Biosynthesis of phenylpropanoid compounds

Abstract

The present invention relates to the field of production of phenylpropanoid compounds, in particular to the field of genetically modified strains for the production of phenylpropanoid compounds. In particular, the present invention relates to a genetically modified strain of Pseudomonas putida containing a mutant AroF-I gene encoding 3-deoxy-D-arabino-heptulosonic acid-7-phosphate (DAHP) and its use for the synthesis of phenylpropanoid compounds, in particular coumaric acid or furanbinone.

Description

[Technical field]

[0001] The present invention relates to the field of phenylpropanoid compound production, in particular to genetically modified strains for the production of phenylpropanoid compounds such as coumaric acid or furanbinone. [Background technology]

[0002] For many years, the biological production of "natural" flavors and fragrances has been an important area of ​​industrial research to meet the needs of consumers who strive to be increasingly environmentally responsible. Although synthetic biology, particularly using microorganisms, makes this natural production possible, yields are not always sufficient for large-scale production.

[0003] The flavor of raspberries (Rubus idaeus) has been associated with over 200 compounds, with the natural phenolic compounds furanbinone being the most influential compound and defining the characteristic raspberry flavor (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 in raspberries in small quantities (1-4 mg / kg of fruit), natural furanbinone is extremely expensive (Larsen et al., 1991). However, because its natural availability is limited, it is highly desirable to produce furanbinone by biotechnology.

[0005] In this context, the biosynthetic pathway of phenylpropanoid compounds, and in particular furanvinone, can be reproduced in microorganisms by inserting heterologous genes encoding some of the key enzymes of this pathway.

[0006] Tyrosine is an important amino acid for the biosynthesis of phenylpropanoid compounds, in particular since it is the precursor of coumaric acid or furanbinone.

[0007] In fact, furanbinone can be obtained from the aromatic amino acid L-tyrosine as the initial substrate via a four-step biosynthetic pathway.

[0008] In the first step, tyrosine is deaminated by tyrosine ammonia lyase (TAL, EC 4.3.1.23) to form coumaric acid. A molecule of coenzyme A (CoA) is attached to coumaric acid, catalyzed by 4-coumaric acid:CoA ligase (4CL, EC 6.2.1.12). Coumaroyl-CoA is then converted to 4-hydroxybenzalacetone by benzalacetone synthase (BAS, EC 2.3.1.212). This reaction is a decarboxylative condensation, using one unit of malonyl-CoA as a cosubstrate. The final step is the reduction of 4-hydroxybenzalacetone to furanbinone by benzalacetone reductase.

[0009] Therefore, the development of strains overproducing tyrosine as a platform for the production of phenylpropanoid compounds, especially furanvinone, is an important starting point for the biosynthesis of these compounds.

[0010] However, the production of tyrosine in microorganisms is as complex as it is tightly regulated, because some enzymes in the tyrosine production pathway, also known as the shikimate pathway, are regulated in a negative feedback loop by this amino acid, which inhibits their activity by binding to inhibitory sites when its concentration in the cell becomes too high. This regulatory system is called feedback inhibition when too much tyrosine is present in the microorganism.

[0011] Despite this, it should be possible by mutagenesis to obtain enzymes that are "resistant" to this inhibition and thus deregulate the tyrosine production pathway. These mutants that are resistant to tyrosine feedback inhibition are referred to as fbr mutants, for "feedback resistance".

[0012] Numerous papers have described the deregulation of this pathway in E. coli, and one enzyme has been particularly well studied: DAHP synthase (DAHP = 3-deoxy-D-arabino-heptulosonic acid-7-phosphate). The first reaction in the shikimate pathway consists of the condensation of phospho(enol)pyruvate (PEP) with erythrose-4-phosphate (E4P) to yield DAHP.

[0013] This DAHP synthase activity is carried out in E. coli by three isoenzymes: AroG (feedback inhibited by phenylalanine), AroF (feedback inhibited by tyrosine) and AroH (feedback inhibited by tryptophan). The structures of these proteins are known and the substructures involved in the feedback inhibition (and also the amino acids involved) have been thoroughly described in the literature.

[0014] Kikuchi et al., 1997 and Cui et al., 2019 identified, described and studied mutants AroGfbr and AroFfbr in E. coli that are resistant to phenylalanine and tyrosine feedback inhibition, respectively. An fbr mutant of protein TyrA (mutant tyrAfbr) has also been described in E. coli by Lutke-Eversloh and Stephanopoulos (2005) (Lutke-Eversloh and Stephanopoulos, Appl. Environ Microbiol. 2005 November;71(11):7224-8).

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

[0016] A review of the modifications made to obtain tyrosine-overproducing E. coli strains is given in the paper “Modular engineering of L-tyrosine production in E. coli” (Juminaga et al., 2011).

[0017] Phenylpropanoid compounds have also been synthesized from Saccharomyces cerevisiae strains overexpressing tyrosine as described by Rodriguez et al., 2015.

[0018] Document GB2416769 describes the possibility of producing furanbinone using a microorganism containing genes encoding the enzymes 4CL and BAS, at least one of which originates from a heterologous source. A preferred microorganism is E. coli (strain BL21), which may further contain sequences encoding BAR, C4H, PAL and / or CHS, including a sequence encoding BAR that is advantageously endogenous.

[0019] However, E. coli or S. cerevisiae do not have good resistance to the toxicity of phenylpropanoid compounds and therefore do not appear to be the most suitable microorganisms for their production.

[0020] Bacteria of the genus Pseudomonas, and in particular the bacterium Pseudomonas putida, are relatively resistant to these highly toxic molecules (Calero et al., 2017). However, the enzymes involved in the production of aromatic amino acids in P. putida have not been described in detail.

[0021] As a result, overproduction of shikimate pathway derivatives such as tyrosine in P. putida involves the use of AroGfbr and TyrAfbr mutants from E. coli, which are not particularly effective ( Calero et al., 2016 ).

[0022] Overproduction of tyrosine and phenol has been described in the bacterium Pseudomonas taiwanensis VLB120 by carrying out point mutations in three 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, especially coumaric acid and furanbinone. [Prior art documents] [Patent documents]

[0024] [Patent Document 1] GB2416769 [Non-patent literature]

[0025] [Non-Patent Document 1] Klesk et al., 2004, J. Agric. Food Chem. 52, 5155-61 [Non-Patent Document 2] Larsen et al., 1991, Acta Agric. Scand. 41, pp. 447 - 54

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[0026] The strains which have been developed so far which overproduce tyrosine are primarily Escherichia coli and S. cerevisiae strains, however these strains do not have good resistance to the toxicity of phenylpropanoid compounds and are therefore not the most suitable microorganisms for their production.

