Genetically modified strain of Pseudomonas putida to express a benzalacetone reductase

Abstract

The present invention relates to the field of production of phenylbutanone compounds or phenylbutanone derivatives such as frambinone, and in particular to genetically modified strains to express a benzalacetone reductase.

Description

Title of the invention: Genetically modified strain of Pseudomonas putida to express a benzalacetone reductase technical field

[0001] The present invention relates to the field of production of phenylbutanone compounds or phenylbutanone derivatives such as frambinone, and in particular to that of genetically modified strains to express a benzalacetone reductase. Previous technique

[0002] The bioproduction of "natural" aromas and fragrances has been an important research focus for the industry for many years, in order to meet the demands of increasingly eco-conscious consumers. Synthetic biology, particularly through the use of microorganisms, enables this natural production, but yields are not always sufficient for large-scale production.

[0003] The flavor of raspberry (Rubus idaeus) is linked to more than 200 compounds, but frambinone, a natural phenolic compound, is the compound that has the most impact, defining its characteristic taste (Klesk et al., 2004, J. Agric. Food Chem. 52, 5155-61; Larsen et al., 1991, Acta Agric. Scand. 41, 447-54).

[0004] Since it is present only in small quantities in raspberries (1-4 mg per kg of fruit), natural frambinone is of great value (Larsen et al., 1991, Acta Agriculturae Scandinavica (Sweden); Beekwilder et al., Biotechnol J. 2007 Oct;2(10): 1270-9). However, as its natural availability is limited, its biotechnological production is highly desirable.

[0005] In this context, the biosynthesis pathway of phenylpropanoid compounds, in particular frambinone, can be reconstituted within a microorganism through the insertion of heterologous genes encoding certain key enzymes of said pathway.

[0006] Tyrosine is the precursor of coumaric acid which is metabolized into frambinone in three steps.

[0007] According to the first step, tyrosine is deaminated by tyrosine ammonia lyase (TAL, EC 4.3.1.23) to form coumaric acid. Catalyzed by 4-coumarate:CoA ligase (4CL, EC 6.2.1.12), a molecule of Coenzyme A (CoA) is grafted onto the coumaric acid. The coumaroyl-CoA is then converted by benzalacetone synthase (BAS, EC 2.3.1.212) to 4-hydroxybenzalacetone. This reaction is a decarboxylative condensation and uses a malonyl-CoA unit. as a co-substrate. The final step is the reduction of 4-hydroxybenzalacetone to frambinone by a benzalacetone reductase.

[0008] This last step involves a reduction of the double bond of an unsaturated α-[3] ketone to a ketone, which can be catalyzed by an enzyme belonging to the oxidoreductase family, NADPH dehydrogenase (EC 1.6.99.1), specifically called benzalacetone reductase or BAR. Benzalacetone reductase belongs to the MDR (medium chain dehydrogenase / reductase leukotriene B4 dehydrogenase) enzyme subfamily.

[0009] Within this family, there are several subclasses studied for the production of pharmaceutical, chemical, or agrochemical products, with varying degrees of selectivity. Among these, the enzymes of the Old Yellow Enzymes family are the most frequently described, although their physiological functions have not yet been fully studied.

[0010] The Rubus idaeus (raspberry) BAR, known in English as "Raspberry Ketone / Zingerone Synthase" (RKS or RZS), was characterized by a Japanese team in 2011 (Koekuda et al., Biochem Biophys Res Commun. 2011 Aug 19;412(1): 104-8). RZS is a 37 kDa enzyme, and only isoform 1 has been shown to be active on the natural precursor of frambinone, 4-hydroxybenzalacetone (HBA).

[0011] Frambinone production has been achieved in E. coli and S. cerevisiae (Beekwilder et al., Biotechnol J. 2007 Oct;2(10): 1270-9; Lee et al., Microb Cell Fact. 2016 Mar 4; 15:49). In these studies, the final step of the reduction of 4-hydroxybenzalacetone to frambinone is endogenous, and the enzymes responsible for this reaction have not been identified. Frambinone biosynthesis thus leads to a mixture of frambinone and its precursor with a low production yield that is particularly unsuitable for large-scale production. Therefore, the frambinone production yield needs to be improved.

[0012] Moore et al. developed a cell-free in vitro platform for frambinone production using the raspberry RKS Rubus idaeus (Moore et al., 2017; doi: https: / / doi.org / 10.1101 / 202341). Frambnone synthesis was also investigated in E. coli, expressing the R. palmatum BAS and the raspberry RKS (Wang et al. Appl Microbiol Biotechnol 103, 3715-3725, 2019).

[0013] Recently, the E. coli enzyme CurA (curcumin / dihydrocurcumin reductase, NADPH-dependent) was used to catalyze the final step of frambinone synthesis in Corynebacterium glutamicum (Milke et al. Microb Cell Fact (2020) 19:92). This enzyme was identified as a BAR, based on the structural similarity of its substrate to frambinone (Hassaninasab A, et al. Proc Natl Acad Sci. 2011; 108:6615–20). Although the E. coli CurA reductase exhibits good activity for the production of FBO in the bacterium Corynebacterium glutamicum, the reaction remains incomplete after 72h of culture with 500 mg / L of HBA. Technical problem

[0014] Few enzymes capable of catalyzing the final step of the reduction of 4-hydroxybenzalacetone to frambinone have been characterized. These enzymes have been studied primarily in E. coli and S. cerevisiae strains. However, these strains do not tolerate the toxicity of phenylpropanoid compounds well and are therefore not the most suitable microorganisms for their production.

[0015] Thus, there is a particular need to characterize the enzymes capable of catalyzing the last step of the reduction of phenylbuten-2-one to phenylbutanone or a derivative of phenylbutanone, in particular 4-(4-hydroxyphenyl)-but-3-en-2-one (HBA) to frambinone and to develop new strains of microorganisms allowing the efficient production of phenylpropanoid compounds.

[0016] Bacteria of the genus Pseudomonas appear to be more tolerant to these highly toxic molecules, particularly the bacterium Pseudomonas putida (Calero et al., Biotechnol Bioeng. 2018 Mar; 115(3):762-774). In contrast, the enzymes involved in the production of aromatic amino acids in P. putida are poorly described, and no enzyme capable of catalyzing the reduction of hydroxybenzalacetone to frambinone in P. putida has been characterized.

[0017] The development of efficient reductase enzymes for the alpha double bond hydroxylation of a ketone and enabling the production of aromatic compounds such as frambinone within microorganisms tolerant to the synthesis of phenylpropanoids is therefore crucial. Summary

[0018] The inventors in this disclosure have identified and characterized enzymes capable of efficiently catalyzing this reaction in Pseudomonas putida and enabling the complete conversion of HBA to frambinone leading to efficient frambinone production.

[0019] One aspect of the present invention relates to a genetically modified strain of Pseudomonas putida characterized in that it expresses a recombinant gene encoding: a) a benzalacetone reductase selected from the group consisting of: - the NADPH-dependent 2-alkenal reductase (AER) of Arabidopsis thaliana defined by the sequence SEQ ID NO: 1, - ene-reductase (ERED) from Zingiber officinale defined by the sequence SEQ ID NO: 2, - NADPH-dependent curcumin reductase (CurA) from Pseudomonas Putida defined by the sequence SEQ ID NO: 3, - the NADP-dependent alkenal double bond reductase (DBR) of Olimarabidopsis pumila defined by the sequence SEQ ID NO: 4, - the NADP(+)-dependent 2-alkenal reductase (DBR) of Nicotiana tabacum defined by the sequence SEQ ID NO: 5, and - 2-alkenal reductase (NADP(+) dependent) (Red) from Capsicum annuum defined by the sequence SEQ ID NO: 6 or b) a functional variant of a benzalacetone reductase having an amino acid sequence having at least 80% identity with one of the sequences chosen from SEQ ID NO: 1 to 6. Another aspect of the invention relates to a method for synthesizing a compound of formula (I): [Chem 1] where RI, R2 and R3 are chosen independently of each other from among a hydrogen, an OH group and OCH3; and R4 is a methyl or aryl group, by implementing a genetically modified strain according to the invention of Pseudomonas putida.

[0020] Finally, the invention also relates to the use of a genetically modified strain of Pseudomonas putida for the synthesis of a compound of formula (I): [Chem 1] where RI, R2 and R3 are chosen independently of each other from among a hydrogen, an OH group and OCH3; and R4 is a methyl or aryl group.

[0021] The features described in the following paragraphs may optionally be implemented. They may be implemented independently of each other or in combination with each other. Brief description of the figures Fig. 1

[0022] [Fig.1] Acrylamide gel to visualize the overexpression of reductase enzymes of interest in P seudomonas putida. Fig. 2

[0023] [Fig.2] Total concentration of frambinone (FBO) produced by the different P. seudomonas putida strains overexpressing the reductase enzymes of interest. Description of embodiments

[0024] The inventors have identified and characterized NADPH dehydrogenases (EC 1.6.99.1) capable in P. putida of asymmetrically catalyzing the reduction of an activated alkene via the cofactor NADPH. In particular, the NADPH dehydrogenase according to this disclosure is a benzalacetone reductase (BAR), also called benzylidenacetone reductase, capable of producing a phenylbutanone or phenylbutanone derivative from a phenylbuten-2-one according to the following reaction:

[0025] [Diagram 1] where RI, R2 and R3 are chosen independently of each other from among a hydrogen, an OH group or OCH3; and R4 is a CH3 or aryl group.

[0026] Advantageously, the Applicant has developed a strain of Pseudomonas putida capable of expressing a benzalacetone reductase and efficiently producing a phenylbutanone or phenylbutanone derivative from a phenylbuten-2-one according to the reaction as described above.

