EP4337767A1

EP4337767A1 - Biosynthesis of phenylpropanoid compounds

Info

Publication number: EP4337767A1
Application number: EP22727965.0A
Authority: EP
Inventors: William MERRÉ; Ricardo DE ANDRADE; Caroline RANQUET
Original assignee: Bgene Genetics
Current assignee: Bgene Genetics
Priority date: 2021-05-12
Filing date: 2022-05-09
Publication date: 2024-03-20
Also published as: JP2024517485A; FR3122882A1; WO2022238645A1; CN117730147A

Abstract

The present invention relates to the field of the production of phenylpropanoid compounds, especially that of genetically modified strains for the production of phenylpropanoid compounds. In particular, the 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), and to the use thereof for the synthesis of phenylpropanoid compounds, in particular coumaric acid or frambinone.

Description

Title: Biosynthesis of phenylpropanoid compounds

Technical area

The present invention falls within the field of the production of phenylpropanoid compounds, and in particular that of strains genetically modified for the production of phenylpropanoid compounds such as coumaric acid or frambinone.

Prior technique

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

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

[0004] Insofar as it is only present in small quantities in raspberries (1-4 mg per kg of fruit), natural frambinone is of great value (Larsen et al., 1991). However, as its natural availability is limited, its biotechnological production is highly desirable.

[0005] In this context, the pathway for the biosynthesis of phenylpropanoid compounds, in particular frambinone, can be reconstituted within a microorganism thanks to 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 in particular the precursor of coumaric acid or frambinone. [0007] In fact, frambinone can be obtained from the aromatic amino acid L-tyrosine as an initial substrate via a 4-step biosynthetic pathway.

[0008] According to the first step, the 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 molecule of Coenzyme A (CoA) is grafted onto coumaric acid. Coumaroyl-CoA is then converted by a benzalacetone synthase (BAS, EC 2.3.1.212) into 4-hydroxy benzalacetone. This reaction is a decarboxylative condensation and uses a malonyl-CoA unit as a co-substrate. The final step is the reduction of 4-hydroxy benzalacetone to frambinone by a benzalacetone reductase.

[0009] Thus, the development of a strain that overproduces tyrosine as a platform for the production of phenylpropanoid compounds, in particular frambinone, is a crucial starting point for the biosynthesis of these compounds.

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

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

[0012] Numerous articles describe the deregulation of this pathway in E. coli and one enzyme has been particularly extensively studied: DAHP synthase (DAHP=3-Deoxy-D-arabino-Heptulodonate 7-phosphate). It 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) to DAHP. 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 retro-regulation of phenylalanine and tyrosine. Fbr mutants of the TyrA protein (tyrAfbr mutant) have also been 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 strains of E. coli overproducing tyrosine obtained by combining these different mutated enzymes. These strains have been 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 made to obtain strains of E. coli that overproduce tyrosine 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 Saccharomyces cerevisiae strains which overexpress tyrosine, as described by Rodriguez et al., 2015.

[0018] Document GB 2416 769 describes the possibility of producing frambinone using microorganisms containing genes coding for the enzymes 4CL and BAS, at least one of which is from a heterologous source. The preferred microorganism is E. coli (strain BL21) and may also comprise a sequence encoding BAR, C4H, PAL and/or CHS, the sequence encoding BAR being advantageously endogenous. [0019] However, it appears that the E. coli or S. cerevisiae strains do not tolerate the toxicity of phenylpropanoid compounds well and are therefore not the microorganisms best suited for their production.

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

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

The overproduction of tyrosine and phenol has been described in the bacterium Pseudomonas taiwanensis VLB120 thanks to the implementation of point mutations in 3 genes: trpEP290S, aroF-l P148L and pheAT310I (Wynands et al., Metab Eng. 2018 May; 47:121-133).

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, in particular coumaric acid and frambinone.

Technical problem

[0024] The tyrosine-overproducing strains 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] This disclosure improves the situation. Summary

One of the aspects of the present invention relates to a genetically modified strain of Pseudomonas putida comprising a mutated AroF-1 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 P160L/Q164A double mutation, or a P160L/S193A double mutation, preferably a P160L/S193A double mutation.

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.

