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Escherichia coli recombinant bacterium for denovo synthesis of vitamin B12, and construction method and application of escherichia coli recombinant bacterium

A technology of Escherichia coli and B12, applied in the field of Escherichia coli recombinant bacteria and its construction, can solve the problems of low output of vitamin B12 and inability to meet industrial production and the like

Active Publication Date: 2019-06-18
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Synthesis of vitamin B via the aerobic synthesis pathway has been reported in Escherichia coli 12 There is no precedent for a rigorous approach to vitamin B 12 identification, but detection of vitamin B by auxotrophic strains 12
This test method can produce false positives
Simultaneously, in the prior art, Escherichia coli is used to synthesize vitamin B 12 The output is not high and cannot meet the needs of industrial production

Method used

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  • Escherichia coli recombinant bacterium for denovo synthesis of vitamin B12, and construction method and application of escherichia coli recombinant bacterium
  • Escherichia coli recombinant bacterium for denovo synthesis of vitamin B12, and construction method and application of escherichia coli recombinant bacterium
  • Escherichia coli recombinant bacterium for denovo synthesis of vitamin B12, and construction method and application of escherichia coli recombinant bacterium

Examples

Experimental program
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Effect test

Embodiment 1

[0124] Construction of recombinant bacteria for the synthesis of Hydrogenobyrinic acid (HBA)

[0125]The phage-derived T7 RNA polymerase was integrated into the lacZ site of E. coli MG1655 by Red recombination method, and the strain was named MG1655(DE3).

[0126] The operon for synthesizing HBA on the pET3a-AIGJMFKLH plasmid is controlled by the constitutive T7 promoter. In order to achieve the purpose of inducing the synthesis of HBA, the pET3a-AIGJMFKLH plasmid was digested with Xba I and BamH I, and the cobAIGJMFKLH fragment with a length of about 8.1K was gelled. It was recovered and inserted into the Xba I and BamH I restriction sites of the pET28a plasmid to obtain the pET28-HBA plasmid. The plasmid was transformed into Escherichia coli MG1655 (DE3) to obtain HBA synthesis recombinant bacterium FH001.

Embodiment 2

[0128] Construction of recombinant bacteria for the synthesis of Hydrogenobyrinic acid a,c-diamide (HBAD)

[0129] Construction of pCDF-RccobB plasmid: Use the pCDFDuet-1 plasmid as a template to amplify the pCDFDuet-1 backbone. Using the R. capsulatus SB1003 genome as a template, the RCcobB fragment was amplified, and connected with the pCDFDuet-1 backbone through GoldenGate to obtain the plasmid pCDF-RccobB. The pCDF-RccobB plasmid was transformed into FH001 to obtain HBAD producing strain FH109.

Embodiment 3

[0131] Construction of cobalt(Ⅱ) yrinic acid a,c-diamide (CBAD) synthetic recombinant bacteria

[0132] The construction of pCDF-RccobB-BmcobN-BmcobS-BmcobT plasmid: using the B. melitensis 16M genome as a template, using BMcobS-F-Gibson, BMcobS-R-Gibson primers to amplify the BmcobS-Gibson fragment, using BMcobT-F-Gibson, BMcobT - R-Gibson primer amplifies the BmcobT-Gibson fragment. Using the pACYCDuet-1 plasmid as a template, the PACYC-Gibson fragment was amplified with PACYC-F-Gibson and PACYC-R-Gibson primers as the plasmid backbone, and the pACYC-BmcobS plasmid was obtained by Gibson assembly with the BmcobS-Gibson fragment. Using the pACYC-BmcobS plasmid as a template, the pCDF-RccobB-Gibson-F and pCDF-RccobB-Gibson-R primers were used to amplify to obtain the plasmid backbone, and the BmcobT-Gibson fragment was obtained by Gibson assembly to obtain the pACYC-BmcobS-BmcobT plasmid. Then, using the pACYC-BmcobS-BmcobT plasmid as a template, T7-cobST-F and T7-cobST-R wer...

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Abstract

The invention provides an escherichia coli recombinant bacterium for denovo synthesis of a vitamin B12, and a construction method and application of the escherichia coli recombinant bacterium. Specifically, escherichia coli serves as a starting strain, the engineering bacterium containing an HBA gene module, an HBAD gene module, a CBAD gene module and a Cbi gene module is constructed, the engineering bacterium efficiently and rapidly produces the B12 (the yield is at least 50 times higher than that of the prior art), and simple compounds can even be used as raw materials to conduct denovo synthesis on the vitamin B12.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically to the de novo synthesis of vitamin B 12 Escherichia coli recombinant bacteria and its construction method and application Background technique [0002] Vitamin B 12 Also known as cyanocobalamin, it is a stable form of cobalamin used in industrial production. Cobalamins mainly include adenosylcobalamin (deoxyadenosylcobalamin), methylcobalamin, hydroxycobalamin and cyanocobalamin. The chemical structure of adenosylcobalamin includes a central cobalt ring, an upper ligand (adenosine group) and a lower ligand (5,6-dimethylbenzimidazole, DMBI). [0003] Limited to the extremely high cost of chemical synthesis, vitamin B 12 It can only be obtained through microbial fermentation. Vitamin B 12 De novo synthesis pathways include aerobic and anaerobic pathways. Vitamin B present 12 Industrial production strains mainly include Pseudomonas denitrifican, Sinorhizobium meliloti, Propion...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P19/42C12R1/19
Inventor 张大伟房欢
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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