be modified, in order to optimize the growth of cells and therefore the metabolic pathways, for example: volume of the culture, temperature, growth medium, source of carbon, management of the toxic effect of vanillin, etc.

Inventors present here genetically modified yeast cells and specific culture conditions, both allowing a significant improvement of vanilloid production from a source of carbon, by fermentation.

SUMMARY OF THE INVENTION

The present invention is related to a genetically modified yeast, optimized for the production of a vanilloid or a precursor thereof from a source of carbon, comprising enzymes required for production of a vanilloid or a precursor thereof, and wherein the said genetically modified yeast has a reduced ability to decarboxylate phenylacrylic acids. Advantageously, the genetically modified yeast of the invention presents also a decreased ability for degrading aldehydes, and ensures an improved availability of aromatic amino acids.

The present invention is also related to an improved method for producing a vanilloid or a precursor thereof by fermentation. Culture conditions such as carbon sources, biphasic media byconversion and volume of the culture were explored to optimize vanillin production.

The present invention is also related to a method for producing a vanilloid or a precursor thereof from a specific source of carbon, by fermentation of genetically modified yeasts.

The present invention is also related to a method for producing a vanilloid or a precursor thereof, and separating it from the culture medium by biphasic culture media to avoid the toxic effect of vanillin. In particular the step of the yeast culture is performed in an appropriate medium comprisi g two phases, an aqueous phase and a water immiscible solvent. Thus, the present invention is also related to a method for producing a vanilloid or precursor thereof in a unicellular microorganism able to produce said vanilloid or precursor thereof, comprising a step of cultivating said microorganism in an appropriate medium comprising an aqueous phase and a water immiscible phase, said water immiscible phase preferably consistin of polymer of propylene glycol, fatty alcohol, fatty acid, fatty ester or an oil.

In addition, the present invention is related to a composition comprising a vanilloid or a precursor thereof obtainable by the method as disclosed above, and to the use of said composition as a flavoring in the human and animal nutrition field, in pharmacy, and as a fragrance in the cosmetics, perfumery and detergency industries.

FIGURES

FIGURE 1A, IB: Biosynthetic pathways of vanillin from aromatic amino acids. The figure shows the schematic diagram wherewith carbon source is converted into vanillin. Phenylalanine undergoes several reactions: deamination, hydroxylation of 4 position of the phenyl ring, reduction chain reaction and hydroxylation of 3 position of the phenyl ring, and O-methylation of the 3 position of the phenyl ring.

FIGURE 2 A, 2B: Global reaction scheme of vanillin biosynthesis from glucose. The shikimic acid pathway (pathways 1 & 2 - Figure 2A) and the aromatic amino acid pathway (pathway 3 - Figure 2B) are represented. PAL relates to a phenylalanine ammonia lyase. C4H relates to a cinnamic acid hydroxylase. 4CL relates to a 4- coumarate-CoA ligase. HBH relates to a hydroxybenzoic acid hydroxylase. ECH relates to an enoyl-CoA hydratase / crotonase.

FIGURE 3: 3 A: conversion of 4-hydroxybenzaldehyde by deletion strains Aald2, Agre3, Aald6 and Aadh6 and a wild-type (WT) strain; 3B: conversion of 3.4- dihydroxybenzaldehyde by the same strains than figure 3 A; 3C: conversion of vanillin by the same strains than figure 3 A; 3D: conversion of vanillin by the strain YOG. Y00 Aadh6 and the multi-deleted strains Y00 Aadh6 Agre3 and Y00 Aadh6 Aald2.

FIGURE 4: Assembly of vanillin pathway by fragments containing homologous gene sequences. This figure shows the co-transformation of 9 fragments comprising the 6 genes for vanillin production starting from phenylalanine and 1 gene coding for the carboxylie acid reductase. HISS and LEU2 are the flanking markers enabling the double selection of the recombinant pathway.

FIGURE 5: Grey bars: Metabolites total concentration from vanillin pathway according to then host strain (cinnamic acid, coumaric acid, 4-hydroxybenzoic acid, 3 ,4-dihydroxybenzoic acid, 3,4-dihydroxybenzaldehyde, vanillin, vanillic acid, vanillyl alcohol). Striped bars: Total concentration of final products from vanillin pathway according to then host strain (vanillin, vanillic acid and vanillyl alcohol). Black bars: vanillin concentration.

FIGURE 6: Metabolite profile of supernatant of culture from strains comprising a recombinant pathway according to figure 4, and the following deletions: Aadh6Aaro 1 OAfdc 1 and Aadh6Aarol OAfdcl Apdcl in 2m L culture volume. 34AD = 3,4- dihydroxybenzaldehydc; 34 AC = 3 ,4-dihydroxybenzoic acid; VANAL = vanillyl alcohol; VAN = vanillin; VANAC = vanillic acid.

FIGURE 7: Vanillin production of a strain comprising a recombinant pathway according to figure 4, and the following deletions: Aadh6Afdc 1 Apad 1 in a 50 mL Erlenmeyer flask.

FIGURE 8: Cytotoxicity assays with vanillin toward yeast in the presence or absence of PPG 1200 done with a strain comprising a recombinant pathway according to figure 4, and the following deletions: Aadh6Apdc 1 Apad 1.

FIGURE 9: Extractive byconversion of vanillin using 30% polypropylene glycol 1200 (PPG). Diagram presents concentration of vanillin (VAN), vanillic acid (VANAC), vanillyl alcohol (VANAL), 3 ,4-dihydroxybenzoic acid (34hbzAC) and 3,4-dihydroxybenzaldehyde (34hbzAD) in control culture (YPGal) and in biphasic culture (YPAGal/PPGl 200) from culture supernatant of a strain comprising a recombinant pathway according to figure 4, and the following deletions: Aadh6Apad 1 Afdc 1.

FIGURE 10: Vanillin producing cells (strains comprising a recombinant pathway according to figure 4, and the following deletions: Aadh6Apdc 1 Afdc 1 ) were precul tared in YPD (10 g/1 bacto-yeast extract. 20 g 1 bacto-peptone and 2% dextrose) or YPGal (10 g/1 bacto-ycast extract. 20 g/1 bacto-peptone and 3% galactose) medium in a 10 mL for 24h, and used to inoculate 50 mL appropriate medium in a 250 mL Erlenmeyer flask. Vanillin concentration (uM) was monitored after 3 days of culture depending on carbon source used for yeast growth. YPAD = YPGal; YPF (10 g/1 bacto-yeast extract. 20 g/1 bacto-peptone and 2% fructose); YPAGal (10 g/1 bacto-yeast extract, 20 g/1 bacto- peptone, 30g/l galactose); YPGE (10 g/1 bacto-yeast extract, 20 g/1 bacto-peptone, 2% glycerol and 2% ethanol); YPG (10 g/1 bacto-yeast extract, 20 g/1 bacto-peptone, 2% glycerol): YPE (10 g/1 bacto-yeast extract. 20 g/1 bacto-peptone and 2% ethanol); YPDE (10 g/1 bacto-yeast extract, 20 g/1 bacto-peptone, 2% dextrose and 2% ethanol).

DETAILED SPECIFICATION

The present invention is related to a recombinant yeast, optimized for the production of a vanilloid or a precursor thereof, comprising a set of exogenous nucleic acids, with each nucleic acid encoding a polypeptide involved in the biosynthesis pathway of a vanilloid, said set of exogenous nucleic acids encoding a set of polypeptides selected in a group comprising:

- a set of enzymes allowing conversion of an aromatic amino acid into a vanilloid; and

- a set of enzymes allowing conversion of an dehydroshikimic acid into a vanilloid;

and wherein said recombinant yeast is devoid of a functional gene adh6 encoding for an alcohol dehydrogenase,

and wherein said recombinant yeast has a reduced ability to decarboxylate phenylacrylic acids, which encompasses preferentially that said yeast does not comprise a fonctional fdcl gene and/ or a functional pad J gene.

This invention also pertains to a recombinant yeast, optimized for the production of a vanilloid or a precursor thereof, expressing a set of polypeptides involved in the biosynthesis pathway of a vanilloid. said set of polypeptides being encoded by one or more exogenous nucleic acids, and said set of polypeptides being selected in a group comprising:

- a set of enzymes allowing conversion of an aromatic amino acid into a vanilloid; and

- a set of enzymes allowing conversion of an dehydroshikimic acid into a vanilloid;

and wherein said recombinant yeast is devoid of a functional gene adh6 encoding for an alcohol dehydrogenase,

and wherein said recombinant yeast has a reduced ability to decarboxylate phenylacrylic acids, which encompasses that said yeast does not comprise a functional fdcl gene and/ or a functional pad I gene.

The following terms are used in the present application and are defined hereunder.

Yeasts are eukaryotic unicellular microorganisms. Yeasts are chemoorganotrophs. as they use organic compounds as a source of energy and do not require sunlight to grow. Over 1 ,500 species are currently known. An extensively studied genus of yeast is Saccharomyces.

By "recombinant yeast" or "genetically modified yeast" is meant a yeast organism whose genetic material has been modified by genetic engineering methods, either by suppression or inactivation of genes, and/or by addition of exogenous genetic material.

By "vanilloid" is meant a chemical compound which possesses a vanillyl group whose chemical tructure is shown below:

wherein R is selected from the group consisting of H, -C¾, -CH ₂ CH ₃ , an aldehyde and a earboxylic acid. Optionally, the OH group can be substituted with an O- glycosyl group. By extension, vanilloids also preferably include chemical compounds possessing a group whose chemical structure is shown below:

wherein R has the same meaning as defined above.

The present invention relates to a method for producing a vanilloid of formula ( I) or of formula (11), preferably of formula (I):

R ¹ being selected from the group consisting of -CHO; - COOH; -COOR ³ - C H ₂ OI I ; - CI I ₂ COOH; -C(=Q)CH ₃ ; -CR ³ (OH)COOH and CHR ³ COOH, wherein R ³ is a lower alkyl,

R ² being different from a methyl (-CH ₃ ), in particular R ² being selected from the group consisting of H, sulfate, phosphate and a glycoside. According to a preferred embodiment, R can be H.

Vanilloids include, in a non limitative manner, vanillyl alcohol, vanillin, vanillic acid, vanillin-glycoside, acetovanillon, vanillylmandclic acid, homovanillic acid, capsaicin and ethylvanillin.