[0027] Thus, a first object of the invention relates to a genetically modified strain of Pseudomonas putida characterized in that it expresses a recombinant gene encoding a benzalacetone reductase capable of producing a phenylbutanone or phenylbutanone derivative.

[0028] Preferably, the Pseudomonas putida strain according to this application is capable of producing a phenylbutanone or a phenylbutanone derivative selected from the group consisting of the products listed in [Table 1] from a corresponding phenylbuten-2-one selected from the group consisting of the substrates listed in [Table 1]. [Tables 1] Substrates Products [Chem 2] p ôch3 4-(4-Hydroxy-3-methoxyphenyl)-3-buten-2-one (vanillylidenacetone; dehydrozingerone) [Chem 3] O il YY'''' OCHs 4-(4-hydroxy-3-methoxyphenyl)butan-2-one (zingerone) [Chem 4] 0 4-Phenylbut-3-en-2-one (Benzalacetone, Benzylideneacetone, Methylstyryl ketone) [Chem 5] 0 At ch3 4-phenylbutan-2-one (benzylacetone) [chem 6] O HO-7—zzzzz---— 4-(4-hydroxyphenyl)-but-3-en-2-one (Hydroxybenzylidene acetone; 4-hydroxybenzalacetone) (HBA) [Chem 7] q 4-(4-hydroxyphenyl)-2-butanone (frambino ne) [Chem 8] [Chem 9] HO, -, f iï Y ' o 1 -(3,4-Dihydroxyphenyl)butan-2-one 4-(3,4-dihydroxyphenyl)-3-buten-2-one [Chem 10] 4-(3,4-dimethoxyphenyl)-3-buten-2-one [Chem 11] J rrr 0 1 -(3,4-Dimethoxyphenyl)butan-2-one) [Chem 12] 4-(4-Methoxyphenyl)-3-buten-2-one [Chem 13] 0 H3CO' 4-(4-methoxyphenyl)-2-butanone ; (Anisyl ketone) [Chem 14] 0 1,3-diphenylprop-2-en-l-one (chalcone, benzylidenacetophenone or phenylstyryl ketone )• [chem 15] 0 . x O' x.-''' 1,3-diphenylpropan-l-one ; dihydrochalcon e [Chem 16] 0 OH .... P ï I 1 H3ccr OH [Chem 17] OH Ç> HO'""' " '''•A" 1 CHj- l-(2,4-dihydroxy-6-methoxyphenyl)-3-phen ylpropan-l-one (Uvangoletin) 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenylprop-2-en-l-one (Alpinetin chalcone, Car damonin) [Chem 18] [Chem 19] 4-3,4,5-trimethoxyphenyl)butan-2-one 4-(3,4,5-trimethoxyphenyl)but-3-en-2-one [Table 1]: Examples of phenylbutanones or phenylbutanone derivatives (products) that can be obtained from the Pseudomonas putida strain as described in this application using the corresponding substrate.

[0029] In a preferred embodiment, the strain of Pseudomon as putida according to the present application is capable of producing frambinone from 4-(4-hydroxyphenyl)-but-3-en-2-one (HBA).

[0030] In another preferred embodiment, the Pseudomon as putida strain according to the present application is capable of producing zingerone from 4-(4-Hydroxy-3-methoxyphenyl)-3-buten-2-one.

[0031] In particular, said genetically modified strain of Pseudomonas putida is characterized in that it expresses a recombinant gene encoding a benzalacetone reductase selected from the group consisting of: - Arabidopsis thaliana dependent NADPH 2-alkenal reductase (Uniprot Q39172, updated June 2, 2021), also called AER and defined by the SEQ ID NO sequence: 1, - ene-reductase from Zingiber officinale (Uniprot A0A096LNF0, updated April 7, 2021) defined by the sequence SEQ ID NO: 2, - NADPH-dependent curcumin reductase, also called CurA from Pseudomonas Putida (uniprot Q88K17, updated on December 2, 2020) defined by the sequence SEQ ID NO: 3, - the NADP-dependent alkenal double bond reductase, also called DBR, from Olimarabidopsis pumila (Uniprot A0A1C9CX65, updated on August 12, 2020) defined by the SEQ ID NO sequence: 4, - the NADP(+)-dependent 2-alkenal reductase, also called DBR, from Nicotiana tabacum (Uniprot Q9SLN8; EC 1.3.1.102), defined by the sequence SEQ ID NO: 5, and - 2-alkenal reductase (NADP(+) dependent), also called Capsicum annuum Red (Uniprot A0A1U8GFY1, updated on February 10, 2021) defined by the sequence SEQ ID NO: 6.

[0032] In a particular embodiment, the invention relates to a genetically modified strain of Pseudomonas putida characterized in that it expresses a recombinant gene encoding a functional variant of a benzalacetone reductase described previously.

[0033] By functional variant of a benzalacetone reductase according to this disclosure, we mean a polypeptide sequence which is derived from the polypeptide sequence of one of the benzalacetone reductase enzymes defined by one of the sequences chosen from SEQ ID NO: 1 to 6, in particular a polypeptide sequence which includes a modification, i.e. substitution, insertion and / or deletion of one or more amino acids but which retains the activity of the benzalacetone reductase and in particular the ability to produce in the P. putida strain a phenylbutanone or a phenylbutanone derivative from phenylbuten-2-one as described above.

[0034] The activity of a functional variant of benzalacetone reductase can be evaluated by any method known to those skilled in the art, in particular as illustrated in the examples by expressing in a strain of Pseudomonas putida a recombinant gene encoding the functional variant of benzalacetone reductase, preferably cloned in a plasmid downstream of a promoter allowing its expression in the strain, and by culturing the strain in the presence of a phenylbutanone or a phenylbutanone derivative as described above, preferably HBA, and by measuring by HPLC the total concentration of phenylbuten-2-ones, preferably frambinone, produced by the strain after 24h. In a specific embodiment, the variant maintains a benzalacetone reductase activity of at least 50%, 60%, 70%, 80%, 90% or at least 95% of the activity measured with its unmodified equivalent (e.g., one of the sequences chosen from SEQ ID NO: 1 to 6).

[0035] Preferably, a functional variant corresponds to a polypeptide sequence exhibiting at least 80%, 85%, 90%, 95% and, in particular, at least 98% identity with one of the sequences chosen from among the SEQ ID NO sequences: 1 to 6.

[0036] For the purposes of the present invention, the percentage of identity refers to the percentage of identical residues in a nucleotide sequence or amino acids on a given fragment after alignment and comparison with a reference sequence. For the comparison, an alignment algorithm is used, and the sequences to be compared are entered with the corresponding parameters of the algorithm. The algorithm's default parameters may be used.

[0037] Preferably, for nucleic acid or polypeptide sequence comparison and determination of a percentage of identity, the blastn or blastp algorithm as described in https: / / blast.ncbi.nlm.nih.gov / Blast.cgi with default parameters is used

[0038] In particular, the functional variant refers to a polypeptide that has an amino acid sequence that differs from one of the sequences selected from SEQ ID NO: 1 to 6 by fewer than 50, 40, 30, 20, 10, 5, 4, 3, 2 or 1 substitutions, insertions or deletions.

[0039] In another particular mode, the functional variant refers to a polypeptide that has an amino acid sequence that differs from one of the sequences chosen from SEQ ID NO: 1 to 6 by fewer than 50, 40, 30, 20, 10, 5, 4, 3, 2 or 1 substitutions, the substitutions preferably being conservative substitutions.

[0040] The term "conservative substitution", as used herein, refers to the replacement of one amino acid residue by another, without altering the conformation or enzymatic activity of the polypeptide thus modified, including, but not limited to, the replacement of one amino acid by another having similar properties (such as, for example, polarity, hydrogen bonding potential, acidity, basicity, shape, hydrophobicity, aromaticity and others).

[0041] Examples of conservative substitutions are found in the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (methionine, leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and small amino acids (glycine, alanine, serine and threonine).

[0042] Wild strains of P. putida KT2440 are available for example in the NBRC Strain Bank (National Institute of Technology and Evaluation Biological Resource center https: / / www.nite.go.jp / en / nbrc / , NBRC100650).

[0043] In addition, strains of Pseudomonas putida or Pseudomonas taiwanensis optimized for tyrosine production are known to those skilled in the art, who can use them as starting strains to obtain the genetically modified strains according to the invention (Calero et al., ACS Synth Biol. 2016 Jul 15;5(7):741-53; Wierckx et al., Appl Environ Microbiol. 2005 Dec;71(12):8221-7, Appl Environ Microbiol. 71(12):8221-7; Wynands et al., 2018; Otto et al. 2019, Front Bioeng Biotechnol Nov 20;7:312).

[0044] For the purposes of this description, the expressions "genetically modified strain of Pseudomonas putida", "modified strain of Pseudomonas putida", "genetically modified strain", or "modified strain" are considered synonymous with each other.

[0045] In particular, a "genetically modified strain" is understood to mean a strain that comprises either (i) at least one recombinant nucleic acid, or transgene, stably integrated into its genome and / or present on a vector, for example, a plasmid vector, or (ii) one or more non-natural mutations by insertion, substitution, or deletion of nucleotides, said mutations being obtained by transformation techniques or by gene-editing techniques known to those skilled in the art. In a particular embodiment, a genetically modified strain is a strain having stably integrated into its genome at least one exogenous nucleic acid, that is, one not naturally present in P. putida, for example, a nucleic acid from another species.

[0046] For the purposes of the present invention, a "recombinant gene encoding a benzalacetone reductase" means an exogenous nucleic acid comprising at least one region encoding a benzalacetone reductase according to the invention as described above. In addition to the region encoding the benzalacetone reductase, the recombinant gene may be under the control of a promoter allowing its expression in the strain, preferably a promoter allowing its expression in the P. putida strain.

