Engineering bacteria for producing maleic acid, and construction method and applications thereof

A technology of engineering bacteria and maleic acid, applied in the field of bioengineering, can solve problems such as environmental pollution and non-renewable energy

Active Publication Date: 2021-02-19
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, 100% of the industrial production of maleic acid adopts chemical methods, mainly n-butane oxidation, aromatic benzene oxidation and unsaturated olefin oxidation, etc. The raw materials used are all non-renewable energy sources, and chemical production will inevitably cause A certain degree of environmental pollution

Method used

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  • Engineering bacteria for producing maleic acid, and construction method and applications thereof
  • Engineering bacteria for producing maleic acid, and construction method and applications thereof
  • Engineering bacteria for producing maleic acid, and construction method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Recombinant plasmid pZE-NagAaGHAb, pCS-EP-Mps-Hbzf

[0040] The recombinant plasmid pZE-NagAaGHAb provided in this example is mainly obtained by linking the gene NagAaGHAb to the Escherichia coli expression vector pZE12-luc. pCS-EP-Mps-Hbzf is mainly obtained by linking genes EntC, PchB, Mps, Hbzf to E. coli expression vector pCS27.

[0041] The method for constructing the above-mentioned recombinant plasmid provided in this example specifically includes the following steps. Screening of genes encoding isochorisate synthase (EntC), isochorisate pyruvate lyase (PchB), salicylate-5-hydroxylase (NagAaGHAb), maleylpyruvate from bacteria, fungi or protein engineering Genes for isomerase (Mps) and maleylpyruvate lyase (Hbzf). By combining isochoristate synthase (EntC), isochoristate pyruvate lyase (PchB), salicylate-5-hydroxylase (NagAaGHAb), maleylpyruvate isomerase (Mps) and After the target gene of acetylpyruvate lyase (Hbzf) is amplified by PCR to obtain the target fra...

Embodiment 2

[0043] Engineering bacteria producing maleic acid: Recombinant Escherichia coli BW1

[0044] The maleic acid-producing engineering bacteria provided by the present invention have no special requirements on the type of host strain used to construct the expression plasmid. The embodiment of the present invention uses the BW25113 (F') strain as the initial host for constructing the plasmid.

[0045] First pick fresh BW25113(F’) colonies and inoculate them into 4mL LB medium, incubate at 37°C for 8-12h, then inoculate 1mL into 100mL LB medium, and incubate at 37°C until OD 600 When the growth reaches 0.6, centrifuge at 6000rpm at 4°C for 10min to collect the bacteria, wash with 10mL of 10% glycerin solution (mass ratio, the same below) pre-cooled to 4°C, centrifuge at 6000rpm for 10min, repeat the glycerin washing step, and centrifuge Finally, pour off the remaining glycerol as much as possible, and finally add 300 μL of 10% glycerol solution to resuspend the cells to obtain compe...

Embodiment 3

[0047] Application of Recombinant Escherichia coli BW1: Production of Maleic Acid by Fermentative Culture

[0048] Pick fresh recombinant Escherichia coli BW1 engineered single colonies on the plate and inoculate them into 4 mL LB test tubes containing ampicillin and kana. After culturing at 37°C for 8 hours, transfer them to shake flasks containing 50 mL medium containing ampicillin and kana for fermentation For cultivation, the inoculum size is 2% by volume, the fermentation temperature is 30° C. or 37° C., and the rotation speed is 200 rpm. Wherein, the medium: 2g·L -1 MOPS, 20g·L -1 Glucose, 10g·L -1 Glycerin, 5g·L -1 Yeast powder, 6g·L -1 NaHPO 4 , 0.5g·L -1 NaCl, 3g·L -1 K H 2 PO 4 , 2g·L -1 NH 4 Cl, 246.5mg L -1 MgSO 4 , 14.7mg·L -1 CaCl 2 , and add ampicillin and kanamycin.

[0049] The induction agent IPTG with a final concentration of 0.5mM was initially added to the fermentation, and part of the fermentation broth was taken out every 12 hours to mea...

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Abstract

The invention discloses engineering bacteria for producing maleic acid, and a construction method and applications thereof, and relates to the technical field of bioengineering. The provided engineering bacteria include genes that can be co-expressed and code isochorismate synthase, isochorismate pyruvate lyase, salicylic acid-5-hydroxylase, maleylpyruvate isomerase and maleylpyruvate lyase in a host. The above engineering bacteria also include the genes that are over-expressed and code shikimate kinase, DAHP synthetase, PEP synthetase and transketolase A in the host so as to realize the increased production of maleic acid. A construction method and applications of the engineering bacteria for producing the maleic acid are also provided. Through the utilization of the engineering bacteriato produce the maleic acid by taking glucose and/or glycerol as carbon source, the de novo synthesis of the maleic acid can be realized.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to an engineering bacterium producing maleic acid and its construction method and application. Background technique [0002] Maleic acid, also known as maleic acid, is a monoclinic colorless crystal in appearance. It has a wide range of applications in food, medicine, cosmetics, materials, etc. At present, its main use is to synthesize unsaturated polyester resin. In addition, it can also be used as a raw material to produce compounds such as tartaric acid, succinic acid, and fumaric acid. At the same time, it can also be used as a food flavoring agent, and is widely used as a sour taste agent. [0003] Maleic acid has a broad market prospect. Maleic anhydride can be obtained by heating maleic acid to 135°C. In 2007, the global market for maleic anhydride reached 1,807,000 tons, and according to forecasts, the global market for maleic anhydride will reach 2.2 million tons ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P7/46C12R1/19
CPCC12Y504/04002C12Y402/99021C12Y502/01004C12N9/0073C12Y207/01071C12Y207/0104C12Y202/01001C12N9/90C12N9/88C12N9/1205C12N9/1022C12N15/70C12P7/46
Inventor 王佳袁其朋盛华康申晓林孙新晓
Owner BEIJING UNIV OF CHEM TECH
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