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A method for improving xylose utilization ability of recombinant Saccharomyces cerevisiae strain and its mutant

A technology of Saccharomyces cerevisiae strain and recombinant Saccharomyces cerevisiae, which is applied in the field of bioengineering, can solve problems such as no work proof Tec1p, etc., and achieve the effects of shortening fermentation time, increasing utilization rate, and increasing ethanol yield

Active Publication Date: 2022-07-08
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, no work has demonstrated that Tec1p is directly related to carbon metabolism, and no work has shown that regulating Tec1p can affect the utilization of xylose by Saccharomyces cerevisiae

Method used

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  • A method for improving xylose utilization ability of recombinant Saccharomyces cerevisiae strain and its mutant
  • A method for improving xylose utilization ability of recombinant Saccharomyces cerevisiae strain and its mutant
  • A method for improving xylose utilization ability of recombinant Saccharomyces cerevisiae strain and its mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Construction and acquisition of Saccharomyces cerevisiae strain BSGX001

[0027] The main construction process is as follows:

[0028] (1) Overexpression of the xylulokinase gene XKS1 of the strain CEN.PK113-5D.

[0029] Using plasmid pUG6 (GenBank: AF298793.1) as a template, primers XKKans and Kana were used to amplify the G418 resistance gene KanMX4 fragment LoxP-KanMX4-LoxP with LoxP sites at both ends.

[0030] Using the Saccharomyces cerevisiae genome as a template, primers XKTEF1ps and XKTEF1pa were used to amplify the fragment containing the promoter TEFp. Then, the fusion amplification of the promoter and the DNA fragment LoxP-KanMX4-LoxP was performed by fusion PCR to obtain the LoxP-KanMX4-LoxP-TEF1p integration fragment.

[0031] The integrated fragment was transformed into Saccharomyces cerevisiae CEN.PK113-5D (MATa SUC2MAL2-8cura3-52) using the lithium acetate transformation method. For details, see the literature [25Yeast Genetic Strain and Pl...

Embodiment 2

[0048] Example 2: Obtainment of Saccharomyces cerevisiae Cre-LoxP genetic manipulation system tool plasmid YEp-CH

[0049] When the Cre enzyme is expressed in the cellular environment, the Cre enzyme recognizes the specific DNA sequence LoxP and catalyzes site-specific recombination. This recombination process will cause the sequence between the two direct LoxP site sequences to be deleted from the DNA. . YEp-CH is a tool plasmid with Cre-LoxP genetic manipulation system used in Saccharomyces cerevisiae, and its Cre enzyme gene is expressed under galactose induction conditions. The construction method of YEp-CH plasmid is obtained by cloning construction, DNA sequence synthesis and other methods known in the art.

[0050] Specifically, the specific source of each sequence of YEp-CH is: the backbone is YEp24 (GenBank: L09156.1), the Cre expression cassette is cloned from 2066 to 4256 bp of plasmid pSH47 (GenBank: AF298782.1), the length is 2019 bp, and inserted into YEp24 At ...

Embodiment 3

[0052] Example 3: In situ mutation of the TEC1 gene on the chromosome of Saccharomyces cerevisiae

[0053] The DNA fragment used to mutate the TEC1 gene in situ on the chromosome was obtained by fusion PCR technology, and the annealing temperature in the amplification conditions was 52 °C, the operation reference Figure 4 shown, the specific steps are as follows:

[0054] Using the chromosomal DNA of Saccharomyces cerevisiae BSGX001 as a template, amplify with primer tec1-1 (SEQ ID NO:39) and primer tec1-2 (SEQ ID NO:40) to obtain DNA fragment 1: TEC1-T273M-1 (839bp); Using primers tec1-3 (SEQ ID NO: 41) and tec1-4 (SEQ ID NO: 42) as primers, DNA fragment 2: TEC1-T273M-2 (687bp) was obtained, fragment 2 was downstream of fragment 1 and upstream of KanMX4, respectively There are homologous parts.

[0055] Using plasmid pUG6 (GenBank: AF298793.1) as a template, using primer tec1-5 (SEQ ID NO:43) and primer tec1-6 (SEQ ID NO:44) to amplify to obtain DNA fragment 3:KanMX4 (1642...

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Abstract

The invention discloses a method for improving the xylose utilization ability of a recombinant Saccharomyces cerevisiae strain by using a transcription factor Tec1p mutant or using xylose-containing raw materials to produce ethanol. To improve the ability of strain xylose utilization; the transcription factor Tec1p mutant is named as Tec1p T273M , is formed by mutating the threonine at position 273 of the wild-type transcription factor Tec1p whose amino acid sequence is SEQ ID NO:1 to methionine, and its amino acid sequence is shown in SEQ ID NO:2. Experiments show that the growth of the mutant strain is significantly better than that of the starting strain BSGX001 in the co-fermentation of glucose and xylose by the method of the present invention, and the utilization of xylose and the production rate of ethanol are significantly higher than those of the control strain.

Description

technical field [0001] The invention relates to a method for improving the xylose utilization ability of a recombinant Saccharomyces cerevisiae strain by using a transcription factor Tec1p mutant and a mutant thereof. It belongs to the field of bioengineering technology. Background technique [0002] There are many kinds of lignocellulose resources and huge annual output. Its comprehensive utilization can not only bring economic benefits, but also reduce environmental pollution caused by improper treatment such as incineration. The content of xylose component in lignocellulose raw materials is as high as about 30%, second only to the glucose component, and its comprehensive utilization can significantly improve the utilization rate of raw materials. Saccharomyces cerevisiae is a microorganism widely used in food and industrial production, which has the characteristics of strong robustness, strong metabolism and food-grade safety. Through genetic engineering, by introducing...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12P7/06C12R1/865
CPCC12P7/06C07K14/395Y02E50/10
Inventor 沈煜魏闪杨梦丹侯进刘巍峰鲍晓明
Owner SHANDONG UNIV
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