EP3799600A1 - Micro-organismes et procédé pour la production d'acide glycolique à partir de pentoses et d'hexoses - Google Patents
Micro-organismes et procédé pour la production d'acide glycolique à partir de pentoses et d'hexosesInfo
- Publication number
- EP3799600A1 EP3799600A1 EP19756423.0A EP19756423A EP3799600A1 EP 3799600 A1 EP3799600 A1 EP 3799600A1 EP 19756423 A EP19756423 A EP 19756423A EP 3799600 A1 EP3799600 A1 EP 3799600A1
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- Prior art keywords
- phosphate
- microorganism
- gene
- activity
- coli
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/42—Hydroxy-carboxylic acids
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
- C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
- C12Y102/01012—Glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) (1.2.1.12)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
- C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
- C12Y102/01021—Glycolaldehyde dehydrogenase (1.2.1.21)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/02—Aldehyde-lyases (4.1.2)
- C12Y401/02013—Fructose-bisphosphate aldolase (4.1.2.13)
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- C12Y—ENZYMES
- C12Y503/00—Intramolecular oxidoreductases (5.3)
- C12Y503/01—Intramolecular oxidoreductases (5.3) interconverting aldoses and ketoses (5.3.1)
- C12Y503/01013—Arabinose-5-phosphate isomerase (5.3.1.13)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/34—Sugars
Definitions
- the present invention belongs to the technical field of bioconversion of a carbon source into at least one metabolite of interest and in particular of glycolic acid.
- the present invention relates to a microorganism whose central carbon metabolism has been modified so as to convert the hexoses and pentoses, originating from a carbon source and, in particular, from plant biomass into glycolic acid.
- the present invention also relates to a process for producing glycolic acid from hexoses and pentoses contained in plant biomass using such a microorganism.
- Glycolic acid also makes it possible to produce polymers such as thermoplastic resins comprising poly (glycolic acid). Such polymers exhibit remarkable gas barrier properties and have the ability to hydrolyze in aqueous environments in a gradual and controllable manner, making these polymers good candidates for packaging materials or absorbable materials useful in the biomedical field. .
- glycolic acid can be obtained from extract of sugar cane, beetroot or grape, its production on an industrial scale comes from the chemical synthesis.
- Several of these syntheses use formaldehyde, which is an irritant and carcinogenic compound, as a starting reagent, which excludes all traces of this substance in preparations benefiting from Marketing Authorization.
- poly (glycolic acid) and polymers containing poly (glycolic acid) could represent a new generation of packaging bio-plastics and bio-absorbable polymers [1] ⁇
- poly (glycolic acid) and polymers containing poly (glycolic acid) could represent a new generation of packaging bio-plastics and bio-absorbable polymers [1] ⁇
- Glycolic acid can be naturally produced in small amounts via the reduction of glyoxylate in bacteria and molds, including yeast [2].
- the first engineering projects were inspired by natural metabolism.
- the maximum theoretical yield that can be achieved by this natural route is 2 moles of glycolic acid (AG) per mole of hexose and 1.66 mole per mole of pentoses (or 0.84 g AG / g sugar).
- Y E maximum energy efficiency
- the yield of the pathway depends on the pathway or metabolic network and is calculated from the stoichiometry of the pathway considered.
- Y p is equal to 2 and we find that Y E equals Y p .
- This equality is not found in all cases.
- the conversion of glucose to acetate results in a yield Y p of 2 but the maximum energy yield Y E is 3.
- the team of Liao et al showed that it was possible to build viable metabolic pathways leading to the production of 3 moles of acetate per mole of glucose [10].
- the inventors set themselves the goal of to propose a microorganism and a process making it possible to produce, in a simple, industrializable manner from plant biomass and in particular from the hexoses and pentoses contained in the latter, glycolic acid with improved yields compared to the processes of l prior art.
- Table 1 presents the theoretical yields of the routes of the prior art for the production of glycolic acid (mol / mol) according to the carbon source used.
- the present invention makes it possible to solve the technical problems of the methods of the prior art as defined above and to achieve the goal which the inventors have set themselves.
- D-arabinose-5-phosphate isomerase an enzyme of the lipopolysaccharide synthesis pathway which converts D-ribulose-5-phosphate into D-arabinose-5-phosphate [11];
- fructose-6-phosphate aldolase (FSA) which catalyzes the cleavage of D-arabinose-5-phosphate into D-glyceraldehyde-3-phosphate and glycolaldehyde; note that this enzyme was initially identified as catalyzing the aldolytic cleavage of fructose-6-phosphate into dihydroxyacetone and glyceraldehyde-3-phosphate, but showed aldolytic activity on D-arabinose-5-phosphate with an affinity 10 times more higher than for fructose-6-phosphate [12] and iii) aldehyde dehydrogenase (AldA) which oxidizes glycolaldehyde to glycolate [13].
- FSA fructose-6-phosphate aldolase
- the first substrate of this new path namely D-ribulose-5-phosphate
- PP pentose phosphates
- the activity of the FSA on D-arabinose-5-phosphate generates 3P (C3) glyceraldehyde which is taken up by oxidative glycolysis.
- C3 3P
- this molecule C3 can give glycolaldehyde but with loss of C0 2 at the level of the reaction catalyzed by pyruvate dehydrogenase.
- the inventors have modified the central carbon metabolism in order to optimize the conservation of carbon by recovering this C3 in the PP path and this, by attenuating and even by inactivating the gapA gene encoding glyceraldehyde-3- phosphate dehydrogenase.
- glyceraldehyde-3-phosphate enters the PP pathway and participates in the synthesis of D-xylulose-5-phosphate, precursor of D-ribulose-5-phosphate.
- the maximum theoretical yield is then 2.5 moles of glycolic acid per mole of pentose.
- the present invention makes it possible to carry out the synthesis of glycolic acid from hexoses and pentoses by a single unnatural path, which makes it possible to make the best use of renewable carbon.
- this approach removes the stoichiometric constraint imposed by the metabolism of f. coli.
- the calculations on the theoretical yields of the unnatural way of assimilation of pentoses and hexoses described in the present invention, for the production of glycolic acid make it possible to foresee a significant improvement of the yields compared to the biosynthesis processes. based on the optimization of natural and / or semi-synthetic routes of the prior art.
- the present invention relates to a recombinant microorganism which has
- micro-organism any organism which exists in the form of a microscopic cell belonging both to the field of prokaryotes and to that of eukaryotes. Therefore, the term “microorganism” includes prokaryotic microalgae, bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as plant cells, eukaryotic microalgae, yeasts and mushrooms. The term also includes cell cultures of any species that can be grown for the production of glycolic acid.
- bacteria which can be used in the context of the present invention, mention may be made of bacteria of the families Burkholderiaceae, Enterobacteriaceae, Brevibacteriaceae, Clostridiaceae, Bacillaceae, Moraxellaceae, Sphingomonadaceae, Lactobacillaceae, Streptomycetaceae, Streptococcaceae, Methylobeaceae.
- bacteria which can be used in the context of the present invention, mention may be made of Brevibacterium flavum, Brevibacterium lactofermentum, Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Corynebacterium efficiens, Zymomonas mobilis, Ralstonia eutropha, Clostridium acetobutylicum and Lactococcus lactis.
- fungi which can be used in the context of the present invention, mention may be made of fungi of the genera Penicillium, Aspergillus, Chrysosporium and Trichoderma.
- mushrooms which can be used in the context of the present invention, mention may be made of Penicillium notatum, Penicillium chrysogenum, Aspergillus niger, Chrysosporium pannorum and Trichoderma reesei.
- the microorganism used in the context of the present invention is a bacterium of the E. coli type or a yeast of the Saccharomyces cerevisiae type.
- recombinant microorganism is meant a microorganism as defined above which is not found in nature and which is genetically different from its equivalent in nature.
- the terms “equivalent in nature”, “unmodified microorganism”, “natural microorganism” and “wild type microorganism” are equivalent and can be used interchangeably.
- the recombinant microorganism is modified by introduction, deletion and / or modification of genetic elements.
- the recombinant microorganism usable in the present invention can be modified to modulate the level of expression of an endogenous gene.
- endogenous gene is meant a gene which was present in the microorganism before any genetic modification of the wild type microorganism.
- Endogenous genes can be overexpressed by introducing additional heterologous sequences or by replacing endogenous regulatory elements, or by introducing one or more additional copies of the gene into a chromosome or onto one or more plasmids. Endogenous genes can also be modified to modulate their expression and / or activity. For example, mutations can be introduced into the coding sequence to modify the gene product or heterologous sequences can be introduced in addition or to replace endogenous regulatory elements. Modulation of an endogenous gene may result in upregulation and / or an increase in the activity of the endogenous gene product or, alternatively, downregulate and / or decrease the activity of the endogenous gene product.
- Another way to modulate the expression of an endogenous gene is to exchange the endogenous promoter of the latter as the wild type promoter, with a stronger or weaker promoter to regulate the expression up or down. of the endogenous gene.
- These promoters can be homologous or heterologous.
- the recombinant microorganism usable in the present invention can also be modified to express an exogenous gene.
- the recombinant microorganism usable in the present invention can be modified to express exogenous genes if these genes are introduced with all the elements allowing their expression in this microorganism.
- Those skilled in the art know different methods of modifying, transforming or transfecting a microorganism with an exogenous gene.
- this method can be a conjugation; electroporation; lipofection; micro injection; bombardment with particles (or biolistics); a biological transformation of a plant using Agrobacterium tumefasciens; transformation by chemical permeabilization; a transformation by the DEAE-dextran method or an introduction via a virus, a virion or a viral particle.
- exogenous gene means a gene which has been introduced into a microorganism, by means well known to those skilled in the art while this gene is not found naturally in the microorganism.
- Exogenous genes can be integrated into one or the chromosome of the microorganism or be expressed in an extra-chromosomal manner by means of plasmids, vectors, cosmids, bacteriophages or viruses such as a baculovirus.
- An exogenous gene can be a homologous gene.
- homologous gene is meant a gene homologous to a gene coding for a reference protein and which codes for a protein homologous to this reference protein.
- protein homologous to a reference protein is meant a protein having a similar function and / or a similar structure as the reference protein. Thus, when the reference protein is an enzyme, a protein homologous to this reference protein catalyzes the same enzymatic reaction.
- a homolog of a gene A can also be a gene encoding a variant of the protein encoded by the gene A. This homolog can be obtained synthetically.
- a protein (or a gene) homologous to a reference protein (or a reference gene) has at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% and / or at least 99% identity respectively with the amino acid sequence of the reference protein (or the nucleotide sequence of the reference gene).
- percentage of identity between two amino acid sequences (or between two nucleotide sequences) is meant, in the context of the present invention, a percentage of identical amino acid (or nucleotide) residues between the two compared sequences, this percentage being obtained after implementation of the best alignment (optimum alignment) between the two sequences.
- Those skilled in the art know different techniques for obtaining such a percentage of identity and involving homology algorithms or computer programs such as the BLAST program.
