[go: up one dir, main page]

WO2000006762A1 - Production de polymeres sequences de polyhydroxyalcanoates dans des systemes biologiques - Google Patents

Production de polymeres sequences de polyhydroxyalcanoates dans des systemes biologiques Download PDF

Info

Publication number
WO2000006762A1
WO2000006762A1 PCT/US1999/017363 US9917363W WO0006762A1 WO 2000006762 A1 WO2000006762 A1 WO 2000006762A1 US 9917363 W US9917363 W US 9917363W WO 0006762 A1 WO0006762 A1 WO 0006762A1
Authority
WO
WIPO (PCT)
Prior art keywords
pha
block copolymers
hydroxybutyrate
sequence distribution
controlling
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.)
Ceased
Application number
PCT/US1999/017363
Other languages
English (en)
Inventor
Frank A. Skraly
David P. Martin
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.)
Yield10 Bioscience Inc
Original Assignee
Metabolix Inc
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 Metabolix Inc filed Critical Metabolix Inc
Priority to AU52479/99A priority Critical patent/AU5247999A/en
Publication of WO2000006762A1 publication Critical patent/WO2000006762A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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

Definitions

  • PHAs Polyhydroxyalkanoates
  • PHAs are linear polyesters of hydroxyalkanoates. As such, they are head-to-tail polymers with a polyester backbone.
  • the mechanical, thermal, and physical properties of PHA vary greatly as a function of the monomer composition and other factors, such as the molecular weight and distribution, the thermal and processing histories, and, for copolymers, the sequence distribution of monomers.
  • PHAs are biosynthesized through a variety of metabolic pathways. These pathways generally differ in the manner in which the activated monomer is biosynthesized.
  • the final step in all known pathways is the polymerization of hydroxyalkanoate-coenzyme A thioester monomers by the enzyme PHA synthase, and the composition of any one type of PHA, i.e. its hydroxyalkanoate makeup, is dependent on the organism's metabolism and the substrate specificity of the enzymes in its PHA biosynthetic pathway. For example, Pseudomonas spp.
  • these copolymers are random copolymers, that is, the sequence distribution of monomers has no defined order. It would be useful to be able to control the sequence distribution in biologically produced PHA copolymers, so as to produce copolymers of PHAs having an ordered sequence distribution, such as in block copolymers.
  • Methods are provided for producing block copolymers of PHAs in biological systems by controlling the sequence distribution in PHA copolymers.
  • the method of controlling the sequence distribution preferably is achieved by (1) modulating the profile of substrate feeding; or (2) controlling the enzyme activities which supply the activated monomer.
  • the biological systems include PHA-producing organisms that express PHA synthase and metabolic pathways for the synthesis of two or more different activated monomers, i.e. hydroxyalkanoyl-CoA esters. Separate pathways are not required for each of the different monomers, as the composition of the growth medium may be altered during the course of the polymer biosynthesis.
  • Non-natural producers which lack the means to utilize PHA as a storage reserve and therefore do not effectively degrade the PHA and which also express the PHA synthase are preferably used to catalyze a "living polymerization".
  • Different monomers or monomer precursors are supplied to the PHA-producing organism at different times.
  • a block copolymer can be produced via fermentation by the sequential and separate feeding of different substrates, wherein each substrate is fed until the desired extent of polymerization has been achieved, and then is depleted or removed from the medium.
  • block copolymers of PHA are produced in a recombinant, PHA-producing plant, wherein the sequence distribution is selectively varied by controlling the enzyme activities which supply the activated monomer.
  • Block copolymers of PHA are synthesized in biological systems by controlling the sequence distribution of activated monomers during the polymerization process.
  • PHAs Polyhydroxyalkanoates
  • Numerous microorganisms have the ability to accumulate intracellular reserves of PHA polymers.
  • PHA biopolymers have emerged from what was originally considered to be a single homopolymer, poly [(R)-3-hydroxybutyrate] (PHB) into abroad class of polyesters with different monomer compositions and a wide range of physical properties.
  • PHA poly [(R)-3-hydroxybutyrate]
  • PHA polyhydroxybutyrate
  • R-3-hydroxybutyric acid units R-3-hydroxybutyric acid units
  • n is 0 or an integer; and wherein Rl, R 2 , R3, and R 4 are each selected from saturated and unsaturated hydrocarbon radicals; halo- and hydroxy-substituted radicals; hydroxy radicals; halogen radicals; nitrogen-substituted radicals; oxygen-substituted radicals; and hydrogen atoms.
