FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

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.

In the past few years, efforts were made to find alternative routes for adipic acid production from renewable carbon sources. Among the different strategies, 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). The first synthetic route using glucose as carbon source has been established in E. 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). However, even optimized, this genetically modified bacterium does not allow a cost-competitive industrial production process. Alternatively, biotechnological production of ccMA has also been implemented in Saccharomyces cerevisiae by introducing a pathway similar to that previously used in E. coli. However, the production titers if ccMA obtained with S. cerevisiae are much lower than those obtained with E. coli (Weber et al., 2012).

Consequently, there is still a strong need for a much improved process achieving industrially relevant productivity of muconic acid from renewable carbon sources, and in particular from lignocellulosic biomasses.

SUMMARY OF THE INVENTION

Based on their solid knowledge of Deinococcus metabolism and genetics, the inventors demonstrated that Deinococcus bacteria can be genetically modified to produce substantial amounts of catechol and muconic acid.

Accordingly, in a first aspect, 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.

In particular, the 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.

Preferably, 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. More preferably, 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. Preferably, 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.

In particular, 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. Preferably, 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.

Alternatively, 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. In particular, the endogenous gene encoding shikimate dehydrogenase (AroE) may be inactivated. Alternatively, the shikimate dehydrogenase activity of the bacterium may be reduced.

The endogenous biosynthetic pathway converting protocatechuate to oxoadipate may be blocked or reduced. In particular, 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.

In particular, 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.

The recombinant bacterium of the invention may further express a heterologous polypeptide exhibiting catechol-O-methyltransferase activity. Preferably, 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.

In a second aspect, 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.

In another aspect, 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. In a further aspect, 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.

In the methods of the invention, 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.

In the methods of the invention, 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. In another aspect, 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. In another aspect, 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. In a further aspect, 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. BRIEF DESCRIPTION OF THE DRAWINGS

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). DETAILED DESCRIPTION OF THE INVENTION

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).

Definitions

In the context of the invention, 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. aquaticus, D. aquatilis, D. aquiradiocola, D. caeni, D. cellulosilyticus, D. citri, D. claudionis, D. daejeonensis , D. depolymerans, D. desertii, D. enclensis, D. ficus, D. frigens, D. geothermalis, D. gobiensis, D. grandis, D. guangriensis, D. guilhemensis, D. hohokamensis, D. hopiensis, D. humi , D. indicus, D. maricopensis, D. marmoris, D. misasensis, D. murrayi, D. navajonensis, D. papagonensis, D. peraridiUtoris, D. phoenicis, D. pimensis, D. piscis, D. proteolyticus, D. puniceus, D. radiodurans, D. radiomollis, D. radiophilus, D. radiopugnans, D. radioresistens, D. radiotolerans, D. reticulitermitis, D. roseus, D. sahariens, D. saxicola, D. soli, D. sonorensis, D. swuensis, D. wulumuqiensis, D. xinjiangensis and D. yavapaiensis bacterium, or any combinations thereof. Preferably, 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. Examples of 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. frigens, D. marmoris, D. ficus, D. apachensis, D. aquatilis, D. pimensis, D. peraridiUtoris, D. puniceus, D.phoenicis, D. swuensis and D. actinosclerus. Examples of thermophilic Deinococcus bacteria 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. The term "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. Preferably, the control sequences are suitable for Deinococcus host cells.

As used herein, the term "expression vector" means a DNA or RNA molecule that comprises an expression cassette. Preferably, the expression vector is a linear or circular double stranded DNA molecule.

The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to a coding sequence, in such a way that the control sequence directs expression of the coding sequence. The term "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.

As used herein, 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...

As used herein, the term "sequence identity" or "identity" refers to the number (%) of matches (identical amino acid residues) in positions from an alignment of two polypeptide sequences. The sequence identity is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. In particular, 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. Smith and Waterman algorithm (Smith and Waterman, 1981) or Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)). 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. For purposes herein, % amino acid sequence identity values refers to values generated using the pair wise sequence alignment program EMBOSS Needle that creates an optimal global alignment of two sequences using the Needleman-Wunsch algorithm, wherein all search parameters are set to default values, i.e. Scoring matrix = BLOSUM62, Gap open = 10, Gap extend = 0.5, End gap penalty = false, End gap open = 10 and End gap extend = 0.5

The term "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).

The term "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).

The term "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).

The term "AroE" 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).

The terms "AroK" 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).

The term "AroA" 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).

The term "AroC" refers to the enzyme chorismate synthase (also named 5- enolpyruvylshikimate-3-phosphate phospholyase, EC 4.2.3.5) encoded by aroC gene, that converts EPSP to chorismate (CHA).

The terms "AroG", "AroF" and AroH" refer 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).

The term "AroB" refers to the enzyme 3-dehydroquinate synthase (EC 4.2.3.4) encoded by aroB gene, that converts DAHP to 3-dehydroquinate (DHQ).

