EP0781339A2 - Procede de preparation d'acides gras polyhydroxyliques et souches bacteriennes recombinees pour la mise en uvre du procede - Google Patents

Procede de preparation d'acides gras polyhydroxyliques et souches bacteriennes recombinees pour la mise en uvre du procede

Info

Publication number
EP0781339A2
EP0781339A2 EP95931149A EP95931149A EP0781339A2 EP 0781339 A2 EP0781339 A2 EP 0781339A2 EP 95931149 A EP95931149 A EP 95931149A EP 95931149 A EP95931149 A EP 95931149A EP 0781339 A2 EP0781339 A2 EP 0781339A2
Authority
EP
European Patent Office
Prior art keywords
acid
mol
polyhydroxy fatty
approx
hydroxyhexanoic
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP95931149A
Other languages
German (de)
English (en)
Inventor
Alexander Steinbüchel
Matthias Liebergesell
Henry Valentin
Andreas Pries
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Buck Chemisch Technische Werke GmbH and Co
Buck Werke GmbH and Co
Monsanto Co
Original Assignee
Buck Chemisch Technische Werke GmbH and Co
Buck Werke GmbH and Co
Monsanto Co
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 Buck Chemisch Technische Werke GmbH and Co, Buck Werke GmbH and Co, Monsanto Co filed Critical Buck Chemisch Technische Werke GmbH and Co
Publication of EP0781339A2 publication Critical patent/EP0781339A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
    • C07C59/01—Saturated compounds having only one carboxyl group and containing hydroxy or O-metal groups
    • C07C59/10—Polyhydroxy carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C12P7/625—Polyesters of hydroxy carboxylic acids