- the recombinant microorganism according to the invention has one or more increased enzymatic activity (s) compared to the same unmodified microorganism and at least one reduced enzymatic activity compared to the same unmodified microorganism.
- the intracellular activity of certain enzymes is increased compared to the same microorganism unmodified and the intracellular activity of at least one other enzyme is decreased compared to the same unmodified microorganism.
- the term "activity" of an enzyme is used interchangeably with the term “function” and denotes the reaction which is catalyzed by the enzyme.
- Those skilled in the art know different techniques for measuring the enzymatic activity of a given enzyme.
- the experimental part below presents various enzymatic tests which can be used to measure the activity of enzymes involved in the present invention.
- the expression “increased activity” applied to an enzyme denotes a specific catalytic activity of the increased enzyme and / or an increased quantity or availability of the enzyme in the cell.
- an increased enzymatic activity in the recombinant microorganism must be understood as an enzymatic activity which is increased by a factor of at least 2, in particular at least 5, in particular, at least 10 and, more particularly, at least 20 relative to the enzymatic activity of the same unmodified microorganism.
- an increase in the enzymatic activity can be obtained (i a ) by increasing the number of copies of the gene coding for the enzyme in the microorganism, (ii a ) by increasing the expression of the gene coding for the enzyme in the microorganism, for example, by modifying the promoter, the regulatory regions and / or the ribosome binding site, (iii a ) by modifying the sequence of the gene encoding the enzyme so as to obtain a more active form or more resistant to inhibition and, optionally, (iv a ) by combining at least two of the alternatives (i a ), (ii a ) and (iii a ).
- the gene can be coded chromosomally or extrachromosomally.
- the gene When the gene is located on the chromosome, several copies of the gene can be introduced onto the chromosome by recombination methods, known to those skilled in the art (including via gene replacement).
- the gene When the gene is located extra-chromosomally, it can be carried by a recombinant expression vector.
- recombinant expression vector is meant a nucleic acid suitable for the expression, in a microorganism, of at least one enzyme encoded by a nucleotide sequence contained in this vector.
- the expression vector according to the present invention comprises, in addition to the nucleotide sequence coding for an enzyme of interest, one (or more) element (s) which allow (s) expression ie transcription and translation of this sequence nucleotide.
- the expression vector used in the present invention is advantageously chosen from a plasmid, a cosmid, a bacteriophage and a virus such as a baculovirus.
- the vector of the invention is a vector with autonomous replication comprising elements allowing its maintenance and its replication in the microorganism as an origin of replication.
- the vector may include elements allowing its selection in the microorganism. These elements are also known as "selection markers".
- selection markers are well known to those skilled in the art and widely described in the literature.
- An expression vector can also present one or more element (s) chosen from a promoter, an amplifier also known by the English term of “enhancer”, a 3 ′ UTR signal (for “UnTranslated Region”), an IRES signal (for “Internai Ribosome Entry Site”), a ribosome binding site (in English RBS, for “Ribosome Binding Site”), a transcription termination signal comprising a cleavage site and a polyA signal (for "Polyadenylation signal”).
- the expression vector according to the invention can comprise 2, 3 or 4 elements listed above. A person skilled in the art is able to choose, from this list, the additional element (s) that the expression vector may include as a function of the microorganism in which the expression must be carried out.
- selection marker is meant a marker chosen from a selection marker usable in prokaryotes or in eukaryotes such as a gene bacterial antibiotic resistance and a metabolism gene for use with an auxotrophic microorganism ie a selection gene which provides complementation with the respective gene deleted at the genome of the host microorganism.
- the expression vector according to the present invention may contain a bacterial gene for resistance to an antibiotic such as amoxicillin, ampicillin, phleomycin, kanamycin, chloramphenicol, neomycin, hygromycin, or geneticin (or G418), carboxin, nursesothricin or triclosan.
- genes for metabolism mention may be made of the trpl gene to be used with a microorganism devoid of the enzyme phosphoribosylanthranilate isomerase such as a trpl yeast or the URA3 gene to be used with a eukaryotic organism devoid of l orotidine 5-phosphate decarboxylase enzyme such as ura3 yeast.
- promoter is meant, in the context of the present invention, both a promoter, constitutive or inducible, suitable for any microorganism as defined above as a promoter, constitutive or inducible, specific for a group of particular microorganisms.
- the promoter which can be used can be homologous or heterologous.
- a constitutive promoter which can be used in the context of the present invention is in particular chosen from the proD promoter, the proC promoter, the 35S promoter, the 19S promoter and the TEV promoter (for “Tobacco Etch Virus”).
- An inducible promoter which can be used in the context of the present invention may be the galactose inducible GAL1 promoter, the methanol inducible promoter AOX1, the isopropyl bDl-thiogalactopyranoside (IPTG) inducible promoter PAllac0-l, the hybrid promoter pTac inducible by IPTG; the MET15 promoter inducible by methionine depletion or the CUP1 promoter inducible by copper ions.
- the promoter can be associated with one or more transcriptional regulatory sequences which are the enhancers.
- the expression vector used in the present invention comprises, operatively linked to each other, a promoter, a nucleotide sequence coding for an enzyme of interest and a transcription termination signal comprising a cleavage site and / or a polyA signal.
- operatively linked to one another means elements linked to one another so that the operation of one of the elements is affected by that of another.
- a promoter is operably linked to a coding sequence when it is able to affect the expression of the latter.
- the regulatory elements for the transcription, translation and maturation of the peptides that the vector can understand are known to those skilled in the art and the latter is capable of choosing them as a function of the host microorganism in which the expression or the cloning must be performed.
- plasmids which differ by their origin of replication and therefore by their number of copies in the cell.
- these plasmids are present in the microorganism in 10 to 15 copies, or in approximately 30 to 50 copies, or even up to 100 copies, depending on the nature of the plasmid: plasmids with low number of copies with close replication, plasmids with number using copies or plasmids with a large number of copies.
- plasmids which can be used in the context of the present invention, mention may be made of the plasmids pSC101, RK2, pACYC, pRSFIO10, pZ and pSK bluescript II.
- the alternative (ii a ) above consists in using a promoter inducing a high level of expression of the endogenous gene.
- a promoter inducing a high level of expression of the endogenous gene.
- the alternative (ii a ) may be to attenuate the activity or expression of a transcription repressor, specific or non-specific for the endogenous gene.
- the expression “reduced activity” or “reduced activity” applied to an enzyme designates a specific catalytic activity of the reduced enzyme and / or a reduced quantity or availability of the enzyme in the cell.
- a reduced enzymatic activity in the recombinant microorganism must be understood as an enzymatic activity which is reduced by a factor of at most 0.5, in particular at most 0.1, in in particular, at most 0.01 and, more particularly, at most 0.001 with respect to the enzymatic activity of the same unmodified microorganism.
- Those skilled in the art know different techniques of microbiology and molecular biology which can be used to obtain, in a given microorganism, the reduction of an enzymatic activity.
- a decrease in the enzymatic activity can be obtained (i d ) by decreasing the expression of the gene encoding the enzyme in the microorganism, for example, by modifying the promoter, the regulatory regions and / or the site for fixing the ribosome, (ii d ) by modifying the sequence of the gene encoding the enzyme so as to obtain a reduced expression of the gene and / or the expression of an enzyme whose activity is reduced, (iii d ) using destabilizing elements the mRNA obtained following the transcription of the gene and, optionally, (iv d ) by inactivating the gene in particular by total or partial deletion of said gene, by total or partial deletion of the promoter preventing any expression of the gene and / or by inserting an external gene element into the coding region of the gene or into the promoter region.
- the recombinant microorganism according to the invention exhibits an activity for converting D-ribulose-5-phosphate into D-arabinose-5-phosphate, increased by comparison with the same unmodified microorganism.
- the intracellular activity of the enzyme which converts D-ribulose-5-phosphate to D-arabinose-5-phosphate in the recombinant microorganism is increased compared to the same unmodified microorganism.
- the enzyme (E,) which converts D-ribulose-5-phosphate into D-arabinose-5-phosphate is in the form of a D-arabinose-5-phosphate isomerase (EC 5.3.1.13).
- D-arabinose-5-phosphate aldose-ketose-isomerase is also known as "D-arabinose-5-phosphate aldose-ketose-isomerase”, “arabinose phosphate isomerase”, “D-arabinose-5-phosphate ketol-isomerase” and "phosphoarabinoisomerase”. All these designations refer to the same enzyme and can be used interchangeably.
- the recombinant microorganism according to the invention has an activity of converting D-ribulose-5-phosphate into D-arabinose-5-phosphate catalyzed by an enzyme consisting of a D-arabinose-5-phosphate isomerase which converts the D-ribulose-5-phosphate to D-arabinose-5-phosphate, increased compared to the same unmodified microorganism.
- the enzyme (E,) is encoded by the kdsD gene of E. coli or by a homolog of such a gene.
- a homolog of the E. coli kdsD gene encodes a protein capable of converting D-ribulose-5-phosphate to D-arabinose-5-phosphate.
- E. coli kdsD gene encodes a protein capable of converting D-ribulose-5-phosphate to D-arabinose-5-phosphate.
- coli kdsD gene mention may be made of the gene of the strain K12 MG1655, the coding sequence of which is between nucleotides 2829813 and 2830778 in the sequence NC_000913.3 accessible on the NCBI website (for "National Center for Biotechnology Information") https: //www.ncbi, nim.nib.gov/ and corresponding to the complete genome of the strain K12 MG1655.
- the 321 amino acid protein sequence encoded by this gene is referenced on the NCBI website, sequence NP_417188.4 and corresponds to the sequence SEQ. ID NO: 1 in the attached sequence listing.
- the increase in the activity of conversion of D-ribulose-5-phosphate to D-arabinose-5-phosphate catalyzed by an enzyme consisting of D-arabinose-5 -phosphate isomerase is obtained by increasing the number of copies of a gene encoding this enzyme in the microorganism and / or by increasing the expression of a gene encoding this enzyme in the microorganism.
- the recombinant microorganism which is the subject of the present invention exhibits an overexpression of a gene encoding a D-arabinose-5-phosphate isomerase which converts D-ribulose-5-phosphate to D-arabinose-5-phosphate.
- the recombinant microorganism which is the subject of the present invention exhibits an overexpression of the kdsD gene of E. coli or of a homolog thereof.
- the recombinant microorganism according to the invention exhibits an aldolic cleavage activity of D-arabinose-5-phosphate into D-glyceraldehyde-3-phosphate and glycolaldehyde, increased by comparison with the same unmodified microorganism.
- the intracellular activity of the enzyme which catalyzes the cleavage of D-arabinose-5-phosphate into D-glyceraldehyde-3-phosphate and glycolaldehyde in the recombinant microorganism is increased compared to the same micro- unmodified organism.
- the enzyme (E n ) which catalyzes the cleavage of D-arabinose-5-phosphate into D- glyceraldehyde-3-phosphate and glycolaldehyde is in the form of a fructose-6-phosphate aldolase (EC 4.1.2.-) [14].
