Biosynthesis of phenylpropanoid compounds

Abstract

The present invention relates to the field of phenylpropanoid compound production, and in particular to genetically modified strains for the production of phenylpropanoid compounds. Specifically, the invention relates to a genetically modified strain of Pseudomonas putida comprising a mutated AroF-I gene encoding 3-Deoxy-D-arabino-Heptulodonate 7-phosphate (DHAP), and its use for the synthesis of phenylpropanoid compounds, in particular coumaric acid or frambinone.

Description

Title of the invention: Biosynthesis of phenylpropanoid compounds Technical field

[0001] The present invention relates to the field of the production of phenylpropanoid compounds, and in particular that of genetically modified strains for the production of phenylpropanoid compounds such as coumaric acid or frambinone. Prior art

[0002] The bioproduction of "natural" aromas and fragrances has been an important area of ​​research for the industry for many years in order to respond to consumers who are increasingly concerned about being eco-responsible. Synthetic biology, particularly through the use of microorganisms, allows this natural production, but the yields are not always sufficient for large-scale production.

[0003] The flavor of raspberry (Rubus idaeus) is related 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 amounts in raspberries (1-4 mg per kg of fruit), natural frambinone is of great value (Larsen et al., 1991). However, since its natural availability is limited, its biotechnological production is highly desirable.

[0005] In this context, the biosynthetic pathway of phenylpropanoid compounds, in particular frambinone, can be reconstituted within a microorganism by means of the insertion of heterologous genes coding for certain key enzymes of said pathway.

[0006] Tyrosine is an important amino acid for the biosynthesis of phenylpropanoid compounds because it is notably the precursor of coumaric acid or frambinone.

[0007] Indeed, frambinone can be obtained from the aromatic amino acid L-tyrosine as the initial substrate via a 4-step biosynthetic pathway.

[0008] In the first step, tyrosine is deaminated by a tyrosine ammonia lyase (TAL, EC 4.3.1.23) to form coumaric acid. Catalyzed by a 4-coumarate:CoA ligase (4CL, EC 6.2.1.12), a Coenzyme A (CoA) molecule is grafted onto coumaric acid. Coumaroyl-CoA is then converted by a benzalacetone synthase (BAS, EC 2.3.1.212) to 4-hydroxy benzalacetone. This reaction is a decarboxylative condensation and uses a malonyl-CoA unit as a cosubstrate. The final step is the reduction of 4-hydroxy benzalacetone to frambinone by a benzalacetone reductase.

[0009] Thus, the development of tyrosine-overproducing strains as a platform for the production of phenylpropanoid compounds, notably frambinone, is a crucial starting point for the biosynthesis of these compounds.

[0010] However, tyrosine production in microorganisms is both complex and finely regulated. Indeed, certain enzymes in the tyrosine production pathway, also known as the shikimate pathway, are negatively regulated by this amino acid, which inhibits their activity by binding to an inhibition site when the concentration becomes too high in the cell. This regulatory system when tyrosine is present in too great a quantity in the microorganism is called negative feedback regulation.

[0011] Despite everything, it should be possible to obtain by mutagenesis enzymes "insensitive" to this inhibition and therefore to deregulate the tyrosine production pathway. These mutants insensitive to the negative feedback regulation of tyrosine are called fbr mutants for "feedback resistant".

[0012] Many articles describe the deregulation of this pathway in E. coli and one enzyme has been particularly studied: DAHP synthase (DAHP = 3-Deoxy-D-arabino-Heptulodonate 7-phosphate). This is the first reaction of the shikimate pathway which consists of the condensation of a phospho(enol)pyruvate (PEP) and an erythrose-4-phosphate (E4P) into DAHP.

[0013] This DAHP synthase activity is carried out via 3 isoenzymes in E. coli: AroG (retro-regulated by phenylalanine), AroF (retro-regulated by tyrosine) and AroH (retro-regulated by tryptophan). The structures of these proteins are known and the substructures (as well as the amino acids involved) related to retro-regulation are also very well described in the literature.

[0014] Kikuchi et al., 1997 and Cui et al., 2019, identified, described and studied in E. coli respectively AroGfbr and AroFfbr mutants insensitive to the feedback regulation of phenylalanine and tyrosine. fbr mutants of the TyrA protein (tyrAfbr mutant) were also described in E. coli by Lütke-Eversloh and Stephanopoulos, (2005) (Lütke-Eversloh and Stephanopoulos, Appl. Environ Microbiol. 2005 Nov; 71(11): 7224-8).

[0015] In 2007, Lütke-Eversloh and Stephanopoulos (Lütke-Eversloh and Stephanopoulos, Appl. Environ Microbiol. 2007 Nov; 75(1): 103-10) also described tyrosine-overproducing E. coli strains obtained by combining these different mutated enzymes. These strains were used to produce phenylpropanoids such as coumaric acid (Kang et al., 2012), or naringenin via, in particular, a mutation of the rpoA gene in addition to the aroGfbr and tyrAfbr mutations (Santos et al., 2011).

[0016] An overview of the modifications carried out to obtain tyrosine-overproducing E. coli strains is well described by the article “Modular engineering of L-tyrosine production in E. coli” (Juminaga et al., 2011).

[0017] Phenylpropanoid compounds have also been synthesized from Saccharomycescerevisiae strains that overexpress tyrosine, as described by Rodriguez et al., 2015.

[0018] GB 2 416 769 describes the possibility of producing frambinone using microorganisms containing genes encoding the enzymes 4CL and BAS, at least one of which is of heterologous source. The preferred microorganism is E. coli (strain BL21) and may further comprise a sequence encoding BAR, C4H, PAL and / or CHS, the BAR coding sequence advantageously being endogenous.

[0019] However, it appears that E. coli or S. cerevisiae strains do not tolerate the toxicity of phenylpropanoid compounds well and are therefore not the best-suited microorganisms for their production.

[0020] Bacteria of the genus Pseudomonas are more tolerant to these highly toxic molecules, notably the bacterium Pseudomonas putida (Calero et al., 2017). On the other hand, the enzymes involved in the production of aromatic amino acids in P. putida are poorly described.

[0021] Therefore, overproduction in P. putida of shiikimate pathway derivatives, such as tyrosine, involves the use of AroGfbr and TyrAfbr mutants from E. coli, which is not very efficient (Calero et al., 2016).

[0022] Overproduction of tyrosine and phenol has been described in the bacterium Pseudomonas taiwanensis VLB 120 through the implementation of point mutations in 3 genes: trpEP290S, aroF-I P148L and pheAT310I (Wynands et al., Metab Eng. 2018 May; 47:121-133).

[0023] However, there is still a need to develop new enzymes and new strains of Pseudomonas putida that overproduce tyrosine allowing the production of phenylpropanoid compounds, notably coumaric acid and frambinone. Technical problem

[0024] The overproducing strains of tyrosine developed to date are essentially E. coli and S. Cerevisiae strains. However, these strains do not tolerate the toxicity of phenylpropanoid compounds well and are therefore not the best-suited microorganisms for their production.

[0025] Thus, there is a particular need to develop new strains that overproduce tyrosine allowing the production of phenylpropanoid compounds.

[0026] The present disclosure improves the situation. Summary

[0027] One aspect of the present invention relates to a genetically modified strain of Pseudomonas putida comprising a mutated AroF-I gene encoding 3-Deoxy-D-arabino-Heptulodonate 7-phosphate (DAHP) synthase having the sequence SEQ ID NO: 1 and having at least one P160L mutation, a double P160L / Q164A mutation, or a double P160L / S193A mutation, preferably a double P160L / S193A mutation.

[0028] Another aspect of the invention relates to a method for synthesizing a phenylpropanoid compound or a phenylpropanoid derivative by using a genetically modified strain according to the invention of Pseudomonas putida.

[0029] Finally, the invention also relates to the use of a genetically modified strain of Pseudomonas putida for the synthesis of phenylpropanoid compounds.

[0030] The features set out in the following paragraphs may optionally be implemented. They may be implemented independently of one another or in combination with one another: Brief description of the figures

[0031] Other characteristics, details and advantages will appear on reading the detailed description below, and on analyzing the attached drawings, in which: Fig.l

[0032] [Fig.l] Graph of PEP consumed in mM in the presence or absence of aromatic amino acid by the wild-type (WT) AroF-I protein. The results presented are those of three independent replicates. Fig. 2

[0033] [Fig.2] Graph representing the proportion of PEP consumed in mM in the presence or absence of aromatic amino acid by the aroF-I WT protein compared to the simple mutants AroF-I-G191, AroF-I-P160 and AroF-LS193. The results presented are those of three independent replicates. Fig. 3

[0034] [Fig.3] Graph representing the proportion of PEP consumed in mM in the presence or absence of aromatic amino acid by the aroF-I WT protein compared to the double mutants AroF-LP160_G191, AroF-LP160L_Q164, AroF-I-P160_S190 and AroF-LP160_S193. The results presented are those of three independent replicates. Fig. 4

[0035] [Fig.4] Graph representing the proportion of PEP consumed in mM in the presence or absence of aromatic amino acid by the aroF-I WT protein compared to the single mutants AroF-LP160 and AroF-LS193 and the double mutant AroF-LP160_S193. The results presented are those of three independent replicates. Fig. 5

[0036] [Fig.5] presents the conjugation protocol that can be used to transform and genetically modify P. putida. Fig. 6

[0037] [Fig.6] Graph showing the production of coumaric acid (PCA) and cinnamic acid (CA) by Pseudomonas putida strains expressing the enzymes AroF-I WT / TAL (aroF-I WT) and aroF-I fbr P160L / S193A / TAL (aroF-I fbr). Fig. 7

[0038] [Fig.7] Graph showing the production of total phenolics and the proportion of coumaric acid among the total phenolics (%PCA) by Pseudomonas putida strains expressing the enzymes AroF-I WT / TAL (aroF-I WT), AroF-I WT / TAL + empty plasmid (aroF-I WT + control), AroF-I WT / TAL + plasmid phhA / B (aroF-I WT + phhA / B), aroF-I fbr P160L / S193A / TAL (aroF-I fbr), aroF-I fbr P160L / S193A / TAL + empty plasmid (aroF-I fbr + control), and aroF-I fbr P160L / S193A / TAL + plasmid phhA / B (aroF-I fbr + phhA / B). Expression of phhA / B genes from the araC / pBAD promoter is induced or not. Description of the embodiments

[0039] A first subject of the invention therefore relates to a genetically modified strain of Pseudomonas putida comprising a mutated AroF-I gene coding for 3-Deoxy-D-arabino-Heptulodonate 7-phosphate (DAHP) synthase, the sequence of which has at least 80% identity with the sequence SEQ ID NO: 1 and having at least one P160L mutation, a double P160L / Q164A mutation, or a double P160L / S193A mutation, preferably a double P160L / S193A mutation.