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

[0030] The characteristics described in the following paragraphs can optionally be implemented. They can be implemented independently of each other or in combination with each other:

Brief description of figures

[0031] Other characteristics, details and advantages will appear on reading the detailed description below, and on analyzing the appended drawings, in which: FIG. 1

[0032] [Fig. 1] Graph of PEP consumed in mM in the presence or absence of aromatic amino acid by the wild-type AroF-1 protein (WT). The results shown are from 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-1 WT protein compared with the simple mutants AroF-1-G191, AroF-1-P160 and AroF-1-S193. The results shown are from three independent replicates. Fig. 3

[0034] [Fig. 3] Graph representing the share of PEP consumed in mM in the presence or absence of aromatic amino acid by the aroF-l WT protein compared to the double mutants AroF-l-P160_G191, AroF-l-P160L_Q164, AroF-l-P160_S190 and AroF -l-P160_S193. The results shown are from three independent replicates.

Fig. 4

[0035] [Fig. 4] Graph representing the share of PEP consumed in mM in the presence or absence of aromatic amino acid by the aroF-l WT protein compared to the simple mutants AroF-l-P160 and AroF-l-S193 and to the double mutant AroF-l- P160_S193. The results shown are from 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 strains of Pseudomonas putida expressing the enzymes AroF-l WT/TAL (aroF-l WT) and aroF-l fbr P160L/S193A /TAL (aroF-l fbr). Fig. 7

[0038] [Fig. 7] Graph presenting the production of total phenolics and the proportion of coumaric acid among total phenolics (%PCA) by strains of Pseudomonas putida expressing the enzymes AroF-l WT/TAL (aroF-l WT), AroF-l WT /TAL + empty plasmid (aroF-l WT + control), AroF-l WT/TAL + phhA/B plasmid (aroF-l WT + phhA/B), aroF-l fbr P160L/S193A/TAL (aroF-l fbr ), aroF-1 fbr P160L/S193A/TAL + empty plasmid (aroF-1 fbr + control), and aroF-1 fbr P160L/S193A/TAL + phhA/B plasmid (aroF-1 fbr + phhA/B). The expression of the phhA/B genes from the araC/pBAD promoter is induced or not.

Description of embodiments A first object of the invention therefore relates to a genetically modified strain of Pseudomonas putida comprising a mutated AroF-l gene coding for 3-Deoxy-D-arabino-Heptulodonate 7-phosphate (DAHP) synthase whose sequence present at the identity with at least 80% with the sequence SEQ ID NO: 1 and exhibiting at least one P160L mutation, a P160L/Q164A double mutation, or a P160L/S193A double mutation, preferably a P160L/S193A double mutation.

Within the meaning of the present description, the expressions “genetically modified strain of Pseudomonas putida”, “modified strain of Pseudomonas putida”, “genetically modified strain”, or even “modified strain” are considered to be synonyms of one another.

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 unnatural 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 editing of genes 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.

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. 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.

The 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) .

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 a starting strain to obtain the genetically modified strains according to the invention (Calera 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).

Within the meaning 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. Default algorithm settings 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 settings is used

For the purposes of the present invention, the term “mutated AroF1 gene” means a nucleic acid comprising at least one part encoding a mutated version of 3-Deoxy-D-arabino-Heptulodonate 7-phosphate (DAHP) synthase under control of a promoter allowing its expression in the genetically modified strain.

For the purposes of the present invention, the term 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) to DAHP. According to a particular embodiment, the genetically modified strain of Pseudomonas putida comprises a mutated AroF-1 gene coding for DAHP synthase, the amino acid sequence of which has at least 80%, 85%, 90%, 95% and most particularly, at least 98% identity with the sequence SEQ ID NO: 1, and exhibiting at least one P160L mutation, a P160L/Q164A double mutation, or a P160L/S193A double mutation, preferably a P160L/ S193A.

The AroF-l gene endogenous to Pseudomonas putida codes for the DAFIP 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-l gene, at least one mutated AroF-l 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.

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

According to a variant of this embodiment, the modified strain of Pseudomonas putida comprises a mutated AroF-1 gene coding for DAFIP synthase, the sequence of which has at least 80%, 85%, 90%, 95% and very particularly , at least 98% identity with the sequence SEQ ID NO: 2 and contains the P160L mutation.

According to another particular embodiment, the modified strain of Pseudomonas putida comprises a mutated AroF-1 gene coding for DAFIP synthase and exhibiting at least one P160L/Q164A double mutation. According to this embodiment, the mutated AroF-l gene codes for DAFIP synthase comprising the double mutation P160L/Q164A defined by the amino acid sequence SEQ ID NO: 3.