The term "AroD" or "AroQ" refers to the enzyme 3-dehydroquinate dehydratase (EC 4.2.1.10) encoded by aroD gene, that converts DHQ to DHS. The term "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 term "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 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 term "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 term "PpsA" refers to a Phosphoenolpyruvate (PEP) synthase (EC 2.7.9.2) encoded by the ppsA gene, that converts pyruvate to PEP.

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.

Heterologous catechol and muconic acid biosynthetic pathway

In a first aspect, 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.

As used herein, the term "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. In particular, the term "muconic acid biosynthetic pathway" refers to a biochemical pathway allowing the production of muconic acid, in particular czVds-muconic acid. In preferred embodiments, 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. All or part of the biochemical pathway converting DHS to ccMA may be heterologous. Thus, 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).

The term "catechol biosynthetic pathway" refers to a biochemical pathway allowing the production of catechol. In preferred embodiments, 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. Thus, 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). In a preferred embodiment, 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 (AroZ) may be any known DHS dehydratase, in particular selected from known fungal or bacterial DHS dehydratases. Preferably, 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. 2002) and Pseudomonas putida H8234 (Molina et al., 2013; SEQ ID NO: 6), Klebsiella pneumonia (Draths et al., 1995; Niu et al, 2002), Acinetobacter calcoaceticus (quiC, Elsemore et al., 1995), Acinetobacter sp. 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). More preferably, 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. Even more preferably, 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). In a particular embodiment, 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). In particular, 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.

In a particular embodiment, 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. Preferably, 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. More preferably, 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 (AroY) may be any known PCA decarboxylase, in particular selected from known fungal or bacterial PCA decarboxylases. Preferably, 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. 2012; three subunits: SEQ ID NO: 10, 12 and 14), Enterobacter cloacae (Yoshida et al., 2010; SEQ ID NO: 23), Sedimentibacter hydroxybenzoicus (He et al., 1996; Weber et al., 2012; three subunits: SEQ ID NO: 17, 19 and 21) and Aerobacter aerogenes (Grant and Patel, 1969; Curran et al., 2013). More preferably, 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. In particular, 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.

In a particular embodiment, 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

15 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, 14, 17, 19, 21 or 23. Preferably, 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:

20 10, 12 or 14. More preferably, the AroY enzyme is a PCA decarboxylase comprising three subunits of SEQ ID NO: 10, 12 and 14.

In embodiments wherein 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, or 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 (CatA) may be any known catechol 1,2-dioxygenase, in particular selected from known fungal or bacterial catechol 1,2-dioxygenases. Preferably, 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: A0A0A6MK79 and P86029; Tsai and Li, 2007; SEQ ID NO: 29), Bulkholderia mallei (Uniprot accession number: Q62E52 ; SEQ ID NO: 33), Bulkholderia xenovorans (Weber et al., 2012; SEQ ID NO: 31), Pseudomonas putida (Cao et al., 2008; SEQ ID NO: 36; Genbank accession number: ABS86779.1), Stenotrophomonas maltophilia KB2 (SEQ ID NO: 34; Genbank accession number: ABS86780.1), Cupriavidus metallidurans CH34 previously known as Ralstonia eutropha and Alcaligenes eutrophus (SEQ ID NO: 35 and 40; Genbank accession number: YP_587012.1 and ABF08660), Burkholderia sp. 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). More preferably, 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), Bulkholderia xenovorans (Weber et al., 2012; SEQ ID NO: 31) and Pseudomonas putida (Cao et al., 2008). More preferably, 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). Even more preferably, 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). In a particular embodiment, the polypeptide exhibiting catechol 1,2-dioxygenase activity is from Acinetobacter calcoaceticus. In particular, 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. Preferably, 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. More preferably, 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. Even more preferably, 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. In a particular embodiment, 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. In a preferred embodiment, 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. In an embodiment, 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). As used herein, the term "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. In the present document, the following terminology is used to designate a substitution: G72A denotes that amino acid residue at position 72 of SEQ ID No. 41 (glycine, G) is changed to an alanine (A). In a particular embodiment, 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).

In an embodiment, the recombinant Deinococcus bacterium comprises a heterologous aroZ gene, a heterologous aroY gene or a heterologous catA gene. In another embodiment, 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. In a preferred embodiment, the recombinant Deinococcus bacterium comprises a heterologous aroZ gene, a heterologous aroY gene and a heterologous catA gene. In a particular embodiment, 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). In another particular embodiment, 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). In another particular embodiment, 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). In a preferred embodiment, 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.

In a particular embodiment, 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

- one or several heterologous nucleic acid sequences encoding a PCA decarboxylase selected from the group consisting of a PCA decarboxylase of SEQ ID

5 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 one or several heterologous nucleic acid sequences encoding the PCA decarboxylase of SEQ ID NO: 10, 12 and 14; and

- optionally, 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: 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, preferably a heterologous nucleic acid sequence encoding the catechol 1,2-dioxygenase of SEQ ID NO: 25, 29 or 31, preferably 15 of SEQ ID NO: 25.

