WO2016207403A1 - Procédé de production d'acide muconique - Google Patents

Procédé de production d'acide muconique Download PDF

Info

Publication number
WO2016207403A1
WO2016207403A1 PCT/EP2016/064752 EP2016064752W WO2016207403A1 WO 2016207403 A1 WO2016207403 A1 WO 2016207403A1 EP 2016064752 W EP2016064752 W EP 2016064752W WO 2016207403 A1 WO2016207403 A1 WO 2016207403A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
catechol
activity
cis
recombinant
Prior art date
Application number
PCT/EP2016/064752
Other languages
English (en)
Inventor
Jean-Paul Leonetti
Patrick HIVIN
Original Assignee
Deinove
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Deinove filed Critical Deinove
Publication of WO2016207403A1 publication Critical patent/WO2016207403A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12P7/44—Polycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12P7/44—Polycarboxylic acids
    • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid

Definitions

  • the present invention relates to the field of microbiology. More particularly, the present invention relates to the field of production of catechol and muconic acid from renewable carbon resources using genetically modified bacteria.
  • Adipic acid is the most important commercial aliphatic dicarboxylic acid in the chemical industry. It is mainly a precursor used for the production of nylon, lubricants, coating, plastics and plasticizers. To date, almost all of the commercial adipic acid is still derived from petrochemistry based precursors. Mainly, the process starts with the oxydation of benzene- derived cyclohexane resulting in a cyclohexanol-cyclohexanone mixture that is further oxidized by nitric acid (Musser, 2005). However, this process depends on finite fossil resources and has a heavy environmental impact due to a high energy input, the production of large amount of the greenhouse gas N 2 0 and the toxicity of the chemical intermediates.
  • a possible replacement precursor is the metabolic intermediate ds-c/s-muconic acid (ccMA) which can be efficiently converted to adipic acid via hydrogenation (Niu et al., 2002).
  • ccMA metabolic intermediate ds-c/s-muconic acid
  • coli and is based on the expression of three heterologous genes that encode a 3-dehydroshikimate dehydratase (AroZ) and a protocatechuate decarboxylase (AroY) from Klebsiella pneumoniae and a catechol 1,2-dioxygenase (CatA) from Acinetobacter calcoaceticus (Niu et al., 2002).
  • AroZ 3-dehydroshikimate dehydratase
  • AuY protocatechuate decarboxylase
  • CatA catechol 1,2-dioxygenase
  • biotechnological production of ccMA has also been implemented in Saccharomyces cerevisiae by introducing a pathway similar to that previously used in E. coli.
  • the production titers if ccMA obtained with S. cerevisiae are much lower than those obtained with E. coli (Weber et al., 2012).
  • the present invention relates to a recombinant Deinococcus bacterium expressing a heterologous polypeptide exhibiting 3- dehydroshikimate dehydratase activity and a heterologous polypeptide exhibiting protocatechuate decarboxylase activity, and optionally further expressing a heterologous polypeptide exhibiting catechol 1,2-dioxygenase activity.
  • the present invention also relates to a recombinant Deinococcus bacterium comprising a heterologous nucleic acid sequence encoding a polypeptide exhibiting 3-dehydroshikimate dehydratase activity and a heterologous nucleic acid sequence encoding a polypeptide exhibiting protocatechuate decarboxylase activity, and optionally a heterologous nucleic acid sequence encoding a polypeptide exhibiting catechol 1,2-dioxygenase activity.
  • the polypeptide exhibiting 3-dehydroshikimate dehydratase activity may be selected, for example, from the group consisting of 3-dehydroshikimate dehydratases from Bacillus thuringiensis, Podospora anserina, , Klebsiella pneumoniae, Acinetobacter calcoaceticus, Acinetobacter sp.
  • ADP1 Acinetobacter baylyi, Neurospora crassa, Aspergillus nidulans, Gluconobacter oxydans and Pseudomonas putida, in particular Pseudomonas putida KT2440 and Pseudomonas putida H8234, preferably selected from the group consisting of Bacillus thuringiensis, Podospora anserina, Pseudomonas putida and Acinetobacter sp.
  • ADP1 more preferably from Bacillus thuringiensis, Podospora anserina and Acinetobacter sp.
  • ADP1 and even more preferably from Bacillus thuringiensis and Acinetobacter sp. ADP1.
  • polypeptide exhibiting 3-dehydroshikimate dehydratase activity may be selected from the group consisting of 3-dehydroshikimate dehydratases of SEQ ID NO: 4, 2, 6 and 8, and polypeptides exhibiting 3-dehydroshikimate dehydratase activity and having at least 60 % identity to SEQ ID NO: 4, 2, 6 or 8, preferably from the group consisting of 3-dehydroshikimate dehydratases of SEQ ID NO: 4 and 8, and polypeptides exhibiting 3-dehydroshikimate dehydratase activity and having at least 60 % identity to SEQ ID NO: 4 or 8.
  • the polypeptide exhibiting protocatechuate decarboxylase activity may be selected, for example, from the group consisting of 3-protocatechuate decarboxylases from Klebsiella pneumoniae, Enterobacter cloacae and Sedimentibacter hydroxybenzoicus, preferably from Klebsiella pneumoniae.
  • the polypeptide exhibiting protocatechuate decarboxylase activity is selected from the group consisting of (i) a PCA decarboxylase comprising polypeptides of SEQ ID NO: 10, 12 and 14, (ii) a PCA decarboxylase comprising polypeptides of SEQ ID NO: 17, 19 and 21, (iii) the PCA decarboxylase of SEQ ID NO: 23, and (iv) PCA decarboxylases having at least 60 % identity to SEQ ID NO: 10, 12, 14, 17, 19, 21 or 23.
  • the polypeptide exhibiting protocatechuate decarboxylase activity is selected from the group consisting of a PCA decarboxylase comprising polypeptides of SEQ ID NO: 10, 12 and 14, and PCA decarboxylases having at least 60 % identity to SEQ ID NO: 10, 12 or 14.
  • the polypeptide exhibiting catechol 1 ,2-dioxygenase activity may be selected, for example, from the group consisting of catechol 1 ,2-dioxygenases from Acinetobacter radioresistens, Acinetobacter calcoaceticus, Candida albicans, Bulkholderia mallei, Bulkholderia xenovorans, Pseudomonas putida, Stenotrophomonas maltophilia KB2, Cupriavidus metallidurans CH34, Burkholderia sp. TH2, Rhodococcus opacus, Rhodococcus erythropolis and Acinetobacter sp. ADP1.
  • the polypeptide exhibiting catechol 1,2-dioxygenase activity is selected from the group consisting of catechol 1,2-dioxygenases from Acinetobacter radioresistens, Acinetobacter calcoaceticus, Candida albicans, Bulkholderia mallei, Bulkholderia xenovorans and Pseudomonas putida, more preferably from Acinetobacter radioresistens, Acinetobacter calcoaceticus, Candida albicans, Bulkholderia mallei and Bulkholderia xenovorans.
  • the polypeptide exhibiting catechol 1 ,2-dioxygenase activity may be selected from the group consisting of catechol 1,2-dioxygenases of SEQ ID NO: 25, 27, 29, 31, 33, 34, 35, 36, 37, 38, 39, 40 and 41 and polypeptides exhibiting catechol 1 ,2- dioxygenase activity and having at least 60 % identity to SEQ ID NO: 25, 27, 29, 31, 33, 34, 35, 36, 37, 38, 39, 40 or 41.
  • the polypeptide exhibiting catechol 1 ,2- dioxygenase activity is selected from the group consisting of catechol 1,2-dioxygenases of SEQ ID NO: 25, 29 and 31 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 60 % identity to SEQ ID NO: 25, 29 or 31. More preferably, the polypeptide exhibiting catechol 1 ,2-dioxygenase activity is selected from the group consisting of catechol 1,2-dioxygenase of SEQ ID NO: 25 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 60 % identity to SEQ ID NO: 25.
  • the polypeptide exhibiting catechol 1,2-dioxygenase activity may be selected from the group consisting of catechol 1,2-dioxygenase of SEQ ID NO: 41 and a variant thereof comprising at least one substitution at position corresponding to residue G72, L73 or P76, preferably at least one substitution or combination of substitutions selected from G72A, L73F, P76A, G72A+L73F, G72A+P76A, P76A+L73F, G72A+L72F+P76A, more preferably selected from G72A, P76A, L73F and L73F+P76A.
  • the endogenous biosynthetic pathway of the bacterium converting 3- dehydroshikimate to chorismate may be blocked or reduced.
  • the endogenous gene encoding shikimate dehydrogenase (AroE) may be inactivated.
  • the shikimate dehydrogenase activity of the bacterium may be reduced.
  • the endogenous biosynthetic pathway converting protocatechuate to oxoadipate may be blocked or reduced.
  • this pathway may be blocked by inactivation one or several endogenous genes selected from genes encoding protocatechuate 3,4- dioxygenase, 3-carboxy-cis,cis-muconate cycloisomerase and 3-oxoadipate enol- lactonase.
  • One or several of the enzymes involved in the conversion of phosphoenolpyruvate and erythrose 4-phosphate to 3-dihydroshikimate preferably selected from AroF, AroG, AroH, AroB and AroD, may be overexpressed and/or may be feedback inhibition resistant enzymes.
  • the recombinant bacterium may express a feedback inhibition resistant DAHP synthase, preferably a variant of the Deinococcus DAHP synthase set forth in SEQ ID NO: 46 comprising at least one substitution at position corresponding to residue N13, P156 or S 186 of SEQ ID NO: 46, preferably selected from N13 , P156L, S186F, N13 + P156L, N13K+S 186F, P156L +S186F and N13K+ P156L +S 186F.
  • a feedback inhibition resistant DAHP synthase preferably a variant of the Deinococcus DAHP synthase set forth in SEQ ID NO: 46 comprising at least one substitution at position corresponding to residue N13, P156 or S 186 of SEQ ID NO: 46, preferably selected from N13 , P156L, S186F, N13 + P156L, N13K+S 186F, P156L +S186F and N13K+ P156L +S
  • the recombinant bacterium of the invention may further express a heterologous polypeptide exhibiting catechol-O-methyltransferase activity.
  • said polypeptide is selected from the group consisting of COMT from Mycobacterium vanbaalenii (SEQ ID NO: 54) and any polypeptide exhibiting COMT activity and having at least 60 % identity to SEQ ID NO: 54.
  • the present invention also relates to a method of producing cis- cis muconic acid comprising culturing a recombinant Deinococcus bacterium of the invention under conditions suitable to produce cis-cis muconic acid, and optionally recovering said cis-cis muconic acid.
  • the present invention also relates to a method of producing catechol comprising culturing a recombinant Deinococcus bacterium of the invention under conditions suitable to produce catechol, and optionally recovering said catechol.
  • the present invention also relates to a method of producing cis- cis muconic acid comprising (i) producing catechol according to the method of producing catechol of the invention, (ii) enzymatically converting catechol to cis-cis muconic acid, and optionally (iii) recovering said cis-cis muconic acid.
  • the culture of the recombinant Deinococcus bacterium under conditions suitable to produce cis-cis muconic acid may be performed at a temperature comprised between 37 and 55°C, preferably at about 48°C.
  • the culture of the recombinant Deinococcus bacterium under conditions suitable to produce catechol may be performed at a temperature comprised between 37 and 55°C, preferably at about 37°C.
  • the present invention relates to a method of producing adipic acid comprising producing cis-cis muconic acid according to the method of the invention and reducing said cis-cis muconic acid to produce adipic acid, and optionally recovering said adipic acid.
  • the present invention relates to a method of producing cis-trans and/or trans-trans muconic acid comprising producing cis-cis muconic acid according to the method of the invention and isomerizing said cis-cis muconic acid to produce cis- trans and/or trans-trans muconic acid, and optionally recovering said cis-trans and/or trans-trans muconic acid.
  • the present invention also relates to a method of producing gaiacol comprising (i) culturing a recombinant Deinococcus bacterium of the invention expressing a heterologous polypeptide exhibiting catechol-O-methyltransferase activity under conditions suitable to produce gaiacol, and optionally (ii) recovering said gaiacol.
  • the present invention further relates to a method of producing vanillin comprising (i) culturing a recombinant Deinococcus bacterium of the invention expressing a heterologous polypeptide exhibiting catechol-O-methyltransferase activity under conditions suitable to produce gaiacol, (ii) converting gaiacol to vanillin and, optionally (iii) recovering said vanillin.
  • the method may further comprise recovering gaiacol produced in step (i) before conversion.
  • Figure 1 Expression cassette for the production of catechol comprising AroZ of Bacillus thuringiensis, aroY of Klebsiella pneumonia and a gene encoding bleomycin resistance. All these genes were placed under the control of strong constitutive promoters.
  • the cassette further comprised two flanking sequences of 1500bp that are homologous to the sequence upstream and downstreal the chromosomic target aroE gene.
  • Figure 2 Titers of DHS, PCA and catechol of recombinant D. geothermalis comprising the cassette of Figure 1. Wild-type strain does not produce PCA or catechol (data not shown).
  • Figure 3 Expression cassette for the production of catechol comprising quiC of Acinetobacter sp. ADP1, aroY of Klebsiella pneumonia and a gene encoding bleomycin resistance. All these genes were placed under the control of strong constitutive promoters. The cassette further comprised two flanking sequences of 1500bp that are homologous to the sequence upstream and downstreal the chromosomic target aroE gene.
  • Figure 4 HPLC analysis of the culture sample of the recombinant Deinococcus geothermalis comprising the expression cassette of Figure 1 and an expression cassette comprising catA gene from Acinetobacter calcoaceticus .
  • the wild-type strain does not produce muconic acid (data not shown).
  • Figure 5 Titers of DHS, catechol and muconic acid of recombinant D. geothermalis strains comprising the expression cassette of Figure 1 (in grey) or Figure 3 (in white) and an expression cassette comprising catA gene from Acinetobacter calcoaceticus. Wild-type strains do not produce catechol or muconic acid (data not shown).
  • Deinococcus bacteria are non-pathogen bacteria that were firstly isolated in 1956 by Anderson and collaborators. These extremophile organisms have been proposed for use in industrial processes or reactions using biomass (see e.g., WO2009/063079; WO2010/094665 or WO2010/081899). Based on their solid knowledge of Deinococcus metabolism and genetics, the inventors found that Deinococcus bacteria can be genetically modified to produce substantial amounts of muconic acid and exhibit specific properties that are particularly useful for industrial production of this compound. Indeed, Deinococcus bacteria are viable at a pH comprised between 4 and 9, they are thus resistant to the decrease of pH values induced by the production of organic acid.
  • Deinococcus bacteria are also able to grow on and/or transform a very large variety of organic substrates, including cellulosic biomass (see e.g. the international patent application WO 2010/130812), thus allowing industrial production from biorenewables. Furthermore, Deinococcus bacteria may exhibit a natural balance between the oxidative pentose phosphate pathway and the glycolysis pathway which promotes the adequate supplying of molecules that are precursors of 3-dehydroshikimate such as phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P).
  • PEP phosphoenolpyruvate
  • E4P D-erythrose 4-phosphate
  • the term "Deinococcus" includes wild type or natural variant strains of Deinococcus, e.g., strains obtained through accelerated evolution, mutagenesis, by DNA-shuffling technologies, or recombinant strains obtained by insertion of eukaryotic, prokaryotic and/or synthetic nucleic acid(s).