[0027] Therefore, there is a particular need to develop new tyrosine overproducing strains that allow the production of phenylpropanoid compounds.

[0028] This disclosure will remedy this situation. [Means for solving the problem]

[0029] One aspect of the present invention relates to a genetically modified strain of Pseudomonas putida having the sequence of SEQ ID NO:1 and comprising a mutant AroF-I gene encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase having at least one P160L mutation, a P160L / Q164A double mutation, or a P160L / S193A double mutation, preferably a P160L / S193A double mutation.

[0030] Another aspect of the present invention relates to a method for synthesizing a phenylpropanoid compound or a phenylpropanoid derivative by utilizing the genetically modified strain of Pseudomonas putida according to the present invention.

[0031] Finally, the present invention also relates to the use of a genetically modified strain of Pseudomonas putida for the synthesis of phenylpropanoid compounds.

[0032] The features described in the following paragraphs may be implemented as options. They may be implemented independently or in combination with each other.

[0033] Other features, details and advantages will become apparent upon reading the following detailed description and upon examining the accompanying drawings. [Brief description of the drawings]

[0034] [Figure 1] Figure 1 shows a graph of PEP consumed in mM by wild-type (WT) AroF-I protein in the presence or absence of aromatic amino acids. Results presented are from three independent replicates. [Diagram 2] Figure 1 shows a graph depicting the percentage of PEP consumed in mM by AroF-I WT protein compared to the single mutants AroF-I-G191, AroF-I-P160 and AroF-I-S193 in the presence or absence of aromatic amino acids. Results presented are from three independent replicates. [Diagram 3]Figure 1 shows a graph depicting the percentage of PEP consumed in mM by AroF-I WT protein compared to double mutants AroF-I-P160_G191, AroF-I-P160L_Q164, AroF-I-P160_S190 and AroF-I-P160_S193 in the presence or absence of aromatic amino acids. Results presented are from three independent repeats. [Figure 4] Figure 1 shows a graph depicting the percentage of PEP consumed in mM by AroF-I WT protein compared to the single mutants AroF-I-P160 and AroF-I-S193 and the double mutant AroF-I-P160_S193 in the presence or absence of aromatic amino acids. Results presented are from three independent repeats. [Diagram 5] FIG. 1 depicts a conjugation protocol that can be used to transform and genetically modify P. putida. [Figure 6] FIG. 1 shows a graph depicting 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). [Figure 7] Figure 1 shows graphs representing the production of total phenols and the percentage of coumaric acid (%PCA) in total phenols by Pseudomonas putida strains expressing the enzymatic enzymes AroF-I WT / TAL (aroF-I WT), AroF-I WT / TAL+empty plasmid (aroF-I WT+control), AroF-I WT / TAL+phhA / B plasmid (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+phhA / B plasmid (aroF-I fbr+phhA / B). Expression of the phhA / B genes from the araC / pBAD promoter is either induced or not induced. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Thus, a first subject of the present invention relates to a genetically modified strain of Pseudomonas putida comprising a mutated AroF-I gene encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, the sequence of which has at least 80% identity to the sequence of SEQ ID NO: 1 and which has at least one P160L mutation, a P160L / Q164A double mutation or a P160L / S193A double mutation, preferably a P160L / S193A double mutation.

[0036] For the purposes of this description, the expressions "genetically modified strain of Pseudomonas putida", "modified strain of Pseudomonas putida", "genetically modified strain" and "modified strain" are considered to be synonymous.

[0037] In particular, a "genetically modified strain" refers to a strain that contains either: (i) at least one recombinant nucleic acid, or transgene, that is stably integrated into its genome and / or is present on a vector, e.g., a plasmid vector; or (ii) one or more non-naturally occurring mutations by nucleotide insertion, substitution or deletion, obtained by transformation or gene editing techniques known to those skilled in the art.

[0038] Indeed, techniques for 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 Martinez-Garcia and de Lorenzo, "Pseudomonas putida in the quest of programmable chemistry", Current Opinion in Biotechnology, 59:111-121, 2019.

[0039] Preferably, the mutagenesis techniques described in Example 2 will be used to integrate the mutated or recombinant genes in Pseudomonas putida.

[0040] In particular, the genetically modified strain can contain nucleic acids that alter the expression of one or more genes that are naturally expressed in Pseudomonas putida.

[0041] According to certain embodiments, the genetically modified strain can include nucleic acids encoding one or more enzymes that are not naturally expressed in Pseudomonas putida.

[0042] A wild-type strain of P. putida KT2440 is available, for example, at the NBRC strain collection (National Institute of Technology and Evaluation Biological Resource center https: / / www.nite.go.jp / en / nbrc / , NBRC100650).

[0043] Furthermore, 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 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, November 20;7:312).

[0044] For the purposes of the present invention, percentage identity refers to the percentage of identical residues in a nucleotide or amino acid sequence over a given fragment after alignment and comparison with a reference sequence. An alignment algorithm is used for the comparison, and the sequences to be compared are input with the corresponding parameters of the algorithm. Default algorithm parameters can be used.

[0045] Preferably, for comparing nucleic acid sequences and determining percentage identity, the BLAST algorithm as described at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi is used with default parameters.

[0046] For the purposes of the present invention, a "mutated AroF1 gene" refers to a nucleic acid comprising at least a portion encoding a mutant form of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase under the control of a promoter allowing its expression in a genetically modified strain.

[0047] For the purposes of the present invention, 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase refers to an enzyme (EC 2.5.1.54) capable of carrying out the first reaction in the shikimate pathway in bacteria, consisting of the condensation of phospho(enol)pyruvate (PEP) with erythrose-4-phosphate (E4P) to yield DAHP.

[0048] According to a particular embodiment, the genetically modified strain of Pseudomonas putida comprises a mutated AroF-I gene encoding a DAHP synthase, the amino acid sequence of which has at least 80%, 85%, 90%, 95% and most particularly at least 98% identity to the sequence of SEQ ID NO:1, and having at least one P160L mutation, a P160L / Q164A double mutation, or a P160L / S193A double mutation, preferably a P160L / S193A double mutation.