Chemical structures of vanillin, isovanillin, vanillic acid and isovanillic acid are respectively given here below:

As used herein, an "exogenous nucleic acid encoding a polypeptide" means a nucleic acid comprising a one or more expression regulatory sequences and an Open Reading Frame (which may also be termed "ORF" or "coding sequence ^' "), that has been initially introduced into a yeast cell by a genetic engineering method, thus allowing the said yeast cell to express a polypeptide encoded by the said ORF. Most preferably, an exogenous nucleic acid also comprises at least a transcription terminator. Nucleic acids are exogenous to the host cell. i.e. are introduced in said host cell; they can be naturally absent or present in said host cell, but in all cases they are introduced by human manipulation. Most preferably, an "exogenous nucleic acid encoding a polypeptide" is not naturally present in the genome of the said yeast cell. An example of an exogenous nucleic acid is a nucleic acid which originates from a yeast species different from the host yeast cell species. Another example of an exogenous nucleic acid is a construct comprising an endogenous ORF which is placed under the control of a promoter which is not the naturally-occurring promoter of the said endogenous ORF.

As used herein, a "set" of exogenous nucleic acids means a plurality of exogenous nucleic acids as defined above, the said plurality f exogenous nucleic acids allowing when expressed in yeast cells wherein they have been introduced, the production of the corresponding set of polypeptides. The examples herein illustrate embodiments of yeast cells in which a set of exogenous nucleic acids has been introduced, the said set of exogenous nucleic acids encoding a set of polypeptides selected in a group comprising:

- a set of enzymes allowing conversion o an aromatic amino acid into a vanilloid; and

- a set of enzymes allowing conversion of an dehydroshikimic acid into a vanilloid.

In some embodiments, a set of exogenous nucleic acids is comprised in a unique polynucleotide, which unique polynucleotide has been introduced in the yeast organism, e.g. within a specific location in a yeast chromosome.

In other embodiments, a set of exogenous nucleic acids is initially comprised in a plurality of polynucleotides which are then fused before introduction in the yeast organism, e.g. within a specific location in a yeast chromosome.

In still other embodiments, a set of exogenous nucleic acids is comprised in a plurality of polynucleotides, each polynucleotide being introduced in the yeast organism, e.g. within a plurality of locations in a yeast chromosome.

As used herein, a recombinant yeast organism "comprises" exogenous nucleic acids when the said exogenous nucleic acids are part of the genetic material thereof, either (i) because the said exogenous nucleic acids are integrated into the chromosomal genetic material of the said yeast organism, (ii) or because the said exogenous nucleic acids are included within the genetic material of the said yeast organism outside chromosomes, which encompasses inclusion of the exogenous nucleic acids into nucleic acid vectors, e.g. plasmids. The term "biosynthesis pathway" as used in the present application refers to the different enzymes involved in specific biochemical steps and intermediates enabling the biosynthesis of products from a substrate in vivo by recombinant host cells. A biosynthesis pathway may be termed "artificial" when one or more of the enzymes comprised therein are encoded by exogenous nucleic acids, i.e. nucleic acids that have been introduced 'artificially' into a host cell, most preferably by using genetic engineering methods.

The term "aromatic amino acid" designates an amino acid that includes an aromatic ring. Among the twenty standard amino acids, four of them consist of aromatic amino acids: phenylalanine, tryptophan, histidine and tyrosine.

The term "dehydroshikimic acid" also called 3-DHS designates the compound of the systemic name (4S,5R)-4,5-D-dihydroxy-3-oxocyclohexene-l-carboxylic acid, whose complete formula is shown in figure 2. Recombinant yeasts with genetic modifications optimizing the synthesis of vanilloids

Different genetic modifications are proposed for optimizing the biosynthesis of vanilloids in recombinant yeasts comprising exogenous nucleic acids coding for polypeptides involved in the biosynthesis of vanilloids.

As previously reported, prevention of the reduction of vanillin into vanillyl alcohol can be achieved by the inactivation or the deletion of at least one gene coding for host alcohol dehydrogenases, in particular by the inactivation or the deletion of the gene adh6.

In the present invention, the recombinant yeast is devoid of a functional gene adh6.

The expression "a functional gene is absent" or "said yeast does not comprise a functional gene" or "said yeast is devoid of a functional gene" means that the said gene does not allow the production of the expected functional polypeptide, e.g. does not allow the production of the expected active enzyme. In some embodiments of a yeast cell that is devoid of a functional given gene, the said gene is not expressed in the yeast cell. e.g. the said gene is not transcribed or the corresponding transcription product is not translated into a protein, e.g. into an enzyme, having its usual activity. In some embodiment, the said gene may be 'inactivated'. The man skilled in the art knows various means to obtain such absence of functionality/inactivation of a gene, such as:

- introduction of a mutation into the gene, in particular generation of a stop codon inducing the expression of a non-functional, truncated protein: - introduction of an 'insert' into the gene, inactivating its correct transcription; e.g. interruption of the gene sequence by introduction of one or more exogenous nucleic acids, which encompasses introduction of a set of exogenous nucleic acids,

- replacement of the natural promoter of the gene by a non-functional promoter, or complete suppression of the promoter.

- deletion, complete or partial of the coding sequence of the gene.

In some embodiments, the introduction of a mutation into the gene may be achieved by mutagenesis, which includes random and directed mutagenesis.

In some preferred embodiments, "absence of a functional gene" refers to an endogenous gene having been inactivated by introduction of a D A insert, or refers to a gene whose coding sequence has been partially of completely deleted.

Phenylacrylic acids include cinnamic acid, coumaric acid and ferulic acid, all of them present in numerous plants. These carboxylic acids are intermediates in the pathway of catabolism of aromatic amino acids. Mukai et al. have shown that genes encoding PAD] and FDC1 are essential for the decarboxylation of phenylacrylic acids in S. cerevisiae (Mukai N, Masaki K, Fujii T, Kawamukai M. Iefuji H. "PADl and FDC1 are essential for the decarboxylation of phenylacrylic acids in Saccharomyces cerevisiae. " J Biosci Bioeng. 2010 Jun;109(6):564-9).

The expression "reduced ability to decarboxylatc phenylacrylic acids" means, for the purpose of the present description, that the intermediates in the vanilloid biosynthesis pathway, such as cinnamic acid, coumaric acid, and ferulic acid, are less converted by decarboxylases than in a wild-type corresponding yeast, and therefore that the availability of these compounds for the biosynthesis pathway of vanilloid is increased.

In particular, the intermediates in the vanilloid pathway may comprise or consist of cinnamic acid, and/or p-coumaric acid, and/or ferulic acid.

According to some embodiments, the yeast cell is genetically modified so as to possess a low capacity of dccarboxylating phenylacrylic acids, because of:

- the absence of at least one functional gene coding for a Phenylacrylic acid decarboxylase (PAD) and/or a Ferulic acid Decarboxylase (FDC); and/or

- the decreased or null enzymatic activity of the encoded proteins PAD and/or FDC; and/or - the decreased expression of at least one nucleic acid coding for said PAD and/or said PDC.

According to one preferred embodiment of the invention, the yeast cell is genetically modified so as to possess a low capacity of decarboxylating phenylacrylic acids, because of the absence of at least one functional gene coding for a Phenylacrylic acid decarboxylase (PAD) and/or a Ferulic acid Decarboxylase (FDC), and most preferably, coding for the decarboxylases Padl p (Phenylacrylic acid decarboxylase) and/or Fdcl p (Ferulic acid Decarboxylase). Respective contribution of the Pad l and Fdclp proteins to the decarboxylation of phenylacrylic acids is disclosed in the article of Mukai et al. (2010), previously cited.

According to one embodiment, a yeast having reduced ability to dccarboxylate phenylacrylic acids encompasses a recombinant yeast that does not comprise a functional gene coding for the decarboxylase Pad l p.

According to one embodiment, a yeast having reduced ability to dccarboxylate phenylacrylic acids encompasses a recombinant yeast that does not comprise a functional gene coding for the decarboxylase Fdcl p.

According to one embodiment, a yeast having reduced ability to dccarboxylate phenylacrylic acids encompasses a recombinant yeast that does not comprise a functional gene coding for the decarboxylase Padl p and a functional gene coding for the decarboxylase Fdcl p.

In a specific embodiment of the invention, the genetically modified yeast is further modified to decrease the consumption of aromatic amino acids in other biosynthesis pathways than the vanilloid biosynthesis pathway. In particular, the expression and/or activity of enzymes using aromatic amino acids as substrates could be decreased, except for the enzymes phenylalanine ammonia lyase and tyrosine ammonia lyase.

Advantageously, the genetically modified yeast does not comprise a functional gene arolO. ArolO gene encodes a phenylpyruvate decarboxylase that is involved in the consumption of phenylalanine and tyrosine (Vuralhan Z. Morais MA, Tai SL, Piper MD. Prank XT. « Identification and characterization of phenylpyruvate decarboxylase genes in Saccharomyces cerevisiae » Appl Environ Microbiol. 2003 Aug: 69(8):4534-41). The disruption of arolO causes an improved availability of phenylalanine for the biosynthesis pathway towards vanilloids.

In vanillin pathway, many intermediate precursors are aldehydes, which are known to be substrates of many endogenous enzymes leading to their corresponding alcohol or acid derivative. In recombinant yeasts according to the invention, aldehydes tend to be oxidized immediately after the reduction chain reaction, and as a consequence no vanillin is detected. In order to lower the conversion of aldehydes into their corresponding acid derivatives, a carboxylic acid reductase protein may be added, that is able to convert aromatic carboxylic acid into corresponding aldehyde. The CAR enzyme requires activation by phosphopantetheinylation (this post-translation modification is done by PPTase).

In a preferred embodiment of the invention, the genetically modified yeast does not comprise a functional gene ald6, coding for an aldehyde dehydrogenase. Its disruption causes an improved availability of aldehyde intermediates in the biosynthesis pathway towards vanilloids.

Advantageously, the genetically modified yeast is further devoid of a functional gene pdcl. Pdcl gene encodes a pyruvate decarboxylase, converting pyruvate into acetaldehyde. Pyruvate is a key metabolite in S. cerevisiae metabolism and the branch point between respiratory and fermentation metabolism. It has been shown that deletion of pdcl results in a reduction of 30% of total pyruvate decarboxylase activity; leading to an increased production of vanillin (Brochado et al., 2010, previously cited).