  • PHAs with long side chains are semi-crystalline thermoplastic materials, whereas PHAs with long side chains are more elastomeric. PHAs of microbial origin containing both (R)- 3 -hydroxy butyric acid units and longer side chain units from C 5 to C 1 have been identified (Wallen & Rohificat, Environ. Sci. Technol, 8: 576-79 (1974)). A number of bacteria which produce copolymers of (R)-3- hydroxybutyric acid and one or more long side-chain hydroxyacid units containing from five to sixteen carbon atoms have been identified
  • the molecular weight of a PHA depends on the production system (typically a microorganism in nature) and growth conditions. In general, PHAs isolated from natural PHA producers vary in molecular weight from about 100,000 to 800,000, with polydispersities of about 2.
  • block copolymer refers to polymers composed of two or more connected sequences (blocks) of homopolymers.
  • the block copolymers can be AB-type or other block structures.
  • Representative blocks include 3-hydroxybutyrate and 4-hydroxybutyrate units.
  • PHAs in biological systems control the sequence distribution in PHA copolymers.
  • the method of controlling the sequence distribution preferably is achieved by (1) modulating the profile of substrate feeding; and/or (2) controlling the enzyme activities which supply the activated monomer.
  • Non- natural producers which lack the means to utilize PHA as a storage reserve and therefore do not effectively degrade PHA and different monomers or monomer precursors are supplied to the PHA-producing organism at different times in the preferred embodiment.
  • a "living polymer” remains active if there are no chain cleavage, transfer, or termination events.
  • Chain transfer or termination processes would stop the propagation reaction of a particular reaction site and “kill" the polymer but not necessarily the catalyst, while chain cleavage would produce more than one polymer per catalyst.
  • a living polymerization reaction can produce polymers of very high molecular weight.
  • Polymerization reactions with purified PHA synthase of R. eutropha have demonstrated that PHAs of very high molecular mass (>1 million g/mol) are produced in vitro (Gerngross, et al, Proc. Natl. Acad. Sci. 92: 6279-83 (1995)).
  • Studies of recombinant, non- natural PHA producers also have shown that PHAs of very high molecular mass (> 1 million g/mol) can be produced in vivo (Kusaka, et al. , Appl. Microbiol. Biotech.
  • the molecular weight of the polymer formed typically is a linear function of the monome ⁇ catalyst ratio. For example, doubling the monomer: catalyst ratio typically results in a doubling of the polymer molecular weight. This linear relationship between polymer molecular mass and monomer to PHA synthase ratio has been demonstrated (Sim, et al, Nature Biotech. 15: 63-67 (1997)). Additionally, a "living polymerization” reaction often is shown to yield a narrow molecular weight distribution (i.e. polydispersity, M w /M n ⁇ 1.5).
  • a narrow polydispersity indicates that the molecular weights of the individual polymer chains are very similar to each other, i.e., similar to what was predicted. Although not an absolute requirement for a "living polymerization,” a narrow polydispersity is observed when all of the polymerization catalyst is simultaneously activated (i.e. when the rate of catalyst initiation is faster than propagation). Therefore, it is possible to vary the monomeric sequence distribution of a "living polymer” via sequential polymerization of different monomers, resulting in the production of a polymer with a defined monomeric sequence, such as a block copolymer.
  • a block copolymer of PHA is produced in vivo in a fermentation process, wherein the composition of the block copolymer is controlled by the profile of substrate feeding.
  • Different PHA compositions can be produced via fermentation by varying the composition of the feed.
  • Copolymers are produced when more than one substrate is fed or when one substrate is fed, but more than one activated monomer can be produced from it by the microorganism. In natural PHA producers, these copolymers are random.
  • the monomer or monomer precursor must be taken up by the cell. This step may occur via a specific uptake system, a nonspecific uptake system, or simply by diffusion of the compound into the cell.
  • the monomer or monomer precursor must be enzymatically converted to the activated monomer, which in all currently known cases is a hydroxyacyl-CoA species. This may consist simply of esterification of monomer with coenzyme A, or it may involve several steps, one of which includes coenzyme A transfer to some precursor of the activated monomer.
  • the activated monomer must be added to the growing polymer chain by PHA synthase. Each of the aforementioned steps occurs at a rate which depends upon the identity of the substrate fed to the cells. Therefore, exposure of cells to different substrates, even at the same concentration for the same duration, typically yields different extents of polymerization.
