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Method for improving the content of lactic acid composition in escherichia coli synthesized poly(3-hydroxybutyrate-co-lactic acid)

A technology of hydroxybutyric acid and Escherichia coli, applied in the field of metabolic engineering, can solve the problems of low cell productivity and slow growth

Active Publication Date: 2019-10-01
EAST CHINA UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The respiratory chain is an important pathway for cellular energy metabolism. When the level of the respiratory chain is high, the cell production rate is high, and the accumulation of reducing equivalent NADH in the cell is small, but many reduced products are commercially profitable. The high level of the respiratory chain is not conducive to the reduced form. Accumulation of products; when the level of respiratory chain is low, cell productivity is low and growth is slow (MetabolicEngineering, 2015, 28:159-168)

Method used

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  • Method for improving the content of lactic acid composition in escherichia coli synthesized poly(3-hydroxybutyrate-co-lactic acid)
  • Method for improving the content of lactic acid composition in escherichia coli synthesized poly(3-hydroxybutyrate-co-lactic acid)
  • Method for improving the content of lactic acid composition in escherichia coli synthesized poly(3-hydroxybutyrate-co-lactic acid)

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Effect test

Embodiment 1

[0034] Example 1. Construction of recombinant Escherichia coli

[0035] Using MG1655 as the starting strain, red recombination technology is used to knock out the ubiX gene in the production process of coenzyme Q8. The specific operation is as follows:

[0036] The gene ubiX was knocked out by electroporation. The temperature-sensitive plasmid pKD46 is introduced into the host bacteria through calcium transduction to express exo, bet, and gam genes that assist homologous recombination, and are used to prepare electroporation competent. Using the primer ubiX-F with the sequence of SEQ ID NO: 1 and the primer ubiX-R with the sequence of SEQ ID NO: 2, the genome of the single-gene deletion bacteria containing the kanamycin resistance gene fragment was used as a template to PCR clone about 2000bp DNA fragments. The voltage of electroconversion is 1.8KV, and the current is 5ms. After transformation is completed, use kanamycin (kan) resistance to screen out recombinants, use the ...

Embodiment 2

[0046] Example 2.pct cp Constitutive promoters of genes optimize expression

[0047] Use the ldhA promoter derived from MG1655 to replace pBADpct cp Central original arabinose promoter. First use the primer PldhA-F with the sequence of SEQ ID NO:9 and the primer PldhA-R with the sequence of SEQ ID NO:10, use MG1655 as a template to PCR a gene of about 300bp containing the ldhA promoter, and use the sequence as SEQ ID Primer pBADpct of NO:11 cp -F and the sequence are primers pBADpct of SEQ ID NO: 12 cp -R to pBADpct cp The plasmid was used as a template for PCR amplification of the 6600bp gene except for the arabinose promoter. Use Clone MultiS One Step CloningKit for one-step connection. Use sequence is the primer PldhA-y-F of SEQ ID NO:13 and the primer PldhA-y-R bacterium colony PCR verification that sequence is SEQ ID NO:14 whether replaces successfully. For the strains with correct colony PCR, the plasmid was extracted and sent for sequencing. The sequencing resu...

Embodiment 3

[0055] Example 3.M9 culture medium fermentation production polymer

[0056] Add 20g / L glucose to M9 medium, add 0.1mM IPTG to induce the expression of phaA, phaB and phaCm, add 50mM L-arabinose (L-arabinose) to induce pct cp Gene expression. Incubate at 30°C for 48 hours to evaluate the performance of recombinant bacteria. The recombinant bacteria MG-01, JX04-01 and JX041-01 were used for fermentation. As shown in Table 7, compared with MG-01, the molar percentage of lactic acid component in P(3HB-co-LA) in the polymer produced by recombinant bacteria JX04-01 increased from 5.1% to 9.1%. An increase of 78.4%. And the polymer content is maintained above 75wt%. The recombinant strain JX041-01 was used for fermentation. Compared with MG-01 and JX04-01, the molar percentage of lactic acid component in P(3HB-co-LA) in the polymer was further increased to 14.1%. Compared with MG-01, it increased by 176.4%, compared with JX04-01, it increased by 54.9%, and the polymer content re...

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Abstract

The invention provides a method for improving the content of the lactic acid component in escherichia coli synthesized poly(3-hydroxybutyrate-co-lactic acid). On the basis of recombinant escherichia coli E.coli MG-01, a flavin isopentenyl transferase gene ubiX is knocked out, and / or a D-lactic dehydrogenase lactate gene dld is knocked, and / or a constitutive ldhA promoter is replaced with a promoter expressing the propionyl coenzyme A transferase pctcp. Accordingly, by analyzing metabolic pathways and control, by means of the genetic engineering means, escherichia coli is transformed, and underthe aerobic condition, obtained strains can improve the mole percent of the lactic acid composition in the poly(3-hydroxybutyrate-co-lactic acid) synthesized in the escherichia coli.

Description

technical field [0001] The present invention relates to the technical field of metabolic engineering, and more specifically, relates to a method and application of finely regulating and controlling the monomer components in microbial synthesis of polyester. Background technique [0002] Polyhydroxyalkanoate (PHA) is a biosynthetic polymer linear polyester that can be used as an intracellular energy and carbon source storage substance. It mostly exists in the form of hydrophobic particles, and its molecular weight generally ranges from tens of thousands to several million Daltons (Biotechnol. Bioeng, 1996, 54:450-472). PHA has attracted extensive attention due to its excellent material properties, such as biorenewability, biodegradability, thermoplasticity, and biocompatibility (Metab Eng, 2016, 35:1-8). [0003] Polyhydroxybutyric acid lactate [P(3HB-co-LA)] is a new member of the PHA family in recent years, and its polymerization monomers are derived from 3-hydroxybutyric ...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12P7/62C12N1/21C12R1/19
CPCC12N9/0006C12N9/1085C12N15/70C12P7/625C12Y101/01028
Inventor 吴辉陆静娴李志敏叶勤
Owner EAST CHINA UNIV OF SCI & TECH
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