Genetically engineered bacterium for producing polyhydroxybutyric acid-valerate as well as construction method and application of genetically engineered bacterium

An amino acid and coding gene technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problems that the performance of strains cannot reach commercial production, increase production costs, and complicate the control process, etc., and achieve fermentation process Ease of control, low cost of raw materials, and safe strains

Active Publication Date: 2015-03-25
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The control process of the production of PHBV in the prior art is complex and increases the production cost, which limits the application
In recent years, researchers have used genetic engineering technology to transform strains, but the performance of the strains is far from meeting the requirements for commercial production, and the host bacteria may be pathogenic bacteria

Method used

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  • Genetically engineered bacterium for producing polyhydroxybutyric acid-valerate as well as construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing polyhydroxybutyric acid-valerate as well as construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing polyhydroxybutyric acid-valerate as well as construction method and application of genetically engineered bacterium

Examples

Experimental program
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Effect test

preparation example Construction

[0049] Preparation method of basal medium: each liter of basal medium contains 6.7g Na 2 HPO 4 2H 2 O, 1.5g KH 2 PO4, 1g(NH 4 ) 2 SO 4 , 0.2g MgSO 4 ·7H 2 O, 1ml of trace elements and 20g of glucose.

[0050] Contains 0.3g H per liter of trace elements 3 BO 3 , 0.2g CoCl 2 , 0.1g ZnSO 4 ·7H 2 O, 0.03g MnCl 2 4H 2 O, 0.02g NaMoO 4 2H 2 O, 0.02g NiCl 2 ·6H 2 O, 0.01g CuSO 4 ·5H 2 O, 0.01g CaCl 2 2H 2 O and 0.06g Fe(NH 4 ) 2 (SO 4 ) 2 .

Embodiment 1

[0051] The construction of the gene knockout vector of embodiment 1, prpC1 and prpC2

[0052] 1. Construction of prpC1 gene knockout vector

[0053] Using the genomic DNA of Ralstonia eutropha H16 as a template, PCR amplification was performed using prpC1df / prpC1dr primers. The primer sequences are as follows:

[0054] prpC1df: 5'-TAGATCTCAAGCGCGCCGGCTACCGGGCC-3';

[0055] prpC1dr: 5'-TTCTAGACAGGTCGAACTTCAGCACGCGCT-3'.

[0056] The amplified fragment with a size of 3222bp was connected to the plasmid pMDT19-simple, and the resulting recombinant vector was designated as pMD19-prpC1. The recombinant vector pMD19-prpC1 was transformed into E. coli DH5α, single colonies were picked, and the plasmid was extracted for sequencing. Sequencing results show that the sequence of the PCR amplification product is shown as sequence 1 in the sequence listing, which contains DNA fragments of the prpC1 gene and its two homologous arms. The nucleotide sequence of the prpC1 gene is shown in ...

Embodiment 2

[0066] Example 2, Construction of prpC1 and prpC2 gene knockout strains

[0067] 1. Construction of prpC1 knockout strain

[0068] Gene knockout was performed by the method of conjugal transfer. The pJQ200mp18Tc::ΔprpC1 knockout vector was transformed into E.coli S17-1, spread on the LB plate containing tetracycline, and the correct transformant was selected and named ΔprpC 1pJQ200 / S17-1.

[0069] ΔprpC 1pJQ200 / S17-1 was used as the donor bacteria for conjugative transfer and the recipient bacteria Rem-1 for conjugative transfer. The process of conjugative transfer is as follows: first, the recipient bacteria Rem-1 was inoculated in PG medium, and cultured at 30°C until the OD value was 1.0; the donor bacteria ΔprpC 1pJQ200 / S17-1 was inoculated in LB culture based on 37°C culture, and the growth When the OD value is 0.4, take 100 μl of the culture solution of the recipient bacteria and the donor bacteria, and centrifuge at 3000 rpm to remove the supernatant, suspend and mix ...

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Abstract

The invention discloses a genetically engineered bacterium for producing polyhydroxybutyric acid-valerate as well as a construction method and application of the genetically engineered bacterium. The genetically engineered bacterium is a gene-deleted strain of ralstonia eutropha or is obtained by introducing a coding gene yliK of methylmalonyl-coA mutase, a coding gene argK of GTP kinase and a coding gene ygfG of methylmalonyl-coA decarboxylase to the ralstonia eutropha or the gene-deleted strain of the ralstonia eutropha; and the gene-deleted strain of the ralstonia eutropha is obtained by deleting at least one of a coding gene of 2-methyl citrate synthase-1 of the ralstonia eutropha and a coding gene of 2-methyl citrate synthase-2. In production of the polyhydroxybutyric acid-valerate, the recombinant bacterium constructed by the invention has the following advantages that the genetically engineered bacterium is safe in strain and relatively cheap in raw material, fermentation process is easy to control, and large-scale production is easy to realize.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a genetically engineered bacterium for producing polyhydroxybutyrate-valerate and its construction method and application. Background technique [0002] Due to the dependence of the plastic industry on petroleum and the seriousness of environmental pollution caused by plastic waste, people are forced to look for environmentally friendly and sustainable materials that can replace chemical plastics. Poly-3-hydroxybutyrate (hereinafter referred to as polyhydroxybutyrate, PHB) is a biodegradable polymer material based on bio-based production, and its performance is similar to that of plastic. However, PHB is brittle, and post-processing has certain difficulties. If 3-hydroxyvaleric acid (3HV) components are mixed into PHB, the obtained poly-3-hydroxybutyric acid copolymerized 3-hydroxyvaleric acid ester (hereinafter referred to as poly Hydroxybutyrate-valerate, PHBV) can imp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12P7/62C12R1/01
CPCC12N9/90C12P7/625C12Y504/99002
Inventor 丁久元张英姿刘桂明翁维琦刘双江
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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