In another embodiment, 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

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; and

- one or several heterologous nucleic acid sequences encoding a 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,

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

- optionally, a heterologous nucleic acid sequence encoding a catechol 1,2- dioxygenase selected from the group consisting of catechol 1,2-dioxygenases of SEQ ID

30 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 or 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. In a further embodiment, 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

- one or several heterologous nucleic acid sequences encoding a 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

- optionally, 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. In the literature, the catX gene is also called "orfl" (Neidle and Ornston, 1986). Preferably, 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. In particular, the recombinant Deinococcus bacterium of the invention may comprise an expression cassette comprising aroZ and aroY genes. Preferably, 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. 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.

Preferably, 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. Preferably, an expression cassette comprising said gene(s) is integrated into the genome. More preferably, an expression cassette comprising aroZ, aroY and catA genes is integrated into the genome. Optionally, 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. In a particular embodiment, 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. In another particular embodiment, 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. Alternatively, or in addition, 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). In particular, 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. Examples of suitable promoters 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. Preferably, 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).

Optionally, the expression cassette may also comprise a selectable marker that permits easy selection of recombinant bacteria. Typically, the selectable marker is a gene encoding antibiotic resistance or conferring autotrophy.

In a particular embodiment, 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. Optionally, the bacterium may further comprise another expression cassette comprising catA gene operably linked to a strong constitutive promoter. In another particular embodiment, the recombinant Deinococcus bacterium of the invention comprises an expression cassette comprising aroZ, aroY and catA genes operably linked to a strong constitutive promoter.

The expression level of each gene may be also controlled by modulating the strength of the ribosome binding site (RBS) installed in front of the gene.

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.

Inactivation of the endogenous biosynthetic pathway converting DHS to chorismate

In some embodiments, 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.

The term "AroE" refers to the enzyme shikimate dehydrogenase (EC 1.1.1.25) encoded by aroE gene, that converts DHS to shikimate (SHK). Examples of 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. maricopensis (Uniprot accession number: E8UA95) and D. proteolytics (Uniprot accession number: F0RN73).The gene encoding AroE 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 AroE enzyme.

The term "AroK" refers to the enzyme shikimate kinase (EC 2.7.1.71) encoded by aroK gene. This enzyme converts SHK to 3-phosphoshikimate (S3P). Examples of 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. phoenicis (Uniprot accession number: A0A016QMJ7) and D. swuensis (Uniprot accession number: AO AO A7 KEPI). 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.

The term "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. gobiensis (Uniprot accession number: H8GWA6), D. phoenicis (Uniprot accession number: A0A016QR42), D. swuensis (Uniprot accession number: A0A0A7KGT9), D. peraridilitoris (Uniprot accession number: L0A580), D. maricopensis (Uniprot accession number: E8U670) and D. proteolyticus (Uniprot accession number: F0RN16). 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.

The term "AroC" refers to the enzyme chorismate synthase (EC 4.2.3.5) encoded by aroC gene, that converts EPSP to chorismate (CHA). Examples of 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. peraridilitoris (Uniprot accession number: L0A5W6), D. maricopensis (Uniprot accession number : E8U7Z9), D. proteolyticus (Uniprot accession number: F0RLE1) and D. swuensis (Uniprot accession number: A0A0A7KJA3). 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.

In an embodiment, 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. In preferred embodiments, all or part of the targeted gene is deleted, for example by gene replacement.

In a particular embodiment, the endogenous biosynthetic pathway converting DHS to chorismate (CHA) is blocked by inactivation of the endogenous aroE gene. In a preferred embodiment, all or part of the aroE gene is deleted. In a particular embodiment, 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.

The inactivation of endogenous biosynthetic pathway converting DHS to chorismate (CHA) 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). As a result, 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. Thus, in another embodiment, one or several enzymatic activities of the pathway converting DHS to chorismate are reduced. Preferably, 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. In an embodiment, 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. 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. Thus the use of a leaky mutant form of aroE would eliminate the dependence on exogenous aromatic amino acids, while still diverting the flow of carbon to ccMA. 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. Alternatively, 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. In an alternative embodiment, 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. In particular, 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.

Inactivation of the endogenous biosynthetic pathway converting protocatechuate to oxoadipate In some embodiments, 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 (EC 1.13.11.3) catalyzes the reaction converting 3,4-dihydroxybenzoate (PCA) to 3-carboxy-cis,cis- muconate. In Deinococcus bacteria, 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; β-subunit: Uniprot accession number: Q1J3Z6) and D. deserti (a-subunit: Uniprot accession number: C1D2D6; β-subunit: Uniprot accession number: C1D2D7). 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 (EC 5.5.1.2) catalyzes the reaction converting 3-carboxy-cis,cis-muconate to γ- 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 (EC 5.5.1.2) catalyzes the reactions converting γ-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. In an embodiment, 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.