[0049] The endogenous Pseudomonas putida AroF-I gene encodes a DAHP synthase with the amino acid sequence of SEQ ID NO: 1. In a particular embodiment, the genetically modified strain according to the invention therefore comprises, in addition to the endogenous AroF-I gene, at least one recombinant mutant AroF-I nucleic acid sequence encoding a mutant protein comprising a P160L mutation, a P160L / Q164A double mutation, or a P160L / S193A double mutation, preferably a P160L / S193A double mutation.

[0050] According to a particular embodiment, the genetically modified strain of Pseudomonas putida comprises a mutated AroF-I gene encoding a DAHP synthase comprising the P160L mutation as defined by the amino acid sequence of SEQ ID NO:2.

[0051] According to a variation of this embodiment, the modified strain of Pseudomonas putida comprises a mutant AroF-I gene encoding a DAHP synthase whose sequence has at least 80%, 85%, 90%, 95% and most particularly at least 98% identity to the sequence of SEQ ID NO:2 and includes a P160L mutation.

[0052] According to another particular embodiment, the modified strain of Pseudomonas putida comprises a mutant AroF-I gene encoding a DAHP synthase and having at least one P160L / Q164A double mutation. According to this embodiment, the mutant AroF-I gene encodes a DAHP synthase containing the P160L / Q164A double mutation defined by the amino acid sequence of SEQ ID NO:3.

[0053] According to a variation of this embodiment, the modified strain of Pseudomonas putida comprises a mutant AroF-I gene encoding a DAHP synthase whose sequence has at least 80%, 85%, 90%, 95% and most particularly at least 98% identity to the sequence of SEQ ID NO:3 and comprises a P160L / Q164A double mutation.

[0054] According to a preferred embodiment, the modified strain of Pseudomonas putida comprises a mutant AroF-I gene encoding a DAHP synthase and having at least one P160L / S193A double mutation. According to this embodiment, the mutant AroF-I gene encodes a DAHP synthase containing a P160L / S193A double mutation defined by the amino acid sequence of SEQ ID NO:4.

[0055] According to a variation of this embodiment, the modified strain of Pseudomonas putida comprises a mutant AroF-I gene encoding a DAHP synthase whose sequence has at least 80%, 85%, 90%, 95% and most particularly at least 98% identity to the sequence of SEQ ID NO: 4 and comprises a P160L / S193A double mutation.

[0056] In one embodiment, which can be combined with the preceding embodiments, the coding sequence of the mutant AroF-I gene is placed under the control of a heterologous promoter, in particular a constitutive or inducible promoter, which allows overexpression of the mutant AroF-I gene in the genetically modified strain according to the invention, for example selected from the promoters ptrc, xyls / pm or araC / pBAD. In a preferred embodiment, the coding sequence of the mutant AroF-I gene in the genetically modified strain according to the invention is inserted in such a way as to render the AroH gene non-functional, for example by disrupting the AroH gene or by deleting all or part of the AroH gene and in particular its coding sequence.

[0057] The AroH gene encodes another DAHP synthase (isoenzyme of AroF). Thus, in one embodiment, a genetically modified strain according to the invention comprises a deleted or disrupted AroH gene and at least one recombinant nucleic acid comprising a mutant AroF-I gene as described above or a coding sequence of a mutant AroF-I gene.

[0058] In a completely advantageous manner, by carrying out specific mutations, the applicant has developed a strain of Pseudomonas putida capable of expressing a recombinant DAHP synthase that is resistant to feedback inhibition by tyrosine, thereby deregulating the tyrosine production pathway. In other words, the invention makes it possible to generate strains that overproduce tyrosine as an end 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 modified strain according to the invention.

[0059] Phenylpropanoid compounds are a class of plant-derived organic compounds that are biosynthesized from phenylalanine or tyrosine. As examples of phenylpropanoid compounds, particular mention may be made of coumaric acid, p-coumaroyl-CoA, 4-hydroxybenzalacetone, furanbinone, zingerone, vanillin, flavonoids and stilbenoids.

[0060] For purposes of the present invention, a "tyrosine overproducing strain" refers to an engineered strain of Pseudomonas putida that is capable of producing more tyrosine, either as an end product or as an intermediate product, than a wild-type strain of Pseudomonas putida that contains the AroF-I gene encoding DAHP synthase of the amino acid sequence of SEQ ID NO:1 (non-mutated gene).

[0061] In order to be able to convert tyrosine into phenylpropanoid compounds such as coumaric acid, the modified strain according to the invention can advantageously further comprise at least one additional recombinant gene.

[0062] For the purposes of the present invention, the expression "additional recombinant gene" means 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, such as a strong promoter, for overexpressing an endogenous Pseudomonas putida gene or a recombinant coding sequence encoding a protein not naturally expressed in Pseudomonas putida.

[0063] According to certain embodiments, the genetically modified strain of Pseudomonas putida comprises at least an additional recombinant gene encoding a polypeptide having phenylalanine hydroxylase (phhA) activity and an additional recombinant gene encoding a polypeptide having tetrahydrobiopterin dehydratase (phhB) activity.

[0064] The applicant has observed that, although present endogenously in Pseudomonas putida, these two enzymes may not be sufficiently active and / or their expression may be insufficient in the context of the use of the strain for the production of phenylpropanoid compounds. According to this embodiment, the activity is therefore optimized by overexpressing these two enzymes phhA and phhB. Overexpression can be obtained, for example, by placing additional recombinant genes for these enzymes under the control of a heterologous promoter, in particular a constitutive or inducible promoter. Examples of such promoters are, in particular, the promoters ptrc, xylS / pm, araC / pBAD.

[0065] Overexpression of the enzymes phhA and phhB is preferably obtained by placing additional recombinant genes for these enzymes under the control of the strong inducible promoter araC / pBADopt (Prior et al., 2010).

[0066] Overexpression of a gene means expression of said gene in a genetically modified strain that is stronger than in the same strain, but in which the gene is expressed only under the control of its native promoter. Overexpression can also be obtained by directly inserting one or more copies of the gene into the genome of the strain, preferably under the control of a strong promoter, or by cloning into a plasmid, in particular a multicopy plasmid, preferably also under the control of a strong promoter.