In a specific embodiment, a recombinant yeast comprising a set of exogenous nucleic acids encoding a set of enzymes allowing conversion of an aromatic amino acid into a vanilloid. presents one of the following phenotypes:

Aahd6Afdcl, Aahd6Afdc lAaro l 0. Aahd6AfdclAald6, Aahd6Afdcl Apdcl, Aahd6Afdcl Aaro 10Aald6, Aahd6Afdcl Aarol OApdcl , Aahd6Afdcl Aald6Apdcl , Aahd6Afdc 1 Aaro 1 ()Aald6Apdc 1 . Aahd6Apad 1 . Aahd6Apad 1 Aaro 10, Aahd6Apadl Aald6, Aahd6Apad 1 Apdc l , Aahd6Apad 1 Aaro 10Aald6, Aahd6Apad 1 Aaro 1 OApdcl ,

Aahd6Apad 1 Aald6Apdc 1 , Aahd6Apad 1 Aaro 10Aald6Apdc 1 , Aahd6Afdc 1 Apad 1 , Aahd6Afdc 1 Apad 1 Aaro 10. Aahd6Afdcl Apad l Aald6, Aahd6Afdc l Apad 1 Apdcl , Aahd6Afdc 1 Apad 1 Aaro 10Aald6. Aahd6Afdc 1 Apad 1 Aaro 1 OApdc 1 , Aahd6AfdclApadlAal d6Apdcl , Aahd6Afdc 1 Apad 1 Aaro 1 0Aald6Apdc 1 or any other combination of these deletions.

In another specific embodiment, a recombinant yeast comprising a set of exogenous nucleic acids encoding a set of enzymes allowing conversion of an dehydroshikimic acid into a vanilloid, presents one of the following phenotypes:

Aahd6Afdcl , Aahd6AfdclAarol0, Aahd6AfdclAald6, Aahd6AfdclApdcl , Aahd6Afdcl Aarol 0Aald6, Aahd6Afdc 1 Aaro 1 OApdcl , Aahd6Afdcl Aald6Apdcl , Aahd6Afdc 1 Aaro 1 ()Aald6Apdc 1 . Aahd6Apadl , Aalid6Apad 1 Aaro 10, Aahd6Apad l Aald6, Aahd6Apad 1 Apdc 1 , Aahd6Apad 1 Aaro 1 ()Aald6 , Aahd6Apad 1 Aaro 1 OApdc 1 , Aahd6Apad 1 Aald6Apdc 1 , Aahd6Apad 1 Aaro 10Aald6Apdc 1 , Aahd6AfdclApadl , Aalid6Afdc 1 Apad 1 Aaro 10. Aahd6Afdcl Apad l Aald6. Aahd6Afdcl ApadlApdcl , Aahd6Afdc 1 Apad 1 Aaro 10Aald6, Aahd6Afdc 1 Apad 1 Aaro 1 OApdc 1 , Aahd6Afdc 1 Apad 1 Aal d6Apdc 1 . Aahd6Afdc 1 Apad 1 Aaro 10Aald6Apdcl or any other combination of these deletions.

Enzymes involved in the biosynthesis pathway of vanilloids from aromatic amino acids

In the present description, enzymes are identified by their specific activities. This definition thus includes all polypeptides that have the defined specific activity also present in other organisms. Enzymes with similar activities can be identified by homology. Using the references given in GenBank for known genes, those skilled in the art are able to determine the equivalent genes in other organisms, bacterial strains, yeasts, plants, etc. This routine work is advantageously done using consensus sequences that can be determined by carrying out sequence alignments with genes derived from other microorganisms, and designing degenerate probes to clone the corresponding gene in another organism. These methods of molecular biology are well known to those skilled in the art, and are described, for example, in Green & Sambrook (Molecular Cloning: a Laboratory Manual. 4 ^th ed. 2012 Cold Spring Harbor Lab., Cold Spring Harbor, New York).

According to a first embodiment of the invention, the artificial vanillin pathway uses endogenous aromatic amino acids (AAA) as primary substrates. Endogenously produced phenylalanine or tyrosine is diverted to the synthesis of vanillin. Exogenous phenylalanine and/or tyrosine can also be added into the culture medium of the yeast. Six enzymes are required for the conversion of L-ph enyl al an i ne/t yrosi n e to vanillin (see figures 1 A and IB).

In particular, phenylalanine is converted to coumarate by the successive actions of the enzymes phenylalanine ammonia lyase (PAL) and cinnamatc-4-hydroxylase (C.41 I). The following step is the reduction chain reaction of the coumaric acid leading to the 4- hydroxybenzaldehyde. The reaction is initiated by the activation of coumaric acid to coumaroyl-CoA provided by 4CL enzyme (4-coumarate coenzyme A ligase), followed by a β-oxidation performed by ECH enzyme (enoyl-CoA hydratase/aldolase, an enzyme of a crotonase family). The next step is the hydroxylation on 3-position of the phenyl ring carried out by HBH enzyme (hydroxybenzoic acid hydroxylase, 3-monooxygenase enzyme family). The final step is the 3-O-methylation leading to vanillin final product. This step is catalyzed by the COMT enzyme (caffeic acid O-methyltransferase, O-methyltransferase enzyme family). In order to lower endogenous conversion of aldehyde intermediate product, carboxylic acid reductase (CAR) protein is preferentially added. The CAR enzyme requires activation by phosphopantetheinylation. and this is achieved by co-expression of a phosphopantetheinyl transferase (PPTase).

According to a specific embodiment of the invention, the aromatic acid is phenylalanine, and the recombinant yeast comprises a set of exogenous nucleic acids coding for the following enzymes: phenylalanine ammonia lyase (PAL), cinnamate-4- hydrolase (C4H), 4-coumarate coenzyme A ligase (4CL), enoyl-c A hydratase/aldolase (ECH). a hydroxybenzoic acid hydroxylase (HBH) and a caffeic acid O-methyltransferase (COMT).

According to another specific embodiment of the invention, the aromatic amino acid is tyrosine, and the genetically modified yeast comprises a set of exogenous nucleic acids coding for the following enzymes: tyrosine ammonia lyase (TAL), cinnamate-4-hydrolase (C4H), 4-coumarate coenzyme A ligase (4CL), enoyl-CoA hydratase/al dol ase (ECH), a hydroxybenzoic acid hydroxylase (HBH). and a caffeic acid O-methyltransferase (COMT).

The phenylalanine ammonia lyase (PAL) is an enzyme catalyzing the tyrosine deamination reaction classified EC 4.3.1.23. The tyrosine ammonia lyase (TAL) is an enzyme catalyzing the phenylalanine deamination reaction classified EC 4.3.1 .24. Another enzyme is known for catalyzing the phenylalanine or tyrosine deamination reaction, classified EC 4.3.1.25. and called PAL/TAL. In a specific embodiment, the PAL enzyme is issued from Petunia species or Populus species. In another specific embodiment, a PAL/TAL enzyme from maize (Rosier, Krekcl, Amrhcin, Schmid. "Maize phenylalanine ammonia-lyase has tyrosine ammonia-lyase activity" Plant Physiol. 1997 January; 1 13(1): 175-179) is introduced into the recombinant yeast. This enzyme is able to convert phenylalanine into einnamic acid and tyrosine into coumaric acid.

The einnamic acid hydroxylase (C4H) is an enzyme catalyzing the cinnamate 4-hydroxylation, classified EC 1.14.13.1 1. It is a P450-dependent enzyme. In a specific embodiment, the C4H enzyme is issued from Petunia species or Glycin species.

The 4-coumarate coenzyme A ligase (4CL) is an enzyme catalyzing the CoA esterification of coumaric acid, classified EC 6.2.1.12. This enzyme belongs to the family of ligases, specifically those forming carbon-sulfur bounds as acid-thiol ligases. In a specific embodiment, the 4CL enzyme is issued from Populus species.

The enoyl-CoA hydratase/aldolase (ECH) is an enzyme catalyzing the hydratation of 2-trans-enoyl-CoA into 3-hydroxyacyl-CoA. belonging to the family of crotonases. and is classified EC 4.2.1.17. In a specific embodiment, the ECH enzyme is issued from Pseudomonas fluorescens or Azotobacter vinelandii.

The hydroxybenzoic acid hydrolase (HBH) is an enzyme catalyzing the hydroxylation of 4-h yd ro yb enzal deh yde. The HBH enzyme is also known as 4- hydroxybenzoate-3-monooxygenase (EC 1 .14.13.2). In a specific embodiment, the HBH enzyme is issued from Pseudomonas fluorescens or Azotobacter vinelandii.

The caffeic acid O-methyltransferase (COMT) is an enzyme catalyzing the chemical conversion of ,4-dihydroxy-trans-cinnamate (caffeate) to 3-methoxy-4-hydroxy-trans- cinnamate (ferulate). This enzyme is also capable of converting protocacheuic aldehyde to vanillin. This enzyme is classified EC 2.1.1.68. Eventually, the same reaction can be catalyzed by the caff eoyl -CoA O-methyl transferase belonging to EC 2.1 .1.104.

Preferentially, the genetically modified yeast further comprises exogenous nucleic acid coding for a carboxyreductase (CAR, EC 1 .2.99.6) and a phosphopantetheinyl transferase (PPTase). In a specific embodiment, the CAR enzyme is issued from the organism Nocardia iowensis or Micobacterium smegmatis and the PPTase is issued from Nocardia iowensis or Micobacterium smegmatis. According to a specific embodiment of the invention, and as described in example 1, the artificial vanillin pathway is introduced into the locus bud 1 of modified yeast cells. Nine synthetic DNA fragments are needed to introduce artificial vanillin pathway in yeast genome (Figure 4). All genes possess one promoter and one terminator. Gene promoters allow constitutive expression of proteins from the pathway. Fragment hybridizes and recombincs together in the region of the entire ORF of each couple of gene. By that way, the whole pathway is assembled in the yeast cell, and then integrated into the chromosome. Tg 5' and Tg 3' correspond to the target sequences on the yeast genome that corresponds to the insertion site in the BUD 31 locus of the yeast chromosome triggering the homologous integration into the desired chromosome site. HIS3 and LEU2 arc the Hanking markers enabling the double selection of the recombinant pathway. After assembly of the fragments by homologous recombination in yeast, a functional complete pathway of 30Kbp is reconstituted and the double selection permits the isolation of recombinants.

In a particular embodiment of the invention, the genetically modified yeast further comprises exogenous nucleic acids coding for a vanillyl alcohol oxidase (EC 1.1 .3.38) that catalyzes the chemical conversion of vanillyl alcohol and 0 ₂ into vanillin and Η ₂ 0 ₂ . Presence of this enzyme allows a better yield of vanillin since all vanillyl alcohol produced via the artificial biosynthetic pathway can be converted into vanillin. A specifically preferred vanillyl alcohol oxidase is the enzyme described in patent US 5,721,125.