  • Deinococcus bacteria can designate any bacterium of the genus Deinococcus, such as without limitation, D. aerius, D. aerolatus, D. aerophilus, D. aetherius, D. alpinitundrae, D. altitudinis, D. antarcticus, D. apachensis, D. aquaticus, D.
  • the term "Deinococcus" refers to D. geothermalis, D. cellulolysiticus, D. deserti, D. murrayi, D. maricopensis or D. radiodurans. More preferably, the term “Deinococcus” refers to D. geothermalis.
  • mesophilic Deinococcus bacteria include, but are not limited to, D. radiodurans, D. grandis, D. cellulolysiticus, D. depolymerans, D. aquaticus, D. deserti. D. wulumuqiensis, D. proteolyticus, D. gobiensis misasensis, D.
  • thermophilic Deinococcus bacteria examples include, but are not limited to, D. geothermalis, D. maricopensis and D. murrayi.
  • the term "recombinant bacterium” or “genetically modified bacterium” designates a bacterium that is not found in nature and which contains a modified genome as a result of either a deletion, insertion or modification of genetic elements.
  • a "recombinant nucleic acid” therefore designates a nucleic acid which has been engineered and is not found as such in wild type bacteria.
  • the term "gene” designates any nucleic acid encoding a protein.
  • the term gene encompasses DNA, such as cDNA or gDNA, as well as RNA.
  • the gene may be first prepared by e.g., recombinant, enzymatic and/or chemical techniques, and subsequently replicated in a host cell or an in vitro system.
  • the gene typically comprises an open reading frame encoding a desired protein.
  • the gene may contain additional sequences such as a transcription terminator or a signal peptide.
  • expression cassette denotes a nucleic acid construct comprising a coding region, i.e. a gene, and a regulatory region, i.e. comprising one or more control sequences, operably linked.
  • the control sequences are suitable for Deinococcus host cells.
  • expression vector means a DNA or RNA molecule that comprises an expression cassette.
  • the expression vector is a linear or circular double stranded DNA molecule.
  • control sequences means nucleic acid sequences necessary for expression of a gene. Control sequences may be native, homologous or heterologous. Well-known control sequences and currently used by the person skilled in the art will be preferred. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. Preferably, the control sequences include a promoter and a transcription terminator.
  • the term “native” or “endogenous” refers to a genetic element or a protein from the non modified Deinococcus bacterium or from a Deinococcus bacterium of the same species.
  • the term “homologous” refers to a genetic element or a protein from a Deinococcus bacterium of another species than the recombinant Deinococcus bacterium.
  • the term “heterologous” refers to a genetic element or a protein from a non Deinococcus origin such as other bacteria, microorganisms, plants, viruses, ect...
  • sequence identity refers to the number (%) of matches (identical amino acid residues) in positions from an alignment of two polypeptide sequences.
  • sequence identity is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • sequence identity may be determined using any of a number of mathematical global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithms (e.g. Needleman and Wunsch algorithm; Needleman and Wunsch, 1970) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g.
  • Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software available on internet web sites such as http://blast.ncbi.nlm.nih.gov/ or http://www.ebi.ac.uk/Tools/emboss/). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • AroZ or "DHS dehydratase” refers to the enzyme 3-dehydroshikimate dehydratase (EC 4.2.1.118) encoded by aroZ gene, that converts 3-dehydroshikimate (DHS) to protocatechuate (PCA).
  • AroY or "PCA decarboxylase” refers to the enzyme protocatechuate decarboxylase (also named 3,4-dihydroxybenzoate decarboxylase or protocatechuate carboxylyase, EC 4.1.1.63) encoded by aroY gene, that converts protocatechuate (also named 3,4-dihydroxybenzoic acid) to catechol (also named Benzene- 1,2-diol, Pyrocatechol, 2-hydroxyphenol, or 1,2-dihydroxybenzene).
  • protocatechuate decarboxylase also named 3,4-dihydroxybenzoate decarboxylase or protocatechuate carboxylyase, EC 4.1.1.63
  • aroY gene encoded by aroY gene, that converts protocatechuate (also named 3,4-dihydroxybenzoic acid) to catechol (also named Benzene- 1,2-diol, Pyrocatechol, 2-hydroxyphenol, or 1,2-dihydroxybenzene).
  • CatA refers to the enzyme catechol 1,2-dioxygenase (also named catechase or pyrocatechase, EC 1.13.11.1) encoded by catA gene, that converts catechol to ds, ds-muconic acid (ccMA).
  • RhoE refers to the enzyme shikimate dehydrogenase (also named 5- dehydroshikimate reductase, EC 1.1.1.25) encoded by aroE gene, that converts DHS to shikimate (SHK).
  • Alignin refers to the enzyme shikimate kinase (also named ATPrshikimate 3-phosphotransferase, EC 2.7.1.71) encoded by aroK gene. This enzyme converts SHK to 3 -phospho shikimate (S3P).
  • Alignin refers to the enzyme 3 -phospho shikimate 1- carboxyvinyltransferase (also named 5-enolpyruvylshikimate-3-phosphate synthase, 3- enol-pyruvoylshikimate-5-phosphate synthase or EPSP synthase, EC 2.5.1.19) encoded by aroA gene, that converts S3P to 5-enolpyruvylshikimate-3-phosphate (EPSP).
  • ESP 5-enolpyruvylshikimate-3-phosphate
  • chorismate synthase also named 5- enolpyruvylshikimate-3-phosphate phospholyase, EC 4.2.3.5
  • aroC aroC gene
  • AroG refers to DAHP synthases (also named 3- deoxy-7-phosphoheptulonate synthase or Phospho-2-dehydro-3-deoxyheptonate aldolase, EC 2.5.1.54) encoded by aroG, aroF and aroH genes, respectively, that convert phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P) to 3-deoxy-D-arabino- hept-2-ulosonate 7-phosphate (DAHP).
  • DAHP DAHP synthases
  • AlignB refers to the enzyme 3-dehydroquinate synthase (EC 4.2.3.4) encoded by aroB gene, that converts DAHP to 3-dehydroquinate (DHQ).
  • AlignD or “AroQ” refers to the enzyme 3-dehydroquinate dehydratase (EC 4.2.1.10) encoded by aroD gene, that converts DHQ to DHS.
  • Rpe refers to ribulose-phosphate 3-epimerase (EC 5.1.3.1) encoded by the rpe gene, that converts D-ribulose 5-phosphate to D-xylulose 5-phosphate.
  • Rpi refers to ribose-5-phosphate isomerase (EC 5.3.1.6) encoded by the rpi gene, that converts D-ribose 5-phosphate to D-ribulose 5-phosphate.
  • TalB refers to transaldolase (EC 2.2.1.2) encoded by the talB gene, that converts sedoheptulose 7-phosphate and D-glyceraldehyde 3-phosphate to D- erythrose 4-phosphate and D-fructose 6-phosphate.
  • TktA refers to transketolase (EC 2.2.1.1) encoded by the tktA gene, that converts sedoheptulose 7-phosphate and D-glyceraldehyde 3-phosphate to D-ribose 5-phosphate and D-xylulose 5-phosphate.
  • PEP Phosphoenolpyruvate
  • the term "COMT” refers to an enzyme exhibiting catechol-O-methyltransferase activity, i.e. catalyzing the reaction converting catechol to gaiacol (EC 2.1.1.6). According to the organism, the nomenclature of the above identified enzymes and encoding genes may vary. However, for the sake of clarity, in the present specification, these terms are used independently from the origin of the enzymes or genes.
  • the present invention relates to a recombinant Deinococcus bacterium comprising a heterologous muconic acid biosynthetic pathway.
  • the present invention also relates to a recombinant Deinococcus bacterium comprising a heterologous catechol biosynthetic pathway.
  • biosynthetic pathway refers to a biochemical pathway comprising one or several enzymes and converting a substrate to a product through one or several biochemical reactions.
  • miconic acid biosynthetic pathway refers to a biochemical pathway allowing the production of muconic acid, in particular czVds-muconic acid.
  • this term refers to a biochemical pathway converting 3-dehydroshikimate (DHS) to czs-c/s-muconic acid (ccMA), and in particular a biochemical pathway converting DHS to protocatechuate (PCA), PCA to catechol and catechol to ccMA.
  • DHS 3-dehydroshikimate
  • PCA protocatechuate
  • the recombinant Deinococcus bacterium of the invention may comprise a heterologous nucleic acid sequence encoding a polypeptide exhibiting 3-dehydroshikimate dehydratase activity ⁇ aroZ gene), a heterologous nucleic acid sequence encoding a polypeptide exhibiting protocatechuate decarboxylase activity (aroY gene), and/or a heterologous nucleic acid sequence encoding a polypeptide exhibiting catechol 1,2-dioxygenase activity (catA gene).
  • catechol biosynthetic pathway refers to a biochemical pathway allowing the production of catechol.
  • this term refers to a biochemical pathway converting 3-dehydroshikimate (DHS) to protocatechuate (PCA) and PCA to catechol. All or part of the biochemical pathway converting DHS to catechol may be heterologous.
  • the recombinant Deinococcus bacterium of the invention comprising a heterologous catechol biosynthetic pathway may comprise a heterologous nucleic acid sequence encoding a polypeptide exhibiting 3-dehydroshikimate dehydratase activity (aroZ gene) and/or a heterologous nucleic acid sequence encoding a polypeptide exhibiting protocatechuate decarboxylase activity (aroY gene).
  • the recombinant Deinococcus bacterium of the invention comprising a heterologous catechol biosynthetic pathway does not exhibit any catechol 1,2- dioxygenase activity.
  • the polypeptide exhibiting DHS dehydratase activity may be any known DHS dehydratase, in particular selected from known fungal or bacterial DHS dehydratases.
  • the polypeptide exhibiting DHS dehydratase activity is selected from the group consisting of DHS dehydratases from Bacillus thuringiensis (AsbF, Fox et al., 2008; SEQ ID NO: 4), Podospora anserina (also known as Podospora pauciseta; Hansen et al. 2009; SEQ ID NO: 2), Pseudomonas putida, in particular Pseudomonas putida KT2440 (Jimenez et al.
  • ADP1 and Acinetobacter baylyi (quiC, SEQ ID NO: 8), Neurospora crassa (Qa-4, Rutledge et al., 1984), Aspergillus nidulans (QutC, Lamb et al., 1992) and Gluconobacter oxydans (DSD, Shinagawa et al., 2010).
  • the polypeptide exhibiting DHS dehydratase activity is selected from the group consisting of DHS dehydratases from Bacillus thuringiensis, Podospora anserina, Pseudomonas putida H8234 and KT2440, Klebsiella pneumonia, Acinetobacter baylyi, Acinetobacter calcoaceticus, Acinetobacter sp. ADP1 and Neurospora crassa.
  • the polypeptide exhibiting DHS dehydratase activity is selected from the group consisting of DHS dehydratases from Bacillus thuringiensis (SEQ ID NO: 4), Podospora anserina (SEQ ID NO: 2), Pseudomonas putida H8234 (SEQ ID NO: 6) and Acinetobacter sp. ADP1 (SEQ ID NO: 8).
  • the polypeptide exhibiting DHS dehydratase activity is selected from the group consisting of DHS dehydratases from Bacillus thuringiensis (SEQ ID NO: 4) and Acinetobacter sp. ADP1 (SEQ ID NO: 8).
  • the AroZ enzyme may be any polypeptide exhibiting DHS dehydratase activity and having at least 60 %, preferably at least 65, 70, 75, 80 or 85%, and more preferably at least 90 or 95%, identity to any DHS dehydratase listed above.
  • the AroZ enzyme is selected from the group consisting of DHS dehydratases of SEQ ID NO: 2, 4, 6 and 8 and polypeptides exhibiting DHS dehydratase activity and having at least 60 %, preferably at least 65, 70, 75, 80 or 85%, and more preferably at least 90 or 95% identity to SEQ ID NO: 2, 4, 6 or 8.
  • the AroZ enzyme is selected from the group consisting of DHS dehydratases of SEQ ID NO: 2, 4 and 8 and polypeptides exhibiting DHS dehydratase activity and having at least 60 %, preferably at least 65, 70, 75, 80 or 85%, and more preferably at least 90 or 95% identity to SEQ ID NO: 2, 4 or 8.
  • the AroZ enzyme is selected from the group consisting of DHS dehydratases of SEQ ID NO: 4 and 8 and polypeptides exhibiting DHS dehydratase activity and having at least 60 %, preferably at least 65, 70, 75, 80 or 85%, and more preferably at least 90 or 95% identity to SEQ ID NO: 4 or 8. Even more preferably, the AroZ enzyme is selected from the group consisting of DHS dehydratases of SEQ ID NO: 4 and 8.
  • the polypeptide exhibiting PCA decarboxylase activity may be any known PCA decarboxylase, in particular selected from known fungal or bacterial PCA decarboxylases.
  • the polypeptide exhibiting PCA decarboxylase activity is selected from the group consisting of PCA decarboxylases from Klebsiella pneumonia (Niu et al., 2002; Weber et al.
  • the polypeptide exhibiting PCA decarboxylase activity is selected from the group consisting of PCA decarboxylases from Klebsiella pneumonia (SEQ ID NO: 10, 5 12 and 14), Enterobacter cloacae (SEQ ID NO: 23) and Sedimentibacter hydroxybenzoicus (SEQ ID NO: 17, 19 and 21). Even more preferably, the polypeptide exhibiting PCA decarboxylase activity is from Klebsiella pneumonia.
  • the AroY enzyme may be any polypeptide exhibiting PCA decarboxylase activity and having at least 60 %, preferably at least 65, 70, 75, 80 or 85%, and more preferably at least 90 or 10 95%, identity to any PCA decarboxylase listed above.
  • the AroY enzyme is selected from the group consisting of (i) a PCA decarboxylase comprising polypeptides of SEQ ID NO: 10, 12 and 14, (ii) a PCA decarboxylase comprising polypeptides of SEQ ID NO: 17, 19 and 21, (iii) the PCA decarboxylase of SEQ ID NO: 23, and (iv) PCA decarboxylases having at
  • the AroY enzyme is selected from the group consisting of a PCA decarboxylase comprising polypeptides of SEQ ID NO: 10, 12 and 14 and PCA decarboxylases having at least 60 %, preferably at least 65, 70, 75, 80 or 85%, more preferably at least 90 or 95% identity to SEQ ID NO:
  • the AroY enzyme is a PCA decarboxylase comprising three subunits of SEQ ID NO: 10, 12 and 14.
  • the PCA decarboxylase comprises several (e.g. three) subunits
  • all subunits may be encoded by the same heterologous nucleic acid
  • each subunit may be encoded by distinct heterologous nucleic acid
  • several (e.g. two) subunits may 25 be encoded by a heterologous nucleic acid while the other(s) is (are) encoded by another heterologous nucleic acid.
  • the polypeptide exhibiting catechol 1,2-dioxygenase activity may be any known catechol 1,2-dioxygenase, in particular selected from known fungal or bacterial catechol 1,2-dioxygenases.
  • the polypeptide exhibiting catechol 1,2- 30 dioxygenase activity is selected from the group consisting of catechol 1,2-dioxygenases from Acinetobacter radioresistens (Uniprot accession numbers: Q9F103 (isoB also called catA B ) and Q93SY8 (isoA also called catA A ; SEQ ID NO: 27); Capioso et al, 2002; Weber et al., 2012), Acinetobacter calcoaceticus (Uniprot accession numbers: A0A0A8XEH7 and F0KF43 ; Neidle and Ornston, 1986; SEQ ID NO: 25), Candida albicans (Uniprot accession numbers: A0A0A6M
  • TH2 (SEQ ID NO: 37; Genbank accession number: BAC16779.1), Rhodococcus opacus (SEQ ID NO: 38; Genbank accession number: CAA67941.1), Rhodococcus erythropolis (SEQ ID NO: 39; Genbank accession number: BAA11859.1) and Acinetobacter sp. ADP1 (Uniprot accession number: P07773, SEQ ID NO:41).
  • the polypeptide exhibiting catechol 1,2-dioxygenase activity is selected from the group consisting of catechol 1 ,2- dioxygenases from Acinetobacter radioresistens (Uniprot accession numbers: Q9F103 (isoB also called catA B ) and Q93SY8 (isoA also called catA A ; SEQ ID NO: 27); Capioso et al., 2002; Weber et al., 2012), Acinetobacter calcoaceticus (Uniprot accession numbers: A0A0A8XEH7 and F0KF43 ; Neidle and Ornston, 1986; SEQ ID NO: 25), Candida albicans (Uniprot accession numbers: A0A0A6MK79 and P86029; Tsai and Li, 2007; SEQ ID NO: 29), Bulkholderia mallei (Uniprot accession number: Q62E52 ; SEQ ID NO: 33), Bulkholderi