Definitions

  • the present invention relates to a process for the preparation of polyhydroxy acids according to claim 1, a recombinant bacterial strain according to claim 19, a polyhydroxy fatty acid according to claim 23 and a DNA fragment according to claim 33.
  • a major disadvantage of organic synthetic plastics is that many of these plastics have enormous biological half-lives or cannot be disposed of in landfills or in waste incineration plants in a harmless manner, but rather aggressive gases are frequently generated, as is the case, for example of polyvinyl chloride, which releases hydrogen chloride gas during combustion.
  • a first step towards success on environmentally compatible materials was achieved with the synthetic plastics, for example with the paraffin-like polymers polyethylene, polypropylene, since these essentially only release CO 2 and water during combustion.
  • renewable raw materials such as plants that contain a lot of polysaccharide, such as potatoes, maize, wheat, beans, peas or the like, to obtain the polysaccharides that occur naturally in these plants and to use them to produce polymers that can be used in plastics technology to produce which are biodegradable.
  • hydroxy fatty acids were used as building blocks of naturally occurring polyhydroxy fatty acids (PHF) wrote.
  • the hydroxyl group of this PHF is usually in the 3 'position.
  • the aliphatic side chains are either saturated or mono- or di-unsaturated. They are thus unbranched or branched and they can be substituted with functional groups such as halogen atoms, preferably bromine, iodine, chlorine, cyano groups, ester groups, carboxyl groups or also with cyclically aliphatic and even aromatic groups.
  • the hydroxyl group is also in the 4 'or 5' position.
  • poly (3-hydroxybutyric acid) has been detected in eukaryotes as the only PHF.
  • This polyester comes in yeasts such as Saccharomyces cere visiae, various plants, for example cauliflower, various organs from animals, for example liver, and also in humans, for example in blood plasma, before [Reusch, RN 1992, Biological complexes of polyhydroxybutyrate, FEMS Microbiol. Rev. 103: 119-130].
  • the proportion of poly (3-hydroxybutyric acid) in eukaryotes is at most 0.1% by weight. Inclusions in the form of grana, as they occur in prokaryotes, are not known in eukaryotes.
  • the eukaryotic PHF are usually not in free form, but the polyester is either bound to other proteins or as a complex spanning the cytoplasmic membrane together with calcium ions and polyphosphate molecules.
  • the biosynthesis of PHF in bacteria can be divided into three phases:
  • phase I the carbon source offered to the bacteria in the medium is first absorbed into the bacterial cell.
  • Either special uptake transport systems have to exist for the corresponding carbon source or the cells are cultivated under conditions which create a certain artificial permeability of the cytoplasmic membrane for the carbon source.
  • Some non-ionic carbon sources for example fatty acids in non-dissociated form, can also get into the cells by passive diffusion.
  • phase II the absorbed carbon source is converted into a suitable substrate for the enzyme that is able to produce PHF.
  • This enzyme is commonly referred to as polyhydroxy fatty acid synthase.
  • Phase III involves the linking of monomeric precursors to the polyester. This reaction is catalyzed by the enzyme PHF synthase, which is the key enzyme in PHF biosynthesis. These enzymes are bound to the PHF-Grana and they are there on the surface. Due to the very low specificity of most of the PHF synthases investigated to date, which occur in different species, the biosynthesis of a large number of different PHFs is possible. So far, only the coenzyme A thioesters of hydroxy fatty acids have been detected as monomeric biosynthetically active precursors. As shown above, PHF synthase is the key enzyme in PHF synthesis.
  • Type I is represented by the PHF synthase from the bacterium Alcaligenes eutrophus, which has been most closely examined with regard to the PHF metabolism and has a molecular weight of approximately 63,940 and catalyzes the synthesis of PHF from short-length hydroxy fatty acids.
  • Type II is represented by the Pseudomonas oleovarans PHF synthase.
  • the size of this enzyme is similar to that of Type I PHF synthases (molecular mass 62400), but it differs considerably in terms of substrate specificity from Type I PHF synthases. Only 3-hydroxy fatty acids of medium chain length can be incorporated into PHF. In contrast, 4- and 5-hydroxyfatty acids and 3-hydroxybutyric acid are not incorporated into the biosynthesized polyesters. However, the specificity of the enzyme is still so wide that approx. 50 different 3-hydroxy fatty acids can be processed as substrates.
  • Type III is represented by Chromatium vinosum PHF synthase. In terms of substrate specificity, this enzyme is similar to Type I PHF synthases. However, it has a significantly lower molecular mass (approx. 39730) and requires a second protein in order to be catalytically active.
  • PHF poly(2-hydroxybutyric acid)
  • poly (3HB) poly (3HB)
  • copolyester poly (3-hydroxybutyric acid-co-3-hydroxyvalerianic acid) poly (3HB-co-3HV)
  • these polymers can be prepared using conventional injection molding processes , Extrusion blow molding and injection blow molding processes as well as by fiber spinning techniques.
  • the first stage is used to grow the bacterial cells to high densities and takes about 48 hours and only glucose is offered as a substrate.
  • the cells have grown into a phosphate limitation and, after a further 40 to 50 hours of cultivation, cell densities of more than 100 g of dry cell mass per liter with a PHF with glucose and with propionic acid as a precursor for the building block 3-hydroxyvaleric acid. Share of more than 70 wt .-% reached.
  • the cells are then removed using an enzyme cocktail consisting essentially of lysozyme, proteases and other hydrolyti see enzymes is treated, which releases the PHF grana.