Enzymes involved in the biosynthesis pathway of vanittoids from dehydroshikimic acid

According to a second embodiment of the invention, the artificial vanillin pathway derives from the acid dehydroshikimic pathway, such as disclosed in Hansen et al, 2009 (cited above). In particular, the genetically modified yeast comprises a set of exogenous nucleic acids coding for the following enzymes able to convert an acid dehydroshikimic into a vanilloid: a 3-dchydroshikimate dehydratase (3DSD), an aromatic carboxylic acid reductase (AGAR) and a catechol O-methyltransferase or a caffeic acid O- methyltransferase (both designed as COMT). Preferentially, the genetically modified yeast further comprises a phosphopantetheinyl transferase (PPTase). In a specific aspect of the invention, and for all disclosed modified yeast, the exogenous nucleic acid encoding the caffeic acid O-methyltransferase (COMT) is preferentially issued from the plants Medicago sativa, Vanilla planifolia or Rosa chinensis. Interestingly, these enzymes have a high selectivity for mcthylation. and their final product is pure vanillin (100% of selectivity) instead of a mixture of vanillin and isovanillin.

Recombinant yeasts

In the sense of the present invention, it is understood that exogenous nucleic acids encoding polypeptides that are introduced into the yeast: are "codon-optimized" to be expressed efficiently into the yeast. The man skilled in the art knows some 'Codon Optimization Tools ^' that allows the preparation of synthetic genes from one host organism for an optimized expression in another, in particular in the yeast according to the invention.

Exogenous nucleic acids encoding the heterologous polypeptides are introduced into the yeast by transformation and this manipulation results into incorporation and expression of exogenous genetic material. Transformation of yeast can be performed by means well known by the man skilled in the art : yeast cells may be treated with enzymes to degrade their cell walls, may be exposed to alkali cations such as lithium acetate, or to polyethylene glycol. Electroporation using electric shock is also a technique that allows exogenous DNA to enter into yeasts. Some of useful techniques are reviewed in the review (Kawai S, Hashimoto W, Murata K. "Transformation of Saccharomyces cerevisiae and other fungi: methods and possible underlying mechanism. " Bioeng Bugs. 2010 Nov-Dec: 1 (6):395-403).

In a preferred embodiment of the invention, transformations of competent yeast cells are performed as described by Gietz and Woods (Gietz and Woods. "Transformation of yeast by the LiAc/ss Carrier DNA/PEG method. " Meth. Enzymol., 350, 87-96).

An equimolar mix of fragments is used to transform yeast.

According to a specific aspect of the invention, the polypeptides are encoded by exogenous genes stably integrated into the yeast genome. Preferentially, the nucleic acids encoding heterologous polypeptides are integrated into the yeast genome by the technique of homologous recombination. An example of integration into genome of the yeast is shown in figure 4. Preferably, in a set of exogenous nucleic acids as described herein, the exogenous nucleic acids are assembled into a cluster and said cluster is integrated into the yeast genome. In particular, polynucleotides encoding a series of enzymes are provided as full-length polynucleotides of different origin, wherein :

- the 5 '-terminal sequence is of the polynucleotide encoding the first enzyme in the series, and

- the 3 ^' -terminal sequence is of the polynucleotide encoding the last enzyme in the series.

Most preferably, each nucleic acid encoding a polypeptide included in an exogenous nucleic acid as described herein comprises, from its 5' -end to its 3 '-end, (1) a regulatory sequence comprising one or more promoter sequences, the said regulatory sequence being functional in the yeast organism wherein it has been introduced, (ii) an ORF encoding a polypeptide of interest and (iii) one or more transcription terminator sequences.

The polynucleotides encoding the enzymes may be in the order of the consecutive enzymatic reactions required for the biosynthesis pathway which is under consideration, or in any other order, provided that the required enzymes are actually produced.

The invention also relates to a recombinant yeast having a reduced alcohol dehydrogenase activity, in the genome of which has been inserted a D A construct comprising :

- one or more nucleic acids encoding a phenylalanine ammonia lyase (PAL) or a tyrosine ammonia lyase (TAL),

- one or more nucleic acids encoding a cinnamate-4-hydrolase (C4H).

- one or more nucleic acids encoding a 4-coumarate coenzyme A ligase (4CL),

- one or more nucleic acids encoding a enoyl-CoA hydratase/aldolase (ECU),

- one or more nucleic acids encoding a hydroxybenzoic acid hydroxylase

(HBH) and - one or more nucleic acids encoding a caffeic acid O-methyltransferase

(COMT),

and wherein in said recombinant yeast the decarboxylation of phenylacrylic acids is attenuated, in particular said yeast does not comprise a functional gene fdcl and/or padl.

The invention also relates to a recombinant yeast such as described above, in the genome of which has been inserted a DNA construct comprising:

- one or more exogenous nucleic acids encoding a carboxyreductase (CAR) and

- one or more exogenous nucleic acids encoding a heterologous phosphopantetheinyl transferase (PPTase).

The invention also relates to a recombinant yeast having a reduced alcohol dehydrogenase activity, in the genome of which has been inserted a DNA construct comprising:

- one or more nucleic acids encoding a 3 -dehydroshikimate dehydratase

(3DSD),

- one or more nucleic acids encoding an aromatic carboxylic acid reductase

(AGAR)

- one or more nucleic acids encoding a phosphopantetheinyl transferase (PPTase), and

- one or more nucleic acids encoding a caffeic acid O-methyltransferase

(COMT),

and wherein said recombinant yeast does not comprise a functional gene ald6. According to the invention, the yeast is from the genera Saccharomyces or Schizosaccharomyces. In particular the modified yeast belongs to the following species:

Saccharomyces cerevisiae Schizosaccharomyces pombe, Pichia pastoris, Yarrowia lipolytica.

Culture process for producing a vanilloid or a precursor thereof The present invention is also related to a method for producing a vanilloid or a precursor thereof, comprising the following successive steps: a) providing a recombinant yeast comprising enzymes necessary for vanilloid biosynthesis,

b) cultivating said yeast in an appropriate medium comprisin a source of carbon chosen from glucose, fructose, galactose, arabinose, lactose, ethanol, glycerol, and mixtures thereof; and

c) isolating said vanilloid or precursor thereof from the medium.

The phrase "recombinant yeast comprising enzymes necessary for vanilloid biosynthesis" designates a genetically modified yeast, expressing enzymes allowing the biosynthesis of a vanilloid in said yeast by any biosynthesis pathway.

In particular, exogenous nucleic acids are inserted into said yeast, said nucleic acids coding for enzymes involved in the biosynthesis pathway of a vanilloid, being chosen among the following groups of enzymes:

- enzymes able to convert an aromatic amino acid into a vanilloid; or

- enzymes able to convert an acid dehydroshikimic into a vanilloid. In particular, said recombinant yeast does not comprise a functional gene adh6. More particularly, said yeast does not comprise a functional gene adh6 neither a functional gene fdcl. More particularly, said yeast does not comprise a functional gene adh6 neither a functional gene padl.

In a preferred aspect of the invention, said recombinant yeast does not comprise functional genes adli6, pdcl, fdcl .

In another preferred aspect of the invention, said recombinant yeast does not comprise functional genes adli6. padl. fdcl.

In another preferred aspect of the invention, said recombinant yeast does not comprise functional genes adh6, padl, pdcl.

In another preferred aspect of the invention, said recombinant yeast does not comprise functional genes adh6, fdcl, padl and arolO.

In a specific aspect of the invention, at least one of the genetically modified yeasts described in the present application is cultivated in this method.

Terms 'biosynthesis ^' , "bioconversion" 'fermentative production' and 'production' have the same meaning in the sense of the invention, and designates the production of at least one vanilloid by a recombinant yeast cultivated in appropriate conditions. The term "precursor" as used herein refers to a substrate molecule that is subject to enzymatic reactions and can be converted into a product by this specific reaction. By "a precursor of vanilloid" is meant a precursor or an intermediate in the biosynthesis pathway of a vanilloid, which is in particular a hydroxybcnzaldehyde or a respective acid or a respective alcohol, an aromatic amino acid, or an acid dehydroshikimic. This term includes in particular: protocatechuic aldehyde, 4- hydroxy aldehyde, protocacheuic alcohol, 4-hydroxybenzoic acid. 4-hydroxybenzyl alcohol, protocacheuic acid, protocacheuic alcohol, 4-hydroxyaldehyde, 4-hydroxyl alcohol, cinnamic acid, coumaric acid, eaffeic acid and ferulic acid.

According to the invention the term 'cultivating' is used to denote the growth of yeast. The term "appropriate medium" designates a medium (e.g., a sterile, liquid media) comprising nutrients essential or beneficial to the maintenance and/or growth of the cell such as carbon sources or carbon substrates, nitrogen sources, urea, ammonium sulfate, ammonium chloride, ammonium nitrate and ammonium phosphate; phosphorus sources; metal salts, for example magnesium salts, cobalt salts and/or manganese salts; as well as growth factors such as amino acids and vitamins. Typical medium is composed of 10 g/1 of yeast extract, 20 g 1 of peptone and 20 g/1 of carbon source.

The term "source of carbon" or "carbon substrate" according to the present invention denotes any source of carbon that can be used by those skilled in the art to support the normal growth of a yeast. Yeasts use sugars as their main carbon and energy source, but non-conventional carbon sources can be accepted too. The major source for energy production in the yeast is glucose and glycolysis is the general pathway for conversion of glucose to pyruvate, whereby production of energy in form of ATP is coupled to the generation of intermediates and reducing power in form of NADH for biosynthetic pathways. Fructose is also a hexose that can be uptake and metabolized by yeast. Galactose is a 'non-conventional' nutrient for yeast, which however can be used as a sole carbon source when glucose is absent from the medium. In yeast cells supplied with glucose, the GAL genes are repressed. They are activated a thousand fold in cells that are starved for glucose, and this one of the few pathways in yeast which is regulated in a nearly 'all-or-nothing' mode. Enzymes implied in conversion of galactose to glucose 1 -phosphate are: galactose kinase, galactose- 1 -phosphate-urydiltransf erase, and UDP-glucose-4- epimerase. Glycerol functions as a compatible solute in osmoregulation in osmotolerant yeasts that are capable of growing in high sugar or salt environments. Many types of yeast can grow on glycerol as a sole carbon source under aerobic conditions, but glycerol is a non-fermentable carbon source for many yeast species, including S. cerevisiae. To serve as a carbon source, glycerol after internalization has to convert by glycerol kinase to glycerol-3-phosphate, which is then transformed into dihydroxyacetone phosphate by gl ycerol-3 -phosphate dehydrogenase that is a substrate in gluconeogenesis.

Many types of yeast have the capability of metabolizing ethanol or methanol. In presence of ethanol ADH2, the enzyme that converts ethanol back into acctaldehyde, is expressed. Acctaldehyde is subsequently converted into acetyl-CoA, the substrate for the citric acid cycle.

The source of carbon is selected among the group consisting of glucose, fructose, mannose, xylose, arabinose, galactose, lactose, cthanol, cellobiose. glycerol and polysaccharides such as cellulose, and mixtures thereof. An especially preferred substrate is ethanol.