  • the polypeptide exhibiting catechol 1,2-dioxygenase activity is selected from the group consisting of catechol 1 ,2- dioxygenases from Acinetobacter radioresistens (SEQ ID NO: 27), Acinetobacter calcoaceticus (SEQ ID NO: 25), Candida albicans (SEQ ID NO: 29), Bulkholderia mallei (SEQ ID NO: 33) and Bulkholderia xenovorans (SEQ ID NO: 31).
  • the polypeptide exhibiting catechol 1 ,2-dioxygenase activity is selected from the group consisting of catechol 1 ,2-dioxygenases from Acinetobacter calcoaceticus (SEQ ID NO: 25), Candida albicans (SEQ ID NO: 29) and Bulkholderia xenovorans (SEQ ID NO: 31).
  • the polypeptide exhibiting catechol 1,2-dioxygenase activity is from Acinetobacter calcoaceticus.
  • the CatA enzyme may be any polypeptide exhibiting catechol 1,2-dioxygenase activity and having at least 60 %, preferably at least 65, 70, 75, 80 or 85%, more preferably at least 90 or 95%, identity to any catechol 1,2-dioxygenase listed above.
  • the CatA enzyme is selected from the group consisting of catechol 1,2-dioxygenases of SEQ ID NO: 25, 27, 29, 31, 33, 34, 35, 36, 37, 38, 39, 40 and 41 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 60 %, preferably at least 65, 70, 75, 80 or 85%, more preferably at least 90 or 95% identity to SEQ ID NO: 25, 27, 29, 31, 33, 34, 35, 36, 37, 38, 39, 40 or 41.
  • the CatA enzyme is selected from the group consisting of catechol 1,2-dioxygenases of SEQ ID NO: 25, 27, 29, 31, 33 and 41, preferably of SEQ ID NO: 25, 27, 29, 31 and 33, and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 60 %, preferably at least 65, 70, 75, 80 or 85%, more preferably at least 90 or 95% identity to SEQ ID NO: 25, 27, 29, 31, 33 or 41, preferably to SEQ ID NO: 25, 27, 29, 31 or 33.
  • the CatA enzyme is selected from the group consisting of catechol 1,2-dioxygenases of SEQ ID NO: 25, 29 and 31, and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 60 %, preferably at least 65, 70, 75, 80 or 85%, more preferably at least 90 or 95% identity to SEQ ID NO: 25, 29 or 31.
  • the CatA enzyme is selected from the group consisting of catechol 1,2-dioxygenase of SEQ ID NO: 25 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 60 %, preferably at least 65, 70, 75, 80 or 85%, more preferably at least 90 or 95% identity to SEQ ID NO: 25.
  • the CatA enzyme is a catechol 1,2-dioxygenase of SEQ ID NO: 25.
  • the CatA enzyme may be a variant of any catechol 1,2-dioxygenase listed above, said variant exhibiting improved properties such as improved activity.
  • the CatA enzyme is selected from the group consisting of catechol 1,2-dioxygenase of SEQ ID NO: 41 and a variant of the enzyme set forth in SEQ ID NO: 41 comprising at least one substitution at position corresponding to residue G72, L73 or P76 of SEQ ID NO: 41 (Han et al., 2015).
  • substitution refers to the replacement of an amino acid residue by another selected from the naturally-occurring standard 20 amino acid residues (G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S and T).
  • the sign "+" indicates a combination of substitutions.
  • G72A denotes that amino acid residue at position 72 of SEQ ID No. 41 (glycine, G) is changed to an alanine (A).
  • the CatA enzyme is a variant of the enzyme set forth in SEQ ID NO: 41 and comprises at least one substitution at position corresponding to residue G72, L73 or P76 of SEQ ID NO: 41, preferably at least one substitution or combination of substitutions selected from G72A, L73F, P76A, G72A+L73F, G72A+P76A, P76A+L73F, G72A+L72F+P76A, more preferably selected from G72A (SEQ ID NO: 42), P76A (SEQ ID NO: 43), L73F (SEQ ID NO: 44) and L73F+P76A (SEQ ID NO: 45).
  • the recombinant Deinococcus bacterium comprises a heterologous aroZ gene, a heterologous aroY gene or a heterologous catA gene.
  • the recombinant Deinococcus bacterium comprises (i) a heterologous aroZ gene and a heterologous aroY gene, (ii) a heterologous aroZ gene and a heterologous catA gene, or (iii) a heterologous aroY gene and a heterologous catA gene.
  • the recombinant Deinococcus bacterium comprises a heterologous aroZ gene, a heterologous aroY gene and a heterologous catA gene.
  • the recombinant Deinococcus bacterium comprises the aroZ gene and the aroY gene from Klebsiella pneumoniae and the catA gene from Acinetobacter calcoaceticus (Niu et al., 2012).
  • the recombinant Deinococcus bacterium comprises the aroZ gene from Bacillus thuringiensis, the aroY gene from Klebsiella pneumoniae and the catA gene from Acinetobacter radioresistens (Weber et al., 2012).
  • the recombinant Deinococcus bacterium comprises the aroZ gene from Podospora anserina, the aroY gene from Enterobacter cloacae and the catA gene from Candida albicans (Curran et al., 2013).
  • the recombinant Deinococcus bacterium comprises the aroZ gene from Bacillus thuringiensis or Acinetobacter sp. ADP1, preferably from Bacillus thuringiensis, the aroY gene from Klebsiella pneumoniae and the catA gene from Acinetobacter calcoaceticus.
  • the recombinant Deinococcus bacterium comprises
  • a heterologous nucleic acid sequence encoding a DHS dehydratase selected from the group consisting of DHS dehydratases of SEQ ID NO: 4 and 8, and polypeptides exhibiting DHS dehydratase activity and having at least 60 %, preferably at least 65, 70, 75, 80 or 85%, more preferably at least 90 or 95% identity to SEQ ID NO: 4 or 8, preferably a heterologous nucleic acid sequence encoding the DHS dehydratase of SEQ ID NO: 4 or 8; and
  • PCA decarboxylases having at least 60 %, preferably at least 65, 70, 75, 80 or 85%, more preferably at least 90 or 95% identity to SEQ ID NO: 10, 12 or 14, preferably one or several heterologous nucleic acid sequences encoding the PCA decarboxylase of SEQ ID NO: 10, 12 and 14; and
  • a heterologous nucleic acid sequence encoding a catechol 1,2- 10 dioxygenase selected from the group consisting of catechol 1,2-dioxygenases of SEQ ID NO: 1
  • the recombinant Deinococcus bacterium comprises
  • a heterologous nucleic acid sequence encoding a DHS dehydratase selected from the group consisting of DHS dehydratase of SEQ ID NO: 4, and polypeptides exhibiting DHS dehydratase activity and having at least 60 %, preferably at least 65, 70, 75, 80 or
  • SEQ ID NO: 4 20 85%, more preferably at least 90 or 95% identity to SEQ ID NO: 4, preferably a heterologous nucleic acid sequence encoding the DHS dehydratase of SEQ ID NO: 4;
  • PCA decarboxylase selected from the group consisting of PCA decarboxylase of SEQ ID NO: 10, 12 and 14, and PCA decarboxylases having at least 60 %, preferably at least 65, 70,
  • SEQ ID NO: 10 25 75, 80 or 85%, more preferably at least 90 or 95% identity to SEQ ID NO: 10, 12 or 14 preferably a heterologous nucleic acid sequence encoding the PCA decarboxylase comprising SEQ ID NO: 10; 12 and 14 and
  • a heterologous nucleic acid sequence encoding a catechol 1,2- dioxygenase selected from the group consisting of catechol 1,2-dioxygenases of SEQ ID NO: 1
  • the recombinant Deinococcus bacterium comprises
  • a heterologous nucleic acid sequence encoding a DHS dehydratase selected from the group consisting of DHS dehydratase of SEQ ID NO: 8, and polypeptides exhibiting DHS dehydratase activity and having at least 60 %, preferably at least 65, 70, 75, 80 or 85%, more preferably at least 90 or 95% identity to SEQ ID NO: 8, preferably a heterologous nucleic acid sequence encoding the DHS dehydratase of SEQ ID NO: 8; and
  • PCA decarboxylase selected from the group consisting of PCA decarboxylase of SEQ ID NO: 10, 12 and 14, and PCA decarboxylases having at least 60 %, preferably at least 65, 70, 75, 80 or 85%, more preferably at least 90 or 95% identity to SEQ ID NO: 10, 12 or 14 preferably a heterologous nucleic acid sequence encoding the PCA decarboxylase comprising SEQ ID NO: 10; 12 and 14 and
  • a heterologous nucleic acid sequence encoding a catechol 1,2- dioxygenase selected from the group consisting of catechol 1 ,2-dioxygenases of SEQ ID NO: 25, 29 and 31, preferably of SEQ ID NO: 25, and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 60 %, preferably at least 65, 70, 75, 80 or 85%, more preferably at least 90 or 95% identity to SEQ ID NO: 25, 29 and 31, preferably to SEQ ID NO: 25, even more preferably a heterologous nucleic acid sequence encoding the catechol 1,2-dioxygenase of SEQ ID NO: 25, 29 or 31, preferably of SEQ ID NO: 25.
  • the recombinant bacterium of the invention may further comprise a heterologous catX gene encoding a protein of unknown function but that enhances CatA activity.
  • the catX gene is also called “orfl” (Neidle and Ornston, 1986).
  • the catX gene is from Acinetobacter calcoaceticus. In Acinetobacter calcoaceticus, the catX gene lies lkbp upstream from the catA gene.
  • Nucleic acid sequences encoding heterologous genes may be comprised in one or several expression cassettes.
  • Each expression cassette may comprise aroZ, aroY and/or catA genes.
  • the recombinant Deinococcus bacterium of the invention may comprise an expression cassette comprising aroZ and aroY genes.
  • the recombinant Deinococcus bacterium of the invention comprises an expression cassette comprising aroZ, aroY and catA genes. These genes may be expressed under the control of a single promoter or under the control of two promoters (with two of these genes under the control of the same promoter). Alternatively, each gene may be expressed under the control of a distinct promoter.
  • expression cassettes may be integrated into the genome of the bacterium or may be maintained in an episomal form into an expression vector.
  • the expression vector may be present in the bacterium in one or several copies, depending on the nature of the origin of replication.
  • the expression cassette(s) is(are) integrated into the genome of the bacterium.
  • One or several copies of aroZ, aroY and/or catA genes may be introduced into the genome by methods of recombination, known to the expert in the field, including gene replacement.
  • an expression cassette comprising said gene(s) is integrated into the genome.
  • an expression cassette comprising aroZ, aroY and catA genes is integrated into the genome.
  • additional copies of expression cassettes comprising aroZ, aroY and/or catA genes, preferably catA gene may be further integrated in the genome.
  • the expression cassette(s) may be integrated into the genome in order to inactive target genes.
  • the expression cassette is integrated in the sequence encoding AroE, AroK, AroL, AroA or AroC, preferably in the sequence encoding AroE, in order to block the chorismate pathway.
  • the expression cassette is integrated in a sequence encoding endogenous protocatechuate 3,4-dioxygenase, 3-carboxy-cis,cis-muconate cycloisomerase or 3- oxoadipate enol-lactonase, in order to block the endogenous biosynthetic pathway converting protocatechuate to oxoadipate.
  • the expression cassette(s) may be integrated into the genome in a non-coding sequence, e.g. an insertion sequence (IS) (Makarova et al. 2001).
  • Expression cassettes useful in the present invention comprise at least one gene selected from the group consisting of aroZ, aroY and catA genes, preferably all of them, operably linked to one or more control sequences, typically a transcriptional promoter and a transcription terminator, that direct the expression of said gene(s).
  • expression cassettes useful in the present invention may also comprise aroZ, aroY and catA genes, each of them operably linked to one or more control sequences, typically a transcriptional promoter and a transcription terminator, that direct the expression of said genes.
  • the control sequence may include a promoter that is recognized by the host cell.
  • the promoter contains transcriptional control sequences that mediate the expression of the enzyme.
  • the promoter may be any polynucleotide that shows transcriptional activity in the Deinococcus bacterium.
  • the promoter may be a native, homologous or heterologous promoter. Preferred promoters are native or homologous. In this regard, various promoters have been studied and used for gene expression in Deinococcus bacteria.
  • promoters examples include VtufA and VtufB promoters from the translation elongation factors Tu genes tufA (DR0309) and tufB (DR2050), the promoter of the resll gene located in pI3, the promoter region PgroESL of the groESL operon (Lecointe et al, 2004; Meima et al, 2001), or derivatives of such promoters.
  • the promoter is a strong constitutive promoter.
  • the control sequence may also be a transcription terminator, which is recognized by Deinococcus bacteria to terminate transcription.
  • the terminator is operably linked to the 3'-terminus of the gene. Any terminator that is functional in Deinococcus bacteria may be used in the present invention such as, for example, the terminator terml 16 described in Lecointe et al (2004).
  • the expression cassette may also comprise a selectable marker that permits easy selection of recombinant bacteria.
  • the selectable marker is a gene encoding antibiotic resistance or conferring autotrophy.
  • the recombinant Deinococcus bacterium of the invention comprises an expression cassette comprising aroZ and aroY, each of them operably linked to a distinct strong constitutive promoter.
  • the bacterium may further comprise another expression cassette comprising catA gene operably linked to a strong constitutive promoter.
  • the recombinant Deinococcus bacterium of the invention comprises an expression cassette comprising aroZ, aroY and catA genes operably linked to a strong constitutive promoter.
  • each gene may be also controlled by modulating the strength of the ribosome binding site (RBS) installed in front of the gene.
  • RBS ribosome binding site
  • the Deinococcus host cell may be transformed, transfected or transduced in a transient or stable manner.
  • the recombinant Deinococcus bacterium of the invention may be obtained by any method known by the skilled person, such as electroporation, conjugation, transduction, competent cell transformation, protoplast transformation, protoplast fusion, biolistic "gene gun” transformation, PEG-mediated transformation, lipid-assisted transformation or transfection, chemically mediated transfection, lithium acetate-mediated transformation or lipo some-mediated transformation.
  • the term "recombinant Deincoccus bacterium” also encompasses the genetically modified host cell as well as any progeny that is not identical to the parent host cell, in particular due to mutations that occur during replication.
  • the endogenous biosynthetic pathway converting DHS to chorismate (CHA) is blocked or reduced to improve the flow of carbon towards ccMA.
  • This biosynthetic pathway involves five enzymes, namely AroE, AroK, AroA and AroC. One or several of these enzymatic activities may be inactivated or reduced.
  • AroE refers to the enzyme shikimate dehydrogenase (EC 1.1.1.25) encoded by aroE gene, that converts DHS to shikimate (SHK).
  • AroE include, but are not limited to, AroE of D. geothermalis (Uniprot accession number: Q1IYW7), D. radiodurans (Uniprot accession number: Q9RY73 and Q9RV57), D. deserti (Uniprot accession number: C1CUV3), D. phoenicis (Uniprot accession number: A0A016QLF8), D. peraridilitoris (Uniprot accession number: K9ZXB2), D.
  • AroK refers to the enzyme shikimate kinase (EC 2.7.1.71) encoded by aroK gene. This enzyme converts SHK to 3-phosphoshikimate (S3P).
  • AroK include, but are not limited to, AroK of D. geothermalis (Uniprot accession number: Q1IXK8), D. radiodurans (Uniprot accession number: Q9RW93), D. peraridilitoris (Uniprot accession number: L0A7T6), D. maricopensis (Uniprot accession number: E8U7Z8), D. proteolyticus (Uniprot accession number: F0RLU5), D.