  • the granas sediment on the bottom of the reactor and are collected, washed, dried, melted, extruded and granulated from there.
  • This PHF is currently produced with a production volume of approx. 300 tons per year.
  • poly (3HB) and poly (3HB-co-3HV) have good properties and can be processed using the methods commonly used in plastics technology, their production is still relatively expensive on the one hand and on the other hand only contains them two monomeric subunits, so that the overall properties of the resulting polymer can only be controlled by means of these two parts, and thus a fine control with regard to flexibility, processability in plastics technology systems, resistance to certain solvents etc. cannot be controlled in fine steps .
  • 3-hydroxyvaleric acid gives a PHF good flexibility and processability
  • the building block 4-hydroxyvaleric acid which can also occur in the PHF synthesized by bacteria, gives the resulting biopolymer a significantly higher degree of flexibility confers than is the case with 3-hydroxyvaleric acid alone.
  • the above object is achieved by the features of patent claim 1.
  • the object is achieved according to the features of patent claim 19.
  • the above object is achieved by the features of patent claim 23.
  • the DNA fragment according to claim 33 also achieves the above object.
  • it is possible for the first time to produce polyesters containing 4HV, starting from levulinic acid.
  • the chemical structure of levulinic acid is shown in the following formula:
  • levulinic acid is a 4-oxopentanoic acid that is easily soluble in water, alcohol and ether.
  • the PHF-free mutants GPpl04 from Pseudomonas putida (Huismann, GW, Wonink, E., Meima, R., Kazemier, W., Trpstra, P. and Witholt, B. (1991) J .Biol.Chem.
  • strains have the special property that they contain essentially exactly those DNA fragments which contain and express the genes phaE and phaC; because the gene products phaE and phaC together are able to develop a PHF synthase activity.
  • nucleotide sequences which carry the phaE and p ⁇ aC genes can additionally have common control regions, for example promoters, S / D sequences, or the like.
  • the inventors of the present invention found that the recombinant bacterial strains which contain and express at least one fragment of the Thiocapsa pfenigii PHF synthase gene are also capable of 5-hydroxyhexanoic acid, its salts, esters and lactones into a copolyester biosynthesized by these bacteria.
  • 5-Hydroxyhexanoic acid was prepared starting from 4-acetoacetic acid, which was quantitatively reduced with NaBH4.
  • 5-Hydroxyhexanoic acid (5HHx) as a building block was detected by gas chromatography after methanolysis both in lyophilized cells and in isolated and purified polyester.
  • the isolated and purified polyester was subjected to 13 C-NMR and ⁇ H-NMR analysis and the incorporation of 5 HHx was thereby confirmed.
  • analysis of the polyester accumulated by the bacterial cells showed a polyester content of over 40% by weight of the dry cell mass, a typical polymer containing: Approx. 71 mol% 3-hydroxybutyric acid, approx. 4 mol% 3-hydroxyhexanoic acid, approx. 23 mol% 5-hydroxyhexanoic acid and approx. 2 mol%. 3-hydroxyoctanoic acid.
  • 4-Hydroxyheptanoic acid was prepared by saponifying ⁇ -heptalactone with NaOH.
  • the analysis of the polymer accumulated by the recombinant cells showed a polyester content of approximately 40% by weight of the dry cell mass.
  • a typical polymer contained approx. 43 mol% 3-hydroxybutyric acid, approx. 16 mol% 3-hydroxyvaleric acid, approx. 27 mol% 3-hydroxyhexanoic acid, approx. 5 mol% 3-hydroxyheptanoic acid, approx. 6 mol%. 4-hydroxyheptanoic acid and approx. 6 mol% 3-hydroxyoctanoic acid.
  • 4-Hydroxyoctanoic acid is prepared by saponifying ⁇ -lactone with NaOH.
  • the bacterial cells accumulated the synthesized copolyester to a content of approximately 20% by weight of the dry cell mass.
  • the new copolyesters produced by means of the process according to the invention likewise had thermoplastic properties and could be processed without problems using the techniques customary in plastics technology.
  • thermoplastic copolymers which had a high degree of biocompatibility, which makes these materials appear to be applicable for use in medical technology - for example as implants - or as sutures or the like.
  • the duration of the cultivation of the microorganisms used for the purposes of the present invention depends on the culture conditions, which are mainly dependent on the temperature, the oxygen content of the medium (aerobic conditions) and the medium itself, amount of the carbon source, the mineral salts, the trace elements and / or the pH value.
  • the amount of substrate used depends on the particular microorganism. However, concentrations in the range of approx. 0.1% (w / v) to 10% (w / v) can correspond to 100g / l, in particular 0.2 (w / v) to 5% (w / v).
  • the cell harvest can generally take place during the log phase up to the stationary phase, preferably it should take place in the stationary phase.
  • the bacterial cells can either be obtained from the medium in their entirety after a single culture (batch or fed-batch process) or can be obtained continuously via a continuous culture, for example by conventional centrifugation or filtration processes.
  • the harvested cells can, if appropriate after washing, for example with a buffer, preferably with a phosphate buffer, particularly preferably with a sodium phosphate buffer in the neutral range of about pH 7.0, be frozen, lyophilized or treated by spray drying.
  • a buffer preferably with a phosphate buffer, particularly preferably with a sodium phosphate buffer in the neutral range of about pH 7.0, be frozen, lyophilized or treated by spray drying.