- the recombinant microorganism according to the invention exhibits an aldolic cleavage activity from D-arabinose-5-phosphate to D-glyceraldehyde-3-phosphate and glycolaldehyde catalyzed by an enzyme consisting of a fructose-6-phosphate aldolase which catalyzes the cleavage of D-arabinose-5-phosphate into D-glyceraldehyde-3-phosphate and glycolaldehyde, increased compared to the same unmodified microorganism.
- the enzyme (En) is encoded by the E. coli gene / sa or by a homolog of such a gene.
- an E. coli gene / sa homolog encodes a protein capable of catalyzing the cleavage of D-arabinose-5-phosphate into D-glyceraldehyde-3-phosphate and glycolaldehyde .
- the E. coli gene / sa there may be mentioned:
- FSAA 220 amino acid protein
- FSAB 220 amino acid protein
- homologs of an E. coli gene / sa mention may be made of the genes encoding the fructose-6-phosphate aldolases variants FSAA L107Y / A129G and FSAA A129T / A165G described by Szekrenyi et al, 2014 [15].
- the increase in the aldolic cleavage activity of D-arabinose-5-phosphate into D-glyceraldehyde-3-phosphate and glycolaldehyde catalyzed by an enzyme consisting of a fructose- 6-phosphate aldolase is obtained by increasing the number of copies of a gene encoding this enzyme in the microorganism and / or by increasing the expression of a gene encoding this enzyme in the microorganism.
- the recombinant microorganism which is the subject of the present invention exhibits an overexpression of a gene encoding a fructose-6-phosphate aldolase which catalyzes the cleavage of D-arabinose-5-phosphate into D-glyceraldehyde-3-phosphate and glycolaldehyde .
- the recombinant microorganism which is the subject of the present invention exhibits overexpression of the gene / sa of E. coli or of a homolog thereof.
- the recombinant microorganism according to the invention exhibits an activity for oxidizing glycolaldehyde to glycolate, increased by comparison with the same unmodified microorganism.
- the intracellular activity of the enzyme that oxidizes the glycolaldehyde to glycolate in the recombinant microorganism is increased compared to the same unmodified microorganism.
- the enzyme (EM,) which oxidizes glycolaldehyde to glycolate is in the form of a glycolaldehyde dehydrogenase and in particular a glycoaldehyde dehydrogenase whose activity requires the presence of the cofactor NAD + (EC 1.2.1.21).
- the recombinant microorganism according to the invention exhibits an activity of oxidation of glycolaldehyde to glycolate catalyzed by an enzyme consisting of a glycolaldehyde dehydrogenase which oxidizes glycolaldehyde to glycolate, increased compared to the same unmodified microorganism.
- the enzyme (EM,) is encoded by the aldA gene of E. coli or by a homolog of such a gene.
- a homolog of the E. coli aldA gene codes for a protein capable of oxidizing glycolaldehyde to glycolate in the presence of a cofactor such as, for example, NAD + .
- a cofactor such as, for example, NAD + .
- an E. coli aldA gene mention may be made of the gene of the strain K12 MG1655, the coding sequence of which is between nucleotides 1488232 and 1489671 in the sequence NC_000913.3 accessible on the NCBI website.
- the sequence of the protein of 479 amino acids, encoded by this gene is referenced, on the NCBI website, sequence NP_415933.1 and corresponds to the sequence SEQ. ID NO: 4 in the attached sequence listing.
- the increase in the oxidation activity of glycolaldehyde to glycolate catalyzed by an enzyme consisting of glycolaldehyde dehydrogenase is obtained by increasing the number of copies of a gene encoding this enzyme in the microorganism and / or by increasing the expression of a gene encoding this enzyme in the microorganism.
- the recombinant microorganism which is the subject of the present invention exhibits an overexpression of a gene encoding a glycolaldehyde dehydrogenase which oxidizes the glycolaldehyde to glycolate. More particularly, the recombinant microorganism which is the subject of the present invention exhibits overexpression of the aldA gene of E. coli or of a homolog thereof.
- the recombinant microorganism according to the present invention comprises:
- plasmids of identical nature but having compatible origins of replication or of different nature, one having two sequences, optionally cloned as an operon, chosen from the sequence of the E. coli kdsD gene or of a homolog of this, the sequence of the E. coli fsa gene or a homolog thereof and the sequence of the E. coli aldA gene or a homolog thereof and the other plasmid the third of these sequences.
- plasmids identical or different, each having a different sequence chosen from the sequence of the kdsD gene of E. coli or of a homolog thereof, the sequence of the gene / sa of E. coli or a homolog thereof and the sequence of the aldA gene from E. coli or a homolog thereof.
- the recombinant microorganism which is the subject of the invention comprises:
- said first and second promoters being identical or different.
- the recombinant microorganism according to the invention exhibits an oxidation activity of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate, reduced by comparison with the same unmodified microorganism.
- the intracellular activity of the enzyme which oxidizes glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate in the recombinant microorganism is decreased compared to the same unmodified microorganism.
- the enzyme (Ej V ) which oxidizes glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate is in the form of a glyceraldehyde-3-phosphate dehydrogenase and in particular of a glyceraldehyde-3-phosphate dehydrogenase whose cofactor is NAD + (EC 1.2.1.12).
- Such an enzyme is also known as "D-glyceraldehyde-3-phosphate: NAD + oxidoreductase".
- the recombinant microorganism according to the invention has an activity of oxidizing glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate catalyzed by an enzyme consisting of glyceraldehyde-3-phosphate dehydrogenase which oxidizes glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate, decreased compared to the same unmodified microorganism.
- the enzyme (E, n ) is encoded by the gapA gene from E. coli or by a homolog of such a gene.
- a homolog of the E. coli gapA gene encodes a protein capable of oxidizing glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate in the presence of a cofactor such as, for example, NAD + .
- a cofactor such as, for example, NAD + .
- a gapA gene from E. coli mention may be made of the gene of the strain K12 MG1655, the coding sequence of which is between nucleotides 1862771 and 1863766 in the sequence NC_000913.3 accessible on the NCBI website.
- the protein amino acid sequence of 331 encoded by this gene is referenced, on the NCBI website, sequence NP_416293.1 and corresponds to the sequence SEQ. ID NO: 5 in the attached sequence listing.
- the reduction in the oxidation activity of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate catalyzed by an enzyme consisting of glyceraldehyde-3-phosphate dehydrogenase is obtained by decreasing the expression of the gene encoding this enzyme and / or by inactivating the gene encoding this enzyme.
- the expression of the gapA gene of E. coli or of a homolog thereof is reduced but not inactivated with respect to the micro- unmodified organism.
- the attenuation of the gapA activity allows growth and, at the same time, the production of glycolic acid.
- maintaining a residual glycolysis does not make it possible to reach the maximum yield.
- the expression of the gapA gene of E. coli or of a homolog thereof is inactivated relative to the unmodified microorganism .
- the growth and production of glycolic acid are decoupled.
- Inactivation of gapA requires that the recombinant microorganism according to the invention has a growth substrate, containing, in addition to pentoses and / or hexoses, C2, C3 or C4 compounds such as acetate, pyruvate, malate or succinate or which enter into metabolism after glyceraldehyde-3-phosphate (GAP).
- GAP glyceraldehyde-3-phosphate
- the recombinant microorganism according to the present invention is modified at the level of glucose transport in the sense that the phosphotransferase system (PTS), which depends on phosphoenolpyruvate (PEP) is inactivated, while a glucose transport activity coded by galP of E. coli or g If of Zymomonas mobilis or one of their counterparts and an activity for converting glucose into glucose- 6-phosphase are increased compared to the same unmodified microorganism.
- PTS phosphotransferase system
- PEP phosphoenolpyruvate
- the phosphotransferase system which depends on phosphoenolpyruvate (PEP) is the most efficient system for transporting glucose.
- PTS phosphotransferase system
- PEP phosphoenolpyruvate
- the activity of the PTS system has an effect on the distribution of carbon flux and plays a key role in the suppression of carbon catabolism.
- the cytoplasmic PTS system is coded by the operon ptsHIcrr. Deletion of the ptsHIcrr operon, particularly in E. coli, is the most commonly used strategy to inactivate the PTS system.
- the PTS phenotype is characterized by a very limited capacity for glucose transport and phosphorylation.
- PEP is not necessary for glucose phosphorylation.
- Glucokinase catalyzes glucose-dependent phosphorylation-ATP in the cytoplasm.
- the overexpression of galactose permease (GalP) for glucose transport and glucokinase (Glk) for phosphorylation restores the PTS + phenotype [18].
- Another strategy is to overexpress the genes gifz m and glkz m respectively coding for an easier transporter of glucose (Gif) and the glucokinase of Zymomonas mobilized in E. coli [19].
- an activity of transport and phosphorylation of glucose from phosphoenolpyruvate by the phosphoenolpyruvate dependent phosphotransferase system is quenched by comparison with the same unmodified microorganism.
- inactivation of the cytoplasmic PTS system encoded by the ptsHIcrr operon in E. coli leads to the deletion of genes:
- E. coli ptsH of the operon ptsHIcrr encoding the phosphohistidine protein Hpr, phosphate carrier, of the PTS system.
- E. coli ptsH gene there may be mentioned the gene of the strain K12-MG1655, the coding sequence of which is included, on the complementary strand, between nucleotides 2533764 and 2534021 in the sequence NC_000913.3 accessible on the NCBI website.
- the 85 amino acid protein sequence encoded by this gene is referenced, on the NCBI website, sequence NP_416910.1 and corresponds to the sequence SEQ. ID NO: 6 in the attached sequence listing.
- E. coli ptsl of the operon ptsHlcrr encoding the enzyme I of the PTS system (EC 2.7.3.9).
- E. coli ptsl gene mention may be made of the gene of the strain K12-MG1655, the coding sequence of which is included, on the complementary strand, between nucleotides 2534066 and 2535793 in the sequence NC_000913.3 accessible on the NCBI website.
- the 575 amino acid protein sequence encoded by this gene is referenced, on the NCBI website, sequence NP_416911.1 and corresponds to the sequence SEQ ID NO: 7 in the attached sequence listing.
- E. coli crr of the operon ptsHlcrr encoding the MA enzyme of complex II of the PTS system (EC 2.7.1.69).
- E. coli crr gene mention may be made of the gene of the strain K12-MG1655, the coding sequence of which is included, on the complementary strand, between nucleotides 2535834 and 2536343 in the sequence NC_000913.3 accessible on the NCBI website.
- the sequence of the protein of 169 amino acids encoded by this gene is referenced, on the NCBI website, sequence NP_416912.1 and corresponds to the sequence SEQ. ID NO: 8 in the attached sequence listing.
- the deletion of the ptsG gene from the PTS system which does not belong to the ptsHlcrr operon, is also required, since PtsG is strongly involved in catabolic repression in E. coli.
- PtsG mutants are able to consume glucose, arabinose and xylose simultaneously while a wild strain consumes glucose then arabinose and finally xylose sequentially [20].
- the ptsG gene encodes a large hydrophobic II B / C domain of complex II of the PTS system. As a more specific example of the E.