  • the gene encoding AroK in the recombinant Deinococcus bacterium of the invention may be easily identified using routine methods, for example based on homology with the nucleic acid encoding any of the above identified AroK enzyme.
  • AroA refers to the enzyme 3-phosphoshikimate 1- carboxyvinyltransferase (or 5-enolpyruvylshikimate-3-phosphate synthase or EPSP synthase, EC 2.5.1.19) encoded by aroA gene, that converts S3P to 5- enolpyruvylshikimate-3-phosphate (EPSP).
  • Examples of AroA include, but are not limited to, AroA of D. geothermalis (Uniprot accession number: Q1IZN3), D. radiodurans (Uniprot accession number: Q9RVD3), D. deserti (Uniprot accession number: C1D1P6), D.
  • the gene encoding AroA in the recombinant Deinococcus bacterium of the invention may be easily identified using routine methods, for example based on homology with the nucleic acid encoding any of the above identified AroA enzyme.
  • AroC refers to the enzyme chorismate synthase (EC 4.2.3.5) encoded by aroC gene, that converts EPSP to chorismate (CHA).
  • AroC include, but are not limited to, AroC of D. geothermalis (Uniprot accession number: Q1IXK9), D. radiodurans (Uniprot accession number: Q9RW94), D. deserti (Uniprot accession number: C1CX22 ), D. gobiensis (Uniprot accession number: H8GZ58), D. phoenicis (Uniprot accession number: A0A016QND6), D.
  • the gene encoding AroC in the recombinant Deinococcus bacterium of the invention may be easily identified using routine methods, for example based on homology with the nucleic acid encoding any of the above identified AroC enzyme.
  • one or several enzymes of the pathway converting DHS to chorismate are inactivated.
  • AroE, aroK, aroA and/or aroC genes may be inactivated by any method known by the skilled person, for example by deletion of all or part of this gene, by introducing a nonsense codon or a mutation inducing a frameshift, or by insertion of an expression cassette, e.g. an expression cassette comprising aroZ, aroY and/or catA genes.
  • all or part of the targeted gene is deleted, for example by gene replacement.
  • the endogenous biosynthetic pathway converting DHS to chorismate (CHA) is blocked by inactivation of the endogenous aroE gene.
  • all or part of the aroE gene is deleted.
  • the aroE gene is inactivated by gene replacement or by insertion in said gene of an expression cassette comprising aroZ, AroY and/or catA genes, preferable an expression cassette comprising aroZ, AroY and catA genes.
  • endogenous biosynthetic pathway converting DHS to chorismate has the effect of turning the strain into an auxotroph for the aromatic amino acids (phenylalanine, tyrosine, and tryptophan) and vitamins or vitamin-like intermediates made from the shikimate pathway (p-hydroxy benzoic acid, p- amino benzoic acid, and 2,3-dihydroxy benzoic acid).
  • this strain during its growth for the production of ccMA requires the exogenous addition of these six compounds (or a common intermediate), thereby adding substantially to the cost of commercial production of ccMA using such a strain.
  • one or several enzymatic activities of the pathway converting DHS to chorismate are reduced.
  • this reduction increases the flow of carbon towards ccMA while maintaining the prototrophy for the aromatic amino acids and vitamins.
  • This reduction may be obtained using altered or "leaky” enzymes or by decreasing the expression level of the genes encoding enzymes of the pathway converting DHS to chorismate.
  • the recombinant bacterium of the invention comprises a nucleic acid sequence encoding a leaky shikimate dehydrogenase (AroE), i.e. an AroE enzyme that confers prototrophy for the aromatic amino acids and vitamins, but without leading to significant secretion of aromatic compounds.
  • AroE leaky shikimate dehydrogenase
  • the leaky aroE mutant would allow a limited flow of carbon to shikimic acid while accumulating significant amounts of DHS which is then available for the conversion into PCA by the action of an AroZ enzyme.
  • the leaky AroE enzyme may be an endogenous, homologous or heterologous enzyme.
  • Recombinant Deinococcus bacteria comprising a leaky AroE enzyme may be obtained as described in the example 10 of the international patent application WO 2013/116244.
  • the leaky AroE may be in place of the endogenous AroE enzyme.
  • the endogenous aroE gene may be inactivated and the leaky AroE expressed from an expression cassette inserted in another locus of the genome or maintained in an episomal form, preferably inserted into the genome.
  • the recombinant bacterium of the invention comprises AroE, AroK, AroA and/or AroC temperature sensitive (Ts) mutants.
  • Ts mutations are typically missense mutations, which retain the function of a specific essential gene at standard (permissive) low temperature, lack that function at a defined high (non-permissive) temperature, and exhibit partial (hypomorphic) function at an intermediate (semi-permissive) temperature (Ben-Aroya et al., 2010).
  • Culturing the recombinant bacterium of the invention comprising TS mutant AroE, AroK, AroA and/or AroC at semi-permissive temperature thus allows a limited flow of carbon to chorismate and eliminate the dependence on exogenous aromatic amino acids.
  • the reduction may also be obtained by decreasing the expression level of one or several enzymes of the chorismate pathway.
  • the endogenous promoter may be replaced by weaker promoters, such as PlexA or PamyE promoters (Meima et al., 2001), thereby inducing a lower expression and thus a decrease of the chorismate production.
  • the endogenous biosynthetic pathway converting protocatechuate to oxoadipate is blocked or reduced to improve the flow of carbon towards ccMA.
  • This biosynthetic pathway involves three enzymatic activities, namely protocatechuate 3,4-dioxygenase activity, 3-carboxy-cis,cis-muconate cycloisomerase activity and 3-oxoadipate enol-lactonase activity. One or several of these enzymatic activities may be inactivated or reduced.
  • the enzyme exhibiting protocatechuate 3,4-dioxygenase activity catalyzes the reaction converting 3,4-dihydroxybenzoate (PCA) to 3-carboxy-cis,cis- muconate.
  • PCA 3,4-dihydroxybenzoate
  • this enzyme comprises two subunits, i.e. alpha and beta subunits.
  • Examples of Deinococcus protocatechuate 3,4-dioxygenases include, but are not limited to, protocatechuate 3,4-dioxygenases of D. geothermalis (a-subunit: Uniprot accession number: Q1J3Z7; ⁇ -subunit: Uniprot accession number: Q1J3Z6) and D.
  • Protocatechuate 3,4-dioxygenase encoding gene may be easily identified in the recombinant Deinococcus bacterium of the invention using routine methods, for example based on homology with the nucleic acid encoding any of the above identified protocatechuate 3,4-dioxygenases.
  • the enzyme exhibiting 3-carboxy-cis,cis-muconate cycloisomerase activity catalyzes the reaction converting 3-carboxy-cis,cis-muconate to ⁇ - carboxymuconolactone.
  • Examples of Deinococcus 3-carboxy-cis,cis-muconate cycloisomerases include, but are not limited to, 3-carboxy-cis,cis-muconate cycloisomerases of D. geothermalis (Uniprot accession number: Q1J3Z8) and D. deserti (Uniprot accession number: C1D2D5).
  • 3-carboxy-cis,cis-muconate cycloisomerase encoding gene may be easily identified in the recombinant Deinococcus bacterium of the invention using routine methods, for example based on homology with the nucleic acid encoding any of the above identified 3-carboxy-cis,cis-muconate cycloisomerases.
  • the enzyme exhibiting 3-oxoadipate enol-lactonase activity catalyzes the reactions converting ⁇ -carboxymuconolactone to 3-oxoadipate-enol-lactone and 3-oxoadipate-enol-lactone to 3-oxoadipate.
  • Examples of Deinococcus 3-oxoadipate enol-lactonases include, but are not limited to, 3-oxoadipate enol-lactonases of D. geothermalis (Uniprot accession number: Q1J3W1) and D. deserti (Uniprot accession number: C1D2D8).
  • 3-oxoadipate enol-lactonase encoding gene may be easily identified in the recombinant Deinococcus bacterium of the invention using routine methods, for example based on homology with the nucleic acid encoding any of the above identified 3-oxoadipate enol-lactonases.
  • one or several enzymes of the pathway converting protocatechuate to oxoadipate are inactivated.
  • Protocatechuate 3,4-dioxygenase, 3- carboxy-cis,cis-muconate cycloisomerase or 3-oxoadipate enol-lactonase encoding gene may be inactivated by any method known by the skilled person, for example by deletion of all or part of this gene, by introducing a nonsense codon or a mutation inducing a frameshift, or by insertion of an expression cassette, e.g. an expression cassette comprising aroZ, aroY and/or catA genes. In preferred embodiments, all or part of the targeted gene is deleted, for example by gene replacement.
  • the endogenous biosynthetic pathway converting protocatechuate to oxoadipate is blocked by inactivation of genes encoding protocatechuate 3,4-dioxygenase, 3-carboxy-cis,cis-muconate cycloisomerase and 3- oxoadipate enol-lactonase.
  • the upper part of the aromatic amino acid biosynthetic pathway i.e. the part of the pathway before the conversion of DHS to PCA by the DHS dehydratase, can be modified in order to increase to amount of DHS produced by the bacterium and that can be converted to ccMA or cathecol.
  • the activity of one or several enzymes involved in the conversion of phosphoenol pyruvate (PEP) and erythrose 4-phosphate (E4P) to DHS i.e. AroF, AroG, AroH, AroB and AroD
  • PEP phosphoenol pyruvate
  • E4P erythrose 4-phosphate
  • AroF, AroG, AroH, AroB and AroD may be increased compared to the non modified bacterium.
  • two, three, four, five, six or seven of these activities are increased. More preferably, all these activities are increased.
  • the activity of these enzymes may be increased due to the overexpression of their encoding genes.
  • at least one gene selected from the group consisting of aroG, aroF, aroH, aroB and aroD genes is overexpressed.
  • at least two, three, four, five, six or seven of these genes are overexpressed. More preferably, all these genes are overexpressed.
  • the skilled person can used any known techniques such as increasing the copy number of the gene in the bacterium, using a promoter inducing a high level of expression of the gene, i.e. a strong promoter, using elements stabilizing the corresponding messenger RNA or modifying Ribosome Binding Site (RBS) sequences and sequences surrounding them.
  • a promoter inducing a high level of expression of the gene i.e. a strong promoter
  • elements stabilizing the corresponding messenger RNA or modifying Ribosome Binding Site (RBS) sequences and sequences surrounding them e.g., Ribosome Binding Site (RBS) sequences and sequences surrounding them.
  • RBS Ribosome Binding Site
  • the overexpression may be obtained by increasing the copy number of the gene in the bacterium.
  • One or several copies of the gene may be introduced into the genome by methods of recombination, known to the expert in the field, including gene replacement or multicopy insertion in IS sequences (Makarova et al. 2001).
  • an expression cassette comprising the gene is integrated into the genome.
  • the gene may be carried by an expression vector, preferably a plasmid, comprising an expression cassette with the gene of interest.
  • the expression vector may be present in the bacterium in one or several copies, depending on the nature of the origin of replication.
  • the overexpression of the gene may also obtained by using a promoter inducing a high level of expression of the gene.
  • the promoter of an endogenous gene may be replaced by a stronger promoter, i.e. a promoter inducing a higher level of expression.
  • the promoters suitable to be used in the present invention are known by the skilled person and can be constitutive or inducible, and native, homologous or heterologous.
  • the overexpressed genes can be native, homologous or heterologous genes.
  • the terms "AroG”, “AroF” and AroH” refer to DAHP synthases encoded by aroG, aroF and aroH genes, respectively, that convert PEP and E4P to DAHP (Bentley, 1990).
  • the aroG, aroF or aroH gene may be any gene encoding a DAHP synthase, preferably a fungal or bacterial gene, more preferably a bacterial gene.
  • the aroG, aroF or aroH gene is from a Deinococcus bacterium.
  • Examples of "AroG”, “AroF” and AroH” from Deinococcus bacteria include, but are not limited to, the DAHP synthases of D. geothermalis (Uniprot accession numbers: Q1IY17 and Q1IXB8 and NCBI Reference Sequence: WP_039686534.1), D. radiodurans (Uniprot accession numbers: Q9RVM6 and Q9RTE8), D. murrayi (NCBI Reference Sequence: WP_027459838.1), D. misasensis (NCBI Reference Sequence: WP_034334648.1), D. soli (NCBI Reference Sequence: WP_046842818.1), D.
  • maricopensis (Uniprot accession numbers: E8U3H9, E8U723 and E8U704), D. marmoris (NCBI Reference Sequence: WP_029479935.1), D. deserti (Uniprot accession numbers: C1CXB8 and C1D0M7, NCBI Reference Sequence: WP_012693957.1), D. gobiensis (Uniprot accession numbers: H8GWJ1 and H8GZA8, NCBI Reference Sequence: WP_043800762.1), D. proteolyticus (Uniprot accession numbers: F0RP29), D. peraridilitoris (Uniprot accession number : K9ZZI4), D.
  • phoenicis (Uniprot accession number: A0A016QQD9), D. swuensis (Uniprot accession number : A0A0A7KMA6) and D. aquatilis (NCBI Reference Sequence: WP_040380876.1).
  • the gene encoding a DAHP synthase in the recombinant Deinococcus bacterium of the invention may be easily identified using routine methods, for example based on homology with the nucleic acid encoding any of the above identified enzymes. Any polypeptide, preferably from a Deinococcus bacterium, having at least 60%, preferably 80%, more preferably 90%, even more preferably 95% sequence identity to any of the above-identified DAHP synthases may be used.
  • the term "AroB” refers to the enzyme 3-dehydroquinate synthase (EC 4.2.3.4) encoded by aroB gene, that converts DAHP to DHQ.
  • the aroB gene may be any gene encoding a 3-dehydroquinate synthase, preferably a fungal or bacterial gene, more preferably a bacterial gene.
  • the aroB gene is from a Deinococcus bacterium. Examples of "AroB" from Deinococcus bacteria include, but are not limited to, the AroB of D. geothermalis (Uniprot accession numbers: Q1IXK7 and Q1I3M6), D.
  • radiodurans (Uniprot accession number: Q9RW92), D. deserti (Uniprot accession number: C1CX24), D. gobiensis (Uniprot accession number: H8GZ56), D. maricopensis (Uniprot accession number: E8U7Z7), D. peraridilitoris (Uniprot accession number: L0A5T3) and D. swuensis (Uniprot accession number: A0A0A7KER6).
  • the gene encoding AroB in the recombinant Deinococcus bacterium of the invention may be easily identified using routine methods, for example based on homology with the nucleic acid encoding any of the above identified enzymes.
  • Any polypeptide, preferably from a Deinococcus bacterium, having at least 60%, preferably 80%, more preferably 90%, even more preferably 95% sequence identity to any of the above-identified AroB enzymes may be used.
  • the term "AroD” or “AroQ” refers to the enzyme 3-dehydroquinate dehydratase
  • aroD also named AroQ
  • the aroD gene may be any gene encoding a 3-dehydroquinate dehydratase, preferably a fungal or bacterial gene, more preferably a bacterial gene.
  • the aroD or AroQ gene is from a Deinococcus bacterium. Examples of "AroD” or “AroQ” from Deinococcus bacteria include, but are not limited to, the AroD or AroQ of D. geothermalis (Uniprot accession numbers: Q1IXK6 and Q1IXK6), D.
  • radiodurans (Uniprot accession number: Q9RW91), D. deserti (Uniprot accession number: C1CX25), D. gobiensis (Uniprot accession number: H8GZ55), D. phoenicis (Uniprot accession number: A0A016QMR8 ), D. maricopensis (Uniprot accession number: E8U7Z6), D. proteolyticus (Uniprot accession number: F0RLU3 ), D. swuensis (Uniprot accession number: A0A0A7KHD4) and D. peraridilitoris (Uniprot accession number: L0A716).
