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An improved method for producing polyhydroxyalkanoate

A technology for polyhydroxyalkanoates and hydroxybutyrate is applied in the field of improved production of polyhydroxyalkanoates, and can solve the problems of toxicity of precursor cells, difficulty in large-scale application to industrial production, high price and the like

Active Publication Date: 2021-12-14
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Another type is to change the proportion of corresponding monomers in the polymer by directly controlling the addition of specific related precursors, but the related precursors of most monomers are very expensive, and some precursors are even toxic to cells, resulting in Such methods are difficult to apply to industrial production on a large scale

Method used

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  • An improved method for producing polyhydroxyalkanoate
  • An improved method for producing polyhydroxyalkanoate
  • An improved method for producing polyhydroxyalkanoate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1, the production of microbial fermentation synthesis P3Hb is increased by adding acetic acid to the base medium

[0089] First, optimization of medium and culture conditions

[0090] (1) Optimization of glucose concentration

[0091]In Halomonas Halomonas bluephagenesis TD01 P3HB experiment as production strains, to determine the optimal concentration of glucose production P3HB. First single colony was picked to the LB60 20mL (LB containing 60g / L NaCl) of medium at 37 ℃, 200rpm overnight culture; then 5% inoculum (v / v), were inoculated into 50mL containing a series of different concentrations glucose (Table 1) MM60 medium (pH 8.5-9.0, NaCl concentration 60g / L), the at 37 ℃, 200rpm shake flask for 48 hours, to obtain bacteria, the samples were centrifuged, washed with water, dry ice was measured the cell dry weight (CDW; unit g / L), and then detect the content percentage of the dry weight P3HB cells (%) by gas chromatography. Each three sets of parallel cond...

Embodiment 2

[0120] Example 2, a carbon source in the medium supplemented with acetic acid were added to test their effect on Halomonasbluephagenesis TDΔβ and P3HB synthesis.

[0121] First, the engineered bacteria TDΔβ building:

[0122] 1) Construction of plasmid pKObet23 knock addition homology arms

[0123] Extraction Halomonas bluephagenesis Halomonas genome of TD01, and its use as a template for PCR pfu enzyme (NEB, USA) was amplified, respectively beta-H1-F and beta-H1-R, beta-H2-F and beta -H2-R as primers to amplify etf-β (nucleotide sequence is shown in SEQ ID NO: 1; the amino acid sequence shown in SEQ ID NO: 2) the left and right homology arms H1 H2 of homology arm; plasmid pSEVA241 as a template (this plasmid documented in the literature:. Silva-Rocha, Rafael, etal * # * the Standard European Vector Architecture (SEVA):. a coherent platform forthe analysis and deployment of complex prokaryotic phenotypes * # * Nucleic AcidsResearch 41.D1 (2012):. D666-D675) is amplified using prim...

Embodiment 3

[0194] Example 3, the proportion of monomers in the control copolymers in the base medium

[0195] The material properties of the copolymer P3HB3HV and P3HB4HB are largely dependent on the monomer ratio, especially for P3HB3HV. At present, the maximum problem with the production of copolymers in Salt Single Bacillus is a high proportion of 3HB, and the proportion of 3HV and 4HB is low. As a carbon source than glucose reductive, the acetic acid is considered to control the internal oxidative environment of the microorganic acid and glucose to control the metabolic flow path of the acetyl co-enzyme A. Specifically, both copolymers P3HB3HV and P3HB4HB are dimer which are copolymerized from 3-hydroxybutyric acid (3Hb) and 3-hydroxyukoic acid (3HV), and the latter is from 3-hydroxybutyric acid (3HB) and 4-hydroxybutyric acid (4Hb) is copolymerized. 3HB was synthesized from the acetyl concentrate by oxygen-free fermentation, while 3HV and 4HB were through the precursor generated from t...

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Abstract

The present invention provides an improved method for producing polyhydroxyalkanoate (PHA) by microorganisms, the method comprising adding acetic acid, acetate, acetate or other acetic acid derivatives; preferably adding acetic acid or acetate. The method can be used to increase the yield of PHA polymer synthesized by microorganisms and / or control the monomer ratio in the PHA copolymer product.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular, the invention relates to an improved method for producing polyhydroxyalkanoate (PHA). More specifically, the present invention relates to a method for increasing the yield of PHA synthesized by microorganisms and / or controlling the monomer ratio in the PHA copolymer product, which is realized by adding a certain amount of acetic acid in the medium. Background technique [0002] At present, large-scale industrial production of biopolymers usually uses glucose or sodium gluconate as a carbon source, wherein glucose or sodium gluconate first generates pyruvate through the glycolysis pathway, and then generates acetyl-CoA from pyruvate, and then enters Synthetic pathways for the synthesis of various polymer monomers starting from acetyl-CoA. Glycolysis will produce a large amount of NADH and ATP. The process of generating acetyl-CoA from pyruvate will also produce NADH. Under high-density...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P39/00C12P7/62C12N1/21C12R1/19C12R1/38C12R1/07
CPCC12P39/00C12P7/625C12P7/62
Inventor 陈国强凌晨乔冠清帅博闻
Owner TSINGHUA UNIV
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