  • polyesters according to the invention can be obtained by known methods, preferably the solution or the extraction is carried out with organic solvents, in particular by halogenated, preferably chlorinated hydrocarbons, particularly preferably by chloroform or methylene chloride.
  • the copolyesters obtained according to the invention are easy to process as thermoplastics and can be used in a variety of ways.
  • thermoplastics for example in surgery for objects for wound closure, for example as suture material or staples or the like, as a fastening element for bones, for example fixation pins, plates, screws, dowels, as separating, filling or covering material, for example in the form of tissue, fleece, Cotton wool.
  • the polyesters according to the invention can be used in pharmaceutical galenics, for example as auxiliaries, carrier materials, release systems for pharmaceuticals or for the encapsulation and / or microencapsulation of substances and active ingredients.
  • biodegradable packaging materials such as foils, bottles, ampoules, cans, bags, boxes, boxes or the like, is possible using the present invention.
  • the advantage of preculturing the recombinant bacteria according to claim 2 in a complex medium is that a strong increase in the biomass is initially achieved in this way, in order then to biochemically stimulate the bacteria for the biosynthesis of the desired PHF.
  • An additional, growth-promoting carbon source according to claim 3 to add to the nutrient medium for the bacterial culture has the advantage that on the one hand, different subunits can be installed, and on the other hand, that the bacteria sometimes grow considerably faster and at the same time a larger amount of biosynthesize the desired PHF.
  • the measures of claim 4 have the advantage that the method of the present invention can be carried out using the methods customary in industrial biotechnology.
  • the measures of claim 5 have the advantage that an economic relationship between polyester yield and dry bacterial cell mass can be obtained, so that the yield obtained is economically worthwhile.
  • the lower limit for profitability can be in the order of approximately 30% by weight of polyester, based on the dry cell mass of the bacterial cell.
  • the measures of claim 6 have the advantage that in the case of copolyesters with at least two subunits, the chemical, biochemical and physical properties of the polyester can be adjusted in fine steps by varying the different subunits if the appropriate substrates are offered to the bacteria.
  • Allowing the recombinant bacteria to grow up with cell densities of up to 100 g dry cell mass per liter of bacterial nutrient medium according to claim 7 has the advantage that relatively small volumes contain a considerable biomass and thus significantly increase productivity with appropriate dimensioning of large-scale plants than lower cell densities.
  • the measures of claim 10 have the advantage that the entire process can be better controlled in the direction of higher yields by gradually increasing the concentration of the substrate carbon source.
  • the measures of claims 11, 12 and 13 reflect advantageous process conditions for the biotechnological production of PHF according to the present invention.
  • the measures of claims 14 and 15 have the advantages that all methods common in biotechnology for breaking down bacteria can be used to obtain the biotechnologically produced PHF. Since these are generally heavier than the surrounding nutrient medium and the cell debris, the PHF can easily be separated and obtained by accelerated sedimentation, for example by centrifugation.
  • the measures of claim 16 have the advantage that the PHF is precipitated by introducing a PHF product dissolved by means of an organic solvent into water or a lower alcohol, preferably ethanol, and a cleaning step is thereby achieved which is a recrystallization process in organic chemistry for cleaning up the desired product.
  • an enzyme cocktail according to claims 17 and 18 has the advantage that specifically the bacterial cell wall and the membrane are destroyed enzymatically, so that the grana, which contain the PHF, fall out of the cytoplasm and sediment to the bottom of the reactor.
  • proteolytic and lytic enzymes for example lysozyme or lipases
  • the entire biotechnological approach essentially consists of macromolecules - namely the desired synthesized polyester - and a large number of smaller molecules which are formed by enzymatic cleavage of the nucleic acids, proteins, glycoproteins, polysaccharides and lipids contained in the cells, as well as the cell wall and cell membrane, and which can thus be easily separated from one another, oh ne that the isolated PHF contain significant impurities from other bacterial compounds.
  • detergents are particularly advantageous here, since proteins and nucleic acids in particular are thereby still solubilized and they are then broken down by the other enzymes, as if they were suspended in the aqueous solutions as colloid particles.
  • care must be taken that detergents in which the enzymes used are still or even more actively are used.
  • Such a mild detergent is, for example, octylglucoside.
  • it is known, for example, of the V8 protease from Staphylococcus aureus that it still exhibits a strong proteolytic activity in 1 to 2% sodium dodecyl sulfate.
  • Claims 19 to 21 relate to novel recombinant bacterial strains according to the invention which contain and express the genes phaC and phaE from Thiocapsa pfennigii which are relevant for PHF synthesis.
  • the Ba / ⁇ HI fragment B28 contained in the newly constructed bacterial strains according to the invention was obtained after Ba / ⁇ HI digestion of the EcoRI fragment E156 and essentially comprises the two genes phaC and phaE.
  • the bacterial strains according to the invention provide particularly high yields of PHF.