In a particular embodiment, 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.

Enhancement of the flow of carbon through the catechol or muconic acid biosynthetic pathway

To enhance the production of ccMA or catechol, 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.

In the recombinant Deinococcus bacterium of the invention, 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, may be increased compared to the non modified bacterium. Preferably, 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. Thus, in an embodiment, at least one gene selected from the group consisting of aroG, aroF, aroH, aroB and aroD genes, is overexpressed. Preferably, at least two, three, four, five, six or seven of these genes are overexpressed. More preferably, all these genes are overexpressed.

To increase the expression of a gene, 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.

In particular, 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). Preferably, an expression cassette comprising the gene is integrated into the genome.

Alternatively, 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. For instance, 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. In a particular embodiment, 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. In a particular embodiment, 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

(EC 4.2.1.10) encoded by aroD (also named AroQ) gene, that converts DHQ to DHS. The aroD gene may be any gene encoding a 3-dehydroquinate dehydratase, preferably a fungal or bacterial gene, more preferably a bacterial gene. In a particular embodiment, 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.

In a preferred embodiment, 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. In a particular embodiment, in the recombinant bacterium of the invention, at least one of the enzymes involved in the conversion of PEP and E4P to DHS is an improved enzyme.

In a preferred embodiment, 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. Preferably the recombinant bacterium comprises an AroF, AroG and/or AroH feedback resistant mutant. Indeed, these three proteins are subjected to feedback inhibition by one or more metabolites of shikimic acid pathway responsible for aromatic amino acid biosynthesis. In particular, 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. In a more preferred embodiment, AroF, AroG and AroH feedback resistant mutants are overexpressed in the recombinant bacterium of the invention.

In a particular embodiment, the recombinant Deinococcus bacterium of the invention expresses a feedback-resistant DAHP synthase. Preferably, the feedback- resistant DAHP synthase is a variant of a Deinococcus DAHP synthase. More preferably, 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. In a more particular embodiment, 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).

In some embodiments, in the recombinant Deinococcus bacterium of the invention, 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.

In an embodiment, at least one gene selected from the group consisting of rpe, rpi, talB, tktA or ppsA, is overexpressed. Preferably, 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.

The term "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. In a particular embodiment, 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.

The term "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. In a particular embodiment, 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. In a particular embodiment, the talB gene is from a Deinococcus bacterium. Examples of "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. maricopensis (Uniprot accession number: E8U8S1), D. proteolyticus (Uniprot accession number: F0RMW2), D. phoenicis (Uniprot accession number: A0A016QNQ5) and D. swuensis (Uniprot accession number: A0A0A7KDB4). 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. Any polypeptide, preferably from a Deinococcus bacterium, having at least 80%, preferably 90%, more preferably 95% sequence identity to any of the above- identified TalB enzymes may be used. The term "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. In a particular embodiment, the tktA gene is from a Deinococcus bacterium. Examples of "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:

9ZZI7 and L0A0X5), D. maricopensis (Uniprot accession number: E8U550) and D. proteolyticus (Uniprot accession number: F0RKJ3). The gene encoding 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.

The term "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. In a particular embodiment, 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. gobiensis (Uniprot accession number: H8H237), D. deserti (Uniprot accession number: C1CWW0), D. swuensis (Uniprot accession number: A0A0A7KM87), D. phoenicis (Uniprot accession number: A0A016QMU4) and D. proteolyticus (Uniprot accession number: F0RNK8). 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. Optionally, in some embodiments, pyruvate kinase I and/or II (EC 2.7.1.40) that use PEP as substrat, may be inactivated or reduced in the recombinant bacterium of the invention. The pyruvate kinase activity may be blocked or reduced as explained above for the enzymes of the chorismate pathway. In a particular embodiment, 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.

Heterologous catechol-O-methyltransferases

In a further aspect, 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). Preferably, 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). Preferably, 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.

Due to the temperature sensitivity of gaiacol, 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. In particular, 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.

Preferably, 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.

Preferably, 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. Preferably, an expression cassette comprising said gene(s) is integrated into the genome. More preferably, an expression cassette comprising aroZ, aroY and COMT genes is integrated into the genome. Optionally, 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. In a particular embodiment, 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. In another particular embodiment, 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. Alternatively, or in addition, 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. Optionally, the endogenous biosynthetic pathway converting protocatechuate to oxoadipate and/or the endogenous biosynthetic pathway converting DHS to chorismate may be inactivated.

Cell extract

In another aspect, the present invention also relates to a cell extract of the recombinant Deinococcus bacterium of the invention. As used herein, 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. Methods of production

In a further aspect, the present invention relates to a use of a recombinant Deinococcus bacterium of the invention for producing ccMA. In particular, 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

(i) culturing a recombinant Deinococcus bacterium according to the invention comprising a heterologous catechol biosynthetic pathway under conditions suitable to produce catechol, and optionally (ii) recovering said catechol.

