EP4337767A1 - Biosynthese von phenylpropanoidverbindungen - Google Patents

Biosynthese von phenylpropanoidverbindungen

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Publication number
EP4337767A1
EP4337767A1 EP22727965.0A EP22727965A EP4337767A1 EP 4337767 A1 EP4337767 A1 EP 4337767A1 EP 22727965 A EP22727965 A EP 22727965A EP 4337767 A1 EP4337767 A1 EP 4337767A1
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Prior art keywords
sequence seq
genetically modified
arof
modified strain
pseudomonas putida
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French (fr)
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William MERRÉ
Ricardo DE ANDRADE
Caroline RANQUET
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Bgene Genetics
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Bgene Genetics
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C12P7/26—Ketones
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
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    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/16—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced pteridine as one donor, and incorporation of one atom of oxygen (1.14.16)
    • C12Y114/16001—Phenylalanine 4-monooxygenase (1.14.16.1)
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    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01054—3-Deoxy-7-phosphoheptulonate synthase (2.5.1.54)
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    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/01096—4a-Hydroxytetrahydrobiopterin dehydratase (4.2.1.96)
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    • C12Y403/00—Carbon-nitrogen lyases (4.3)
    • C12Y403/01—Ammonia-lyases (4.3.1)
    • C12Y403/01023—Tyrosine ammonia-lyase (4.3.1.23)
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    • C12Y602/00—Ligases forming carbon-sulfur bonds (6.2)
    • C12Y602/01—Acid-Thiol Ligases (6.2.1)
    • C12Y602/01012—4-Coumarate-CoA ligase (6.2.1.12)
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    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/38—Pseudomonas
    • C12R2001/40—Pseudomonas putida

