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Method for producing short, medium and long chain polyhydroxyfatty acid copolymers by microorganisms

A technology of polyhydroxyalkanoate and hydroxyalkanoate, which is applied in the field of microbial production of short, medium and long-chain polyhydroxyalkanoate copolymers, which can solve the problems of difficult to ensure stable material properties, large differences, and incomplete weakening

Active Publication Date: 2019-01-25
TSINGHUA UNIV
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

[0008] (3) The proportion of MCL monomer is difficult to control
Because the β-oxidation cycle of most strains exists or is not completely weakened, the fatty acid as a substrate will lose two carbon atoms every time it goes through a β-oxidation cycle after entering the cell, so the SCL-co-MCL PHA synthesized by the strain The MCL monomer part generally includes multiple monomers such as C6, C8, C10, and C12, and it is rarely possible to synthesize a polymer composed of only one SCL monomer and one MCL monomer
Since the proportion of medium and long-chain monomers (C6:C8:C10:C12) produced by the β-oxidation cycle is not easy to control, the SCL-co-MCL PHA obtained from different batches may have large differences in the proportion of MCL monomers, and the material properties Difficult to get stable guarantee

Method used

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  • Method for producing short, medium and long chain polyhydroxyfatty acid copolymers by microorganisms
  • Method for producing short, medium and long chain polyhydroxyfatty acid copolymers by microorganisms
  • Method for producing short, medium and long chain polyhydroxyfatty acid copolymers by microorganisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Embodiment 1, preparation recombinant pseudomonas

[0098] 1. Preparation of plasmid pSEVA341-Pre-phaC61-3-phaA-phaB-Kan

[0099] The plasmid pSEVA341-Pre-phaC61-3-phaA-phaB-Kan was prepared, as shown in sequence 1 of the sequence listing. The schematic diagram of the structure of plasmid pSEVA341-Pre-phaC61-3-phaA-phaB-Kan is as follows figure 1 Shown is a circular plasmid.

[0100] In sequence 1 of the sequence listing, nucleotides 33-428 are the Pre promoter, nucleotides 464-2143 are the phaC61-3 gene, nucleotides 2228-3409 are the phaA gene, and nucleotides 3484-4224 The first nucleotide is the phaB gene, and the 4780-5574th nucleotide is the kanamycin resistance gene.

[0101] 2. Preparation of P.entomophila LAC31 (pSEVA341-Pre-phaC61-3-phaA-phaB-Kan)

[0102] 1. Introduce the plasmid pSEVA341-Pre-phaC61-3-phaA-phaB-Kan into Escherichia coli S17-1 to obtain recombinant bacteria named E.coli S17-1 (pSEVA341-Pre-phaC61-3-phaA-phaB-Kan ).

[0103] 2. Co-cultivat...

Embodiment 2

[0121] Embodiment 2, application of recombinant Pseudomonas to produce P(3HB-co-3HDD)

[0122] 1. Application of P.entomophila LAC31 (pSEVA341-Pre-phaC61-3-phaA-phaB-Kan)

[0123] 1. Prepare the seed liquid of P. entomophila LAC31 (pSEVA341-Pre-phaC61-3-phaA-phaB-Kan).

[0124] 2. Fermentation with glucose as the sole carbon source

[0125] Take 2.5mL of the seed liquid obtained in step 1, inoculate it into 47.5mL of fermentation medium, and cultivate it with shaking at 30°C and 200rpm for 48 hours. Fermentation medium: LB medium containing 50mg / L kanamycin and 25g / L glucose.

[0126] 3. Fermentation with glucose and lauric acid as carbon sources

[0127] Take 2.5mL of the seed liquid obtained in step 1, inoculate it into 47.5mL of fermentation medium, and cultivate it with shaking at 30°C and 200rpm for 48 hours. Fermentation medium: LB medium containing 50mg / L kanamycin, 20g / L glucose and 5g / L lauric acid.

[0128] 4. Determination of dry cell weight, PHA content of bacte...

Embodiment 3

[0146] Example 3, Synthesis of P(3HB-co-3HDD) in different proportions with P.entomophila LAC32

[0147] 1. Prepare the seed solution of P. entomophila LAC32 (pSEVA341-Pre-phaC61-3-phaA-phaB-Kan).

[0148] 2. Fermentation

[0149] Take 2.5mL of the seed liquid obtained in step 1, inoculate it into 47.5mL of fermentation medium, and cultivate it with shaking at 30°C and 200rpm for 48 hours.

[0150] The fermentation medium is any of the following:

[0151] Fermentation medium 20G: LB medium containing 50mg / L kanamycin and 20g / L glucose;

[0152] Fermentation medium 20G+0.5C12: LB medium containing 50mg / L kanamycin, 20g / L glucose and 0.5g / L lauric acid;

[0153] Fermentation medium 20G+1C12: LB medium containing 50mg / L kanamycin, 20g / L glucose and 1g / L lauric acid;

[0154] Fermentation medium 15G+2C12: LB medium containing 50mg / L kanamycin, 15g / L glucose and 2g / L lauric acid;

[0155] Fermentation medium 5G+5C12: LB medium containing 50mg / L kanamycin, 5g / L glucose and 5g / L...

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Abstract

The invention discloses a method for producing short, medium and long chain polyhydroxyfatty acid copolymer by microorganism. The recombinant bacterium of the invention is obtained by modifying the starting bacterium as follows (a1), (a2) and (a3): (a1) introducing a low-specific PHA polymerase gene; (a) introducing an encoding gene of a key protein of the PHB synthesis pathway; (a3) knocking outthat encoding gene of the key enzyme of the fatty acid de novo synthesis pathway. The invention also protects the recombinant bacteria mentioned above in preparing SCL-Co-MCL PHA. The invention can prepare SCL by adjusting the proportion of carbon source controllable SCL-Co-MCL PHA. Poly (3HB-Co-MCL 3HA) random copolymer or block copolymer can also be produced by controlling the addition time of the carbon source. The invention is applicable to SCL with controllable production ratio SCL-Co-MCL PHA is of great significance.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for microorganisms to produce short, medium and long chain polyhydroxy fatty acid copolymers. Background technique [0002] Polyhydroxyalkanoate (PHA) is a natural energy storage material for many microorganisms, and its chemical essence is a kind of polymer polyester. Due to its characteristics of complete biodegradability and complete biosynthesis, PHA is considered to be the most potential biomaterial to solve global plastic pollution. After decades of production exploration, a variety of PHAs have been successfully synthesized and put into industrial production, including poly 3-hydroxybutyrate (PHB), poly 3-hydroxybutyrate-3-hydroxyvalerate (PHBV) , Poly 3-hydroxybutyrate-3-hydroxyhexanoate (PHBHHx), poly 3-hydroxybutyrate-4-hydroxyvalerate (P3HB4HB). Compared with traditional non-degradable petroleum-based plastics, the application of PHA still faces diff...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P7/62C12R1/38C12R1/01
CPCC07K14/195C07K14/21C12P7/625
Inventor 陈国强李梦怡陈祥斌
Owner TSINGHUA UNIV
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