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Recombinant yarrowia lipolytica genetically engineered bacterium biotransformed by methanol as well as construction method and application of recombinant yarrowia lipolytica genetically engineered bacterium

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

Active Publication Date: 2022-03-01
NANJING UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Purpose of the invention: The purpose of the present invention is to provide a synthetic biology method to construct a strain that can metabolize methanol, and use the strain to ferment and produce citric acid, which solves the problem of high cost of traditional citric acid production

Method used

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  • Recombinant yarrowia lipolytica genetically engineered bacterium biotransformed by methanol as well as construction method and application of recombinant yarrowia lipolytica genetically engineered bacterium
  • Recombinant yarrowia lipolytica genetically engineered bacterium biotransformed by methanol as well as construction method and application of recombinant yarrowia lipolytica genetically engineered bacterium
  • Recombinant yarrowia lipolytica genetically engineered bacterium biotransformed by methanol as well as construction method and application of recombinant yarrowia lipolytica genetically engineered bacterium

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Construction and identification of genetically engineered bacteria Y001.

[0031] The specific steps for constructing plasmid 113-GPD-TEF-das-dak-pki-aox-cta are:

[0032]The genes methanol oxidase gene aox, catalase gene cta, dihydroxyacetone synthase gene das, and dihydroxyacetone kinase gene dak were amplified from the genome of Pichia pastoris GS115 by polymerase chain reaction (PCR) technique . Among them, genes aox and das, TEF promoter and CYC terminator constitute TEF-aox1-CYC1t and TEF-das-tCYC1 expression cassettes. Gene cta, PDC promoter and TDH terminator constitute the PDC1p-cta-TDH2t expression cassette. Gene dak, promoter GPD and terminator TXPR constitute pGPD-dak-TXPR2 expression box. Through the method of multi-fragment cloning, the four expression cassettes were connected with the 113 plasmid and transformed into E.coli DH5α.

[0033] The 113-das-dak-cta-aox recombinant plasmid was digested with Not I restriction endonuclease, and the d...

Embodiment 2

[0043] Embodiment 2: Analysis and detection method.

[0044] (1) Determination of cell density

[0045] During the fermentation period, samples were regularly taken every day, the bacterial solution was diluted to an appropriate multiple (A600 value between 0.2-0.8), and the absorbance value was measured at 600nm with an ultraviolet-visible spectrophotometer. The turbidity of the bacterial solution OD600=A600×dilution factor. In order to ensure the accuracy of the experimental data, the determination of the density of the bacteria requires the current measurement.

[0046] (2) Detection of fermentation products

[0047] Through high performance liquid chromatography (HPLC High Performance Liquid Chromatography), the conditions of Yarrowia lipolytica fermentation products were explored, and it was finally determined that the fermentation products of Yarrowia lipolytica were mainly citric acid. Therefore, high performance liquid chromatography was mainly used in this project t...

Embodiment 3

[0051] Embodiment 3: the optimization of the methanol and xylose co-substrate fermentation conditions of recombinant bacterial strain Y003.

[0052] figure 1 It is the metabolic map of genetically engineered bacteria Y003. First, methanol is catalyzed by methanol oxidase (Aox) to formaldehyde in the organelle peroxisome of Yarrowia lipolytica, and at the same time, the toxic substance hydrogen peroxide is catalyzed by catalase to produce non-toxic water and oxygen , and then formaldehyde and the metabolic precursor xylulose pentaphosphate are catalyzed by dihydroxyacetone synthase (Das) and dihydroxyacetone kinase (Dak) to generate dihydroxyacetone phosphate and then enter the central metabolic pathway to produce citric acid. Xylose reductase (Xyr), xylitol dehydrogenation (Xdh), xylulokinase (Xyk), 1,6-fructose bisphosphatase (Fbp), 1,6-diphosphate fructose aldolase (fba) and enhanced expression of transaldolase (tal) can provide a cyclic supply of the precursor xylulose pe...

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Abstract

The invention discloses a recombinant yarrowia lipolytica genetically engineered bacterium by utilizing methanol biotransformation, which is prepared by introducing a methanol oxidase gene aox, a catalase gene cta, a dihydroxyacetone synthase gene das, a dihydroxyacetone kinase gene dak, a xylose reductase gene xyr, a xylitol dehydrogenation gene xdh, a xylulokinase gene xyk, 1, 2, 3, 4-tetramethyl-1, 3-pentanediol monoisobutyrate gene xyr into a genome of host bacteria yarrowia lipolytica. The genetically engineered bacterium is obtained after a 1, 6-fructose diphosphatase gene fbp, a 1, 6-fructose diphosphatase aldolase gene fba and a transaldolase gene tal are synthesized, and a construction method and application of the genetically engineered bacterium are provided at the same time. According to the method, a methanol assimilation pathway, a xylose utilization pathway and a xylose pentaphosphate circulation pathway in peroxidase are introduced into the yarrowia lipolytica by utilizing a synthetic biology method, so that the purpose that the yarrowia lipolytica produces citric acid by taking non-food-grade raw materials, namely methanol and xylose, as a co-substrate carbon source is realized; and the production cost is reduced to a certain extent, and great significance and economic value are achieved.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a recombinant Yarrowia lipolytica genetic engineering bacterium utilizing methanol biotransformation and its construction and application. Background technique [0002] With the rapid development of metabolic engineering and the rise of synthetic biology, the ability of humans to transform microorganisms as cell factories for biomanufacturing has been significantly improved. In recent years, with the depletion of fossil resources, methanol, as a renewable biomass raw material, has great commercial value and market potential in the field of biotransformation. The development of biotransformation and utilization of non-traditional carbon source methanol is increasingly valued by researchers at home and abroad, which can not only reduce the raw material cost of industrial biotransformation, but also make better use of methanol resources rationally. At present, the biosynth...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/53C12N15/54C12N15/55C12N15/60C12N15/81C12P7/48C12R1/645
CPCC12N9/0006C12N9/0065C12N9/1205C12N9/16C12N9/88C12N9/1085C12N9/1022C12N15/815C12N1/16C12P7/48C12Y111/01006C12Y207/01029C12Y101/01307C12Y101/01009C12Y207/01017C12Y301/03011C12Y401/02013C12Y205/01026C12Y202/01002Y02E50/10
Inventor 章文明张尚杰姜岷蒋羽佳董维亮信丰学方艳马江锋周杰
Owner NANJING UNIV OF TECH
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