- coli ptsG gene mention may be made of the gene of the strain K12-MG1655, the coding sequence of which is included, on the complementary strand, between nucleotides 1157869 and 1159302 in the sequence NC_000913.3 accessible on the NCBI website.
- the 477 amino acid protein sequence encoded by this gene is referenced, on the NCBI website, sequence NP_415619.1 and corresponds to the sequence SEQ ID NO: 9 in the attached sequence listing.
- the E. coli galP gene encodes a galactose permease. Based on the definition of “homolog” previously provided, a homolog of the E. coli galP gene codes for a protein of the galactose permease type.
- the galP gene of E. coli there may be mentioned the gene of the strain K12 MG1655, the coding sequence of which is between nucleotides 3088284 and 3089678 in the sequence NC_000913.3 accessible on the NCBI website.
- the 464 amino acid protein sequence encoded by this gene is referenced, on the NCBI website, sequence NP_417418.1 and corresponds to the sequence SEQ ID NO: 10 in the attached sequence listing.
- the Z mobilis glf gene codes for a glucose transporter. Based on the definition of "homolog" previously provided, a homolog of the glf gene from Z. mobilis codes for a glucose transporter. As a particular example of the Z mobilis glf gene, mention may be made of the gene of the ATCC 31821 / ZM4 / CP4 strain which codes for the 473 amino acid protein referenced on the UniProtKB site (http: //www.uniprot. org /), sequence P21906 and corresponding to the sequence SEQ. ID NO: 11 in the attached sequence listing.
- the recombinant microorganism according to the invention exhibits an activity for converting glucose into glucose-6-phosphase, increased by comparison with the same unmodified microorganism.
- the intracellular activity of the enzyme which transforms glucose into glucose-6-phosphase in the recombinant microorganism is increased compared to the same unmodified microorganism.
- the enzyme that turns glucose into glucose-6-phosphase is in the form of a glucokinase (EC 2.7.1.2).
- the recombinant microorganism according to the invention exhibits an activity for converting glucose into glucose-6-phosphase catalyzed by an enzyme consisting of a glucokinase which transforms glucose into glucose-6-phosphase, increased compared to the same micro- unmodified organism.
- this enzyme is coded by the glK gene of E. coli or by a homolog of such a gene.
- a homolog of the E. coli glK gene encodes a protein capable of transforming glucose into glucose-6-phosphase.
- an E. coli glK gene mention may be made of the gene of strain K12 MG1655, the coding sequence of which is between nucleotides 2508461 and 2509426 in the sequence NC_000913.3 accessible on the NCBI website.
- the protein sequence of 321 amino acids, encoded by this gene is referenced, on the NCBI website, sequence NP_416889.1 and corresponds to the sequence SEQ. ID NO: 12 in the attached sequence listing.
- the increase in glucose transport catalyzed by an enzyme consisting of a glucose transporter or a galactose permease is obtained by increasing the number of copies of a gene encoding this enzyme in the microorganism and / or by increasing the expression of a gene encoding this enzyme in the microorganism.
- the recombinant microorganism which is the subject of the present invention exhibits an overexpression of a gene coding for a glucose transporter and / or an overexpression of a gene coding for a galactose permease which catalyzes the transport of glucose.
- the increase in the transformation of glucose into glucose-6-phosphate catalyzed by an enzyme consisting of a glucokinase which transforms glucose into glucose-6-phosphase is obtained by increasing the number of copies of a gene encoding this enzyme in the microorganism and / or by increasing the expression of a gene encoding this enzyme in the microorganism.
- the recombinant microorganism which is the subject of the present invention exhibits an overexpression of a gene encoding a glucokinase which transforms glucose into glucose-6-phosphase.
- the recombinant microorganism which is the subject of the present invention exhibits an overexpression of the aldA gene of E. coli or of the glf gene of Z. mobilis or one of their counterparts and an overexpression of the glK gene of E. coli or d 'a counterpart of it.
- the genes above listed are under the dependence of a strong constitutive promoter such as the proD promoter.
- the microorganism recombinant object of the present invention may have at least one of the following characteristics:
- the recombinant microorganism according to the invention has an activity of oxidizing glycolate to glyoxylate, reduced by comparison with the same unmodified microorganism.
- the intracellular activity of the enzyme which oxidizes glycolate to glyoxylate in the recombinant microorganism is decreased compared to the same unmodified microorganism.
- the enzyme (E v ) which oxidizes glycolate to glyoxylate is in the form of a glycolate dehydrogenase and in particular of a glycolate dehydrogenase whose cofactor is NAD + (EC 1.1.99.14).
- Such an enzyme is also known as "glycolate oxidoreductase” and “glycolate oxidase”. All these designations refer to the same enzyme and can be used interchangeably.
- the recombinant microorganism according to the invention exhibits an activity of oxidation of glycolate to glyoxylate catalyzed by an enzyme consisting of a glycolate dehydrogenase which oxidizes glycolate to glyoxylate, decreased compared to the same unmodified microorganism.
- the glycolate dehydrogenase activity of E. coli is encoded by the glcDEF genes or by a homolog of such genes.
- a homolog of the E. coli glcD, glcE and glcF genes encodes a protein capable of oxidizing glycolate to glyoxylate in the presence of a cofactor such as, for example, NAD + .
- E. coli glcD gene As a particular example of an E. coli glcD gene, mention may be made of the gene of the strain K12 MG1655, the coding sequence of which is included, on the complementary strand, between nucleotides 3126522 and 3128021 in the sequence NC_000913.3 accessible from the NCBI website.
- the 499 amino acid protein sequence encoded by this gene is referenced on the NCBI website, sequence NP_417453.1 and corresponds to the sequence SEQ ID NO: 13 in the attached sequence listing.
- E. coli glcE gene As a particular example of an E. coli glcE gene, there may be mentioned the gene of the strain K12 MG1655, the coding sequence of which is included, on the complementary strand, between nucleotides 3125470 and 3126522 in the sequence NC_000913.3 accessible from the NCBI website.
- the sequence of the protein of 350 amino acids, coded by this gene is referenced, on the site of the NCBI, sequence YP_026191.1 and corresponds to the sequence SEQ. ID NO: 14 in the attached sequence listing.
- E. coli glcF gene As a specific example of an E. coli glcF gene, mention may be made of the gene of strain K12 MG1655, the coding sequence of which is included, on the complementary strand, between nucleotides 3124236 and 3125459 in the sequence NC_000913.3 accessible from the NCBI website.
- the 407 amino acid protein sequence encoded by this gene is referenced, on the NCBI website, sequence YP_026190.1 and corresponds to the sequence SEQ ID NO: 15 in the attached sequence listing.
- the decrease in the oxidative activity of the glycolate to glyoxylate catalyzed by an enzyme consisting of a glycolate dehydrogenase is obtained by decreasing the expression of the gene encoding this enzyme and / or by inactivating the gene encoding this enzyme.
- the expression of the glcD, glcE and glcF genes of E. coli or of homologs thereof is reduced compared to the unmodified microorganism.
- the E. coli glcD, glcE and glcF genes or the homologs thereof are deleted in the recombinant microorganism object of the present invention in order to allow the accumulation of glycolate [21 ].
- the recombinant microorganism according to the invention exhibits increased aerobic respiratory activity by comparison with the same unmodified microorganism, this by reducing the repression of the genes involved in the regulation of aerobic respiratory metabolism. This repression does not call on an enzyme but on a transcriptional regulator.
- the recombinant microorganism according to the invention has a repression of the genes involved in the regulation of aerobic respiratory metabolism induced by a transcriptional regulator capable of repressing the genes involved in the regulation of aerobic respiratory metabolism, reduced compared to the same microorganism not modified.
- this transcriptional regulator is encoded by the arcA gene from E. coli or by a homolog of such a gene.
- a homolog of the E. coli arcA gene encodes a protein capable of repressing the genes involved in aerobic respiratory metabolism
- the arcA gene from E. coli mention may be made of the gene of the strain K12 MG1655, the coding sequence of which is included, on the complementary strand, between nucleotides 4639590 and 4640306 in the sequence NC_000913.3 accessible from the NCBI website.
- the 238 amino acid protein sequence encoded by this gene is referenced on the NCBI website, sequence NP_418818.1 and corresponds to the sequence SEQ. ID NO: 16 in the attached sequence listing. All the variants previously envisaged for decreasing an enzymatic activity can be used, mutatis mutandis, for decreasing the activity of the transcriptional regulator repressing the genes involved in aerobic respiratory metabolism.
- the reduction in the repression of the genes involved in the regulation of aerobic respiratory metabolism induced by a transcriptional regulator is obtained by decreasing the expression of the gene coding for this regulator and / or by inactivating the gene encoding this regulator.
- the expression of the arcA gene of E. coli or of a homolog thereof is reduced compared to the unmodified microorganism.
- the arcA gene of E. coli or the homolog thereof is deleted in the recombinant microorganism object of the present invention.
- the recombinant microorganism according to the invention exhibits internalization of the glycolate, reduced by comparison with the same unmodified microorganism. This internalization calls for proteins important glycolate [23, 24, 25]
- the recombinant microorganism according to the invention exhibits internalization of the glycolate induced by at least one important protein glycolate, reduced compared to the same unmodified microorganism.
- proteins are encoded by the glcA, HdP or yjcG genes of E. coli or by homologs of such genes. Based on the definition of "homolog" previously provided, homologs of the E. coli gicA, HdP or yjcG genes encode proteins capable of internalizing the glycolate.
- E. coli glcA gene As a specific example of an E. coli glcA gene, mention may be made of the gene of the strain K12 MG1655, the coding sequence of which is between nucleotides 1157869 and 1159302 in the sequence NC_000913.3 accessible on the NCBI website. The sequence of the protein of 477 amino acids, coded by this gene is referenced, on the site of NCBI, sequence NP_415619.1 and corresponds to the sequence SEQ ID NO: 17 in the attached sequence listing.
- E. coli lldP gene As a particular example of the E. coli lldP gene, mention may be made of the gene of the strain K12 MG1655, the coding sequence of which is between nucleotides 3777399 and 3779054 in the sequence NC_000913.3 accessible on the NCBI website.
- the 551 amino acid protein sequence encoded by this gene is referenced on the NCBI website, sequence NP_418060.1 and corresponds to the sequence SEQ. ID NO: 18 in the attached sequence listing.
- E. coli yjcG gene As a specific example of an E. coli yjcG gene, mention may be made of the gene of strain K12 MG1655, the coding sequence of which is included, on the complementary strand, between nucleotides 4283253 and 4284902 in the sequence NC_000913.3 accessible from the NCBI website.
- the 549 amino acid protein sequence encoded by this gene is referenced on the NCBI website, sequence NP_418491.1 and corresponds to the sequence SEQ ID NO: 19 in the attached sequence listing.
- the decrease in the internalization of the glycolate induced by one or more important protein (s) the glycolate is obtained by decreasing the expression of the gene (s) encoding this or these protein (s) and / or by inactivating the gene (s) encoding this or these protein (s).