  • the gene encoding AroD in the recombinant Deinococcus bacterium of the invention may be easily identified using routine methods, for example based on homology with the nucleic acid encoding any of the above identified enzymes. Any polypeptide, preferably from a Deinococcus bacterium, having at least 60%, preferably 80%, more preferably 90%, even more preferably 95% sequence identity to any of the above-identified AroD enzymes may be used.
  • At least a gene encoding the DAHP synthase and/or a gene encoding AroB are overexpressed in the recombinant bacterium of the invention.
  • the activity of the enzymes involved in the conversion of PEP and E4P to DHS may also be increased by overexpressing endogenous enzymes, or expressing or overexpressing improved enzymes, i.e. enzymes that possess at least one mutation in their sequence, in comparison with the amino acid sequence of the wild-type enzyme, said mutation leading to an increase of their activity ,an increased specific catalytic activity, an increased specificity for the substrate, an increased protein or RNA stability and/or an increased intracellular concentration of the enzyme, or leading to a feedback resistant mutant.
  • at least one of the enzymes involved in the conversion of PEP and E4P to DHS is an improved enzyme.
  • the recombinant bacterium of the invention comprises at least one of the enzymes involved in the conversion of PEP and E4P to DHS which is a feedback resistant mutant.
  • the recombinant bacterium comprises an AroF, AroG and/or AroH feedback resistant mutant.
  • these three proteins are subjected to feedback inhibition by one or more metabolites of shikimic acid pathway responsible for aromatic amino acid biosynthesis.
  • the endogenous aroG gene may be replaced by a modified aroG gene which codes for an AroG protein that is resistant to feedback inhibition by one or more metabolites of the aromatic amino acid pathway within the microbial cell, including the aromatic amino acid themself .
  • Feedback resistant mutants of all three enzymes are well known (e.g. Draths et al., 1992; Lutke-Eversloh and Stephanopoulos, 2007; Hu et al, 2003; and Shumilin et al, 1999).
  • Feedback resistant mutants may be mutated endogenous enzymes or homologous or heterologous enzymes.
  • AroF, AroG and AroH feedback resistant mutants are overexpressed in the recombinant bacterium of the invention.
  • the recombinant Deinococcus bacterium of the invention expresses a feedback-resistant DAHP synthase.
  • the feedback- resistant DAHP synthase is a variant of a Deinococcus DAHP synthase.
  • the feedback-resistant DAHP synthase is a variant of the Deinococcus DAHP synthase set forth in SEQ ID NO: 46 and comprises at least one substitution at position corresponding to residue N13, P156 or S186 of SEQ ID NO: 46.
  • the feedback-resistant DAHP synthase is a variant of the enzyme set forth in SEQ ID NO: 46 and comprises at least one substitution or combination of substitutions selected from N13K (SEQ ID NO: 47), P156L (SEQ ID NO: 48), S186F (SEQ ID NO: 49), N13K+ P156L (SEQ ID NO: 50), N13K+S186F (SEQ ID NO: 51), P156L +S 186F (SEQ ID NO: 52) and N13K+ P156L +S 186F (SEQ ID NO: 53).
  • the activity of one or several enzymes involved in the production of PEP or E4P is increased compared to the non modified bacterium.
  • the activity of these enzymes may be increased due to the overexpression of their encoding genes and/or the use of feedback resistant mutants.
  • At least one gene selected from the group consisting of rpe, rpi, talB, tktA or ppsA is overexpressed.
  • at least two, three, four or five of these genes are overexpressed. More preferably, all these genes are overexpressed.
  • the overexpressed genes can be native, homologous or heterologous genes.
  • Rpe refers to ribulose-phosphate 3-epimerase (EC 5.1.3.1) encoded by the rpe gene, that converts D-ribulose 5-phosphate to D-xylulose 5-phosphate.
  • the rpe gene may be any gene encoding a ribulose-phosphate 3-epimerase, preferably a fungal or bacterial gene, more preferably a bacterial gene.
  • the rpe gene is from a Deinococcus bacterium. Examples of "Rpe" from Deinococcus bacteria include, but are not limited to, the Rpe of D. geothermalis (Uniprot accession numbers: Q1IYR9), D.
  • radiodurans (Uniprot accession number: Q9RUI5), D. deserti (Uniprot accession number: C1D1F4), D. gobiensis (Uniprot accession number: H8GVN2), D. phoenicis (Uniprot accession number: A0A016QL52), D. peraridilitoris (Uniprot accession number: 9ZW42), D. maricopensis (Uniprot accession number: E8UAB 1), D. proteolyticus (Uniprot accession number: F0RMS8) and D. swuensis (Uniprot accession number: A0A0A7KIC0).
  • the gene encoding Rpe in the recombinant Deinococcus bacterium of the invention may be easily identified using routine methods, for example based on homology with the nucleic acid encoding any of the above identified enzymes. Any polypeptide, preferably from a Deinococcus bacterium, having at least 80%, preferably 90%, more preferably 95% sequence identity to any of the above-identified Rpe enzymes may be used.
  • Rpi refers to ribose-5-phosphate isomerase (EC 5.3.1.6) encoded by the rpi gene, that converts D-ribose 5-phosphate to D-ribulose 5-phosphate.
  • the rpi gene may be any gene encoding a ribose-5-phosphate isomerase, preferably a fungal or bacterial gene, more preferably a bacterial gene.
  • the rpi gene is from a Deinococcus bacterium. Examples of "Rpi” from Deinococcus bacteria include, but are not limited to, the Rpi of D. geothermalis (Uniprot accession numbers: Q1IYX3), D.
  • radiodurans (Uniprot accession number: Q9RW24), D. deserti (Uniprot accession number: C1CUX0), D. maricopensis (Uniprot accession number: E8UAB6), D. proteolyticus (Uniprot accession number: F0RLG0), D. phoenicis (Uniprot accession number: A0A016QLR1), D. gobiensis (Uniprot accession number: H8GT08), D. peraridilitoris (Uniprot accession number: K9ZYM6) and D. swuensis (Uniprot accession number: A0A0A7KIY3).
  • the gene encoding Rpi in the recombinant Deinococcus bacterium of the invention may be easily identified using routine methods, for example based on homology with the nucleic acid encoding any of the above identified enzymes. Any polypeptide, preferably from a Deinococcus bacterium, having at least 80%, preferably 90%, more preferably 95% sequence identity to any of the above- identified Rpi enzymes may be used.
  • the term "TalB” refers to transaldolase (EC 2.2.1.2) encoded by the talB gene, that converts sedoheptulose 7-phosphate and D-glyceraldehyde 3-phosphate to D- erythrose 4-phosphate and D-fructose 6-phosphate.
  • the talB gene may be any gene encoding a transaldolase, preferably a fungal or bacterial gene, more preferably a bacterial gene.
  • the talB gene is from a Deinococcus bacterium.
  • "TalB" from Deinococcus bacteria include, but are not limited to, the TalB of D. geothermalis (Uniprot accession numbers: Q1IZD4), D. radiodurans (Uniprot accession number: Q9RUP6), D. deserti (Uniprot accession numbers: C1D3N5 and C1CV54), D. gobiensis (Uniprot accession number: H8GUX5), D.
  • the gene encoding TalB in the recombinant Deinococcus bacterium of the invention may be easily identified using routine methods, for example based on homology with the nucleic acid encoding any of the above identified enzymes.
  • TktA refers to transketolase (EC 2.2.1.1) encoded by the tktA gene, that converts sedoheptulose 7-phosphate and D-glyceraldehyde 3-phosphate to D-ribose 5-phosphate and D-xylulose 5-phosphate.
  • the tktA gene may be any gene encoding a transketolase, preferably a fungal or bacterial gene, more preferably a bacterial gene.
  • the tktA gene is from a Deinococcus bacterium.
  • "TktA" from Deinococcus bacteria include, but are not limited to, the TktA of D. geothermalis (Uniprot accession numbers: Q1IW07), D. radiodurans (Uniprot accession number: Q9RS71), D. phoenicis (Uniprot accession numbers: A0A016QSC5), D. deserti (Uniprot accession numbers: C1CXQ1), D. peraridilitoris (Uniprot accession numbers:
  • TktA in the recombinant Deinococcus bacterium of the invention may be easily identified using routine methods, for example based on homology with the nucleic acid encoding any of the above identified enzymes. Any polypeptide, preferably from a Deinococcus bacterium, having at least 80%, preferably 90%, more preferably 95% sequence identity to any of the above-identified TktA enzymes may be used.
  • PpsA refers to a Phosphoenolpyruvate synthase (EC 2.7.9.2) encoded by the ppsA gene, that converts pyruvate to PEP.
  • the ppsA gene may be any gene encoding a transketolase, preferably a fungal or bacterial gene, more preferably a bacterial gene.
  • the ppsA gene is from a Deinococcus bacterium. Examples of "PpsA" from Deinococcus bacteria include, but are not limited to, the PpsA of D. geothermalis (Uniprot accession numbers: Q1J0R1), D. radiodurans (Uniprot accession number: 083026), D.
  • the gene encoding PpsA in the recombinant Deinococcus bacterium of the invention may be easily identified using routine methods, for example based on homology with the nucleic acid encoding any of the above identified enzymes.
  • any polypeptide preferably from a Deinococcus bacterium, having at least 80%, preferably 90%, more preferably 95% sequence identity to any of the above-identified PpsA enzymes may be used.
  • pyruvate kinase I and/or II EC 2.7.1.40
  • the pyruvate kinase activity may be blocked or reduced as explained above for the enzymes of the chorismate pathway.
  • the recombinant Deinococcus bacterium of the invention naturally provides sufficient amounts of PEP and E4P and is not genetically modified in order to increase the production of these precursors.
  • the present invention also relates to a recombinant Deinococcus bacterium comprising (i) a heterologous catechol biosynthetic pathway, i.e. converting 3-dehydroshikimate (DHS) to protocatechuate (PCA) and PCA to catechol, and (ii) a heterologous nucleic acid sequence encoding a polypeptide exhibiting catechol- O-methyltransferase activity, i.e. catalyzing the reaction converting catechol to gaiacol (EC 2.1.1.6).
  • said recombinant Deinococcus bacterium does not exhibit any catechol 1,2 dioxygenase activity.
  • the polypeptide exhibiting catechol-O-methyltransferase activity may be any known catechol-O-methyltransferase (COMT).
  • the polypeptide exhibiting COMT activity is selected from the group consisting of COMT from Mycobacterium vanbaalenii (Uniprot accession number: A1TA78; SEQ ID NO: 54) and any polypeptide exhibiting COMT activity and having at least 60 %, preferably at least 65, 70, 75, 80, 85, 90 or 95%, identity to said COMT. More preferably, the polypeptide exhibiting COMT activity is COMT from Mycobacterium vanbaalenii (SEQ ID NO: 54). An example of nucleic acid sequence encoding said COMT is set forth in SEQ ID NO: 55.
  • the recombinant Deinococcus bacterium expressing a heterologous COMT is preferably a mesophilic strain, more preferably D. grandis, D. cellulolysiticus, D. depolymerans, D. aquaticus, D. deserti. D. wulumuqiensis, D. proteolyticus, D. gobiensis misasensis, D. frigens, D. marmoris, D. ficus, D. apachensis, D. aquatilis, D. pimensis, D. peraridilitoris, D. puniceus, D.phoenicis, D. swuensis or D. actinosclerus, and even more preferably D. depolymerans or D. aquaticus.
  • Nucleic acid sequences encoding heterologous genes may be comprised in one or several expression cassettes.
  • Each expression cassette may comprise aroZ, aroY and/or COMT genes.
  • the recombinant Deinococcus bacterium of the invention may comprise (i) an expression cassette comprising aroZ gene, an expression cassette comprising aroY gene and an expression cassette comprising COMT gene; (ii) an expression cassette comprising aroZ and aroY genes and an expression cassette comprising COMT gene; (iii) an expression cassette comprising aroZ and COMT genes and an expression cassette comprising aroY gene; (iv) an expression cassette comprising aroY and COMT genes and an expression cassette comprising aroZ gene, or (v) an expression cassette comprising aroZ, aroY and COMT genes.
  • the recombinant Deinococcus bacterium of the invention comprises an expression cassette comprising aroZ, aroY and COMT genes. These genes may be expressed under the control of a single promoter or under the control of two promoters (with two of these genes under the control of the same promoter). Alternatively, each gene may be expressed under the control of a distinct promoter.
  • These expression cassettes may be integrated into the genome of the bacterium or may be maintained in an episomal form into an expression vector. In embodiments wherein the expression cassette(s) is(are) maintained in an episomal form, the expression vector may be present in the bacterium in one or several copies, depending on the nature of the origin of replication.
  • the expression cassette(s) is(are) integrated into the genome of the bacterium.
  • One or several copies of aroZ, aroY and/or COMT genes may be introduced into the genome by methods of recombination, known to the expert in the field, including gene replacement.
  • an expression cassette comprising said gene(s) is integrated into the genome.
  • an expression cassette comprising aroZ, aroY and COMT genes is integrated into the genome.
  • additional copies of expression cassettes comprising aroZ, aroY and/or COMT genes may be further integrated in the genome.
  • the expression cassette(s) may be integrated into the genome in order to inactive target genes.
  • the expression cassette is integrated in the sequence encoding AroE, AroK, AroL, AroA or AroC, preferably in the sequence encoding AroE, in order to block the chorismate pathway.
  • the expression cassette is integrated in a sequence encoding endogenous protocatechuate 3,4-dioxygenase, 3-carboxy-cis,cis-muconate cycloisomerase or 3- oxoadipate enol-lactonase, in order to block the endogenous biosynthetic pathway converting protocatechuate to oxoadipate.
  • the expression cassette(s) may be integrated into the genome in a non-coding sequence, e.g. an insertion sequence (IS) (Makarova et al. 2001).
  • the recombinant Deinococcus bacterium comprising (i) a heterologous catechol biosynthetic pathway, i.e. converting 3-dehydroshikimate (DHS) to protocatechuate (PCA) and PCA to catechol, and (ii) a heterologous nucleic acid sequence encoding a polypeptide exhibiting catechol-O-methyltransferase activity, may further comprise an enhancement of the carbon flow to catechol production i.e. the upper part of the aromatic amino acid biosynthetic patway before the conversion of DHS to PCA as described above.
  • the endogenous biosynthetic pathway converting protocatechuate to oxoadipate and/or the endogenous biosynthetic pathway converting DHS to chorismate may be inactivated.
  • the present invention also relates to a cell extract of the recombinant Deinococcus bacterium of the invention.
  • the term "cell extract” refers to any fraction obtained from a host cell, such as a cell supernatant, a cell debris, cell walls, DNA extract, enzymes or enzyme preparation or any preparation derived from host cells by chemical, physical and/or enzymatic treatment, which is essentially or mainly free of living cells such as whole broken cell extract.
  • the present invention relates to a use of a recombinant Deinococcus bacterium of the invention for producing ccMA.
  • the present invention relates to a method of producing ccMA comprising (i) culturing a recombinant Deinococcus bacterium according to the invention comprising a heterologous muconic acid biosynthetic pathway under conditions suitable to produce ccMA and optionally (ii) recovering said ccMA.
  • the present invention also relates to a method of producing catechol comprising
