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Recombinant microorganism capable of efficiently utilizing methanol and application thereof

A technology of recombinant microorganisms and methanol dehydrogenase, applied in the fields of genetic engineering and biological fermentation, can solve the problems of grain competition and price competition, and achieve the effect of increasing biomass, improving biological efficiency and broad application prospects.

Pending Publication Date: 2022-03-04
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the microbial production of chemicals relies on sugar-based raw materials, and there are problems such as competition with others for food and price competition

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1 introduces methanol conversion pathway in Escherichia coli

[0025] In this example, the chassis strain X1 / mdh-das capable of utilizing methanol was obtained by introducing the plasmid pTrc99a-mdh-das into Escherichia coli W3110 (purchased from China Industrial Microorganism Culture Collection Center).

[0026] The construction method of plasmid pTrc99a-mdh-das: artificially synthesize mdh and das genes, the gene sequences of which are shown in SEQ ID No: 1 and SEQ ID No: 2, respectively. The mdh and das genes were inserted into the EcoRI / SmaI restriction site of plasmid pTrc99a (purchased from addgene) by Gibson assembly method to obtain plasmid pTrc99a-mdh-das.

[0027] The construction method of Escherichia coli X1 / mdh-das: The plasmid pTrc99a-mdh-das was electrotransformed into Escherichia coli W3110, and the resistant strain was obtained on the LB plate containing 100mg / L ampicillin, named X1 / mdh-das.

[0028] Escherichia coli X1 / mdh-das was cultured ...

Embodiment 2

[0029] Example 2 Further improving methanol utilization by transforming the metabolism of Escherichia coli itself

[0030] Methanol utilization pathways are limited by the original intracellular formaldehyde dissimilation pathway, most of which are converted to carbon dioxide instead of entering central carbon metabolism. In this embodiment, the formaldehyde dissimilation gene frmAB is firstly knocked out by Red recombination technology (the nucleic acid sequence of the frmA gene is shown in SEQ ID No: 3, and the nucleic acid sequence of the frmB gene is shown in SEQ ID No: 4), which can improve the integration of methanol into the central metabolic cycle Proportion. In addition, by knocking out the 6-phosphofructokinase gene pfkAB (the nucleic acid sequence of the pfkA gene is shown in SEQ ID No: 5, and the nucleic acid sequence of the pfkB gene is shown in SEQ ID No: 6) and the α-ketoglutarate dehydrogenase gene sucA (The nucleic acid sequence of the sucA gene is shown in S...

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Abstract

The invention relates to the technical field of genetic engineering and biological fermentation, and particularly discloses a recombinant microorganism capable of efficiently utilizing methanol and application of the recombinant microorganism. The invention provides a recombinant microorganism which has increased expression and / or enzymatic activity of methanol dehydrogenase and dihydroxyacetone synthetase and reduced expression quantity of a formaldehyde dissimilatory gene frmAB and expression and / or enzymatic activity of 6-phosphofructokinase compared with an original strain, and the recombinant microorganism has the advantages that the expression quantity of the methanol dehydrogenase and the dihydroxyacetone synthetase is increased, and the expression quantity of the formaldehyde dissimilatory gene frmAB and the expression quantity of 6-phosphofructokinase are reduced; the starting strain is escherichia coli. Specifically, after a formaldehyde dissimilatory gene frmAB, a 6-phosphofructokinase gene pfkAB and an alpha-ketoglutarate dehydrogenase gene sucA are knocked out at the same time, and a 3-phosphoglyceraldehyde dehydrogenase gene gapA is inhibited, the utilization rate of methanol can be further improved, and accumulation of intermediate formaldehyde is reduced.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and biological fermentation, in particular to a recombinant microorganism capable of efficiently utilizing methanol and its application. Background technique [0002] The development of green biomanufacturing technology is one of the effective ways to solve energy and environmental problems and achieve the goal of "carbon neutrality". Its core is to build microbial cell factories to efficiently synthesize bulk and high-value chemicals. important applications in the field. However, most of the microbial production of chemicals relies on sugar-based raw materials, and there are problems such as competition with others for food and price competition. As a basic chemical raw material, liquid methanol can be prepared from natural gas, shale gas, etc., with low price, simple storage and transportation conditions, and has great potential as a raw material for biomanufacturing. Moreover, th...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P7/04C12R1/19
CPCC12N9/0006C12N9/1205C07K14/245C12N15/70C12P7/04C12Y101/01244C12Y207/01029C12Y207/01011Y02E50/10
Inventor 陈振孙青
Owner TSINGHUA UNIV
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