- the expression of the glcA, lldP or yjcG genes of E. coli or of homologs thereof is reduced compared to the unmodified microorganism.
- the gicA, lldP or yjcG genes of E. coli or of homologs thereof are deleted relative to the unmodified microorganism.
- the recombinant microorganism according to the invention exhibits an irreversible methylglyoxal formation activity from dihydroxyacetone phosphate (DHAP), reduced by comparison with the same unmodified microorganism. In other words, the intracellular activity of the enzyme which forms, irreversibly, methylglyoxal, cytotoxic compound, from DHAP in the recombinant microorganism is decreased compared to the same unmodified microorganism.
- DHAP dihydroxyacetone phosphate
- the enzyme (E Vm ) which irreversibly forms methylglyoxal from DHAP is in the form of a methylglyoxal synthase (EC 4.2.3.3) [26].
- the recombinant microorganism according to the invention has an activity of irreversible formation of methylglyoxal from DHAP catalyzed by an enzyme consisting of methylglyoxal synthase which forms, irreversibly, methylglyoxal from DHAP, decreased compared to same unmodified microorganism.
- the enzyme (E Vm ) is encoded by the mgsA gene of E. coli or by a homolog of such a gene.
- a homolog of the mgsA gene from E. coli encodes a protein capable of irreversibly forming methylglyoxal from DHAP.
- E. coli mgsA gene mention may be made of the gene of strain K12 MG1655, the coding sequence of which is included, on the complementary strand, between nucleotides 1026557 and 1027015 in the sequence NC_000913.3 accessible from the NCBI website.
- the 152 amino acid protein sequence encoded by this gene is referenced on the NCBI website, sequence NP_415483.2 and corresponds to the sequence SEQ. ID NO: 20 in the attached sequence listing.
- the reduction in irreversible formation of methylglyoxal from DHAP catalyzed by an enzyme consisting of methylglyoxal synthase is obtained by decreasing the expression of the gene encoding this enzyme and / or by inactivating the gene encoding this enzyme.
- the expression of the mgsA gene of E. coli or of a homolog thereof is reduced compared to the unmodified microorganism.
- the mgsA gene of E. coli or the homolog thereof is deleted in the recombinant microorganism object of the present invention.
- the recombinant microorganism according to the invention exhibits an activity for converting fructose-6-phosphate into fructose-1,6-biphosphate, reduced by comparison with the same unmodified microorganism.
- the intracellular activity of the enzyme which converts fructose-6-phosphate into fructose-1,6-biphosphate in the recombinant microorganism is decreased compared to the same unmodified microorganism.
- the enzyme (E, c) that converts fructose-6-phosphate to fructose-1,6-biphosphate is in the form of a phosphofructokinase (EC 2.7.1.11).
- the recombinant microorganism according to the invention has an activity of converting fructose-6-phosphate into fructose-1,6-biphosphate catalyzed by an enzyme consisting of a phosphofructokinase which converts fructose-6-phosphate into fructose-l , 6-biphosphate, decreased compared to the same unmodified microorganism.
- the enzyme (E, c ) is encoded by the pfKA gene from E. coli or by a homolog of such a gene.
- a homolog of the E. coli pfKA gene encodes a protein capable of converting fructose-6-phosphate to fructose-1,6-biphosphate.
- the E. coli pfKA gene mention may be made of the gene of the strain K12 MG1655, the coding sequence of which is included, on the complementary strand, between nucleotides 4107552 and 4108514 in the sequence NC_000913.3 accessible from the NCBI website.
- sequence of the protein of 320 amino acids, coded by this gene is referenced, on the site of the NCBI, sequence NP_418351.1 and corresponds to the sequence SEQ. ID NO: 21 in the attached sequence listing. All the variants previously envisaged for decreasing an enzymatic activity can be used to decrease the activity of the enzyme (Ej X ) which converts fructose-6-phosphate into fructose-1,6-biphosphate.
- the decrease in the activity of converting fructose-6-phosphate into fructose-1,6-biphosphate catalyzed by an enzyme consisting of a phosphofructokinase is obtained by decreasing the expression of the gene encoding this enzyme and / or by inactivating the gene encoding this enzyme.
- the expression of the pfKA gene of E. coli or of a homolog thereof is reduced compared to the unmodified microorganism.
- the pfKA gene of E. coli or the homolog thereof is deleted in the recombinant microorganism object of the present invention and this, to avoid a futile cycle with fructose-1, 6 -biphosphate [27].
- the recombinant microorganism according to the invention has an activity for producing D-ribose-1-phosphate from dihydroxyacetone phosphate and glycolaldehyde, reduced by comparison with the same unmodified microorganism.
- the intracellular activity of the enzyme which produces D-ribose-1-phosphate from dihydroxyacetone phosphate and glycolaldehyde in the recombinant microorganism is decreased compared to the same unmodified microorganism .
- the enzyme (E x ) which produces D-ribose-1-phosphate from dihydroxyacetone phosphate and glycolaldehyde is in the form of an L-fuculose-phosphate aldolase (EC 4.1.2.17) [28].
- the recombinant microorganism according to the invention has an activity for producing D-ribose-1-phosphate from dihydroxyacetone phosphate and glycolaldehyde catalyzed by an enzyme consisting of L-fuculose-phosphate aldolase which produces D- ribose-1-phosphate from dihydroxyacetone phosphate and glycolaldehyde, decreased compared to the same unmodified microorganism.
- the enzyme (E x ) is encoded by the fucA gene from E. coli or by a homolog of such a gene. Based on the definition of “homolog” previously provided, a homolog of the E.
- coli fucA gene encodes a protein capable of producing D-ribose-1-phosphate from dihydroxyacetone phosphate and glycolaldehyde.
- the fucA gene from E. coli mention may be made of the gene of the strain K12 MG1655, the coding sequence of which is included, on the complementary strand, between nucleotides 2933041 and 2933688 in the sequence NC_000913.3 accessible on the NCBI website.
- the sequence of the protein of 215 amino acids, coded by this gene is referenced, on the NCBI website, sequence NP_417280.1 and corresponds to the sequence SEQ. ID NO: 22 in the attached sequence listing.
- the decrease in the activity of producing D-ribose-1-phosphate from dihydroxyacetone phosphate and glycolaldehyde catalyzed by an enzyme consisting of an L-fuculose-phosphate aldolase is obtained by decreasing the expression of the gene encoding this enzyme and / or by inactivating the gene encoding this enzyme.
- the expression of the E. coli fucA gene or of a homolog thereof is reduced compared with the unmodified microorganism.
- the E. coli fucA gene or the homolog thereof is deleted in the recombinant microorganism object of the present invention.
- the recombinant microorganism according to the invention has an activity of production of 6-phospho-D-glucono-1,5-lactone from D-glucose-6-phosphate decreased or increased compared to the same unmodified microorganism .
- the recombinant microorganism according to the invention exhibits an activity for the production of 6-phospho-D-glucono-1,5-lactone from D-glucose-6-phosphate decreased by comparison with the same unmodified microorganism.
- the intracellular activity of the enzyme (E Xi ) which produces 6-phospho-D-glucono-1,5-lactone from D-glucose-6-phosphate, in the recombinant microorganism is decreased compared to the same unmodified microorganism.
- the enzyme (E Xj ) which produces 6-phospho-D-glucono-1,5-lactone from D-glucose-6-phosphate is in the form of a NADP + dependent glucose-6-phosphate dehydrogenase ( EC 1.1.1.49) [29].
- the recombinant microorganism according to the invention has an activity of producing 6-phospho-D-glucono-1,5-lactone from D-glucose-6-phosphate catalyzed by an enzyme consisting of glucose-6 cofactor-dependent phosphate dehydrogenase which produces 6-phospho-D-glucono-1,5-lactone from D-glucose-6-phosphate, decreased compared to the same unmodified microorganism.
- the enzyme (E Xi ) is coded by the zwf gene of E. coli or by a homolog thereof.
- a homolog of the E. coli zwf gene encodes a protein capable of producing 6-phospho-D-glucono-1,5-lactone from D-glucose- 6-phosphate in the presence of a cofactor such as, for example, NADP + .
- a cofactor such as, for example, NADP + .
- the E. coli zwf gene mention may be made of the gene of the strain K12 MG1655, the coding sequence of which is included, on the complementary strand, between nucleotides 1934839 and 1936314 in the sequence NC_000913.3 accessible from the NCBI website.
- sequence of the protein of 491 amino acids, coded by this gene is referenced, on the site of the NCBI, sequence NP_416366 corresponds to the sequence SEQ. ID NO: 23 in the attached sequence listing.
- the expression of the E. coli zwf gene or of a homolog thereof is reduced compared to the unmodified microorganism.
- the E. coli zwf gene or the homolog thereof is deleted in the recombinant microorganism object of the present invention.
- the recombinant microorganism according to the invention exhibits an activity for the production of 6-phospho-D-glucono-1,5-lactone from D-glucose-6-phosphate increased by comparison with the same unmodified microorganism.
- the intracellular activity of the enzyme (E Xi ) which produces 6-phospho-D-glucono-1,5-lactone from D-glucose-6-phosphate, in the recombinant microorganism is increased compared to the same unmodified microorganism.
- the recombinant microorganism according to the invention has an activity of producing 6-phospho-D-glucono-1,5-lactone from D-glucose-6-phosphate catalyzed by an enzyme consisting of glucose-6 cofactor-dependent phosphate dehydrogenase which produces 6-phospho-D-glucono-1,5-lactone from D-glucose-6-phosphate, increased compared to the same unmodified microorganism.
- the increase in the activity of producing 6-phospho-D-glucono-1,5-lactone from D-glucose-6-phosphate catalyzed by an enzyme consisting of glucose-6-phosphate Cofactor-dependent dehydrogenase is obtained by increasing the number of copies of a gene encoding this enzyme in the microorganism and / or by increasing the expression of a gene encoding this enzyme in the microorganism.
- the recombinant microorganism which is the subject of the present invention exhibits an overexpression of a gene encoding a cofactor-dependent glucose-6-phosphate dehydrogenase which produces 6-phospho-D-glucono-1,5-lactone from D-glucose 6-phosphate.
- the recombinant microorganism which is the subject of the present invention exhibits an overexpression of the zwf gene of E. coli or of a homolog thereof.
- the recombinant microorganism which is the subject of the invention has the following characteristics:
- the recombinant microorganism according to the invention exhibits a 2-dehydroxy-3-deoxy-D-gluconate-6-phosphate formation activity reduced by comparison with D-gluconate-6-phosphate. to the same unmodified microorganism.
- the intracellular activity of the enzyme which forms 2-dehydroxy-3-deoxy-D-gluconate-6-phosphate from D-gluconate-6-phosphate in the recombinant microorganism is decreased compared to the same unmodified microorganism.
- the enzyme (E XÜ ) which produces 2-dehydroxy-3-deoxy-D-gluconate-6-phosphate from D-gluconate-6-phosphate is in the form of a phosphogluconate dehydratase (EC 4.2.1.12) [30].