According to another preferred embodiment of the invention, the volume of the culture is at least one liter. Indeed, when the culture volume is low (about 1 ml), vanillin is produced but is quickly converted to vanillic acid. This conversion is not observed in stable continuous culture in larger volume, of at least one liter.

The cultivation process comprises a fermentation process (batch, fed-batch or continuous mode) under controlled conditions of pH. temperature. p0 ₂ and aeration, conditions well known to a person skilled in the art.

In a specific embodiment, the vanilloid isolated from the medium is selected from the group consisting of: vanillin, vanillic acid, vanillyl alcohol and vanillin- glycoside.

In another specific embodiment, the vanilloid precursor isolated from the medium is selected from the group consisting of protoeatechuic aldehyde, protocacheuic acid, protocacheuic alcohol. 4-hydrox yal dehyd e, 4-hydrobenzoic acid. 4-hydroxyl alcohol, cinnamic acid, coumaric acid, caffeic acid and ferulic acid.

According to a specific embodiment, the present invention is related to a method for producing a vanilloid comprising the following successive steps:

a) providing a recombinant yeast comprising enzymes necessar for vanilloid biosynthesis, b) cultivating said yeast in an appropriate medium comprising a source of carbon chosen from glucose, fructose, galactose, arabinose, lactose, ethanol, glycerol, and mixtures thereof;

c) eventually, isolating said vanilloid from the medium;

and further comprising a step d:

d) converting precursors of vanilloid remaining in the medium into vanilloid.

Culture process using bi-phase culture

Vanillin cytotoxicity is a bottleneck in the microbial bioconversion including unicellular microorganisms such as yeasts. We proceeded to an extractive bioconversion of vanillin using polypropylene glycol. In the two-phase process, the bioconversion is performed in a medium comprising an aqueous phase containing the biocatalyst (cells) and hydrophobic organic solvent (water-immiscible phase) containing organic compounds as substrates and / or products. The organic solvent acts as a reservoir for hydrophobic compounds. The biphasic system enables continuous supply of substrate depending on the metabolic demand and without inhibiting the enzymatic reaction. Moreover, the product of the reaction is progressively extracted into the organic phase. A major advantage is to reduce the cytotoxicity of substrates and/or products of the bioconversion. This system allows raising yields of bioconversion.

PPG is a polymer of propylene glycol also called propane- 1 ,2-diol or 1 ,2- propanediol. When aqueous solutions (culture medium) and PPG are mixed, the mixture becomes turbid and when it is left for a while, phase separation occurs. Aqueous two- phase system therefore provides mild conditions for cells and proteins. The interfacial tension is very low compared with that of organic solvent /water system. The phase system forms a finely dispersed emulsion with large surface area and short diffusion distances from one phase to the other. This means that conditions are very favorable for mass transfer.

This method is particularly convenient for improving the production of vanilloids or precursors thereof in such unicellular microorganisms.

The phrases "tw o-phase system", or "bi-phase medium", or ''bi-phase culture", or "bi-phasic system" are considered herein to have the same meaning.

Thus, the present invention is also related to a method for producing a vanilloid or precursor thereof in a unicellular microorganism able to produce said vanilloid or precursor thereof, comprising a step of cultivating said microorganism in an appropriate medium comprising an aqueous phase and a water immiscible phase, said water immiscible phase preferably comprising or consisting of a polymer of propylene glycol, a fatty alcohol, a fatty acid, a fatty ester or an oil. and mixtures thereof.

For example, a fatty acid may be selected from a group comprising or consisting of octanoic acid and oleic acid.

For example, a fatty alcohol may be selected from a group comprising or consisting of n-octanol, lauryl alcohol, and oleyl alcohol.

The phrase "unicellular microorganism" designates any unicellular microorganism, including bacteria, archaea, protozoa, unicellular algae and unicellular fungi, that is either prokaryotic or cukaryotic; which also includes wild-type, natural ly- occuring, non-naturally-occuring, genetically modified or recombinant microorganisms such as recombinant yeasts.

A "genetically modified microorganism" includes microorganisms obtained or obtainable by random or directed mutagenesis.

Examples of unicellular microorganisms include strains belonging to the order of Actinomycetales, which includes a suborder selected from the group consisting of Actinomycineae, Actinopolysporineae, Catenulisporineae, Corynebacterineae, Frankineae, Glycomycineae, Kineosporiineae, Micrococcineae, Micromonosporineae, Propionibacterineae, Pseudonocardineae, Streptomycineae and Streptosporanginea; which includes recombinant strains from the genus Amycolatopsis or Streptomyces; which includes strains Amycolatopsis sp. ATCC 39116, HR167 and DSMZ 9992.

Still, and in a non-limitative manner, other examples of unicellular microorganisms include yeasts, lactic acid bacteria and Escherichia Coli strains.

Some of the ab ve-m ent i on ed unicellular microorganisms are naturally able to produce a vanilloid or precursor thereof, while others require to be genetically modified and/or cultivated in a manner that is suitable for expressing enzymes allowing the biosynthesis of said vanilloid or precursor.

The phrase "unicellular microorganism able to produce a vanilloid or precursor thereof designates, in particular, unicellular microorganisms expressing enzymes allowing the biosynthesis of a vanilloid or precursor thereof by any biosynthesis pathway.

According to on preferred embodiment, the unicellular microorganism is a recombinant yeast comprising enzymes necessary for vanilloid biosynthesis.

According to another preferred embodiment, the unicellular microorganism is a strain belonging to the order of Actinomycetales.

Strains belonging to the order of Actinomycetales which are specifically considered are strains wherein at least one gene encoding an enzyme having vanillin reductase activity is non-functional.

The phrase "recombinant yeast comprising enzymes necessary for vanilloid biosynthesis" designates a genetically modified yeast, expressing enzymes allowing the biosynthesis of a vanilloid in said yeast by any biosynthesis pathway.

In particular, exogenous nucleic acids are inserted into said microorganism, including said yeast, said nucleic acids coding for enzymes involved in the biosynthesis pathway of a vani lloid. being chosen among the following groups of enzymes:

- enzymes able to convert an aromatic amino acid into a vanilloid: or

- enzymes able to convert an acid dehydroshikimic into a vanilloid.

In particular, said recombinant yeast does not comprise a functional gene adh6. More particularly, said yeast does not comprise a functional gene adh6 neither a functional gene (del. More particularly, said yeast does not comprise a functional gene adh6 neither a functional gene (del . More particularly, said yeast does not comprise a functional gene adh6 neither functional genes fdcl nor pad!.

In another preferred aspect of the invention, said recombinant yeast does not comprise functional genes adh6, pad}, pdcl.

In another preferred aspect of the invention, said recombinant yeast does not comprise functional genes adh6, fdcl, pad 1 and arolO.

In a specific aspect of the invention, the genetically modified yeasts described in the present application are cultivated according to this method, in a bi-phase medium, wherein said medium comprises two phases, an aqueous phase and a water immiscible phase, said water immiscible phase preferably consisting of: polymer of propylene glycol, fatty alcohol, fatty acid, fatty ester or an oil. Thus, the present invention is also related to a method for producing a vanilloid or a precursor thereof, and/or as defined above, comprising the following steps:

a) providing a recombinant yeast comprising enzymes necessary for vanilloid biosynthesis,

b) cultivating said yeast in an appropriate medium comprising a precursor chosen from glucose, fructose, galactose, arabinose, lactose, ethanol, glycerol, and mixtures thereof; and

c) isolating said vanilloid or precursor thereof from the medium,

wherein the step b) is performed in an appropriate medium comprising two phases, an aqueous phase and a water immiscible phase, said water immiscible phase preferably comprising or consisting of polymer of propylene glycol, fatty alcohol, fatty acid, fatty ester or an oil.

According to some embodiments, the vanilloid or precursor thereof in step c) is isolated from the aqueous phase only, from the water immiscible phase only, or from a mixture thereof.

The appropriate medium comprises precursors of the step b) or mixtures of thereof, either in pure grade or technical grade.

The cultivation process comprises a fermentation process (batch, fed-batch or continuous mode) under controlled conditions of pH, temperature. p0 ₂ and aeration, conditions well known to a person skilled in the art.

According to one exemplary embodiment, said water immiscible phase comprises or consists of polymers of polypropylene glycol (PPG). A polymer of polypropylene glycol in the sense of the invention may include polymers of PPG having an average molecular weight ranging for instance from about 200 to about 4000 g/mol, and mixtures thereof: which includes 250; 425; 725: 1000; 1200: 2000; and 2700 g/mol.

In a non-limitative manner, such polymers of PPG s are commercially available under the DOW-P reference (The Dow Chemical Company©), and/or commercialized by Sigma Aldrich©.

One example of such polymer of PPG is PPG 1200. Still, in a non-limitative manner, the water-immiscible phase is obtained by mixing a solution comprising a hydrophobic organic solvent, such as a polymer of polypropylene glycol, with an appropriate medium in a manner suitable for phase separation to occur; thereby forming in said appropriate medium an aqueous phase and a water immiscible phase.

In a non-limitative manner, a precursor can be added to the appropriate medium either before the occurence of phase separation, during phase separation or after separation. In view of the above, and still in a non-limitative manner, the precursor can be added before mixing or after mixing the solution comprising said hydrophobic organic solvent.

According to said embodiment, the precursor is preferably chosen from: glucose, fructose, galactose, arabinose, lactose, ethanol, glycerol, and mixtures thereof.

The hydrophobic organic solvent, such as the polymer of polypropylene glycol, may be present in the solution, prior to mixing, in varying amounts ranging from 0.1 % to 90% in volume percent of the total volume of said solution; which includes 0.1%; 1 %; 5%; 10%; 20%; 30%; 40%; 50%; 60%; 70%; 80% and 90% in volume percent of the total volume of said solution.

According to one preferred embodiment, the present invention is related to a method for producing a vanilloid or a precursor thereof in said unicellular microorganism, including said recombinant yeast, comprising a step of cultivating said microorganism and/or yeast in an appropriate medium comprising at least two phases:

- an aqueous phase comprising a precursor chosen from glucose, fructose, galactose, arabinose, lactose, ethanol. glycerol, and mixtures thereof; and

- a water immiscible phase, comprising or consisting of a hydrophobic organic solvent such as a solvent selected from: a polymer of propylene glycol, a fatty alcohol, a fatty acid, a fatty ester or an oil. and mixtures thereof.

According to one exemplary embodiment, the present invention is related to a method for producing a vanilloid or a precursor thereof in said microorganism and/or yeast, comprising a step of cultivating said microorganism and/or yeast in an appropriate medium comprising at least two phases: - an aqueous phase comprising a precursor chosen from glucose, fructose, galactose, arabinose, lactose, ethanol, glycerol, and mixtures thereof; and

- a water immiscible phase, comprising or consisting of a polymer of propylene glycol, such as PPG 1200;

wherein said appropriate medium is optionally obtained by mixing a solution comprising or consisting of said polymer of polypropylene glycol, with an appropriate medium in a manner suitable for phase separation to occur; thereby forming said appropriate medium said aqueous phase and said water immiscible phase.