  • the present invention further relates to a method of producing ccMA comprising (i) culturing a recombinant Deinococcus bacterium according to the invention comprising a heterologous catechol biosynthetic pathway under conditions suitable to produce catechol, (ii) enzymatically converting catechol to ccMA, and optionally (iii) recovering said ccMA.
  • catechol produced in step (i) may be recovered before enzymatic conversion.
  • Catechol produced by the recombinant bacterium of the invention is secreted in the culture supernatant.
  • the enzymatic conversion of catechol to ccMA may be carried out using any enzyme exhibiting catechol 1,2-dioxygenase, in particular CatA enzymes disclosed above.
  • This enzyme may be provided by another strain co-cultured with the recombinant Deinococcus bacterium of the invention and secreting CatA enzyme.
  • the enzyme may be added to the culture medium or reaction medium comprising the catechol.
  • the enzyme may be purified or may be comprised in a cell extract, in particular a cell extract of the recombinant Deinococcus bacterium of the invention exhibiting CatA activity.
  • the present invention further relates to a method of producing gaiacol comprising (i) culturing a recombinant Deinococcus bacterium according to the invention comprising a heterologous catechol biosynthetic pathway and a heterologous nucleic acid sequence encoding a polypeptide exhibiting catechol-O-methyltransferase activity, under conditions suitable to produce gaiacol, and optionally (ii) recovering said gaiacol.
  • the present invention further relates to a method of producing vanillin comprising (i) culturing a recombinant Deinococcus bacterium according to the invention comprising a heterologous catechol biosynthetic pathway and a heterologous nucleic acid sequence encoding a polypeptide exhibiting catechol-O-methyltransferase activity, under conditions suitable to produce gaiacol, (ii) converting gaiacol to vanillin and, optionally (iii) recovering said vanillin.
  • gaiacol produced in step (i) may be recovered before conversion.
  • the conversion of gaiacol to vanillin may be carried out using any method known by the skilled person, e.g. the method described by Mottern (Mottern, 5 1934).
  • Conditions suitable to produce ccMA, catechol or gaiacol may be easily determined by the skilled person according to the recombinant Deinococcus bacterium used.
  • the carbon source may be selected from the group consisting of C5 sugars such as xylose and arabinose, C6 sugars such as glucose, cellobiose, saccharose0 and starch.
  • ccMA, catechol or gaiacol is produced from renewable, biologically derived carbon sources.
  • more complex carbon sources can be used such as cellulosic biomass.
  • cellulosic biomass refers to any5 biomass material, preferably vegetal biomass, comprising cellulose, hemicellulose and/or lignocellulose, preferably comprising cellulose and hemicellulose.
  • Cellulosic biomass includes, but is not limited to, plant material such as forestry products, woody feedstock (softwoods and hardwoods), agricultural wastes and plant residues (such as corn stover, shorghum, sugarcane bagasse, grasses, rice straw, wheat straw, empty fruit bunch from0 oil palm and date palm, agave bagasse, from tequila industry), perennial grasses (switchgrass, miscanthus, canary grass, erianthus, napier grass, giant reed, and alfalfa); municipal solid waste (MSW), aquatic products such as algae and seaweed, wastepaper, leather, cotton, hemp, natural rubber products, and food and feed processing by-products.
  • plant material such as forestry products, woody feedstock (softwoods and hardwoods), agricultural wastes and plant residues (such as corn stover, shorghum, sugarcane bagasse, grasses, rice straw, wheat straw, empty fruit bunch from0 oil palm and date palm, agave bagasse, from tequila industry
  • the cellulosic biomass comprises lignocellulose
  • this biomass is pre-5 treated before hydrolysis.
  • This pretreatment is intended to open the bundles of lignocelluloses in order to access the polymer chains of cellulose and hemicellulose.
  • Pretreatment methods are well known by the skilled person and may include physical pretreatments (e.g. high pressure steaming, extrusion, pyrolysis or irradiation), physicochemical and chemical pretreatments (e.g. ammonia fiber explosion, treatments0 with alkaline, acidic, solvent or oxidizing agents) and/or biological pretreatments.
  • Temperature conditions can also be adapted depending on the use of mesophilic or thermophilic Deinococcus bacteria.
  • the Deinococcus bacterium is a thermophilic Deinococcus, such as for example D. geothermalis, and the culture of the recombinant Deinococcus bacterium under conditions suitable to produce ccMA, catechol or gaiacol is performed at a temperature comprised between 35 and 60°C, preferably 37 and 55°C, more preferably at about 48°C.
  • the Deinococcus bacterium is a mesophilic Deinococcus and the culture of the recombinant Deinococcus bacterium under conditions suitable to produce ccMA, catechol or gaiacol is performed at a temperature comprised between 30 and 42°C, preferably between 35 and 40°C, more preferably at about 37°C.
  • the present invention relates to a use of a recombinant Deinococcus bacterium of the invention for producing cis-trans and/or trans-trans muconic acid (ctMA and ttMA, respectively).
  • the present invention also relates to a method of producing ctMA and/or ttMA comprising (i) producing ccMA according to the method of the invention and as described above and (ii) isomerizing said ccMA to produce ctMA and/or ttMA, and (iii) optionally recovering said ctMA and/or ttMA.
  • the method may further comprise an additional step of recovering ccMA before isomerization.
  • ccMA may be isomerized to produce ctMA and ctMA may be further isomerized to produce ttMA.
  • the isomerization may be carried out by any method known by the skilled person such as for example the method disclosed in the US patents 8,809,583, 8,426,639 or 8,367,858.
  • the present invention relates to a use of a recombinant Deinococcus bacterium of the invention for producing adipic acid.
  • the present invention also relates to a method of producing adipic acid comprising (i) producing ccMA according to the method of the invention and as described above and (ii) reducing said ccMA to produce adipic acid, and (iii) optionally recovering said adipic acid.
  • the method may further comprise an additional step of recovering ccMA before reduction.
  • the reduction may be carried out by any method known by the skilled person such as for example the method disclosed in Niu et al., 2002.
  • the present invention relates to a use of a recombinant Deinococcus bacterium of the invention for producing teraphtalic acid and/or trimetillitic acid.
  • the present invention also relates to a method of producing terephthalic acid and/or trimellitic acid comprising (i) producing ccMA, ctMA and/or ttMA according to the method of the invention and as described above and (ii) converting said ccMA, ctMA and/or ttMA to produce terephthalic acid and/or trimellitic acid, and (iii) optionally recovering said terephthalic acid and/or trimellitic acid.
  • the method may further comprise an additional step of recovering ccMA, ctMA and/or ttMA before conversion.
  • the conversion of ccMA, ctMA and/or ttMA to teraphtalic acid and/or trimetillitic acid may be carried out by any method known by the skilled person such as for example the method disclosed in the US patent 8,367,858.
  • Muconic acid, adipic acid, teraphtalic acid or trimetillitic acid produced according to the methods of the invention may be further used in the production various compounds such as, for example, Nylon 6, Nylon-6,6, polyester polyols, polytrimethylene terephthalate, polyethylene terephthalate, dimethyl terephthalate, trimellitic anhydride, industrial plastis, resins, food ingredients, plasticizers, cosmetics, pharmaceuticals or other polymers.
  • the methods of the invention may be performed in a reactor, in particular a reactor of conversion of biomass.
  • reactor is meant a conventional fermentation tank or any apparatus or system for biomass conversion, typically selected from bioreactors, biofilters, rotary biological contactors, and other gaseous and/or liquid phase bioreactors.
  • the apparatus which can be used according to the invention can be used continuously or in batch loads.
  • the method may be conducted under aerobiosis, anaerobiosis or microaerobiosis.
  • a recombinant Deinococcus bacterium expressing a heterologous polypeptide exhibiting 3-dehydroshikimate dehydratase activity and a heterologous polypeptide exhibiting protocatechuate decarboxylase activity.
  • the recombinant bacterium of item 1 wherein said bacterium further expresses a heterologous polypeptide exhibiting catechol 1 ,2-dioxygenase activity.
  • polypeptide exhibiting 3-dehydroshikimate dehydratase activity is selected from the group consisting of 3-dehydroshikimate dehydratases from Bacillus thuringiensis, Podospora anserina, , Klebsiella pneumoniae, Acinetohacter calcoaceticus, Acinetohacter sp.
  • ADPl Acinetohacter baylyi, Neurospora crassa, Aspergillus nidulans, Gluconobacter oxydans and Pseudomonas putida, in particular Pseudomonas putida KT2440 and Pseudomonas putida H8234. 4.
  • polypeptide exhibiting 3-dehydroshikimate dehydratase activity is selected from the group consisting of 3-dehydroshikimate dehydratases from Bacillus thuringiensis, Podospora anserina, Pseudomonas putida and Acinetohacter sp.
  • ADPl preferably from Bacillus thuringiensis, Podospora anserina and Acinetohacter sp.
  • ADPl and more preferably from Bacillus thuringiensis and Acinetohacter sp. ADPl .
  • polypeptide exhibiting 3-dehydroshikimate dehydratase activity is selected from the group consisting of 3-dehydroshikimate dehydratases of SEQ ID NO: 4, 2, 6 and 8, and polypeptides exhibiting 3-dehydroshikimate dehydratase activity and having at least 60 % identity to SEQ ID NO: 4, 2, 6 or 8.
  • polypeptide exhibiting 3-dehydroshikimate dehydratase activity is selected from the group consisting of 3-dehydroshikimate dehydratases of SEQ ID NO: 4 and 8, and polypeptides exhibiting 3-dehydroshikimate dehydratase activity and having at least 60 % identity to SEQ ID NO: 4 or 8.
  • polypeptide exhibiting 3-dehydroshikimate dehydratase activity is selected from the group consisting of 3-dehydroshikimate dehydratase of SEQ ID NO: 4, and polypeptides exhibiting 3-dehydroshikimate dehydratase activity and having at least 60 % identity to SEQ ID NO: 4.
  • polypeptide exhibiting 3-dehydroshikimate dehydratase activity is selected from the group consisting of 3-dehydroshikimate dehydratase of SEQ ID NO: 4, and polypeptides exhibiting 3-dehydroshikimate dehydratase activity and having at least 80 % identity to SEQ ID NO: 4, preferably at least 90 % identity to SEQ ID NO: 4.
  • polypeptide exhibiting 3-dehydroshikimate dehydratase activity is selected from the group consisting of 3-dehydroshikimate dehydratase of SEQ ID NO: 8, and polypeptides exhibiting 3-dehydroshikimate dehydratase activity and having at least 60 % identity to SEQ ID NO: 8.
  • polypeptide exhibiting 3-dehydroshikimate dehydratase activity is selected from the group consisting of 3-dehydroshikimate dehydratase of SEQ ID NO: 8, and polypeptides exhibiting 3-dehydroshikimate dehydratase activity and having at least 80 % identity to SEQ ID NO: 8, preferably at least 90 % identity to SEQ ID NO: 8.
  • polypeptide exhibiting 3-dehydroshikimate dehydratase activity is selected from the group consisting of 3-dehydroshikimate dehydratase of SEQ ID NO: 2, and polypeptides exhibiting 3-dehydroshikimate dehydratase activity and having at least 60 % identity to SEQ ID NO: 2.
  • polypeptide exhibiting 3-dehydroshikimate dehydratase activity is selected from the group consisting of 3-dehydroshikimate dehydratase of SEQ ID NO: 2, and polypeptides exhibiting 3-dehydroshikimate dehydratase activity and having at least 80 % identity to SEQ ID NO: 2, preferably at least 90 % identity to SEQ ID NO: 2.
  • polypeptide exhibiting 3-dehydroshikimate dehydratase activity is selected from the group consisting of 3-dehydroshikimate dehydratase of SEQ ID NO: 6, and polypeptides exhibiting 3-dehydroshikimate dehydratase activity and having at least 60 % identity to SEQ ID NO: 6.
  • polypeptide exhibiting 3-dehydroshikimate dehydratase activity is selected from the group consisting of 3-dehydroshikimate dehydratase of SEQ ID NO: 6, and polypeptides exhibiting 3-dehydroshikimate dehydratase activity and having at least 80 % identity to SEQ ID NO: 6, preferably at least 90 % identity to SEQ ID NO: 6.
  • polypeptide exhibiting protocatechuate decarboxylase activity is selected from the group consisting of 3-protocatechuate decarboxylases from Klebsiella pneumoniae, Enterobacter cloacae and Sedimentibacter hydroxybenzoicus, preferably from Klebsiella pneumoniae.
  • polypeptide exhibiting protocatechuate decarboxylase activity is selected from the group consisting of (i) a PCA decarboxylase comprising polypeptides of SEQ ID NO: 10, 12 and 14, (ii) a PCA decarboxylase comprising polypeptides of SEQ ID NO: 17, 19 and 21, (iii) the PCA decarboxylase of SEQ ID NO: 23, and (iv) PCA decarboxylases having at least 60 % identity to SEQ ID NO: 10, 12, 14, 17, 19, 21 or 23. 21.
  • polypeptide exhibiting protocatechuate decarboxylase activity is selected from the group consisting of a PCA decarboxylase comprising polypeptides of SEQ ID NO: 10, 12 and 14, and PCA decarboxylases having at least 60 % identity to SEQ ID NO: 10, 12 or 14.
  • polypeptide exhibiting protocatechuate decarboxylase activity is selected from the group consisting of a PCA decarboxylase comprising polypeptides of SEQ ID NO: 10, 12 and 14, and PCA decarboxylases having at least 80 % identity to SEQ ID NO: 10, 12 or 14, preferably at least 90 % identity to SEQ ID NO: 10, 12 or 14.
  • PCA decarboxylase 15 consisting of a PCA decarboxylase comprising polypeptides of SEQ ID NO: 17, 19 and 21, and PCA decarboxylases having at least 60 % identity to SEQ ID NO: 17, 19 or 21.
  • PCA decarboxylases having at least 80 % identity to SEQ ID NO: 17, 19 or 21, preferably at least 90 % identity to SEQ ID NO: 17, 19 or 21.
  • the recombinant bacterium according to any of items 1 to 20, wherein said polypeptide exhibiting protocatechuate decarboxylase activity is selected from the group consisting of a PCA decarboxylase of SEQ ID NO: 23, and PCA decarboxylases having at least 80 % identity to SEQ ID NO: 23, preferably at least 90 % identity to SEQ ID NO: 23.
  • polypeptide exhibiting catechol 1,2-dioxygenase activity is selected from the group consisting of catechol 1,2-dioxygenases from Acinetobacter radioresistens, Acinetobacter calcoaceticus, Candida albicans, Bulkholderia mallei, Bulkholderia xenovorans and Pseudomonas putida, preferably from Acinetobacter calcoaceticus, Candida albicans and Bulkholderia xenovorans.
  • polypeptide exhibiting catechol 1,2-dioxygenase activity is selected from the group consisting of catechol 1,2-dioxygenases of SEQ ID NO: 25, 27, 29, 31, 33, 34, 35, 36, 37, 38, 39, 40 and 41 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 60 % identity to SEQ ID NO: 25, 27, 29, 31, 33, 34, 35, 36, 37, 38, 39, 40 or 41.
  • polypeptide exhibiting catechol 1,2-dioxygenase activity is selected from the group consisting of catechol 1,2-dioxygenase of SEQ ID NO: 25 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 60 % identity to SEQ ID NO: 25.
  • polypeptide exhibiting catechol 1,2-dioxygenase activity is selected from the group consisting of catechol 1,2-dioxygenase of SEQ ID NO: 25 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 80 % identity to SEQ ID NO: 25, preferably at least 90 % identity to SEQ ID NO: 25.