According to a variant of this embodiment, the modified strain of Pseudomonas putida comprises a mutated AroF-1 gene coding for DAFIP synthase whose sequence has at least 80%, 85%, 90%, 95% and most particularly at least 98% identity with the sequence SEQ ID NO: 3 and contains the double mutation P160L/Q164A.

According to a preferred embodiment, the modified strain of Pseudomonas putida comprises a mutated AroF-1 gene coding for DAHP synthase exhibiting at least one double P160L/S193A mutation. According to this embodiment, the mutated AroF-l gene codes for DAFIP synthase comprising the double mutation P160L/S193A defined by the amino acid sequence SEQ ID NO: 4.

According to a variant of this embodiment, the modified strain of Pseudomonas putida comprises a mutated AroF-1 gene coding for DAFIP synthase, the sequence of which has at least 80%, 85%, 90%, 95% and most particularly , at least 98% identity with the sequence SEQ ID NO: 4 and contains the double mutation P160L/S193A.

In one embodiment, which can be combined with the previous ones, the coding sequence of the mutated AroF-1 gene is placed under the control of a heterologous promoter, in particular a constitutive or inducible promoter, for example chosen from promoters ptrc, xyls/pm or araC/pBAD, which makes it possible to overexpress the mutated AroF-1 gene in the genetically modified strain according to the invention. In a preferred embodiment, the coding sequence of the mutated AroF-1 gene in the genetically modified strain according to the invention is inserted in such a way as to render the AroFI gene non-functional, for example by disruption of the AroFI gene, or by deletion of the gene AroFI and in particular all or part of its coding sequence.

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

Quite advantageously, by implementing specific mutations, the Applicant has developed a strain of Pseudomonas putida capable of expressing a recombinant DAFIP synthase insensitive to negative retroregulation by tyrosine, thus deregulating the pathway production of the tyrosin. 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.

The term "tyrosine overproducing strain" within the meaning of the present invention, a modified strain of Pseudomonas putida capable of producing tyrosine in a greater quantity compared to a wild-type Pseudomonas putida strain comprising the AroF-1 gene encoding DHAP synthase of amino acid sequence SEQ ID NO: 1 (unmutated gene), either as a final product or as an intermediate product.

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

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-1 gene as defined above. The additional recombinant gene may result from the insertion of a heterologous promoter, for example a strong promoter to overexpress an endogenous gene of Pseudomonas putida, or a recombinant coding sequence coding for a protein not naturally expressed in Pseudomonas putida.

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 ). The Applicant has found that these two enzymes, although present endogenously in Pseudomonas putida, may not be active enough 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 have therefore been optimized by overexpressing these two enzymes phhA and phhB. The 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.

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

The 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 dependence on a strong promoter.

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.

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 most 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 an enzyme with phhB activity, in particular under the control of a heterologous promoter allowing their overexpression.

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.

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-1 gene coding for DAFIP synthase comprising the double mutation P160L/S193A defined by the amino acid sequence SEQ ID NO: 4, or a sequence presenting 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 very particularly, at least 98% d 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 most particularly at least 98% d 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.

According to another particular embodiment which can preferably be combined with the preceding one, the genetically modified strain of Pseudomonas putida comprises an additional recombinant gene coding for a polypeptide with tyrosine ammonia lyase (TAL) activity. A recombinant gene encoding TAL can come from the microorganism Rhodotorula glutinis and be optimized according to the 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 coding for 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 most particularly at least 98% identity with the sequence SEQ ID NO: 7 and coding for an enzyme with TAL activity.

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 enzyme TAL.

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 coding for 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 presenting at least 80%, 85%, 90%, 95% and most particularly, at least 98% identity with the sequence SEQ ID NO: 8 and coding for an enzyme with 4-CL activity.

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

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 coding for a BAS (EC 2.3.1.212) whose sequence is defined by the amino acid sequence SEQ ID NO: 9 or by a sequence presenting at least 80%, 85% , 90%, 95% and most particularly, at least 98% identity with the sequence SEQ ID NO: 9 and coding for an enzyme with BAS activity.

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

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 tetrahydrobiopterine 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 coding for a benzalacetone synthase (BAS), preferably a BAS defined by the sequence SEQ ID NO: 9.