  • PHA synthase catalyzes a "living polymerization,” adding monomer units one at a time to the end of a growing polymer chain.
  • sequence of any polymer chain is a chronological history of the availability of activated monomers to the synthase that produced it. If only one type of activated monomer is available in the cell over a given period of time, the polymer chains produced during that time will be homopolymeric. If a mixture of activated monomers is available in the cell during a given period of time, the polymer chains produced will be random copolymers.
  • each substrate is fed separately until the desired extent of polymerization has been achieved, and then is effectively eliminated from the medium.
  • the abolition of each substrate from the medium may be accomplished by the cells' complete usage of the substrate, or by replacing the medium with a medium containing a different substrate.
  • the substrates need not be fed one at a time and the polymer need not include covalently- linked homopolymeric segments.
  • Such a polymer can be prepared by gradually changing the monomer composition of the medium in which the cells are being cultivated.
  • a large assortment of compositions can be prepared using variations of this basic fermentation method.
  • block copolymers of PHA are produced in a recombinant, PHA-producing organism, most preferably a plant.
  • the sequence distribution is varied by controlling the enzyme activities which supply the activated monomer.
  • many genes are regulated in plants by a circadian mechanism (Takahashi, Curr. Opin. Genet. Dev. 3:301-09 (1993)).
  • Regulatory sequences associated with genes responsible for the synthesis of activated monomers can be engineered such that the synthesis of one monomer type is triggered by light and another monomer by darkness. This strategy should provide blocks of relatively short length. For very long blocks, the regulatory sequences associated with stages of development of the plant (for example, growth, fruiting, etc.) may be used.
  • the substrate feeding profile can be adjusted to produce a large variety of block copolymers.
  • the time periods or amounts of substrates during each phase of the polymer synthesis can be adjusted to vary the composition and/or sequence distribution of monomers. For instance, the synthesis of multi-block copolymers requires multiple changes in the feeding profile.
  • the synthesis of block copolymers containing more than two different monomers requires an appropriate feeding profile. Regardless of the feeding profile, if it is desired to produce block copolymers using a specific feeding profile, it is necessary to maintain the "living polymerization" during the polymer synthesis.
  • the "living" nature of the polymerization is maintained if chain cleavage, transfer or termination events are kept to a minimum and the polymerization system remains active. This can be done by ensuring cell viability, PHA synthase activity, production of active substrate and by minimizing polymer and PHA synthase degradation during polymer synthesis.
  • Controlling the expression of the enzyme PHA synthase is important for controlling the molecular weight, composition and sequence distribution of polymers produced in vivo.
  • the molecular weight of each block in a block copolymer depends on the amounts of PHA synthase and activated substrate reacted in a certain time period. Lower molecular weight blocks are produced, for instance, from lower monomer-to-PHA synthase ratios or from shorter polymerization periods. In these cases, it is necessary to induce a high level of expression of PHA synthase, to introduce low amounts of substrate, or to reduce the time period for polymerization. Also, the "living" nature of the polymerization requires control of PHA synthase expression.
  • the biological systems include PHA-producing organisms that contain at least PHA synthase and metabolic pathways for the synthesis of two or more different activated monomers, i.e. hydroxyalkanoyl-CoA esters. Separate pathways are not required for each of the different monomers, as the composition of the growth medium may be altered during the course of the polymer biosynthesis.
  • the biosynthetic systems include transgenic organisms, which typically are not natural PHA producing species, but have been genetically engineered to produce PHAs. The organisms contain at least a PHA synthase and metabolic pathways for the synthesis of at least two different activated monomers, i.e. hydroxyalkanoyl-CoA esters.
  • Non-natural producers typically lack the means to utilize PHA as a storage reserve and therefore do not effectively degrade it. This characteristic is important, as the rate of chain cleavage, transfer or termination is greatly reduced in these organisms, which allows PHA synthase to function as a "living polymerization catalyst.”
  • natural PHA-producing organisms may be engineered to produce block copolymers by using standard genetic engineering techniques to eliminate the genes which encode proteins responsible for chain cleavage, termination or transfer processes, either by knocking out the gene so that no protein is expressed, or so that an inactive protein is expressed.