[0047] In a particular embodiment, the nucleic acid encoding one of the benzalacetone reductases as described above is chosen from one of the SEQ ID NO: 13 to 18 sequences, or a sequence exhibiting at least 80%, 85%, 90%, 95% and, in particular, at least 98% identity with one of the sequences chosen from the SEQ ID NO: 13 to 18 sequences.

[0048] In one embodiment, which can be combined with the preceding ones, the recombinant gene encoding benzalacetone reductase as described above is placed under the control of a heterologous promoter, in particular a constitutive or inducible promoter, for example chosen from the ptrc, xyls / pm or araC / pBAD promoters, which allows the recombinant gene encoding benzalacetone reductase to be overexpressed in the genetically modified strain according to the invention.

[0049] The techniques of genetic modification by transformation, mutagenesis or gene editing are well known to those skilled in the art and are described for example in "Molecular cloning: a laboratory manual", J. Sambrook, ed. Cold Spring Harbor, "Strategies used for genetically modifying bacterial genome: site-directed mutagenesis, gene inactivation", Journal of Zhejiang Univ-Sci B (Biomed & Biotechnol) 2016 17(2):83-99., and in Martinez-Garcia and de Lorenzo, "Pseudomonas putida in the quest of programmable chemistry", Current Opinion in Biotechnology, 59:111-121, 2019.

[0050] In one embodiment, a genetically modified strain may comprise an expression-modifying nucleic acid, preferably overexpressing the expression of one or more genes naturally expressed in Pseudomonasputida, in particular overexpressing the expression of the NADPH-dependent curcumin / dihydrocurcumin reductase (CurA) of Pseudomonas putida (Uniprot Q88K17) defined by the sequence SEQ ID NO: 3.

[0051] Overexpression of a gene is understood as a higher expression of said gene in a genetically modified strain compared to the same strain in which the gene is expressed solely under the control of the natural promoter. Overexpression can be achieved by inserting one or more copies of the gene directly into the genome of the strain, preferably under the control of a strong promoter, or also by cloning into plasmids, in particular multicopy plasmids, preferably also under the control of a strong promoter.

[0052] In another particular mode, a genetically modified strain may include a nucleic acid encoding one or more enzymes not naturally expressed in Pseudomonas putida.

[0053] Advantageously, the Applicant has developed a strain of Pseudomonas putida capable of expressing a benzalacetone reductase and efficiently producing a phenylbutanone or phenylbutanone derivative of formula (I):

[0054] [Chem.l]

[0055] where RI, R2 and R3 are chosen independently of each other from among a hydrogen, an OH group or OCH3; and R4 is a CH3 or aryl group.

[0056] Preferably, the Pseudomonas putida strain according to the present disclosure expresses a recombinant gene encoding a benzalacetone reductase capable of efficiently producing a phenylbutanone or phenylbutanone derivative of formula (I), preferably selected from the group consisting of: 4-(4-hydroxy-3-methoxyphenyl)butan-2-one (zingerone), 4-phenylbutan-2-one (benzylacetone), 4-(4-hydroxyphenyl)-2-butanone (frambinone), l-(3,4-Dihydroxyphenyl)butan-2-one, l-(3,4-Dimethoxyphenyl)butan-2-one), 4-(4-methoxyphenyl)-2-butanone (Anisylacetone), 1,3-diphenylpropan-l-one; dihydrochalcone, l-(2,4-dihydroxy-6-methoxyphenyl)-3-phenylpropan-l-one; (uvangoletin) and 4-3,4,5-trimethoxyphenyl) butan-2-one, more specifically frambinone or zingerone.

[0057] According to a particular embodiment of the application, the strain of Pseudomonas putida as described above is capable of overproducing a phenylbutanone or a phenylbutanone derivative of formula (I), preferably selected from the group consisting of: 4-(4-hydroxy-3-methoxyphenyl)butan-2-one (zingerone), 4-phenylbutan-2-one (benzylacetone), 4-(4-hydroxyphenyl)-2-butanone (frambinone), l-(3,4-Dihydroxyphenyl)butan-2-one, l-(3,4-Dimethoxyphenyl)butan-2-one), 4-(4-methoxyphenyl)-2-butanone (Anisylacetone), 1,3-diphenylpropan-l-one; dihydrochalcone, l-(2,4-dihydroxy-6-methoxyphenyl)-3-phenylpropan-l-one; (uvangoletin) and 4-3,4,5-trimethoxyphenyl)butan-2-one, more specifically frambinone or zingerone.

[0058] Preferably, the Pseudomonas putida strain according to the present disclosure is capable of overproducing frambinone from HBA or zingerone from 4-(4-Hydroxy-3-methoxyphenyl)-3-buten-2-one.

[0059] For the purposes of this disclosure, "phenylbutanone overproducing strain or phenylbutanone derivative" means a modified strain of Pseudomonas putida capable of producing phenylbutanone or a phenylbutanone derivative as described above, preferably frambinone or zingerone, in greater quantities than a wild-type Pseudomonas putida strain.

[0060] Preferably, the phenylbutanone or phenylbutanone derivative overproducing strain according to this disclosure is capable of producing 2, 3, 4, 5 or 6 times more of a phenylbutanone or a phenylbutanone derivative (e.g. frambinone or zingerone) compared to a wild Pseudomonas putida strain as shown in the examples.

[0061] Preferably, the phenylbutanone overproducing strain or a phenylbutanone derivative according to this disclosure is capable of converting all of the phenylbuten-2-one synthesized in situ or added to the culture medium, into phenylbutanone or a phenylbutanone derivative of formula (I), preferably converting all of the HBA into frambinone or converting all of the 4-(4-Hydroxy-3-methoxyphenyl)-3-buten-2-one into zingerone.

[0062] According to a particular mode, the strain according to the present disclosure is capable of producing a phenylbutanone or a phenylbutanone derivative of formula (I) by adding the corresponding substrate during the culture of the strain.

[0063] According to another particular embodiment, the strain according to the present application is capable of producing the substrate of phenylbutanone or of the phenylbutanone derivative itself (in situ synthesis). The strain may thus further comprise at least one additional recombinant gene enabling the synthesis of the phenylbutanone substrate. or a phenylbetanone derivative of formula (I), preferably allowing the synthesis of the frambinone substrate such as HBA or one of the intermediate products.

[0064] The expression "additional recombinant gene" means, for the purposes of the present invention, any recombinant gene present in the Pseudomonas putida strain in addition to the recombinant gene encoding a benzalacetone reductase as defined above.

[0065] The additional recombinant gene may result from the insertion of a heterologous promoter, for example a strong promoter to overexpress an endogenous gene of Pseudomonas putida, or a recombinant coding sequence encoding a protein not naturally expressed in Pseudomonas putida.

[0066] According to a particular embodiment, the genetically modified strain of Pseudomonas putida includes an additional recombinant gene encoding a polypeptide with tyrosine ammonia lyase (TAL) activity. A recombinant gene encoding TAL can be derived from the microorganism Rhodotorula glutinis and optimized according to Zhou et al., Appl Microbiol Biotechnol. 2016 Dec; 100(24):10443-10452 (three point mutations of this TAL enzyme make it more efficient: S9N; A11T; E518V). This TAL enzyme is called TAL_rg_opt. In particular, the modified strain may include a recombinant gene encoding a tyrosine ammonia lyase (TAL) (EC 4.3.1.23) whose sequence is defined by the amino acid sequence SEQ ID NO: 7 (TAL_rg_opt) or by a sequence exhibiting at least 80%, 85%, 90%, 95% and, in particular, at least 98% identity with the sequence SEQ ID NO: 7 and encoding an enzyme with TAL activity.

[0067] According to this particular embodiment, the genetically modified strain according to the invention is capable of converting tyrosine into coumaric acid via the TAL enzyme.

[0068] According to another particular embodiment that can be combined with the preceding one, the genetically modified strain of Pseudomonas putida comprises an additional recombinant gene encoding 4-coumarate-CoA ligase (4-CL). In particular, the modified strain may comprise a recombinant gene encoding a 4-CL (EC 6.2.1.12) whose sequence is defined by the amino acid sequence SEQ ID NO: 8 or by a sequence having at least 80%, 85%, 90%, 95%, and, in particular, at least 98% identity with the sequence SEQ ID NO: 8 and encoding an enzyme with 4-CL activity.

[0069] According to this particular embodiment, the genetically modified strain is capable of converting coumaric acid into p-coumaryl-coA via the 4-CL enzyme.

[0070] According to another particular embodiment that can be combined with the preceding ones, the genetically modified strain of Pseudomonas putida comprises a additional recombinant gene encoding a polypeptide with benzalacetone synthase (BAS) activity. In particular, the modified strain may include a recombinant gene encoding a BAS (EC 2.3.1.212) whose sequence is defined by the amino acid sequence SEQ ID NO: 9 or by a sequence exhibiting at least 80%, 85%, 90%, 95% and, in particular, at least 98% identity with the sequence SEQ ID NO: 9 and encoding an enzyme with BAS activity.

[0071] According to this particular embodiment, the genetically modified strain is capable of converting p-coumaroyl-coA into 4-hydroxybenzalketone via the BAS enzyme.

[0072] According to a preferred embodiment, the genetically modified strain of Pseudomonas putida comprises several additional recombinant genes, namely the three additional recombinant genes listed below: - a recombinant gene encoding a tyrosine ammonia lyase (TAL_RG_OPT), preferably a TAL_RG_OPT defined by the sequence SEQ ID NO: 7, - a recombinant gene encoding a 4-coumarate-CoA ligase (4-CL), preferably a 4-CL defined by the sequence SEQ ID NO: 8, - a recombinant gene encoding a benzalacetone synthase (BAS), preferably a BAS defined by the sequence SEQ ID NO: 9.

