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Gene-engineering bacterium, construction method of same, and method of producing vanillin

A technology of genetically engineered bacteria and construction methods, applied in the direction of microorganism-based methods, biochemical equipment and methods, enzymes, etc., can solve the problems of many foreign genes, long routes, and cumbersome steps

Active Publication Date: 2017-07-14
BOTON SHANGHAI BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In 1998, Li et al. used recombinant Escherichia coli to produce vanillic acid through the shikimic acid pathway with glucose as a substrate, and produced trace amounts of vanillin under the catalysis of aromatic acid reductase produced by Neurospora crassa in vitro, but the method was cumbersome.
In 2009, Hansen et al. modified Schizosaccharomyces pombe and Saccharomyces cerevisiae by metabolic engineering method. The modified strains can use glucose as substrate respectively, and can produce 65 mg / L and 45 mg / L vanillin. However, on the one hand, this method produces Isovanillin is a by-product that is difficult to separate. On the other hand, due to the strong ability of yeast to degrade vanillin and the toxicity of vanillin to bacteria, vanillin needs to be further derivatized into glucoside-vanillin. for subsequent applications
In 2015, Xu Ping used metabolic engineering methods to transform Escherichia coli, and the constructed strain produced 97.2 mg / L vanillin with tyrosine as a substrate, and 19.3 mg / L and 13.3 mg / L with glucose, xylose and glycerol as substrates, respectively. mg / L and 24.7mg / L vanillin, but this route introduces more foreign genes, the route is longer, and the efficiency needs to be improved

Method used

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  • Gene-engineering bacterium, construction method of same, and method of producing vanillin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Knockout of shikimate dehydrogenase encoding gene aroE in Escherichia coli BL21(DE3)

[0057] The shikimate dehydrogenase encoding gene aroE in Escherichia coli BL21(DE3) was knocked out by PCR Targeting technology, and the obtained strain was named WYV5. The specific operation is as follows:

[0058] Using the BL21(DE3) strain genome as a template, the primer pairs aroE_up_s, aroE_up_a and aroE_dw_s, aroE_dw_a were used to amplify the upstream and downstream homology arms of the aroE gene, and pKD3 was used as a template to amplify the chloride The mycin resistance gene fragment, the three fragments were amplified by overlapping PCR to obtain the aroE gene knockout fragment aroE::cm, the primer sequences are as follows:

[0059] aroE_up_s: 5'-AACGGAAGCCGTTTTCGGTG-3';

[0060] aroE_up_a: 5'-CATTATGTTACCCCTGTCGA-3';

[0061] aroE-cm_s: 5′-AATCCGCGATGCCCTGACGGGTGAACTGTTTCGACAGGGGTAACATAATGGTGTAGGCTGGAGCTGCTTC-3′;

[0062] aroE-cm_a: 5′-ATTCTCGTCCCACTCTTCCCT...

Embodiment 2

[0073] Example 2: Knockout of a possible vanillin-degrading gene cluster yqhC-yqhD-dkgA

[0074] Using PCR Targeting technology, the possible vanillin degradation gene cluster yqhC-yqhD-dkgA was knocked out of the strain obtained in Example 1, and the obtained strain was named WYV18. The specific operation is as follows:

[0075] Using the BL21(DE3) strain genome as a template, the primer pairs yqhC-dkgA_up_s, yqhC-dkgA_up_a and yqhC-dkgA_dw_s, yqhC-dkgA_dw_a were used to amplify the upstream and downstream homology arms of the yqhC-yqhD-dkgA gene cluster, and pKD3 was used as a template, Amplify the chloramphenicol resistance gene fragment with primer pair yqhC-dkgA-cm_s, yqhC-dkgA-cm_a, three fragments are obtained yqhC-yqhD-dkgA gene cluster knockout fragment yqhC-yqhD-dkgA by overlapping PCR amplification: :cm. The primer sequences are as follows:

[0076] yqhC-dkgA_up_s: 5'-TTTTCTGCCTACGATTGC-3';

[0077] yqhC-dkgA_up_a: 5'-GGAAAATCGTCAGGCGTTAC-3';

[0078] yqhC-dkgA...

Embodiment 3

[0089] Example 3: Knockout of a possible vanillin degradation gene yahK

[0090] Using the PCR Targeting technique, the possible vanillin degradation gene yahK was knocked out of the strain WYV18 obtained in Example 2, and the obtained strain was named WYV39. The specific operation is as follows:

[0091] Using the BL21(DE3) strain genome as a template, the primer pair yahK_up_s, yahK_up_a and yahK_dw_s, yahK_dw_a were used to amplify the upstream and downstream homology arms of the yahK gene, and pKD3 was used as a template to amplify the chloride Mycin resistance gene fragments, the three fragments were amplified by overlapping PCR to obtain the yahK gene knockout fragment yahK::cm, the primer sequences are as follows:

[0092] yahK_up_s: 5'-AGATGAACTGGCGAACCGGA-3';

[0093] yahK_up_a: 5′-GAACTTCGAAGCAGCTCCAGCCTACACCATTGTGTTTACTCCTGATTAGC-3′;

[0094] yahk-cm_s: 5′-GCTAATCAGGAGTAAACACAATGGTGTAGGCTGGAGCTGCTTCGAAGTTC-3′;

[0095] yahK-cm_a: 5'-TGTTAAACCACAGGGTATTTTATTAATTT...

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Abstract

The invention belongs to the technical field of bioengineering, and discloses a gene-engineering bacterium, a construction method of same, and a method of producing vanillin. In the gene-engineering bacterium, the metabolic pathway of 3-dehydro-shikimic acid is modified, wherein a shikimic acid dehydrogenase encoding gene is deleted and a vanillin degradation related gene is also deleted, so that the bacterial strain can accumulate a large amount of 3-dehydro-shikimic acid; in addition, expression on a 3-dehydro-shikimic acid dehydratase gene, an O-transmethylase gene and a vanillic acid reductase gene and two genes in the metabolic pathway of the 3-dehydro-shikimic acid are enhanced, so that yield of the vanillin is increased, degradation of the vanillin is reduced and quantity of the vanillin in a fermentation liquid is increased, thus improving the capability of fermenting production of the vanillin by the bacterial strain. A test proves that the gene-engineering bacterium uses glucose or glycerol as a substrate for producing the vanillin, wherein the yield of the vanillin is increased significantly. A vanillin fermentation liquid produced with the method is free of an isovanillin impurity, so that subsequent separation is easy to carry out.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a genetically engineered bacterium and its construction method and a method for producing vanillin, especially a genetically engineered bacterium for producing vanillin and its construction method and the use of the strain to produce A method for producing vanillin from cheap carbon sources such as glucose and glycerol. Background technique [0002] Vanillin, also known as vanillin, chemically named 3-methoxy-4-hydroxybenzaldehyde, is white or slightly yellow crystal, with vanilla aroma and strong milky aroma, and is generally loved by people. The main ingredient of creamy vanilla essence. Vanillin is currently the most produced synthetic fragrance in the world. The synthesis methods of vanillin mainly include chemical synthesis and microbial transformation. Among them, the chemical synthesis method has high yield and low cost, but there are problems such as...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/66C12P7/24C12R1/19
CPCC07K14/245C12N9/0006C12N9/1007C12N9/88C12P7/24C12Y101/01025C12Y401/01001C12Y402/01118
Inventor 吴艳王元彩李斌
Owner BOTON SHANGHAI BIOLOGICAL TECH CO LTD
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