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Saccharomyces cerevisiae engineering bacterium for performing alcohol fermentation by using xylose, and preparation method and application thereof

A kind of Saccharomyces cerevisiae, ethanol fermentation technology, applied in the field of Saccharomyces cerevisiae engineering bacteria and preparation

Inactive Publication Date: 2015-02-04
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no effective solution to these problems at present. Using new exogenous genes to construct the xylose metabolic pathway of Saccharomyces cerevisiae can further understand the metabolic mechanism of other strains. The understanding of microbial metabolic engineering is of great value

Method used

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  • Saccharomyces cerevisiae engineering bacterium for performing alcohol fermentation by using xylose, and preparation method and application thereof
  • Saccharomyces cerevisiae engineering bacterium for performing alcohol fermentation by using xylose, and preparation method and application thereof
  • Saccharomyces cerevisiae engineering bacterium for performing alcohol fermentation by using xylose, and preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0066] Embodiment 1 utilizes xylose to carry out the preparation of the Saccharomyces cerevisiae engineered bacterium of ethanol fermentation

[0067] (1) The primers designed to realize the present invention were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. as shown in Table 1 above.

[0068] (2) Obtaining xylose reductase gene (xyl1) and xylitol dehydrogenase gene (xyl2)

[0069] Candida lusitaniae CICC 1461 of Portugal (China Industrial Microbial Culture Collection Management Center) was inoculated on YPX plate medium (yeast extract 10g / L, peptone 20g / L, xylose 20g / L, agar 15g / L, pH Naturally) at 30°C for activation. The activated yeast was inoculated into (1% (v / v) inoculum amount) YPX liquid medium, and cultured at 30° C. and 200 rpm for 18 hours to obtain cells in logarithmic phase. Add 400 μL of AE buffer (pH 5.2, 50 mM NaAc, 10 mM EDTA), 50 μL of 10% (w / v) SDS and 600 μL of phenol / chloroform mixture into a 1.5 mL centrifuge tube (phenol and chloroform ar...

Embodiment 2

[0117] Example 2 Enzyme Activity Determination of Xylose Reductase and Xylitol Dehydrogenase in Saccharomyces cerevisiae

[0118] (1) Preparation of crude enzyme solution

[0119] Insert yeast strain 004-PGK and Saccharomyces cerevisiae strain 1917 into 5mL YPX liquid medium, shake culture overnight at 30°C, 200rpm, transfer into 50mL fresh YPX medium, and make the initial OD 600 0.1, 30°C, 200rpm shake flask culture, to OD 600 about 1. Use a pre-frozen 50mL centrifuge tube to centrifuge at 4°C and 3000rpm for 15min, collect the cells, discard the supernatant, resuspend the bacteria with an equal amount of pre-cooled triethanolamine buffer (100mM, pH 7.0), wash the cells, centrifuge and discard Supernatant; then resuspend the cells with 10 mL ice-cold triethanolamine buffer, transfer 1.5 mL to a pre-frozen cell disruption tube containing 750 mg glass beads, add 15 μL PMSF (1 mmol) and 7.5 μL DTT (0.5 mmol). Put the cell disruption tube containing the glass beads and cells i...

Embodiment 3

[0128] Example 3 Metabolite Detection of Engineering Bacteria in Xylose as the Only Carbon Source Medium

[0129] (1) Fermentation method

[0130] The fermentation temperature was controlled at 30°C, the optimum growth temperature of yeast, and YPX medium was used. The aerobic fermentation is as follows: use a 250mL conical flask with a liquid volume of 50mL, seal it with gauze, and select a rotation speed of 200rpm. The anaerobic fermentation is as follows: use a 100mL serum bottle with a liquid volume of 50mL, use an anaerobic pumping system to vacuumize the serum bottle and fill it with nitrogen to form anaerobic conditions. The yeast to be tested was streaked on the YPX plate to isolate a single colony, and the bacteria were picked and inoculated into 50mL liquid YPX medium, and cultured at 30°C and 200rpm to OD 600 between 1.0-1.5, centrifuged to collect the bacteria, inoculated into the corresponding fermentation medium, so that the initial OD 600 is 0.1. After sampl...

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Abstract

The invention discloses a Saccharomyces cerevisiae engineering bacterium for performing alcohol fermentation by using xylose, and a preparation method and application thereof. The strain contains the following nucleotide sequences: xylose reductase gene, xylitol dehydrogenase gene and xylulokinase gene; and the xylulokinase gene is overexpressed. The preparation method comprises the following steps: integrating a constitutive strong promoter to the upstream of the xylulokinase gene of the Saccharomyces cerevisiae initial strain genome by homologous recombination to obtain a xylulokinase-overexpressed strain; and transforming into Candida lusitaniae to obtain xylose reductase gene and xylitol dehydrogenase gene, thereby obtaining the Saccharomyces cerevisiae engineering bacterium for performing alcohol fermentation by using xylose. The Saccharomyces cerevisiae engineering bacterium can grow by using the xylose as the unique carbon source, and lays foundation for the subsequent construction of alcohol production by xylose / glucose co-fermentation. The engineering strain can be further screened and transformed to produce alcohol from lignocellulose, thereby greatly lowering the alcohol production cost.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a Saccharomyces cerevisiae engineering bacterium using xylose for ethanol fermentation, a preparation method and an application. Background technique [0002] Lignocellulose is a kind of abundant and cheap renewable resource, and it is the main material for the production of fuel ethanol. After hydrolysis, fermentable sugars mainly glucose and xylose can be obtained. Effective utilization of xylose is one of the key steps in the bioconversion of lignocellulose to ethanol. Saccharomyces cerevisiae has been the first choice for ethanol production since human beings recorded it. It is currently the strain that ferments cornstarch and other sugar compounds to produce ethanol on an industrial scale. It consumes sugar quickly and has strong ethanol tolerance. There is great potential for transformation. However, Saccharomyces cerevisiae cannot utilize xylose. In vi...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/53C12N15/81C12P7/06C12R1/865
CPCC12N9/0006C12N9/1205C12P7/06C12Y101/01009C12Y101/01307C12Y207/01017Y02E50/10
Inventor 朱明军区健发谢文化
Owner SOUTH CHINA UNIV OF TECH
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