[0073] The enzymes TAL, 4-CL and BAS are all enzymes involved in the synthesis of phenylpropanoid compounds. According to this preferred embodiment, the modified strain is capable of producing a multitude of phenylpropanoid compounds, namely coumaric acid, p-coumaroyl-coA, and 4-hydroxybenzalketone. Method for synthesizing a phenylpropanoid compound

[0074] Another object of the invention relates to a process for the synthesis of one or more phenylbutanone compounds or phenylbutanone derivatives of formula (I), preferably one or more products listed in [Table 1], preferably frambinone or zingerone.

[0075] The synthesis process according to the invention includes carrying out a growth step of a genetically modified strain of Pseudomonasputida as defined above in a culture medium under conditions allowing the expression of the recombinant gene encoding benzalacetone reductase.

[0076] Preferably, the culture conditions are the culture conditions classically used in fermenters for the growth of P. putida.

[0077] In a particular embodiment, the process according to this disclosure is characterized in that the culture medium comprises a substrate of formula (II): [Chem 20] Or RI, R2 and R3 are chosen independently of each other from among a hydrogen, an OH group or OCH3; and R4 is chosen from a methyl or aryl group.

[0078] In another particular embodiment, the process according to this disclosure includes carrying out a growth step of a genetically modified strain of Pseudomonas putida as defined above in a culture medium under conditions allowing the expression of the recombinant gene encoding benzalacetone reductase and additional recombinant genes necessary for the synthesis of one or more phenylbutanone compounds or phenylbutanone derivatives as described above.

[0079] According to a particular embodiment, the process comprises a growth step of a genetically modified strain of Pseudomonas putida comprising a recombinant gene encoding a benzalacetone reductase defined by one of the sequences selected from SEQ ID NO: 1 to 6 and comprising the following additional recombinant genes encoding: - a tyrosine ammonia lyase (TAL_RG_OPT), preferably a TAL_RG_OPT defined by the sequence SEQ ID NO: 7, - a 4-coumarate-CoA ligase (4-CL), preferably a 4-CL defined by the sequence SEQ ID NO: 8, - a benzalacetone synthase (BAS), preferably a BAS defined by the sequence SEQ ID NO: 9.

[0080] According to a particular embodiment, the synthesis process according to the invention makes it possible to produce frambinone in large quantities, for example at yields of at least 100 mg / L, preferably at least 500 mg / L, preferably at least 1 g / L, 1.5 g / L or 2 g / L and in particular in quantities produced 2, 3, 4, 5 or 6 times compared with a wild-type Pseudomonas putida strain or one comprising the following additional recombinant genes encoding: - a tyrosine ammonia lyase (TAL_RG_OPT), preferably a TAL_RG_OPT defined by the sequence SEQ ID NO: 7, - a 4-coumarate-CoA ligase (4-CL), preferably a 4-CL defined by the sequence SEQID NO:8, - a benzalacetone synthase (BAS), preferably a BAS defined by the sequence SEQ ID NO:9, but not including a recombinant gene encoding a benzalacetone reductase.

[0081] In one embodiment, the process allows at least 50%, preferably 60%, 70%, 80%, 90%, 95%, or even at least 99% of the HBA synthesized in situ by the strain into frambinone.

[0082] The synthesis process according to the invention may also include a step of purification and / or recovery of the phenylbutanone compound or phenylbutanone derivative of formula (I), such as frambinone. Uses of the strains according to the invention

[0083] Another object of the invention relates to the use of a strain of Pseudomonas putida as defined above for the synthesis of phenylbutanone compound or phenylbutanone derivative of formula (I), preferably chosen from the group consisting of: 4-(4-hydroxy-3-methoxyphenyl)butan-2-one (zingerone), 4-phenylbutan-2-one (benzylacetone), 4-(4-hydroxyphenyl)-2-butanone (frambinone), l-(3,4-Dihydroxyphenyl)butan-2-one, l-(3,4-Dimethoxyphenyl)butan-2-one), 4-(4-methoxyphenyl)-2-butanone (Anisylacetone), 1,3-diphenylpropan-l-one; dihydrochalcone, l-(2,4-dihydroxy-6-methoxyphenyl)-3-phenylpropan-l-one; (uvangoletin) and 4-3,4,5-trimethoxyphenyl)butan-2-one.

[0084] According to a preferred embodiment, the phenylbutanone compound or phenylbutanone derivative of formula (I) is frambinone or zingerone, preferably frambinone.

[0085] Thus, in particular, the invention relates to the use for the synthesis of frambinone or zingerone of a genetically modified strain of P. putida comprising at least one recombinant gene encoding: a) a benzalacetone reductase selected from the group consisting of: - the NADPH-dependent 2-alkenal reductase (AER) of Arabidopsis thaliana defined by the sequence SEQ ID NO: 1, - ene-reductase (ERED) from Zingiber officinale defined by the sequence SEQ ID NO: 2, - NADPH-dependent curcumin reductase (CurA) from Pseudomonas Putida defined by the sequence SEQ ID NO: 3, - the NADP-dependent alkenal double bond reductase (DBR) of Olimarabidopsis pumila defined by the sequence SEQ ID NO: 4, - the NADP(+)-dependent 2-alkenal reductase (DBR) of Nicotiana tabacum defined by the sequence SEQ ID NO: 5, and - 2-alkenal reductase (NADP(+) dependent) (Red) from Capsicum annuum defined by the sequence SEQ ID NO: 6 or b) a functional variant of a benzalacetone reductase having an amino acid sequence having at least 80% identity with one of the sequences chosen from SEQ ID NO: 1 to 6.

[0086] The present invention will be better understood in the light of the following non-limiting examples, which are given purely for illustrative purposes and are not intended to limit the scope of this invention as defined by the claims. Examples 1. In Silico Study

[0087] A first identification of enzymes exhibiting "ene-reductase" activity in Pseudomonas putida KT2440 was carried out by a bioinformatics study by identifying enzymes with an activity bearing an EC number 1.3.1.31. This study made it possible to identify the enzymes XenA (SEQ ID NO: 12) (Uniprot Q9R9V9; Uniprot Q88NF7 in Pseudomonas putida KT2440) and CurA (Uniprot Q88K17).

[0088] The activity of the XenA enzyme has never been verified in the phenylpropanoid biosynthesis pathway, particularly in the reduction of HBA to frambinone. The CurA enzyme from Pseudomonas putida shows only 70.25% identity with the CurA enzyme from E. coli (Uniprot P76113). The activity of these enzymes in Pseudomonas putida has never been studied.

[0089] A second study based on the structure and function of enzymes was carried out in silico. Enzymes with a tertiary structure linked to molecules of the coniferaldehyde family and enzymes exhibiting high structural and sequence similarity to raspberry RKS were identified in the Protein Data Bank database. This study identified the following enzymes: - NADPH-dependent 2-alkenal reductase (AER) from A. thaliana (Uniprot Q39172) (SEQ ID NO: 1) - the NADP(+)-dependent 2-alkenal reductase (DBR) of N. tobacco (Uniprot Q9SLN8) (SEQ ID NO: 5), - 2-alkenal reductase NADPH dependent (ERED) from Zingiber officinale (Uniprot A0A096LNF0), also called ene reductase from Zingiber officinale (SEQ ID NO: 2), - putative NADP(+) dependent type 2-alkenal reductase enzyme (Red) from Solanum chacoense (Uniprot A0A0V0I4S3) (SEQ ID NO: 10) - the alkenal double bond reductase NADP dependent (DBR) from Olimarabidopsis pumila (Unprot A0A1C9CX65) (SEQ ID NO: 4) - 2-Alkenal reductase (NADP(+) dependent) (Red) from Capsicum annuum (Uniprot A0A1U8GFY1) (SEQ ID NO: 6)

[0090] The reductase activity of the identified enzymes was tested for the reduction of benzalacetone reductase (HBA) to frambinone in the bacterium Pseudomonas putida in comparison with the RZS of raspberry (Rubus idaeus) in the following examples. 2. Cloning and gene expression

[0091] The genes encoding these reductases were synthesized and cloned into a pBBRl plasmid downstream of a promoter allowing overexpression of the genes in E. coli S 17.1 and in Pseudomonas putida.

[0092] The expression of the different genes was tested in a wild-type P. putida KT 2440 strain [Fig.1]. 3. Production of raspberry from HBA

[0093] The different strains containing the plasmids constructed above and producing the potential reductases were cultured in the presence of the precursor of frambinone (FBO), benzalacetone reductase (HBA), and the frambinone produced after a few hours of culture is detected by HPLC.

[0094] The different constructed P. putida strains are cultured in 50 mL of MPpu medium + 5 g / L glucose + 5 mM HBA (810 mg / L) with a ΔO600 = 0.05. The cultures are incubated at 30°C with shaking at 140 rpm for 24 h. After 24 h, the cultures are centrifuged, and the supernatant is collected for HPLC analysis. The pellet is resuspended in 3 mL of supernatant, and the cells are lysed by sonication. The mixture is centrifuged, and the supernatant is collected for HPLC analysis. 4. SDS-PAGE and HPLC results

[0095] Figure 2 shows that the Pseudomonas putida WT strain, cultivated in the presence The precursor HBA already naturally produces a small amount of frambinone. Pseudomonas putida possesses two genes encoding potential reductases (XenA and CurA), which are certainly involved in this low production. It does not appear that XenA is responsible for this production, as it remains quite low even when XenA is overexpressed (P. putida / pC2F04-6). The P. putida / pC2F047 strain overproduces the raspberry reductase, which is the most commonly used for FBO bioproduction. However, under culture conditions, the inventors have shown that this reductase is only slightly more efficient than XenA for FBO production.

