Full-biosynthesis method of pimelic acid

A technology for pimelic acid and cell production of pimelic acid, applied in the field of bioengineering, can solve the problem that the synthesis mechanism is not further elucidated, and achieve the effects of clear reaction mechanism, increased yield and optimized process

Active Publication Date: 2021-12-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above two pathways both produce a small amount of pimelic acid, but the synthesis mechanism has not been further elucidated

Method used

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  • Full-biosynthesis method of pimelic acid
  • Full-biosynthesis method of pimelic acid
  • Full-biosynthesis method of pimelic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Knockout of lactate dehydrogenase gene (ldhA), acetyl-CoA acetyltransferase gene (atoB) and succinyl-CoA synthetase α subunit gene (sucD) in BL21 (DE3)

[0043] The gene number of ldhA is ECD_01352, the gene number of atoB is ECD_02151, and the gene number of sucD is ECD_00688.

[0044] Using CRISPR / Cas9 technology, primers were designed according to the nucleotide sequence of the target gene published on the KEGG website, and the ldhA gene was knocked out. The pTarget plasmid required for the knockout was designed and provided by Suzhou Hongxun Company.

[0045] Obtaining of upstream and downstream homology arm fragments: using ldhAUP500-F, ldhAUP500-R and ldhADOWN500-F, ldhADOWN500-R as two pairs of primers, using the Escherichia coli BL21 (DE3) genome as a template, the size of A 515bp DNA fragment, which is a homologous sequence with the upstream and downstream 515bp of the ldhA gene, was recovered and purified by gel, and the purified upstream and downst...

Embodiment 2

[0052] Embodiment 2: Construction of recombinant plasmid and acquisition of recombinant Escherichia coli

[0053] The NCBI accession number of gene Tfu_0875 is MH157180; the NCBI accession number of gene Tfu_2399 is MH157181; the NCBI accession number of gene Tfu_0067 is MH157182; the NCBI accession number of gene Tfu_1647 is MH157183; for MH157194.

[0054] Plasmid pRSFDuet-1 was digested with EcoRI and HindⅢ, the vector fragment (3798bp) was recovered and purified by gel, and the plasmid pUC57-Tfu_0875 was digested with the same method, and the target gene fragment Tfu_0875 (shown in SEQID NO.1) was obtained by gel recovery and purification ), and then use T4 DNA ligase to connect the two fragments, transform into JM109 competent cells, pick the transformants for colony PCR verification, and extract the plasmids from the positive transformants for verification, and the verified plasmid is named pRSF-0875.

[0055] Recombinant plasmid pRSF-0875 was digested with BglⅡ and Kpn...

Embodiment 3

[0058] Embodiment 3: shake flask fermentation and result analysis of recombinant escherichia coli

[0059] Shake flask fermentation system 50mL.

[0060] Fermentation medium:

[0061] SOB medium, the composition is 5g L -1 Yeast powder, 20g· L-1 Tryptone, 5g·L -1 NaCl, 2.03g L - 1 MgCl 2 ·6H 2 O, 0.186g L -1 KCl, 4g L -1 Glucose, 6.6g·L -1 Glutaric acid, 50μg·mL -1 Kanamycin Sulfate, 50μg·mL -1 Ampicillin, 50 μg mL -1 streptomycin.

[0062] Seed liquid preparation: Streak the strains preserved in glycerol on the plate, pick a single colony and inoculate it in a triangular flask containing 50ml LB medium, 37°C, 250rpm·min -1 Shake the flask overnight. The next day, take 1ml of the bacterial solution and transfer it to 50ml of LB liquid medium, at 37°C, 250rpm·min -1 Incubate for 12-16 hours.

[0063] Fermentation conditions: the strain was inoculated in the fermentation medium at an inoculum size of 2% (2mL / 100mL), 37°C, 250rpm·min -1 Add 1mM IPTG to induce re...

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Abstract

The invention discloses a full-biosynthesis method of pimelic acid, and belongs to the field of bioengineering. Escherichia coli BL21(DE3) with a lactate dehydrogenase gene (ldhA), an acetyl-CoA acetyltransferase gene (atoB) and a succinyl-CoA synthetase alpha subunit gene (sucD) knocked out is used as a host, genes of beta-ketothiolase, 3-hydroxyacyl-coenzyme A dehydrogenase, 3-hydroxyadipyl dehydrogenase, 5-carboxyl-2-pentenoyl coenzyme A reductase and adipyl coenzyme A synthetase from thermopsis fusca are overexpressed in modules, and the yield of pimelic acid can reach 252.60mg.L < -1 > through fermentation optimization of added inorganic salt in a culture medium. A new route is provided for synthesis of pimelic acid in the Escherichia coli host, and a new idea is provided for industrial fermentation production of pimelic acid.

Description

technical field [0001] The invention relates to a total biosynthesis method of pimelic acid, which belongs to the field of bioengineering. Background technique [0002] Pimelic acid, Heptanedioic acid, also known as pyradolic acid, 1,5-pentane dicarboxylic acid, is a seven-carbon dicarboxylic acid, widely used in synthetic lubricants, plasticizers, copolymers , surfactants, etc., are important chemical raw materials and intermediates in the chemical industry. [0003] At present, the industrial production of pimelic acid mainly relies on chemical methods. Reduction with isoamyl alcohol and cleavage of salicylic acid with sodium crystallized pimelic acid in 43-50% yield. The laboratory preparation method of pimelic acid also includes using crown ether as a phase transfer catalyst to catalyze 1,5-pentanediol, phosphorus tribromide and potassium cyanide to synthesize pimelic acid. These methods have various techniques and long reaction times, which are unfavorable for large-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/44C12N1/21C12N9/04C12N9/10C12N9/00C12N15/53C12N15/52C12N15/54C12R1/19
CPCC12P7/44C12N9/0006C12N9/1029C12N9/93C12Y203/01009C12Y602/01005C12Y203/01174C12Y101/01035Y02A50/30
Inventor 邓禹李国辉鲍青青支睿李顺毛银赵运英周胜虎
Owner JIANGNAN UNIV
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