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Production method for C8:0/C10:0/C12:0/C14:0 medium-chain fatty acid and ethyl ester thereof

A medium-chain fatty acid, 0-C10 technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as high price and limited sources, improve low-temperature performance, simplify production processes, and reduce costs. Effect

Inactive Publication Date: 2012-07-18
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, medium-chain fatty acids are mostly extracted from plant seeds, the source is limited, and the price is relatively high

Method used

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  • Production method for C8:0/C10:0/C12:0/C14:0 medium-chain fatty acid and ethyl ester thereof
  • Production method for C8:0/C10:0/C12:0/C14:0 medium-chain fatty acid and ethyl ester thereof
  • Production method for C8:0/C10:0/C12:0/C14:0 medium-chain fatty acid and ethyl ester thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] 1 Cloning of genes related to fatty acid regulation-thioesterase in Calyxa plants and construction of recombinant vectors

[0043] 1.1 Cloning of thioesterase gene from Calyxa plant

[0044] The thioesterase gene DNA (acyl-acylcarrier protein (ACP) thioesterase) in Cuphea hookeriana was synthesized by the method of total gene synthesis. The chFatB in Calyxa plants encodes a thioesterase gene, which can specifically hydrolyze C8:0-C10:0 fatty acyl ACP into fatty acids, so that medium-chain fatty acids can be obtained in recombinant Escherichia coli.

[0045] Using the thioesterase gene DNA of the above-mentioned synthetic Calyxa plant as a template, design primers according to the GenBank sequence, and add corresponding restriction sites (Nde I and EcoR V) and protective bases, PCR amplifies thioesterase The primers used for gene chFatB are as follows:

[0046] Upstream primer: 5'-GGAATTC CATATG GTTGCTGCGGCTGCCAGC-3'

[0047] Downstream primer: 5'-CGG GATATC TTAGC...

Embodiment 2

[0057] 1 Cloning of fatty acid regulation-related gene-thioesterase in laurel tree and construction of recombinant vector

[0058] 1.1 Cloning of thioesterase gene in laurel

[0059] The thioesterase gene DNA (acyl-acylcarrier protein (ACP) thioesterase) in laurel (Umbellularia californica) was synthesized by the method of total gene synthesis. The ucFatB in laurel tree encodes a thioesterase gene, which can specifically hydrolyze C12:0 fatty acyl ACP into fatty acids, so that a single medium-chain fatty acid can be obtained in recombinant E. coli.

[0060] With the above-mentioned synthetic laurel thioesterase gene DNA as template, design primers according to the GenBank sequence, and add corresponding enzyme cutting sites (Nde I and EcoR V) and protective bases, PCR amplification thioesterase gene ucFatB primers as follows:

[0061] Upstream primer: 5'-GGAATTC CATATG GCTACCACCTCTCTGGCCAG-3'

[0062] Downstream primer: 5'-CGG GATATCTTAAACACGCGGTTCCGC-3'

[0063] The P...

Embodiment 3

[0072] 1 Cloning of fatty acid regulation-related gene-thioesterase in camphor and construction of recombinant vector

[0073] 1.1 Cloning of thioesterase gene in camphor

[0074] The thioesterase gene DNA (acyl-acylcarrier protein (ACP) thioesterase) in Cinnamomum camphora was synthesized by the method of total gene synthesis. ccFatB in Cinnamomum camphora encodes a thioesterase gene, which can specifically hydrolyze C14:0 fatty acyl ACP into fatty acids, so that medium-chain fatty acids can be obtained in recombinant Escherichia coli.

[0075] Using the thioesterase gene DNA in the camphor camphora synthesized above as a template, primers were designed according to the GenBank sequence, and corresponding enzyme cutting sites (Nde I and EcoR V) and protective bases were added to PCR amplify the thioesterase gene ccFatB. Primers are as follows:

[0076] Upstream primer: 5'-GGAATTC CATATG GCCACTACCAGCCTGGCCA-3'

[0077] Downstream primer: 5'-CGG GATATC TTACACGCTGCTTTCCGC...

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Abstract

The invention relates to a production method for synthesizing C8:0 / C10:0 / C12:0 / C14:0 medium-chain fatty acid and ethyl ester thereof by using engineering Escherichia coli. The production method comprises the following steps of: cloning thioesterase genes with different specificities chFatB / ucFatB / ccFatB in cuphea / laurel / camphorwood into an expression vector pETDuet-1 to obtain a corresponding recombinant plasmid; transferring the recombinant plasmid into Escherichia coli BL21, performing isopropyl-beta-d-thiogalactoside (IPDT) induction expression, and specifically hydrolyzing acyl carrier protein (ACP) with specific chain length by using the expressed thioesterase to obtain medium-chain fatty acid; cloning acyltransferase gene (atfA) in acinetobacter into expression vectors pETDuet-chFatB / pETDuet-ucFatB / pETDuet-ccFatB to obtain corresponding recombinant plasmids; and transferring the recombinant plasmids into the Escherichia coli BL21, performing IPTG induction expression, performing acyl transfer reaction on exogenously fed ethanol and acyl-CoA formed by the medium-chain fatty acid intracellularly by using the co-expressed acyltransferase to obtain fatty acid ethyl ester.

Description

technical field [0001] The invention belongs to the field of biochemical industry, and in particular relates to a production method of C8:0 / C10:0 / C12:0 / C14:0 medium-chain fatty acids and ethyl esters thereof. Background technique [0002] As an alternative to fossil energy, biodiesel has the advantages of safety, biodegradability, and complete combustion, and has been highly concerned and valued by many countries. In 2010, the global production of biodiesel was about 17 million tons, and it showed a continuous growth trend. At present, biodiesel mainly uses animal and vegetable oils, leftovers after oil refining, oil sludge, urban hogwash oil, waste oil after fried food, etc., after modification, it will be combined with short-chain alcohols such as methanol or ethanol under the catalysis of a catalyst. Composite and so on. The main components of biodiesel are fatty acid short-chain esters, such as fatty acid methyl ester, fatty acid ethyl ester, etc. [0003] Raw materia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/64C12P7/62C12R1/19
Inventor 邓利范丽苹刘军锋刘珞王芳谭天伟
Owner BEIJING UNIV OF CHEM TECH
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