WO2010003463A1 - Procédé de polymérisation de l’acide glycolique avec des micro-organismes - Google Patents

Procédé de polymérisation de l’acide glycolique avec des micro-organismes Download PDF

Info

Publication number
WO2010003463A1
WO2010003463A1 PCT/EP2008/059067 EP2008059067W WO2010003463A1 WO 2010003463 A1 WO2010003463 A1 WO 2010003463A1 EP 2008059067 W EP2008059067 W EP 2008059067W WO 2010003463 A1 WO2010003463 A1 WO 2010003463A1
Authority
WO
WIPO (PCT)
Prior art keywords
microorganism
glycolic acid
gene
coa
glycolyl
Prior art date
Application number
PCT/EP2008/059067
Other languages
English (en)
Inventor
Philippe Soucaille
Wanda Dischert
Original Assignee
Metabolic Explorer
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Metabolic Explorer filed Critical Metabolic Explorer
Priority to PCT/EP2008/059067 priority Critical patent/WO2010003463A1/fr
Priority to US13/003,297 priority patent/US20110118434A1/en
Priority to JP2011517171A priority patent/JP2011527367A/ja
Priority to KR1020117003130A priority patent/KR20110033265A/ko
Priority to CA2730220A priority patent/CA2730220A1/fr
Priority to MX2011000351A priority patent/MX2011000351A/es
Priority to RU2011103721/10A priority patent/RU2011103721A/ru
Priority to CN2009801354481A priority patent/CN102171356A/zh
Priority to PCT/EP2009/058836 priority patent/WO2010004032A1/fr
Priority to EP09793964A priority patent/EP2310518A1/fr
Priority to ARP090102633A priority patent/AR072501A1/es
Publication of WO2010003463A1 publication Critical patent/WO2010003463A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C12P7/625—Polyesters of hydroxy carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/34—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from hydroxy compounds or their metallic derivatives
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/02—Phosphotransferases with a carboxy group as acceptor (2.7.2)
    • C12Y207/02007—Butyrate kinase (2.7.2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y208/00—Transferases transferring sulfur-containing groups (2.8)
    • C12Y208/03—CoA-transferases (2.8.3)
    • C12Y208/03008—Acetate CoA-transferase (2.8.3.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y602/00—Ligases forming carbon-sulfur bonds (6.2)
    • C12Y602/01—Acid-Thiol Ligases (6.2.1)
    • C12Y602/01001—Acetate-CoA ligase (6.2.1.1)