Composition comprising vaniUoid or a precursor thereof

The present invention is also related to a composition comprising a vanilloid or a precursor thereof obtainable by the method as disclosed above, and to the use of said composition as a flavoring in the human and animal nutrition field, in pharmacy, and as a fragrance in the cosmetics, perfumery and dctergency industries.

Vanillin is a compound well-known for its aromatic properties. However, the flavor profile of an aromatic composition is dependent on for instance byproducts and impurities, and consequently on the preparation process. The composition comprising a vanilloid or a precursor thereof obtainable by the method of the invention may thus show aromatic notes different from other aromatic composition.

Thus, the present invention also relates to a composition comprising a vanilloid obtainable or obtained by one of the methods as disclosed above, from said microorganism and or yeast, or from the culture supernatant thereof, which includes clarified culture supernatant.

A composition comprising said vanilloid, in the sense of the invention, may include pharmaceutical compositions, cosmetic compositions and/or compositions which are suitable for human and animal nutrition, including oral and topical administration.

According to some embodiments the composition is a culture supernatant or a clarified culture supernatant.

According to some embodiments, the composition comprising said vanilloid is obtainable or obtained by cultivating said microorganism and or yeast in an appropriate medium comprising an aqueous phase and a water immiscible phase as previously described, wherein said composition is collected from the aqueous phase and/or the water immiscible phase.

EXAMPLES

Example 1. Introduction of a vanillin biosynthesis pathway into Aadh6 yeast strain

All of the Saccharomyces cerevisiae strains used in this work were isogenic haploids from BY4741 and were obtained from EUROSCARF (haploid a-mater BYOO or a-mater BY 10). Yeast strain BY47 derived from a strain collection that contains knock outs of auxotrophic (-ura3, -leu2, -his3) marker genes. Enrichment and propagation of clones were made in YPD liquid cultures (10 g/1 bacto-yeast extract. 20 g/1 bacto-peptone and 2% dextrose) at 30°C. Recombinants were selected on dropout agar plates (YNB + CSM) in the absence of uracil, leucine or histidine. The gene defects in uracil, histidine and leucine biosynthetic pathway result in auxotrophy. All ORF used for the pathway were synthesized by GeneArt (Germany) and then amplified by PGR. Amplification was performed using high fidelity PhusionTaq (New England Biolabs). Amplicons were cleaned up by using the Wizard PGR Clean-up System (Promega) and used for transformation assays.

The following genes were used:

Table 1 : Reference identities of the genes/proteins used in this example

The complete pathway was assembled using a somatic in vivo DNA assembly. Eight fragments containing (Fl to F8) the 6 genes of the vanillin pathway were designed by computational analysis. Fragments hybridize and reeombine together in the region of the entire ORF of each couple of gene. That way enabled the whole pathway assembly in the yeast cell, and its integration into the chromosome. T 5' and T 3' correspond to the bud3 1 target sequences on the yeast genome allowing homologous integration onto the chromosome locus. Overlapping sequences correspond to the 5' part and the 3" part of the marker genes. HIS 6 and LEU 2 are the flanking markers enabling the double selection of the recombinant pathway. Each gene is under the control of one promoter and one terminator sequences allowing its expression in yeast cells. After assembly of the fragments by homologous recombination in yeast, a functional complete pathway is reconstituted and the double selection permits the isolation of recombinants.

Transformations of competent yeast cells were performed as described by Gietz and Woods (Transformation of yeast by the LiAc/ss Carrier DNA/PEG method. Meth. Enzymol., 350, 87-96). Analysis of clones revealed that the genes had assembled, resulting in correct ORFs.

HPLC was used to measure the production of vanillin and pathway intermediates in S. cerevisiae cultures. Analysis showed that chromatograms from cells expressing vanillin pathway genes contained additional peaks compared to a yeast control. These peaks were identified as c inn ami c acid, coumaric acid, 4- hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, vanillic acid and vanillin were identified. When the culture is not supplemented by exogenous phenylalanine, the PAL protein uses endogenous phenylalanine. The pathway is fully functional as 3,4- dihydroxybenzoi c acid and vanillic acid were detected but the yield is reduced 4 times when compared to the phenylalanine supplemented medium. Only 1 μΜ of vanillin was detected in the non-adapted strain.

A carboxylic acid reductase protein (CAR) was added with its activating coupling protein phosphopantetheinyi transferase (PPTase). The bicistronic construction was integrated to yeast genome by homologous recombination using URA as selection marker into ADH6 locus. Prevention of reduction of vanillin t vanillyl alcohol was achieved by knockout of the host alcohol dehydrogenase ADH6. In order to take off selection marker, flanking repeated sequences were added to URA3 gene in order to permit URA3 gene excision. Recombinant cells were selected on -LIRA selective medium and the right integration was verified by PCR.

Example 2-Improvcmcnt of aldehyde stability in S. cerevisiae

Vanillin biosynthesis pathway used in our production process implies three aldehydes including the final product vanillin. One way to increase vanillin production yield during a bioproduction using S. cerevisiae is to reduce aldehyde oxidase or aldehyde reductase endogenous activity in the production strain. A large number of Euroscarf deletion strains have been screened; these strains were firstly selected on the basis of a biochemical study and rational targeting of potentially interesting genes such as ADH (alcohol dehydrogenases), ALD (aldehyde dehydrogenases), GRE (aldehyde reductases), OYE, AAD (aryl alcohol dehydrogenase) & ARI (aldehyde reductase).

Table 2: Euroscarf deletion strains

Gene Systematic Euroscarf

Description Alias(es)

Name Name Acc N°

Alcohol

dehydrogenase alcohol dehydrogenase

ADH5 YBR145W Y03284 isoenzyme V ADH 5

ADH 7 NADPH-dependent YCR 105W ADH VII Y05821 medium chain

alcohol

dehydrogenase

SFA1 Bifunctional alcohol YDL 1 68W Y03866

ADH 5. bifunctional alcohol dehydrogenase dehydrogenase/S- and formaldehyde

(hydroxymethyl)glutathione

dehydrogenase dehydrogenase Putative aryl-alcohol

AAD 14 YNL33 1 C 10565 A dehydrogenase

NADPH-dependent YGL157W carbonyl reductase (NADPH- Y04524 aldehyde dependent) ARI1

ARI 1

reductase

A screening has been performed to evaluate stability of the three aldehydes of the vanilloid pathway with these 22 strains and results have been compared with the same approach with a wild-type S. cerevisiae strain. Principle of the assay is to grow yeasts for a first 24h in rich-medium in a 96- well microplates. Then cells are two fold diluted using the same quantity of medium and let grown 24h more in presence of 5()()μΜ of the aldehyde to be evaluated (4-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde and vanillin).

Figure 3 shows the titers obtained for three oxidative states (aldehyde, acid, alcohol) related to the feeding.

Looking at these results, the strongest impact is given by adh6 deletion. ALD6 deletion also has an impact on the quantity of acid formed (more acid than the wild type cells). ALD6 could work on the conversion of aldehyde to alcohol. The third strong effect concerns ADH3. Actually, alcohol concentration is higher in ,\adh3 cells than in the WT. ADH3 probably catalyzes the conversion of aldehyde into its acid form. A very small effect has been detected for Agre3 and Aald2. These genes were then assayed in combination with the most promising ones. Looking at all these results, the goal was then to combine gene deletions in the same cell. An expression of the van pathway in such a genetic context could favor vanillin synthesis.

Figures 3 show that the major effect is given by ADH6 deletion in WT cells, as expected. However a little effect is also detected when ADH3 deletion is added to that of ADH6. Indeed, alcohol concentration is a little lower in this strain.

Aald6 is another deletion that significantly disturbs the equilibrium between the three oxidation states of 4HB and 3.4diHB. Since it doesn't show a strong impact on vanillin, we did not try it during the first assay of multi-deletion. However, the low impact of the other double deletions convinced us to try a deletion of ald6 in combination with Aadh6. Example 3. Effect of metabolic engineering on vanillin pathway flux - Multideletion of padl, fdcl & arolO genes

The deletion of three genes coding for enzymes involved in the catabolism of phenylpropanoid compounds {[del, pad I & arolO) were tested. Each new gene deletion and combinations thereof were evaluated for the production of vanillin and intermediates of the vanillin pathway. Taking into account the already obtained results in the investigation of metabolic engineering, the tested gene deletions were: arolO. fdcl, padl, adh6, ald6, adh3.

ArolO deletion increases the intrinsic concentration of phenylalanine, which is the branching point of our pathway. Fdcl and padl deletions prevent the decarboxylation of intermediate metabolites, such as cinnamic and coumaric acids.

Gene deletion is performed using a CreLox deletion system. First, a deletion cassette is obtained by PGR on an appropriate plasmid (pUG72 or pUG73 from Euroscarf). The forward Primers used for amplification of the deletion K7 are designed to add upstream of the 5' side of the selection marker a track of 50 bp homologous to the last 50 bp of the promoter of the gene to be deleted. The reverse ones are designed to add downstream of the 3' side of the selection marker a track of 0 bp homologous to the first 50 bp of the terminator of the gene to be deleted. Once transformed by an appropriate deletion K7. the selection marker (Ura3 or Leu 2) is integrated in the yeast genome and it strictly replaces the ORF of the targeted gene. The selection marker is framed by two identical sequences (LoxP) that allow marker excision under the action of a Cre enzyme obtained by a new transformation of the new strain using a pSH62 plasmid (Euroscarf). His3 selection marker of the pSH62 is then removed by a little growth of the transformed cells in a rich medium. This deletion system has been used to successively delete genes and to retrieve selection markers for next transformations. Each new gene deletion is followed by evaluation of cinnamic and coumaric acids production capacities after a transformation of the cells by a ZmPAL expression cassette.

Several modified yeast strains were evaluated for vanillin production; results are shown in figure 5:

Y I O

Y \ ()Aadh 6ApadlAfdcl ; Y l 0Aarol 0Aadh6Afdcl ;

Yl 0Aarol0Aadh6AfdclAadh3;

Y\0Aarol0Aadh6AfdclAadh3Aald6\

Recombinant modified clones containing vanillin pathway were cultured in rich medium with galactose as carbon source using 100ml Erlenmeyer. After three days, clarified supernatants were analyzed by HPLC and concentration of metabolites form the pathway was evaluated. Total concentration of metabolites from vanillin pathway was evaluated.