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. Optionally, 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. Alternatively, 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. Optionally, 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. In particular, 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.

Preferably, ccMA, catechol or gaiacol is produced from renewable, biologically derived carbon sources. In particular, when the recombinant Deinococcus bacterium exhibits cellulolytic and/or xylanolytic activity, more complex carbon sources can be used such as cellulosic biomass. As used herein, the term "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.

Preferably, if 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.

In an embodiment, 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.

In another embodiment, 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.

In a further aspect, 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. Optionally, 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.

In a further aspect, 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. In a further aspect, 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. By "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. Depending on the cells used, the method may be conducted under aerobiosis, anaerobiosis or microaerobiosis.

The following items are herein described:

1. A recombinant Deinococcus bacterium expressing a heterologous polypeptide exhibiting 3-dehydroshikimate dehydratase activity and a heterologous polypeptide exhibiting protocatechuate decarboxylase activity. 2. The recombinant bacterium of item 1, wherein said bacterium further expresses a heterologous polypeptide exhibiting catechol 1 ,2-dioxygenase activity.

3. The recombinant bacterium according to item 1 or 2, wherein said 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. The recombinant bacterium according to any of items 1 to 3, wherein said 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 .

5. The recombinant bacterium according to any of items 1 to 4, wherein said 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.

6. The recombinant bacterium according to any of items 1 to 5, wherein said 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.

7. The recombinant bacterium according to any of items 1 to 6, wherein said 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. 8. The recombinant bacterium according to any of items 1 to 7, wherein said 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.

9. The recombinant bacterium according to any of items 1 to 8, wherein said polypeptide exhibiting 3-dehydroshikimate dehydratase activity is a 3-dehydroshikimate dehydratase of SEQ ID NO: 4.

10. The recombinant bacterium according to any of items 1 to 6, wherein said 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.

11. The recombinant bacterium according to any of items 1 to 6, wherein said 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.

12. The recombinant bacterium according to any of items 1 to 6, wherein said polypeptide exhibiting 3-dehydroshikimate dehydratase activity is a 3-dehydroshikimate dehydratase of SEQ ID NO: 8.

13. The recombinant bacterium according to any of items 1 to 5, wherein said 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.

14. The recombinant bacterium according to any of items 1 to 5, wherein said 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.

15. The recombinant bacterium according to any of items 1 to 5, wherein said polypeptide exhibiting 3-dehydroshikimate dehydratase activity is a 3-dehydroshikimate dehydratase of SEQ ID NO: 2.

16. The recombinant bacterium according to any of items 1 to 5, wherein said 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.

17. The recombinant bacterium according to any of items 1 to 5, wherein said 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.

18. The recombinant bacterium according to any of items 1 to 5, wherein said polypeptide exhibiting 3-dehydroshikimate dehydratase activity is a 3-dehydroshikimate dehydratase of SEQ ID NO: 6.

19. The recombinant bacterium according to any of items 1 to 18, wherein said 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.

20. The recombinant bacterium according to any of items 1 to 19, wherein said 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. 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 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.

5 22. 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 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.

10 23. The recombinant bacterium according to any of items 1 to 20, wherein said polypeptide exhibiting protocatechuate decarboxylase activity is a PCA decarboxylase comprising polypeptides of SEQ ID NO: 10, 12 and 14.

24. The recombinant bacterium according to any of items 1 to 20, wherein said polypeptide exhibiting protocatechuate decarboxylase activity is selected from the group

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.

25. 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 comprising polypeptides of SEQ ID NO: 17, 19 and

20 21, and 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.

26. The recombinant bacterium according to any of items 1 to 20, wherein said polypeptide exhibiting protocatechuate decarboxylase activity is a PCA decarboxylase comprising polypeptides of SEQ ID NO: 17, 19 and 21.

25 27. 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 60 % identity to SEQ ID NO: 23. 25. 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.

26. The recombinant bacterium according to any of items 1 to 20, wherein said polypeptide exhibiting protocatechuate decarboxylase activity is a PCA decarboxylase of SEQ ID NO: 23.

27. The recombinant bacterium according to any of items 2 to 26, wherein said 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, Pseudomonas putida, Stenotrophomonas maltophilia KB2, Cupriavidus metallidurans CH34, Burkholderia sp. TH2, Rhodococcus opacus, Rhodococcus erythropolis and Acinetobacter sp. ADP1.

28. The recombinant bacterium according to any of items 2 to 27, wherein said 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.

29. The recombinant bacterium according to any of items 2 to 27, wherein said 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.

30. 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-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.

31. The recombinant bacterium according to any of items 2 to 30, wherein said 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.

32. The recombinant bacterium according to any of items 2 to 31, wherein said 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.

33. The recombinant bacterium according to any of items 2 to 32, wherein said polypeptide exhibiting catechol 1 ,2-dioxygenase activity is a catechol 1 ,2-dioxygenase of SEQ ID NO: 25. 34. 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 60 % identity to SEQ ID NO: 27.