  • the biological system described herein allows for the synthesis of a very large number of polymer compositions and sequence distributions. It will be obvious to those skilled in the art that the growth conditions, feeding profile and synthase expression levels can be adjusted to produce a large variety of materials.
  • block copolymers of PHA described herein are useful in a wide variety of applications.
  • the block copolymers can be used as blending and compounding agents, adhesives, in the manufacture of plastic articles, such as diapers, in paints, screen binders, and in biomedical applications, such as implantable controlled delivery devices.
  • the cells were grown in 250 mL of a medium containing 25 g/L LB broth powder (Difco; Detroit, Michigan) and 100 ⁇ g/mL ampicillin at 37 °C overnight with shaking at 225 rpm.
  • the cells were removed from this medium by centrifugation (2000 x g, 10 minutes) and resuspended in 250 mL of a medium containing the following quantities per liter: 5 g LB broth powder; 50 mmol potassium phosphate, pH 7; 10 g 3- hydroxybutyric acid (adjusted to neutral pH with sodium hydroxide); 2 g glucose; 100 ⁇ g ampicillin; and 0.1 mmol isopropyl- ⁇ -D- thiogalactopyranoside (IPTG). The cells were incubated in this medium with shaking at 150 rpm at 33 °C for 24 hours.
  • IPTG isopropyl- ⁇ -D- thiogalactopyranoside
  • the cells were isolated by centrifugation as described above, washed once with water and lyophilized. The cells in the remaining 150 mL were isolated by centrifugation as described above.
  • the cells were resuspended in 150 mL of a medium identical to that of the second phase, except that the 3 -hydroxybutyric acid was replaced by 4- hydroxybutyric acid. The cells were incubated in this medium with shaking at 150 rpm at 33 °C for 22 hours. The cells then were removed by centrifugation as described above, washed once with water, and lyophilized.
  • GC analysis was carried out on the lyophilized cell mass isolated following the second and third phases. About 20 mg of lyophilized cell mass was subjected to simultaneous extraction and butanolysis at 110 °C for 3 hours in 2 mL of a mixture containing (by volume) 90% 1-butanol and 10% concentrated hydrochloric acid, with 2 mg/mL benzoic acid added as an internal standard. The water-soluble components of the resulting mixture were removed by extraction with 3 mL water.
  • the organic phase (1 ⁇ L at a split ratio of 1 : 50 at an overall flow rate of 2 mL/min) was analyzed on an HP 5890 GC with FID detector (Hewlett- Packard Co, Palo Alto, CA) using an SPB-1 fused silica capillary GC column (30 m; 0.32 mm ID; 0.25 ⁇ m film; Supelco; Bellefonte, Pennsylvania) with the following temperature profile: 80 °C, 2 min; 10 °C per min to 250 °C; and 250 °C, 2 min.
  • FID detector Hewlett- Packard Co, Palo Alto, CA
  • SPB-1 fused silica capillary GC column (30 m; 0.32 mm ID; 0.25 ⁇ m film; Supelco; Bellefonte, Pennsylvania
  • the standard used to test for the presence of 4-hydroxybutyrate units in the polymer was ⁇ -butyrolactone, which, like poly(4-hydroxybutyrate), forms n-butyl 4-hydroxybutyrate upon butanolysis.
  • the standard used to test for 3-hydroxybutyrate units in the polymer was PHB.
  • the lyophilized cell mass was composed (by weight) of 69.5% biomass, 30.5% polymerized 3- hydroxybutyrate, and no detectable polymerized 4-hydroxybutyrate.
  • the lyophilized cell mass was composed (by weight) of 34.3% biomass, 17.2% polymerized 3-hydroxybutyrate, and 48.5% polymerized 4-hydroxybutyrate.
  • the polymeric material was composed of 73.8% 4-hydroxybutyrate units and 26.2% 3-hydroxybutyrate units.
  • the polymer was extracted from the lyophilized cell mass obtained after both the second and third phases.
  • lyophilized cell mass was mixed with about three times its own volume of chloroform and incubated with mild shaking in a closed tube at 37 °C for 16 hours.
  • the viscosity of the resulting slurry was reduced by the addition of chloroform until the slurry was thin enough to filter through coarse filter paper.
  • the large particles of cell mass were first removed by passing the slurry through glass wool, and smaller particles were removed by passing the slurry through coarse filter paper.
  • DSC analysis can discern between a random copolymer and a block copolymer, but not between a block copolymer and a blend of two separate homopolymers, especially when the block copolymer has very long blocks of one type of monomer as in the present example, because the crystalline domains of two separate homopolymers will closely resemble those of such a block copolymer.
  • DSC analysis (see Table 1 below) indicated two distinct glass-transition temperatures (-52 °C and -2 °C) and two distinct melting temperatures (51 °C and 169 °C) for the block copolymer, which were nearly the same as those of a blend of PHB and poly(4-hydroxybutyrate). Thus the polymeric material isolated after the third phase does not have the thermal characteristics expected for a random copolymer.