[0096] Among all the enzymes tested, some have little or no activity on HBA, such as XenA, RZS 1 (raspberry), and the reductase (Red) from Solanum chacoense. In contrast, the best conversion activity is obtained with the NADPH-dependent 2-alkenal reductase (AER) from Arabidopsis thaliana and ERED from Zingiber. officinale, CurA from P seudomonas putida, NADP-dependent alkenal double bond reductase (DBR) from Olimarabidopsis pumila and Nicotiana tobacco, 2-alkenase reductase (NADP(+)-dependent) (Red) from Capsicum annuum.

[0097] In addition to a high production of FBO, these reductases allow 100% conversion of the HBA precursor.

[0098] This study identified and characterized new enzymes, most of which were not previously known to have reductase activity on the HBA precursor. Expression of these enzymes in P. putida results in improved conversion activity and efficient production of frambinone compared to raspberry RKS. Thus, the use of these enzymes represents a substantial improvement over previously published results. These new enzymes will be very useful for the natural bioproduction of FBO by microorganisms. Free text for the sequence list

[0099] In this application, reference is made to the sequence listings whose identifiers (or “SEQ ID NO”) are listed in the table below. Regardless of the form in which the listings are provided, they form part of this application.

[0100] [Tables2] SEQ ID NO: 1 AA 2-alcénal réductase N ADPH dépendante d' Arabidopsis thaliana ( Uniprot Q39172) MTATNKQVILKDYVSGFPTESDFDFTTTTVELRVPEGTNSVLVK NLYLSCDPYMRIRMGKPDPSTAALAQAYTPGQPIQGYGVSRIIES GHPDYKKGDLLWGIVAWEEYSVITPMTHAHFKIQHTDVPLSYY TGLLGMPGMTAYAGFYEVCSPKEGETVYVSAASGAVGQLVGQ LAKMMGCYVVGSAGSKEKVDLLKTKFGFDDAFNYKEESDLTA ALKRCFPNGIDIYFENVGGKMLDAVLVNMNMHGRIAVCGMISQ YNLENQEGVHNLSNIIYKRIRIQGFVVSDFYDKYSKFLEFVLPHIR EGKITYVEDVADGLEKAPEALVGLFHGKNVGKQVVVVARE SEQ ID NO: 2 AA 2-alcénal reductase N ADPH dependent Zi ngiber officinale (Uni pratA0A096LNF0) MASAEDVVVVNKQVLLKHFIPEGAPKETDMELVTTGTIRLRVPE GSNAVLLKNLYLSCDPYMRMRMTKHEEASYVDDFVPGAPITGF GVGKVVDSSHPDFKTGDYVWGLIGWEEYSLITKPQGLFKIHHTE IPLSYYTGILGMVGLTAYVGFYDICSPKKGERVFVSAAAGAVGQ IKEEKIKYLEDIVEGLENAPAALIGLFEGRNVGKQVVVVSRE SEQ ID NO: 3 AA MPQSKLINRRVVLASRPHGAPLEANFRIEQSPIPEPAEGQVLLRTVYLSLDPYMRGRMSDAPSYAAPVEIGGVIVGGTVCRVEASKNPA YKVGDWVLSFAGWQDYTLSDGSDLTALGESPAHPSYALGIFGM curcumin réductase PGFTAYMGLLDIGRPQAGETLVVAAATGPVGATVGQIGKIKGCH NADPH dependent (Cura) of Pseudomon as putida(\3nvpmt Q8 8K17) VVGVAGGAEKCRHAVEVLGFDACLDHRAPDFAEQLAKACPAG IDIYFENVGGKVFDAVLPLLNTKARVPVCGIIAHYNDTALPNGPD RLPALMGSILRKRIHVQGFIIFDDYGHRYNEFFNDMSSWFAQGRI KYREELVSGLEEAPKAFIGLLEGRNFGKLVVRVSED SEQ ID N0:4 AA MTATATNKQVILKDYVSGFPKESDFDFTTTTVELKVPEGSNSVL VKNLYLSCDPYMRTRMGKPKPSTALAQAYALGQPIYGYGVSRV Alcénal double liaison IESGHPDYKKGDLLWGIVGWEEYSVITPTPDVHFKIQHTHVPLSY reductase NADP deep ndante (DBR) from Arabidopsis pumilaQJ niprot A0A1C9CX65) YTGLLGMPGMTAYAGFYEICSPKEGETVYVSAASGAVGQLVGQ FAKMMGCYVVGSAGSKEKVDLLKTKFGFDDAFNYKEEPDLSA ALKRCFPKGVDIYFENVGGKMLDAVLLNMNVHGRIAVCGMISQ YNLENQEGVHNLSNIIYKRIRIQGFAVFDFYDKYSKFLEFVLPQIK EGKIKYVEDVADGLEKGPEALVGLFHGKNVGKQVVVIARE SEQ ID NO:5 AA MAEEVSNKQVILKNYVTGYPKESDMEIKNVTIKLKVPEGSNDVV VKNLYLSCDPYMRSRRMKIEGSYVESFAPGSPITGYGVAKVLES 2-alcénal ductase N GDPKFQKGDLVWGMTGWEEYSIITPTQTLFKIHDKDVPLSYYTG ADP(+) dependent (DBR) from Nicotiana to bacco(Uniprot Q9SL N8) ILGMPGMTAYAGFHEVCSPKKGETVFVSAASGAVGQLVGQFAK MLGCYVVGSAGSKEKVDLLKSKFGFDEAFNYKEEQDLSAALKR YFPDGIDIYFENVGGKMLDAVLVNMKLYGRIAGMISQYNLE QTEGVHNLFCLITKRIRMEGFLVFDYHLYPKYLEMVIPQIKAGK VVYVEDVAHGLESAPTALVGLFSGRNIGKVVMVSRE SEQ ID NO: 6 AA MAEEVSNKQVILKHYVTGYPKEPDMEIKNGTIKLKVPEGSNAVL LKNLYLSCDPYMRSRMQKKEGSYVDSFTPGSPITGYGVAKVLES 2-alcenal reductase (N ADP(+) dependent from Capsicum annuum ( Uniprot A0A1U8GFY D GDSNFKKGDLVWGMTGWEEYSIFTAIHTLFLSYDVPLSYTGDG ILGMPGMTAYAGFYVCSPKKGETVFVSAASGAVGQLVGQFAK ILGCYVVGSAGSKEKVDLLKSKFGFDEAFNYKEEQDFAAALKR YFPDGIDIYFENVGGKMLDAVLLNMKLHGRIAVCGMISQYNLD KTEGVHNLFCLITKFLIGKHYPKYKYPKYKYKY VVYMEDVAEGLESAPSALVGLFSGRNVGKQVVMISHE SEQ ID NO:7 AA MAPRPTSQNQTRTCPTTQVTQVDIVEKMLAAPTDSTLELDGYSL NLGDVVSAARKGRPVRVKDSDEIRSKIDKSVEFLRSQLSMSVYG VTTGFGGSADTRTEDAISLQKALLEHQLCGVLPSSFDSFRLGRGL tyrosine ammonia lyas e (TAL_RG_OPT) ENSLPLEVVRGAMTIRVNSLTRGHSAVRLVVLEALTNFLNHGITP IVPLRGTISASGDLSPLSYIAAAISGHPDSKVHKVARKEGVYGEAMALFNLEPVVLGPKEGLGLVNGTAVSASMATLALHDAHMLS LLSQSLTAMTVEAMVGHAGSFHPFLHDVTRPHPTQIEVAGNIRK LLEGSRFAVHHEEEVKVKDDEGILRQDRYPLRTSPQWLGPLVSD LIHAHAVLTIEAGQSTTDNPLIDVENKTSHHGGNFQAAAVANTM EKTRLGLAQIGKLNFTQLTEMLNAGMNRGLPSCLAAEDPSLSYH CKGLDIAAAAYTSELGHLANPVTTHVQPAEMANQAVNSLALISA RRTTESNDVLSLLATHLYCVLQAIDLRAIVFEFKKQFGPAIVSLI DQHFGSAMTGSNLRDELVEKVNKTLAKRLEQTNSYDLVPRWH DAFSFAAGTVVEVLSTSTSLSLAAVNAWKVAAAESAISLTRQVRE TFWSAASTSSPALSYLSPRTQILYAFVREELGVKARRGDVFLGKQ EVTIGSNVSKIYEAIKSGRINNVLLKMLA SEQ ID NO: 8 AA MNNEARSGSTDPGQRPRYRQVAIGHPQVVSHVDDVLRMQPVE PLAPLPARLLERLVHWAQVRPDTTFIAARQADGAWRSISYVQML ADVRTIAANLLGLGLSAERPLALLSGNDIEHLQIALGAMYAGIAY 4-coumarate-CoA ligand (4-CL) CPVSPAYALLSQDFAKLRHVCEVLTPGVVFVSDSQPFQRAFEAV LDDSVGVISVRGQVAGRPHISFDSLQPGDLAAADAAFAATGPD TIAKFLFTSGSTKLPKAVITTQRMLCANQQMLLQTFPTFAEEPPV LVDWLPWNHTFGSHNLGIVLYNGGSFYLDAGKPTPQGFAETL RNLREISPTAYLTVPKGWEELVKALEQDPALREVFFARIKLFFFA AAGLSQSVWDRLDRIAEQHCGERIRMMAGGLGMTEASPSCTFTTT GPLSMAGYVGLPAPGCEVKLVPVGDKLEARFRGPHIMPGYWRS PQQTAEAFDEEGFYCSGDALKLADARQPELGLMFDGRIAEDFKL SSGVFVSVGPLRNRAVLEGSPYVQDIVVTAPDRECGLLVFPRLP ECRRLAGLAEDASDARVLANDTVRSWFADWLERLNRDAQGNASRIEWLSLLAEPPSIDAGEITDKGSINQRAVLQRRAAQVEALYRG EDPDALHAKVRP SEQ ID NO :9 AA MATEEMKKLATVMAIGTANPPNCYYQADFPDFYFRVTNSDHLI NLKQKFKRLCENSRIEKRYLHVTEEILKENPNIAAYEATSLNVRH KMQVKGVAELGKEAALKAIKEWGQPKSKITHLIVCCLAGVDMP benzalacétone synthas e (BAS) GADYQLTKLLDLDPSVKRFMFYHLGCYAGGTVLRLAKDIAENN KGARVLIVCSEMTTTCFRGPSETHLDSMIGQAILGDGAAAVIVG ADPDLTVERPIFELVSTAQTIVPESHGAIEGHLLESGLSFHLYKTV