Definitions

  • the present invention relates to a method for making polygly colic acid polymers called PGA. More specifically, the invention relates to a method comprising the steps of:
  • a genetically engineered microorganism with a suitable carbon source, including or not gly colic acid, said microorganism expressing a gene encoding an enzyme that converts gly co late into glycolyl-CoA and a gene encoding an enzyme involved in PHA synthesis and
  • PLA and PGA polymers are biodegradable thermoplastic materials, with a broad range of industrial and biomedical applications (Williams and Peoples, 1996, CHEMTECH 26, 38-44).
  • polyesters play important roles not only as industrial plastics but also as medical biopolymers in applications such as drug delivery carriers (Drug delivery and targeting. Nature 392, 5-10 (1998), hanger, R.), biomaterial scaffolds and medical devices (Biodegradable polyesters for medical and ecological applications. Macromol. Rapid Commum. 21, 117-132 (2000), Ikada, Y. & Tsuji, H.; Sterilization, toxicity, biocompatibility and clinical applications of polylactic acid/polygly colic acid copolymers. Biomaterials 17, 93-102 (1996), Athanasiou, K. A. et at).
  • PGA is a polyester resin which has good properties: a very high gas impermeability even under 80% humidity, biodegradability, high mechanical strength, and good moldability ⁇ Poly (glycolic acid) In polymer data handbook (ed. Mark, J. E.) 566-569 (Oxford University Press, New York, 1999) Lu, L; & Mikos, A. G). This unique combination of properties makes PGA ideally suited for high performance packaging and industrial applications.
  • PET polyethylene terephthalate
  • PGA is being prepared by two different chemical routes, either the ring- opening polymerization of cyclic diesters or the poly condensation of ⁇ -hydroxycarboxylic acids.
  • Ring-opening polymerization of cyclic diesters is in three steps: (i) poly condensation of ⁇ -hydroxycarboxylic acids, (ii) the synthesis of cyclic diesters by a thermal unzipping reaction and (iii) ring-opening polymerization of the cyclic diester (Preparative Methods of Polymer Chemistry 2 nd edition, Interscience Publishers Inc, New York 1963, Sorensen, W. R. & Campbell, T. W.; Controlled Ring-opening Polymerization of Lactide and Glycolide. Chem. Rev.
  • low-molecular- weight PGA can be produced by the direct polycondensation of glycolic acid.
  • the attainment of only low-molecular- weight polymers is largely due to the difficulty in removing water, the by-product during polymerization, which favors depolymerization (Synthesis of polylactides with different molecular weights. Biomaterials 18, 1503-1508 (1997), Hyon, S. -H. et al.,). Therefore, ring-opening polymerization of cyclic diesters using coordination initiators is preferred for the synthesis of high- molecular- weight polymers.
  • PHAs - are stored as intracellular granules as a result of a metabolic stress upon imbalanced growth due to a limited supply of an essential nutrient and the presence of an excess of a carbon source (Lenz and Marchessault 2005; Lenz 1993; Sudesh et al., 2000; Sudesh and Doi 2005; Steinb ⁇ chel and F ⁇ chtenbusch 1998; Steinb ⁇ chel and Valentin 1995; Steinb ⁇ chel 1991).
  • PHAs are naturally synthesized by a wide range of different Gram-positive and Gram-negative bacteria, as well as by some Archaea. PHAs have attracted considerable attention in recent decades due to similarity in the physical properties of this biopolymer to conventional petrochemical-based polypropylene in terms of their tensile strength and stiffness (Sudesh et al., 2000). Unlike conventional plastics, however, PHAs are biodegradable and recyclable in nature thus, making this class of polymer friendly to the environment.
  • PHAs Two types according to the length of the side chain are distinguished.
  • One type is consisting of short-chain-length hydroxyalkanoic acids, sc/PHA, with short alkyl side chains (3-5 carbon atoms) that are produced by Ralstonia eutropha (Lenz and Marchessault 2005).
  • the second type is consisting of medium-chain-length hydroxyalkanoic acids, mc/PHA, with long alkyl side chains (6-14 carbon atoms) that are produced by Pseudomonas oleovorans and other Pseudomonas (Timm and Steinb ⁇ chel, 1990) (Nomura, C. T. & Taguchi, S., 2007; Steinb ⁇ chel, A. & Hazer, B., 2007). Although the most well-studied PHA is poly(3-hydroxybutyrate) (PHB), a polymer of 3-hydroxybutyrate (3HB), there are over 150 constituents monomers (Steinb ⁇ chel A. Valentin AE.
  • polyester synthases are key enzymes of polyester biosynthesis and catalyse the conversion of (i?)-(>3)-hydroxyacyl-CoA thioesters to polyesters with the concomitant release of CoA. These polyester synthases have been biochemically characterized. An overview of these recent findings is provided in (Rehm, 2003). There are 4 major classes of PHA synthases according to their sequence, their substrate specificity, and their subunit composition (Rhem B. H. A. Biochem J.