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.

36. The recombinant bacterium according to any of items 2 to 29, wherein said polypeptide exhibiting catechol 1 ,2-dioxygenase activity is a catechol 1 ,2-dioxygenase of

SEQ ID NO: 27.

37. 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: 29 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 60 % identity to SEQ ID NO: 29.

38. 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: 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.

39. The recombinant bacterium according to any of items 2 to 29, wherein said polypeptide exhibiting catechol 1 ,2-dioxygenase activity is a catechol 1 ,2-dioxygenase of SEQ ID NO: 29.

40. 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: 31 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 60 % identity to SEQ ID NO: 31. 41. 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: 31 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 80 % identity to SEQ ID NO: 31, preferably at least 90 % identity to SEQ ID NO: 31. 42. The recombinant bacterium according to any of items 2 to 29, wherein said polypeptide exhibiting catechol 1 ,2-dioxygenase activity is a catechol 1 ,2-dioxygenase of SEQ ID NO: 31.

43. 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: 33 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 60 % identity to SEQ ID NO: 33.

44. 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: 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.

48. The recombinant bacterium according to any of items 2 to 29, wherein said polypeptide exhibiting catechol 1 ,2-dioxygenase activity is a catechol 1 ,2-dioxygenase of SEQ ID NO: 33.

49. 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: 34 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 60 % identity to SEQ ID NO: 34. 50. 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: 34 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 80 % identity to SEQ ID NO: 34, preferably at least 90 % identity to SEQ ID NO: 34. 51. The recombinant bacterium according to any of items 2 to 29, wherein said polypeptide exhibiting catechol 1 ,2-dioxygenase activity is a catechol 1 ,2-dioxygenase of SEQ ID NO: 34.

52. 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: 35 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 60 % identity to SEQ ID NO: 35.

53. 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: 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.

54. The recombinant bacterium according to any of items 2 to 29, wherein said polypeptide exhibiting catechol 1 ,2-dioxygenase activity is a catechol 1 ,2-dioxygenase of SEQ ID NO: 35. 55. 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: 36 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 60 % identity to SEQ ID NO: 36. 56. 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: 36 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 80 % identity to SEQ ID NO: 36, preferably at least 90 % identity to SEQ ID NO: 36. 57. The recombinant bacterium according to any of items 2 to 29, wherein said polypeptide exhibiting catechol 1 ,2-dioxygenase activity is a catechol 1 ,2-dioxygenase of SEQ ID NO: 36.

58. 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: 37 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 60 % identity to SEQ ID NO: 37.

59. 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: 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.

60. The recombinant bacterium according to any of items 2 to 29, wherein said polypeptide exhibiting catechol 1 ,2-dioxygenase activity is a catechol 1 ,2-dioxygenase of SEQ ID NO: 37. 61. 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: 38 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 60 % identity to SEQ ID NO: 38. 62. 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: 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.

63. The recombinant bacterium according to any of items 2 to 29, wherein said polypeptide exhibiting catechol 1 ,2-dioxygenase activity is a catechol 1 ,2-dioxygenase of SEQ ID NO: 38.

64. 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: 39 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 60 % identity to SEQ ID NO: 39.

65. 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: 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.

66. The recombinant bacterium according to any of items 2 to 29, wherein said polypeptide exhibiting catechol 1 ,2-dioxygenase activity is a catechol 1 ,2-dioxygenase of SEQ ID NO: 39.

67. 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: 40 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 60 % identity to SEQ ID NO: 40. 68. 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: 40 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 80 % identity to SEQ ID NO: 40, preferably at least 90 % identity to SEQ ID NO: 40. 69. The recombinant bacterium according to any of items 2 to 29, wherein said polypeptide exhibiting catechol 1 ,2-dioxygenase activity is a catechol 1 ,2-dioxygenase of SEQ ID NO: 40.

70. 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: 41 and polypeptides exhibiting catechol 1,2-dioxygenase activity and having at least 60 % identity to SEQ ID NO: 41.

71. 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: 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.

72. The recombinant bacterium according to any of items 2 to 29, wherein said polypeptide exhibiting catechol 1 ,2-dioxygenase activity is a catechol 1 ,2-dioxygenase of SEQ ID NO: 41.

73. 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: 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.

74. The recombinant bacterium according to any of items 2 to 29, wherein said polypeptide exhibiting catechol 1,2-dioxygenase activity is a variant of the catechol 1,2- dioxygenase of SEQ ID NO: 41 comprising at least one substitution or combination of substitutions selected from G72A, P76A, L73F and L73F+P76A.

75. The recombinant bacterium according to any of items 1 to 74, wherein the endogenous biosynthetic pathway converting 3-dehydroshikimate to chorismate is blocked or reduced. 76. The recombinant bacterium according to item 75, wherein the endogenous gene encoding shikimate dehydrogenase (AroE) is inactivated.

