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Method for producing 1, 6-hexanediol through whole-cell catalysis, recombinant microorganism and application of recombinant microorganism

A technology for recombining microorganisms and catalytic systems, applied in the direction of microorganism-based methods, microorganisms, biochemical equipment and methods, etc., to achieve the effects of short reaction time, high yield, and less intermediate operations

Active Publication Date: 2022-06-10
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003]In industry, 1,6-hexanediol is mainly produced through esterification and hydrogenation of 1,6-hexanediol. The production equipment is a high-pressure Class A explosion-proof equipment. Usually faced with high equipment requirements, serious equipment corrosion problems, poor catalyst stability and other problems

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1 introduces adipic acid conversion pathway in escherichia coli

[0026] Escherichia coli itself cannot directly utilize adipic acid to synthesize 1,6-hexanediol. For this reason, the carboxylic acid reductase MpCAR (amino acid sequence shown in SEQ ID NO.1) from Mycolicibacterium paratuberculosis is introduced into Escherichia coli. 4'-phosphopantetheyltransferase sfp of Bacillus subtilis (the amino acid sequence is shown in SEQID NO.2), and overexpress the original aldehyde reductase yahk in E. Escherichia coli recombinant strain W03 / MpCAR-sfp-yahk of hexanediol.

[0027] Construction method of recombinant strain W03 / MpCAR-sfp-yahk:

[0028] The plasmid PCDFDuet-MpCAR-sfp-yahk was electrotransformed into Escherichia coli W03 (Escherichia coli W3110 utilizes CRISPR-Cas9 technology to knock out the pta-ackA gene, ldhA gene, adhE gene) in a competent state, and contained 100mg / L spectinomycin The correct recombinant strain was obtained on the LB plate and na...

Embodiment 2

[0032] Embodiment 2 Whole cell catalyzes the method for synthesizing 1,6-hexanediol

[0033] In order to increase the yield of 1,6-hexanediol, the present invention has developed a method for whole-cell catalytic synthesis of 1,6-hexanediol. The specific steps are as follows:

[0034] (1) Escherichia coli strain W03 / MpCAR-sfp-yahk was cultured overnight at 37°C on an LB plate containing 100 mg / L spectinomycin. Then inoculate into 30mL LB medium containing 100mg / L spectinomycin, cultivate at 37°C for 12h to obtain seed liquid; insert the seed liquid into 200mL LB medium containing 100mg / L spectinomycin according to the inoculation amount of 1% volume , fermented at 37°C for 12 hours, centrifuged at 4000rpm, collected the cells, washed the cells with PBS buffer (10mM, pH 7.2), resuspended the cells with sterile water after centrifugation, and obtained a recombinant E. coli cell suspension.

[0035] (2) Add 10mL of catalytic system to a 250mL shake flask with baffles, which cont...

Embodiment 3

[0037] This example provides a method for whole-cell catalyzed synthesis of 1,6-hexanediol. The specific method is the same as in Example 2, the only difference being that the addition of disodium adipate in step (2) of Example 2 is changed. amount, so that the concentrations of adipic acid in the catalytic system are 40g / L, 10g / L, 60g / L respectively, and other reaction conditions remain unchanged, the concentration of 1,6-hexanediol in the reaction solution after 24h is 5.37g / L respectively L, 6.18g / L, 3.54g / L.

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PUM

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Abstract

The invention relates to the technical field of biochemical engineering, and particularly discloses a method for producing 1, 6-hexanediol through whole-cell catalysis, a recombinant microorganism and application of the recombinant microorganism. Compared with an original strain, the recombinant microorganism provided by the invention has the advantages that aldehyde reductase yahk is overexpressed, carboxylic acid reductase MpCAR and 4 '-phosphoric acid panthenyl aminotransferase sfp are expressed, and the original strain is escherichia coli. The recombinant Escherichia coli obtained by introducing exogenous carboxylic acid reductase and 4 '-phosphoric acid panthenotransferase into Escherichia coli and overexpressing aldehyde reductase is used for whole-cell catalysis, 1, 6-hexanediol can be produced from 1, 6-adipic acid under mild conditions, and the method has a very wide application prospect.

Description

technical field [0001] The invention relates to the technical field of biochemical industry, in particular to a method for catalyzing the production of 1,6-hexanediol by whole cells, recombinant microorganisms and applications thereof. Background technique [0002] 1,6-Hexanediol is an important chemical that can be used as a raw material to synthesize a series of high value-added products with important applications, such as high-grade polyurethane adhesives and polyurethane elastomers with special properties. Its applications cover polyurethane, Polyester, paint, dyestuff, medicine and many other fields have broad market prospects. At present, the high price of 1,6-hexanediol limits its production capacity and application, but the demand for this product in the global market is growing at an annual growth rate of 5% to 8%. [0003] In industry, 1,6-hexanediol is mainly produced through esterification and hydrogenation of 1,6-hexanediol. The production device is a high-pre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/18C12R1/19
CPCC12P7/18C12N9/0006C12N9/0008C12N9/1096C12Y101/01021C12Y102/99006C12Y206/01
Inventor 陈振朱强
Owner TSINGHUA UNIV
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