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

According to this preferred embodiment, the modified strain is capable of producing a multitude of phenylpropanoid compounds, namely in particular coumaric acid, p-coumaroyl-coA, 4-hydroxybenzalketone. Another subject of the invention relates to a genetically modified strain of Pseudomonas putida comprising an additional recombinant gene AroF-1 coding for 3-Deoxy-D-arabino-Heptulodonate 7-phosphate (DHAP) synthase, the sequence of which is present 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

Another object of the invention relates to a process for the synthesis of one or more phenylpropanoid compounds.

The phenylpropanoid compounds can be defined above.

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

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

According to a variant of this embodiment, the method may comprise a step of growing a genetically modified strain of Pseudomonas putida comprising a mutated AroF-1 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.

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 I. According to this embodiment, the method comprises a step of growing a genetically modified strain of Pseudomonas putida comprising a mutated AroF-1 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.

The synthesis process according to the invention may also comprise a step for purifying and/or recovering the phenylpropanoid compound, such as coumaric acid or frambinone

According to a particular embodiment, the strain is a genetically modified strain of Pseudomonas putida comprising an additional recombinant gene AroF-1 coding for 3-Deoxy-D-arabino-Fleptulodonate 7-phosphate (DFIAP) 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

Another object of the invention relates to the use of a strain of Pseudomonas putida as defined above for the synthesis of phenylpropanoid compounds.

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.

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

[0109] Example 1: Development of a mutant allowing the production of 3-Deoxy-D-arabino-Heptulodonate 7-phosphate (DHAP) insensitive to the negative retro-regulation of tyrosine [0100] A. In Silico Study

The starting enzyme used is the AroF-1 enzyme identified as 3-Deoxy-D-arabino-Heptulodonate 7-phosphate (DAHP) in P. putida (Uniprot identifier Q88KG6).

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 oerevisiae. The region which characterizes tyrosine retro-regulation has been identified in the literature via a homologous protein in E. coli. Thanks to an alignment of tertiary structures of these two enzymes, the region corresponding to this retro-regulation on AroF-1 has been identified.

[0104] Next, the “docking” of a tyrosine molecule with the AroF-1 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 Computanional Chemistry, 2010).

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 in positions 160 (P160), 164 (Q164), 190 (S190), 191 (G191), 193 (S193) and 225 (I225).

[0106] By research in the literature but also in the NCBI and Uniprot databases, we have identified protein sequences similar to AroF-1, but presenting a certain diversity of amino acids, in particular in the positions corresponding to those involved in the dock. This approach has made it possible to identify amino acids which can replace the amino acids naturally found in the “docking” pocket of the AroF-1 protein without modifying its structure or its activity.

Thus, only tyrosine binding is inhibited by the mutations demonstrated. Table 1 below lists the positions and the original amino acids as well as the surrogates that characterize the mutations that could potentially disable the feedback regulation function of AroF-1.

[0109] [Table 1] Among the various 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). The chosen subset is as follows: P160L, Q164A, S190A, G191 K, S193A, and I225P.

[0112] B. Experiments

[0113] Materials and methods

[0114] Cloning of the genes coding for the AroF-1 enzyme and the derived mutants

In order to express and purify the AroF-1 enzyme and the resulting mutants, 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).

To perform 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

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/l

- tryptone: 10 g/l

- Na ₂ SC>4: 0.7 g/l

- NH ₄ CI: 2.7 g/l

- KH2PO4: 3.4 g/l

- Na2HPC>4: 4.45 g/l

[0120] b) Solution of sugars (concentrated 50 times):

- Glycerol: 250 g/l

- Glucose: 25 g/l

- Lactose: 100 g/l [0121] c) Solution of metals (concentrated 5000 times): [Table 2]

[0122] d) Preparation of the complete medium: [0123] [Table 3] 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 at 140 rpm, 5 hours at 25°C then 19 hours at 15°C.

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

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

The cells are then lysed by sonication (Omni Sonic Ruptor 400, Omni International, power 30%, pulses 40 for 8 minutes). After adding 0.22% of 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)

Finally, the purified proteins are concentrated ~10 times using Amicon Ultra-430Kd centrifugal filters (Merck) and the elution buffer is replaced with 0.1 M Tris-HCI pH7.5 + 10% Glycerol . Purified proteins are stored at -80°C.

[0129] Enzyme activity test

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

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

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

The enzymatic activity tests are first centrifuged for 10 minutes at 15000×g in order to precipitate the proteins. The supernatant obtained is filtered on a 0.22 μm membrane before HPLC analysis.