[0067] In another embodiment, which can be combined with the preceding embodiments, the endogenous phhA and phhB coding sequences are placed under the control of heterologous promoters as defined above, e.g., to overexpress the corresponding endogenous coding sequences in the genetically modified strain according to the invention.

[0068] In particular, the modified strain may comprise additional recombinant genes encoding phenylalanine hydroxylase (phhA) (EC 1.14.16.1), the sequence of which is defined by the amino acid sequence of SEQ ID NO: 5 or by a sequence having at least 80%, 85%, 90%, 95% and most particularly at least 98% identity to the sequence of SEQ ID NO: 5 and which encode an enzyme having phhA activity, and additional recombinant genes encoding tetrahydrobiopterin dehydratase (phhB) (EC 4.2.1.96), the sequence of which is defined by the amino acid sequence of SEQ ID NO: 6 or by a sequence having at least 80%, 85%, 90%, 95% and most particularly at least 98% identity to the sequence of SEQ ID NO: 6 and which encode an enzyme having phhB activity, in particular under the control of heterologous promoters allowing their overexpression.

[0069] According to this embodiment, the genetically modified strain according to the invention is capable of overproducing tyrosine and is also capable of converting phenylalanine to tyrosine via the phhA and phhB enzymes.

[0070] According to certain embodiments, the additional recombinant genes encoding phenylalanine hydroxylase (phhA) and 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 a DAHP synthase comprising a P160L / S193A double mutation defined by the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 85%, 90%, 95% and most particularly at least 98% identity to the sequence of SEQ ID NO: 4, and two additional recombinant genes: - a recombinant gene encoding a phenylalanine hydroxylase (phhA), preferably phhA defined by the sequence SEQ ID NO: 5 or a sequence having at least 85%, 90%, 95% and most particularly at least 98% identity to the sequence SEQ ID NO: 5, and - a recombinant gene encoding a tetrahydrobiopterin dehydratase (phhB), preferably phhB defined by the sequence SEQ ID NO: 6 or a sequence having at least 85%, 90%, 95% and most particularly at least 98% identity to the sequence SEQ ID NO: 6; - Preferably, said genes phhA and phhB are under the control of a heterologous promoter allowing their overexpression.

[0071] According to another particular embodiment, which may be preferably combined with the preceding embodiment, the genetically modified strain of Pseudomonas putida comprises an additional recombinant gene encoding a polypeptide having tyrosine ammonia lyase (TAL) activity. The recombinant gene encoding the TAL originates from the microorganism Rhodotorula glutinis and can be optimized according to reference Zhou et al., 2015 (three point mutations in this TAL enzyme make it more effective: S9N; A11T; E518V). This TAL enzyme is called TAL_rg_opt. In particular, the modified strain can comprise a recombinant gene encoding a tyrosine ammonia lyase (TAL) (EC 4.3.1.23) whose sequence is defined by the amino acid sequence of SEQ ID NO: 7 (TAL_rg_opt) or by a sequence having at least 80%, 85%, 90%, 95% and most particularly at least 98% identity to the sequence of SEQ ID NO: 7, and which encodes an enzyme having TAL activity.

[0072] According to a particular embodiment, the genetically modified strain according to the invention is capable of overproducing tyrosine and also converting it to coumaric acid via the TAL enzyme.

[0073] According to another specific embodiment, which may be combined with the preceding embodiments, 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 4-CL (EC 6.2.1.12), the sequence of which is defined by the amino acid sequence of SEQ ID NO: 8 or by a sequence having at least 80%, 85%, 90%, 95% and most particularly at least 98% identity to the sequence of SEQ ID NO: 8, and which encodes an enzyme having 4-CL activity.

[0074] According to this particular embodiment, the genetically modified strain is capable of converting coumaric acid to p-coumaroyl-CoA via the 4-CL enzyme.

[0075] According to another particular embodiment, which may be preferably combined with the preceding embodiments, the genetically modified strain of Pseudomonas putida comprises an additional recombinant gene encoding a polypeptide having 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 of SEQ ID NO: 9 or by a sequence having at least 80%, 85%, 90%, 95% and most particularly at least 98% identity to the sequence of SEQ ID NO: 9 and which encodes an enzyme having BAS activity.

[0076] According to this particular embodiment, the genetically modified strain is capable of converting p-coumaroyl-CoA to 4-hydroxybenzalcetone via the BAS enzyme.

[0077] According to another particular embodiment, the genetically modified strain of Pseudomonas putida comprises a plurality of additional recombinant genes, namely the following five additional recombinant genes: - a recombinant gene encoding a phenylalanine hydroxylase (phhA), preferably the phhA defined by the sequence SEQ ID NO: 5, in particular under the control of a heterologous promoter allowing its overexpression, - a recombinant gene encoding tetrahydrobiopterin dehydratase (phhB), preferably phhB defined by the sequence SEQ ID NO: 6, in particular under the control of a heterologous promoter allowing its overexpression, - a recombinant gene encoding a tyrosine ammonia lyase (TAL_RG_OPT), preferably a TAL_RG_OPT as defined by the sequence SEQ ID NO: 7, - a recombinant gene encoding 4-coumarate-CoA ligase (4-CL), preferably 4-CL as defined by the sequence SEQ ID NO: 8, - a recombinant gene encoding a benzalacetone synthase (BAS), preferably a BAS as defined by the sequence SEQ ID NO: 9.

[0078] The enzymes TAL, 4-CL and BAS are all enzymes involved in the synthesis of phenylpropanoid compounds.

[0079] According to this preferred embodiment, the modified strain is capable of producing a number of phenylpropanoid compounds, namely, in particular, coumaric acid, p-coumaroyl-CoA and 4-hydroxybenzalacetone.

[0080] Another subject of the present invention relates to a genetically modified strain of Pseudomonas putida, which comprises an additional recombinant AroF-I gene encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, the sequence of which has at least 80% identity to the sequence of SEQ ID NO: 1, and in which the genes encoding the enzymes phhA and phhB are overexpressed.

[0081] Methods for synthesizing phenylpropanoid compounds Another subject of the present invention relates to a method for synthesizing one or more phenylpropanoid compounds.

[0082] The phenylpropanoid compound may be as defined above.