  • the organic phase (1 ⁇ L at a split ratio of 1:50 at an overall flow rate of 2 mL/min) was analyzed on an SPB-1 fused silica capillary GC column (30 m; 0.32 mm ID; 0.25 ⁇ m film; Supelco; Bellefonte, Pa.) with the following temperature profile: 80 °C, 2 min.; 10 C° per min. to 250 °C; 250 °C, 2 min.
  • the standard used to test for the presence of 4-hydroxybutyrate units in the polymer was g- butyrolactone, which, like poly(4-hydroxybutyrate), forms n-butyl 4-hydroxybutyrate upon butanolysis.
  • the standard used to test for 3-hydroxybutyrate units in the polymer was poly(3-hydroxybutyrate).
  • the thermal program used was as follows: 25°C, 2 min.; heat to 195°C at 10 C° per min.; hold at 195°C 2 min.; cool to -80°C at 300 C° per min.; hold at -80°C for 2 min.; heat to 195°C at 10 C° per min. Tg, Tx and Tm were determined during the second heating cycle.
  • Example 1 In order to generate a relatively large quantity of block copolymer material, the experiment in Example 1 was repeated on a larger scale in two 2800-mL Erlenmeyer flasks. The procedures described below were used for both flasks.
  • the cells were grown in 1500 mL of a medium containing 25 g/L LB broth powder (Difco; Detroit, Michigan) and 100 ⁇ g/mL ampicillin at 37 °C overnight with shaking at 225 rpm.
  • the cells In the second phase, the cells were removed from this medium by centrifugation (2000 x g,
  • the cells in the remaining 1400 mL were isolated by centrifugation, and resuspended, to start the third phase, in 1500 mL of a medium identical to that of the second phase, except that the sodium 3-hydroxybutyrate was replaced by 4-hydroxybutyrate.
  • the cells were incubated in this medium with shaking at 225 rpm at 30 °C for 22.5 hours, and then removed by centrifugation, washed once with water, and lyophilized.
  • the polymer was extracted from the lyophilized cell mass obtained after the second and third phases as in Example 1, except that 1,2- dichloroethane, rather than chloroform, was used as the solvent.
  • GC analysis was done on the extracted polymers from the second and third phases as in Example 1.
  • the extracted polymer isolated after the second phase weighed 0.19 g, and that isolated after the third phase weighed 6.8 g.
  • the polymer isolated after the second phase was composed entirely of PHB, which was expected because the cells had been exposed only to 3-hydroxybutyrate until the end of the second phase. This PHB accounted for 51.3% of the dry cell weight.
  • the polymer consisted of 57.4% 4- hydroxybutyrate units and 42.6% 3-hydroxybutyrate units and accounted for 78.6% of the dry cell weight.
  • Polymer samples were fractionated by precipitation from chloroform solution into acetone. Four samples were tested for comparison: PHB, poly(4-hydroxybutyrate), a 1 :1 blend of PHB and poly(4-hydroxybutyrate), and the block copolymer from Example 2 (3HB/4HB 43:57). Samples were dissolved in chloroform (25 mg/mL) with heating and gentle shaking. After dissolution, the solutions were filtered through glass wool to remove particulates, and then added dropwise to 10 volumes of acetone with rapid stirring. The mixtures were allowed to stand for 2 hours at room temperature. The precipitated material was collected by suction filtration onto pre-weighed filter paper. The filtrate was evaporated to yield a film (see Table 2 below).
  • DSC data for the block copolymer (poly(3-hydroxybutyrate-b-4- hydroxybutyrate)) and the blend of PHB and poly(4-hydroxybutyrate) were nearly identical.
  • the blend was separable into its component homopolymers by acetone fractionation, while the block copolymer sample yielded an acetone-insoluble fraction that contained a poly(4- hydroxybutyrate) fraction.
  • This poly(4-hydroxybutyrate) fraction is part of a block copolymer with 3-hydroxybutyrate, since the homopolymer, poly(4- hydroxybutyrate), would be soluble in acetone and would not precipitate.
  • DSC analysis also demonstrated that this acetone-insoluble fraction was not a random copolymer, but rather demonstrated a thermal profile consistent with the block copolymer, poly(3-hydroxybutyrate-b-4-hydroxybutyrate).
  • DSC analysis indicated two distinct glass-transition temperatures (-52 °C and 1 °C), crystallization temperatures (-20 °C and 40 °C) and melting temperatures (53 °C and 177°C) for the block copolymer which were nearly the same as those for a blend of PHB and poly(4-hydroxybutyrate) (Tg -52 °C, 1 °C: Tx -21°C, -41°C: and Tm 54 °C, 176 °C).
  • This P3HB was purchased from a commercial source.
  • This P4HB was prepared by Metabolix, Inc. in a previous fermentation
  • Examples 1 and 2 can be made to modify the polymer composition and/or change the percentage of block copolymer produced in vivo. These changes relate to the growth conditions, substrate feeding profile and expression of the enzyme PHA synthase, as described above.