- the recombinant microorganism according to the invention exhibits a 2-dehydroxy-3-deoxy-D-gluconate-6-phosphate activity from D-gluconate-6-phosphate catalyzed by an enzyme consisting of a phosphogluconate dehydratase which produces 2-dehydroxy-3-deoxy-D-gluconate-6-phosphate from D-gluconate-6-phosphate, decreased compared to the same unmodified microorganism.
- the enzyme (E XÜ ) is encoded by the edd gene of E. coli or by a homolog thereof.
- a homolog of the E. coli edd gene encodes a protein capable of producing 2- dehydroxy-3-deoxy-D-gluconate-6-phosphate from D- gluconate-6-phosphate.
- the edd gene from E. coli mention may be made of the gene of the strain K12 MG1655, the coding sequence of which is included, on the complementary strand, between nucleotides 1932794 and 1934604 in the sequence NC_000913.3 accessible from the NCBI website.
- the sequence of the protein of 603 amino acids, coded by this gene is referenced, on the site of the NCBI, sequence CAA45221 and corresponds to the sequence SEQ. ID NO: 24 in the attached sequence listing.
- the reduction in the oxidation activity of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate catalyzed by an enzyme consisting of glyceraldehyde-3-phosphate dehydrogenase is obtained by decreasing the expression of the gene encoding this enzyme and / or by inactivating the gene encoding this enzyme.
- the expression of the edd gene of E. coli or of a homolog thereof is reduced compared to the unmodified microorganism.
- the edd gene of E. coli or the homolog thereof is deleted in the recombinant microorganism object of the present invention.
- the recombinant microorganism according to the invention has an activity for producing D-glyceraldehyde-3-phosphate and pyruvate from 2-dehydroxy-3-deoxy-D-gluconate-6-phosphate decreased compared to the same unmodified microorganism.
- the intracellular activity of the enzyme that produces D-glyceraldehyde-3-phosphate and pyruvate to from 2-dehydroxy-3-deoxy-D-gluconate-6-phosphate, in the recombinant microorganism is decreased compared to the same unmodified microorganism.
- the enzyme (Exm) which produces D-glyceraldehyde-3-phosphate and pyruvate from 2-dehydroxy-3-deoxy-D-gluconate-6-phosphate is in the form of a 2-dehydroxy-3- deoxy-phosphogluconate aldolase [31].
- the recombinant microorganism according to the invention exhibits an activity for producing D-glyceraldehyde-3-phosphate and pyruvate from 2-dehydroxy-3-deoxy-D-gluconate-6-phosphate catalyzed by a consistent enzyme into a 2-dehydroxy-3-deoxy-phosphogluconate aldolase which produces D-glyceraldehyde-3-phosphate and pyruvate from 2-dehydroxy-3-deoxy-D-gluconate-6-phosphate, decreased compared to the same micro - unmodified organism.
- the enzyme (E Xiü ) is encoded by the eda gene of E. coli or by a homolog thereof.
- a homolog of the E. coli edd gene encodes a protein capable of producing D-glyceraldehyde-3-phosphate and pyruvate from 2-dehydroxy-3-deoxy -D-gluconate- 6-phosphate.
- an eda gene from E. coli mention may be made of the gene of the strain K12 MG1655, the coding sequence of which is included, on the complementary strand, between nucleotides 1932115 and 1932756 in the sequence NC_000913.3 accessible from the NCBI website.
- the sequence of the 213 amino acid protein, encoded by this gene is referenced, on the NCBI website, sequence NP_416364.1 and corresponds to the sequence SEQ. ID NO: 25 in the attached sequence listing.
- the decrease in the activity of producing D-glyceraldehyde-3-phosphate and pyruvate from 2-dehydroxy-3-deoxy-D-gluconate-6-phosphate catalyzed by an enzyme consisting of a 2-dehydroxy-3-deoxy-phosphogluconate aldolase is obtained by decreasing the expression of the gene encoding this enzyme and / or by inactivating the gene encoding this enzyme.
- the expression of the eda gene of E. coli or of a homolog thereof is reduced compared to the unmodified microorganism.
- the E. coli eda gene or the homolog thereof is deleted in the recombinant microorganism which is the subject of the present invention.
- the present invention also relates to the use of a recombinant microorganism as defined above for the production of glycolic acid from a culture medium comprising, as carbon source, at least one pentose and / or at least one hexose.
- the present invention relates to a process for producing glycolic acid comprising the steps consisting in:
- the production process which is the subject of the present invention uses steps and devices conventionally used in the field of biofermentation.
- the microorganism in step (a) of the method according to the present invention, can be cultivated in a culture medium according to the usual techniques used to cultivate this type of microorganism.
- the culture medium can be a commercial medium or a medium prepared extemporaneously.
- the culture medium used in the process of the invention is in the form of a sterile liquid containing a carbon source, a nitrogen source, a phosphate source, trace elements and optionally a sulfur source .
- the carbon source comprises at least one pentose and / or at least one hexose.
- this carbon source can comprise at least two different pentoses and at least one hexose, in particular at least xylose, arabinose and glucose and, in particular, D-xylose, L-arabinose and D-glucose.
- These pentoses and hexose are typically derived from a renewable carbon material such as plant biomass and, in particular from lignocellulosic biomass.
- the plant biomass is chosen from the group made up of agricultural products such as dedicated products called "energy” such as miscanthus, switchgrass (Panicum virgatum or switchgrass) and very short rotation coppices such as, for example, poplar or willow; agricultural production residues such as cereal straw, corn cane and sugar cane stalks; forest production; forest production residues such as wood processing residues.
- energy such as miscanthus, switchgrass (Panicum virgatum or switchgrass) and very short rotation coppices such as, for example, poplar or willow
- agricultural production residues such as cereal straw, corn cane and sugar cane stalks
- forest production forest production residues such as wood processing residues.
- Lignocellulose is composed of 75% carbohydrates and its hydrolysis releases fermentable sugars, mainly D-glucose, D-xylose and L-arabinose [32].
- the recombinant microorganism cultured is a recombinant microorganism as defined above and in which the expression of the gapA gene from E. coli or a counterpart thereof is decreased but not inactivated compared to the unmodified microorganism.
- the carbon source used may comprise only one element chosen from D-glucose, D-xylose, L-arabinose and one of their mixtures.
- mixture is meant a mixture of D-glucose and D-xylose, a mixture of D-glucose and L-arabinose, a mixture of D-xylose and L-arabinose and a mixture of D-glucose, D-xylose and L-arabinose.
- the recombinant microorganism cultured is a recombinant microorganism as defined above and in which the expression of the gapA gene from E. coli or a counterpart thereof is inactivated relative to the unmodified microorganism.
- the carbon source used comprises, in addition to D-xylose and / or L-arabinose and / or D-glucose, one or more chosen C2, C3 or C4 compounds among malate, pyruvate, succinate, acetate and one of their mixtures.
- the glycolic acid production process is done in two phases, with a first phase of biomass production, followed by a production phase which is triggered when the C2, C3 or C4 compounds arrive with exhaustion contributing to the bioconversion of hexoses and pentoses into glycolic acid.
- the carbon source used can also comprise at least one other carbon element such as galactose, xylose, fructose, lactose, sucrose, maltose, molasses, starch and a starch hydrolyzate.
- at least one other carbon element such as galactose, xylose, fructose, lactose, sucrose, maltose, molasses, starch and a starch hydrolyzate.
- suitable nitrogen sources include ammonia, ammonium salts such as ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, other compounds containing nitrogen; a peptone such as tryptone; meat extract, yeast extract, corn liquor; a casein hydrolyzate; soy flour and soy flour hydrolyzate.
- step (a) of the process according to the invention Those skilled in the art know different examples of sulfur sources, phosphate sources and trace elements which can be used in step (a) of the process according to the invention. He will be able to choose, without any inventive effort, the sources and trace elements best suited to the recombinant microorganism cultivated and the culture conditions.
- the temperature during the culture of step (a) is typically between 15 and 45 ° C, and the pH during this culture is typically maintained at a value between 3.0 and 9.0.
- the pH can be adjusted using, for example, an inorganic or organic acid, an alkaline solution, urea or calcium carbonate.
- the recombinant microorganism is, in step (a) under conditions in which there are production of glycolic acid from the carbon source as defined above.
- this culture is carried out under aerobic conditions, ie in the presence of oxygen.
- the recombinant microorganisms are cultivated in the form of a suspension.
- a "suspension" of cells is generally understood to include all types of suspended or dispersed cell cultures.
- the expression “in the form of a suspension” is thus used to distinguish cells which are not cultivated in a liquid medium, such as cells cultured by adhering to a petri dish.
- the term “suspension” includes both freely dispersed cells and agglomerated cells, regardless of whether agglomeration can occur spontaneously or not.
- the recombinant microorganisms can be cultivated in the form attached to a solid phase such as microbeads, beads, capillaries, hollow fibers, these various elements being typically made of a material compatible with the microorganism such as, for example. for example, dextran, gelatin, glass and cellulose.
- the culture methods which can be used during step (a) of the method according to the present invention include, but are not limited to, a batch culture, a continuous culture or a "fed-batch" culture.
- a “continuous (cell) culture” is a cell culture characterized by both a continuous supply of a nutritious liquid supply and a continuous flow of liquid.
- a continuous culture may constitute an "infusion culture", in which case the flow of liquid contains a culture medium which is substantially free of cells, or a cell concentration substantially lower than that in the bioreactor.
- cells can be retained, for example, by filtration, centrifugation or sedimentation.
- a “fed-batch” culture is a discontinuous cell culture to which a substrate, in concentrated form, solid or liquid, is added periodically or continuously during the analysis.
- a “fed-batch” culture is initiated by inoculating cells into the medium, but, unlike a staple crop, there is a subsequent influx of nutrients, such as through a concentrated nutrient diet.
- Unlike a continuous culture there is no systematic elimination of liquid originating from the culture or from the cells in a “fed-batch” culture.
- Step (a) of the method according to the invention can be implemented in any container suitable for cell culture.
- any container suitable for cell culture there may be mentioned an Erlenmeyer flask, a bioreactor or a biofermenter of different capacities. Additional information on these different containers can be found in [5].
- steps (a) and (b) can take place one after the other or, on the contrary, simultaneously.
- the glycolic acid is recovered from the culture medium.
- Figure 3 is a schematic representation of the enzymatic test to verify the activity of arabinose-5P isomerase (KdsD), fructose-6P aldolase (FSA) and aldehyde dehydrogenase (aldA).
- KdsD, FSA and aldA were purified, triose phosphate isomerase (Tpi) and glycerol-3P dehydrogenase (G3PDH) were ordered from Sigma.
- Figure 4 shows the system comprising 7 purified enzymes (AraA, AraB, AraD, RPE, KdsD, FSA, AldA) which catalyze the conversion of L-arabinose to glycolic acid (Figure 4A).
- the production of glycolic acid with this system in the presence of L- arabinose was compared to that with a system without AraA; the enzymatic reaction is based on the NADH assay at 340 nm ( Figure 4B).
