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Genetically engineered bacterium for producing lipase by using methanol and xylose co-substrate and application of genetically engineered bacterium

A technology of genetically engineered bacteria and lipase, applied in the field of bioengineering, can solve the problem of high cost of producing lipase, and achieve the effect of reducing production cost, great significance and economic value

Active Publication Date: 2020-06-19
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a synthetic biology method to construct a bacterial strain that can utilize methanol and xylose co-substrates for metabolism, and use the bacterial strain to ferment and produce lipase, which solves the problem of high cost of traditional lipase production

Method used

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  • Genetically engineered bacterium for producing lipase by using methanol and xylose co-substrate and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing lipase by using methanol and xylose co-substrate and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing lipase by using methanol and xylose co-substrate and application of genetically engineered bacterium

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Experimental program
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Embodiment 1

[0037] Example 1: Obtaining expressed genes

[0038] Pichia pastoris Pichia pastoris Using the genome as a template, design primers to amplify methanol oxidase aox 1. Dihydroxyacetone synthase gene das , catalase gene cta , dihydroxyacetone kinase gene dak .

Embodiment 2

[0039] Embodiment 2: Utilize the method construction of synthetic biology Candida - aox 1- das - cta - dak

[0040] In order to quickly and effectively realize the co-expression of multiple genomes and ensure the stability of gene expression, the gene expression cassette was integrated into the Candida antarctica genome by using the method of DNA assembly.

[0041] (1) Design primers for amplification, add promoter and terminator homology arms at both ends of each gene, design upstream and downstream primers, and obtain expression cassettes. The gene and primer sequences are shown in Table 1.

[0042] Table 1 Gene and primer sequences

[0043]

[0044] (2) Perform multi-fragment cloning to form a promoter-gene-terminator expression cassette TEF- aox 1-CYC1t, TEF- das -tCYC1, PDC1p- cta -TDH2t, pGPD- dak - TXPR2; the expression cassette of the above-mentioned promoter-gene-terminator is to combine and connect the indicated genes to form an expression fragment b...

Embodiment 3

[0059] Embodiment 3: the fermentation experiment of recombinant bacterial strain

[0060] (1) Test tube seed culture: inoculate recombinant Candida antarctica at 1% (v / v) inoculation amount from the cryopreservation tube into the test tube seed medium, the liquid volume in the test tube is 5 mL, and cultivate aerobically at 24°C for 18-22 h, to obtain the test tube seed culture solution.

[0061] Wherein, the formula of the seed medium is as follows: peptone 6 g / L, hydrolyzed casein 4 g / L, yeast powder 3 g / L, beef extract 1.5 g / L, glucose 1 g / L.

[0062] (2) Shake flask seed culture: Inoculate the test tube seed culture solution from the test tube culture medium to the shake flask seed culture medium according to the inoculum amount of 1% (v / v). The liquid volume in a 250 mL Erlenmeyer flask is 50 mL, and cultivate aerobically at 24 °C After 18-22 hours, the shake flask seed culture solution was obtained.

[0063] (3) Production of lipase by fermentation: Inoculate the shake...

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Abstract

The invention relates to a genetically engineered bacterium for producing lipase by using a methanol and xylose co-substrate and application of the genetically engineered bacterium. A methanol oxidasegene aox1, a dihydroxyacetone synthase gene das, a catalase gene cta and a dihydroxyacetone kinase gene dak are introduced into host bacteria; and the host bacteria are Candida antarctica capable ofproducing the lipase by utilizing xylose. According to the invention, a methanol metabolic pathway is introduced into the Candida antarctica by utilizing a synthetic biology way, so that the Candida antarctica can produce the lipase by taking non-food-grade raw materials, namely the methanol and the xylose, as a co-substrate, the production cost is reduced to a certain extent, and the applicationhas great significance and economic value.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to a genetically engineered bacterium which utilizes methanol and xylose co-substrate to produce lipase and its application. Background technique [0002] There are many kinds of microbial lipases, which widely exist in organisms. They have been widely studied because of their wider action pH and action temperature range than animal lipases, as well as their role in enzyme theoretical research and practical applications. Lipases can catalyze The decomposition, synthesis, transesterification and other reactions of ester compounds have high chemical, regio and stereoselectivity. In recent years, they have been widely used in organic synthesis, pharmaceuticals, detergents, biosurfactants and other fields. [0003] Candida antarctica lipase is an important lipase with many excellent properties, and because it is a new type of non-specific enzyme, it has strong stability both in solution an...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N9/20C12R1/72
CPCC12N9/0065C12N9/0051C12N9/1205C12Y207/01029C12Y111/01006C12N15/81C12N9/20C12Y301/01003
Inventor 章文明姜岷杨桥信丰学马江锋李艳董维亮周杰
Owner NANJING UNIV OF TECH
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