Genetic engineering bacteria for producing PHA (polyhydroxyalkanoates) from acetic acid and propionic acid as well as construction method and application of genetically engineering bacteria

A technology of polyhydroxyalkanoates and engineering bacteria, which is applied in the fields of biotechnology, genetic engineering and fermentation engineering, and can solve the problems of restricting the large-scale industrial production and application of PHA, high production costs, etc.

Active Publication Date: 2018-08-03
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PHA has similar material properties to petroleum-derived plastics and can be used as packaging materials and tissue engineering materials, but its high production cost is the main factor limiting the large-scale industrial production and application of PHA.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Embodiment 1, construction of recombinant bacteria E.coliJM109 (pUC19-phaCAB+pMCS-pta-ackA)

[0084] 1. Construction of recombinant plasmid pMCS-pta-ackA

[0085] 1. The artificially synthesized DNA shown in Sequence 1 in the sequence list contains the pta-ackA expression cassette, the upstream is the EcoRI site, and the downstream is the XbaI site, wherein the 9th-51st nucleotide is the promoter sequence, and the 70th-51st nucleotide is the promoter sequence. Nucleotide 1272 is the ackA gene sequence, and 1347-3491 nucleotides are the pta gene sequence.

[0086] 2. Digest the DNA sequence synthesized in step 1 with EcoRI and XbaI, and recover a DNA fragment with a size of 3489bp; use EcoRI and XbaI to double digest the plasmid pBBR1MCS-2, and recover a DNA fragment with a size of 5114bp; the above 3489bp and 5114bp The two DNA fragments were ligated, and the ligated product was introduced into E.coli JM109 by chemical transformation method, spread on LB solid medium c...

Embodiment 2

[0102] Example 2, Construction of recombinant bacteria E.coliJM109SG (pUC19-phaCAB-orfZ+pMCS-pta-ackA-sucD-4hbD)

[0103] 1. Construction of recombinant plasmid pMCS-pta-ackA-sucD-4hbD

[0104] 1. The DNA shown in sequence 3 in the sequence list is artificially synthesized, which contains the sucD-4hbD expression cassette, the upstream is the XbaI site, and the downstream is the SacI site, wherein the 9th-51st nucleotides are the promoter sequence, and the 70th-4hbD Nucleotide 1431 is the sucD gene sequence, and 1468-2583 nucleotides are the 4hbD gene sequence.

[0105] 2. Digest the DNA sequence synthesized in step 1 with XbaI and SacI, and recover a DNA fragment with a size of 2585bp; use XbaI and SacI to double digest the plasmid pBBR1MCS-2, and recover a DNA fragment with a size of 5116bp; the above 2585bp and 5116bp The two DNA fragments were ligated, and the ligated product was introduced into E.coliJM109 by chemical transformation method, spread on LB solid medium cont...

Embodiment 3

[0148] Example 3, Construction of recombinant bacteria E.coli JM109 (pUC19-phaCAB-pct+pMCS-pta-ackA)

[0149] 1. Construction of recombinant plasmid pUC19-phaCAB-pct

[0150] 1. The artificially synthesized DNA shown in Sequence 5 in the sequence table contains the pct expression cassette, the upstream is the BamHI site, and the downstream is the EcoRI site, wherein the 9th-51st nucleotide is the promoter sequence, and the 70th-1623rd nucleotide Nucleotides are pct gene sequences.

[0151] 2. Digest the DNA sequence synthesized in step 1 with BamHI and EcoRI, and recover a DNA fragment with a size of 1621bp; use BamHI and EcoRI to double digest the recombinant plasmid pUC19-phaCAB, and recover a DNA fragment with a size of 6553bp; the above 1621bp and Two DNA fragments of 6553 bp were ligated, and the ligated product was introduced into E.coliJM109 by chemical transformation method, spread on LB solid medium containing ampicillin, and cultured at 37°C for 16 hours to obtain t...

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PUM

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Abstract

The invention discloses genetic engineering bacteria for producing PHA (polyhydroxyalkanoates) from acetic acid and propionic acid as well as a construction method and an application of the genetic engineering bacteria. The method for preparing the engineering bacteria for producing PHA comprises the following steps: improving expression and/or activity of acetokinase, phosphotransacetylase, PHA synthetase, beta-ketothiolase, acetoacetyl-CoA reductase, succinate hemiacetal dehydrogenase, 4-hydroxybutyrate dehydrogenase, 4-hydroxybutyl CoA:CoA-transferase and propionyl CoA transferase in recipient bacteria, and reducing expression and/or activity of non-CoA-cycle succinate hemiacetal dehydrogenase in the recipient bacteria to obtain the engineering bacteria for producing PHA, wherein the recipient bacteria can grow with acetic acid as a carbon source. The prepared engineering bacteria are used for producing PHA with acetic acid as the carbon source, PHA yield can reach a higher level, and the bacteria have better industrial application prospect. The genetic engineering bacteria have great application value.

Description

technical field [0001] The invention belongs to the fields of biotechnology, genetic engineering and fermentation engineering, and specifically relates to a genetically engineered bacterium for producing polyhydroxyalkanoate by using acetic acid and propionic acid, a construction method and application thereof. Background technique [0002] Renewable biomass resources based on agriculture and planting, including glucose, xylose, starch, and cellulose, are carbon sources and energy materials commonly used by microorganisms in the fermentation industry. In 2014, the total output of major products in my country's bio-fermentation industry was 24.2 million tons (the development status and development trend of my country's bio-fermentation industry, Industrial Microbiology, 2015, 45: 62-66), and a large amount of agricultural and sideline products such as starch were consumed in the production process , there is a problem of "competing with people for food and land with food". Wi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/62C12R1/19
CPCC12N9/0006C12N9/0008C12N9/1029C12N9/1217C12N9/13C12N9/93C12P7/625C12Y101/01036C12Y101/01061C12Y102/01C12Y203/01008C12Y203/01009C12Y207/02001C12Y208/03
Inventor 李正军陈静李微笪央央李良康
Owner BEIJING UNIV OF CHEM TECH
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