  • Claim 23 relates to a PHF as can be obtained by a process according to one of Claims 1 to 18, in particular the following PHF or polyester or copolyester could be prepared according to Claim 24 by the process according to the invention: (A) 3-hydroxybutyric acid, 3-hydroxyvaleric acid, and 4-hydroxyvaleric acid;
  • Claims 25 to 32 indicate preferred quantitative compositions of the PHF, as obtained with the method according to the invention. All PHF obtained can be processed as thermoplastic and are extremely flexible.
  • Claims 33 and 34 relate to a DNA fragment which carries the genes phaC and phaE from Thiocapsa pfennigii. The phaC gene encodes the phaC protein and the phaE gene encodes the phaE protein.
  • FIG. 1 shows the DNA sequence of a DNA fragment according to the invention from Thiocapsa pfennigii and the amino acid sequence of the phaC and phaE proteins.
  • the 2.8 kb DNA fragment shown in FIG. 1 is obtained by BamHI digestion of a 15.6 kb EcoRI fragment from Thiocapsa pfennigii. 1 shows the assignment of the amino acid sequences of the phaC and phaE proteins (in the IUPAC one letter code) to their corresponding genes phaC (DNA sequence section 1322 to 2392) and phaE (DNA sequence section 180 to 1280).
  • example 1 shows the assignment of the amino acid sequences of the phaC and phaE proteins (in the IUPAC one letter code) to their corresponding genes phaC (DNA sequence section 1322 to 2392) and phaE (DNA sequence section 180 to 1280).
  • Example 2 The procedure was as given in Example 1, except that instead of Pseudomonas putida GPpl04 (pHP1014 :: E156) the strain Alcali ⁇ enes eutrophus PHB ⁇ 4 (pHP1014 :: E156) was used, and that instead of neutralized octane acid 0.3% neutralized gluconic acid was offered as a carbon source in addition to levulinic acid.
  • polyesters had the analytical data shown below, where "A" represents the 13 C-NMR spectrum and "B" the 1 H-NMR spectrum.
  • the signal assignment results from the numbering given in the structural formula of poly (3HB-CO-3HV-CO-4HV) shown. The gas chromatogram after methanolysis of this polyester is then shown.
  • Example 2 The procedure was as described in Example 2, except that the volume of the inoculum from the preculture was only 3 ml and that was inoculated with 50 ml of the above-mentioned mineral salt medium in a 500 ml Erlenmeyer flask as the main culture. The flasks were then shaken aerobically for 72 hours before cell harvesting.
  • the analysis of the polymer accumulated by the cells showed a polyester content of approx. 25 percent by weight of the dry cell mass.
  • the polymer consisted of approximately 61 mol% 3-hydroxybutyric acid, approximately 32 mol% 3-hydroxyvaleric acid and approximately 7 mol% 4-hydroxyvaleric acid.
  • the analysis of the polymer accumulated by the cells showed a polyester content of approx. 37 percent by weight of the cell dry matter.
  • the polymer consisted of approx. 37 mol% 3-hydroxybutyric acid, approx. 50 mol% 3-hydroxyvaleric acid and approx. 13 mol% 4-hydroxyvaleric acid.
  • 5HHx as a building block was detected by gas chromatography after methanolysis both in lyophilized cells and on isolated and cleaned polyester.
  • the isolated and purified polyester was also subjected to 13 C-NMR and 1 H-NMR analysis, and the incorporation of 5 HHx was confirmed thereby.
  • 5-Hydrohexanoic acid was prepared starting from 4-acetoacetic acid, which was reduced quantitatively with NaBH 4 .
  • the analysis of the polymer accumulated by the cells showed a polyester content of approx. 36 percent by weight of the dry cell mass.
  • the polymer consisted of approx. 71 mol% 3-hydroxybutyric acid, approx. 4 mol% 3-hydroxyhexanoic acid and approx. 23 mol% 5-hydroxyhexanoic acid and approx. 2 mol% 3-hydroxyoctanoic acid (also a minimal amount of 4-hydroxyoctanoic acid).
  • polyesters had the analytical data shown below, where "A” represents the 13 C-NMR spectrum and "B" the 1 H-NMR spectrum.
  • the signal assignment is based on the numbering that is shown in the structural formula of poly (3HB-co-3HHx- co-5HHx-3HO).
  • the GC analysis after methanolysis is then shown.
  • 4-Hydroxyheptanoic acid was prepared by saponifying ⁇ -heptalactone with NaOH.
  • the polymer consisted of 43 mol% 3-hydroxybutyric acid, 16 mol% 3-hydroxyvaleric acid, 27 mol% 3-hydroxyhexanoic acid, 5 mol% 3-hydroxyheptanoic acid, 6 mol% 4-hydroxyheptanoic acid and 3-hydroxyoctanoic acid.
  • the analysis of the polymer accumulated by the cells showed a polyester content of approx. 18 percent by weight of the dry cell mass.
  • the polymer consisted of approx. 75 mol% of approx. 3-hydroxybutyric acid, approx. 22 mol% of 3-hydroxyhexanoic acid, approx. 1.5 mol% of 3-hydroxyoctanoic acid and approx. 3 mol% of 4-hydroxy-octanoic acid.
  • P. putida GPp104 PHF accumulated to a maximum of approx. 50% of the dry cell mass, which consists of 3-hydroxybutyric acid (approx. 70 to 90 mol%), 3-hydroxyvaleric acid (approx. 1 to 5 mol%), 3-hydroxyhexanoic acid (approx. 10 to 20 mol%), 3-hydroxyoctanoic acid (approx. 1 to 5 mol%) and 4-hydroxyoctanoic acid (approx. 0.5 to 4 mol%).
  • Example 2 The procedure was as described in Example 1, except that the volume of the inoculum from the preculture was only 3 ml, and that was inoculated with 50 ml of the mineral salt medium mentioned above in a 500 ml Erlenmeyer flask as the main culture. The flasks were then shaken aerobically for 72 hours before cell harvesting.
  • a copolyester of the formula poly (3HB-co-3HHx-co-4HHx) could be produced, the analytical data of which are reproduced below.
  • the polyesters had the analytical data shown below, where "A” represents the 13 C-NMR spectrum and "B” the * H-NMR spectrum.
  • the signal assignment results from the numbering given in the structural formula of poly (3HB-co-3HHx-co-4HHx) shown.
  • the gas chromatogram after methanolysis is then shown.
  • 3HB 3-hydroxybutyric acid
  • 3HHx 3-hydroxyhexanoic acid
  • 4HHx 4-hydroxyhexanoic acid.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Polyesters Or Polycarbonates (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

