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A xylose fermentation strain tolerant to inhibitors and its construction method and application

A fermentation strain and inhibitor technology, applied in the field of yeast, can solve the problems of complex construction steps and lack of Saccharomyces cerevisiae strains, and achieve the effects of superior inhibitor tolerance, improved inhibitor tolerance, and good fermentation results

Active Publication Date: 2021-08-10
CHINA PETROLEUM & CHEM CORP +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Chinese patent CN105985915A discloses a genetically engineered strain in which a GRE3 gene having the nucleotide sequence shown in SEQ ID NO: 1 is deleted in Saccharomyces cerevisiae 1308-P. The construction method of the engineered strain is: GRE3 gene knockout component 1. Transfer the GRE3 gene knockout component into yeast, knock out the GRE3 gene\remove the resistance marker, and finally get a recombinant strain lacking a GRE3 gene, which can improve the tolerance to inhibitors and efficiently co-ferment glucose and wood sugar, but the construction steps of this Saccharomyces cerevisiae strain are complicated, and its tolerance to the inhibitor furfural is only improved, and its tolerance to mixed inhibitors is not involved

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  • A xylose fermentation strain tolerant to inhibitors and its construction method and application
  • A xylose fermentation strain tolerant to inhibitors and its construction method and application
  • A xylose fermentation strain tolerant to inhibitors and its construction method and application

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Embodiment 1

[0040] This embodiment is a method for constructing xylose fermentation strain SEB11 tolerant to inhibitors. The starting strain used in this embodiment is Saccharomyces cerevisiae SEB5 (CGMCC No.11325, General Microbiology Center of China Committee for Culture Collection of Microorganisms), which has Excellent performance of fermenting xylose to produce ethanol. The construction method includes the following steps:

[0041] (1) plasma mutagenesis treatment;

[0042]Select a loop of SEB5 strain into 2% YPD liquid medium, put it into a 30°C constant temperature shaker at 160rpm for pre-cultivation. After 16 hours of pre-cultivation, 1 mL of the bacterial liquid was taken, centrifuged at 10,000 rpm for 3 minutes, the bacterial cells were collected, transferred to 2% YPD, and placed in a constant temperature shaker at 160 rpm at 30°C. When cultured for 8 hours, the starting yeast strain SEB5 was in the transition period between the logarithmic growth phase and the stationary ph...

Embodiment 2

[0047] The present embodiment is a fermentation experiment in which xylose is the sole carbon source under the condition of acetic acid (80mmol / L), which adopts the following steps:

[0048] SEB11 was inoculated on 2% YPD solid plates and activated in a 30°C incubator for 24 hours. A ring of activated yeast was selected and inoculated into 100 mL of 5% YPD liquid medium, and incubated at a constant temperature for 16 hours (30° C., 160 rpm). The pre-cultured bacterial liquid was centrifuged to collect the thalline, inoculated 2.5g of wet thallus into 100mL of 5% YPX medium containing 80mmol / L acetic acid, and fermented in a constant temperature water bath (200rpm, 35°C). The results were as follows: figure 1 shown.

[0049] figure 1 Part A is the fermentation result of SEB11, figure 1 Part B of is the fermentation result of the starting strain SEB5. Compared with the original strain, the xylose consumption rate of SEB11 was significantly increased. The consumption rate of ...

Embodiment 3

[0051] The present embodiment is a fermentation experiment in which xylose is the only carbon source under the condition of the presence of 5-HMF (15mmol / L), and it adopts the following steps:

[0052] SEB11 was inoculated on 2% YPD solid plates and activated in a 30°C incubator for 24 hours. A ring of activated yeast was selected and inoculated into 100 mL of 5% YPD liquid medium, and incubated at a constant temperature for 16 hours (30° C., 160 rpm). The pre-cultured bacterial liquid was centrifuged to collect the bacterial cells, inoculated 2.5g of wet bacterial cells into 100mL of 5% YPX medium containing 15mmol / L 5-HMF, and fermented in a constant temperature water bath (200rpm, 35°C), the results Such as figure 2 shown.

[0053] figure 2 Part A is the fermentation result of SEB11 under this condition, figure 2 Part B is the fermentation result of the starting strain SEB5 under this condition. 15mmol / L 5-HMF had no obvious inhibitory effect on SEB11, showing bette...

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Abstract

The invention discloses a xylose fermentation strain tolerant to inhibitors, which is named SEB11, and its preservation number is CGMCC No.16570; the invention also relates to a construction method of the above xylose fermentation strain, which includes the following steps: Select the starting strain SEB5; carry out plasma mutagenesis treatment on the starting strain, and select colonies with better or similar growth than the starting strain; carry out plate primary screening and re-screening of the actual saccharification solution for the selected bacterial strains, and obtain tolerance to inhibitors. Xylose fermenting strain SEB11; the present invention also relates to the application of the above xylose fermenting strain in producing ethanol by using xylose in lignocellulose. The present invention takes industrial strains that can utilize xylose as starting strains, utilizes atmospheric pressure and room temperature plasma mutagenesis technology to carry out mutagenesis, and according to the growth conditions in the plate culture medium containing inhibitors and the actual saccharification liquid fermentation re-screening, obtains The xylose fermentation mutant strain SEB11 with excellent inhibitor tolerance has a significantly increased xylose consumption rate, showing better fermentation results.

Description

technical field [0001] The invention relates to a yeast, in particular to a xylose fermentation strain resistant to inhibitors and its construction method and application. Background technique [0002] With the depletion of fossil resources such as petroleum and the intensification of the greenhouse effect, it has become the focus of people's attention to find alternatives to petroleum resources. Using lignocellulose to produce fuel ethanol to replace fossil fuels can effectively solve the energy crisis and environmental pollution caused by fossil fuels. However, several obstacles exist in the conversion of lignocellulosic ethanol, including inhibitor tolerance and low xylose utilization. Inhibitors formed by acid-catalyzed hydrolysis of lignocellulose (mainly including furan derivatives, weak acids, and phenolic compounds) reduce the growth rate and fermentation of S. cerevisiae, thereby reducing ethanol production. Although there are many reports on the ability of Saccha...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/18C12N15/01C12N13/00C12P7/10C12R1/865
CPCY02E50/10
Inventor 缪晡程华陈栋汤岳琴吴娅箐丁伟军
Owner CHINA PETROLEUM & CHEM CORP
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