[0083] The synthesis method according to the present invention comprises the performance of the steps of growing a genetically modified strain of Pseudomonas putida as defined above in a culture medium under conditions allowing the expression of the mutant genes and / or additional recombinant genes required for the synthesis of one or more phenylpropanoid compounds.

[0084] According to a particular embodiment, the synthesis method according to the invention makes it possible to produce large amounts of coumaric acid.

[0085] According to a variant of this embodiment, the method may comprise growing a genetically modified strain of Pseudomonas putida comprising a mutant AroF-I gene encoding 3-deoxy-D-arabino-heptulosonic acid-7-phosphate (DAHP) synthase, for example of the sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, and at least additional recombinant genes encoding the following enzymes: - a phenylalanine hydroxylase (phhA) of sequence SEQ ID NO:5, - tetrahydrobiopterin dehydratase (phhB) of sequence SEQ ID NO: 6, - and a tyrosine ammonia lyase (TAL_RG_OPT) of sequence SEQ ID NO: 7.

[0086] According to another particular embodiment, the synthesis process according to the invention makes it possible to produce furanbinone, in particular in industrial quantities thereof, in a yield equal to at least 20 g / L of furanbinone, in particular in a fermenter of at least 500 L.

[0087] According to this embodiment, the method comprises growing a genetically modified strain of Pseudomonas putida that contains a mutant AroF-I gene encoding 3-deoxy-D-arabino-heptulosonic acid-7-phosphate (DAHP) synthase of sequence SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, and that contains additional recombinant genes encoding the following enzymes: - tyrosine ammonia lyase (TAL_RG_OPT), preferably TAL_RG_OPT defined by the sequence SEQ ID NO: 7, - 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.

[0088] The synthesis method according to the invention can also include a step of purifying and / or recovering the phenylpropanoid compound, such as coumaric acid or furanvinone.

[0089] According to a particular embodiment, the strain is a genetically modified strain of Pseudomonas putida, comprising an additional recombinant AroF-I gene encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, the sequence of which has at least 80% identity to the sequence of SEQ ID NO:1, and in which the genes encoding the enzymes phhA and phhB are overexpressed.

[0090] Uses of the strains according to the invention Another subject of the invention relates to the use of a strain of Pseudomonas putida as defined above, for the synthesis of phenylpropanoid compounds.

[0091] According to a preferred embodiment, the phenylpropanoid compound is selected from coumaric acid, p-coumaroyl-CoA, 4-hydroxybenzalacetone and furanbinone, preferably from coumaric acid and / or furanbinone.

[0092] The present invention will be better understood in light of the following non-limiting examples, which are provided for illustrative purposes only and are not intended to limit the scope of the invention as defined by the claims. EXAMPLES

[0093] Example 1 Development of a mutant that allows production of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) that is resistant to tyrosine feedback inhibition A. In silico research The starting enzyme used is the AroF-I enzyme, identified in P. putida as 3-deoxy-D-arabino-heptulosonic acid-7-phosphate (DAHP) (Uniprot identifier Q88KG6).

[0094] The tertiary structure of this enzyme (in PDB format) was reconstructed from its protein sequence using 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 base structure selected by sequence homology was that of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Saccharomyces cerevisiae.

[0095] The region that characterizes tyrosine feedback inhibition has been identified in the literature through the homologous protein in E. coli. Using an alignment of the tertiary structures of the two enzymes, we identified the region in AroF-I that corresponds to this feedback inhibition.

[0096] Next, docking of the tyrosine molecule with the AroF-I enzyme was performed using 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 Chemistry, 2010).

[0097] This method made it possible to identify the amino acids involved in the formation of chemical bonds with phenylalanine in the docking pocket; namely, inter alia, the amino acids at positions 160 (P160), 164 (Q164), 190 (S190), 191 (G191), 193 (S193) and 225 (I225).

[0098] By searching in the literature and also in the NCBI and Uniprot databases, the inventors identified protein sequences similar to AroF-I but with a certain amount of amino acid diversity, especially at positions corresponding to those involved in docking.

[0099] This approach made it possible to identify amino acids that could be substituted for those naturally found in the docking pocket of the AroF-I protein by modifying its structure or activity.

[0100] Thus, only binding to tyrosine is inhibited by the indicated mutations. Table 1 below lists the positions and original and alternative amino acids that characterize possible mutations that may inactivate AroF-I feedback inhibitory function.

[0101] [Table 1]

[0102] Among the various mutations identified in table 1, a subset of mutations was selected according to the properties of the alternative amino acids and also according to the effects of those mutations on evolutionarily related 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)).

[0103] The selected subset is as follows: P160L, Q164A, S190A, G191K, S193A, and I225P.

[0104] B. Experiment Materials and Methods Cloning of the genes encoding the AroF-I enzyme and derived mutants To express and purify the AroF-I enzyme and mutants derived therefrom, the corresponding genes are cloned into the pET28_b(+) plasmid with a His tag attached to the N-terminal part of the protein (upstream of the ATG of the gene).

[0105] To generate these constructs, the Gibson assembly method (Gibson et al., 2009) is used. The reaction products are transformed into E. coli strain BL21(DE3) (Novagen). The transformed cells are plated on agar media containing antibiotics for selection. The resulting colonies are then confirmed using PCR and sequenced to verify them.

[0106] Enzyme expression and purification The E. coli BL21(DE3) strain containing the plasmid is cultivated for 24 h in ZYM autoinduction medium with the following composition:

[0107] a) Saline-based: - Yeast extract: 5g / L - Tryptone: 10g / L - Na2SO4: 0.7g / L - NH4Cl: 2.7g / L - KH2PO4: 3.4g / L - Na2HPO4: 4.45g / L

[0108] b) Sugar solution (50 times concentrated): - Glycerol: 250g / L - Glucose: 25g / L - Lactose: 100g / L

[0109] c) Metal solution (5000 times concentrated)

[0110] [Table 2]

[0111] d) Preparation of complete medium:

[0112] [Table 3]

[0113] The medium is inoculated at OD=0.05 from an overnight preculture. The cultivation is carried out in a final volume of 50 mL in 250 mL Erlenmeyer flasks. The cultivation parameters are as follows: 5 h at 25° C., followed by 19 h at 15° C. with agitation at 140 rpm.

[0114] All chemicals used to prepare the media are obtained from Sigma-Aldrich, Roth or Euromedex.