Definitions

  • 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.
  • 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.
  • Tyrosine is an important amino acid for the biosynthesis of phenylpropanoid compounds because it is in particular the precursor of coumaric acid or frambinone.
  • frambinone can be obtained from the aromatic amino acid L-tyrosine as an initial substrate via a 4-step biosynthetic pathway.
  • the tyrosine is deaminated by a tyrosine ammonia lyase (TAL, EC 4.3.1.23) to form coumaric acid.
  • TAL tyrosine ammonia lyase
  • a 4-coumarate:CoA ligase (4CL, EC 6.2.1.12)
  • CoA Coenzyme A
  • Coumaroyl-CoA is then converted by a benzalacetone synthase (BAS, EC 2.3.1.212) into 4-hydroxy benzalacetone.
  • BAS benzalacetone synthase
  • 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.
  • DAHP 3-Deoxy-D-arabino-Heptulodonate 7-phosphate
  • PEP phospho(enol)pyruvate
  • E4P erythrose-4-phosphate
  • 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) .
  • AroG retro-regulated by phenylalanine
  • AroF retro-regulated by tyrosine
  • AroH retro-regulated by tryptophan
  • Phenylpropanoid compounds have also been synthesized from Saccharomyces cerevisiae strains which overexpress tyrosine, as described by Rodriguez et al., 2015.
  • 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.
  • 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.
  • Bacteria of the Pseudomonas genus are more tolerant to these highly toxic molecules, in particular the bacterium Pseudomonas putida (Calera et al., 2017).
  • the enzymes involved in the production of aromatic amino acids in P. putida are poorly described.
  • 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.
  • 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.
  • DAHP 3-Deoxy-D-arabino-Heptulodonate 7-phosphate
  • 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.
  • the invention also relates to the use of a genetically modified strain of Pseudomonas putida for the synthesis of phenylpropanoid compounds.
  • FIG. 1 A block diagram illustrating an exemplary computing environment in accordance with the present disclosure.
  • FIG. 1 A block diagram illustrating an exemplary computing environment in accordance with the present disclosure.
  • 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 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 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 Graph representing the proportion of PEP
  • 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 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 presents the conjugation protocol that can be used to transform and genetically modify P. putida.
  • 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).
  • PCA coumaric acid
  • CA cinnamic acid
  • 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 + p
  • 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.
  • DAHP 3-Deoxy-D-arabino-Heptulodonate 7-phosphate
  • 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.
  • the mutagenesis technique described in Example 2 will be used.
  • a genetically modified strain may comprise a nucleic acid modifying the expression of one or more genes naturally expressed in Pseudomonas putida.
  • 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) .
  • 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).
  • 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.
  • 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.
  • blastn algorithm as described in https://blast.ncbi.nlm.nih.gov/Blast. cgi with default settings is used
  • 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.
  • DAHP 3-Deoxy-D-arabino-Heptulodonate 7-phosphate
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the modified strain of Pseudomonas putida comprises a mutated AroF-1 gene coding for DAHP synthase exhibiting at least one double P160L/S193A mutation.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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 .
  • 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.
  • 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.
  • this overproduction is particularly advantageous for the production of phenylpropanoid compounds by the strains modified according to the invention.
  • 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.
  • 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.
  • the modified strain according to the invention may also advantageously comprise at least one additional recombinant gene.
  • 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.
  • 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 ).
  • phhA phenylalanine hydroxylase activity
  • phhB tetrahydrobiopterin dehydratase activity
  • 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.
  • a heterologous promoter in particular a constitutive or inducible promoter.
  • promoters are in particular the ptrc, xylS/pm, araC/pBAD promoters.
  • 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.
  • 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.
  • 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.
  • phhA phenylalanine hydroxylase
  • 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.
  • 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).
  • 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:
  • phhA phenylalanine hydroxylase
  • SEQ ID NO: 5 a phenylalanine hydroxylase
  • phhB tetrahydrobiopterin dehydratase
  • the genetically modified strain of Pseudomonas putida comprises an additional recombinant gene coding for a polypeptide with tyrosine ammonia lyase (TAL) activity.
  • TAL tyrosine ammonia lyase
  • 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.
  • 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.
  • TAL tyrosine ammonia lyase
  • 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.
  • the genetically modified strain of Pseudomonas putida comprises an additional recombinant gene encoding 4-coumarate-CoA ligase (4-CL).
  • 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.
  • the genetically modified strain is capable of converting coumaric acid into p-coumaryl-coA via the enzyme 4-CL.
  • the genetically modified strain of Pseudomonas putida comprises an additional recombinant gene encoding a polypeptide with benzalacetone synthase (BAS) activity.
  • 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.
  • the genetically modified strain is capable of converting p-coumaryl-coA into 4-hydroxybenzalketone via the BAS enzyme.
  • the genetically modified strain of Pseudomonas putida comprises several additional recombinant genes, namely in particular the five additional recombinant genes below:
  • phhA phenylalanine hydroxylase
  • SEQ ID NO: 5 a phenylalanine hydroxylase
  • phhB tetrahydrobiopterine dehydratase
  • phhB tetrahydrobiopterine dehydratase
  • TAL_RG_OPT tyrosine ammonia lyase
  • 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.
  • 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.
  • DHAP 3-Deoxy-D-arabino-Heptulodonate 7-phosphate
  • 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.
  • the synthesis process according to the invention makes it possible to produce coumaric acid in large quantities.
  • 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:
  • DAHP 3-Deoxy-D-arabino-Heptulodonate 7- phosphate
  • phhA a phenylalanine hydroxylase
  • TAL_RG_OPT a tyrosine ammonia lyase
  • 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.
  • 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:
  • TAL_RG_OPT a tyrosine ammonia lyase
  • 4-CL 4-coumarate-CoA ligase (4-CL), preferably a 4-CL defined by the sequence SEQ ID NO: 8,
  • BAS benzalacetone synthase
  • 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
  • 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.
  • DFIAP 3-Deoxy-D-arabino-Fleptulodonate 7-phosphate
  • Another object of the invention relates to the use of a strain of Pseudomonas putida as defined above for the synthesis of phenylpropanoid compounds.
  • the phenylpropanoid compound is chosen from coumaric acid, p-coumaroyl-coA, 4-hydroxybenzalacetone and frambinone, preferably from coumaric acid and/or frambinone.
  • 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]
  • DHAP 3-Deoxy-D-arabino-Heptulodonate 7-phosphate
  • 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).
  • DAHP 3-Deoxy-D-arabino-Heptulodonate 7-phosphate
  • 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 (2016)).
  • 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.
  • 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).
  • 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).
  • 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.
  • 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:
  • 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)
  • 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.
  • 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.
  • PEP phospho(enol)pyruvate
  • 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.
  • E4P erythrose-4-phosphate
  • 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 ⁇ m membrane before HPLC analysis.
  • 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.
  • 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.
  • Example 2 Pseudomonas putida strains genetically modified for the synthesis of phenylpropanoid compound
  • 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:
  • 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.
  • DHAP 3-Deoxy-D-arabino-Heptulodonate 7-phosphate
  • 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.
  • SEQ ID NO: 11 sequence of the gene for 3-Deoxy-D-arabino-
  • DHAP Heptulodonate 7-phosphate (DHAP) synthase with double mutation P160L/Q164A (Pseudomonas putida KT2440) encoding the DHAP synthase of sequence SEQ ID NO:3.
  • SEQ ID NO: 12 sequence of the gene for 3-Deoxy-D-arabino-
  • DHAP Heptulodonate 7-phosphate
  • SEQ ID NO: 13 phenylalanine hydroxylase (phhA) gene (Pseudomonas putida KT2440) encoding phhA of sequence SEQ ID NO: 5.
  • SEQ ID NO: 14 tetrahydrobiopterin dehydratase (phhB) gene (Pseudomonas putida KT2440) encoding the phhB of sequence SEQ ID NO: 6.
  • 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.
  • SEQ ID NO: 17 benzalacetone synthase (BAS) gene (Rheum palmatum) encoding the BAS of sequence SEQ ID NO: 9.
  • BAS benzalacetone synthase
  • Example 3 In vivo activity of a genetically modified Pseudomonas putida strain according to the invention
  • 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.
  • TAL_RG_OPT heterologous tyrosine ammonia lyase
  • 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).
  • 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 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.
  • 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.
  • - 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.
  • - 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)
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EP0488424B1 (de) * 1990-11-30 1997-03-05 Ajinomoto Co., Inc. Rekombinante DNS-Sequenzen kodierend für Enzyme frei von Feedback Inhibition, Plasmide diese Sequenzen enthaltend, transformierte Mikroorganismen nützlich für Produktion von aromatischen Aminosäuren, und deren Verfahren zur Herstellung durch Fermentation
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