[0040] 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 to be synonymous with each other.

[0041] In particular, the term "genetically modified strain" means a strain which 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.

[0042] Indeed, 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 “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.

[0043] Preferably, for the integration of mutated or recombinant genes in Pseudomonas putida, the mutagenesis technique described in Example 2 will be used.

[0044] In particular, a genetically modified strain may comprise a nucleic acid modifying the expression of one or more genes naturally expressed in Pseudomonas putida.

[0045] According to a particular embodiment, a genetically modified strain may comprise a nucleic acid encoding one or more enzymes, not naturally expressed in Pseudomonas putida.

[0046] 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).

[0047] In addition, strains of Pseudomonas putida or Pseudomonas taiwanensis optimized for the production of tyrosine 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., 2016; Wierckx et al., 2005, Appl Environ Microbiol. 71(12):8221-7; Wynands et al., 2018; Otto et al. 2019, Front Bioeng Biotechnol Nov 20;7:312).

[0048] For the purposes of the present invention, the percentage of identity refers to the percentage of identical residues in a nucleotide or amino acid sequence 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 default parameters of the algorithm can be used.

[0049] Preferably, for nucleic acid sequence comparison and determination of percent identity, the blastn algorithm as described in https: / / blast.ncbi.nlm.nih.gov / Blast.cgi with default parameters is used.

[0050] For the purposes of the present invention, the term “mutated AroFl gene” means a nucleic acid comprising at least one part coding for a mutated version of 3-Deoxy-D-arabino-Heptulodonate 7-phosphate (DAHP) synthase under the control of a promoter allowing its expression in the genetically modified strain.

[0051] For the purposes of the present invention, 3-Deoxy-D-arabino-Heptulodonate 7-phosphate (DAHP) synthase means the enzymes (EC 2.5.1.54) capable of carrying out in bacteria the first reaction of the shikimate pathway which consists of the condensation of a phospho(enol)pyruvate (PEP) and an erythrose-4-phosphate (E4P) into DAHP.

[0052] According to a particular embodiment, the genetically modified strain of Pseudomonas putida comprises a mutated AroF-I gene coding for DAHP synthase whose amino acid sequence has at least 80%, 85%, 90%, 95% and very particularly, at least 98% identity with the sequence SEQ ID NO: 1, and having at least one P160L mutation, a double P160L / Q164A mutation, or a double P160L / S193A mutation, preferably a double P160L / S193A mutation.

[0053] The AroF-I gene endogenous to Pseudomonas putida codes for DAHP synthase of amino acid sequence SEQ ID NO: 1. In a particular embodiment, the genetically modified strain according to the present invention may therefore comprise, in addition to the endogenous AroF-I gene, at least one mutated AroF-I recombinant nucleic acid sequence encoding a mutated protein comprising the P160L mutation, the P160L / Q164A double mutation, or the P160L / S193A double mutation, preferably the P160L / S193A double mutation.

[0054] According to a particular embodiment, the genetically modified strain of Pseudomonas putida comprises a mutated AroF-I gene coding for DAHP synthase comprising a P160L mutation as defined by the amino acid sequence SEQ ID NO: 2.

[0055] According to a variant of this embodiment, the modified strain of Pseudomonas putida comprises a mutated AroF-I gene coding for DAHP synthase, the sequence of which has at least 80%, 85%, 90%, 95% and, in particular, at least 98% identity with the sequence SEQ ID NO: 2 and contains the P160L mutation.

[0056] According to another particular embodiment, the modified strain of Pseudomonas putida comprises a mutated AroF-I gene encoding DAHP synthase and having at least one double mutation P160L / Q164A. According to this embodiment, the mutated AroF-I gene encodes DAHP synthase comprising the double mutation P160L / Q164A defined by the amino acid sequence SEQ ID NO: 3.

[0057] According to a variant of this embodiment, the modified strain of Pseudomonas putida comprises a mutated AroF-I gene coding for DAHP synthase, the sequence of which has at least 80%, 85%, 90%, 95% and, in particular, at least 98% identity with the sequence SEQ ID NO: 3 and contains the double mutation P160L / Q164A.

[0058] According to a preferred embodiment, the modified strain of Pseudomonas putida comprises a mutated AroF-I gene encoding DAHP synthase having at least one double mutation P160L / S193A. According to this embodiment, the mutated AroF-I gene encodes DAHP synthase comprising the double mutation P160L / S193A defined by the amino acid sequence SEQ ID NO: 4.

[0059] According to a variant of this embodiment, the modified strain of Pseudomonas putida comprises a mutated AroF-I gene coding for DAHP synthase, the sequence exhibits at least 80%, 85%, 90%, 95% and in particular, at least 98% identity with the sequence SEQ ID NO: 4 and contains the double mutation P160L / S193A.

[0060] In one embodiment, which can be combined with the previous ones, the coding sequence of the mutated AroF-I gene 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 makes it possible to overexpress the mutated AroF-I gene in the genetically modified strain according to the invention. In a preferred embodiment, the coding sequence of the mutated AroF-I gene in the genetically modified strain according to the invention is inserted so as to render the AroH gene non-functional, for example by disruption of the AroH gene, or by deletion of the AroH gene and in particular of all or part of its coding sequence.

[0061] The AroH gene encodes another DAHP synthase (AroF isoenzyme). Thus, in one embodiment, the genetically modified strain according to the present invention comprises the deleted or disrupted AroH gene and at least one recombinant nucleic acid comprising the mutated AroF-I gene as described above or a coding sequence of the mutated AroF-1 gene.

[0062] In a highly advantageous manner, by implementing specific mutations, the Applicant has developed a strain of Pseudomonas putida capable of expressing a recombinant DAHP synthase insensitive to negative feedback regulation by tyrosine, thus deregulating the tyrosine production pathway. In other words, the present invention allows the production of strains that overproduce tyrosine, as a final product or as an intermediate product in the synthesis of phenylpropanoid compounds. In particular, this overproduction is particularly advantageous for the production of phenylpropanoid compounds by the strains modified according to the invention.

[0063] Phenylpropanoid compounds are a class of organic compounds, derived from plants, and biosynthesized from phenylalanine or tyrosine. Examples of phenylpropanoid compounds include coumaric acid, p-coumaroyl-coA, 4-hydroxybenzalacetone, frambinone, zingerone, vanillin, flavonoids, and stilbenoids.

[0064] For the purposes of the present invention, the term "tyrosine-overproducing strain" means a modified strain of Pseudomonas putida capable of producing tyrosine in greater quantities compared to a wild-type Pseudomonas putida strain comprising the AroF-I gene encoding DHAP synthase of amino acid sequence SEQ ID NO: 1 (non-mutated gene), either as a final product or as an intermediate product.

[0065] In order to be able to convert tyrosine into a phenylpropanoid compound, such as the acid coumaric, the modified strain according to the invention may further advantageously comprise at least one additional recombinant gene.

[0066] The expression "additional recombinant gene" is understood to mean, within the meaning of the present invention, any recombinant gene present in the Pseudomonas putida strain in addition to the mutated AroF-I gene as defined above. The additional recombinant gene may result from the insertion of a heterologous promoter, for example a strong promoter for overexpressing an endogenous Pseudomonas putida gene, or a recombinant coding sequence coding for a protein not naturally expressed in Pseudomonas putida.

[0067] According to a particular embodiment, the genetically modified strain of Pseudomonas putida comprises at least one additional recombinant gene coding for a polypeptide with phenylalanine hydroxylase activity (phhA) and one additional recombinant gene coding for a polypeptide with tetrahydrobiopterin dehydratase activity (phhB).

[0068] The Applicant has found that these two enzymes, although present endogenously in Pseudomonas putida, may not be sufficiently active and / or their expression may be insufficient in the context of use of the strain for the production of phenylpropanoid compounds. According to this embodiment, the activities were therefore optimized by overexpressing these two enzymes phhA and phhB. Overexpression can for example be obtained by placing the additional recombinant genes of these enzymes under the control of a heterologous promoter, in particular a constitutive or inducible promoter. Examples of such promoters are in particular the ptrc, xylS / pm, araC / pBAD promoters.

[0069] Preferably, overexpression of the phhA and phhB enzymes is achieved by placing the additional recombinant genes of these enzymes under the control of a strong inducible promoter araC / pBADopt (Prior et al., 2010).

[0070] 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 only under the control of the natural promoter. Overexpression can be obtained 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.

[0071] In another embodiment, which can be combined with the previous one, the endogenous coding sequences phhA and phhB are placed under the control of a heterologous promoter as defined previously, for example in order to overexpress the corresponding endogenous coding sequences in the genetically modified strain according to the invention.

[0072] In particular, the modified strain may comprise an additional recombinant gene coding for a phenylalanine hydroxylase (phhA) (EC 1.14.16.1) whose sequence is defined by the amino acid sequence SEQ ID NO: 5 or by a sequence having at least 80%, 85%, 90%, 95% and very particularly, at least 98% identity with the sequence SEQ ID NO: 5 and coding for an enzyme with phhA activity, and an additional recombinant gene coding for a tetrahydrobiopterin dehydratase (phhB) (EC 4.2.1.96) whose sequence is defined by the amino acid sequence SEQ ID NO: 6 or by a sequence having at least 80%, 85%, 90%, 95% and very particularly, at least 98% identity with the sequence SEQ ID NO: 6 and coding for a enzyme with phhB activity, in particular under the control of a heterologous promoter allowing their overexpression.

[0073] According to this embodiment, the genetically modified strain according to the invention is capable of overproducing tyrosine but also of transforming phenylalanine into tyrosine via the enzymes phhA and phhB.

[0074] According to a particular embodiment, the additional recombinant genes coding for phenylalanine hydroxylase (phhA) and for tetrahydrobiopterin dehydratase (phhB) comprise the corresponding coding sequences of Pseudomonas fluorescens (phhA VVN86558.1 / phhB: AYF50180.1) or Pseudomonas aeruginosa (phhA AAA25936.1 / phhB AAA25937.1). According to a preferred embodiment, the modified strain of Pseudomonas putida comprises a mutated AroF-I gene encoding DAHP synthase comprising the double mutation P160L / S193A defined by the amino acid sequence SEQ ID NO: 4, or a sequence having at least 85%, 90%, 95% and most particularly, at least 98% identity with the sequence SEQ ID NO: 4, and the two additional recombinant genes below: - a recombinant gene coding for a phenylalanine hydroxylase (phhA), preferably a phhA defined by the sequence SEQ ID NO: 5, or a sequence having at least 85%, 90%, 95% and in particular, at least 98% identity with the sequence SEQ ID NO: 5, and - a recombinant gene coding for a tetrahydrobiopterin dehydratase (phhB), preferably a phhB defined by the sequence SEQ ID NO: 6, or a sequence having at least 85%, 90%, 95% and in particular, at least 98% identity with the sequence SEQ ID NO: 6, preferably said phhA and phhB genes being placed under the control of a heterologous promoter allowing their overexpression.

[0075] According to another particular embodiment which can preferably be combined with the previous one, the genetically modified strain of Pseudomonas putida comprises an additional recombinant gene coding for a polypeptide with activity Tyrosine ammonia lyase (TAL). A recombinant gene encoding TAL may originate from the microorganism Rhodotorula glutinis and be optimized according to reference Zhou et al., 2015 (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 comprise 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 having 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.

[0076] According to this particular embodiment, the genetically modified strain according to the invention is capable of overproducing tyrosine but also of converting it into coumaric acid via the TAL enzyme.

[0077] According to another particular embodiment which can be combined with the previous ones, 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 very particularly, at least 98% identity with the sequence SEQ ID NO: 8 and encoding an enzyme with 4-CL activity.

[0078] According to this particular embodiment, the genetically modified strain is capable of converting coumaric acid into p-coumaryl-coA via the enzyme 4-CL.

[0079] According to another particular embodiment which can be combined with the previous ones, the genetically modified strain of Pseudomonas putida comprises an additional recombinant gene encoding a polypeptide with benzalacetone synthase (BAS) activity. In particular, the modified strain may comprise 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 having at least 80%, 85%, 90%, 95% and very particularly, at least 98% identity with the sequence SEQ ID NO: 9 and encoding an enzyme with BAS activity.

[0080] According to this particular embodiment, the genetically modified strain is capable of converting p-coumaryl-coA into 4-hydroxybenzalketone via the BAS enzyme.

[0081] According to a preferred embodiment, the genetically modified strain of Pseudomonas putida comprises several additional recombinant genes, namely in particular the five additional recombinant genes below: - a recombinant gene coding for a phenylalanine hydroxylase (phhA), preferably a phhA defined by the sequence SEQ ID NO: 5, in particular under the control of a heterologous promoter allowing its overexpression, - a recombinant gene coding for a tetrahydrobiopterin dehydratase (phhB), preferably a phhB defined by the sequence SEQ ID NO: 6, in particular under the control of a heterologous promoter allowing its overexpression, - a recombinant gene coding for 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.

[0082] The enzymes TAL, 4-CL and BAS are all enzymes involved in the synthesis of phenylpropanoid compounds.

[0083] According to this preferred embodiment, the modified strain is capable of producing a multitude of phenylpropanoid compounds, namely in particular coumaric acid, p-coumaroyl-coA, 4-hydroxybenzalketone.

[0084] Another subject of the invention relates to a genetically modified strain of Pseudomonas putida comprising an additional recombinant gene AroF-I coding for 3-Deoxy-D-arabino-Heptulodonate 7-phosphate (DHAP) synthase whose sequence has at least 80% identity with the sequence SEQ ID NO: 1, and in which the genes coding for the enzymes phhA and phhB are overexpressed.

[0085] Process for the synthesis of a phenylpropanoid compound

[0086] Another subject of the invention relates to a process for the synthesis of one or more phenylpropanoid compounds.

[0087] Phenylpropanoid compounds can be defined previously.

[0088] The synthesis method according to the invention comprises the implementation of a step of growing a genetically modified strain of Pseudomonas putida as defined previously in a culture medium under conditions allowing the expression of the mutated gene and / or additional recombinant genes necessary for the synthesis of one or more phenylpropanoid compounds.

[0089] According to a particular embodiment, the synthesis method according to the invention makes it possible to produce coumaric acid in large quantities.

[0090] According to a variant of this embodiment, the method may comprise a step of growing a genetically modified strain of Pseudomonasputida comprising a mutated AroF-I gene coding for 3-Deoxy-D-arabino-Heptulodonate 7-phosphate (DAHP) synthase, for example of sequence SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, and at least the additional recombinant genes below coding for: - a phenylalanine hydroxylase (phhA) of sequence SEQ ID NO: 5, - a tetrahydrobiopterin dehydratase (phhB) of sequence SEQ ID NO: 6, and - a tyrosine ammonia lyase (TAL_RG_OPT) of sequence SEQ ID NO: 7.

[0091] According to another particular embodiment, the synthesis process according to the invention makes it possible to produce frambinone, in particular in industrial quantities, and in particular with a yield at least equal to 20 g / l of frambinone in a fermenter of at least 500 l.

[0092] According to this embodiment, the method comprises a step of growing a genetically modified strain of Pseudomonas putida comprising a mutated AroF-I gene coding for 3-Deoxy-D-arabino-Heptulodonate 7-phosphate (DHAP) synthase of sequence SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. and comprising the following additional recombinant genes coding for: - 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.

[0093] The synthesis process according to the invention may also comprise a step of purification and / or recovery of the phenylpropanoid compound, such as coumaric acid or frambinone.

[0094] According to a particular embodiment, the strain is a genetically modified strain of Pseudomonas putida comprising an additional recombinant gene AroF-I coding for 3-Deoxy-D-arabino-Heptulodonate 7-phosphate (DHAP) synthase whose sequence has at least 80% identity with the sequence SEQ ID NO: 1, and in which the genes coding for the enzymes phhA and phhB are overexpressed.

[0095] Uses of the strains according to the invention

[0096] Another subject of the invention relates to the use of a strain of Pseudomonas putida as defined above for the synthesis of phenylpropanoid compounds.

[0097] According to a preferred embodiment, the phenylpropanoid compound is chosen from coumaric acid, p-coumaroyl-coA, 4-hydroxybenzalacetone and frambinone, preferably from coumaric acid and / or frambinone.

[0098] The present invention will be better understood in 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 which is defined by the claims. Examples

[0099] Example 1: Development of a mutant allowing the production of 3-Deoxy-D-arabino-Heptulodonate 7-phosphate (DHAP) insensitive to negative feedback regulation of tyrosine

[0100] A. In Silico Study

[0101] The starting enzyme used is the AroF-I enzyme identified as 3-Deoxy-D-arabino-Heptulodonate 7-phosphate (DAHP) in P. putida (Uniprot identifier Q88KG6).

[0102] The tertiary structure (in PDB format) of this enzyme was reconstructed from its protein sequence by the methodology described in Waterhouse, A. et al., (SWISS-MODEL: homology modeling of protein structures and complexes. Nucleic Acids Res. 46(W1), W296-W303 (2018)). The basic structure selected by sequence homology was that of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase from Saccharomyces cerevisiae.

[0103] The region that characterizes the retroregulation at tyrosine was identified in the literature via a homologous protein in E. coli. Through an alignment of tertiary structures of these two enzymes, the region corresponding to this retroregulation on AroF-I was identified.

[0104] Then, the “docking” of a tyrosine molecule with the AroF-I enzyme was carried out according to the methodology described by O. Trott et al., (“AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading”, Journal of Computational Chemestry, 2010).

[0105] This method made it possible to identify the amino acids involved in the formation of chemical bonds with phenylalanine in the docking pocket, namely, among others, the amino acids at positions 160 (P160), 164 (Q164), 190 (S190), 191 (G191), 193 (S193) and 225 (1225).

[0106] By searching the literature but also in the NCBI and Uniprot databases we identified protein sequences similar to AroF-I, but presenting a certain diversity of amino acids, in particular in the positions corresponding to those involved in docking.

[0107] This approach made it possible to identify amino acids that could replace the amino acids naturally found in the docking pocket of the AroF-I protein without modifying its structure or its activity.

[0108] Thus, only tyrosine binding is inhibited by the mutations highlighted. Table 1 below lists the positions and the original amino acids as well as the substitutes that characterize the mutations that could potentially deactivate the retroregulatory function of AroF-I.

[0109] [Tableauxl] Original amino acid (AroF-1) Position in FASTA Substitute amino acids P 160 A, L, T, V, MQ 164 A, RS 190 A, FG 191 KS 193 A, GI 225 P, V, T

[0110] Among the different mutations identified in Table 1, a subset of mutations was chosen according to the properties of the substitute amino acids as well as according to the impact of these mutations in evolutionarily close proteins already described in the literature ((Cui, D et al., Molecular basis for feedback inhibition of tyrosine-regulated 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase from Escherichia coli, 2019), (Ding, R. et al. Introduction of two mutations into AroG increases phenylalanine production in Escherichia coli. Biotechnol Lett 36, 2103-2108 (2014)), (Kikuchi Y et al. Mutational analysis of the feedback sites of phenylalanine-sensitive 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase of Escherichia coli. Appl Environ Microbiol 63:761-762 (1997). [YES] The chosen subset is: P160L, Q164A, S190A, G191K, S193A, and I225P.

[0112] B. Experiments

[0113] Materials and methods

[0114] Cloning of the genes coding for the AroF-I enzyme and derived mutants

[0115] In order to express and purify the AroF-I enzyme and the mutants derived therefrom, the The corresponding genes are cloned into a plasmid pET28_b(+) with a His-Tag grafted onto the N-terminal part of the protein (upstream of the ATG of the gene).

[0116] To carry out these constructions, the Gibson Assembly method is used (Gibson et al., 2009). The reaction product is transformed into E. coli BL21(DE3) strains (Novagen). The transformed cells are plated on agar medium with antibiotic for selection. The colonies obtained are then verified by PCR and sequenced in order to validate them.

[0117] Expression and purification of enzymes

[0118] The E. coli BL21(DE3) strains containing the plasmids are cultured for 24 hours in an auto-inducing ZYM medium whose composition is as follows:

[0119] a) Saline base: - Yeast extract: 5 g / 1 - tryptone: 10 g / l - Na2SO4: 0.7 g / 1 - NH4C1: 2.7 g / 1 - KH2PO4: 3.4 g / 1 - Na2HPO4: 4.45 g / l

[0120] b) Sugar solution (concentrated 50 times): - Glycerol: 250 g / 1 - Glucose: 25 g / 1 - Lactose: 100 g / 1

[0121] c) Metal solution (concentrated 5000 times): [Tables 2] Metal Solution Molar Mass (g / mol) Concentration (g / l) Concentration (mM) Final Concentration (mM) Per 100 ml(g) FeCl3 162.2 40.55 250 0.050 4.05 CaCl2.2H2O 147.01 14.70 100 0.100 1.47 MnCl2 125.84 6.29 50 0.050 0.63 ZnSO4.7H2O 287.56 14.38 50 0.050 1.44 CoCl2 129.84 1.30 10 0.010 0.13 CuCl2 134.45 1.35 10 0.010 0.13 NiCl2 129.60 1.30 10 0.010 0.13 Na2MoO4.2H2O 241.95 2.42 10 0.010 0.24 Na2SeO3 172.94 1.73 10 0.010 0.17 H3BO3 61.83 0.62 10 0.010 0.06

[0122] d) Preparation of the complete medium:

[0123] [Tables3] ZYM-5052 Per 50 ml ZYM saline base (ml) 49.5 MgSO4 IM (in pl) 100 5000x Metals Solution (in pl) 10 5052 50x Solution (in pl) 1 Antibiotic 50 mg / ml (in pl) 50

[0124] The medium is inoculated at D0=0.05 from an overnight preculture. The culture is carried out in a final volume of 50ml in a 250ml Erlenmeyer flask. The culture parameters are as follows: stirring 140rpm, 5 hours at 25°C then 19 hours at 15°C.

[0125] All chemicals used for the preparation of the medium come from Sigma-Aldrich, Roth or Euromedex.

[0126] After incubation, the 50ml of culture are centrifuged for 10 minutes at 5000xg 4°C. The supernatant is discarded and the pellet is taken up in 3ml of LEW buffer (Macherey-Nagel, see below), 1mg / ml of lyzosyme are added and the suspension is left on ice for 30 minutes.

[0127] The cells are then lysed by sonication (Omni Sonic Ruptor 400, Omni International, power 30%, pulse 40 for 8 minutes). After adding 0.22% Streptomycin, the samples are then centrifuged for 30 minutes at 15000xg 4°C. The supernatant is recovered and the purification of soluble proteins is carried out according to the supplier's recommendations using the Protino® Ni-TED 2000 kit (Macherey-Nagel)

[0128] Finally, the purified proteins are concentrated ~10 times using Amicon Ultra-4 30Kd centrifuge filters (Merck) and the elution buffer is replaced with 0.1M Tris-HCl pH7.5 + 10% Glycerol. The purified proteins are stored at -80°C.

[0129] Enzyme activity test

[0130] The activity of the enzymes is measured by monitoring the disappearance of the substrate with detection by HPLC (High Pressure Liquid Chromatography).

[0131] The reaction is carried out in a final volume of 200 μl with 40 mM phosphate buffer pH 7, 300 μM phospho(enol)pyruvate (PEP), 20 μg of purified enzyme, 1 mM tyrosine (or other aromatic amino acid) or 3 μM HCl, and the volume is made up with ultrapure water.

[0132] After pre-incubation for 2 minutes at 30°C, the reaction is started by adding 300pM of erythrose-4-phosphate (E4P) and the reaction mixture is incubated for 60 minutes at 30°C.

[0133] Finally, the mixture is heated for 5 minutes at 80°C in order to stop the reaction and precipitate the proteins.

[0134] Measurement of PEP consumption by HPLC

[0135] The enzymatic activity tests are first centrifuged for 10 minutes at 15000xg in order to precipitate the proteins. The supernatant obtained is filtered through a 0.22pm membrane before HPLC analysis.

[0136] The method used is described below: - System: Agilent 1100 (Agilent) - Column: Luna OMEGA polar Cl8 (Phenomenex) - Mobile phase: Phosphoric acid IrnM - Flow rate: 0.3ml / min - UV detection: 220nm - Injection: 5pl - Analysis time: 15 minutes

[0137] In order to quantify the amount of PEP present in the samples, a range of standards is analyzed with PEP concentrations from 0.1mM to 1mM.

[0138] Measurement of PC A and CA concentration by HPLC The column used is: Kinetex® 5pm F5 100A 150x4.6mm Mobile Phases: 0.1% Formic Acid and Acetonitrile Gradient: 5 min: 100% Formic acid 5min to 25min: 75% formic acid - 25% acetonitrile 25min to 30min: 62% Formic acid - 38% Acetonitrile 30min to 35min: 100% Formic acid Flow rate: Iml / min Oven temperature: 40°C Sample injection: lOpl Coumaric acid detection: 315nm Cinnamic acid detection: 280nm

[0139] Cloning of phaA / B genes into an arabinose-dependent expression plasmid The phhA / B genes were amplified by PCR directly from gDNA of Pseudomonas putida strain KT2440 and cloned into a pBBRl-MCS2 plasmid, under the control of the araC / pBAD promoter.

[0140] To carry out these constructions, the Gibson Assembly method is used (Gibson et al., 2009). The reaction product is transformed into the E. coli BL21(DE3) strain (Novagen). The transformed cells are plated on agar medium with antibiotic for selection. The colonies obtained are then verified by PCR and the plasmids sequenced in order to validate them.

[0141] The expression plasmid is then transformed into the donor E. coli strain S 17.1 in order to be transferred into the Pseudomonas putida strains of interest by conjugation.

[0142] Results

[0143] The activity of the wild-type AroF-I enzyme of Pseudomonas putida is indeed inhibited by tyrosine ([Fig.l]). The introduction of the P160L mutation makes the enzyme partially insensitive to this inhibition by tyrosine ([Fig.2]), and the effect can be further slightly improved by the introduction of a double mutation P160L / Q164A ([Fig.3]). On the other hand, the double mutations P160L / G191K and P160L / S190 have a deleterious effect on the AroF-I enzyme, which no longer has any activity, whether in the presence of tyrosine or not ([Fig.3]).

[0144] On the other hand, and in a particularly interesting and surprising manner, the introduction of a double mutation P160L / S193A restores almost all of the enzymatic activity and makes the AroF-I enzyme almost completely insensitive to tyrosine inhibition ([Fig.3]).

[0145] These results are confirmed by [Fig.4] which clearly shows the synergistic effect of this double mutation on the negative feedback regulation exerted by tyrosine compared to the single P160L or S193A mutation.

[0146] Example 2: Genetically modified strains of Pseudomonas putida for the synthesis of phenylpropanoid compounds

[0147] In order to enable the biosynthesis of phenylpropanoid compounds, several genetic modifications are carried out within the Pseudomonas putida strain. For this, additional recombinant genes encoding enzymes for the synthesis of phenylpropanoid compounds such as coumaric acid or frambinone are integrated into the Pseudomonas putida chromosome according to the following protocol.

[0148] Protocol for genetic mutagenesis of Pseudomonas putida:

[0149] Genetic mutagenesis in Pseudomonasputida is carried out by means of suicide plasmids which integrate into the chromosome and exit leaving the desired gene deletion or insertion. The suicide plasmid used is pK18mobsacB (Schafer A, Tauch A, Jâger W, Kalinowski J, Thierbach G, Pühler A. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Coryne-bacterium glutamicum. Gene 1994 Jul 22; 145(1):69-73). This plasmid carries a resistance cassette to the antibiotic Kanamycin and the counter-selection cassette sacB. It also has an origin of replication that only functions in the bacterium E. coli, and an origin of transfer oriT that allows it to be transferred by conjugation from E. coli to another bacterial strain such as Pseudomonas putida.

[0150] The genes to be inserted are cloned into this plasmid, as well as the homology zones at the chosen insertion site on the bacterial chromosome. These homology zones are cloned on either side of the gene to be inserted, and must have a size of 800 pairs of minimum bases.

[0151] Cloning is done in a strain of Escherichia coli capable of conjugation (generally the strain S 17.1, Simon, R., Priefer, U. and A. Pülher, A broad host range mobilization System for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Nature BioTechnology volume 1, pages 784-791 (1983)).

[0152] The conjugation protocol is as follows:

[0153] A drop of S17.1 / suicide plasmid culture (donor strain) is placed on a rich LB Agar medium (Luria-Miller, Roth, reference X968.2 containing 1.5% agar), and a drop of Pseudomonas putida strain culture (recipient strain) is placed on the first drop. The culture dish is incubated overnight at 30°C. The drop is then diluted in 10 mM MgSO4 and the protocol is carried out as described in [Fig.6].

[0154] The conjugation plasmid pK18mobsacB is an integrative plasmid capable of integrating into the genome of the recipient bacterial strain in order to produce transconjugants. Since the plasmid is resistant to Kanamycin, the transconjugant selection medium is LB Agar containing Ampicillin 100 mg / ml (Pseudomonas putida is naturally resistant to this antibiotic), and Kanamycin at 50 mg / ml (Kanamycin, ROTH reference T832.4; Ampicillin, EUROMEDEX, reference EU0400-D). This medium therefore makes it possible to select Pseudomonas putida bacteria in which a plasmid has integrated.

[0155] The rest of the protocol includes the following steps: - Excision of the suicide plasmid: take 8 Kn+AmpR clones and culture them in 10ml of LB+Amp 100 mg / ml medium (dilution 1 / 2000) at 30°C with shaking. Incubate for 12 to 24 hours. - sacB counter-selection on Sucrose 25%: streak the clone pool culture onto YT agar and YT+Sucrose 25% agar. Incubate at 30°C until the next morning. Subculture 20-25 Sucrose+ clones onto YT agar, YT+Sucrose 25% agar and YT agar +Kn 50 mg / ml. Incubate at 30°C until the next morning. YT: Yeast extract tryptone - PCR verification of gene insertion

[0156] Primers hybridizing on either side of the chromosomal insertion zone make it possible to verify the mutant clones by carrying out colony PCRs for the KnS and Sucrose+ clones.

[0157] Genes to be inserted into Pseudomonas putida

[0158] One or more of the genes listed below are inserted into Pseudomonas putida to enable the biosynthesis of phenylpropanoid compounds, such as coumaric acid or frambinone.

[0159] These genes have been specifically identified and selected in microorganisms known. Genes are synthesized and codons are optimized for maximum expression in Pseudomonas putida.

[0160] Some genes are endogenous to Pseudomonas putida, but it is recommended to test the activity of the proteins for which they code and if these enzymes are not active enough or their expression insufficient, these activities can be optimized.

[0161] Genes encoding a mutated enzyme 3-Deoxy-D-arabino-Heptulodonate 7-phosphate (DHAP) synthase:

[0162] SEQ ID NO: 10: sequence of the 3-Deoxy-D-arabino-Heptulodonate 7-phosphate (DHAP) synthase gene with P160L mutation (Pseudomonas putida KT2440) encoding the DHAP synthase of sequence SEQ ID NO: 2.

[0163] SEQ ID NO: 11: Sequence of the 3-Deoxy-D-arabino-Heptulodonate 7-phosphate (DHAP) synthase gene with double mutation P160L / Q164A (Pseudomonas putida KT2440) encoding the DHAP synthase of sequence SEQ ID NO: 3.

[0164] SEQ ID NO: 12: 3-Deoxy-D-arabino-Heptulodonate 7-phosphate (DHAP) synthase gene sequence with double mutation P160L / S193A (Pseudomonas putida KT2440) encoding DHAP synthase of sequence SEQ ID NO: 4.

[0165] Additional genes enabling the synthesis of phenylpropanoid compounds

[0166] SEQ ID NO: 13: phenylalanine hydroxylase (phhA) gene (Pseudomonas putida KT2440) encoding phhA of sequence SEQ ID NO: 5.

[0167] SEQ ID NO: 14: gene (Pseudomonas putida KT2440) of tetrahydrobiopterin de-shydratase (phhB) encoding phhB of sequence SEQ ID NO: 6.

[0168] SEQ ID NO: 15: tyrosine ammonia lyase gene (Rhodotorula glutinis) encoding beta-xylosidase of sequence SEQ ID NO: 7.

[0169] SEQ ID NO: 16: gene (fcs gene Pseudomonas putida KT2440) of 4-coumarate-CoA ligase (4-CL) encoding 4-CL of sequence SEQ ID NO: 8.

[0170] SEQ ID NO: 17: benzalacetone synthase (BAS) gene (Rheum palmatum) encoding BAS of sequence SEQ ID NO: 9.

[0171] Example 3: In vivo activity of a genetically modified Pseudomonasputida strain according to the invention

[0172] Preparation of an AroF-I-fbr P160L / S193A mutant

[0173] A Pseudomonas putida KT2440 strain was genetically modified as described in Example 1 so as to express the AroF-I gene encoding DHAP synthase comprising the double mutation P160L / S193A defined by the amino acid sequence SEQ ID NO: 4, and to express the additional recombinant gene encoding a heterologous tyrosine ammonia lyase (TAL_RG_OPT) of sequence SEQ ID NO: 7.

[0174] This strain is called “mutant AroF-I-fbr P160L / S193A”.

[0175] The activity of the AroF-I-fbr P160L / S193A mutant on the production of coumaric acid (CPA) or cinnamic acid (CA) was determined by measuring the amounts produced from these acids compared to a Pseudomonas putida KT2440 strain genetically modified to express the wild-type AroF-I enzyme and which serves as a control (Mutant AroF-I-WT).

[0176] Results

[0177] Cinnamic acid production increases significantly in a P. putida strain expressing the AroF-I fbr enzyme having the double mutation P160L / S193A ( [Fig.6]).

[0178] Optimization of the AroF-I-fbr P160L / S193A mutant for the production of coumaric acid

[0179] The AroF-I-fbr P160L / S193A mutant was modified to express additional recombinant genes encoding a phenylalanine hydroxylase (phhA) of sequence SEQ ID NO: 5 and a tetrahydrobiopterin dehydratase (phhB) of sequence SEQ ID NO: 6. The genes encoding these enzymes were cloned into a plasmid and expressed under the control of the inducible promoter araC / pBADopt, induction being carried out with 0.5% arabinose: plasmid pC2F387.

[0180] This strain is called “optimized AroF-I-fbr mutant”.

[0181] The production of coumaric acid (CPA) or cinnamic acid (CA) of the optimized AroF-I-fbr mutant was measured and then compared to that obtained with the AroF-I-fbr P160L / S193A mutant, and that obtained with two control strains, namely: - a Pseudomonasputida KT2440 strain expressing the wild AroF-I enzyme (Mutant AroF-I-WT), and - a Pseudomonasputida KT2440 strain expressing the wild-type AroF-I enzyme and the two enzymes phhA and phhB of sequences SEQ ID NO: 5 and SEQ ID NO: 6 respectively (Mutant AroF-I-WT phhA / B).

[0182] Results

[0183] The results presented in [Fig.6] show that overexpression of the phhA and phhB genes in the AroFLWT mutant strain results in greater production of phenolic acids (PCA + CA).

[0184] Of particular interest, the optimized AroF-I-fbr mutant allows for a much higher production of total phenolic acids than the AroF-I-fbr P160L / S193A mutant and the AroF-I-WT phhA / B mutant. Proportionally, coumaric acid represents almost all of the total phenolics produced and this is advantageous because cinnamic acid is not a compound that can be exploited industrially for the production of phenylpropanoid compounds.

[0185] These results clearly demonstrate that the optimized AroF-I-fbr mutant is particularly suitable for the production of phenylpropanoid compounds, and particularly coumaric acid. Free text of sequence listing

[0186] In the present 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 the present application.

[0187] [Tableaux4] SEQIDNO:1 AA AroF-I sauvage LAAGTRDLTSNTMADLPIDDLNVASNETLITPDQLKKEIPL SAKALQTVTAGREVVRNILDGKDHRLFVVVGPCSIHDIKA AHEYAERLKVLAEEVSDTLYLVMRVYFEKPRTTVGWKG LINDPYLDDSFKIQDGLHIGRKLLLDLAEMGLPTATEALD PISPQYLQDLISWSAIGARTTESQTHREMASGLSSAVGFKN GTDGGLTVAINALQSVSKPHRFLGINQEGGVSIVTTKGNP YGHVVLRGGNGKPNYDSVSVALCEQDLAKAKIKANIMV DCSHANSNKDPALQPLVMENVANQILEGNQSIIGLMVES HLNWGCQSIPKNLDDLQYGVSITDACIDWSATEKTLRSM HAKLKDVLPQRKRG SEQ ID NO:2 AA AroF-I_P160L LAAGTRDLTSNTMADLPIDDLNVASNETLITPDQLKKEIPL SAKALQTVTAGREVVRNILDGKDHRLFVVVGPCSIHDIKA AHEYAERLKVLAEEVSDTLYLVMRVYFEKPRTTVGWKG LINDPYLDDSFKIQDGLHIGRKLLLDLAEMGLPTATEALD LISPQYLQDLISWSAIGARTTESQTHREMASGLSSAVGFK NGTDGGLTVAINALQSVSKPHRFLGINQEGGVSIVTTKGN PYGHVVLRGGNGKPNYDSVSVALCEQDLAKAKIKANIM VDCSHANSNKDPALQPLVMENVANQILEGNQSIIGLMVE SHLNWGCQSIPKNLDDLQYGVSITDACIDWSATEKTLRS MHAKLKDVLPQRKRG SEQ ID NO:3 AA AroF-I_P160L / Q16 4A LAAGTRDLTSNTMADLPIDDLNVASNETLITPDQLKKEIPL SAKALQTVTAGREVVRNILDGKDHRLFVVVGPCSIHDIKA AHEYAERLKVLAEEVSDTLYLVMRVYFEKPRTTVGWKGLINDPYLDDSFKIQDGLHIGRKLLLDLAEMGLPTATEALD LISPAYLQDLISWSAIGARTTESQTHREMASGLSSAVGFK NGTDGGLTVAINALQSVSKPHRFLGINQEGGVSIVTTKGN PYGHVVLRGGNGKPNYDSVSVALCEQDLAKAKIKANIM VDCSHANSNKDPALQPLVMENVANQILEGNQSIIGLMVE SHLNWGCQSIPKNLDDLQYGVSITDACIDWSATEKTLRS MHAKLKDVLPQRKRG SEQ ID NO:4 AA AroF-I_P160L / S19 3A LAAGTRDLTSNTMADLPIDDLNVASNETLITPDQLKKEIPL SAKALQTVTAGREVVRNILDGKDHRLFVVVGPCSIHDIKA AHEYAERLKVLAEEVSDTLYLVMRVYFEKPRTTVGWKG LINDPYLDDSFKIQDGLHIGRKLLLDLAEMGLPTATEALD LISPQYLQDLISWSAIGARTTESQTHREMASGLASAVGFK NGTDGGLTVAINALQSVSKPHRFLGINQEGGVSIVTTKGN PYGHVVLRGGNGKPNYDSVSVALCEQDLAKAKIKANIM VDCSHANSNKDPALQPLVMENVANQILEGNQSIIGLMVE SHLNWGCQSIPKNLDDLQYGVSITDACIDWSATEKTLRS MHAKLKDVLPQRKRG SEQ ID N0:5 AA MKQTQYVAREPDAHGFIDYPQQEHAVWNTLITRQLKVIE GRACQEYLDGIDQLKLPHDRIPQLGEINKVLGATTGWQV phénylalanine hydroxylase (phhA) ARVPALIPFQTFFELLASKRFPVATFIRTPEELDYLQEPDIF HEIFGHCPLLTNPWFAEFTHTYGKLGLAATKEQRVYLAR LYWMTIEFGLMETAQGRKIYGGGILSSPKETVYSLSDEPE tetrahydrobiopterin e deshydratase TEWGKVTVTWWSHSIKGLHRND FIMCARTDK-VAETAEGRK (phhB) SEQ ID N0:7 AA MAPRPTSQNQTRTCPTTQVTQVDIVEKMLAAPTDSTLEL DGYSLNLGDVVSAARKGRPVRVKDSDEIRSKIDKSVEFLR tyrosine ammonia lyase (TAL_RG_OPT) SQLSMSVYGVTTGFGGSADTRTEDAISQLQLQLEKHLCGG VLPSSFDSFRLGRGLENSLPLEVVRGAMTIRVNSLTRGHS AVRLVVLEALTNFLNHGITPIVPLRGTISASGDLSPLSYIAA AISGHPDSKVHVHEGKILYAREAMALFNLEPVLGP KEGLGLVNGTAVSASMATLALHDAHMLLSQSLTAMT VEAMVGHAGSFHPFLHDVTRPHPTQIEVAGNIRKLLEGSR FAVHHEEEVKVKDDEGILRQDRYPLRTSPQWLGPLVSDLI HAHAVLTIEAGQSTTDNPLIDVENKTSHGGNFQAAAVA NTMECTRLGLAQIGKLNFTQLTEMLGLPSCLAGMNAGM EDPSLSYHCKGLDIAAAAYTSELGHLANPVTTHVQPAEM ANQAVNSLALIS ARRETES ND VLSLLLATHLYCVLQAIDL RAIVFEFKKFGPAIVSLIDQHFGSAMTGSNLRDELVEKV NKTLAKRLEQTNSYDLVPRWHDALSFAGFSSTVSTVSTV LSLAAVNAWKVAAAESAISLTRQVRETFWSAASTSSPALS YLSPRTQILYAFVREELGVKARRGDVFLGKQEVTIGSNVS KIYEAIKSGRINNVLLKMLA SEQ ID NO: 8 AA MNNEARSGSTDPGQRPRYRQVAIGHPQVQVSHVDDVDDVLR MQPVEPLAPLPARLLERLVHWAQVRPDTTFIAARQADGA 4-coumarate-CoA WRSISYVQMLADVRTIAANLLGLGLSAERPLALLSGNDIE ligase (4-CL) HLQIALGAMYAGIAYCPVSPAYALLSQDFAKLRHVCEVL TPGVVFVSDSQPFQRAFEAVLDDSVGVISVRGQVAGRPHI SFDSLLQPGDLAAADAAFAATGPDTIAKFLFTSGSTKLPK AVITTQRMLCANQQMLLQTFPTFAEEPPVLVDWLPWNHT FGGSHNLGIVLYNGGSFYLDAGKPTPQGFAETLRNLREIS PTAYLTVPKGWEELVKALEQDPALREVFFARIKLFFFAAA GLSQSVWDRLDRIAEQHCGERIRMMAGLGMTEASPSCTF TTGPLSMAGYVGLPAPGCEVKLVPVGDKLEARFRGPHIM PGYWRSPQQTAEAFDEEGFYCSGDALKLADARQPELGL MFDGRIAEDFKLSSGVFVSVGPLRNRAVLEGSPYVQDIVV TAPDRECLGLLVFPRLPECRRLAGLAEDASDARVLANDT VRSWFADWLERLNRDAQGNASRIEWLSLLAEPPSIDAGEI TDKGSINQRAVLQRRAAQVEALYRGEDPDALHAKVRP SEQ ID NO :9 AA MATEEMKKLATVMAIGTANPPNCYYQADFPDFYFRVTN SDHLINLKQKFKRLCENSRIEKRYLHVTEEILKENPNIAAY benzalacétone EATSLNVRHKMQVKGVAELGKEAALKAIKEWGQPKSKI synthase (BAS) THLIVCCLAGVDMPGADYQLTKLLDLDPSVKRFMFYHLG CYAGGTVLRLAKDIAENNKGARVLIVCSEMTTTCFRGPSE THLDSMIGQAILGDGAAAVIVGADPDLTVERPIFELVSTA QTIVPESHGAIEGHLLESGLSFHLYKTVPTLISNNIKTCLSD AFTPLNISDWNSLFWIAHPGGPAILDQVTAKVGLEKEKLK VTRQVLKDYGNMSSATVFFIMDEMRKKSLENGQATTGE GLEWGVLFGFGPGITVETVVLRSVPVIS SEQIDNO :10 Nt 5 ’-TTGGCGGCGGCACCCGTGACCTGACGAGTAACAG ATGGCTGATTTACCGATCGATGACTTGACGTTGCCTC AroF-I_P160L CAACGAGACCCTGATCACCCCTGATCAGCTCAAGAAGG AAATCCCCTCAGCGCCAAGGCCCTGCAGACCGTGACT GCCGGCCGTGAAGTGGTGCCGCAATATTCTCGACGGCAA GGACCATCGCCCTGTTCGTCGTCTGGCCCTTGCTCCAT CCACGACATCAAGGCAGCCCACGAATACGCCGAGCGC CTGAAAGTGCTGGCCGAAGAGTGTCCGATACGCTGA CCTGGTCATGCGCGTGTACTTCGAAAAGCCGCGCCACCA CCGTCGGCTGGGAAAGGCCTGATCAACGATCCGTACCTG GATGACTCGTTCAAGATCCAGGACGGCCTGCACATCGG CCGCAAGTTGCTGCTGGACCTGGCCGAAATGGGCCTGC CGACCGCCACCGAAGCGCTGACCTGATTTCGCCGCAG TACCTGCAAGACCCTGATCAGCTGGTCGGCCATCGGTGC CCGCACCACCGAATCGCAAACACACCGGAGATGGCC TCGGGCCTGTCCTCGGCGGTGGGTTCAAGAACGGTAC CGATGGCGGCCTGACCGTTGCCATCAATGCCCTGCAGT CGGTGTCCAAGCCGCACCGCTTCCTGGGCATCAACCAG GAAGGCGGCGTGTCGATCGTCACCACCAAGGGCAACC CATACGGCCACGTGGTACTGCGCGGCGCAATGGCAA GCCGAACTACGACTCGGTCAGCTCCCGTGCGAAC AGGACCTGGCCAAGGCCAAAGATCAAGGCCAACATCAT GGTCGACTGCAGCCATGCCAACTCCAAAGGACCCG GCCCTGCAACCGCTGGTGATGGAGAACGTCGCCAACCA GATTCTCGAAGGCAACCAGTCGATCATCGGCCTGATGTCGAAAGCCACCTGAACTGGGGCTGTCAGTCCATTCCG AAAAACCTGGACGATTTGCAGTATGGCGTGTCGATCAC GGACGCCTGCATCGACTGGTCGGCTACCGAGAAAACCC TGCGCAGCATGCATGCCAAGCTCAAGGATGTGCTGCCG CAGCGT AAGCGCGGCTGA-3 ' SEQIDNO :11 5 ' -TTGGCGGCGGCACCCGTGACCTGACGAGTAACACG Nt ATGGCTGATTTACCGATCGATGACTTGAACGTTGCCTC AroF-I_P16L / Q164 CAACGAGACCCTGATCACCCCTGATCAGCTCAAGAAGG A AAATCCCCCTCAGCGCCAAGGCCCTGCAGACCGTGACT GCCGGCCGTGAAGTGGTGCGCCAATATTCTCGACGGCAA GGACCATCGCCTGTTCGTCGTGGTCCGCCCTTGCTCCAT CCACGACATCAAGGCAGCCCACGAATACGCCGAGCGC CTGAAAGTGCTGGCCGAAGAAGTGTCCGATACGCTGTA CCTGGTCATGCGCGTGTACTTCGAAAAGCCGCGCACCACCGTCGGCTGGAAAGGCCTGATCAACGATCCGTACCTG GATGACTCGTTCAAGATCCAGGACGGCCTGCACATCGG CCGCAAGTTGCTGCTGGACCTGGCCGAAATGGGCCTGC CGACCGCCACCGAAGCGCTCGACCTGATTTCGCCGGCC TACCTGCAAGACCTGATCAGCTGGTCGGCCATCGGTGC CCGCACCACCGAATCGCAAACACACCGCGAGATGGCC TCGGGCCTGTCCTCGGCGGTGGGTTTCAAGAACGGTAC CGATGGCGGCCTGACCGTTGCCATCAATGCCCTGCAGT CGGTGTCCAAGCCGCACCGCTTCCTGGGCATCAACCAG GAAGGCGGCGTGTCGATCGTCACCACCAAGGGCAACC CATACGGCCACGTGGTACTGCGCGGCGGCAATGGCAA GCCGAACTACGACTCGGTCAGCGTCGCCCTGTGCGAAC AGGACCTGGCCAAGGCCAAGATCAAGGCCAACATCAT GGTCGACTGCAGCCATGCCAACTCCAACAAGGACCCG GCCCTGCAACCGCTGGTGATGGAGAACGTCGCCAACCA GATTCTCGAAGGCAACCAGTCGATCATCGGCCTGATGG TCGAAAGCCACCTGAACTGGGGCTGTCAGTCCATTCCG AAAAACCTGGACGATTTGCAGTATGGCGTGTCGATCAC GGACGCCTGCATCGACTGGTCGGCTACCGAGAAAACCC TGCGCAGCATGCATGCCAAGCTCAAGGATGTGCTGCCG CAGCGT AAGCGCGGCTGA-3 ' SEQIDNO :12 Nt AroF-I_P160L / S19 3A 5 ' -TTGGCGGCCGGCACCCGTGACCTGACGAGTAACACG ATGGCTGATTTACCGATCGATGACTTGAACGTTGCCTC CAACGAGACCCTGATCACCCCTGATCAGCTCAAGAAGG AAATCCCCCTCAGCGCCAAGGCCCTGCAGACCGTGACT GCCGGCCGTGAAGTGGTGCGCAATATTCTCGACGGCAA GGACCATCGCCTGTTCGTCGTGGTCGGCCCTTGCTCCAT CCACGACATCAAGGCAGCCCACGAATACGCCGAGCGC CTGAAAGTGCTGGCCGAAGAAGTGTCCGATACGCTGTA CCTGGTCATGCGCGTGTACTTCGAAAAGCCGCGCCACCA CCGTCGGCTGGAAAGGCCTGATCAACGATCGTACCTG GATGACTCGTTCAAGATCCAGGACGGCCTGCACATCGG CCGCAAGTTGCTGCTGGACCTGGCCGAAATGGGCCTGC CGACCGCCACCGAAGCGCTCGACCTGATTTCGCCGCAG TACCTGCAAGACCTGATCAGCTGGTCGGCCATCGGTGC CCGCACCACCGAATCGCAAACACACCGCGAGATGGCC TCGGGTTTGGCATCGGGCGGTGGGTTTCAAGAACGGTACCGATGGCGGCCTGACCGTTGCCATCAATGCCCTGCAGT CGGTGTCCAAGCCGCACCGCTTCCTGGGCATCAACCAG GAAGGCGGCGTGTCGATCGTCACCACCAAGGGCAACC CATACGGCCACGTGGTACTGCGCGGCGGCAATGGCAA GCCGAACTACGACTCGGTCAGCGTCGCCCTGTGCGAAC AGGACCTGGCCAAGGCCAAGATCAAGGCCAACATCAT GGTCGACTGCAGCCATGCCAACTCCAACAAGGACCCG GCCCTGCAACCGCTGGTGATGGAGAACGTCGCCAACCA GATTCTCGAAGGCAACCAGTCGATCATCGGCCTGATGG TCGAAAGCCACCTGAACTGGGGCTGTCAGTCCATTCCG AAAAACCTGGACGATTTGCAGTATGGCGTGTCGATCAC GGACGCCTGCATCGACTGGTCGGCTACCGAGAAAACCC TGCGCAGCATGCATGCCAAGCTCAAGGATGTGCTGCCG CAGCGT AAGCGCGGCTGA-3 ’ SEQIDNO :13 5 ’ - ATGAAAC AGACGC AATACGTGGCACGCGAGCCCGAT Nt GCGCATGGTTTTATCGATTACCCGCAGCAAGAGCATGC phénylalanine hydroxylase (phhA) GGTGTGGAACACCCTGATCACCCGCCAGCTGAAAGTGA TCGAAGGCCGTGCGTGCCAGGAATACCTGGACGGCATC GACCAGCTGAAATTGCCGCATGACCGCATTCCGCAACT GGGCGAGATCAACAAGGTGCTGGGTGCCACCACCGGC TGGCAGGTTGCCCGGGTTCCGGCGCTGATCCCCTTCCA GACCTTCTTCGAATTGCTGGCCAGCAAGCGCTTTCCGG TCGCCACCTTCATCCGCACCCCGGAAGAGCTGGACTAC CTGCAAGAGCCGGATATCTTCCACGAGATCTTCGGCCA CTGCCCGCTGCTGACCAATCCCTGGTTCGCCGAATTCA CCCACACCTACGGCAAGCTCGGCCTGGCCGCGACCAAG GAACAACGTGTGTACCTGGCACGCTTGTACTGGATGAC CATCGAGTTTGGCCTGATGGAAACCGCGCAAGGCCGCA AAATCTATGGTGGTGGCATCCTCTCGTCGCCGAAAGAG ACCGTCTACAGTCTGTCTGACGAGCCTGAGCACCAGGC CTTCGACCCGATCGAGGCCATGCGTACACCCTACCGCA TCGACATTCTGCAACCGGTGTATTTCGTACTGCCGAAC ATGAAGCGCCTGTTCGACCTGGCCCACGAGGACATCAT GGGCATGGTCCATAAAGCCATGCAGCTGGGTCTGCATG CACCGAAGTTTCCACCCAAGGTCGCTGCCTGA-3’ SEQIDNO :14 5 ’ - ATGAATGCCTTGAACCAAGCCCATTGCGAAGCCTGC NtCGCGCCGACGCACCGAAAGTCACCGACGAAGAGCTGG T étrahy drobiopterin e deshydratase (phhB) CCGAGCTGATCCGCGAAATCCCGGACTGGAACATCGA AGTACGTGACGGCCACATGGAGCTGGAGCGCGTGTTCC TGTTCAAGAACTTCAAGCACGCCCTGGCGTTCACCAAT GCCGTGGGCGAAATTGCCGAAGCCGAAGGCCACCACC CAGGGCTGCTGACTGAATGGGGCAAGGTCACCGTGAC CTGGTGGAGCCACTCGATCAAAGGCCTGCACCGCAACG ACTTCATCATGTGCGCACGCACTGACAAGGTGGCGGAA ACGGCTGAAGGCCGGAAGTAA-3 ’ SEQIDNO :15 5 ’ - ATGGCCCCTCGCCCT ACCTCAC AGAACCAAACCCGC Nt ACATGCCCGACGACGCAGGTTACTCAAGTTGATATAGT tyrosine ammonia lyase TGAGAAGATGCTCGCTGCACCAACTGACAGCACCCTAG AGCTCGACGGGTATTCACTAAATCTTGGGGACGTCGTT (TAL_RG_OPT) TCAGCTGCAAGGAAAGGAAGACCTGTAAAGGAA ATAGTGATGAAATTCGGAGTAAAAATAGATAAGTCCGTA GAGTTTTTAAGGTCACAACTTAGCATGTCCGTATACGG GGTCACTACCGGGTTCGCGGTTCCGCCGACACCCGCA CCGAGGACGCTATATCATTGCAGAAAGCTCTTCTAGAG CATCAGCCTTGCGGGCGTTCTTCCAAGTTCCTTCGATTCG TTTAGGCTGGGCGGGCGGGCTTGAGAACTCTCTGCCCCT AGAAGTGGTAAGGGCGCTATGACAATACGGGTGTAAC AGTCTAACAAGAGGTCCACAGCGCGGTTAGACTAGTTGT ACTTGAAGCTCTGACTAACTTCTTAAACCACGGGATTA CCCCGATTGTCCCACTCCGGGGAGCATCAGTGCGTCC GGTGACCTATCGCCCCTCTCATATTTGCGGCAGCTAT ATCAGGACATCCAGATTCAAAGGTTCATGTAGTACATG AAGGAAAAGGAAAATACTTTACGCCACGGCGAGCCAT GGCCCTTTTTAACCTGAGCCCGTGGTACTTGGTCCGA AAGAGGGCCTCGGACTAGTTAACGGTACTGCCGTCAGT GCCTCAATGGCTACGCTTGCACTCCACGATGCGCACAT GCTGAGCCTGCTAAGTCAAAGTCTCACAGCGATGACCG TGGAGCCATGGTGGGCATGCGGGTCATTTCATCCA TTTTTGCATGATGTCACTCGTCCGCATCCTACGCAGATT GAGGTAGCAGGCAACATTCCGCAAGCTTCTCAGGGA GTCGTTTCGCCGTCCATCATGAGGAAGAAGTAAAAGTA AAGGATGACGAAGGAATATTAAGGCAAGACCGATACC CGCTCCGCACGTCACGCGCAATGGTTGGGTCCCACTGGTT TCAGACCTCATCCACGCCACGCTCTTAACTATTGAAGCAGGGCAATCGACGACAGACAATCCTCTCATCGAC GTAGAGAATAAGACCTCGCATCATGGAGGAAATTTTCA AGCTGCAGCTGTCGCGAACACAATGGAAAAGACACGT CTCGGCCTGGCGCAAATAGGGAAACTGAATTTCACCCA GCTCACGGAAATGCTGAACGCCGGCATGAACCGCGGC CTGCCGTCTTGTCTCGCCGCGGAAGATCCTTCTTTATCA TATCACTGTAAGGGTTTAGATATCGCGGCAGCTGCATA TACGTCCGAACTAGGTCATCTGGCTAACCCTGTCACGA CCCACGTACAACCGGCGGAGATGGCTAATCAAGCAGTT AACTCCCTTGCACTAATTTCCGCCCGCCGGACAACAGA GAGTAACGACGTGTTATCACTGCTGCTCGCTACCCACT TATACTGCGTCTTGCAGGCTATCGACTTACGCGCAATC GTGTTCGAATTTAAGAAGCAATTCGGGCCAGCTATTGT GTCCCTAATTGATCAGCACTTCGGAAGCGCCATGACTG GGTCTAATCTTCGAGACGAGCTAGTCGAAAAAGTAAAT AAGACACTCGCAAAGAGGCTGGAACAGACTAACAGCT ACGACCTAGTTCCACGGTGGCACGACGCCTTTAGTTTT GCAGCGGGAACGGTAGTAGAGGTATTGTCATCGACTTC GTTGTCGTTGGCTGCTGTCAACGCGTGGAAAGTTGCAG CTGCAGAGTCAGCAATTTCGCTGACGCGGCAAGTACGC GAAACATTTTGGAGCGCTGCTTCGACAAGCTCGCCAGC CCTTTCTTACCTGTCCCCACGTACGCAGATCTTGTACGC ATTCGTAAGAGAGGAGTTAGGAGTCAAAGCCCGAAGG GGTGACGTATTCCTTGGAAAGCAAGAAGTTACAATTGG ATCCAACGTTTCAAAGATCTATGAGGCCATTAAGAGTG GGCGCATAAATAACGTCCTGTTGAAGATGCTGGCCTGA -3’ SEQIDNO :16 Nt 4-coumarate-CoA ligase (4-CL) 5 ’ -GTGAATAACGAAGCCCGCTCAGGGTCGACCGACCCT GGCCAACGTCCGCGCTACCGCCAGGTGGCCATCGGGCA TCCCCAGGTGCAGGTCAGTCACGTCGACGACGTGCTGC GCATGCAACCTGTCGAGCCACTGGCGCCGCTGCCGGCG CGCCTGCTCGAGCGCCTGGTGCATTGGGCCCAGGTGCG CCCGGACACCACTTTCATCGCGGCACGCCAGGCAGACG GTGCCTGGCGTTCGATCAGCTACGTGCAGATGCTCGCC GATGTGCGCACCATCGCCGCCAACTTGCTAGGACTGGG CCTCAGTGCCGAGCGCCCGCTGGCGCTGCTTTCCGGCA ACGACATCGAACACCTGCAAATCGCCCTCGGCGCCATG TATGCCGGTATTGCCTATTGCCCGGTGTCGCCGGCCTACGCGCTGTTGTCGCAAGACTTCGCCAAGTTGCGCCATG TCTGCGAGGTGCTCACCCCCGGAGTGGTCTTCGTCAGC GACAGCCAGCCGTTCCAGCGCGCCTTCGAGGCGGTGCT GGACGATTCGGTCGGCGTGATCAGCGTGCGTGGCCAGG TCGCAGGTCGCCCCCATATAAGCTTCGACAGCCTGTTG CAACCGGGTGACCTGGCGGCGGCCGATGCGGCTTTCGC CGCCACCGGGCCGGACACCATCGCCAAATTCCTCTTCA CCTCGGGCTCGACCAAGCTGCCCAAGGCGGTGATCACC ACCCAGCGCATGCTGTGCGCCAATCAGCAGATGCTTCT GCAGACTTTTCCGACGTTCGCCGAGGAGCCGCCGGTGC TGGTGGACTGGCTGCCGTGGAACCACACGTTCGGCGGT AGCCACAACCTCGGCATCGTGCTTTACAACGGGGGCAG TTTCTACCTGGACGCCGGCAAGCCGACCCCGCAAGGCT TCGCCGAGACCTTGCGCAATCTGCGCGAGATTTCCCCC ACGGCCTACCTCACCGTACCCAAGGGCTGGGAGGAACT GGTCAAGGCACTGGAGCAGGACCCCGCGCTACGCGAG GTGTTCTTTGCCCGCATCAAGCTGTTCTTCTTTGCCGCC GCAGGCCTGTCGCAAAGCGTCTGGGACCGGCTGGACC GCATTGCCGAGCAACACTGTGGCGAACGCATCCGCATG ATGGCCGGCCTTGGCATGACCGAAGCCTCGCCATCGTG CACCTTCACCACCGGGCCTTTGTCGATGGCCGGCTATG TCGGGCTGCCGGCACCTGGCTGCGAAGTGAAGCTGGTG CCGGTGGGCGACAAGCTCGAGGCGCGCTTCCGTGGCCC GCATATCATGCCGGGCTACTGGCGCTCGCCGCAGCAGA CCGCCGAGGCGTTCGACGAGGAGGGCTTCTACTGTTCG GGCGACGCGTTGAAGCTGGCCGATGCCAGGCAGCCCG AGCTTGGCCTGATGTTCGATGGCCGTATCGCTGAGGAC TTCAAACTTTCGTCCGGGGTATTCGTCAGTGTCGGGCC GCTGCGCAACCGCGCAGTGCTGGAGGGCTCGCCTTACG TACAGGACATCGTGGTCACCGCGCCGGACCGTGAATGC CTGGGCCTGCTGGTGTTCCCGCGTCTGCCCGAGTGTCG GCGCCTGGCCGGGCTGGCAGAGGATGCCAGCGATGCG CGGGTGCTGGCCAACGACACCGTGCGCAGTTGGTTCGC TGACTGGCTGGAGCGCTTGAACCGCGATGCCCAAGGCA ACGCCAGCCGTATCGAATGGCTGTCGCTGCTGGCCGAG CCGCCGTCGATCGACGCCGGTGAAATCACCGACAAGG GCTCGATCAATCAGCGCGCCGTGCTGCAGCGGCGCGCCGCTCAGGTCGAGGCGCTGTACCGTGGCGAAGACCCCG ACGCATTGCACGCCAAGGTGCGGCCTTGA-3’ SEQIDNO :17 5 ’ - ATGGC AACTGAGGAGATGAAGAAATTGGCCACCGTG Nt ATGGCCATTGGCACGGCCAACCCTCCGAACTGCTACTA benzalacétone CCAGGCCGACTTTCCCGACTTCTACTTCCGCGTCACCA synthase (BAS) ACAGCGACCACCTCATCAACCTCAAGCAAAAGTTCAAG CGCCTTTGTGAAAACTCAAGGATTGAGAAGCGTTACCT TCATGTGACCGAAGAGATTCTCAAGGAAAACCCAAAC ATTGCTGCCTACGAGGCAACCTCGTTGAATGTAAGACA CAAAATGCAAGTGAAAGGAGTTGCAGAGCTTGGGAAA GAGGCTGCCCTCAAGGCCATCAAAGAATGGGGCCAAC CCAAGTCCAAGATCACACATCTCATCGTGTGTTGCCTA GCCGGCGTTGACATGCCCGGCGCGGATTATCAACTCAC TAAGCTTCTTGACCTTGACCCTTCCGTCCAAGCGTTTTAT GTTTTACCACCTAGGATGCTACGCTGGTGGCACTGTCC TTCGCCTTGCAAAGGACATAGCGGAGAACAACAAGGG AGCTCGTGTTCTCATCAGTTGATCCAACTCAA CTTGTTTTCGTGGGCCATCTGAAACCCATCTGGACTCCA TGATAGGCCAAGCAATATTAGGCGATGGGGCTGCAGCT GTCATAGTTGGCGCAGATCCAACCTAACCGTTGAGAG GCCCATATTCGAGTTGGTTTCCACAGCCCAGACTATG TACCCGAATCCATGGTGGATTGGCCGATT GAATCTGGACTCAGTTTCCATTTGTACAAGACCGTTCCT ACACTAATCTCTAACAACATTAAAACTTGCCTTTCTGAT GCTTTCACTCCTCTAAACATTAGCGATTGGAACTCTCTT TTCTGGATCCGCACACCCTGGTGGTCCTGCCATCCTAGA CCAAGTTACTAGGATTAGGATTGGATTGATT TCAAGGTAACTAGACAAGTGTTGAAGGACTATGGAAA CATGTCGAGTGCTACGGTGTTTTTCATCATGGATGAGA TGAGGAAGAAGTCACTCGAAAACGGTCAAGCAACCAC TGGAGAAGGGCTCGAGTGGGGTGTTTTGGGGTTCG GGCCTGGAATCACCGTTGAAGGACTATGTTGACTGGTTAGTTGGTTAGTT AG-3 ' List of papers cited Patent documents

[0188] For any useful purpose, the following patent document(s) is cited: - patcitl: FR 1234567 (deposit number); - patcit2: US2004230550 (publication number); and - patcit3: FR2795457 (publication number). Non-patent literature

[0189] For all useful purposes, the following non-patent elements are cited: - nplcitl: Klesk et al., 2004, J. Agric. Food Chem. 52, 5155-61; - nplcite2: Larsen et al., 1991, Acta Agric. Scand. 41, 447-54). - nplcite3: Kikuchi Y et al. “Mutational analysis of the feedback sites of pheny-lalanine-sensitive 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase of Es-cherichia coli.” Appl Environ Microbiol 63:761-762 (1997).): P160L, Q164A, S190A, G191K, S193A, I225P. - nplcit4: Cui, Di et al., “Molecular basis for feedback inhibition of tyrosine-regulated 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase from Escherichia coli”, J Struct Biol. 2019 Jun 1; 206(3):322-334. - nplcit5: Lütke-Eversloh and Stephanopoulos, 2005 - nplcitô : Lütke-Eversloh and Stephanopoulos, 2007. “L-Tyrosine production by de-regulated strains of Escherichia coli”. - nplcit7 : Kang et al., 2012 “Artificial biosynthesis of phenylpropanoic acids in a tyrosine overproducing Escherichia coli strain » - nplcit8 : Santos et al., 2011. “Optimization of a heterologous pathway for the production of flavonoids from glucose », - nplcit9 : Juminaga et al., 2011. « Modular engineering of L-tyrosine production in E. coli ». - nplcitlO : Rodriguez et al., 2015, “Establishment of a yeast platform strain for production of p-coumaric acid through metabolic engineering of aromatic amino acid biosynthesis.” - nplcitl 1 : Calero et al., 2016. « Broad-host range ProUSER vectors enable fast cha-racterization of inducible promoters and optimization of p-coumaric acid production in Pseudomonas putida KT2440”. - nplcitl2 : Calero et al., 2017. « Genome-wide identification of tolérance mechanisms toward p-coumaric acid in Pseudomonas putida ». - nplcitl3 : Koeduka, T., et al. (2011). “Characterization of raspberry ketone / zingerone synthase, catalyzing the alpha, beta-hydrogenation of phenylbutenones in raspberry fruits ». Biochemical and Biophysical Research Communications 412, 104-108. - nplcitl4 : Beekwilder et al., 2007. “Microbial production of natural raspberry ketone”. - nplcitl5 : Lee et al., 2017. “Heterologous production of raspberry ketone in the wine yeast Saccharomyces cerevisiae via pathway engineering and synthetic enzyme fusion”. - nplcitlô : Waterhouse, A. et al., « SWISS-MODEL: homology modelling of protein structures and complexes ». Nucleic Acids Res. 46(W1), W296-W303 (2018) - nplcitl7 : O. Trott et al., « AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading », Journal of computanional chemestry, 2010). - nplcitl8 : Ding, R. et al. “Introduction of two mutations into AroG increases pheny-lalanine production in Escherichia coli”. Biotechnol Lett 36, 2103-2108 (2014). - nplcitl9 : (Gibson et al., 2009). - nplcit20 : Schafer A, et al., “Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19 : sélection of defined délétions in the chromosome of Corynebacterium glutamicum”. Gene 1994 Jul 22 ; 145(1) :69-73) - nplcit21 : Simon, R., et al. “A broad host range mobilization System for in vivo genetic engineering : transposon mutagenesis in gram négative bacteria”. Nature Bio-Technology volume 1, pages784-791 (1983)). - nplcit22 : O. Trott et al., 2010. « AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading », Journal of computanional chemestry. - nplcit23: Zhou et al., “Characterization of mutants of a tyrosine ammonia-lyase from Rhodotorula glutinis”. Appl. Microbiol. Biotechnol, 2015. - nplcit23: Prior et al., “Broad-host-range vectors for protein expression across gram négative hosts”. Biotechnol Bioeng, 2010 Jun 1; 106(2):326-32.

Claims

Claims

1. Genetically modified strain of Pseudomonas putida characterized in that it comprises a mutated AroF-I gene coding for 3-Deoxy-D-arabino-Heptulodonate 7-phosphate (DAHP) synthase whose sequence has at least 90% identity with the sequence SEQ ID NO: 1 and having at least one double mutation P160L / S193A.

2. Genetically modified strain according to claim 1, characterized in that it comprises an additional recombinant gene coding for a phenylalanine hydroxylase (phhA) whose sequence has at least 80% identity with the sequence SEQ ID NO: 5, and an additional recombinant gene coding for a tetrahydrobiopterin de-shydratase (phhB) whose sequence has at least 80% identity with the sequence SEQ ID NO: 6, preferably said recombinant genes phhA and phhB being placed under the control of a heterologous promoter allowing their overexpression.

3. Genetically modified strain according to claim 1 or 2, characterized in that it expresses a DAHP synthase having an amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 2, the sequence SEQ ID NO: 3, or the sequence SEQ ID NO:

4.

4. Genetically modified strain according to claim 3, characterized in that it expresses a DAHP synthase having an amino acid sequence defined by the sequence SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:

4.

5. Genetically modified strain according to one of claims 1 to 4, characterized in that it further comprises one or more additional recombinant genes chosen 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.

6. A method of synthesizing a phenylpropanoid compound, characterized in that it comprises a step of growing the genetically modified strain according to claim 5, in a culture medium under conditions allowing the expression of the recombinant genes necessary for the synthesis of said phenylpropanoid, said phenylpropanoid compound being synthesized by said genetically modified strain.

7. Method according to claim 6, characterized in that it also comprises a step of recovering the phenylpropanoid compound in the culture medium.

8. Use of the strain according to one of claims 1 to 5, for the synthesis of a phenylpropanoid compound or a phenylpropanoid derivative, preferably coumaric acid or frambinone.

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