Landscapes

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

Abstract

On décrit des procédés de production de copolymères séquencés de polyhydroxyalcanoates (PHA) dans des systèmes biologiques dans lesquels on régule la distribution des séquences dans les copolymères PHA. Le procédé de régulation de la distribution des séquences est de préférence effectué de la manière suivante: (1) on module le profil de l'alimentation en substrat; et/ou on contrôle les activités des enzymes qui alimentent le monomère activé. Les systèmes biologiques comprennent les organismes producteurs de PHA qui contiennent la synthase PHA et les mécanismes métaboliques nécessaires pour synthétiser au moins deux monomères activés différents, c'est-à-dire les esters hydroxyalcanoyle-CoA. Dans le procédé préféré, un copolymère séquencé est produit par fermentation avec des micro-organismes, ceci permettant à un ou plusieurs gènes codant des protéines responsables de la terminaison de clivage de la chaîne PHA, ou le processus de transfert d'être inactivés ou de coder des protéines inactives, au moyen de l'alimentation séquentielle ou séparée de différents substrats, chaque substrat étant alimenté jusqu'à ce que la polymérisation désirée soit terminée, puis appauvri ou éliminé du milieu. Dans une autre forme de réalisation, des copolymères séquencés sont produits dans une plante de recombinaison produisant du PHA, la distribution des séquences étant sélectivement modifiée au moyen du contrôle des activités enzymatiques qui alimentent le monomère activé.
PCT/US1999/017363 1998-07-30 1999-07-30 Production de polymeres sequences de polyhydroxyalcanoates dans des systemes biologiques Ceased WO2000006762A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU52479/99A AU5247999A (en) 1998-07-30 1999-07-30 Production of block copolymers of polyhydroxyalkanoates in biological systems