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: 5mI

- Analysis time: 15 minutes

In order to quantify the quantity of PEP present in the samples, a range of standards is analyzed with PEP concentrations of 0.1 mM to 1 mM.

[0138] Measurement of PCA and CA concentration by HPLC

The column used is as follows: Kinetex® 5pm F5 100A 150x4.6mm Mobile phases: Formic acid 0.1% and Acetonitrile Gradient:

5min: 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: 1ml/min

Oven temperature: 40°C

Sample injection: 10mI Coumaric acid detection: 315nm Cinnamic acid detection: 280nm

Cloning of the phaA/B Genes in an Arabinose-Dependent Expression Plasmid The phhA/B genes are amplified by PCR directly from gDNA of the Pseudomonas putida KT2440 strain and cloned into a pBBR1-MCS2 plasmid, dependent on of the araC/pBAD promoter.

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

The expression plasmid is then transformed into the strain of E. coli donor S17.1 in order to be transferred into the strains of Pseudomonas putida of interest by conjugation.

[0142] Results

The activity of the wild-type AroF-l enzyme of Pseudomonas putida is well inhibited by tyrosine (FIG. 1). The introduction of the P160L mutation makes it possible to render the enzyme partially insensitive to this inhibition by tyrosine (Figure 2), and the effect can be further improved slightly by the introduction of a double P160L/Q164A mutation (Figure 3 ). On the other hand, the double mutations P160L/G191K and P160L/S190 have a deleterious effect on the AroF-l enzyme which no longer has any activity, whether in the presence of tyrosine or not (Figure 3).

On the other hand, and in a particularly interesting and surprising manner, the introduction of a double P160L/S193A mutation restores almost all of the enzymatic activity and renders the AroF-1 enzyme almost completely insensitive to inhibition. tyrosine (Figure 3). These results are confirmed by FIG. 4, which clearly shows the synergistic effect of this double mutation on the negative retro-regulation exerted by tyrosine compared with the single P160L or S193A mutation.

Example 2: Pseudomonas putida strains genetically modified for the synthesis of phenylpropanoid compound

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

Protocol for genetic mutagenesis of Pseudomonas putida:

[0149] Genetic mutagenesis in Pseudomonas putida is carried out by means of suicide plasmids which integrate into the chromosome and emerge from it, leaving the desired deletion or insertion of genes. The suicide plasmid used is pK18mobsacB (Schàfer A, Tauch A, Jàger 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 Corynebacterium glutamicum Gene 1994 Jul 22;145(1):69-73). This plasmid carries a resistance cassette to the antibiotic Kanamycin and the sacB counter-selection cassette. It also has an origin of replication that only works in E. coli bacteria, 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.

The genes to be inserted are cloned into this plasmid, as well as the regions of homology at the chosen insertion site on the bacterial chromosome. These areas of homology are cloned on either side of the gene to be inserted, and must be at least 800 base pairs in size.

The cloning is done in a strain of Escherichia coli capable of conjugating (generally the strain S17.1, 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)).

The conjugation protocol is as follows:

A drop of culture of S17.1/suicide plasmid (donor strain) is deposited on a rich LB Agar medium (Luria-Miller, Roth, reference X968.2 containing 1.5% agar), and a drop of culture of the Pseudomonas putida strain (recipient strain) is deposited 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 performed as described in Figure 6.

The conjugation plasmid pK18mobsacB is an integrative plasmid capable of integrating into the genome of the recipient bacterial strain in order to produce transconjugants. The plasmid being 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 the Pseudomonas putida bacteria in which a plasmid has integrated.

The remainder of the protocol comprises the following steps:

- Excision of the suicide plasmid: take 8 Kn+AmpR clones and put them in culture in 10ml of LB+Amp 100 mg/ml medium (1/2000 dilution) at 30° C. with shaking. Incubate between 12h and 24h.

- SacB counter-selection on Sucrose 25%: streak the culture of the pool of clones on YT agar and YT+Sucrose 25% agar. Incubate at 30°C overnight. Subculture 20-25 Sucrose+ clones onto YT agar, YT+Sucrose 25% agar and YT agar +Kn 50 mg/ml. Incubate at 30°C overnight.