- Figure 5 shows the system comprising 6 purified enzymes (XylA, XylB, RPE, KdsD, FSA, AldA) which are capable of converting D-xylose to glycolic acid (Figure 5A).
- the production of glycolic acid with this system in the presence of D-xylose was compared with that with a system without XylA; the enzymatic reaction is based on the NADH assay at 340 nm ( Figure 5B).
- Figure 6 shows the system comprising 7 purified enzymes (Hxk, Pgi, Tkt, RPE, KdsD, FSA, AldA) which are capable of converting D-glucose into glycolic acid (Figure 6A).
- Hxk purified enzymes
- Pgi Pgi
- Tkt RPE
- KdsD KdsD
- FSA AldA
- FIG. 7 shows the demonstration of the in vivo functionality of the non-natural route according to the invention in E. coli MG1655 AtktA AtktB AglcD, with in bold: the non-natural route according to the invention, in dotted lines, the deletions.
- Xu5P xylulose-5-phosphate
- Ru5P ribulose-5-phosphate
- R5P ribose-5-phosphate
- S7P sedoheptulose-7- phosphate
- F6P fructose-6-phosphate
- F16BP Fructose-1, 6-bisphosphate
- G6P glucose-6-phosphate
- DHAP dihydroxyacetone phosphate
- Glyald glycolaldehyde
- E4P erythrose-4-phosphate
- G3P glyceraldehyde-3-phosphate.
- Figure 8 shows the production of glycolic acid from the f strain.
- coli MG1655 AtktA AtktB AglcD expressing the non-natural pathway dependent on kdsD-fsa-aldA according to the invention from D-xylose or L-arabinose, at 37 ° C., 100 h.
- Figure 9 shows the production of glycolic acid from the strain f. coli WC3G AgapA AglcD AarcA AmgsA AfucA Apkf proD-galP expressing the non-natural pathway dependent on kdsD-fsa-aldA according to the invention from glucose, xylose and arabinose, at 37 ° C., 50 h. DETAILED PRESENTATION OF PARTICULAR EMBODIMENTS
- the oligonucleotides used for the amplification of the ORF of the enzymes of E. coli are listed in Table 3 below, the sequences SEQ. ID NO: referring to the sequence listing in the appendix.
- the genomic DNA of E. coli which served as a matrix is that of the strain K12-MG1655.
- XylA, XylB, AraA, AraB, AraD, AraB, Rpe, KdsD, FSA, AldA were constructed by HiFi assembly ® (NEB) with pET28a as recipient vector.
- pET28a was previously linearized with the restriction enzymes HindIII and BamHI (NEB) (Table 4).
- the strain of E. coli NEB5 ® derived from DH5 alpha, was used for the cloning and storage of the various plasmids.
- the competent cells of f. coli BL21 have been used for the expression of labeled proteins since these cells express T7 RNA polymerase, and are therefore compatible with the T7 expression system of the vector pET28a.
- the pET28a plasmids previously verified by sequencing were transformed into BL21 (DE3) according to the NEB protocol.
- the strains obtained are stored in 50% glycerol at -80 ° C (Table 5).
- All of the expressed proteins are soluble and were produced from the expression vector pET28a transformed into a strain of f. coli BL21 (DE3).
- a preculture in LB medium for “Luria-Bertani”) added with the antibiotic kanamycin is carried out overnight at 37 ° C.
- the preculture is used to seed a fresh culture of 200 ml of LB-Kanamycin at an optical density at 600 nm (DOeoo) of 0.1 (37 ° C., 200 rpm).
- DOeoo optical density at 600 nm
- the expression of the protein of interest is induced by the addition of IPTG to 1 mM final. Proteins are expressed overnight at 16 ° C.
- the cells are collected in 50 ml fractions and centrifuged at 4800 rpm for 15 min at 4 ° C.
- the cell pellets are stored at -20 ° C.
- the purification of the proteins is carried out from the cell pellets obtained during the production stage. All steps are done cold to avoid degradation of proteins by proteases.
- the cell pellets are resuspended in 1.5 ml of washing buffer (50 mM HEPES, pH 7.5; 0.3 M NaCl) and then sonicated on ice. A centrifugation step at 13,000 rpm, for 15 min at 4 ° C makes it possible to separate the cellular debris from the cytoplasmic liquid.
- the clarified lysates are deposited on 600 ⁇ l of cobalt resin (Clontech) previously equilibrated with the washing buffer.
- the tubes are centrifuged (700 rcf, 3 min, 4 ° C). The supernatant is removed, the resin is brought into contact with 3 ml of washing buffer for 10 min in order to eliminate non-specific interactions. After centrifugation (700 rcf, 3 min, 4 ° C) and removal of the supernatant, 3 mL of a 15 mM solution of imidazole are brought into contact with the resin for 5 min. The supernatant is separated from the resin by centrifugation, replaced by 500 mI of 200 mM imidazole. Imidazole elutes proteins carrying a polyhistidine label. To promote the stability of proteins at their optimal pH, the buffer has been modified.
- the method used to measure the concentration of proteins in solution is based on the Bradford method.
- the Protein assay ® reagent sold by BioRad is diluted to 1 ⁇ 4, the reaction mixture comprises 160 ⁇ l of reagent and 40 ml of diluted eluate (io th and 20 th ).
- a standard range is produced with BSA from 12.5 to 100 mg / ml.
- the hexokinase enzymes of Saccaromyces cerevisiae (Hxk), Escherichia coli transketolase (Tkt), Escherichia coli pyruvate kinase (PK) and Escherichia coli lactate dehydrogenase (LDH), and triose phosphate isomerase (Tpi) and glycerol -3-phosphate dehydrogenase (G3PDH) were ordered from Sigma.
- Escherichia coli phosphoglucose isomerase (Pgi) from Megazyme.
- AldA aldehyde dehydrogenase
- FSA fructose-6P aldolase
- D-arabinose-5-phosphate isomerase catalyzes the interconversion of D-ribulose-5-phosphate into D-arabinose-5-phsphate.
- the activity of KdsD on D-ribulose-5-phosphate was determined by adding FSA, aldA in excess in the presence of 3 mM of NAD + .
- Ribulose-5P is a metabolite common to the catabolism of arabinose, xylose and glucose in Escherichia coli.
- the unnatural way of converting D-ribulose-5P into glycolic acid which is the subject of the present invention consists of the enzymes KdsD (D-arabinose 5P isomerase), FSA (Fructose-6P aldolase) and AldA (glycolaldehyde dehydrogenase) .
- KdsD D-arabinose 5P isomerase
- FSA Fetose-6P aldolase
- AldA glycolaldehyde dehydrogenase
- the catalytic constants of these 3 enzymes are presented in Table 6.
- the proteins labeled polyhistidine KdsD, FSA and aldA are active.
- the reaction mixture containing KdsD, FSA and AldA in the presence of 5 mM of D-ribulose-5P was analyzed by HPLC, 1.5 mM of glycolic acid were quantified.
- the production of glycolic acid measured corresponds to the initial amount of NAD + in the reaction medium, indicating that the reaction has been complete.
- the enzymes catalyzing the conversion of L-arabinose, D-xylose and D-glucose to glycolic acid have been purified (AraA, AraB, AraD, XylA, XylB, Rpe, KdsD, Fsa, AldA) or ordered (Tkt, Glk, Pgi ) in order to demonstrate the feasibility of producing glycolic acid from these 3 substrates in vitro. i. Conversion of L-arabinose to glycolic acid.
- Table 8 Enzymatic activity measured for the conversion of D-xylose to glycolic acid at 340 nm, 37 ° C.
- One unit of enzyme activity (U) is defined as the conversion of one micromole of substrate per minute.
- the negative control is devoid of xylulose isomerase activity catalyzed by XylA.
- Table 9 Enzymatic activity measured for the conversion of D-glucose into glycolic acid at 340 nm, 37 ° C. One unit of enzyme activity is defined as the conversion of 1 micromole of substrate per minute. The negative control is devoid of hexokinase (Hxk) activity.
- Hxk hexokinase
- the carbohydrates L-arabinose, D-xylose and D-glucose are naturally assimilated and converted to D-ribulose-5P in Escherichia coli, the unnatural conversion of D-ribulose-5P to glycolic acid enabled by the overexpression of KdsD, FSA and aldA has been demonstrated.
- the implementation of the unnatural KdsD-FSA-aldA pathway is thermodynamically favorable, the complete pathways for assimilation and conversion of the reconstructed pentoses and hexoses have enabled glycolic acid synthesis in vitro.
- the series of pZ vectors (Expressys) has the advantage of being modular: it is easy to change the origin of replication, the resistance marker and the promoter of the vectors by restriction / ligation.
- the vectors pZA23, pZA33, pZE23 and pZS23 have the promoter PAllacO-1 which is a promoter derived from the promoter of the lactose operon including the operator o.
- PAllac0-l is under the control of the lacl repressor: in its active form, the lacl repressor binds to the operator o and inhibits transcription whereas, complexed with IPTG, it changes conformation and is no longer able to attach to site o, transcription then becoming possible.
- the PAllacO-1 promoter is said to be inducible to IPTG. Even if E.
- coli naturally has a copy of the lacl gene in its genome upstream of the lac operon, the majority of IPTG-inducible bacterial expression vectors carry the lacl gene in order to ensure total inhibition of the transcription of genes that are under its dependence.
- the pZ vectors have the particularity of having a light structure of the lacl gene which gives them a small size (2358 to 3764 bp).
- the HiFi assembly ® (NEB) method was used to construct the vectors used below. This method allows the assembly of several fragments. She was validated for fragments of different sizes with variable regions of overlap (15-80 bp). In a single step, the fragments can be assembled, it is a method commonly used for its simplicity and flexibility.
- the commercial mixture supplied by New England Biolabs contains different enzymes: (a) an exonuclease which creates 3 'single stranded ends, which facilitates the assembly of fragments which share sequence complementarity; (b) a polymerase which fills the voids after the fragments have assembled; and (c) a ligase which binds the fragments together.
- kdsD, fsa and aldA were amplified by PCR from the genome DNA extracted from f. coli K12 MG1655 and inserted by HiFi assembly ® in pZ vectors previously linearized by PCR with primers hybridizing to either side of the MCS. All plasmids were checked by sequencing before use. iii. Expression vectors for overexpression of kdsD, fsa, aldA
- KEIO strains carrying a single deletion and a resistance cassette to the antibiotic Kanamycin were infected with Plvir and high titer lysates 1 were obtained [35].
- Donor strains (KEIO) were grown overnight in LB at 37 ° C. The following day, 5 ml of LB containing 0.2% of glucose and 5 mM of CaCl 2 were inoculated with 200 ml of the donor strain and cultured for 30 min at 37 ° C. Then, 100 ⁇ l of Plvir lysate ( ⁇ 5 x 10 8 phages / ml) was added to each donor culture and incubated again at 37 ° C for 2 to 3 hours until the culture was clear and the cells are completely lysed. Lysates were filtered using sterile syringe filters of 25 mm with a Supor ® membrane of 0.2 pm (Pall) and stored at 4 ° C.