  • PHA synthase synthesizes PHA using (>3)-hydroxyacyl-CoA as a substrate. Therefore, the first step of polymerization is the obtention of (>3)-hydroxyacyl-coA thioesters, substrates of the synthases. Accordingly, conversion of hydroxy acid to (R)- (>3)-hydroxyacyl-CoA thioesters is an essential step for the biosynthesis of polyesters.
  • the following enzymes are known as enzymes capable of generating 3- hydroxyacyl-CoA; ⁇ -ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), cloned from Ralstonia eutropha, 3-hydroxydecanoyl-ACP:CoA transferase (PhaG) cloned from Pseudomonas, (R)-specific enoyl-CoA hydratase (PhaJ) derived from Aeromonas caviae and Pseudomonas aeruginosa (Fukui et al., J. Bacteriol.
  • the propionyl coenzymeA synthetase encoding gene from Salmonella enterica was cloned in 2000 and named PrpE; see for reference (Valentin et al., 2000).
  • Reported substrates of this enzyme are propionate, acetate, 3-hydroxypropionate, and butyrate. This enzyme catalyzes the transformation of these substrates into their corresponding coenzyme A esters.
  • this enzyme is co-expressed with a PHA synthase from Ralstonia eutropha in a recombinant E. coli, formation of a PHA copolymer is observed.
  • acetyl-coA synthetase encoding gene from Escherichia coli was cloned in 2006 and named acs; see for reference (Lin et al., 2006).
  • this enzyme reduces the acetate accumulation into the microorganism, by transforming said acetate into acetyl-coA.
  • US 2007/0277268 (Cho et al.) relates the bioproduction of polylactate (PLA) or its copolymers by cells or plants.
  • WO 2004/038030 shows the formation of co-polymers containing monomers of glycolyl-CoA and at least one other monomer selected from the group consisting of 3-hydroxybutyric acid, 3-hydroxypropionic acid, 3-hydroxyvaleric acid, etc....
  • the substrate glycolyl-CoA is obtained via the 4-hydroxybutyryl-CoA molecule and a reaction requiring FadE, AtoB and thiolase II.
  • PGA gly colic acid
  • inventors have developed a method to produce high-molecular- weight PGA using microorganisms. As disclosed herein, the inventors describe that polyglycolic acid homopolymers is produced by culturing recombinant microorganisms transformed with a PHA synthase gene and a gene encoding an enzyme that converts glycolate into gly co IyI- CoA, in a production medium containing a suitable carbon source. DESCRIPTION OF THE INVENTION
  • This method is based on the use of a recombinant microorganism, expressing: 1. a gene encoding for an heterologous polyhydroxyalkanoate (PHA) synthase, and 2. a gene encoding for an enzyme transforming the glycolic acid into glycolyl-coA.
  • PHA polyhydroxyalkanoate
  • FIG. 1 shows the successive reactions for production of polyglycolic acid polymer, PGA.
  • the first reaction is the formation of glycolyl-CoA, substrate of the second reaction of polymerisation catalyzed by the PHA synthase.
  • FIG. 2 is a schematic diagram showing the pathway for synthesizing polyglycolate using cells cultivated on a medium containing glucose plus glycolate.
  • FIG. 3 is a schematic diagram showing the pathway for synthesizing polyglycolate using cells cultivated on a medium containing glucose without any exogenous glycolate.
  • the present invention is related to a method for obtaining the polymerisation of glycolic acid into PGA with a microorganism, comprising the steps of: - cultivating a microorganism expressing a gene encoding for an heterologous polyhydroxyalkanoate (PHA) synthase, in a medium comprising a carbon source, and recovering the polymerised glycolic acid (PGA), wherein the microorganism further expresses a gene encoding for an enzyme transforming the glycolic acid into glycolyl-coA.
  • PHA polyhydroxyalkanoate
  • PGA polymerised glycolic acid
  • polymerization or “homopolymerization” means a chemical reaction in which the molecules of a monomer are linked together to form large molecules whose molecular weight is a multiple of that of the original substance.
  • PGA designates the polyglycolic acid, also called polyglycolate consisting of glycolic acid-recurring unit represented on the Figure n°l and by the formula I below: - ( - O - CH 2 - CO - ) - PGA is a homopolymer comprising at least 55wt.% of the above-mentioned glycolic acid-recurring unit (also called glycolate).
  • the content of the above-mentioned glycolic acid-recurring unit in the PGA resin is at least 55 wt.%, preferably at least 70 wt.%, more preferably 90 wt.%.
  • PGA may preferably have a weight-average molecular weight in a range of 10,000
  • the terms 'culture' or 'fermentation' are used interchangeably to denote the growth of bacteria on an appropriate growth medium containing a carbon source.
  • the sentence "recovering the polymerised glycolic acid from the culture medium” designates the action of recovering PGA such as well known by the man skilled in the art.
