Construction method of synthetic strain for generating fengycin by transforming xylose through regulating and controlling Dahms pathway

A construction method, xylose technology, applied in the field of industrial microorganisms, can solve the problems of reduced biomass of strains, poor growth of strains, cytotoxicity, etc.

Active Publication Date: 2021-10-12
TIANJIN UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, in the study of converting xylose using the Dahms pathway, it was found that the expression of the xylose metabolic pathway would reduce the biomass of the strain. For example, So Young Choi et al. introduced the genes xylB and xylC successfully constructed a complete Dahms metabolic pathway. During the fermentation process, it was observed that the growth of the strain was worse than that of the original strain, and a certain amount of lactic acid was accumulated (Choi SY, Park SJ, Kim WJ, et al. One-stepfermentative production of poly(lactate-co-glycolate) from carbohydrates in Escherichia coli[J].Nature Biotechnology,2016,34(4):435-+.)
At the same time, the Dahms pathway also produces the toxic metabolite glycolaldehyde, which brings toxic effects to cells

Method used

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  • Construction method of synthetic strain for generating fengycin by transforming xylose through regulating and controlling Dahms pathway
  • Construction method of synthetic strain for generating fengycin by transforming xylose through regulating and controlling Dahms pathway
  • Construction method of synthetic strain for generating fengycin by transforming xylose through regulating and controlling Dahms pathway

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Construction of Dahms module expression vector pHY300P03

[0066] (1) Promoter P 43 (SEQ ID NO:1) and gene xylB (SEQ ID NO:2), xylC (SEQ ID NO:3), yjhG (SEQ ID NO:4), yjhH (SEQ ID NO:5) gene amplification primer design and synthesis. There are few multiple cloning sites available in the shuttle vector pHY300PLK, so when designing P 43 When using primers, add SphI and XhoI endonuclease recognition sequences to the 3 ends of the primers. According to the nucleotide sequences of genes xylB, xylC, yjhG, and yjhH, the amplification primers containing RBS sequences were designed as follows (the underline indicates the HindIII restriction site, the double underline wavy line indicates the XhoI cleavage site, the single wavy line indicates the SphI, The dotted line indicates SalI, and the double underline indicates the RBS sequence), the plasmid introduction design is as follows, and the primers were synthesized by Qingke Biological Co., Ltd.

[0067] P43-F: AACG...

Embodiment 2

[0105] Example 2: Electric shock transformation of recombinant vectors pHY300P03 and pHP13P03 to BSU00

[0106] (1) Electroporation Competent Preparation of Bacillus subtilis

[0107] Streak culture on the solid LB plate of Bacillus subtilis stored at -80°C for 16 hours, pick a single colony and inoculate it in 50mL liquid LB medium, culture overnight at 220rpm, 37°C; inoculate fresh Cultivate in LBS growth medium at 37°C and 220rpm until the OD600 is between 0.85 and 0.95; collect the bacterial solution in a 50mL clean and sterilized centrifuge tube, pre-cool on ice for 30min; centrifuge at 6000rpm at 4°C for 10min, pour Discard the supernatant; wash and resuspend the bacteria with 20mL washing medium, pay attention to slow and gentle blowing, 6000rpm, 4°C for 10min, pour off the supernatant; repeat the previous step 2-3 times; use 1mL electroporation Resuspend the obtained bacteria in the suspension medium, and put each 100 μL aliquot into a clean and sterile 1.5mL centrifu...

Embodiment 3

[0112] Example 3: Fermentation experiment of recombinant strain BSU02 xylose as carbon source to produce fengycin

[0113] (1) Culture medium composition:

[0114] Solid medium: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L, agar 20g / L;

[0115] Seed medium: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L;

[0116] Fermentation medium: xylose 15g / L, soybean cake powder 21.9g / L, NaNO 3 3g / L, MnSO 4 0.2g / L;

[0117] (2) Fermentation experiment: the bacterial strain BSU02 constructed by the present invention was cultured and activated in a solid medium at 37°C for 20h, and a single colony was picked and placed in a 250mL shaker flask containing 50mL seed medium, cultivated overnight at 37°C and 220rpm, and press 5% of the inoculum was taken to ferment the seed liquid into the fermentation medium, 30°C, 200rpm conditional culture for 60h, and the fermentation ended.

[0118] (3) Extraction and determination of fengycin: Take 20mL of the fermentation broth ...

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Abstract

The invention provides a construction method of a synthetic strain for generating fengycin by transforming xylose through regulating and controlling a Dahms pathway. The construction method comprises the following steps of: connecting a promoter gene P43, xylose dehydrogenase xylB, xylC, a xylosic acid lactonase gene yjhG and a 2-ketone-3-deoxyxylosic acid dehydratase gene yjhH in series to prepare a Dahms xylose pathway module into a shuttle vector pHY300PLK; meanwhile, starting from metabolite glycolaldehyde of the Dahms pathway, connecting an aldehyde dehydrogenase gene aldA, malic acid synthetase aceB, malic acid dehydrogenase mdh and the promoter P43 in series to prepare an glycolaldehyde back-complement TCA regulating and controlling module into a shuttle vector pHP13; co-expressing the two shuttle vectors in a chassis strain to construct a fengycin high-quality strain BSU02 which is synthesized by co-expressing and transforming the xylose through the Dahms module and the glycolaldehyde back-complement TCA regulating and controlling module.

Description

technical field [0001] The invention relates to a method for constructing a synthetic bacterial strain for expressing converted xylose and synthesizing fengycin by combining a Dahms xylose pathway module and a TCA module for regulating glycolaldehyde replenishment, which belongs to the technology of industrial microbes. Background technique [0002] Lignocellulose is the most abundant renewable biomass resource in nature, and the global annual production of lignocellulose is about 210 11 metric tons, widely derived from woody biomass such as wood, agricultural residues such as rice straw, energy crops such as switchgrass, and various cellulosic wastes such as waste from wood mills, pulp mills, etc., mainly composed of cellulose (30%-50% ), hemicellulose (20%-35%) and lignin (15%-20%), they can be used as valuable chemical raw materials, or be used for microbial fermentation after pretreatment (Kim JS, Lee YY, Kim TH.A review on alkalinepretreatment technology for bioconvers...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N15/60C12N15/55C12N15/54C12N15/53C12P21/02C07K7/06C12R1/125
CPCC12N15/75C12N9/88C12N9/1025C12N9/0006C12N9/18C12N9/0008C12N15/52C12Y402/01082C12Y203/03009C12Y101/01037C12Y101/01175C12Y102/01003C12P21/02C07K7/06
Inventor 闻建平高文婷靳佳琦
Owner TIANJIN UNIV
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