L'invention concerne un procédé de préparation d'acides gras polyhydroxyliques et des souches bactériennes recombinées utiles pour mettre en ÷uvre ce procédé. L'invention concerne en outre de nouveaux acides gras polyhydroxyliques, de nouveaux substrats pour la préparation d'acides gras polyhydroxyliques nouveaux et classiques, un fragment d'ADN codant pour un composant PhaE et un composant PhaC de la synthase d'acide gras polyhydroxylique issue de Thiocapsa pfennigii, et la synthase correspondante d'acide gras polyhydroxylique.
EP95931149A 1994-09-16 1995-09-15 Procede de preparation d'acides gras polyhydroxyliques et souches bacteriennes recombinees pour la mise en uvre du procede Withdrawn EP0781339A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE4433134 1994-09-16
DE4433134A DE4433134A1 (de) 1994-09-16 1994-09-16 Verfahren zur Herstellung von Polyhydroxyfettsäuren sowie rekombinanter Bakterienstämme zur Durchführung des Verfahrens
PCT/DE1995/001279 WO1996008566A2 (fr) 1994-09-16 1995-09-15 Procede de preparation d'acides gras polyhydroxyliques et souches bacteriennes recombinees pour la mise en ×uvre du procede

Publications (1)

Publication Number Publication Date
EP0781339A2 true EP0781339A2 (fr) 1997-07-02

Family

ID=6528458

Family Applications (1)

Application Number Title Priority Date Filing Date
EP95931149A Withdrawn EP0781339A2 (fr) 1994-09-16 1995-09-15 Procede de preparation d'acides gras polyhydroxyliques et souches bacteriennes recombinees pour la mise en uvre du procede

Country Status (5)

Country Link
US (2) US6011144A (fr)
EP (1) EP0781339A2 (fr)
CA (1) CA2203264A1 (fr)
DE (1) DE4433134A1 (fr)
WO (1) WO1996008566A2 (fr)