- the strain to be deleted was infected with Plvir containing a donor gene deletion cassette with resistance to kanamycin.
- the receptor strain was cultured in 5 ml of LB medium at 37 ° C. The cells were centrifuged at 1,500 g for 10 min and resuspended in 1.5 ml of 10 mM MgSO 4 and 2 to 5 mM CaCl 2 .
- Donor strain lysate (0.1 ml) is added to the cell suspension which is incubated for 30 min. Then 0.1 ml of 1 M sodium citrate is added to the cell and Plvir mixture. Then, 1 ml of LB is added to the homogenized suspension before a 1 h incubation at 37 ° C, 200 rpm.
- the cell suspensions are spread on a solid LB medium with the appropriate antibiotic and then the colonies are screened by PCR to demonstrate successful transduction events.
- the cells were transformed with a plasmid pCP20 carrying the FLP recombinase. Each step was checked by PCR.
- the deleted strain is sensitive to kanamycin after removal from the cassette, it can again be used as a recipient strain in order to add a new deletion from a new phage lysate.
- the competent non-commercial strains are prepared according to the protocol of Chung et al, 1989 slightly modified [36].
- a preculture is carried out in LB overnight to inoculate the next day with a fresh culture of LB at a DO 6 oo of 0.1.
- EOO 0.3 to 0, 5 the bacteria can be made competent to a D0 6 oo 1
- an amount equivalent cell culture in a 6 oo D0 unit is removed and centrifuged (8000 rpm, 2 min). The supernatant is removed while the pellet is taken up in 300 mI of TSS buffer (2.5% ( W t / voi) PEG 3350, 1 M MgCl 2 , 5% ( VO i / voi) DMSO).
- the mixture is incubated for 10 min on ice.
- the plasmid can then be added to the competent cells. After an additional 30 min of incubation on ice, a thermal shock is carried out at 42 ° C for 90 seconds.
- the transformed cells are placed in ice for 10 min. 400 mI of LB are added and the culture is incubated at 200 rpm for 1 h, at a suitable temperature (the temperature cannot exceed 30 ° C. in the case of a transformation with a plasmid whose origin of replication is thermosensitive).
- the bacterial culture is centrifuged at 8000 rpm for 2.5 min. 600 ml of supernatant are removed, the remaining volume is inoculated on a solid LB dish with the appropriate antibiotic.
- NEB5 fhuA2 A (argF-iacZ) U169 phoA glnV44 F80 A (lacZ) M15 NEB
- the cells are cultured on the LB medium for the stages of molecular biology (cloning, deletions, transformation).
- This rich medium is composed of 10 g / L of tryptone, 5 g / L of yeast extracts and 5 g / L of NaCl. 15 g / L of agar are added to obtain a solid medium.
- the LB, with or without agar, is sterilized by autoclaving 20 min at 110 ° C before use.
- glycolic acid The cultures for the production of glycolic acid are carried out in an M9 mineral medium (Table 12) containing a carbon source (glucose, xylose or arabinose) at a concentration of 10 g / L or 20 g / L and traces of LB so to reduce the latency phase (2 g / L tryptone, 1 g / L of yeast extracts and 1 g / L of NaCl).
- a carbon source glucose, xylose or arabinose
- the M9 medium with LB destroys is supplemented with 500 mM L-phenylalanine, 250 pM L-tyrosine, 200 pM L- tryptophan, 6 pM p-aminobenzoate, 6 pM p-hydroxydenzoate and 280 pM from shikimate.
- the medium was made up with 0.4 g / L of malic acid adjusted to pH 7 with KOH beforehand.
- the medium is buffered by the addition of 20 g / L of 3- (N-morpholino) propanesulfonic acid (MOPS) at pH 7 and then filtered through membranes at 0.2 ⁇ m to obtain a sterile medium.
- MOPS 3- (N-morpholino) propanesulfonic acid
- the appropriate antibiotics were added to the medium (ampicillin 100 pg / mL, kanamycin 50 pg / mL, chloramphenicol 25 pg / mL).
- kanamycin 50 pg / mL kanamycin 50 pg / mL
- chloramphenicol 25 pg / mL chloramphenicol 25 pg / mL
- All products have been ordered from Sigma.
- the cultures are placed in a shaker (Infors) at 200 rpm, 37 ° C. for the time of the experiment.
- the strains are taken up from a glycerol stock stored at -80 ° C in 10 mL of LB, at 37 ° C overnight.
- the precultures are centrifuged the following day at 4000 rpm for 5 min and resuspended in 20 mL of 1% M9 xylose or arabinose medium in 100 mL Erlenmeyer flasks.
- An adaptation phase lasting 24 hours allows the strains to adapt to the use of pentose.
- the cells are then centrifuged (4000 rpm, 5 min) and taken up again in the final culture conditions: at an initial ⁇ qeoo of 0.5 in 50 ml of the medium of composition identical to that used during the adaptation phase (M9 xylose or arabinose 1%). 250 ml culture flasks with baffles are used for optimal oxygenation. The cultures are agitated at 200 rpm, at 37 ° C.
- the GA23 strain is taken up from a glycerol stock stored at -80 ° C in 10 mL of LB containing the following antibiotics: tetracycline (10 pg / ml), kanamycin (50 pg / ml) and chloramphenicol (25 pg / ml). The preculture is incubated at 37 ° C, 200 rpm overnight.
- the process is divided into 2 phases: a growth phase dedicated to the production of biomass and a production phase dedicated to the production of glycolic acid.
- the growth phase is carried out in 50 mL of M9 medium at pH 7 containing 1.5 g / L of xylose, 5 g / L of succinate, 73 mg / L of L-methionine, 73 mg / L of L-tryptophan and lg / L of casamino acids inoculated with the preculture at an ⁇ qeoo of 0.2 in a 250 ml baffled erlenmeyer flask.
- the cells After 24 hours of culture at 37 ° C., 200 rpm, the cells are centrifuged, washed and taken up in 50 ml of M9 at pH 7 containing 10 g / L of lignocellulosic sugar (glucose, xylose or arabinose), 73 mg / L of L-methionine, 73 mg / L of L-tryptophan and lg / L of casamino acids in a 250 ml baffled Erlenmeyer flask.
- the middle used for production is devoid of succinate and does not allow growth, the carbon source (glucose, xylose or arabinose) is used for the production of glycolic acid.
- the production phase lasts 48 hours at 37 ° C, 200 rpm, samples are taken regularly in order to measure the pH, the optical density and follow the sugar consumption and the production of GA by HPLC.
- the LB precultures of the strains containing a vector with an inducible promoter are cultured at an initial ⁇ qeoo of 0.1 in 30 ml of 1% M9 glucose.
- ⁇ qeoo reaches 0.6
- 0.1 mM of IPTG are added to induce the expression of genes under the control of the promoter inducible to IPTG (plac or ptac).
- the cells are centrifuged, washing with sterile water makes it possible to remove the traces of glucose, and resuspended in 20 ml of the medium chosen for the study (M9 xylose or arabinose 1%, 0.1 mM IPTG ) for the adaptation phase.
- the rest of the culture protocol is identical to that for the strains with constitutive promoters. iii. Fermentation process in 2L BIOSTAT ® B Startorius bioreactor
- the culture medium M9 (Table 12) contains 10 g / L of xylose or glucose, as well as LB (10%) and 0.4 g / L of malic acid.
- the cultures were carried out at 37 ° C., with a agitation (300-1500 rpm) and aeration keeping the dissolved oxygen above 20% of the air flow.
- the pH was maintained at 7 with a basic KOH solution.
- the culture was carried out in batch mode for 40 hours.
- the HPLC system is equipped with a cation exchange column (Aminex, HPX87H - 300 x 7.8 mm, 9 pm, BioRad), an automatic injector (WPS-3000RS, Dionex), an IR detector (RID 10A, Shimadzu) and a UV detector (SPD-20A, Shimadzu).
- the mobile phase is a 1.25 mM sulfuric acid solution at a flow rate of 0.5 mL / min.
- the samples are kept at 4 ° C. and the injection of 20 ⁇ l is carried out in the column at 35 ° C.
- the inventors have demonstrated that the overexpression in E. coli of the 3 enzymes of the non-natural pathway according to the invention, ie arabinose-5-phosphate isomerase (KdsD), fructose-6-phosphate aldolase (FSA) and aldehyde dehydrogenase (AldA) is necessary and sufficient for the synthesis of glycolic acid from lignocellulosic monosaccharides.
- KdsD arabinose-5-phosphate isomerase
- FSA fructose-6-phosphate aldolase
- AldA aldehyde dehydrogenase
- transketolases are major enzymes in the pentose phosphate pathway. In their absence, growth on pentose is impossible, the pentose phosphate intermediates (D-ribose-5-phosphate and D-Xylulose-5-phosphate) accumulate and cannot be converted into glyceraldehyde-3phosphate for growth [38] This strain needs the non-natural route according to the invention to grow from pentoses.
- the 3 genes were cloned into operons and expressed in a strain of f. coli MG1655 AtktA AtktB AglcD. To do this, an expression system with two vectors was used, the latter having been accepted by the strain without causing deleterious modifications for the expression of the synthetic route according to the invention.
- the synthetic pathway expression system according to the invention therefore comprises a vector with a medium number of copies carrying kdsD-fsa co-expressed with a vector with a low number of copies carrying aldA.
- Expression systems 9 and 23 were transformed into the strain of f. coli WC3G AgapA AglcD AarcA AmgsA AfucA Apkf proD-galP (GA00) designed for the production of glycolic acid from hexoses and pentoses with optimal carbon conservation, generating strains GA09 and GA23, respectively.
- the bacterial cultures were carried out in an Erlenmeyer flask at 37 ° C. on D-glucose, L-arabinose and D-xylose for 46 h. Overexpression of the kdsD, fsa and aldA genes is necessary for the production of glycolic acid.
- the GA00 strain with the constitutive expression system No. 23 or GA23 showed a production of glycolic acid from D-glucose (0.29 g / L), D-xylose (0.41 g / L) and L-arabinose (0.07 g / L).
- the production of glycolic acid with the inducible expression system No. 9 or GA09 is less important on D-glucose (0.1 g / L), D-xylose (0.05 g / L) and L-arabinose ( 0.03 g / L) ( Figure 9).
- the expression system comprising constitutive promoters is favorable.
- the yield of the GA23 strain in glycolic acid is 0.09 g / g (0.21 mol / mol) from glucose, 0.18 g / g (0.36 mol / mol) from xylose and 0 , 16 g / g (0.32 mol / mol) from arabinose.
- the yield of the GA23 strain in glycolic acid is 0.096 g / g
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FR1856511A FR3083804B1 (fr) | 2018-07-13 | 2018-07-13 | Micro-organismes et procede pour la production d'acide glycolique a partir de pentoses et d'hexoses |
PCT/FR2019/051760 WO2020012138A1 (fr) | 2018-07-13 | 2019-07-12 | Micro-organismes et procédé pour la production d'acide glycolique à partir de pentoses et d'hexoses |
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BR112021000517A2 (pt) | 2021-04-06 |
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