Figure 5 shows the impact of deletions on the flow of the vanillin pathway.

The total metabolites concentration (grey bars) comprises cinnamic acid, cou marie acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 3 , 4 -d ihydroxybenzal dehyde, vanillin, vanillic acid and vanillyl alcohol concentrations. Amount of final products (vanillin, vanillyl alcohol and vanillic acid) are indicated with striped bars and vanillin concentration is indicated with black bars.

Ami (I deletion, by increasing the concentration of phenylalanine produced by the yeast, clearly increases the overall flow of the vanillin pathway. The total metabolite concentration is multiplied 4- fold. When the deletion of aid 6 is added, flow is also increased. In contrast, the adhS deletion decreases the flow of the pathway. Vanillin produced equilibrates into acid and alcohol oxidation states. The results show that am 10 deletion has little effect on the final concentration of metabolites (vanillin, vanillic acid & vanillyl alcohol). Besides increasing the flow of the pathway, ald6 deletion significantly increases the concentration of end metabolites.

Figure 6 shows the profile of supernatants of strains presenting the following deletions: Aadh6Aarol OAfdcl and Aadh6Aarol OAfdcl Apdcl . Clearly, the deletion of pad] is beneficial for the vanillin biosynthesis pathway in these strains.

Example 4. Optimization of culture conditions to increase vanillin production - Yeast volume culture

The synthesis of vanillin was evaluated based on the volume of culture for bioconversion. From a pre-culture. YPD medium was inoculated to an OD ₆₀ o of 0.1 . The cells were then distributed into the different kind of media support from 96-well plate to a 1 liter Erlenmeycr flask. The concentration of vanillin was found to be clearly dependent on the volume of the support. Culture in 96- well plate allows good growth of yeast, but very little metabolite pathway vanillin are produced. Using 24-well plates, vanillin is produced, but quickly converted to vanillic acid. In contrast, when the bioconversion takes place in 1 liter Erlenmeyer flask, the synthesis of vanillin is continuous and stable and reaches 250 μΜ after 8 days, and the the concentration drops down (Figure 7).

Example 5. Extractive bioconversion of vanillin using polypropylene glycol (PPG)

The toxicity of vanillin for yeast was evaluated (Figure 8). A yeast culture of

Aadh6iSpdcl padl strain expressing vanillin pathway was inoculated in YPDE to an OD ₆ oo of 0.05 and yeast growth was monitored in the presence of different concentrations of vanillin (0 to 30 mM). The same experiment was carried out by adding 30% PPG 1200. Figure 7 shows that the toxicity threshold is multiplied by a factor 10.

In a second step we evaluated the bioconversion by our pathway in the presence of PPG. To do this, from a preculture in YPGal medium, an optimized cell expressing the pathway of vanillin was inoculated into 50 ml YPAGal in a 250 ml Erlenmeyer containing 50% of PPG. A control was done without PPG. The concentration of each metabolite was monitored in the control YPGal alone. Typically clarified supernatant was removed and analyzed by HPLC. In the biphasic culture, two phases were studied. Phase containing the supernatant was analyzed. PPG phase was taken and diluted to a volume of acetonitrile to be analyzed by HPLC.

In control culture, total metabolite concentration reaches 710 μΜ whereas in biphasic culture concentration reaches 2,800 μΜ, 75% (2.100 μΜ) were located in PPG phase and 25% (700 μΜ) were located in aqueous phase. As to compare to control culture, bioconversion yield is clearly improved when PPG is present and vanillin concentration increased from 100 μΜ to 735 μΜ (Figure 9). All intermediate metabolites of the pathway are also extractable in PPG. but the emulsion is sufficiently high to allow the availability of substrates to the cell while reducing any toxicity. This leads to a signi icant increase in the flux of the pathway (4 fold increase). Vanillin concentration was significantly increased (8 fold increase). Example 6. Optimization of carbon source precursor for yeast growth on vanillin factory cell

Different carbon sources have been tested as substrates to produce vanillin in recombinant modified cells: sugars such as hexoses (glucose, fructose, galactose) as well as alcohol compounds with two carbons (ethanol) or three carbons (glycerol).

Enrichment and propagation of clones were classically made in YPD liquid cultures (10 g/1 bacto-yeast extract, 20 g/1 bacto-peptone and 2% dextrose) at 3 °C.

Several media were used: YPGal (10 g 1 bacto-yeast extract, 20 g/1 bacto- peptone and 3% galactose); YPF (10 g/1 bacto-yeast extract, 20 g/1 bacto-peptone and 2% fructose): YPDE (10 g/1 bacto-yeast extract, 20 g/1 bacto-pcptone, 2% dextrose and 2% ethanol); YPGE (10 g 1 bacto-yeast extract, 20 g/1 bacto-peptone. 2% glycerol and 2% ethanol): YPG (10 g/1 bacto-yeast extract, 20 g/1 bacto-peptone, 2% glycerol); YPE ( 10 g/1 bacto-yeast extract, 20 g/1 bacto-peptone and 2% ethanol).

Vanillin producing cells were precultured in YPD or YPGal medium in a 10 mL for 24h, and used to inoculate 5()mL appropriate medium in a 250 niL Erlenmeyer flask. Metabolites concentrations were monitored.

Figure 10 shows that when yeasts grow on glucose carbon source, vanillin concentration reaches 100 μΜ over 3 days, then concentration decreases in favor of vanillic acid. Results are similar when yeasts grow on galactose carbon source, but vanillin is more stable. Fructose carbon source gives concentration of vanillin smaller than both other sugars. However. YPDE and YPGE give same results, vanillin concentration grows. According to concentration of vanillin when yeasts grow on glyceraldehyde carbon source as to compare to ethanol alone, ethanol was the key factor to increase vanillin concentration. Ethanol is a normal by-product of glucose fermentation, transiently accumulated in the yeast culture medium. But when it is added to medium in high concentration (2% or more), it influences yeast metabolism. Besides being a source of carbon for yeast growth, ethanol influences integrity of cellular membrane and carbon metabolism of S. cerevisiae. Addition f ethanol significantly changes the permeability of the cytoplasmic membrane, and results in a marked decrease in the mitochondrial membrane potential. In the present case, ethanol significantly increases vanillin synthesis. Conclusion

The synthesis of vanillin was significantly enhanced by the use of the process as described. Gene deletions have increased the flow of pathway by promoting the synthesis process and reducing the degradation process. The control of culture parameters has allowed the increase of the bioconversion. These combined changes have allowed the increase of the concentration of the recovered vanillin from 1 to 250 μΜ.

SEQUENCES

PAL pop_715AA (SEQ ID 1)

METVTK GYQNGSLESLCV QRDPLSWGVAAEAMKGSHLDEVKRMVADYRKPWKLGGET LTIAQVASIAGHDTGDVKVELSESARPGVKASSDWVMDSMDKGTDSYGVTTGFGATSHRR TKQGGALQKELIRFLNAGIFGNG ETCHTLPHSATRAAMLVRINTLLQGYSGIRFEILEA ITRLLNNNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKATGPTGEVLDAAEAFKAA GIESGFFELQPKEGLALVNGTAVGSGLASMVLFET VLAVLSELLSAIFAEVM GKPEFT DHLTHKLKHHPGQIEAAAIMEHILDGSAYM AAKKLHETDPLQKP QDRYALRTSPQWLG PQIEVIRFST SIEREINSVNDNPLIDVSRW AIHGGNFQGTPIGVSMDNVRLAIASIGK LLFAQFSELWDFYISESIGLPSNLTASPJNIPSLDYGFKGAEIAMASYCSELQYLA PVTTHVQ SAEQHNQDVNSLGLISSRKTAEAVDILKLMSTTFLVALCQAIDLRHLEENLKSAVKNTVS QVSKRVLTTGANGELHPSRFCEKELL WDREYVFAYVDDPCSATYPLMQ LRQVFVDHA LENGENEK FSTSVFQKIEAFEEELKALLPKEVESARAAYDSGNSAIDNKI ECRSYPLY KFVREELGTVLLTGEKVQSPGEEFDKVFTA CQGKIIDPMLECLGEW GSPLPIC

PALpet_718 A (SEQ ID 2)

MAYVNGTTNGHANGNGLDLCMK EDPLNWGVAAEALTGSHLDEVKRMVAEYRKPWKLEG ETLTISQVAAISARDDSGVKVELSEEARAGVKASSD VMDSMNKGTDSYGVTTGFGATSH RRTKQGGALQKELIRFLNAGIFGSGAEAGW TLPHSATRAAMLVRINTLLQGYSGIRFEI LEAITKFLNH ITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPTGVTLSPEEAF KLAGVEGGFFELQPKEGLALWGTAVGSGMASWLFEANILAVLAEV SAIFAEVMQGKP EFTDHLTH LKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQ WLGPQIEVIRSSTKMIEREINSVNDNPLIDVSRNKAIHGGNFQGSPIGVSMDNTRLAIAA IGKLMFAQFSELWDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLANPVTN HVQSAEQHNQD\NSLGLISSRKTSEAVEIL LMSTTFLVGLCQAIDLRHLEENLKSTVKN TVSQVAKRVLTMGV GELHPSRFCEKDLLRWDREYIFAYIDDPCSATYPLMQKLRETLV EHALNNGDKERNLSTSIFQKIAAFEDELKALLPKEVETARAALESGNPAIPNRIKECRSY PLYRFVREELGTEYLTGEKVRSPGEEFEKVFTAMSKGEIIDPLLECLESWNGAPLPIC

C4Hgly__506AA (SEQ ID 3)

MDLLLLEKTLIGLFLAAWAIAVSTLRGR FKLPPGPLPVPIFGNWLQVGDDLNHR LTD LAKKFGDIFLLRMGQR-SiLVWSSPELAREVLHTQGVEFGSRTRJSrWFDIFTGKGQDMV FT VYGEHWR MRRIMTVPFFTNKWQQYRHGWESEAAAWEDWKNPDAAVSGTVIRRRLQL MMYTN3NMYRIMFDRRFESEEDPIFQRLRALNGERSRLAQSFEYNYGDFIPILRPFLKGY LK ICKEVKETRLKLFKDYFVDERKKLGSTKSTNNN ELKCAIDHILDAQRKGEINEDNVLYI VENI AAIETTL SIEWGIAELVNHPEIQQKLRDEIDRVLGAGHQVTEPDIQKLPYLQA WKETLRLRMAIPLLVPHMNLHDA LGGYDIPAESKILWAWWLAN PAHWKKPEEFRPE RFFEEESLVEANGNDFRYLPFGVGRRSCPGIILALPILGITLGRLVQNFELLPPPGQSQI DTSEKGGQFSLHILKHSTIVAKPRSF

C4Hpet_506AA (SEQ ID 4)

MMDFVLLEKALLGLFIATIVAI ISKLRGKKLKLPPGPIPVPVFGN LQVGDDLNQRNLV DYAKKFGDLFMLRMGQRNLVWSSPELAKDVLHTQGVEFGSRTRNWFDIFTGKGQDMVF TVYSEHWRJERRIMTVPFF NK^A^

LLMYNNMYRIMFDRRFESVDDPLFLKLKALNGERSRLAQSFEYHFGDFIPILRPFLRGYL KLCQEIKDKRLKLFKDYFVDERKKLESIKSVDN SLKCAIDHIIEAQQ GEINED VLYI VENINVAAIETTLWSIEWGI ELVN PEIQ LRHELDTVLGAGVQICEPDVQKLPYLQA VIKETLRYRMAIPLLVPHMNLHDAKLAGYDIPAESKILV AWWLA NPAHW KPDEFRPE RFLEEESKVEANGNDFKYIPFGVGRRSCPGIILALPILGIVIGRLVQNFELLPPPGQSKI DTAEKGGQFSLQILKHSTIVCKPRSL 4CLpop_570AA (SEQ ID 5)

MMSVATVEPPKPELSPPQNQNAPSSHETDHIFRSKLPDITISNDLPLHAYCFENLSDFSD RPCLISGSTG TYSFAETHLISR VAAGLSNLGIKKGDVIMTLLQNCPEFVFSFIGAS I GAVITTA PFYTQSEIFKQFSASRAKLIITQSQYVNKLGDSDCHEMNQKPGEDFIVITID DPPENCLHFNVLVEASESEMPTVSILPDDPVALPFSSGTTGLPKGVILTHKSLI SVAQQ VDGEIPNLYLKQDDWLCVLPLFHIFSLNSVLLCSLRAGSAVLLMQKFEIGSLLELIQKH NVSVAAWPPLVLALA NPLEANFDLSSIRVVLSGAAPLGKELEDALRSRVPQAILGQGY GMTEAGPVLSMCLAFSKQPFPTKSGSCGTWRNAELKVIDPETGRSLGYNQPGEICIRGS QIMKGYLNDAEATANTIDVEGWLHTGDIGYVDDDDEIFIVDRVKEIIKFKGFQVPPAELE ALLVNHPSIADAAWPQKDEVAGEVPVAFWRSDDLDLSEEAVKEYIAKQWFYKKLHKV FFVKSIPKSASG ILRKDLRAKLATATTMS

ECHpf1_276AA (SEQ ID 6)

MSNYEGRWTTVKVEIEDGIAWILmPEKRNAMSPTLNREMIDVLETLEQDPAAGVLVLT GAGEA TAGMDLKEYFREVDAGPEILQEKIRREASQ Q KLLRMYAKPTIAMVNGWCFGG GFSPLVACDLAICADEATFGLSEINWGIPPGNLVSKA ADTVGHRQSLYYIMTGKTFGGQ KAAEMGLVNDSVPLARLREVTIELARNLLEKNPWLRAAKHGFKRCRELTWEQNEDYLYA KLDQSRLLDTEGGREQGMKQFLDDKSIKPGLQAYKR

ECHavi_276AA (SEQ ID 7)

MNKYEGR KTVIVEIEGGIAWVTLNRPD RNAMSPTL REMRDVLETLEQDPAARVLVLT GAGSA TAGMDLKEYFREVDAGPEILQEKIRREACEWQW LLRMYAKPWAMVNG CFGG

GFSPLVACDLAICADEATFGLSEIIWGIPPGNLVSKAMADTVGHRQALYYIMTGKTF DGR QAAEMGLVNQSVPLAQLRETVATLCQDLLD NPWLRAAKNGFKRCRELTWEQNEDYLYA KLDQSRLLDEEGGREEGMRQFLDE SI PGLQ YKR

HBHpae_394AA (SEQ ID 8)

MKTQVAIIGAGPSGLLLGQLLHKAGIDNVILERQTPDYVLGRIRAGVLEQGMVDLLREAG VDRRMARDGLVHEGVEIAFAGQRRRIDLKRLSGGKTV VYGQTEVTRDLMEAREACGATT VYQAAEVRLHDLQGERPYVTFERDGERLRLDCDYIAGCDGFHGISRQSIPAERLKVFERV YPFGWLGLLADTPPVSHELIYANHPRGFALCSQRSATRSRYYVQVPLTEKVEDWSDERFW TELKARLPAEVAEKLVTGPSLE SIAPLRSFWEPMQHGRLFLAGDAAHIVPPTGAKGLN LAASDVSTLYRLLLKAYREGRGELLERYSAICLRRIWKAERFSWWMTSVLHRFPDTDAFS QRIQQTELEYYLGSEAGLA IAE YVGLPYESIE

KBHavi_39 A (SEQ ID 9)

MKTQVAIIGAGPSGLLLGQLLHKAGID VILERHSPDYVLGRIRAGVLEQGWDLLREAG VAERMDREGLVHEGIELACSGRRIRLDLKALSGGKTVMVYGQTEVTRDLMDARRASGAPI VYEAQNVRLSGLKDGMPHVTYEKDGQTHRLDCDYIAGCDGFHGVSRQSIPAEALSHYERV YPFGWLGLLSDTPPVHEELIYAHTDLGFVLCSQRSPTRSRYYLQVPLSDRVED SDERFW NELKRRLPGDVANRLVTGPSLEKSIAPLRSYWEPMQYGRLFLVGDAAHIVPPTGA GLN LAGSDVCYLYRILLKVYREGRTELLEKYSELALRRV KGERFSWFMTNLLHDFEGSDAFD RRMQLADRDYYLDSEAGRVTIAENYVGLPYEEIA COM msa_365AA (SEQ ID 10)

MGSTGETQITPTHISDEEA LFAMQLASASVLPMILKSALELDLLEIIAKAGPGAQISPI EIASQLPTTNPDAPVMLDRMLRLLACY ILTCSVRTQQDGKVQRLYGLA VAKYLVKNED GVSISALNLMWQDKVLMESWYHLKDAVLDGGIPFNKAYGMTAFEYHGTDPRFNKVFNKGM SDHSTITMKKILETYTGFEGLKSLVDVGGGTGAVINTIVSKYPTIKGINFDLPHVIEDAP SYPGVEHVGGDMFVSIP ADAVFMKWICHDWSDEHCLKFL NCYEALPDNGKVIVAECIL PVAPDSSLATKGWHIDVIMLAHNPGGKERTQ EFEDLA GAGFQGFKVHCNAFNTYIME FLKKV COMTvpl_365AA (SEQ ID 11)

MATWVEHQQQQNGSKDVDEEACMYAMQLSSMWLPMTLRVAVELGILEQIQAGGPDSYLT AEDLAARLGNSNPLAPVMIERILRLLTSYSILNFTDTVDGEGRTVRSYGAAHVCKYLTPN QDGVSmPLVLl TDKVLMESWYHMKDAVTlSIGGIPFNLAYGMTAFEYHGKDLRFlSIKVFNE GMKNNSIIITKKILERYKRFEDVNVLIDVGGGIGG I SMI AKYPHIHGINFDLPHWS E APPFQGVEHVGGNMFESVPIGDAIFIKWILHDWSDEHCLKLLRNCAKSLPDKGKVIWEC ILPDAPLVTPEAEGVFHLDMIMLAHNPGGKERTKKEFKELAMLSGFSNFKALFSYAlxrV WV MEFN Carboxylic acid reductase_l 174AA { SEQ ID 12)

MAVDSPDERLQRRIAQLFAEDEQWAARPLEAVSAAVSAPGmLAQIAA VMAGYADRPA AGQRAFELNTDDATGRTSLRLLPRFETITYRELWQRVGEVAAA HHDPENPLRAGDFVAL LGFTSIDYATLDLADIHLGAVTVPLQASAAVSQLIAILTETSPRLLASTPEHLDAAVECL LAGTTPERLWFDYHPEDDDQRAAFESARRRLADAGSLVIVETLDAVRARGRDLPAAPLF VPDTDDDPLALLIYTSGSTGTPKGAMYTNRLAATMWQGNSMLQGNSQRVGINLNYMPMSH IAGRISLFGVLARGGTAYFAAKSDMSTLFEDIGLVRPTEIFFVPRVCDMVFQRYQSELDR RSVAGADLDTLDREVKADLRQ YLGGRFLVAWGSAPLAAEM TFMESVLDLPLHDGYGS TEAGASVLLDNQIQRPPVLDYKLVDVPELGYFRTDRPHPRGELLLRAETTIPGYYKRPEV TAEIFDEDGFYKTGDIVAELEHDRLVYVDRRM LKLSQGEFVTVAHLEAVFASSPLIRQ IFIYGSSERSYLLAVIVPTDDALRGRDTATLKSALAESIQRIAKDA LQPYEIPRDFLIE TEPFTIANGLLSGIAKLLRPNLKERYGAQLEQMYTDLATGQADELLALRREAADLPVLET VSRAAKAMLGVASADMRPDAHFTDLGGDSLSALSFSNLLHEIFGVEVPVGVWSPANELR DLA YIEAERNSGAKRPTFTSVHGGGSEIRAADLTLDKFIDARTLAAADSIPHAPVPAQT VLLTGANGYLGRFLCLEWLERLDKTGGTLICWRGSDAAAARKRLDSAFDSGDPGLLEHY QQLAARTLEVLAGDIGDPNLGLDDAT QRLAETTOLIVHPAALVNHVLPYTQLFGPNWG TAEIVRLAITARRKPVTYLSTVGVADQVDPAEYQEDSDVREMSAVRWRESYA GYGNSK AGEVLLREAHDLCGLPVAVFRSDMILAHSRYAGQL VQDVFTRLILSLVATGIAPYSFY RTDAEXGNRQRAHYDGLPADFTAAAI ALGIQATSGFRTYDVLNPYDDGISLDEFVD LVE SGHPIQRITDYSDWFHRFETAIRALPEKQRQASVLPLLDAYRNPCPAVRGAILPAKEFQA AVQTAKIGPEQDIPHLSAPLIDKYVSDLELLQLL

PPTase„222AA (SEQ ID 13)

MIETILPAGVESAELLEYPEDLKAHPAEEHLIAKSVEKRRRDFIGARHCARLALAELGEP PVAIG GERGAPI PRGWGSLTHCDGYRAAAVAHKMRFRSIGIDAEPHATLPEGVLDSV SLPPEREWLKTTDSALHLDRLLFCA EATY AWWPLTARWLGFEEAHITFEIEDGSADSG

NGTFHSELLVPGQTNDGGTPLLSFDGRWLIADGFILTAIAYA