Families Citing this family (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000189170A (ja) * 1998-05-08 2000-07-11 Daicel Chem Ind Ltd 光学活性４―ハロ―３―ヒドロキシ酪酸エステルの製造方法
EP1255846B1 (fr) 2000-02-11 2010-01-06 Metabolix, Inc. Constructions a expression de genes mutiples contenant des inteines modifiees
CA2428713C (fr) * 2000-11-17 2012-04-17 Metabolix, Inc. Production de polyhydroxyalcanoates a chaine de longueur moyenne a partir de voies de biosynthese d'acides gras
US7141400B2 (en) * 2002-01-16 2006-11-28 University Of Hawaii Production of biodegradable thermoplastic materials from organic wastes
US8735113B2 (en) 2003-10-15 2014-05-27 Newlight Technologies, Llc Methods and systems for production of polyhydroxyalkanoate
US8071342B2 (en) * 2003-10-15 2011-12-06 Newlight Technologies, Llc Process for the treatment of methane emissions
US7579176B2 (en) 2003-10-15 2009-08-25 Newlight Technologies, Llc Method for the production of polyhydroxyalkanoic acid
US7745197B1 (en) 2003-10-15 2010-06-29 Newlight Technologies, Llc Process for the utilization of ruminant animal methane emissions
CA2600882C (fr) 2005-03-16 2013-02-26 Metabolix, Inc. Expression inductible chimiquement de voies biosynthetiques
WO2008115840A2 (fr) 2007-03-16 2008-09-25 Genomatica, Inc. Compositions et procédés pour la biosynthèse de 1,4-butanediol et de ses précurseurs
US7947483B2 (en) 2007-08-10 2011-05-24 Genomatica, Inc. Methods and organisms for the growth-coupled production of 1,4-butanediol
WO2010030711A2 (fr) 2008-09-10 2010-03-18 Genomatica, Inc. Microorganismes pour la production de 1,4-butanediol
CA2754261A1 (fr) 2009-03-05 2010-09-10 Metabolix, Inc. Plantes stables et fertiles produisant des niveaux eleve de polyhydroxybutyrate et procedes d'obtention
BRPI1012685A2 (pt) * 2009-03-05 2015-09-15 Metabolix Inc método para melhorar as características de uma planta, método para propagação de plantas transgênicas, método para supertransformação de plantas transgênicas, planta trasngênica produzida pelo método, semente da planta transgênica e matéria-prima de biorefinaria
CN102459138A (zh) * 2009-06-04 2012-05-16 基因组股份公司 分离发酵液成分的方法
MY187676A (en) 2009-06-04 2021-10-08 Genomatica Inc Microorganisms for the production of 1,4-butanediol and related methods
US20120165500A1 (en) 2009-08-27 2012-06-28 Newlight Technologies, Llc Process for the production of polyhydroxyalkanoates
CA2773707A1 (fr) 2009-09-15 2011-03-24 Metabolix, Inc. Generation d'oleagineux produisant du polyhydroxybutrate haut
CA2777459A1 (fr) * 2009-10-13 2011-04-21 Genomatica, Inc. Microorganismes pour la production de 1,4-butanediol, 4-hydroxybutanal, 4-hydroxybutyryl-coa, putrescine et de composes associes, et procedes afferents
WO2011066076A1 (fr) 2009-11-25 2011-06-03 Genomatica, Inc. Micro-organismes et procédés pour la coproduction de 1,4-butanediol et de gamma-butyrolactone
US8048661B2 (en) * 2010-02-23 2011-11-01 Genomatica, Inc. Microbial organisms comprising exogenous nucleic acids encoding reductive TCA pathway enzymes
US8445244B2 (en) * 2010-02-23 2013-05-21 Genomatica, Inc. Methods for increasing product yields
WO2012122343A2 (fr) 2011-03-08 2012-09-13 Newlight Technologies, Llc Procédé de production de polyhydroxyalkanoate
US8986977B2 (en) * 2011-12-30 2015-03-24 Alliance For Sustainable Energy, Llc Disruption of cell walls for enhanced lipid recovery
US20200347417A1 (en) 2012-03-29 2020-11-05 Newlight Technologies, Inc Polyhydroxyalkanoate production methods and materials and microorganisms used in same
US9085784B1 (en) 2012-03-29 2015-07-21 Newlight Technologies, Llc Polyhydroxyalkanoate production methods and materials and microorganisms used in same
TW201410865A (zh) 2012-06-04 2014-03-16 Genomatica Inc 製造４－羥基丁酸酯、１，４－丁二醇及相關化合物之微生物及方法
WO2016081902A1 (fr) 2014-11-20 2016-05-26 Full Cycle Bioplastics Inc. Production de copolymères de polyhydroxyalcanoates à partir de déchets organiques
KR20210049865A (ko) * 2018-08-24 2021-05-06 모하람 벤처스 인크. 생분해성 폴리머 조성물 및 이를 생산하는 방법