77. The recombinant bacterium according to item 75 or 76, wherein the endogenous shikimate dehydrogenase activity is reduced. 78. The recombinant bacterium according to any of items 1 to 77, wherein the endogenous biosynthetic pathway converting protocatechuate to oxoadipate is blocked or reduced.

79. The recombinant bacterium according to item 78, wherein the endogenous biosynthetic pathway converting protocatechuate to oxoadipate is 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.

80. The recombinant bacterium according to any of items 1 to 79, wherein 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, are overexpressed and/or are feedback inhibition resistant enzymes.

81. The recombinant bacterium according to item 80, wherein the recombinant bacterium expresses a feedback inhibition resistant DAHP synthase.

82. The recombinant bacterium according to item 81, wherein 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.

83. The recombinant bacterium of any of items 1 to 82, further expresses a heterologous polypeptide exhibiting catechol-O-methyltransferase activity.

84. 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. 85. 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. 86. A method of producing catechol comprising culturing a recombinant

Deinococcus bacterium according to any of items 1 to 82 under conditions suitable to produce catechol, and optionally recovering said catechol.

87. 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.

88. The method according to any of items 85 to 87 wherein the culture of the recombinant Deinococcus bacterium under conditions suitable to produce cis-cis muconic acid or catechol is performed at a temperature comprised between 37 and 55°C.

89. 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.

90. 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.

91. 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.

92. 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.

93. The method of item 92, further comprising recovering gaiacol produced in step (i) before conversion. Further aspects and advantages of the present invention will be described in the following examples, which should be regarded as illustrative and not limiting.

EXAMPLES

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.

For the expression of 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).

The genetic constructs of the cassettes for the production of catechol were as follow: 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).

To make seed cultures, individual colonies were picked to inoculate 25 ml of 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 μΜ ; Na2SO4.10H2O 0.276 mM ; MgC12.6H20 0.528 mM ; (NH4)6(Mo7)024.4H20 3 nM ; H3B03 0.4 μΜ ; CoC12.6H20 30 nM ; CuS04.5H20 10 nM ; MnC12 0.25 μΜ ; ZnS04.7H20 10 nM ; D-Biotin 1 μg/L ; Niacin (nicotinic acid) 1 μg/L ; B6 vitamin 1 μg/L ; B l vitamin ; FeC13 20 μΜ ; Sodium Citrate.2H20 20 μΜ ; K2HP04 5,7 mM) containing 1% glucose as the main carbon source, and cultured at 37 °C and 250 rpm overnight.

Seed from log phase of growth was then inoculated into 1 ml of the mineral medium (0.4 g/L of yeast extract; (NH4)2S04 < 100 mM ; NaH2P04.H20 < 10 mM ; KC1 < 10 mM ; Na2S04 < 10 mM ; Acide citrique < 30 mM ; MgC12.6H20 < 10 mM ; CaC12.2H20 < 10 mM ; ZnC12 < 50 mg/L ; FeS04.7H20 <50 mg/L ; MnC12.4H20 <50 mg/L; CuS04 < 50 mg/L; CoC12.6H20 < 50 mg/L ; H3B03 < 5 mg/L ; MES < 200 mM ; (NH4)6Mo7024.4H20 < 0,5 mM ; Glucose < 30 g/L (166 mM)) at an initial optical density at 600 nm (OD600) of 0.4. The cultures were performed in deepwell at 48°C at 250 rpm. At 72h of growth, aliquots of 500 μΐ of culture were taken and filtrated using 0.22 μηι membrane filter MF (Millipore). The filtrate was then injected in HPLC.

Results:

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.

Exemple 2: Muconic acid production by recombinant Deinococcus geothermalis

Materiel & Methods:

Deinococcus geothermalis strains were genetically engineered to produce muconic acid.

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.

For the expression of 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).

The genetic constructs of the cassettes for the production of muconic acid were as follow: quiC of Acinetobacter sp. ADP1 (Ac_quiC; SEQ ID NO: 7) or aroZ of Bacillus thuringiensis (Bt_aroZ; SEQ ID NO: 3) both 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). 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 Acinetobacter calcoaceticus (Ac.cjCatA, SEQ ID NO: 24) under the control of strong constitutive promoter, in an IS sequence (IS66).

To make seed cultures, individual colonies were picked to inoculate 25 ml of 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 μΜ ;

Na2SO4.10H2O 0.276 mM ; MgC12.6H20 0.528 mM ; (NH4)6(Mo7)024.4H20 3 nM ;

H3B03 0.4 μΜ ; CoC12.6H20 30 nM ; CuS04.5H20 10 nM ; MnC12 0.25 μΜ ;

ZnS04.7H20 10 nM ; D-Biotin 1 μg/L ; Niacin (nicotinic acid) 1 μg/L ; B6 vitamin 1 μg/L ; B l vitamin ; FeC13 20 μΜ ; Sodium Citrate.2H20 20 μΜ ; K2HP04 5,7 mM) containing 1% glucose as the main carbon source, and cultured at 37°C and 250 rpm overnight.