PTLISNNIKTCLSDAFTPLNISDWNSLFWIAHPGGPAILDQVTAKV GLEKEKLKVTRQVLKDYGNMSSATVFFIMDEMRKKSLENGQAT TGEGLEWGVLFGFGPGITVETVVLRSVPVIS SEQ ID NO : 10 AA MKKFEGGYVESFTPGSPITGYGVAKVLESDDSNFQKGDLVLGRT GWEEYSIVTATPTLFKIHDKDVPLSYYTGILGMPGLTAYAGFYE enzyme de type 2-alc énal réductase putativ e NADP(+) dépendan VCSPKKGETVFVSAASGAVGQLVGQFAKMLGCYVVGSAGSKE KVDRLKSTFGFDEAFNYKEEQDLDAALKRYFPDGIDIYFENVGG KMLDAVLLNMKIHGRIAVCGMISQYNLEQTEGVHNLFCLISKRI te de Solarium chacoe CMEGFLVFDYYHLYPKYLEMIIPQIKAGKVVYVEDVAEGLESAP n.sA Uniprot A0A0V0 SALVGLFSGRNIGKQVVMVSRE I4S3) SEQIDNO: 11 MASGGEMQVSNKQVIFRDYVTGFPKESDMELTTRSITLKLPQGS AA TGLLLKNLYLSCDPYMRARMTNHHRLSYVDSFKPGSPIIGYGVA Ketone / zingerone synth hase 1 (Uniprot G1FC GO) ;ZS1 RVLESGNPKFNPGDLVWGFTGWEEYSVITATESLFKIHNTDVPLS YYTGLLGMPGMTAYAGFYEICSPKKGETVYVSAASGAVGQLVG QFAKLTGCYVVGSAGSKEKVDLLKNKFGFDEAFNYKEEADLDA ALRRYFPDGIDIYFENVGMLGWEEYSVITATESLFKIHNTDVPLS NLEQPEGVRNLMALIVKQVRMEGFMVFSYHLYGKFLETVLPYI KQGKITYVEDVVDGLDNAPAALIGLYSGRNVGKQVVVVSRE SEQ ID NO: 12 MSALFEPYTLKDVTLRNRIAIPPMCQYMAEDGMINDWHHVHHA GLARGGAGLLVVEATAVAPEGRITPGCAGIWSDAHAQAFVPVV XENA (Uniprot Q88 NF7) QAIKAAGSVPGIQIAHAGRKASANRPWEGDDIAADDARGWET IAPSAIAFGAHLPKVPREMTLDDIARVKQDFVDAARRARDAGFE WIELHFAHGYLGQSFFSEHSNKRTDAYGGSFDNRSRFLLETLAA VREVWPENLPLTARFGVLEYDGRDEQTLEESIELARRFKAGLD LLSVSVGFTIPDTNIPWGPAFMGPIAERVRREAKLPVTSAWGFGT PQLAEAALQANQLDLVSVGRAKHLADPHWAKVEKVEKGLD WTLPAPYAHWLERYR SEQ ID NO: 13 5 ' - ATGACCGCAACC AACAAAC AGGTC ATCCTGAAGGACTACG Nt TCAGCGGCTTCCCCACCGAATCGGACTTCGACTTCACGACCAC 2-alcenal reductase NGACCGTTGAGTTGCGCGTCCCGGAGGGGACAAACAGTGTCCT ADPH dépendante d’ Arabidopsis thaliana ( Uniprot Q39172) GGTCAAGAACCTGTACCTGTCATGCGACCCGTACATGCGCAT TCGCATGGGCAAGCCGGACCCGAGCACCGCGGCACTGGCGCA GGCGTACACCCCGGGCCAACCCATCCAAGGCTACGGCGTGTC CCGTATCATCGAATCCGGCCACCCGGACTATAAAAAGGGTGA TCTGCTGTGGGGGATCGTCGCCTGGGAGGAATACTCTGTGAT CACGCCCATGACACACGCCCACTTCAAGATCCAACACACTGA CGTGCCGCTGTCCTACTACACCGGTCTCCTGGGGATGCCGGGT ATGACCGCATACGCTGGCTTCTACGAGGTGTGCAGCCCTAAG GAAGGCGAAACCGTGTACGTGAGCGCCGCCAGCGGCGCCGTC GGCCAGCTCGTAGGCCAGCTGGCGAAGATGATGGGATGCTAC GTGGTAGGCAGCGCTGGTAGCAAAGAGAAGGTGGACCTGCTG AAGACCAAGTTCGGTTTCGACGATGCCTTCAACTACAAGGAG GAGAGCGACCTCACCGCGGCCCTAAAGCGCTGCTTTCCTAAC GGCATAGACATCTATTTTGAAAACGTCGGTGGGAAGATGTTG GACGCCGTGCTGGTAAACATGAATATGCACGGCCGCATCGCG GTGTGTGGTATGATCTCGCAGTACAACCTGGAGAACCAGGAG GGCGTCCATAACCTGTCGAATATCATCTATAAACGTATCCGG ATTCAGGGGTTCGTGGTGTCGGATTTGATAAGTATTCGA AGTTCGACCGCCGATTGATTGATT TTACTTATGTGGAAGATGTTGCCGATGGCTTGGAAAAAGCCC CAGAAGCCCTTTGTGGGCCTTTTTCATGGCAAAAATGTTGGCA AACAGGTGGTGGTGGTAGCCCGGGAATGA-3' SEQ ID NO: 14 5 ' - ATGGCTTCGGCCGA AGATGTGGTGGTCAAGGGTCAAGGGGGGAATGA-3' SEQ ID NO: 14 5 ' TCCTGCTGAAGCATTTCATCCCAGAAGGCGCTCCGAAGGAAA 2-alcenal reductase N CGGATATGGAGCTGGTGACCACCGGTACCATCCGCCTCCGCG ADPH dépendante Zz ngiber officinale (Uni protA0A096LNF0) TGTCGTGCGACCATACATGCGCATGCGGATGACCAAGCACG AGGAAGCCTCATACGTGGACGACTTCGTGCCAGGCGCCCCCA TCACCGGCTTCGGAGTCGGCAAGGTGGTCGACTCGTCGCACC CGGATTTTAAGACCGGCGACTACGTGTGGGGCCTGATTGGCT GGGAAGAGTATTCGCTGATCACAAAGCCACAGGGCCTGTTCA AGATCCACCACACGGAAATCCCCCTCTCCTACTACACCGGCA TCCTGGGTATGGTAGGTCTGACCGTATGTCGGCTTCTATGA CATCTGCTCGCCGAAGAAAGGCGAACGCGTGTTCGTGTCCGCGGCTCGCCGGCCGAGCCGAGCCGAGCAGTTCGGCTGTTACGTTGTCGGCAGCGCAGTAGCGACGA GAAGGTAAACCTGCTGGAAGACGAAGTTCGCTTTGATGAAGC GTTCAACTACAAGAAGGAGCCGGACCTGAAAAAGCGCTGA AACGCTACTTCCCGGAGGGTATCGATATCTCTCGAGAAGCG TTGGTGGCCCGATGTGGAAGCCGTCCTTCACAACATGCGCA TCAAAGGTCGGATCGCGCGTGTGGCATGATCAGCCAGTACA ACCTGGAGAAGCCTGAGGGCGTCCATAACCTGTTTCCTGATCAGTA CTACGGGTCGTACCCCGAGTTCGAGGAAAAGGTCGTTCAGCT GATTAAGGAGGAAAAAATTAAGTACCTGGAAGATATCGTGGA AGGTTTGGGAGAACGCCCCTGCAGCCCTGAGACCCTGATAGGGCCCTGTTTGA AGGCCGCAACGTGGGCAAGCAAGTTGTAGTGGTGAGGCCGCGA ATGA-3' SEQ ID NO: 15 5' - ATGCCCCAATCAAAGCTAATCAATCGGCGCGTCGTTCTGGC Nt CTCACGTCCCCACGGTGCGCCTCTCGAAGCGAATTTTCGCATT GAGCAAAGCCCCATTCCTGAGCCAGCAGAAGGGCAGGTTCTG curcumin reductase TTGCGTACCGTTTACCTCTCACTTGACCCTTACATGCGTGGTC NADPH dependent ( CurA) of Pseudomon GCATGAGTGATGCACCGTCCTATGCTGCGCCGGTGGAAATCG GTGGAGTGATTGTGGGTGGCACTGTATGCCGTGTGGATTGTGGGTGGCACTGTATGCCGTGTGTGGATTGGGGTGGCACTGTATGCCGTGTGGATTGGTGGGTGGCACTGTGCCGTGTGGATTGGTGGGTGGCACTGTATGCCGTGTGGGATTGGGGTGGGCACTGTGCCGTGTGGATTGGGGTGGGCACTGTGGGAAATCG CGAAAAACCCGGCCTACAAGGTCGGTGACTGGGTGCTGTCCT TTGCTGGCTGGCAGGACTACACGCTGTCCGATGGAAGCGATC TGACCGCGTTGGGTGAGTCGCCGGCATCCTTCTTTATGCCTT GGGCATCTTTGGCATGCCGGGCTTCACCTT CTCGATATTGGCCGGCCGCAGGCGGGTGAAACCCTGGTGGTG GCGGCGGCCACCGGGCCGGTCGGCGCGACTGTGGGGCAGATC GGCAAGATCAAGGGCTGCCATGTGGTCGGTGTCGCCGGTGGC GCGGAAAAATGCCGGCATGCGGTCGAGGTGCT GCCTGTCTAGATCACCGTGCGCCGGACTTCGCCGAGCAACTG GCCAAGGCCTGCCCGGCAGGTATCGACATCTACTTCGAGAAT GTCGGCGGCAAGGTCTTCGATGCGGTGCTGCCGCTGCTCAAT ACCAAGGCCCGAGTGCCCGTCTGCGGCATTATTGCGCCACTACTAGCCCTAGCCTTGCCTT TGATGGGCAGCATTCTGCGCAAGCGTATTCATGTGCAGGGTT TCATTATTTTTGATTACGGCCACCGCTACAACGAGTTCTT TAACGATATGTCGAGCTGGTTTGCGCAGGGCCGGATTAAATA CCGTGAAGAATTAGTGAGTGGTCTGGAGGAGGCGCCTAAGGC CTTTATCGGCCTGCTCGAGGGGCGAAATTTTGGCAAGTTAGTGGTTCGCGTCAGTGAGGACTG A-3 ' SEQ ID NO: 16 5 ' - ATGACAGCCACTGCAACCAACAAGCAGGT AATCCTGAAGG Nt ACTATGTCTCAGGCTTCCCGAAAGAGTCGGACTTCGACTTCAC Alcenal double bond CACGACCACCGTGGAACTGAGGAGGAGGAGGACTCAGGTGACTCAG ndante (DBR) of Olim GGTCTTGGTGAAGAACCTGTACCTCTCGTGCGACCCCTACATG CGCACCCGTATGGGTAAGCCTAAGCCGAGCACGGCGTTGGCG arabidopsis pumilaQJ niprot A0A1C9CX65) CAGGCCTACGCTCTCGGCCAGTCGTTGTTGTT CGCGCGTCATCGAAAGCGGTCACCCAGACTACAAGAAGGGTG ATTTGCTGTGGGGCATCGTGGGGTGGGAAGAATACTCCGTGA TCACTCCGACGCCGGACGTTCATTTCAAGATTCAACACACCCA CGTGCCCTTGAGCTACTATACCGGGCTGCTGGGCATGCCGGGG CATGACCGCGTACGCGGGCTTCTACGAGATCTGCAGTCCTAA GGAGGGGGAGACCGTCTACGTTAGCGCCGCCAGCGGCGCGGT CGGACAGCTGGTGGGCCAGTTCGCCAAGATGGGCTGCTA TGTGGTAGGGTCCGCTGGCAGCAAGGAAAAGGTGGACCTGCT GAAAACCAAGTTCGGTTTCGACGACGCGTTTAATTACAAGGA GGAGCCCGATCTGAGCGCAGCCCTGAAGCGCTGCTTTCCGAA AGGGGTCGACATCTACTTTGAATGTAGGCGGCAAGATGCT GGACGCAGTGCTGCTGAACATGAACGTGCACGGCCGCATCGC CGTGTGTGGCATGATTTCCCAATAACCTGGAAAACAGGA