YT: Yeast extract tryptone

- Verification by PCR of the insertion of the gene

Primers hybridizing on either side of the chromosomal insertion zone make it possible to verify the mutant clones by carrying out PCR on the colony for the KnS and Sucrose+ clones. [0157] Genes to insert into Pseudomonas putida

One or more of the genes listed below are inserted into Pseudomonas putida in order to allow the biosynthesis of phenylpropanoid compounds, such as for example coumaric acid or frambinone. These genes have been specifically identified and selected from known microorganisms. Genes are synthesized and codons are optimized for maximum expression in Pseudomonas putida.

Certain 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.

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

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

[0163] SEQ ID NO: 11: sequence of the gene for 3-Deoxy-D-arabino-

Heptulodonate 7-phosphate (DHAP) synthase with double mutation P160L/Q164A (Pseudomonas putida KT2440) encoding the DHAP synthase of sequence SEQ ID NO:3.

[0164] SEQ ID NO: 12: sequence of the gene for 3-Deoxy-D-arabino-

Heptulodonate 7-phosphate (DHAP) synthase with double mutation P160L/S193A (Pseudomonas putida KT2440) encoding the DHAP synthase of sequence SEQ ID NO: 4. [0165] Additional genes allowing 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: tetrahydrobiopterin dehydratase (phhB) gene (Pseudomonas putida KT2440) encoding the 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.

SEQ ID NO: 16: gene (fcs Pseudomonas putida KT2440 gene) for 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 the BAS of sequence SEQ ID NO: 9.

Example 3: In vivo activity of a genetically modified Pseudomonas putida strain according to the invention

[0172] Preparation of an AroF-1-fbr P160L/S193A mutant

A Pseudomonas putida KT2440 strain was genetically modified as described in example 1 so as to express the AroF-1 gene coding for 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 coding for a heterologous tyrosine ammonia lyase (TAL_RG_OPT) of sequence SEQ ID NO: 7.

This strain is called “AroF-1-fbr P160L/S193A mutant”.

The activity of the AroF-l-fbr P160L/S193A mutant on the production of coumaric acid (CPA) or cinnamic acid (CA) was determined by measuring the quantities produced of these acids compared to a Pseudomonas strain putida KT2440 genetically modified so as to express the wild-type AroF-1 enzyme and which serves as a control (Mutant AroF-1-WT).

[0176] Results

The production of cinnamic acid increases significantly in a strain of P. putida expressing the enzyme AroF-1 fbr having the double mutation P160L/S193A (FIG. 6). [0178] Optimization of the AroF-1-fbr P160L/S193A mutant for the production of coumaric acid

The AroF-1-fbr P160L/S193A mutant was modified in order to express the additional recombinant genes coding for a phenylalanine hydroxylase (phhA) of sequence SEQ ID NO: 5 and a tetrahydrobiopterin dehydratase (phhB) of sequence SEQ ID NO: 6. The genes coding for these enzymes were cloned into a plasmid and expressed under the control of the inducible araC/pBADopt promoter, the induction being carried out with 0.5% arabinose: plasmid pC2F387.

This strain is called “optimized AroF-1-fbr mutant”.

The production of coumaric acid (CPA) or cinnamic acid (CA) of the optimized AroF-l-fbr mutant was measured and then compared with that obtained with the AroF-l-fbr mutant P160L/S193A, and that obtained with two control strains, namely:

- a Pseudomonas putida KT2440 strain expressing the wild-type AroF-l enzyme (Mutant AroF-l-WT), and

- a Pseudomonas putida KT2440 strain expressing the wild-type AroF-l enzyme and the two phhA and phhB enzymes of sequences SEQ ID NO: 5 and SEQ ID NO: 6 respectively (Mutant AroF-l-WT phhA/B).

[0182] Results

The results presented in Figure 6 show that the overexpression of the phhA and phhB genes in the AroFI-WT mutant strain leads to a greater production of phenolic acid (PCA+CA).

Of particular interest, the optimized AroF-l-fbr mutant makes it possible to obtain a much greater production of total phenolic acids than the AroF-l-fbr P160L/S193A mutant and the AroF-l-WT phhA mutant. /B. In proportion, coumaric acid represents almost all of the total phenolics produced and this is advantageous because cinnamic acid is not an industrially exploitable compound for the production of phenylpropanoid compounds. These results clearly demonstrate that the optimized AroF-1-fbr mutant is particularly suitable for the production of phenylpropanoid compounds, and particularly coumaric acid.