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US9467098P 1998-07-30 1998-07-30
US60/094,670 1998-07-30

Publications (1)

Publication Number Publication Date
WO2000006762A1 true WO2000006762A1 (fr) 2000-02-10

Family

ID=22246478

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/017363 Ceased WO2000006762A1 (fr) 1998-07-30 1999-07-30 Production de polymeres sequences de polyhydroxyalcanoates dans des systemes biologiques

Country Status (2)

Country Link
AU (1) AU5247999A (fr)
WO (1) WO2000006762A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6479145B1 (en) 1999-09-09 2002-11-12 Regents Of The University Of Minnesota Biopolymers and biopolymer blends, and method for producing same
WO2014032633A1 (fr) 2012-08-27 2014-03-06 Vysoke Uceni Technicke V Brne Procédé de production de polyhydroxyalcanoates (pha) sur la base d'un substrat huileux
CN105296409A (zh) * 2015-07-14 2016-02-03 西安交通大学 一株用于生产固定化碱性果胶酶纳米微球的工程菌及其构建方法与应用

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989000202A2 (fr) * 1987-06-29 1989-01-12 Massachusetts Institute Of Technology Procede de production de nouveaux biopolymeres de polyester
WO1991000917A1 (fr) * 1989-07-10 1991-01-24 Massachusetts Institute Of Technology Procede de production de nouveaux biopolymeres de polyester
EP0533144A2 (fr) * 1991-09-17 1993-03-24 Kanegafuchi Kagaku Kogyo Kabushiki Kaisha Copolymère et son procédé de production
WO1993006225A1 (fr) * 1991-09-27 1993-04-01 Center For Innovative Technology Procede de production de copolymeres du type poly-beta-hydroxyalcanoate
EP0614672A1 (fr) * 1992-12-31 1994-09-14 United States Surgical Corporation Dispositif médical biocompatible
WO1994021810A1 (fr) * 1993-03-24 1994-09-29 Center For Innovative Technology PRODUCTION DE POLY-β-HYDROXYBUTIRATE DANS DES CELLULES-HÔTES PROCARYOTIQUES
WO1996009402A1 (fr) * 1994-09-22 1996-03-28 Monsanto Company Copolyesters
WO1997007153A1 (fr) * 1995-08-14 1997-02-27 University Of Massachusetts Medical Center Procedes pour reguler des structures de polyester microbiennes
WO1997024387A1 (fr) * 1995-12-29 1997-07-10 Minnesota Mining And Manufacturing Company Poly (beta-hydroxyorganoates) fluores

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989000202A2 (fr) * 1987-06-29 1989-01-12 Massachusetts Institute Of Technology Procede de production de nouveaux biopolymeres de polyester
WO1991000917A1 (fr) * 1989-07-10 1991-01-24 Massachusetts Institute Of Technology Procede de production de nouveaux biopolymeres de polyester
EP0533144A2 (fr) * 1991-09-17 1993-03-24 Kanegafuchi Kagaku Kogyo Kabushiki Kaisha Copolymère et son procédé de production
WO1993006225A1 (fr) * 1991-09-27 1993-04-01 Center For Innovative Technology Procede de production de copolymeres du type poly-beta-hydroxyalcanoate
EP0614672A1 (fr) * 1992-12-31 1994-09-14 United States Surgical Corporation Dispositif médical biocompatible
WO1994021810A1 (fr) * 1993-03-24 1994-09-29 Center For Innovative Technology PRODUCTION DE POLY-β-HYDROXYBUTIRATE DANS DES CELLULES-HÔTES PROCARYOTIQUES
WO1996009402A1 (fr) * 1994-09-22 1996-03-28 Monsanto Company Copolyesters
WO1997007153A1 (fr) * 1995-08-14 1997-02-27 University Of Massachusetts Medical Center Procedes pour reguler des structures de polyester microbiennes
WO1997024387A1 (fr) * 1995-12-29 1997-07-10 Minnesota Mining And Manufacturing Company Poly (beta-hydroxyorganoates) fluores

