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Method for producing long-chain binary acid by using recombinant escherichia coli strain

A technology for recombining Escherichia coli and long-chain dibasic acids, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problems of difficult entry into cells, low yield, etc., to increase yield and overcome reaction Harsh effects

Inactive Publication Date: 2017-08-08
河北美邦工程科技股份有限公司
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Problems solved by technology

The growth cycle of Escherichia coli is shorter than that of yeast, but this transformation method requires the addition of exogenous substrates, and it is difficult for aliphatic alkanes or their derivatives with different chain lengths to enter the cells, although there are also ways to express the outer membrane protein alkL gene (Mattijs K et al., 2012, Applied and Environmental Microbiology, 5724–5733) increased cell input of fatty acid methyl esters, but the yield was relatively low

Method used

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  • Method for producing long-chain binary acid by using recombinant escherichia coli strain
  • Method for producing long-chain binary acid by using recombinant escherichia coli strain
  • Method for producing long-chain binary acid by using recombinant escherichia coli strain

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Embodiment 1

[0026] Construction of embodiment 1 recombinant strain MJ1

[0027] a, The precursor of ω-dodecanedibasic acid synthesis is dodecanoic acid, but in the cell fatty acid will be in fatty acyl-CoA synthetase encoded by fadD gene, acyl-CoA dehydrogenase encoded by fadE gene and The enoyl-CoA encoded by the fadB gene is gradually degraded through the β-oxidation pathway. Therefore, it is first necessary to block or weaken the β-oxidation pathway genes so that cells can accumulate fatty acids under the expression of other fatty acid synthase genes.

[0028] The wild-type W3110 strain was used as the basic strain to knock out the fadD gene to weaken the fatty acid degradation pathway. The cell construction process was as follows: using pKD4 as a template, the primer pair fadD-K-F / fadD-K-R amplified the knockout fragment (see Table 1 for the primer sequence), The large W3110 electroporation competent cells were electrotransformed, spread on the Kana-resistant plate for screening, and...

Embodiment 2

[0030] Example 2 Construction of long-chain fatty acid synthesis expression plasmid pEZ07-fatB

[0031] According to SEQ ID NO: 1, the acyl-[ACP]thioesterase gene fatB derived from Umbellularia California was synthesized and connected into the pUC57 vector (purchased from Suzhou Jinweizhi Biotechnology Co., Ltd.) to obtain the plasmid pUC57-fatB. The fatB gene is mainly used to catalyze the synthesis of long-chain fatty acids, especially twelve-carbon fatty acids.

[0032] The expression plasmid pEZ07-fatB was constructed by seamless cloning and recombination technology. pEZ07 is a low-copy plasmid with spectinomycin resistance and Trc as a promoter, constructed in our laboratory using pTrc99A and pCL1920 from Biovector China Plasmid Vector Strain Cell Gene Collection Center. Using the plasmid pUC57-fatB synthesized by the above gene as a template, the primer pair fatB-N-F / fatB-X-R amplifies the gene fatB (see Table 1 for the sequence of the primer pair) containing homologous...

Embodiment 3

[0044] Example 3 Construction of long-chain fatty acid synthesis expression plasmid pEZ07-fatB

[0045] The Escherichia coli fadR gene is a regulator of fatty acid synthesis and degradation-related transcriptional genes. The literature shows that overexpressing fadR helps to increase the amount of fatty acid synthesis. Therefore, the fadR gene is co-expressed on the basis of the expression plasmid pEZ07-fatB in Example 1. In order to increase the production of twelve carbon fatty acids.

[0046] The expression plasmid pEZ07-fatB-fadR was constructed by seamless cloning and recombination technology. Using the MG1655 genome as a template, the primer pair fadR-P-F / fadR-H-R amplified the gene fatR with homologous regions at both ends (see Table 1 for the sequence of the primer pair), and recovered it; The recovered plasmid pEZ07-fatB was digested with PstI and HindIII for EZ clone, transformed into Escherichia coli competent cell TG1, and the construction of plasmid pEZ07-fatB-fa...

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Abstract

The invention discloses a method for producing long-chain binary acid by using a recombinant escherichia coli strain. The method comprises the following steps of knocking out or weakening a fatty acid beta-oxidation and degradation pathway gene in escherichia coli to establish the recombinant escherichia coli; using the recombinant escherichia coli to express fatty acid synthetase and a fatty acid synthesizing regulatory gene, and utilizing sustained carbon source glucose as a raw material to enable a cell to accumulate long-chain fatty acid; finally, expressing fatty acid hydroxylase, fatty alcohol, aldehyde reductase and the like on the basis, and gradually converting the accumulated long-chain fatty acid step by step, so as to obtain the long-chain binary acid. The method has the advantages that the defects of the prior art are overcome; in the whole preparation process, the reaction conditions are mild, the technology is simple, the environment-friendly effect is realized, and the product yield is higher.

Description

technical field [0001] The invention is applicable to the field of biochemical industry, and in particular relates to a method for producing long-chain dibasic acid by recombinant Escherichia coli strains. Background technique [0002] a, ω-long-chain dibasic acid refers to a straight-chain dibasic carboxylic acid containing more than 10 carbon atoms. It is an important fine chemical raw material with a wide range of uses, and is widely used in fragrances, preservatives and polymers. [0003] a, ω-Long-chain dibasic acid does not exist in nature. At present, there are two methods of production according to the number of carbon atoms of long-chain dibasic acid. One is chemical synthesis and the other is biological fermentation. The chemical synthesis method mainly uses petroleum raw materials for biotransformation (Liu et al., 2011, Biomacromolecules, 12:3291–3298), but the use of chemical methods for production has its limitations. First, the chemical synthesis methods have...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P7/64C12R1/19
CPCC12N9/0006C12N9/0073C12P7/6409C12Y101/01002C12Y114/1303
Inventor 刘东江君君胡志浩李丽
Owner 河北美邦工程科技股份有限公司
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