Free text of the 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 this application.

[0187] [Table 4]

List of cited documents

Patent documents

For all practical purposes, the following patent document(s) is (are) cited: - patcitl: FR1234567 (filing number);

- patcit2: US2004230550 (publication number); and

- patcit3: FR2795457 (publication number).

Non-patent literature

For all practical purposes, the following non-patent elements are cited: - nplcitl: 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. 2019 Jun 1; 206(3):322-334.

- nplcit5: Lütke-Eversloh and Stephanopoulos, 2005

- nplcit6: Lütke-Eversloh and Stephanopoulos, 2007. “L-Tyrosine production by deregulated 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”.

- npicitlO: Rodriguez et al., 2015, “Establishment of a yeast platform strain for production of p-coumaric acid through metabolic engineering of aromatic amino acid biosynthesis.”

- npicitH: Calera et al., 2016. “Broad-host range ProUSER vectors enable fast characterization of inducible promoters and optimization of p-coumaric acid production in Pseudomonas putida KT2440”.

- nplcitl 2: Calera et al., 2017. “Genome-wide identification of tolerance mechanisms toward p-coumaric acid in Pseudomonas putida”.

- nplcitl 3: 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.

- nplcitl 4: Beekwilder et al., 2007. “Microbial production of natural raspberry ketone”.

- nplcitl 5: Lee et al., 2017. “Heterologous production of raspberry ketone in the wine yeast Saccharomyces cerevisiae via pathway engineering and synthetic fusion enzyme”.

- nplcitl 6: Waterhouse, A. et al., “SWISS-MODEL: homology modeling of protein structures and complexes”. Nucleic Acids Res. 46(W1), W296-W303 (2018)

- nplcitl 7: O. Trott et al., “AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading”,

Journal of computer chemistry, 2010).

- nplcitl 8: Ding, R. et al. “Introduction of two mutations into AroG increases phenylalanine production in Escherichia coli”. Biotechnol Lett 36, 2103-2108 (2014).

- nplcitl 9: (Gibson et al., 2009). - nplcit20: Schàfer 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 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 negative bacteria”. Nature

BioTechnology volume 1, pages 784-791 (1983)).

- nplcit22: 0. 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 negative hosts”. Biotechnol Bioeng, 2010 Jun 1;106(2):326-32.

Claims

[Claim 1] Genetically modified strain of Pseudomonas putida characterized in that it comprises a mutated AroF-1 gene coding for 3-Deoxy-D-arabino-Heptulodonate 7-phosphate (DAHP) synthase, the sequence of which presents at least 90% of identity with the sequence SEQ ID NO: 1 and exhibiting at least one P160L mutation, a P160L/Q164A double mutation, or a P160L/S193A double mutation, preferably a P160L/S193A double mutation.

[Claim 2] Genetically modified strain of Pseudomonas putida, according to claim 1, characterized in that it is capable of expressing a recombinant DAHP synthase insensitive to retroregulation by tyrosine.

[Claim 3] Genetically modified strain according to claim 1 or 2, 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 dehydratase (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.

[Claim 4] Genetically modified strain according to one of Claims 1 to 3, 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.

[Claim 5] Genetically modified strain according to claim 4, 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.

[Claim 6] Genetically modified strain according to one of Claims 1 to 5, characterized in that it also comprises one or more additional recombinant genes chosen from:

- the recombinant gene encoding a polypeptide with tyrosine ammonia lyase activity (TAL), 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/ Where

- 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.

[Claim 7] Genetically modified strain according to one of Claims 1 to 6, characterized in that it is capable of producing a phenylpropanoid compound, preferably coumaric acid, p-coumaroyl-coA, 4-hydroxybenzalacetone or frambinone.

[Claim 8] Process for the synthesis of a phenylpropanoid compound, characterized in that it comprises a step of growing the genetically modified strain according to claim 7, in a culture medium under conditions allowing the expression of the necessary recombinant genes to the synthesis of said phenylpropanoid, said phenylpropanoid compound being synthesized by said genetically modified strain.

[Claim 9] Process according to claim 8, characterized in that it also comprises a step of recovering the phenylpropanoid compound from the culture medium.

[Claim 10] Use of the strain according to one of Claims 1 to 9, for the synthesis of a phenylpropanoid compound or a phenylpropanoid derivative, preferably coumaric acid or frambinone.