Zymomonas mobilis recombination strain producing isobutanol, construction method and application thereof

A technology of Zymomonas and recombinant strains, which is applied in the field of construction and production of Zymomonas mobilis recombinant strains for producing isobutanol, which can solve the problems of unsatisfactory industrial production and low yield of isobutanol, and achieve high yield, The effect of high conversion efficiency and cost reduction

Active Publication Date: 2019-10-22
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although the above-mentioned method for producing isobutanol based on recombinant Zymomonas mobilis is improved from aerobic fermentation to static anaerobic fermentation; however, the yield of isobutanol is too low, only 0.1g / L, which cannot meet industrialization production requirements

Method used

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  • Zymomonas mobilis recombination strain producing isobutanol, construction method and application thereof
  • Zymomonas mobilis recombination strain producing isobutanol, construction method and application thereof
  • Zymomonas mobilis recombination strain producing isobutanol, construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 The kdcA expression module was introduced into Zymomonas mobilis to obtain the recombinant strain ZM-Q1 capable of inducing the production of isobutanol

[0038] In this example, the dihydroxy-aciddehydratase (KDCA) gene derived from Lactococcus lactis was first integrated into the ZMO0038 site in the genome of the model strain Z. mobilis ZM4 by means of homologous recombination. And use the inducible promoter to control the expression of nuclease, construct the recombinant strain ZM-Q1, the recombination schematic diagram of the recombinant strain is as follows: figure 1 shown.

[0039] 1. Construction of recombinant plasmids

[0040] Use PCR to amplify the kdcA gene sequence, resistance selection marker (spectinomycin), inducible promoter gene sequence (tetracycline-inducible promoter), gene sequence upstream and downstream of the insertion site and reverse amplification of pUC57 for integration the vector sequence. The PCR amplification program was set as ...

Embodiment 2

[0072] Example 2 Overexpression of genes in the L-valine synthesis pathway, and screening of ALS in the metabolic pathway to achieve the purpose of increasing production of isobutanol

[0073] In the isobutanol synthesis pathway, pyruvate is first catalyzed by pyruvate synthase (ALS, EC 2.2.1.6) to generate acetolactate, and then ketoacid reductoisomerase (ILVC, EC 1.1.1.86 ) is reduced to 2,3-dihydroxyisovalerate (2,3-dihydroxyisovalerate). The intermediate 2,3-dihydroxyisovalerate is converted to 2-ketoisovalerate by dihydroxyacid dehydratase (ILVD, EC4.2.1.9). Then, 2-ketoisovalerate can be further converted to isobutyraldehyde by KDCA and then to alcohol by alcohol dehydrogenases (ADHs).

[0074] The recombinant strain ZM-Q1 can produce isobutanol, and the overexpression of Als, ilvC, ilvD in the valine synthesis pathway can transfer the carbon flow from ethanol synthesis to isobutanol production. These three genes were integrated into the shuttle plasmid pEZ15a, and the...

Embodiment 3

[0114] Example 3 Adjusting the expression levels of different genes in the metabolic pathway and constructing a high-yield isobutanol recombinant strain

[0115] The inventor found through experiments in the early stage that the strength of the promoter limits the production of isobutanol to a certain extent, and replaced the inducible promoter on the kdcA expression module with a constitutive promoter. The dihydroxy-acid dehydratase (KDCA) gene derived from Lactococcus lactis was integrated into the ZMO0038 site in the Z. mobilis ZM4 genome by homologous recombination, and the constitutive promoter Pgap was used to control The expression level of nuclease was used to construct the recombinant strain ZMQ3. The three genes in the valine metabolic pathway were integrated into the shuttle plasmid pEZ15a, and the expression of the genes was controlled by an inducible promoter. The recombinant plasmid was transferred into the recombinant strain of ZMQ3, and the production of isobut...

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Abstract

The invention belongs to the technical field of biological genetic engineering, and particularly relates to a zymomonas mobilis recombination strain producing isobutanol, a construction method and application thereof. Zymomonas mobilis ZM4 serves as a model strain, firstly, the strain is transformed by means of genetic engineering, a metabolic intermediate in the synthesis pathway of valine of zymomonas mobilis is utilized for introducing a KDCA gene, 2-ketoisovalerate in the valine pathway is converted into a precursor isobutyraldehyde of isobutanol, and then isobutanol is generated under thehelp of alcohol dehydrogenase; secondly, the purpose of increasing the yield is achieved by overexpressing genes in the L-valine synthesis pathway and screening acetolactate synthase in the metabolicpathway;finally, by adjusting the expression quantity of different genes in the metabolic pathway, the purpose of high yield is finally achieved.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and in particular relates to a Zymomonas mobilis recombinant strain producing isobutanol, a construction method and an application thereof. Background technique [0002] Due to the global energy crisis and serious environmental problems caused by fossil fuels, environmentally friendly and renewable biofuels have attracted widespread attention. With the development and progress of synthetic biology technology, the use of microorganisms for metabolic engineering and systems biology research has received more and more attention. The production of biofuels and value-added chemicals from inexpensive renewable resources using microorganisms as carriers provides an effective alternative to address the shortage of fossil resources and the related environmental problems caused by them. In recent years, a variety of microorganisms for renewable biofuels have been developed through metabolic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/52C12N15/53C12N15/74C12P7/04C12R1/01
CPCC12N9/88C12N9/00C12N15/74C12P7/04C12Y402/01009Y02E50/10
Inventor 杨世辉仇梦月沈威
Owner HUBEI UNIV
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