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. At 72h of growth, aliquots of 500 μΐ of culture were taken and filtrated using 0.22 μηι membrane filter MF (Millipore). The filtrate was then injected in HPLC.

Results:

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).

Exemple 3: Muconic acid production by recombinant Deinococcus geothermalis

Materiel & Methods:

Deinococcus geothermalis strains were genetically engineered to produce muconic acid.

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. For the expression of 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).

The genetic constructs of the cassettes for the production of muconic acid were as follow: quiC of Acinetobacter sp. ADPl {Ac_quiC; SEQ ID NO: 7) or aroZ of Bacillus thuringiensis (Bt_aroZ; SEQ ID NO: 3) both 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).

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).

To make seed cultures, individual colonies were picked to inoculate 25 ml of 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 μΜ ; Na2SO4.10H2O 0.276 mM ; MgC12.6H20 0.528 mM ; (NH4)6(Mo7)024.4H20 3 nM ; H3B03 0.4 μΜ ; CoC12.6H20 30 nM ; CuS04.5H20 10 nM ; MnC12 0.25 μΜ ; ZnS04.7H20 10 nM ; D-Biotin 1 μg/L ; Niacin (nicotinic acid) 1 μg/L ; B6 vitamin 1 μg/L ; Bl vitamin ; FeC13 20 μΜ ; Sodium Citrate.2H20 20 μΜ ; K2HP04 5,7 mM) containing 1% glucose as the main carbon source, and cultured at 37 °C and 250 rpm overnight.

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. At 72h of growth, aliquots of 500 μΐ of culture were taken and filtrated using 0.22 μιη membrane filter MF (Millipore). The filtrate was then injected in HPLC.

Results:

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.

For the expression of 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).

The genetic construct of the cassette for the production of catechol was as follow: 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).

To make seed cultures, individual colonies were picked to inoculate 25 ml of 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 μΜ ; Na2SO4.10H2O 0.276 mM ; MgC12.6H20 0.528 mM ; (NH4)6(Mo7)024.4H20 3 nM ; H3B03 0.4 μΜ ; CoC12.6H20 30 nM ; CuS04.5H20 10 nM ; MnC12 0.25 μΜ ; ZnS04.7H20 10 nM ; D-Biotin 1 μg/L ; Niacin (nicotinic acid) 1 μg/L ; B6 vitamin 1 μg/L ; B l vitamin ; FeC13 20 μΜ ; Sodium Citrate.2H20 20 μΜ ; K2HP04 5,7 mM) containing 1% glucose as the main carbon source, and cultured at 37 °C and 250 rpm overnight.

Seed from log phase of growth was then inoculated into 1 L of the mineral medium pH 6 (0.4 g/L of yeast extract; (NH4)2S04 < 100 mM ; NaH2P04.H20 < 10 mM ; KCl < 10 mM ; Na2S04 < 10 mM ; Acide citrique < 30 mM ; MgC12.6H20 < 10 mM ; CaC12.2H20 < 10 mM ; ZnC12 < 50 mg/L ; FeS04.7H20 <50 mg/L ; MnC12.4H20 <50 mg/L; CuS04 < 50 mg/L; CoC12.6H20 < 50 mg/L ; H3B03 < 5 mg/L ; MES < 200 mM ; (NH4)6Mo7024.4H20 < 0,5 mM ; Glucose < 30 g/L (166 mM)) at an initial optical density at 600 nm (OD600) of 0.4. The cultures were performed in reactor (fermenter) at 48°C. At 64h of growth, aliquots of 500 μΐ of culture were taken and filtrated using 0.22 μιη membrane filter MF (Millipore). The filtrate was then injected in HPLC.

Results

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.

For the expression of 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). The genetic constructs of the cassettes for the production of muconic acid were as follow: 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).

To make seed cultures, individual colonies were picked to inoculate 25 ml of 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 μΜ ; Na2SO4.10H2O 0.276 mM ; MgC12.6H20 0.528 mM ; (NH4)6(Mo7)024.4H20 3 nM ; H3B03 0.4 μΜ ; CoC12.6H20 30 nM ; CuS04.5H20 10 nM ; MnC12 0.25 μΜ ; ZnS04.7H20 10 nM ; D-Biotin 1 μg/L ; Niacin (nicotinic acid) 1 μg/L ; B6 vitamin 1 μg/L ; Bl vitamin ; FeC13 20 μΜ ; Sodium Citrate.2H20 20 μΜ ; K2HP04 5,7 mM) containing 1% glucose as the main carbon source, and cultured at 37 °C and 250 rpm overnight.

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. At 144h of growth, aliquots of 500 μΐ of culture were taken and filtrated using 0.22 μπι membrane filter MF (Millipore). The filtrate was then injected in HPLC.

Results

The recombinant Deinococcus strain was able to produce up to 175 mg/L of muconic acid in flask at 144 hours of cultivation. REFERENCES

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