  • polymer substance accumulated in the strains is recovered using chloroform (Lageveen et al, 1988; Amara et al, 2002).
  • PGA is extracted from lyophilized cells using chloroform extraction and subsequently precipitated in ethanol. The precipitate is obtained by centrifugation, dissolved in chloroform and precipitated again in order to highly purified PGA. Polymers are further analyzed by NMR.
  • microorganism designates a bacterium, yeast or fungus.
  • the microorganism is selected among Enterobacteriaceae, Bacillaceae, Streptomycetaceae and Corynebacteriaceae. More preferentially, the microorganism is a species of Escherichia, Klebsiella, Pantoea, Salmonella or Corynebacterium. Even more preferentially, the microorganism is Escherichia coli.
  • carbon source' denotes any source of carbon that can be used by those skilled in the art to support the normal growth of a microorganism, which can be hexoses (such as glucose, galactose or lactose), pentoses, monosaccharides, disaccharides (such as sucrose, cellobiose or maltose), oligosaccharides, molasses, starch or its derivatives, hemicelluloses, glycerol and combinations thereof.
  • An especially preferred simple carbon source is glucose.
  • Another preferred simple carbon source is sucrose.
  • glycolic acid into glycolyl-CoA designates an enzyme able to activate glycolic acid molecules into glycolyl-CoA, substrate for the PHA synthase in the polymerization process.
  • the glycolic acid is produced by the same microorganism expressing genes encoding a PHA synthase and an enzyme transforming the glycolic acid into glycolyl-CoA.
  • Microorganisms producing high level of glycolic acid by fermentation from a renewable source of carbon have been previously described; see in particular WO 2007/140816 and WO 2007/141316.
  • glycolic acid producing microorganism It would be also advantageous to reduce the exportation of glycolic acid from this glycolic acid producing microorganism.
  • the man skilled in the art knows numerous means to obtain such reduction of transport of a specific metabolite, in particular reducing or inhibiting the activity and/or the expression of a transport protein, able to export glycolic acid from the microorganism to the medium.
  • the glycolic acid is provided to the microorganism exogenously in the culture medium.
  • an amount of at least 2 grams/Liter of glycolic acid is added in the culture medium, preferentially at least 10g/L.
  • the man skilled in the art will adjust the dose in a way to avoid the toxicity of high concentrations of glycolic acid, such as 30g/L.
  • the exportation of glycolic acid may be reduced or even totally prevented in the microorganism according to the invention.
  • the enzyme transforming the glycolic acid into glycolyl-CoA is chosen among acyl-CoA synthetases and acyl-CoA transferases.
  • Acyl-CoA transferases found in anaerobic bacteria are known to catalyze the formation of short- to medium-chain-length CoA-thioesters (Mack, M. and Buckel, W., 1997).
  • the enzyme transforming the glycolic acid into glycolyl-CoA is: a propionyl coenzyme A synthetase from Escherichia coli encoded by the gene prpE; or the acetyl-CoA transferase from E. coli encoded by the gene acs.
  • encoding or “coding” refer to the process by which a polynucleotide, through the mechanisms of transcription and translation, produces an amino-acid sequence.
  • the genetic code is the relation between the sequence of bases in DNA and the sequence of amino-acids in proteins.
  • One major feature of the genetic code is to be degenerate, meaning that one amino-acid can be coded by more than one triplet of bases (one "codon").
  • the direct consequence is that the same amino-acid sequence can be encoded by different polynucleotides.
  • codons can vary according to the organisms. Among the codons coding for the same amino-acid, some can be used preferentially by a given microorganism. It can thus be of interest to design a polynucleotide adapted to the codon usage of a particular microorganism in order to optimize the expression of the corresponding protein in this organism.
  • genes and proteins are identified using the denominations of the corresponding genes in E. coli. However, and unless specified otherwise, use of these denominations has a more general meaning according to the invention and covers all the corresponding genes and proteins in other organisms, more particularly microorganisms.
  • PFAM protein families database of alignments and hidden Markov models; http://www.sanger.ac.uk/Software/Pfam/) represents a large collection of protein sequence alignments. Each PFAM makes it possible to visualize multiple alignments, see protein domains, evaluate distribution among organisms, gain access to other databases, and visualize known protein structures.