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6479145B1 (en) 1999-09-09 2002-11-12 Regents Of The University Of Minnesota Biopolymers and biopolymer blends, and method for producing same
US6723800B2 (en) 1999-09-09 2004-04-20 Regents Of The University Of Minnesota Biopolymers and biopolymer blends, and method for producing same
US7026413B2 (en) 1999-09-09 2006-04-11 Regents Of The University Of Minnesota Biopolymers and biopolymer blends, and method for producing same
WO2014032633A1 (fr) 2012-08-27 2014-03-06 Vysoke Uceni Technicke V Brne Procédé de production de polyhydroxyalcanoates (pha) sur la base d'un substrat huileux
CN105296409A (zh) * 2015-07-14 2016-02-03 西安交通大学 一株用于生产固定化碱性果胶酶纳米微球的工程菌及其构建方法与应用

Also Published As

Publication number Publication date
AU5247999A (en) 2000-02-21

Similar Documents

Publication Publication Date Title
Prieto et al. Synthesis and degradation of polyhydroxyalkanoates
Luengo et al. Bioplastics from microorganisms
Kahar et al. High yield production of polyhydroxyalkanoates from soybean oil by Ralstonia eutropha and its recombinant strain
Doi Microbial synthesis, physical properties, and biodegradability of polyhydroxyalkanoates
Doi et al. Microbial synthesis and characterization of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate)
Lenz et al. Bacterial polyesters: biosynthesis, biodegradable plastics and biotechnology
Chodak Polyhydroxyalkanoates: origin, properties and applications
Verlinden et al. Bacterial synthesis of biodegradable polyhydroxyalkanoates
CA2303070C (fr) Systemes biologiques utilises pour produire des polymeres de polyhydroxyalcanoate contenant des acides 4-hydroxy
Zinn et al. Tailored synthesis of poly ([R]‐3‐hydroxybutyrate‐co‐3‐hydroxyvalerate)(PHB/HV) in Ralstonia eutropha DSM 428
Steinbüchel et al. Biosynthesis of polyesters in bacteria and recombinant organisms
Bonartsev et al. Biosynthesis of poly (3-hydroxybutyrate) copolymers by Azotobacter chroococcum 7B: A precursor feeding strategy
Shi et al. Microbial polyester synthesis: effects of poly (ethylene glycol) on product composition, repeat unit sequence, and end group structure
MXPA00011401A (es) Composiciones de biopolimero de polimero de polihidroxialcanoato.
WO1997007153A1 (fr) Procedes pour reguler des structures de polyester microbiennes
Choi et al. High level production of supra molecular weight poly (3-hydroxybutyrate) by metabolically engineered Escherichia coli
EP1172438B1 (fr) Polyhydroxyalcanoate synthase et gène codant pour cette enzyme
Singh et al. Microbially originated polyhydroxyalkanoate (PHA) biopolymers: an insight into the molecular mechanism and biogenesis of PHA granules
EP1138770B1 (fr) Polyhydroxyalkanoatesynthase et gène codant pour celle-ci
Ramachander et al. Synthesis of PHB by recombinant E. coli harboring an approximately 5 kb genomic DNA fragment from Streptomyces aureofaciens NRRL 2209
EP1149912B1 (fr) Polyhydroxyalcanoate synthase et gène codant pour cette enzyme
US6723800B2 (en) Biopolymers and biopolymer blends, and method for producing same
EP1154021B1 (fr) Polyhydroxyalcanoate synthase et gène codant pour cette enzyme
WO2000006762A1 (fr) Production de polymeres sequences de polyhydroxyalcanoates dans des systemes biologiques
Balakrishna Pillai et al. Bacterial polyhydroxyalkanoates: recent trends in production and applications

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP MX

AL Designated countries for regional patents

Kind code of ref document: A1

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

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase