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Metabolic engineering pseuomonas denitrifican for producing succinic acid as well as construction method and application of pseuomonas denitrifican

A technology of Pseudomonas denitrogenation and metabolic engineering, which is applied in the field of Pseudomonas denitrogenation of metabolic engineering for producing succinic acid, and achieves the effects of wide source of raw materials and low cost

Inactive Publication Date: 2019-03-15
XUZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the direct biosynthesis of succinic acid using Pseudomonas denitrifica as the host and acetic acid as the sole carbon source.

Method used

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  • Metabolic engineering pseuomonas denitrifican for producing succinic acid as well as construction method and application of pseuomonas denitrifican
  • Metabolic engineering pseuomonas denitrifican for producing succinic acid as well as construction method and application of pseuomonas denitrifican

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1. Knockout of genes to obtain succinic acid producing strains

[0019] The gene sdhAB was knocked out using the traceless knockout fund technique. The specific operation is as follows:

[0020] For the knockout of gene sdhAB, at first use the sequence as the primer (F1-sdhAB) of SEQ ID NO.1 and the sequence as the primer (R1-sdhAB) of Seq ID NO.2 with Pseudomonas denitrification ATCC13867 genomic DNA as template PCR clones the DNA fragment sdh US of about 500bp of the upstream sequence of the sdhAB gene, and uses the primer (F2-sdhAB) with the sequence of SEQ ID NO.3 and the primer (R2-sdhAB) with the sequence of Seq ID NO.4 to deaza Genomic DNA of bacteria ATCC13867 was used as a template to PCR clone the DNA fragment sdh DS of about 500 bp downstream of the sdhAB gene, and then the sdh US and sdh DS fragments were fused into one fragment sdh del by fusion PCR method, and the fused fragment was cloned into the pQSAK plasmid to form a recombinant Plasmid pQSA...

Embodiment 2

[0026] Example 2. Overexpression of acetyl-CoA synthetase acs

[0027] The acetyl-CoA synthetase gene acs of Pseudomonas denitrifica origin was overexpressed using the plasmid pUCPK. First, the acs gene was amplified by PCR using genomic DNA as a template. The Escherichia coli-Pseudomonas shuttle plasmid pUCPK was digested with XbaI and HindIII, and the acs fragment was ligated to pUCPK. The obtained recombinant plasmid was named pUCPK-acs and sent for sequencing verification.

Embodiment 3

[0028] Embodiment 3 produces Pseudomonas succinate strain shaking flask fermentation

[0029] Using Pseudomonas ATCC13867 as the starting strain, the sdhAB gene was knocked out, the TCA cycle was blocked, and the succinic acid-producing basic strain ZSP01 was obtained

[0030] On the basis of the single deletion strain, the pta-ackA gene was knocked out to obtain the strain ZSP02.

[0031] After the above strains were constructed, they were stored in glycerol (20%, v / v). The completed plasmid pUCPK-acs was used to transform each deletion strain, and the recombinant bacteria overexpressing the acs gene were obtained, which were named ZSP01-acs and ZSP02-acs. Shake flask fermentation was used to verify the fermentation performance of each strain.

[0032] Pseudomonas strains ZSP01, ZSP02 and recombinant strains ZSP01-acs, zsp-acs were cultured on LB plates overnight. A single colony from the fresh plate was inoculated into a 50 mL Erlenmeyer flask containing 10 mL of seed med...

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Abstract

The invention discloses metabolic engineering pseuomonas denitrifican for producing succinic acid as well as a construction method and the application of the pseuomonas denitrifican and belongs to thefields of genetic engineering and fermentation engineering. The construction method comprises the following steps: knocking out genes sdhAB and / or pta-ackA from genome DNA of pseuomonas denitrifican,over-expressing acetyl coenzyme A synthetase genes acs derived from the pseuomonas denitrifican in the pseuomonas denitrifican, and performing aerobic fermentation on the constructed pseuomonas denitrifican by taking acetic acid as a unique raw material, so as to obtain the succinic acid. According to the method disclosed by the invention, the acetic acid serves as the raw material, and the succinic acid is biologically synthesized by utilizing an acetate metabolism intermediate product acetylcoenzyme A. Compared with the prior art, the metabolic engineering pseuomonas denitrifican disclosedby the invention has the advantages that the acetylcoenzyme A serves as a metabolic intermediate, the raw material source is wide, and the cost is low.

Description

technical field [0001] The invention relates to the fields of molecular biology and genetic engineering, in particular to a metabolic engineering Pseudomonas denitrificans producing succinic acid, a construction method and application thereof. Background technique [0002] Succinic acid, an important C4 platform compound, was identified by the U.S. Department of Energy as one of the most valuable biorefinery products in 2004. It is a precursor for the synthesis of many important intermediate products and industrial chemicals, and is widely used in food, medicine, chemistry and agriculture. Succinic acid can also be used as an intermediate to synthesize many compounds such as γ-butyrolactone, 1,4-butanediamine and tetrahydrofuran with additional application value. [0003] In microbial cells, succinic acid is an intermediate metabolite of the TCA cycle, so succinic acid can be produced by fermentation of biomass raw materials. At present, most of the fermentation substrates...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/78C12P7/46C12R1/38
CPCC12N9/001C12N9/1217C12N9/93C12N15/78C12P7/46C12Y103/99001C12Y207/02001C12Y602/01003
Inventor 周生芳赵梅张春媚
Owner XUZHOU NORMAL UNIVERSITY
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