  • COGs clusters of orthologous groups of proteins; http ://www.ncbi.nlm.nih. gov/COG/ are obtained by comparing protein sequences from 66 fully sequenced genomes representing 30 major phylogenic lines. Each COG is defined from at least three lines, which permits the identification of former conserved domains.
  • the means of identifying homologous sequences and their percentage homologies are well known to those skilled in the art, and include in particular the BLAST programs, which can be used from the website http://www.ncbi.nlm.nih.gov/BLAST/ with the default parameters indicated on that website.
  • the sequences obtained can then be exploited (e.g., aligned) using, for example, the programs CLUSTALW (http://www.ebi.ac.uk/clustalw/) or MULTALIN (http://prodes.toulouse.inra.fr/multalin/cgi-bin/multalin.pl), with the default parameters indicated on those websites.
  • the present invention is also related to an expression cassette comprising a polynucleotide encoding an enzyme transforming the gly colic acid into glycolyl-CoA under the control of regulatory elements functional in a host microorganism.
  • expression refers to the transcription and translation of a gene sequence leading to the generation of the corresponding protein, product of the gene.
  • the gene encoding for an enzyme transforming the glycolic acid into glycolyl-CoA is overexpressed into the microorganism.
  • the terms "increased expression” “enhanced expression” or “overexpression” are used interchangeably in the text and have similar meaning, i.e. that the transcription and translation of the gene is increased compared to a non-recombinant microorgansim, leading to an increased amount of enzyme into the cell.
  • the expert in the field knows different ways to manipulate genes expression.
  • the gene may be expressed using promoters with different strength, which may be inducible. These promoters may be homologous or heterologous. The man skilled in the art knows which promoters are the most convenient, for example, promoters Ptrc, Ptac, Plac or the lambda promoter cl are widely used.
  • the gene may be expressed by a plasmid or vector introduced into the microorganism.
  • Said microorganism is then said a "host microorganism", referring to a microorganism able to receive foreign or heterologous genes or extra copies of its own genes and able to express those genes to produce an active protein product.
  • transformation refers to the introduction of new genes or extra copies of existing genes into a host organism.
  • a method for transferring DNA into a host organism is electroporation.
  • transformation vector refers to any vehicle used to introduce a polynucleotide in a host organism.
  • vehicle can be for example a plasmid, a phage or other elements known from the expert in the art according to the organism used.
  • the transformation vector usually contains in addition to the polynucleotide or the expression cassette other elements to facilitate the transformation of a particular host cell.
  • An expression vector comprises an expression cassette allowing the suitable expression of the gene borne by the cassette and additional elements allowing the replication of the vector into the host organism.
  • An expression vector can be present at a single copy in the host organism or at multiple copies. The man skilled in the art knows different types of plasmids that differ with respect to their origin of replication and thus their copy number in the cell.
  • the present invention provides a transformation vector comprising a gene encoding for an enzyme transforming the glycolic acid into glycolyl-coA.
  • said gene may be integrated into the chromosome of the microorganism.
  • Another mean to obtain an overexpression of the genes is to modify the expression or regulation of the elements stabilizing the corresponding messenger RNA (Carrier et al. Biotechnol Bioeng. 59:666-72, 1998) if translation of the mRNA is optimized, then the amount of available enzyme is increased.
  • the recombinant microorganism used in the invention also expresses a gene encoding for a polyhydroxyalkanoate synthase.
  • Four major classes of PHA can be distinguished (Rhem, B., 2003).
  • Class I and Class II PHA synthases comprise enzymes consisting of only one type of subunit (PhaC). According to their in vivo and in vitro specificity, class I PHA synthases (e.g. in Ralstonia eutropha) preferentially utilize CoA- thioester of various hydroxy fatty acids comprising 3 to 5 carbons atoms, whereas class II PHA synthases (e.g. in Pseudomonas aeruginosa) preferentially utilize CoA-thioester of various hydroxy fatty acids comprising 6 to 14 carbon atoms. Class III synthases (e.g.
  • Allochromatium vinosum comprises enzymes consisting of two different types of subunits: the PhaC and the PhaE subunits. These PHA synthases prefer CoA-thioesters of hydroxy fatty acids comprising 3 to 5 carbons atoms. Class IV PHA synthases (e.g. in Bacillus megaterium) resemble the class III PHA synthases, but PhaE is replaced by PhaR.
  • the gene encoding the heterologous PHA synthase is chosen among phaC, phaEC or phaCR, preferentially among phaC and phaEC and most preferentially the gene selected is phaC encoding an enzyme of Class I PHA synthases.
  • phaC phaC encoding an enzyme of Class I PHA synthases.
  • use of these denominations 'phaC, 'phaEC and 'phaCR' cover all the corresponding genes and proteins in other organisms, more particularly microorganisms. Indeed, using the references given in GenBank for known genes, those skilled in the art are able to determine the equivalent genes in other organisms, bacterial strains, yeasts, fungi, mammals, plants, etc.
  • the overexpression of a gene may be obtained by different ways known by the man skilled in the art; the gene may be expressed by a plasmid / expression vector introduced into the microorganism, or be integrated into the chromosome of said microorganism.
  • the recombinant microorganism used in the method also expresses genes phaP and phaR from W. eutropha encoding respectively for a phasin and its transcriptional expression regulator.
  • the phasin protein, PhaP is likely to be involved in maintenance of the optimal intracellular environment of PHA synthesis and provides guidance during the process of granule formation (Wieczorek, R. et al. 1995).
  • PhaR homologs have been investigated both in vitro and in vivo (Wieczorek R. et al. 1995 and York G.M. et al. 2002) and is proposed to function as a regulator of the ph ⁇ P transcription. Use of these denominations 'phaP' and 'phaR' cover all the corresponding genes and proteins in other microorganisms, which are incorporated herein by reference.
  • the invention is also relative to a polymerised glycolic acid (PGA) obtained by the method according to the invention.
  • PGA polymerised glycolic acid
  • the main advantage of the invention is to produce PGA in an easier and cheaper way than the chemical one that necessitates the use of glycolide, a compound difficult to produce from glycolic acid.
  • the invention is also relative to a microorganism expressing genes encoding for a heterologous PHA synthase and an enzyme transforming the glycolic acid into glycolyl- CoA.
  • said microorganism is an Enterobacteriaceae, more preferentially an Escherichia coli.
  • Table 1 sequences of the oligonucleotides used in the constructions described below.
  • the PCR fragment of acs is digested with Xbal and BgIII and cloned into the vector pSCB (Stratagene Blunt PCR Cloning Kit CAT 240207-5) resulting in plasmid pSCB- ⁇ cs.
  • the PCR fragment of prpE is digested with Xbal and BamHI and cloned into the vector pSCB resulting in plasmid pSCB-prpE.
  • the plasmid carrying the gene phaCl from Ralstonia eutropha is provided by a company that synthesizes the gene with an optimized sequence to get the best transcription rate in E.coli.
  • the relative frequency of codon use varies widely depending on the organism and organelle. Many design programs for synthetic protein coding sequences allow the choice of organism.
  • the codon usage database has codon usage statistics for many common and sequenced organisms like E.coli.
  • the synthetic gene phaCl encoding the PHA synthase is provided ready to use by the company.
  • the gene is cloned under a PtrcOl promoter with operator and RBS sequences located upstream the gene, and a terminator sequence located downstream phaCl, leading to the plasmid pUC19-Ptrc01+O-RBS-/?/z ⁇ C7re-TT02.
  • Plasmids pSCB- ⁇ cs and pSCB-prpE are digested with Xbal and Nhel and the resulting DNA fragments comprising either acs or prpE are cloned into the vector pUC19- Ptrc01+O-RBS-/?/z ⁇ C7-TT02 cut by the same restriction enzymes.
  • the resulting plasmids are named pUC19-Ptrc01+O-RBS01-/?A ⁇ Cire- ⁇ cs-TT02 and pUC19-Ptrc01+O-RBS01- phaClre-prpE-TT02.
  • Vectors pUC19-Ptrc01+O-RBS01-/?A ⁇ Cire- ⁇ cs-TT02 and pUC19-Ptrc01+O-RBS01- phaCl re-prpE '-TT '02 are introduced by electroporation into an E.coli MG1655 wild-type strain, leading to strains MG1655 (pUC19-Ptrc01+O-RBS01-/?/z ⁇ C7re- ⁇ cs-TT02) and MG1655 (pUC19-Ptrc01+O-RBS01-pAaCire-p/p£-TT02) respectively.
  • the resulting strains (FIG.
  • Vectors pUC19-Ptrc01+O-RBS01-/?A ⁇ Cire- ⁇ cs-TT02 and pUC19-Ptrc01+O-RBS01- phaCl re-prpE '-TT '02 are introduced by electroporation into an engineered E.coli strain that produces glycolic acid and which is claimed in the patents WO 2007/141316 and WO 2007/140816.
  • the resulting strains are respectively AaceB Agcl ⁇ g/cDEFGB AaIdA ⁇ zc/R Aedd-eda Apgi AudhA (pMElOl-ycdW) (p ⁇ JC ⁇ 9-Ptvc0 ⁇ +O-RBS0 ⁇ -phaClre-acs- ⁇ 02) and AaceB Agcl ⁇ g/cDEFGB AaIdA ⁇ zc/R Aedd-eda Apgi AudhA (pUC19-Ptrc01+O- RBSO 1 -phaClre-prpE- ⁇ 02).
  • the recombinant strains (FIG.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Polyesters Or Polycarbonates (AREA)
  • Biological Depolymerization Polymers (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

Cette invention concerne un procédé de production et de préparation du polyglycolate (PGA) à partir d’organismes génétiquement modifiés. L’invention concerne plus spécifiquement un procédé comprenant deux étapes : 1) la mise en culture, dans un milieu contenant ou non de l’acide glycolique, de micro-organismes exprimant un gène codant une enzyme convertissant le glycolate en glycolyl-CoA, et un gène codant la polyhydroxyalkanoate (PHA) synthase qui utilise le glycolyl-CoA comme substrat, 2) la récupération du polymère polyglycolate.
PCT/EP2008/059067 2008-07-11 2008-07-11 Procédé de polymérisation de l’acide glycolique avec des micro-organismes WO2010003463A1 (fr)

Priority Applications (11)

Application Number Priority Date Filing Date Title
PCT/EP2008/059067 WO2010003463A1 (fr) 2008-07-11 2008-07-11 Procédé de polymérisation de l’acide glycolique avec des micro-organismes
US13/003,297 US20110118434A1 (en) 2008-07-11 2009-07-10 Method for polymerising glycolic acid with microorganisms
JP2011517171A JP2011527367A (ja) 2008-07-11 2009-07-10 微生物を用いたグリコール酸の重合方法
KR1020117003130A KR20110033265A (ko) 2008-07-11 2009-07-10 미생물에 의해 글리콜산을 중합하는 방법
CA2730220A CA2730220A1 (fr) 2008-07-11 2009-07-10 Procede pour la polymerisation d'acide glycolique avec des micro-organismes
MX2011000351A MX2011000351A (es) 2008-07-11 2009-07-10 Metodo para la polimerizacion del acido glicolico con microorganismos.
RU2011103721/10A RU2011103721A (ru) 2008-07-11 2009-07-10 Способ полимеризации гликолевой кислоты микроорганизмами
CN2009801354481A CN102171356A (zh) 2008-07-11 2009-07-10 用微生物聚合羟基乙酸的方法
PCT/EP2009/058836 WO2010004032A1 (fr) 2008-07-11 2009-07-10 Procédé pour la polymérisation d'acide glycolique avec des micro-organismes
EP09793964A EP2310518A1 (fr) 2008-07-11 2009-07-10 Procédé pour la polymérisation d'acide glycolique avec des micro-organismes
ARP090102633A AR072501A1 (es) 2008-07-11 2009-07-13 Procedimiento para la polimerizacion del acido glicolico con microorganismos

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/EP2008/059067 WO2010003463A1 (fr) 2008-07-11 2008-07-11 Procédé de polymérisation de l’acide glycolique avec des micro-organismes

Publications (1)

Publication Number Publication Date
WO2010003463A1 true WO2010003463A1 (fr) 2010-01-14

Family

ID=39744968

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/EP2008/059067 WO2010003463A1 (fr) 2008-07-11 2008-07-11 Procédé de polymérisation de l’acide glycolique avec des micro-organismes
PCT/EP2009/058836 WO2010004032A1 (fr) 2008-07-11 2009-07-10 Procédé pour la polymérisation d'acide glycolique avec des micro-organismes

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/EP2009/058836 WO2010004032A1 (fr) 2008-07-11 2009-07-10 Procédé pour la polymérisation d'acide glycolique avec des micro-organismes

Country Status (8)

Country Link
JP (1) JP2011527367A (fr)
KR (1) KR20110033265A (fr)
CN (1) CN102171356A (fr)
AR (1) AR072501A1 (fr)
CA (1) CA2730220A1 (fr)
MX (1) MX2011000351A (fr)
RU (1) RU2011103721A (fr)
WO (2) WO2010003463A1 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8852919B2 (en) * 2011-11-17 2014-10-07 Rheonix, Inc. Microfluidic apparatus, method, and applications
FR3033168B1 (fr) * 2015-02-27 2017-03-10 Afyren Procede de production de polyhydroxyalcanoates a partir de precurseurs obtenus par fermentation anaerobie a partir de biomasse fermentescible
JP2018527937A (ja) * 2015-09-10 2018-09-27 サイエンティスト・オブ・フォーチュン・ソシエテ・アノニム ２−ヒドロキシアルデヒドからのアシルリン酸の酵素的産生
CN116814615B (zh) * 2023-08-30 2024-01-19 清华大学 一种细胞形态纤长化的重组菌株及其构建方法和应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004038030A2 (fr) * 2002-05-10 2004-05-06 Metabolix, Inc. Polymere bioabsorbable contenant des monomeres 2-hydroxyacides
US20070277268A1 (en) * 2005-05-24 2007-11-29 Lg Chem, Ltd. Cells or plants producing polylactate or its copolymers and uses thereof
WO2007141316A2 (fr) * 2006-06-09 2007-12-13 Metabolic Explorer Production d'acide glycolique par fermentation à partir de ressources renouvelables

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004038030A2 (fr) * 2002-05-10 2004-05-06 Metabolix, Inc. Polymere bioabsorbable contenant des monomeres 2-hydroxyacides
US20070277268A1 (en) * 2005-05-24 2007-11-29 Lg Chem, Ltd. Cells or plants producing polylactate or its copolymers and uses thereof
WO2007141316A2 (fr) * 2006-06-09 2007-12-13 Metabolic Explorer Production d'acide glycolique par fermentation à partir de ressources renouvelables

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HENRY LIN ET AL: "Acetyl-CoA synthetase overexpression in Escherichia coli demonstrates more efficient acetate assimilation and lower acetate accumulation: a potential tool in metabolic engineering", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, SPRINGER, BERLIN, DE, vol. 71, no. 6, 22 February 2006 (2006-02-22), pages 870 - 874, XP019421965, ISSN: 1432-0614 *
VALENTIN HENRY E ET AL: "Application of a propionyl coenzyme A synthetase for poly(3-hydroxypropionate-co-3-hydroxybutyrate) accumulation in recombinant Escherichia coli.", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 66, no. 12, December 2000 (2000-12-01), pages 5253 - 5258, XP002497013, ISSN: 0099-2240 *
YORK GREGORY M ET AL: "The Ralstonia eutropha PhaR protein couples synthesis of the PhaP phasin to the presence of polyhydroxybutyrate in cells and promotes polyhydroxybutyrate production", JOURNAL OF BACTERIOLOGY, vol. 184, no. 1, January 2002 (2002-01-01), pages 59 - 66, XP002497014, ISSN: 0021-9193 *
YUAN W ET AL: "Class I and III Polyhydroxyalkanoate Synthases from Ralstonia eutropha and Allochromatium vinosum: Characterization and Substrate Specificity Studies", ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, ACADEMIC PRESS, US, vol. 394, no. 1, 12 September 2001 (2001-09-12), pages 87 - 98, XP002979706, ISSN: 0003-9861 *

Also Published As

Publication number Publication date
JP2011527367A (ja) 2011-10-27
CA2730220A1 (fr) 2010-01-14
MX2011000351A (es) 2011-02-22
WO2010004032A1 (fr) 2010-01-14
AR072501A1 (es) 2010-09-01
CN102171356A (zh) 2011-08-31
KR20110033265A (ko) 2011-03-30
RU2011103721A (ru) 2012-08-20

Similar Documents

Publication Publication Date Title
Liu et al. Biosynthesis of poly (3-hydroxydecanoate) and 3-hydroxydodecanoate dominating polyhydroxyalkanoates by β-oxidation pathway inhibited Pseudomonas putida
Możejko-Ciesielska et al. Bacterial polyhydroxyalkanoates: Still fabulous?
Steinbüchel et al. Bacterial and other biological systems for polyester production
Reinecke et al. Ralstonia eutropha strain H16 as model organism for PHA metabolism and for biotechnological production of technically interesting biopolymers
Meng et al. Synthetic biology of polyhydroxyalkanoates (PHA)
Behera et al. Polyhydroxyalkanoates, the bioplastics of microbial origin: Properties, biochemical synthesis, and their applications
Li et al. The production of polyhydroxyalkanoates in recombinant Escherichia coli
Zhou et al. Production of 3-hydroxypropionate homopolymer and poly (3-hydroxypropionate-co-4-hydroxybutyrate) copolymer by recombinant Escherichia coli
EP2376622B1 (fr) Procédé et compositions écologiques pour la production de produits chimiques à base de poly(5hv) et de 5 carbones
US6329183B1 (en) Polyhydroxyalkanoate production from polyols
US8956835B2 (en) Methods for producing polyhydroxyalkanoates from biodiesel-glycerol
CN102307988B (zh) 能够产生聚乳酸或聚乳酸共聚物的重组富氧罗尔斯通氏菌和使用其制备聚乳酸或聚乳酸共聚物的方法
Zhang et al. Engineering of Ralstonia eutropha for the production of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) from glucose
US20130017583A1 (en) Methods for producing polyhydroxyalkanoate copolymer with high medium chain length monomer content
JP2004512825A (ja) ポリオールからのポリヒドロキシルアルカノエートの生成
Yañez et al. Beyond intracellular accumulation of polyhydroxyalkanoates: chiral hydroxyalkanoic acids and polymer secretion
JP5541809B2 (ja) ポリラクテートとその共重合体生成能が改善された変異微生物を利用したポリラクテートとその共重合体の製造方法
Wang et al. Polyhydroxyalkanoates: sustainability, production, and industrialization
Yang et al. Microbial production of lactate‐containing polyesters
Al-Kaddo et al. Production of P (3HB-co-4HB) copolymer with high 4HB molar fraction by Burkholderia contaminans Kad1 PHA synthase
Kutralam-Muniasamy et al. Genome characteristics dictate poly-R-(3)-hydroxyalkanoate production in Cupriavidus necator H16
Shozui et al. Establishment of a metabolic pathway to introduce the 3-hydroxyhexanoate unit into LA-based polyesters via a reverse reaction of β-oxidation in Escherichia coli LS5218
CN116240155A (zh) 一种生产聚（羟基脂肪酸-co-乳酸）的重组菌及其应用
WO2010003463A1 (fr) Procédé de polymérisation de l’acide glycolique avec des micro-organismes
Liu et al. Biopolymer production in microbiology by application of metabolic engineering

Legal Events

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

Ref document number: 08786067

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08786067

Country of ref document: EP

Kind code of ref document: A1