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8513310D0 (en) * 1985-05-28 1985-07-03 Ici Plc Copolymer production
US5229279A (en) * 1987-06-29 1993-07-20 Massachusetts Institute Of Technology Method for producing novel polyester biopolymers
AT393134B (de) * 1989-12-27 1991-08-26 Danubia Petrochemie Verfahren zur freisetzung von poly-3-hydroxycarbonsaeuren
US5512456A (en) * 1990-05-25 1996-04-30 James Madison University Method for the improved production and recovery of poly-β-hydroxybutyrate from transformed Escherichia coli
EP0475785A3 (en) * 1990-09-14 1993-04-14 Mitsubishi Gas Chemical Company, Inc. Process for preparation of copolymer
JP2777757B2 (ja) * 1991-09-17 1998-07-23 鐘淵化学工業株式会社 共重合体およびその製造方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9608566A2 *

Also Published As

Publication number Publication date
US6011144A (en) 2000-01-04
US6770464B2 (en) 2004-08-03
WO1996008566A2 (fr) 1996-03-21
CA2203264A1 (fr) 1996-03-21
US20010031489A1 (en) 2001-10-18
DE4433134A1 (de) 1996-03-21
WO1996008566A3 (fr) 1996-05-17

Similar Documents

Publication Publication Date Title
EP0781339A2 (fr) Procede de preparation d'acides gras polyhydroxyliques et souches bacteriennes recombinees pour la mise en uvre du procede
DE69227878T2 (de) Copolymere und Verfahren zu deren Herstellung
DE69830193T2 (de) Eine methode zur herstellung von hydroxycarboxylsäuren durch selbstdegradation von polyhydroxyalkanoaten
CN101300354A (zh) 具有改良的聚羟基链烷酸产生能力的去调节的细菌
EP0575908B1 (fr) Pseudomonas aeruginosa et son utilisation pour la préparation du L-rhamnose
Akhlaq et al. Polyhydroxybutyrate biosynthesis from different waste materials, degradation, and analytic methods: A short review
DE69005623T2 (de) Mikrobiologische herstellung von polyestern.
EP0149744B1 (fr) Procédé de préparation biotechnologique de l'acide poly-D-(-)-3-hydroxybutyrique
KR20210049865A (ko) 생분해성 폴리머 조성물 및 이를 생산하는 방법
DE69029993T2 (de) Copolymerproduktion
DE102008045237B4 (de) Verfahren zur simultanen Produktion von PHA und kompatiblen Soluten in halophilen Bakterien
DE10258127A1 (de) Verfahen zur fermentativen Herstellung von R-α-Liponsäure
US5273891A (en) Process for the production of microbial cellulose
Chemama et al. Feasibility study for D-lacti c acid production from thai rice by Leuconostoc pseudomesenteroides TC49 and D-lacti c acid purification
DE3885438T2 (de) Verfahren zur Herstellung von mikrobieller Cellulose.
DE69924830T2 (de) Gene von alcaligenes latus, die mit der biosynthese von polyhydroxyalkanoaten assoziiert sind.
WO2000049133A1 (fr) Procede de preparation de l-sorbose
DE69218837T2 (de) Bakterienwachstumsförderndes Material
RU2818688C2 (ru) Биоразлагаемая полимерная композиция и способ ее изготовления
Maheswari et al. A comparative study on the PHB production by Bacillus species under solid-state fermentation by agricultural wastes
RU2148648C1 (ru) Способ получения гетерополисахарида
DE4415127A1 (de) Abbaubare Polymere
DE19962395C1 (de) Verfahren zur Herstellung von Poly-3-hydroxybutyrat
JPH08163992A (ja) εーポリーＬーリジンの製造法
DE102004030385B4 (de) Verfahren zur Herstellung von Poly(3D-Hydroxybuttersäure-co-4-Hydroxybuttersäure) (P3HB/4HB) definierter molarer Zusammensetzung mit Delftia acidovorans P4A

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19970404

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL PT SE

17Q First examination report despatched

Effective date: 20010430

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20020312