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Green process and compositions for producing poly(5HV) and 5 carbon chemicals

A technology of hydroxyvaleric acid and polyhydroxyalkanoic acid, which is applied in the direction of genetic engineering, biochemical equipment and methods, and the use of vectors to introduce foreign genetic materials, etc., can solve the problems of expensive and high cost of 5-carbon petroleum substrates

Active Publication Date: 2012-01-11
CJ CHEILJEDANG CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To date, these materials have been produced by feeding petroleum-derived 5-carbon substrates such as 5-hydroxyvaleric acid (5HV) or 1,5-pentanediol to microorganisms capable of metabolizing these substrates into activated mono PHB5HV and P5HV polymers produced by these methods are only partly made from renewable resources, and due to the cost of 5-carbon petroleum matrix very high and quite expensive

Method used

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  • Green process and compositions for producing poly(5HV) and 5 carbon chemicals
  • Green process and compositions for producing poly(5HV) and 5 carbon chemicals
  • Green process and compositions for producing poly(5HV) and 5 carbon chemicals

Examples

Experimental program
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Effect test

example 1

[0167] Example 1: Biosynthesis of P(5HV) Homopolymer by Sodium 5-Hydroxyvalerate

[0168] In the first demonstration of the ability to synthesize 5HV-containing PHA, sodium 5-hydroxyvalerate (Na5HV) was fed to E. coli strains expressing CoA transferase or CoA synthetase and PHA synthase to determine the 5HV fed Whether the monomer is accepted and incorporated directly into the P(5HV) homopolymer. Na5HV is synthesized by alkaline hydrolysis of delta-valerolactone (DVL). This procedure involves adding 0.1 M NaOH to 50 mL of methanol with stirring until dissolved. Under vigorous stirring, 0.1 molar DVL was added thereto. The resulting precipitate was dried, dissolved in water, adjusted to pH 8.5, and sterile filtered with a 0.2 micron filter. The saline solution was analyzed on a Waters Alliance HPLC to confirm that all DVL had been saponified.

[0169] Different combinations of CoA transferase / synthetase and PHA synthase genes were tested to find the best combination for P(5...

example 2

[0184] Example 2: Biosynthesis of P(3HB-co-5HV) copolymers from sodium 5-hydroxyvalerate

[0185] The next experiments will illustrate the production of P(3HB-co-5HV) copolymers in E. coli strains capable of synthesizing 3HB-CoA and 5HV-CoA monomers and incorporating them into PHA.

[0186] Strain construction

[0187] The strain used in this example was MBX2641, a derivative of MG1655, containing an operon consisting of Ralstonia eutropha H16 bktB-Bacillus megaterium phaB-kan integrated randomly into the chromosome. For operon integration, pCJ022 ( Strain S17-1λpir (Miller and Mekalanos, J.Bacteriol.) 170 (6): 2575-2583 (1988)) and naphthalene of MG1655, as described below Mating of pyroxacid-resistant mutant MBX1987. MBX1987 derivatives carrying the bktB-phaB-kan cassette in the chromosome were selected on LB plates containing 30 μg / ml nalidixic acid (Nl) and 50 μg / ml kanamycin (Km). Show Nl R Km R One such integrant of the phenotype is preserved as MBX2079. Next, ph...

example 3

[0197] Example 3: Tunable 5HV monomer composition in P(3HB-co-5HV) copolymer and its effect on material properties

[0198] The copolymer composition was adjusted by changing the amount of Na5HV and glucose added to the production medium. Alternative ways to achieve this include (1) feeding different amounts of L-lysine into the growth medium of recombinant cells that can produce 5HV from L-lysine, as shown in Example 6; L-lysine pathway genes are deregulated in recombinant cells that can produce 5HV from glucose via L-lysine, as shown in Examples 9 and 10.

[0199]To illustrate the tunable 5HV monomer composition in P(3HB-co-5HV) copolymers, strain MBX2641 expressing enzymes of the 3HB pathway (bktB and phaB) as well as coenzyme A transferase (orfZ) and PHA synthase (phaC) was used [pFS30]. Parallel cultures of MBX2641[pFS30] were grown in decreasing concentrations of glucose (10, 5, 1, 0.5, 0.1, 0 g / L) or Na5HV (10, 5, 1, 0.5, 0.1, 0 g / L) , and the polymer content was ana...

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Abstract

Recombinant hosts for producing polyhydroxyalkanoates and methods of producing polyhydroxyalkanoates from renewable carbon substrates are provided. Certain recombinant hosts that produce 5 carbon chemicals such as 5-amino?entanoate (5AP), 5 -hydroxy valerate (5HV), glutarate, and 1,5 pentanediol (PDO) are also provided. One embodiment provides a recombinant host expressing a gene encoding a heterologous enzyme selected from the group consisting of a polyhydroxyalkanoate synthase and a 5-hydroxyvalerate-CoA (5HV-CoA) transferase, wherein the host produces a polymer containing 5-hydroxyvalerate. Preferably, the host expresses both a polyhydroxyalkanoate synthase and a 5HV-CoA transferase. The host can be prokaryotic or eukaryotic. A preferred prokaryotic host is E. coli. The polymers produced by the recombinant hosts can be homopolymers or copolymers of 5-hydroxyvalerate.; A preferred copolymer is poly(3-hydroxybutyrate-co-5-hydroxyvalerate).

Description

technical field [0001] The present invention generally relates to transgenic organisms producing polyhydroxyalkanoic acids and copolymers thereof containing 5-hydroxyvaleric acid and chemical intermediates (C5 compounds) containing 5 carbon atoms. [0002] Cross References to Related Applications [0003] This application claims the benefit of and priority to US Provisional Patent Application Serial No. 61 / 122,250, filed December 12, 2008, which is hereby permitted to be incorporated by reference in its entirety. Background technique [0004] Polyhydroxyalkanoate (PHA) is a biodegradable plastic useful in the manufacture of (but not limited to) films (e.g. packaging films, agricultural films, mulch films), golf tees, balls Caps and covers, agricultural stands and posts, paper and cardboard coatings (e.g. for cups, plates, boxes, etc.), thermoformed products (e.g. trays, containers, cheese jars, flower pots, noodle bowls, moulds, etc.), Housings (e.g. for electronics), bag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N9/00C12N9/02C12N9/04C12N9/10C12N9/80C12N9/88C12N15/52C12P7/04C12P7/18C12P7/42C12P7/44C12P7/62
CPCC12N9/93C12N9/0006C12P7/625C12N9/0069C12N9/1096C12N9/80C12P7/44C12P7/42C12N9/10C12P7/04C12N9/1029C12N15/52C12N9/88C12N9/0008C12P7/18C12N1/20C12N5/10C12N5/14C12N9/00C12N15/00C12N15/09C12N15/70C12N15/8234C12N15/8241C12P7/00
Inventor W·R·法莫尔J·比克迈耶陆晨峰常东云F·斯科拉里T·M·拉姆塞耶尔
Owner CJ CHEILJEDANG CORP
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