AGACCTGACCATCCATTCGATCGATCGA CATCCAGGGCTTCGCCGTATTCGACTTCTACGATAAGTACAGC AAATTCCTGGAGTTCGTCCTACCGCAGATCAAGGAAGGAAAG ATCAAATACGTCGAAGATGTCGCCGATGGCCTCGAAAAAGGC CCCGAGGCCCTTGTCGGCCTTTCATGGCAAAAATGTGGGTA AACAAGTGGTGGTGGAQGGAQGGAQGTA NO: 17 5 ' - ATGGCAG AAGAGGTCTCAA ATAAAC AGGTGATCCTTAAA A Nt ACTATGTGACTGGCTACCCAAAAGAGAGCGACATGGAGATTA 2-alcenal reductase N AAAACGTCACGATCAAACTGAAGGTGCCGGAGGGCAGCAAT ADP(+) dependent (+) to Nicotian D GACGTCGTGGTGAAGAACCTGTACTTGTCGTGCGATCCGTAC ATGCGCTCGCGCATGCGTAAGATCGAGGGCAGCTACGTCGAA bacco (Uniprot Q9SL N8) AGCTTCGCTCCGGGCAGCCCGATCGGGTTACGGGGTGGCA CCTCGTCTGGGGGATGACCGGGTGGGAAGAGTACAGCATTTAT CACGCCAACCCAGACCCTGTCCAAAATCCATGACAAGGATGT GCCGTTGAGCTACTACACCGGGATCTTGGGCATGCCGGGGATGACCGCCTACGCTGGTTTTCACGAGGTGTGTAGTCCTAAGAA GGGTGAAACCGTCTTCGTCTCGGCCGCGAGCGGCGCCGTAGG TCAGCTGGTGGGCCAGTTTGCGAAGATGCTTGGCTGCTACGTC GTGGGTAGTGCGGGCTCCAAGGAGAAGGTAGACCTGCTGAAG TCGAAGTTCGGTTTCGATGAAGCGTTCAACTACAAGGAGGAA CAGGATCTGTCGGCCGCCCTGAAGCGCTACTTTCCTGATGGA ATCGACATCTATTTCGAAAACGTCGGCGGCAAGATGCTGGAC GCCGTGCTCGTCAACATGAAGCTGTACGGCCGTATTGCGGTG TGCGGCATGATCTCCCAGTACAACCTGGAACAAACCGAGGGC GTGCATAACCTGTTCTGCCTGATCACCAAGCGCATCCGGATG GAGGGCTTCCTGGTTTTCGACTACTATCACCTCTATCCCAAGT ATCTGGAAATGGTTATCCCCCAGATCAAGGCCGGCAAGGTGG TGTATGTGGAAGACGTGGCCCACGGCCTGGAATCGGCCCCCA CAGCCCTGGTTGGCCTATTCTCCGGCCGAAACATAGGCAAGC AGGTGGTTATGGTATCTCGCGAATGA-3 ’ SEQIDNO: 18 5 ’ - ATGGCGG AGGAAGTATCGAAT AAGCAGGTGATCCTCAAAC AA ACTACGTCACCGGCTACCCGAAGGAGCCTGACATGGAGATCA 2-alcenal reductase (N ADP(+) dependent of Capsicum annuum ( Uniprot A0A1U8GFY D AAAACGGCACCATCAAGCTGAAGGTGCCCGAAGGCTCCAACG CGGTTCTGTTGAAAAATCTCTCTGTCGTGCGATCCGTACAT GCTTCACGCCTGGCTCGCCGATTACCGGTTACGGCGTGGCAA AGGTACTGGAGTCGGGCGATTCGAACTTCAAGAAAGGCGACC TGGTGTGGGGCATGACCGGGTGGGAAGAGTAGCATTTTCCA CCGCCATCCACACCCTATTCAAGATTCATGATAAAGACGTGC CCCTCTCCTATTACACCGGCATCTGGGCATGCCGGGCATGAC GGCCTACGCCGGTTTTTACGAAGTCTGTAGCCCGAAGAAGGG CGAAACCGTATTCGTTAGCGCCGTCAGGTGCGGTGGGGCA GCTCGTCGGCCAATTTGCGAAGATCTGGGCTGCTACGTGGTGG GGCAGCGCCGGTTCCAAGGAGAAGGTGGACCTGCTGAAGAG CAAGTTCGGCTTTGACGAGGCCTTGAAACGCTACTTCCCAGACGGCAT CGACATCTATTTCGAGAACGTGGGGCGGCAAGATGCTGGACGC CGTGTTGGACCATGGCTGCTGCTGCTGCTGCTGCCAT CGGGATGATCAGTCAGTATAACCTGGATAAAACTGAAGGAGT GCACAACCTGTTCTGCCTTATCACAAAGCGTATCCGGATGGA GGGGTTCCTGGTTCGAATACTATCACCTGTACCCGAAGTAC CTTGAGATGATCATACCCCACATCAAGGCTGGTAAAGTCGTC TACATGAGATGAGTTGAGCCGACTGAGCCGGCCGCCCTGGTCGGATTGTTCAGCGGTCGAAACGTCGGGAAGCAG GTCGTCATGATCAGCCATGAATGA-3 ’ SEQ ID NO: 19 Nt tyrosine ammonia lyas e (TAL_RG_OPT) 5’- ATGGCCCCTCGCCCTACCTCACAGAACCAAACCCGCACATGCC CG SEQ ID NO: 20 Nt 4-coumarate-CoA liga se (4-CL) 5 ’ -GTGAAT AACGAAGCCCGCTCAGGGTCGACCGACCCTGGCC AACGTCCGCGCTACCGCCAGGTGGCCATCGGGCATCCCCAGG TGCAGGTCAGTCACGTCGACGACGTGCTGCGCATGCAACCTG TCGAGCCACTGGCGCCGCTGCCGGCGCGCCTGCTCGAGCGCC TGGTGCATTGGGCCCAGGTGCGCCCGGACACCACTTTCATCG CGGCACGCCAGGCAGACGGTGCCTGGCGTTCGATCAGCTACG TGCAGATGCTCGCCGATGTGCGCACCATCGCCGCCAACTTGCT AGGACTGGGCCTCAGTGCCGAGCGCCCGCTGGCGCTGCTTTC CGGCAACGACATCGAACACCTGCAAATCGCCCTCGGCGCCAT GTATGCCGGTATTGCCTATTGCCCGGTGTCGCCGGCCTACGCG CTGTTGTCGCAAGACTTCGCCAAGTTGCGCCATGTCTGCGAGG TGCTCACCCCCGGAGTGGTCTTCGTCAGCGACAGCCAGCCGT TCCAGCGCGCCTTCGAGGCGGTGCTGGACGATTCGGTCGGCG TGATCAGCGTGCGTGGCCAGGTCGCAGGTCGCCCCCATATAA GCTTCGACAGCCTGTTGCAACCGGGTGACCTGGCGGCGGCCG ATGCGGCTTTCGCCGCCACCGGGCCGGACACCATCGCCAAAT TCCTCTTCACCTCGGGCTCGACCAAGCTGCCCAAGGCGGTGA TCACCACCCAGCGCATGCTGTGCGCCAATCAGCAGATGCTTCT GCAGACTTTTCCGACGTTCGCCGAGGAGCCGCCGGTGCTGGT GGACTGGCTGCCGTGGAACCACACGTTCGGCGGTAGCCACAA CCTCGGCATCGTGCTTTACAACGGGGGCAGTTTCTACCTGGAC GCCGGCAAGCCGACCCCGCAAGGCTTCGCCGAGACCTTGCGC AATCTGCGCGAGATTTCCCCCACGGCCTACCTCACCGTACCCA AGGGCTGGGAGGAACTGGTCAAGGCACTGGAGCAGGACCCC GCGCTACGCGAGGTGTTCTTTGCCCGCATCAAGCTGTTCTTCT TTGCCGCCGCAGGCCTGTCGCAAAGCGTCTGGGACCGGCTGG ACCGCATTGCCGAGCAACACTGTGGCGAACGCATCCGCATGA TGGCCGGCCTTGGCATGACCGAAGCCTCGCCATCGTGCACCT TCACCACCGGGCCTTTGTCGATGGCCGGCTATGTCGGGCTGCC GGCACCTGGCTGCGAAGTGAAGCTGGTGCCGGTGGGCGACAA GCTCGAGGCGCGCTTCCGTGGCCCGCATATCATGCCGGGCTA CTGGCGCTCGCCGCAGCAGACCGCCGAGGCGTTCGACGAGGA GGGCTTCTACTGTTCGGGCGACGCGTTGAAGCTGGCCGATGCCAGGCAGCCCGAGCTTGGCCTGATGTTCGATGGCCGTATCGC TGAGGACTTCAAACTTTCGTCCGGGGTATTCGTCAGTGTCGGG CCGCTGCGCAACCGCGCAGTGCTGGAGGGCTCGCCTTACGTA CAGGACATCGTGGTCACCGCGCCGGACCGTGAATGCCTGGGC CTGCTGGTGTTCCCGCGTCTGCCCGAGTGTCGGCGCCTGGCCG GGCTGGCAGAGGATGCCAGCGATGCGCGGGTGCTGGCCAACG ACACCGTGCGCAGTTGGTTCGCTGACTGGCTGGAGCGCTTGA ACCGCGATGCCCAAGGCAACGCCAGCCGTATCGAATGGCTGT CGCTGCTGGCCGAGCCGCCGTCGATCGACGCCGGTGAAATCA CCGACAAGGGCTCGATCAATCAGCGCGCCGTGCTGCAGCGGC GCGCCGCTCAGGTCGAGGCGCTGTACCGTGGCGAAGACCCCG ACGCATTGCACGCCAAGGTGCGGCCTTGA-3’ SEQ ID NO : 21 Nt 5 ’ - ATGGCA ACTGAGG AGATGAAGAAATTGGCCACCGTGATGG CCATTGGCACGGCCAACCCTCCGAACTGCTACTACCAGGCCG benzalacétone synthas ACTTTCCCGACTTCTACTTCCGCGTCACCAACAGCGACCACCT e (BAS) CATCAACCTCAAGCAAAAGTTCAAGCGCCTTTGTGAAAACTC AAGGATTGAGAAGCGTTACCTTCATGTGACCGAAGAGATTCT CAAGGAAAACCCAAACATTGCTGCCTACGAGGCAACCTCGTT GAATGTAAGACACAAAATGCAAGTGAAAGGAGTTGCAGAGC TTGGGAAAGAGGCTGCCCTCAAGGCCATCAAAGAATGGGGCC AACCCAAGTCCAAGATCACACATCTCATCGTGTGTTGCCTAG CCGGCGTTGACATGCCCGGCGCGGATTATCAACTCACTAAGC TTCTTGACCTTGACCCTTCCGTCAAGCGTTTTATGTTTTACCAC CTAGGATGCTACGCTGGTGGCACTGTCCTTCGCCTTGCAAAGG ACATAGCGGAGAACAACAAGGGAGCTCGTGTTCTCATCGTTT GCTCAGAGATGACAACAACTTGTTTTCGTGGGCCATCTGAAA CCCATCTGGACTCCATGATAGGCCAAGCAATATTAGGCGATG GGGCTGCAGCTGTCATAGTTGGCGCAGATCCAGACCTAACCG TTGAGAGGCCCATATTCGAGTTGGTTTCCACAGCCCAGACTA TTGTACCCGAATCCCATGGTGCAATTGAGGGCCACTTGCTTGA ATCTGGACTCAGTTTCCATTTGTACAAGACCGTTCCTACACTA ATCTCTAACAACATTAAAACTTGCCTTTCTGATGCTTTCACTC CTCTAAACATTAGCGATTGGAACTCTCTTTTCTGGATCGCACA CCCTGGTGGTCCTGCCATCCTAGACCAAGTTACTGCTAAGGTT GGTCTTGAAAAGGAGAAACTCAAGGTAACTAGACAAGTGTTG AAGGACTATGGAAACATGTCGAGTGCTACGGTGTTTTTCATCATGGATGAGATGAGGAAGAAGTCACTCGAAAACGGTCAAGC AACCACTGGAGAAGGGCTCGAGTGGGGTGTTTTGTTTGGGTT CGGGCCTGGAATCACCGTTGAAACTGTAGTGCTACGCAGTGT GCCCGT AATT AGCT AG-3 ’

Claims

Demands

1. A genetically modified strain of Pseudomonas putida characterized in that it comprises a gene encoding: a) a benzalacetone reductase selected from the group consisting of: - NADPH-dependent 2-alkenal reductase (AER) from Arabidopsis thaliana having an amino acid sequence defined by the sequence SEQIDNO: 1, - NADPH-dependent 2-alkenal reductase from Zingiber officinale having an amino acid sequence defined by the sequence SEQ ID NO: 2, - NADP-dependent alkenal double bond reductase (DBR) from Olimarabidopsis pumila having an amino acid sequence defined by the sequence SEQ ID NO: 4, - NADP(+)-dependent 2-alkenal reductase (DBR) from Nicotiana tobacco having an amino acid sequence defined by the sequence SEQ ID NO: 5, and,- 2-alkenal reductase (NADP(+) dependent) from Capsicum annuum having an amino acid sequence defined by the sequence SEQ ID NO: 6 or b) a functional variant of a benzalacetone reductase having an amino acid sequence having at least 80% identity with one of the sequences chosen from SEQ ID NO: 1 to 2, 4 to 6 retaining benzalacetone reductase activity.

2. Genetically modified strain according to claim 1, characterized in that it further comprises one or more additional recombinant genes selected from: - the recombinant gene encoding a polypeptide with tyrosine ammonia lyase (TAL) activity, in particular a TAL polypeptide having at least 80% identity with the amino acid sequence SEQ ID NO: 7 of the TAL_RG_OPT polypeptide, - the recombinant gene encoding a polypeptide with 4-coumarate-CoA ligase (4-CL) activity, in particular, a 4CL polypeptide having at least 80% identity with the amino acid sequence SEQ ID NO: 8, and / or - the recombinant gene encoding a polypeptide with benzalacetone synthase (BAS) activity, in particular a BAS polypeptide having at least 80% identity with the sequence SEQ ID NO:

9.

3. Synthesis process for a compound of formula (I): [Chem 1]

4. where RI, R2 and R3 are chosen independently from each other from a hydrogen, an OH group and OCH3, and R4 is chosen from a methyl or aryl group, characterized in that it comprises a growth step of the genetically modified strain according to claim 1 or 2, in a culture medium under conditions permitting the expression of the recombinant gene(s) necessary for the synthesis of said compound of formula (I), said compound being synthesized by said genetically modified strain. A method according to claim 3 characterized in that the culture medium comprises a substrate of formula (II): [Chem 2]

5. Or RI, R2 and R3 are chosen independently from each other from a hydrogen, an OH group or OCH3; and R4 is chosen from a methyl or aryl group. A process according to claim 4 for synthesizing frambinone characterized in that the culture medium comprises 4-(4-hydroxyphenyl)-but-3-en-2-one (HBA).

6.

7. A process according to any one of claims 3 to 5, characterized in that it also comprises a step of recovering the compound of formula (I) in the culture medium. Use of the strain according to claim 1 or 2, for the synthesis of a compound of formula (I): [Chem 1] where RI, R2 and R3 are chosen independently from each other from a hydrogen, an OH group and OCH3 and R4 is chosen from a methyl or aryl group, preferably said compound being frambinone.

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