Saccharomyces cerevisiae of overexpression MIG1 genes and preparing method and application of saccharomyces cerevisiae

A kind of Saccharomyces cerevisiae, overexpression technology, applied in biological field

Active Publication Date: 2015-10-14
PETROCHINA CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the performance of mutants overexpressing this gene on mixed sugar fermentation has not been reported

Method used

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  • Saccharomyces cerevisiae of overexpression MIG1 genes and preparing method and application of saccharomyces cerevisiae
  • Saccharomyces cerevisiae of overexpression MIG1 genes and preparing method and application of saccharomyces cerevisiae
  • Saccharomyces cerevisiae of overexpression MIG1 genes and preparing method and application of saccharomyces cerevisiae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1, the preparation of a kind of xylose metabolism engineering bacterium ZQ5-OE035

[0042] Step 1 Construct HO-MIG1-SPSC01 expression vector

[0043] 1.1 Extraction of genomic DNA from flocculating yeast SPSC01

[0044] (1) SPSC01 bacteria (CGMCC No.0587, Bioprocess Engineering Laboratory, School of Life Sciences, Dalian University of Technology) cultured in YPD medium overnight (30°C, 150rpm) was centrifuged (12000rpm, 5min) to collect cells.

[0045] (2) Add 0.1mol / L sodium citrate buffer solution to deflocculate the SPSC01 bacteria and shake evenly.

[0046] (3) Wash the cells twice with sterile water, centrifuge (12000 rpm, 5 min), and collect the cells.

[0047] (4) Resuspend the cells with an appropriate amount of lysate (0.1mol / L Tris-hydrochloric acid, pH 8.0; 50mmol / L ethylenediaminetetraacetic acid, pH 8.0; 1% sodium dodecyl sulfate), and then add clean 0.5mm Glass beads and 25 μL (5mol / L concentration) NaCl solution.

[0048] (5) American SI Vo...

Embodiment 2

[0161] Embodiment 2, xylose metabolism engineering bacterium ZQ5-OE035 can improve the heat resistance of xylose bacterial strain and the growth status on pure xylose plate

[0162] Comparison of growth of ZQ5 empty control strain and xylose metabolism engineered strain ZQ5-OE035 on high temperature plate

[0163] (1) Inoculate the ZQ5 empty control strain and the overexpressed MIG1 strain ZQ5-OE035 in YPD liquid medium (10 g / L yeast extract powder, 20 g / L glucose, 20 g / L peptone) supplemented with G418 100 μg / mL, Cultivate overnight at 30°C, 150rpm.

[0164] (2) As above (same as step (1) in Example 2), 10% volume was transferred to continue the culture, and expanded to 30mLYPD liquid medium to ensure continuous culture and maintain cell viability.

[0165] (3) Measure and use the absorbance value of fresh YPD liquid culture medium at a wavelength of 620nm to be zero, transfer to fresh YPD medium with 10% inoculum size, continue to cultivate for 4h to 5h, and the absorbance ...

Embodiment 3

[0176] Embodiment 3, Shake flask fermentation performance comparison between no-load control strain and xylose metabolism engineering bacteria in mixed sugar and pure xylose

[0177] 1. Comparison of fermentation performance of ZQ5 no-load control strain and xylose metabolism engineered strain ZQ5-OE035 in mixed sugar

[0178] (1) Inoculate the ZQ5 empty control strain and the overexpressed MIG1 strain ZQ5-OE035 in 5 mL of YPD seed medium (10 g / L yeast extract powder, 20 g / L glucose, 20 g / L peptone) supplemented with G418 100 μg / mL Medium, overnight culture, 30°C, 150rpm.

[0179] (2) Same as 1, expanded to 150mLYPD liquid medium.

[0180] (3) Measure and use the absorbance value (OD value) of fresh YPD liquid medium at wavelength 620nm as zero point, transfer to fresh YPD medium with 10% inoculum size respectively, continue to cultivate 6h, make the growth of bacterial strain keep good state.

[0181] (4) Use a sterilized 50mL centrifuge tube, 10000rpm, 5min, to collect th...

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Abstract

The invention provides saccharomyces cerevisiae of overexpression MIG1 genes and a preparing method and application of the saccharomyces cerevisiae. According to the saccharomyces cerevisiae, the genetic engineering technology is mainly used for shifting glycometabolism transcription factors into the industrial saccharomyces cerevisiae so as to prepare xylose metabolism engineering bacteria of the overexpression MIG1 genes; and the xylose metabolism engineering bacteria have the capacity of conducting fermentation in a pure xylose culture medium for generating ethyl alcohol, and therefore the yield of the ethyl alcohol in the process of producing fuel ethanol through glucose and xylose sugar mixture fermentation can be improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a Saccharomyces cerevisiae overexpressing MIG1 gene and its preparation method and application. It mainly uses genetic engineering technology to transfer sugar metabolism transcription factors into industrial Saccharomyces cerevisiae to prepare a xylose metabolism engineering bacterium. In order to achieve the effect of improving the yield of ethanol in the process of producing fuel ethanol through the fermentation of mixed sugars of glucose and xylose. Background technique [0002] Using biotechnology to bioconvert abundant and cheap biomass to produce biofuels and bio-based chemicals has the characteristics of environmental friendliness, resource conservation, and regeneration, and has been widely concerned by researchers at home and abroad. Biomass includes plant stems, branches, leaves and other lignocellulosic substances, agricultural waste (corn stalks, straw, wheat straw, etc.),...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/31C12N15/81C12P7/06C12P19/02C12R1/865
CPCY02E50/10
Inventor 胡世洋赵心清徐友海许建韧岳军程诚王继艳熊亮白凤武
Owner PETROCHINA CO LTD
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