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3-hydroxybutyric acid, 4-hydroxybutyric acid and 3-hydroxyvalerate terpolymer p (3HB-4HB-3HV) and microbial production thereof

A 3HB-4HB-3HV, ​​Alcaligenes technology, applied in the field of biotechnology and biomaterials, can solve the problems of brittle material properties, poor thermal stability, poor toughness and so on

Active Publication Date: 2022-03-04
TSINGHUA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A PHA obtained by monomer polymerization, such as P(3HB), has the problems of low elongation at break, brittle material properties, poor toughness, and unstable molten state
PHA obtained by copolymerization of two monomers, such as P(3HB- co -4HB), P(3HB- co -3HV), P(3HB- co -3HHx), there are problems such as material becoming brittle, low transparency, and poor thermal stability caused by recrystallization

Method used

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  • 3-hydroxybutyric acid, 4-hydroxybutyric acid and 3-hydroxyvalerate terpolymer p (3HB-4HB-3HV) and microbial production thereof
  • 3-hydroxybutyric acid, 4-hydroxybutyric acid and 3-hydroxyvalerate terpolymer p (3HB-4HB-3HV) and microbial production thereof
  • 3-hydroxybutyric acid, 4-hydroxybutyric acid and 3-hydroxyvalerate terpolymer p (3HB-4HB-3HV) and microbial production thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0112] Embodiment 1: Knock out in halophilic bacteria prpC Genes that increase P(3HB- co 3HV ratio in -3HV)

[0113] The present invention uses CRISPR / Cas9 technology to construct a plasmid with pSEVA321 as the backbone and a gRNA with the sequence cgcaccgtcatcatcaggt (SEQ ID NO: 1). Halobacteria Halomonas blue phase The genome of TD01 knocked out 2-methylcitrate synthase (PrpC) to promote the conversion of propionyl-CoA (Propionyl-CoA) to 3-ketopentyl-CoA (3-ketovaleryl-CoA), and Under the action of acetoacetyl coenzyme A (PhaB), more 3HV precursors - 3-hydroxypentyl coenzyme A (3-hydroxyvaleryl-CoA) are synthesized, and finally combined with PHA synthase (PhaC) Halomonas bluephagenesis 3-hydroxybutyryl-CoA (3-hydroxybutyryl-CoA) produced by TD01's own metabolism is copolymerized into P(3HB- co -3HV). The success of genetic manipulation can be judged by adding propionic acid, a carbon source related to 3HV, and whether the conversion rate of propionic acid to 3HV is...

Embodiment 2

[0118] Embodiment 2: introduce into halophilic bacteria scpA , scpB , to increase the proportion of 3HV synthesized from unrelated carbon sources

[0119] The present invention adopts in halophilic bacteria Halomonas bluephagenesis In TD01-D1, methylmalonyl-CoA mutase (ScpA) and methylmalonyl-CoA decarboxylase (ScpB) were introduced at the G49 site by homologous recombination. In the copolymer, if the proportion of 3HB is too high, it will easily lead to the lack of flexibility and ductility of the material performance, and a variety of promoters with different strengths are designed to express scpA and scpB Gene, which is able to convert tricarboxylic acid cycle (TCA cycle) product - succinyl coenzyme A (Succinyl-CoA) into propionyl coenzyme A (Propionyl-CoA), and synthesized under the action of the metabolic pathway described in Example 1 P(3HB- co -3HV). The tricarboxylic acid cycle is the main pathway of glucose metabolism. Related principles see attached fig...

Embodiment 3

[0125] Embodiment 3: introduce into halophilic bacteria ogA , sucD , wxya , orf Z , to increase the proportion of 4HB synthesized from unrelated carbon sources

[0126] The present invention adopts in halophilic bacteria Halomonas bluephagenesisIn TD01, 4-hydroxybutyryl-CoA transferase (OrfZ) was introduced at the G4 site by homologous recombination, and 2-oxoglutarate decarboxylase (OgdA) and succinic semialdehyde were introduced at the G7 site. The promoters of different strengths of dehydrogenase (SucD) and 4-hydroxybutyrate dehydrogenase (4hbd) were combined in tandem to convert the tricarboxylic acid cycle (TCA cycle) product - α-ketoglutarate (α-ketoglutarate) Converted into 4-hydroxybutyryl-CoA (4-hydroxybutyryl-CoA), and 3-hydroxybutyryl-CoA (3-hydroxybutyryl-CoA) produced by its own metabolism under the action of the bacteria's own PHA synthase (PhaC) Copolymerized into P(3HB- co -4HB). The tricarboxylic acid cycle is the main pathway of glucose metabolism...

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Abstract

The invention provides a recombinant bacterium and a method for producing P (3HB-4HB-3HV) through fermentation of the recombinant bacterium, and further provides a method for adjusting the proportion of monomers in the P (3HB-4HB-3HV). According to the method, microorganisms which cannot produce P (3HB-4HB-3HV) originally are transformed to construct recombinant bacteria capable of producing P (3HB-4HB-3HV), meanwhile, the content of 4HB and 3HV in the produced P (3HB-4HB-3HV) can be well increased by culturing the recombinant bacteria, and a P (3HB-4HB-3HV) material with excellent performance is obtained.

Description

technical field [0001] The invention relates to the fields of biotechnology and biomaterials, in particular to 3-hydroxybutyric acid, 4-hydroxybutyric acid and 3-hydroxyvaleric acid copolymer P (3HB-4HB-3HV) and its microbial production. Background technique [0002] The degradable plastic PHA produced by microbial fermentation mainly includes homopolymer P(3HB), copolymer P(3HB- co -4HB), P(3HB- co -3HV) and P(3HB- co -3HHx). In addition to the fact that 3HB monomer can be synthesized by non-related carbon sources such as glucose to reach the production level in some microorganisms (such as Halomonas) (reference: Tan D., et al. Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD01.Bioresource Technology. 2011. 102: 8130-8136), several other monomers have the problem of low production level of microbial native pathways (for example, reference: Li M.Y., et al. Engineering Pseudomonas entomophila for synthesis of copolymers with defined fractio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/625C12R1/01C12R1/40C12R1/19C12R1/05
CPCC12N15/52C12N9/90C12N9/88C12N9/0008C12N9/0006C12N9/13C12N9/001C12N9/1025C12P7/625C12Y504/99002C12Y401/01041C12Y102/01016C12Y102/01024C12Y102/01076C12Y102/01079C12Y401/01071C12Y101/01061C12Y208/03C12Y103/99001C12Y101/01202C12Y102/0101C12Y102/99003C12Y102/99007C12Y203/03005
Inventor 陈国强黄悟哲何宏韬吴赴清兰宇轩
Owner TSINGHUA UNIV