[0115] After incubation, 50 mL of culture is centrifuged at 5000×g for 10 min at 4° C. The supernatant is discarded, the pellet is taken up in 3 mL of LEW buffer (Macherey-Nagel, see below), 1 mg / mL of lyzosyme is added and the suspension is left on ice for 30 min.

[0116] The cells are then lysed using ultrasonic disruption (Omni Sonic Ruptor 400, Omni International, power 30%, pulse 40, 8 min). After addition of 0.22% streptomycin, the samples are then centrifuged at 15000×g for 30 min at 4° C. The supernatant is collected and the soluble proteins are purified using the Protino® Ni-TED 2000 kit (Macherey-Nagel) according to the supplier's recommendations.

[0117] Finally, the purified protein is concentrated approximately 10-fold using an Amicon Ultra-4 30Kd centrifugal filter (Merck) and the elution buffer is replaced with 0.1M Tris-HCl, pH 7.5 + 10% glycerol. The purified protein is stored at -80°C.

[0118] Enzyme activity assay The activity of the enzyme is measured by monitoring the disappearance of the substrate with HPLC (high pressure liquid chromatography) detection.

[0119] Reactions are carried out in a final volume of 200 μL containing 40 mM phosphate buffer, pH 7, 300 μM phospho(enol)pyruvate (PEP), 20 μg of purified enzyme, 1 mM tyrosine (or another aromatic amino acid) or 3 μM HCl, and made up to volume with ultrapure water.

[0120] After a 2 min preincubation at 30° C., the reaction is initiated by the addition of 300 μM erythrose-4-phosphate (E4P) and the reaction mixture is incubated at 30° C. for 60 min.

[0121] Finally, the mixture is heated at 80° C. for 5 min to stop the reaction and precipitate the proteins.

[0122] Measurement of PEP consumption using HPLC The enzyme activity assay is first centrifuged at 15000×g for 10 min to precipitate the proteins, and the resulting supernatant is filtered through a 0.22 μm membrane before HPLC analysis.

[0123] The method used is described below: - System: Agilent 1100 (Agilent) - Column: Luna OMEGA polar C18 (Phenomenex) - Mobile phase: 1mM phosphoric acid - Flow rate: 0.3mL / min - UV detection: 220nm - Injection: 5μL - Duration of analysis: 15 minutes

[0124] To quantify the amount of PEP present in the samples, analyze a standard series with PEP concentrations ranging from 0.1 mM to 1 mM.

[0125] Determination of PCA and CA concentrations using HPLC The column used is: Kinetex® 5 μm F5 100A 150×4.6 mm Mobile phase: 0.1% formic acid and acetonitrile gradient: 5 min: 100% formic acid 5 to 25 minutes: 75% formic acid - 25% acetonitrile 25-30 min: 62% formic acid - 38% acetonitrile 30-35 minutes: 100% formic acid Flow rate: 1mL / min Oven temperature: 40℃ Sample injection: 10 μL Coumaric acid detection: 315 nm Cinnamic acid detection: 280 nm

[0126] Cloning of the phhaA / B genes in an arabinose-dependent expression plasmid. The phhA / B genes were amplified by PCR directly from gDNA of Pseudomonas putida strain KT2440 and cloned into the pBBR1-MCS2 plasmid under the control of the araC / pBAD promoter.

[0127] To generate these constructs, the Gibson assembly method (Gibson et al., 2009) is used. The reaction products are transformed into E. coli strain BL21(DE3) (Novagen). The transformed cells are plated on agar media containing antibiotics for selection. The resulting colonies are then confirmed using PCR and the plasmids are sequenced to verify them.

[0128] The expression plasmid is then transformed into the donor E. coli strain S17.1 for transfer into the target Pseudomonas putida strain by conjugation.

[0129] result The activity of the wild-type Pseudomonas putida AroF-I enzyme is indeed inhibited by tyrosine (Figure 1). The introduction of the P160L mutation makes it possible to make the enzyme partially resistant to this inhibition by tyrosine (Figure 2), and the effect can be improved somewhat by introducing the P160L / Q164A double mutation (Figure 3). However, the P160L / G191K and P160L / S190 double mutations have a deleterious effect on the AroF-I enzyme, which no longer has any activity, regardless of the presence or absence of tyrosine (Figure 3).

[0130] In contrast, and particularly beneficially and surprisingly, introduction of the P160L / S193A double mutation restores nearly all enzymatic activity and renders the AroF-I enzyme almost completely resistant to tyrosine inhibition (FIG. 3).

[0131] These results are confirmed by FIG. 4, which shows the synergistic effect of this double mutation on the feedback inhibition caused by tyrosine, compared to the single P160L or S193A mutations.

[0132] Example 2 Genetically engineered strains of Pseudomonas putida for the synthesis of phenylpropanoid compounds To enable the biosynthesis of phenylpropanoid compounds, multiple genetic modifications are performed in Pseudomonas putida strains. For this purpose, additional recombinant genes encoding enzymes for the synthesis of phenylpropanoid compounds such as coumaric acid or furanbinone are integrated into the Pseudomonas putida chromosome according to the following protocol.

[0133] Protocol for gene mutagenesis in Pseudomonas putida: Genetic mutagenesis in Pseudomonas putida is performed using suicide plasmids that integrate into the chromosome and then detach from it again, resulting in the desired gene deletion or insertion. The suicide plasmid used is pK18mobsacB (Schafer A, Tauch A, Jager W, Kalinowski J, Thierbach G, Puhler A. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 1994 July 22;145(1):69-73). This plasmid carries a kanamycin antibiotic resistance cassette and a sacB counterselection cassette. This plasmid also contains an origin of replication that functions only in the bacterium Escherichia coli, and an origin of transfer oriT that allows for conjugative transfer from E. coli to another bacterial strain, such as Pseudomonas putida.

[0134] The gene to be inserted is cloned into this plasmid as well as homologous regions at the selected insertion site in the bacterial chromosome. These homologous regions are cloned on either side of the gene to be inserted and should have a minimum size of 800 base pairs.

[0135] Cloning is carried out in a conjugative E. coli strain (typically strain S17.1; Simon, R., Priefer, U. and A. Pulher, A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Nature BioTechnology, Vol. 1, pp. 784-791 (1983)).

[0136] The conjugation protocol is as follows: A drop of culture of S17.1 / suicide plasma (donor strain) is placed on a rich LB agar medium (Luria-Miller, Roth, ref. X968.2, containing 1.5% agar) and a drop of culture of a Pseudomonas putida strain (recipient strain) is placed on top of the first drop. The culture dish is incubated overnight at 30 °C. The drop is then diluted in 10 mM MgSO4 and the protocol as described in Figure 6 is followed.

[0137] The pK18mobsacB conjugative plasmid is an integrative plasmid that can integrate into the genome of a recipient bacterial strain to generate exconjugants. Since the plasmid is resistant to kanamycin, the selection medium for exconjugants is LB agar containing 100 mg / mL ampicillin (Pseudomonas putida is naturally resistant to this antibiotic) and 50 mg / mL kanamycin (kanamycin, ROTH, ref. T832.4; ampicillin, EUROMEDEX, ref. EU0400-D). This medium therefore allows to select Pseudomonas putida in which the plasmid has been integrated.

[0138] The remainder of the protocol includes the following steps: - Removal of suicide plasmid: Pick 8 Kn+AmpR clones and culture them in 10mL of 100mg / mL LB+Amp medium (1 / 2000 dilution) at 30°C with agitation. Incubate for 12-24 hours. - Counterselect against 25% sucrose sacB: Spread cultures of pools of clones onto YT agar and YT + sucrose 25% agar. Incubate at 30°C until the following morning. Re-pick 20-25 clones onto sucrose + YT agar, YT + sucrose 25% agar, and YT agar + Kn 50mg / mL. Incubate at 30°C until the following morning. YT: Yeast extract tryptone - Verification of gene insertion using PCR.

[0139] Primers hybridizing on either side of the chromosomal insertion region allow for verification of mutant clones by colony PCR on KnS and sucrose+ clones.

[0140] The gene of interest to be inserted into Pseudomonas putida To allow for the biosynthesis of phenylpropanoid compounds, such as coumaric acid or furanbinone, one or more of the genes listed below are inserted into Pseudomonas putida.

[0141] These genes have been specifically identified and selected in known microorganisms. The genes were synthesized and codon optimized for maximum expression in Pseudomonas putida.

[0142] Although some genes are endogenous to Pseudomonas putida, it is recommended to test the activity of the proteins they encode, so that these activities can be optimized if these enzymes are not sufficiently active or are poorly expressed.

[0143] A gene encoding a mutant 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase enzyme:

[0144] SEQ ID NO: 10: Sequence of the 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase gene with the P160L mutation (Pseudomonas putida KT2440), encoding the DAHP synthase of the sequence of SEQ ID NO: 2.

[0145] SEQ ID NO:11: Sequence of the 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase gene carrying the P160L / Q164A double mutation (Pseudomonas putida KT2440), encoding the DAHP synthase of the sequence of SEQ ID NO:3.

[0146] SEQ ID NO: 12: Sequence of the 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase gene carrying the P160L / S193A double mutation (Pseudomonas putida KT2440), encoding the DAHP synthase of the sequence of SEQ ID NO: 4.

[0147] Additional genes enabling the synthesis of phenylpropanoid compounds

[0148] SEQ ID NO: 13: The gene for phenylalanine hydroxylase (phhA) (Pseudomonas putida KT2440), encoding phhA of the sequence SEQ ID NO: 5.

[0149] SEQ ID NO:14: The gene for tetrahydrobiopterin dehydratase (phhB), encoding phhB of the sequence of SEQ ID NO:6 (Pseudomonas putida KT2440).

[0150] SEQ ID NO: 15: Gene for tyrosine ammonia lyase (Rhodotorula glutinis), encoding the β-xylosidase of sequence SEQ ID NO: 7.

[0151] SEQ ID NO: 16: gene for 4-coumarate-CoA ligase (4-CL) (Pseudomonas putida KT2440 fcs gene), encoding 4-CL of sequence SEQ ID NO: 8.

[0152] SEQ ID NO: 17: gene for benzalacetone synthase (BAS) (Rheum palmatum), encoding the BAS of sequence SEQ ID NO: 9.

[0153] Example 3 In vivo activity of the genetically modified strain of Pseudomonas putida according to the present invention Construction of the AroF-I-fbr P160L / S193A mutant Pseudomonas putida KT2440 strain was genetically modified as described in Example 1 to express the AroF-I gene encoding a DAHP synthase containing the P160L / S193A double mutation defined by the amino acid sequence of SEQ ID NO:4, and to express an additional recombinant gene encoding a heterologous tyrosine ammonia lyase (TAL_RG_OPT) of the sequence of SEQ ID NO:7.

[0154] This strain is referred to as "AroF-I-fbr P160L / S193A mutant."

[0155] The activity of the AroF-I-fbr P160L / S193A mutant for the production of coumaric acid (PCA) or cinnamic acid (CA) was determined by measuring the production of these acids in comparison with a Pseudomonas putida KT2440 strain genetically modified to express the wild-type AroF-I enzyme, with the latter strain serving as a control (AroF-I-WT mutant).

[0156] result Cinnamic acid production is significantly increased in P. putida strains expressing the AroF-I enzyme carrying the P160L / S193A double mutation (Figure 6).

[0157] Optimization of AroF-I-fbr P160L / S193A mutant for coumaric acid production 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 araC / pBADopt inducible promoter, induction was performed with 0.5% arabinose:plasmid pC2F387.

[0158] This strain is called "optimized AroF-I-fbr mutant."

[0159] Subsequently, the production of coumaric acid (PCA) or cinnamic acid (CA) by the optimized AroF-I-fbr mutant was compared with that obtained using the AroF-I-fbr P160L / S193A mutant and two control strains, namely: - Pseudomonas putida strain KT2440 expressing the wild-type AroF-I enzyme (AroF-I-WT mutant), and - Pseudomonas putida strain KT2440 expressing wild-type AroF-I and the two phhA and phhB enzymes of the sequences SEQ ID NO:5 and SEQ ID NO:6, respectively (AroF-I-WT phhA / B mutant). The results were compared with those obtained using

[0160] result The results presented in FIG. 6 show that overexpression of the phhA and phhB genes in the AroF-I-WT mutant strain results in more phenolic acids (PCA+CA) production.

[0161] Of particular interest, the optimized AroF-I-fbr mutant makes it possible to obtain a much higher production of total phenolic acids than the AroF-I-fbr P160L / S193A and AroF-I-WT phhA / B mutants. In comparison, coumaric acid represents almost all of the total phenols produced, which is advantageous since cinnamic acid is not an industrially useful compound for the production of phenylpropanoid compounds.

[0162] These results clearly demonstrate that the optimized AroF-I-fbr mutant is particularly suitable for the production of phenylpropanoid compounds, especially coumaric acid.

[0163] In this application, reference is made to a sequence listing, the identifiers of which ("SEQ ID NO") are listed in the table below. Regardless of the form in which the sequence listings are provided, they form part of this application.

[0164] [Table 4A]

[0165]

Table 4B

[0166]

Table 4C

[0167]

Table 4D

[0168]

Table 4E

[0169] [Table 4F]

[0170] Table 4G

[0171]

Table 4H

[0172]

Table 4I

[0173]

Table 4J

[0174]

Table 4K

[0175] List of cited references Patent Literature The following patent documents are cited where they may be useful: - patcit1:FR1234567(application number); - patcit2:US2004230550 (publication number); and - patcit3:FR2795457(publication number).

[0176] Non-patent literature The following non-patent literature is cited where possible: - nplcit1: Klesk et al., 2004, J. Agric. Food Chem. 52, 5155-61; - nplcit2:Larsen et al., 1991, Acta Agric.Scand.41, 447~54). - nplcit3: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).): 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. June 1, 2019;206(3):322-334. - nplcit5:Lutke-Eversloh and Stephanopoulos, 2005 - nplcit6:Lutke-Eversloh and Stephanopoulos、2007.「L-Tyrosine production by deregulated strains of Escherichia coli」. - nplcit7:Kangら、2012「Artificial biosynthesis of phenylpropanoic acids in a tyrosine overproducing Escherichia coli strain」 - nplcit8:Santosら、2011.「Optimization of a heterologous pathway for the production of flavonoids from glucose」、 - nplcit9:Juminagaら、2011.「Modular engineering of L-tyrosine production in E. coli」. - nplcit10:Rodriguezら、2015、「Establishment of a yeast platform strain for production of p-coumaric acid through metabolic engineering of aromatic amino acid biosynthesis.」 - nplcit11:Caleroら、2016.「Broad-host range ProUSER vectors enable fast characterization of inducible promoters and optimization of p-coumaric acid production in Pseudomonas putida KT2440」. - nplcit12:Caleroら、2017.「Genome-wide identification of tolerance mechanisms toward p-coumaric acid in Pseudomonas putida」. - nplcit13: 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, pp. 104 - 108. - nplcit14: Beekwilder, et al., 2007. "Microbial production of natural raspberry ketone". - nplcit15: Lee, et al., 2017. "Heterologous production of raspberry ketone in the wine yeast Saccharomyces cerevisiae via pathway engineering and synthetic enzyme fusion". - nplcit16: Waterhouse, A., et al., "SWISS-MODEL: homology modelling of protein structures and complexes". Nucleic Acids Res. 46(W1), W296 - W303 (2018) - nplcit17: 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 chemistry, 2010). - nplcit18: Ding, R. et al., "Introduction of two mutations into AroG increases phenylalanine production in Escherichia coli". Biotechnol Lett 36, 2103 - 2108 (2014). - nplcit19: (Gibson et al., 2009). - nplcit20: Schafer A et al., "Small mobilizable multi - purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum". Gene Jul 22, 1994;145(1):69 - 73 - nplcit21: Simon, R. et al., "A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria". Nature BioTechnology, Vol. 1, 784 - 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 computational chemistry. - 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 negative hosts." Biotechnol Bioeng, 2010 Jun 1;106(2):326-32.

Claims

1. A genetically modified strain of Pseudomonas putida, characterized in that it contains a mutated AroF-I gene encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, the sequence of which has at least 90% identity to the sequence of SEQ ID NO:1 and which has at least one P160L mutation, a P160L / Q164A double mutation, or a P160L / S193A double mutation, preferably a P160L / S193A double mutation.

2. 2. The genetically modified strain of Pseudomonas putida according to claim 1, characterized in that it is capable of expressing a recombinant DAHP synthase that is resistant to feedback inhibition by tyrosine.

3. 3. A genetically modified strain according to claim 1 or 2, characterized in that it comprises an additional recombinant gene encoding a phenylalanine hydroxylase (phhA), the sequence of which has at least 80% identity to the sequence of SEQ ID NO: 5, and an additional recombinant gene encoding a tetrahydrobiopterin dehydratase (phhB), the sequence of which has at least 80% identity to the sequence of SEQ ID NO: 6; said recombinant genes phhA and phhB are preferably under the control of heterologous promoters allowing their overexpression.

4. 3. A 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 to the sequence of SEQ ID NO: 2, the sequence of SEQ ID NO: 3, or the sequence of SEQ ID NO:

4.

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

4.

6. The following recombinant genes: - a recombinant gene encoding a polypeptide having tyrosine ammonia lyase (TAL) activity, in particular a TAL polypeptide having at least 80% identity to the amino acid sequence of SEQ ID NO: 7 of the polypeptide TAL_RG_OPT, - a recombinant gene encoding a polypeptide having 4-coumarate-CoA ligase (4-CL) activity, in particular a 4CL polypeptide having at least 80% identity to the amino acid sequence of SEQ ID NO: 8, - a recombinant gene encoding a polypeptide having benzalacetone synthase (BAS) activity, in particular a BAS polypeptide having at least 80% identity to the sequence of SEQ ID NO:

9.

3. The genetically modified strain according to claim 1 or 2, further comprising one or more additional recombinant genes selected from:

7. 2. The genetically modified strain according to claim 1, characterized in that it is capable of producing phenylpropanoid compounds, preferably coumaric acid, p-coumaroyl-CoA, 4-hydroxybenzalacetone or furanbinone.

8. 10. A method for synthesizing a phenylpropanoid compound, comprising growing the genetically modified strain of claim 7 in a culture medium under conditions allowing expression of recombinant genes required for the synthesis of phenylpropanoids, characterized in that the phenylpropanoid compound is synthesized by the genetically modified strain.

9. 9. The method of claim 8, further comprising recovering the phenylpropanoid compound from the culture medium.

10. 3. Use of the strain according to claim 1 or 2 for the synthesis of a phenylpropanoid compound or a phenylpropanoid derivative, preferably coumaric acid or furanbinone.

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