  • 35 The recombinant bacterium according to any of items 2 to 29, wherein said polypeptide exhibiting catechol 1,2-dioxygenase activity is selected from the group consisting of catechol 1,2-dioxygenase of SEQ ID NO: 27 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 80 % identity to SEQ ID NO: 27, preferably at least 90 % identity to SEQ ID NO: 27.
  • polypeptide exhibiting catechol 1,2-dioxygenase activity is selected from the group consisting of catechol 1,2-dioxygenase of SEQ ID NO: 29 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 80 % identity to SEQ ID NO: 29, preferably at least 90 % identity to SEQ ID NO: 29.
  • said polypeptide exhibiting catechol 1 ,2-dioxygenase activity is a catechol 1 ,2-dioxygenase of SEQ ID NO: 31.
  • polypeptide exhibiting catechol 1,2-dioxygenase activity is selected from the group consisting of catechol 1,2-dioxygenase of SEQ ID NO: 33 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 80 % identity to SEQ ID NO: 33, preferably at least 90 % identity to SEQ ID NO: 33.
  • said polypeptide exhibiting catechol 1 ,2-dioxygenase activity is a catechol 1 ,2-dioxygenase of SEQ ID NO: 34.
  • polypeptide exhibiting catechol 1,2-dioxygenase activity is selected from the group consisting of catechol 1,2-dioxygenase of SEQ ID NO: 35 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 60 % identity to SEQ ID NO: 35.
  • polypeptide exhibiting catechol 1,2-dioxygenase activity is selected from the group consisting of catechol 1,2-dioxygenase of SEQ ID NO: 35 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 80 % identity to SEQ ID NO: 35, preferably at least 90 % identity to SEQ ID NO: 35.
  • said polypeptide exhibiting catechol 1 ,2-dioxygenase activity is a catechol 1 ,2-dioxygenase of SEQ ID NO: 36.
  • polypeptide exhibiting catechol 1,2-dioxygenase activity is selected from the group consisting of catechol 1,2-dioxygenase of SEQ ID NO: 37 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 60 % identity to SEQ ID NO: 37.
  • polypeptide exhibiting catechol 1,2-dioxygenase activity is selected from the group consisting of catechol 1,2-dioxygenase of SEQ ID NO: 37 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 80 % identity to SEQ ID NO: 37, preferably at least 90 % identity to SEQ ID NO: 37.
  • polypeptide exhibiting catechol 1,2-dioxygenase activity is selected from the group consisting of catechol 1,2-dioxygenase of SEQ ID NO: 38 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 80 % identity to SEQ ID NO: 38, preferably at least 90 % identity to SEQ ID NO: 38.
  • polypeptide exhibiting catechol 1,2-dioxygenase activity is selected from the group consisting of catechol 1,2-dioxygenase of SEQ ID NO: 39 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 60 % identity to SEQ ID NO: 39.
  • polypeptide exhibiting catechol 1,2-dioxygenase activity is selected from the group consisting of catechol 1,2-dioxygenase of SEQ ID NO: 39 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 80 % identity to SEQ ID NO: 39, preferably at least 90 % identity to SEQ ID NO: 39.
  • said polypeptide exhibiting catechol 1 ,2-dioxygenase activity is a catechol 1 ,2-dioxygenase of SEQ ID NO: 40.
  • polypeptide exhibiting catechol 1,2-dioxygenase activity is selected from the group consisting of catechol 1,2-dioxygenase of SEQ ID NO: 41 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 60 % identity to SEQ ID NO: 41.
  • polypeptide exhibiting catechol 1,2-dioxygenase activity is selected from the group consisting of catechol 1,2-dioxygenase of SEQ ID NO: 41 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 80 % identity to SEQ ID NO: 41, preferably at least 90 % identity to SEQ ID NO: 41.
  • polypeptide exhibiting catechol 1,2-dioxygenase activity is selected from the group consisting of catechol 1,2-dioxygenase of SEQ ID NO: 41 and a variant thereof comprising at least one substitution at position corresponding to residue G72, L73 or P76, preferably at least one substitution or combination of substitutions selected from G72A, L73F, P76A, G72A+L73F, G72A+P76A, P76A+L73F and G72A+L72F+P76A.
  • the feedback inhibition resistant DAHP synthase is a variant of the Deinococcus DAHP synthase set forth in SEQ ID NO: 46 and comprises at least one substitution at position corresponding to residue N13, P156 or S186 of SEQ ID NO: 46, preferably selected from N13K, P156L, S186F, N13 + P156L, N13 +S 186F, P156L +S186F and N13K+ P156L +S 186F.
  • the recombinant bacterium of any of items 1 to 82 further expresses a heterologous polypeptide exhibiting catechol-O-methyltransferase activity.
  • the recombinant bacterium of item 83, wherein the polypeptide exhibiting catechol-O-methyltransferase activity is selected from the group consisting of COMT from Mycobacterium vanbaalenii (SEQ ID NO: 54) and any polypeptide exhibiting COMT activity and having at least 60 % identity to SEQ ID NO: 54.
  • a method of producing cis-cis muconic acid comprising culturing a recombinant Deinococcus bacterium according to any of items 2 to 82 under conditions suitable to produce cis-cis muconic acid, and optionally recovering said cis-cis muconic acid.
  • a method of producing catechol comprising culturing a recombinant
  • a method of producing cis-cis muconic acid comprising (i) producing catechol according to the method of item 86, (ii) enzymatically converting catechol to cis-cis muconic acid, and optionally (iii) recovering said cis-cis muconic acid.
  • a method of producing adipic acid comprising producing cis-cis muconic acid according to the method of item 85, 87 or 88 and reducing said cis-cis muconic acid to produce adipic acid, and optionally recovering said adipic acid.
  • a method of producing cis-trans and/or trans-trans muconic acid comprising producing cis-cis muconic acid according to the method of item 85, 87 or 88 and isomerizing said cis-cis muconic acid to produce cis-trans and/or trans-trans muconic acid, and optionally recovering said cis-trans and/or trans-trans muconic acid.
  • a method of producing gaiacol comprising (i) culturing a recombinant Deinococcus bacterium according to item 83 or 84 under conditions suitable to produce gaiacol, and optionally (ii) recovering said gaiacol.
  • a method of producing vanillin comprising (i) culturing a recombinant Deinococcus bacterium according to item 83 or 84 under conditions suitable to produce gaiacol, (ii) converting gaiacol to vanillin and, optionally (iii) recovering said vanillin.
  • Example 1 Catechol production by recombinant Deinococcus geothermalis Materiel & Methods:
  • a Deinococcus geothermalis strain was genetically engineered to produce catechol.
  • Genomic DNAs were prepared using Dneasy&Blood QIAGEN Kit as indicated by the Manufacturer. Each gene was amplified and assembled by overlapping PCR. Insertion of DNA fragments into the chromosome of D. geothermalis was performed using homologous recombination mechanism. Insertion cassettes comprised a nucleic acid sequence to be inserted into the chromosome flanked by 1500 bp region homologous to the sequence upstream or downstream the chromosomic target aroE gene.
  • heterologous genes strong constitutive promoters were used such as PtufA and PtufB promoters from the translation elongation factors Tu genes tufA (DR0309) and tufB (DR2050), or the promoter region PgroESL of the groESL operon (Lecointe et al, 2004; Meima et al, 2001).
  • aroZ of Bacillus thuringiensis Bt_aroZ; SEQ ID NO: 3 or quiC of Acinetobacter sp.
  • ADP1 (Ac_quiC; SEQ ID NO: 7) encoding DHS dehydratase (EC 4.2.1.118) was placed under the control of a strong constitutive promoter and was followed by aroY of Klebsiella pneumonia (Kp_aroY, SEQ ID NO: 15) comprising the genes encoding the three subunits of the protocatechuate decarboxylase (EC 4.1.1.63) also controlled by a strong constitutive promoter (cf. Figures 1 and 3).
  • the cultures were performed in deepwell at 48°C at 250 rpm. At 72h of growth, aliquots of 500 ⁇ of culture were taken and filtrated using 0.22 ⁇ membrane filter MF (Millipore). The filtrate was then injected in HPLC.
  • Recombinant D. geothermalis strains comprising the genetic construct illustrated in Figure 1, i.e. expressing aroZ of Bacillus thuringiensis and aroY of Klebsiella pneumonia produced PC A and catechol ( Figure 2). The same results were obtained with a recombinant D. geothermalis strain expressing quiC of Acinetobacter sp. ADPl and aroY of Klebsiella pneumonia.
  • Genomic DNAs were prepared using Dneasy&Blood QIAGEN Kit as indicated by the Manufacturer. Each gene was amplified and assembled by overlapping PCR. Insertion of DNA fragments into the chromosome of D. geothermalis was performed using homologous recombination mechanism. Insertion cassettes comprised a nucleic acid sequence to be inserted into the chromosome flanked by 1500 bp region homologous to the sequence upstream or downstream the chromosomic target aroE gene.
  • heterologous genes strong constitutive promoters were used such as Ptuf A and PtufB promoters from the translation elongation factors Tu genes tufA (DR0309) and tufB (DR2050), or the promoter region PgroESL of the groESL operon (Lecointe et al, 2004; Meima et al, 2001).
  • CMG2% medium Peptone 2 g/L ; Yeast Extract 5 g/L ; Glucose 55 mM (20 g/L) ; MOPS acid 40 mM ; NH4C1 20 mM ; NaOH 10 mM ; KOH 10 mM ; CaC12.2H20 0,5 ⁇ ;
  • Seed from log phase of growth was then inoculated into 1 ml of the mineral medium (as described above) at an initial optical density at 600 nm (OD600) of 0.4.
  • the cultures were performed in deepwell either at 37°C or 48°C at 250 rpm.
  • aliquots of 500 ⁇ of culture were taken and filtrated using 0.22 ⁇ membrane filter MF (Millipore). The filtrate was then injected in HPLC.
  • Recombinant D. geothermalis strains comprising an expression cassette comprising (a) quiC oi Acinetobacter sp. ADPl and aroY oi Klebsiella pneumonia, or (b) aroZ of Bacillus thuringiensis and aroY of Klebsiella pneumonia, and an expression cassette comprising catA of Acinetobacter calcoaceticus produced muconic acid ( Figure 4 and 5).
  • Genomic DNAs were prepared using Dneasy&Blood QIAGEN Kit as indicated by the Manufacturer. Each gene was amplified and assembled by overlapping PCR. Insertion of DNA fragments into the chromosome of D. geothermalis was performed using homologous recombination mechanism. Insertion cassettes comprised a nucleic acid sequence to be inserted into the chromosome flanked by 1500 bp region homologous to the sequence upstream or downstream the chromosomic target aroE gene.
  • heterologous genes strong constitutive promoters were used such as Ptuf A and PtufB promoters from the translation elongation factors Tu genes tufA (DR0309) and tufB (DR2050), or the promoter region PgroESL of the groESL operon (Lecointe et al, 2004; Meima et al, 2001).
  • Each recombinant comprising an expression cassette as illustrated in Figure 1 or 3 was further modified by inserting an expression cassette comprising catA encoding catechol 1 ,2-dioxygenase from Candida albicans (SEQ ID NO: 29) or Bulkholderia xenovorans (SEQ ID NO: 31), under the control of strong constitutive promoter, in an IS sequence (IS66).
  • an expression cassette comprising catA encoding catechol 1 ,2-dioxygenase from Candida albicans (SEQ ID NO: 29) or Bulkholderia xenovorans (SEQ ID NO: 31), under the control of strong constitutive promoter, in an IS sequence (IS66).
  • Seed from log phase of growth was then inoculated into 1 ml of the mineral medium (as described above) at an initial optical density at 600 nm (OD600) of 0.4.
  • the cultures were performed in deepwell either at 48°C pH6 at 250 rpm.
  • aliquots of 500 ⁇ of culture were taken and filtrated using 0.22 ⁇ membrane filter MF (Millipore). The filtrate was then injected in HPLC.
  • Recombinant D. geothermalis strains comprising an expression cassette comprising (a) quiC of Acinetobacter sp. ADP1 and aroY of Klebsiella pneumonia, or (b) aroZ of Bacillus thuringiensis and aroY of Klebsiella pneumonia, and an expression cassette comprising catA of Candida albicans or Bulkholderia xenovorans, produced muconic acid.
  • Example 4 Catechol production by recombinant Deinococcus geothermalis cultivated in fermenter A Deinococcus geothermalis strain was genetically engineered to produce catechol as described in example 1 and figure 1.
  • Genomic DNAs were prepared using Dneasy&Blood QIAGEN Kit as indicated by the Manufacturer. Each gene was amplified and assembled by overlapping PCR. Insertion of DNA fragments into the chromosome of D. geothermalis was performed using homologous recombination mechanism. Insertion cassettes comprised a nucleic acid sequence to be inserted into the chromosome flanked by 1500 bp region homologous to the sequence upstream or downstream the chromosomic target aroE gene.
  • heterologous genes strong constitutive promoters were used such as Ptuf A and PtufB promoters from the translation elongation factors Tu genes tufA (DR0309) and tufB (DR2050), or the promoter region PgroESL of the groESL operon (Lecointe et al, 2004; Meima et al, 2001).
  • aroZ of Bacillus thuringiensis (Bt_aroZ; SEQ ID NO: 3) encoding DHS dehydratase (EC 4.2.1.118) was placed under the control of a strong constitutive promoter and was followed by aroY of Klebsiella pneumonia (Kp_aroY, SEQ ID NO: 15) comprising the genes encoding the three subunits of the protocatechuate decarboxylase (EC 4.1.1.63) also controlled by a strong constitutive promoter (cf. Figures 1 and 3).
  • the recombinant Deinococcus strain was able to produce up to 1000 mg/L of catechol in fermenter at 64 hours of cultivation.
  • Example 5 cis,cis muconic acid production by recombinant Deinococcus geothermalis cultivated in fermenter
  • Deinococcus geothermalis strains were genetically engineered to produce muconic acid as described in example 2 and figure 1.
  • Genomic DNAs were prepared using Dneasy&Blood QIAGEN Kit as indicated by the Manufacturer. Each gene was amplified and assembled by overlapping PCR. Insertion of DNA fragments into the chromosome of D. geothermalis was performed using homologous recombination mechanism. Insertion cassettes comprised a nucleic acid sequence to be inserted into the chromosome flanked by 1500 bp region homologous to the sequence upstream or downstream the chromosomic target aroE gene.
  • heterologous genes strong constitutive promoters were used such as Ptuf A and PtufB promoters from the translation elongation factors Tu genes tufA (DR0309) and tufB (DR2050), or the promoter region PgroESL of the groESL operon (Lecointe et al, 2004; Meima et al, 2001).
  • aroZ of Bacillus thuringiensis (Bt_aroZ; SEQ ID NO: 3) encoding DHS dehydratase (EC 4.2.1.118) was placed under the control of strong constitutive promoter and was followed by aroY of Klebsiella pneumonia (SEQ ID NO: 15) encoding protocatechuate decarboxylase (EC 4.1.1.63) controlled by a strong constitutive promoter ( Figures 1 and 3).
  • Recombinant comprising an expression cassette as illustrated in Figure 1, was further modified by inserting an expression cassette comprising catA encoding catechol 1,2-dioxygenase from Acinetobacter calcoaceticus (AccjCatA, SEQ ID NO: 24) under the control of strong constitutive promoter, in an IS sequence (IS66).
  • an expression cassette comprising catA encoding catechol 1,2-dioxygenase from Acinetobacter calcoaceticus (AccjCatA, SEQ ID NO: 24) under the control of strong constitutive promoter, in an IS sequence (IS66).
  • Seed from log phase of growth was then inoculated into 50 ml of the mineral medium (as described above) at an initial optical density at 600 nm (OD600) of 0.4.
  • the cultures was performed in 500ml-flask at pH6, 37°C and at 250 rpm.
  • aliquots of 500 ⁇ of culture were taken and filtrated using 0.22 ⁇ membrane filter MF (Millipore). The filtrate was then injected in HPLC.

Abstract

La présente invention concerne une bactérie Deinococcus de recombinaison comprenant une voie de biosynthèse hétérologue convertissant la 3-déshydroshikimate (DHS) en catéchol et/ou acide cis,cis-muconique et son utilisation pour la production d'acide cis,cis-muconique ou de catéchol, ainsi que des dérivés associés.
PCT/EP2016/064752 2015-06-24 2016-06-24 Procédé de production d'acide muconique WO2016207403A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP15305972.0 2015-06-24
EP15305972 2015-06-24

Publications (1)

Publication Number Publication Date
WO2016207403A1 true WO2016207403A1 (fr) 2016-12-29

Family

ID=53488273

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2016/064752 WO2016207403A1 (fr) 2015-06-24 2016-06-24 Procédé de production d'acide muconique

Country Status (1)

Country Link
WO (1) WO2016207403A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113151053A (zh) * 2021-03-15 2021-07-23 广东工业大学 一株佛里德兰德氏杆菌gdutan10及其应用
EP3789481A4 (fr) * 2018-05-01 2022-02-16 Research Institute Of Innovative Technology For The Earth Transformant d'une bactérie corynéforme et procédé de production de composé utile l'utilisant
WO2022177569A1 (fr) * 2021-02-18 2022-08-25 Zymergen Inc. Voies de biosynthèse modifiées pour la production d'acide 3,4-amino-4-hydroxybenzoïque par fermentation
CN117004547A (zh) * 2023-09-27 2023-11-07 北京化工大学 一种以葡萄糖为底物从头合成顺，顺-粘康酸的基因工程菌及其应用

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009063079A1 (fr) 2007-11-14 2009-05-22 Deinove Utilisation de bactéries pour la production de bioénergie
WO2009113853A2 (fr) * 2008-03-11 2009-09-17 Dsm Ip Assets B.V. Synthèse d’un adipate (ester ou thioester)
WO2010081899A1 (fr) 2009-01-19 2010-07-22 Deinove Méthodes d'isolement de bactéries
WO2010094665A2 (fr) 2009-02-17 2010-08-26 Deinove Compositions et procédés de dégradation de biomasse ligneuse
WO2010130812A1 (fr) 2009-05-14 2010-11-18 Deinove Bactéries métaboliques haute performance
WO2011085311A1 (fr) * 2010-01-08 2011-07-14 Draths Corporation Procédés pour produire des isomères d'acide muconique et de sels de muconate
WO2012106257A1 (fr) * 2011-01-31 2012-08-09 Los Alamos National Security, Llc Production de composés intéressants au plan industriel dans des organismes prokaryotes
US8367858B2 (en) 2009-06-16 2013-02-05 Amyris, Inc. Terephthalic and trimellitic based acids and carboxylate derivatives thereof
US8426639B2 (en) 2009-06-16 2013-04-23 Amyris, Inc. Preparation of trans, trans muconic acid and trans, trans muconates
WO2013116244A1 (fr) 2012-01-30 2013-08-08 Myriant Corporation Obtention d'acide muconique à partir de micro-organismes génétiquement modifiés

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009063079A1 (fr) 2007-11-14 2009-05-22 Deinove Utilisation de bactéries pour la production de bioénergie
WO2009113853A2 (fr) * 2008-03-11 2009-09-17 Dsm Ip Assets B.V. Synthèse d’un adipate (ester ou thioester)
WO2010081899A1 (fr) 2009-01-19 2010-07-22 Deinove Méthodes d'isolement de bactéries
WO2010094665A2 (fr) 2009-02-17 2010-08-26 Deinove Compositions et procédés de dégradation de biomasse ligneuse
WO2010130812A1 (fr) 2009-05-14 2010-11-18 Deinove Bactéries métaboliques haute performance
US8367858B2 (en) 2009-06-16 2013-02-05 Amyris, Inc. Terephthalic and trimellitic based acids and carboxylate derivatives thereof
US8426639B2 (en) 2009-06-16 2013-04-23 Amyris, Inc. Preparation of trans, trans muconic acid and trans, trans muconates
WO2011085311A1 (fr) * 2010-01-08 2011-07-14 Draths Corporation Procédés pour produire des isomères d'acide muconique et de sels de muconate
US8809583B2 (en) 2010-01-08 2014-08-19 Amyris, Inc. Methods for producing isomers of muconic acid and muconate salts
WO2012106257A1 (fr) * 2011-01-31 2012-08-09 Los Alamos National Security, Llc Production de composés intéressants au plan industriel dans des organismes prokaryotes
WO2013116244A1 (fr) 2012-01-30 2013-08-08 Myriant Corporation Obtention d'acide muconique à partir de micro-organismes génétiquement modifiés

Non-Patent Citations (32)

* Cited by examiner, † Cited by third party
Title
BEN-AROYA ET AL., METHODS ENZYMOL., vol. 470, 2010, pages 181 - 204
BENTLEY, CRIT REV BIOCHEM MOL BIOL, vol. 25, no. 5, 1990, pages 307 - 384
CAO, B.; GENG, A.;; LOH, K.C., APPL. MICROBIOL. BIOTECHNOL., vol. 81, 2008, pages 99 - 107
CAPOSIO ET AL., RES MICROBIOL., vol. 153, no. 2, March 2002 (2002-03-01), pages 69 - 74
CURRAN ET AL., METAB ENG, vol. 15, January 2013 (2013-01-01), pages 55 - 66
DRATHS ET AL., JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 114, 1992, pages 3956 - 3962
DRATHS, K.; FROST, J.;, J. AM. CHEM. SOC., vol. 117, 1995, pages 2395 - 2400
ELSEMORE, OURNAL OF BACTERIOLOGY, vol. 177, no. 20, 1995, pages 5971 - 5978
FOX ET AL., BIOCHEMISTRY, vol. 47, no. 47, 25 November 2008 (2008-11-25), pages 12251 - 12253
GRANT DJ; PATEL JC, ANTONIE LEEUWENHOEK, vol. 35, 1969, pages 325 - 343
HAN ET AL., SCI REP., vol. 5, 26 August 2015 (2015-08-26), pages 13435
HANSEN ET AL., APPL ENVIRON MICROBIOL., vol. 75, no. 9, May 2009 (2009-05-01), pages 2765 - 2774
HE, Z.; WIEGEL, J., J. BACTERIOL., vol. 178, 1996, pages 3539 - 3543
HU ET AL., J BASIC MICROBIOL, vol. 43, 2003, pages 399 - 406
JIMENEZ ET AL., ENVIRONMENTAL MICROBIOLOGY, vol. 4, no. 12, 2002, pages 824 - 841
LAMB ET AL., BIOCHEM. J., vol. 284, pages 181 - 187
LECOINTE ET AL., MOL MICROBIOL, vol. 53, 2004, pages 1721 - 1730
LUTKE-EVERSLOH, T.; STEPHANOPOULOS, G., APPL MICROBIOL BIOTECHNOL, vol. 75, 2007, pages 103 - 110
MAKAROVA ET AL., MICROBIOL MOL BIOL REV, vol. 65, 2001, pages 44 - 79
MEIMA ET AL., J BACTERIOL, vol. 183, 2001, pages 3169 - 3175
MOLINA ET AL., GENOME ANNOUNC, vol. 1, no. 4, 18 July 2013 (2013-07-18), pages E00496 - E00513
MOTTERN, J. AM. CHEM. SOC., vol. 56, no. 10, 1934, pages 2107 - 2108
MUSSER, M. T.: "ULLMANN'S Encyclopedia of Industrial Chemistry", 2005, WILEY-VCH, article "Adipic acid."
NEIDLE, E.L.; ORNSTON, L.N., J. BACTERIOL., vol. 168, 1986, pages 815 - 820
NIU ET AL.: "Benzene-free synthesis of adipic acid", BIOTECHNOL. PROG., vol. 18, 2002, pages 201 - 211, XP002568326, DOI: doi:10.1021/BP010179X
NIU WEI ET AL: "Benzene-free synthesis of adipic acid", BIOTECHNOLOGY PROGRESS, AMERICAN INSTITUTE OF CHEMICAL ENGINEERS, US, vol. 18, no. 2, 1 March 2002 (2002-03-01), pages 201 - 211, XP002568326, ISSN: 8756-7938, [retrieved on 20020212], DOI: 10.1021/BP010179X *
RUTLEDGE ET AL., GENE, vol. 32, no. 3, December 1984 (1984-12-01), pages 275 - 287
SHINAGAWA ET AL., BIOSCI BIOTECHNOL BIOCHEM., vol. 74, no. 5, 2010, pages 1084 - 1088
SHUMILIN ET AL., STRUCTURE, vol. 7, 1999, pages 865 - 875
TSAI, S.C.; LI, Y.K., ARCH. MICROBIOL., vol. 187, 2007, pages 199 - 206
WEBER ET AL., APPL.ENVIRON. MICROBIOL., vol. 78, 2012, pages 8421 - 8430
YOSHIDA ET AL., BIOTECHNOL. LETT., vol. 32, 2010, pages 701 - 705

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3789481A4 (fr) * 2018-05-01 2022-02-16 Research Institute Of Innovative Technology For The Earth Transformant d'une bactérie corynéforme et procédé de production de composé utile l'utilisant
US11359217B2 (en) 2018-05-01 2022-06-14 Research Institute Of Innovative Technology For The Earth Transformant of coryneform bacterium and production method for useful compound using same
WO2022177569A1 (fr) * 2021-02-18 2022-08-25 Zymergen Inc. Voies de biosynthèse modifiées pour la production d'acide 3,4-amino-4-hydroxybenzoïque par fermentation
CN113151053A (zh) * 2021-03-15 2021-07-23 广东工业大学 一株佛里德兰德氏杆菌gdutan10及其应用
CN113151053B (zh) * 2021-03-15 2022-11-04 广东工业大学 一株佛里德兰德氏杆菌gdutan10及其应用
CN117004547A (zh) * 2023-09-27 2023-11-07 北京化工大学 一种以葡萄糖为底物从头合成顺，顺-粘康酸的基因工程菌及其应用
CN117004547B (zh) * 2023-09-27 2023-12-22 北京化工大学 一种以葡萄糖为底物从头合成顺，顺-粘康酸的基因工程菌及其应用

Similar Documents

Publication Publication Date Title
US8852899B2 (en) Methods of making nylon intermediates from glycerol
US11685938B2 (en) Muconic acid production from genetically engineered microorganisms
JP2019195330A (ja) 遺伝子操作された微生物からのムコン酸の生成
CA2985481C (fr) Micro-organismes genetiquement modifies de production de produits derives de chorismate
US20230203542A1 (en) Microbial Production of 2-Phenylethanol from Renewable Substrates
US20100248233A1 (en) Acetyl-coa producing enzymes in yeast
US20100081183A1 (en) Enhanced dihydroxy-acid dehydratase activity in lactic acid bacteria
JP2010207094A (ja) プロトカテク酸の製造法
CN104718282A (zh) 用于生产脂肪酸和脂肪酸衍生产物的微生物及方法
EP2993228A2 (fr) Production d'esters d'acides gras
WO2016207403A1 (fr) Procédé de production d'acide muconique
US9719113B2 (en) Microbial production of muconic acid and salicylic acid
KR20150100666A (ko) 재조합 세포, 및 이소프렌의 생산 방법
US9920343B2 (en) Method for producing aniline derivative by fermentation from carbon source
CN106460019A (zh) 生产尼龙的方法
WO2021028993A1 (fr) Bactérie du genre hydrogenophilus transgénique produisant de l'acide lactique
CN113840909A (zh) 从气态底物发酵生产2-苯乙醇
WO2014102280A1 (fr) Organisme microbien utilisé pour produire du téréphtalate à partir de biomasse
DK2173881T3 (en) YET ACETYL-COA-PRODUCING ENZYMS

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16734244

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16734244

Country of ref document: EP

Kind code of ref document: A1