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Method for improving xylose utilization capacity of recombinant saccharomyces cerevisiae strain and mutant strain of recombinant saccharomyces cerevisiae stain

A technology of Saccharomyces cerevisiae strains and recombinant Saccharomyces cerevisiae, which is applied in the field of bioengineering, can solve the problems of no work proof Tec1p, etc., and achieve the effect of shortening the fermentation time

Active Publication Date: 2019-07-23
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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  • Method for improving xylose utilization capacity of recombinant saccharomyces cerevisiae strain and mutant strain of recombinant saccharomyces cerevisiae stain
  • Method for improving xylose utilization capacity of recombinant saccharomyces cerevisiae strain and mutant strain of recombinant saccharomyces cerevisiae stain
  • Method for improving xylose utilization capacity of recombinant saccharomyces cerevisiae strain and mutant strain of recombinant saccharomyces cerevisiae stain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0027] The main process of construction is as follows:

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

[0029] Using the 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 promoter TEFp. Then, fusion amplification of the promoter and the DNA fragment LoxP-KanMX4-LoxP was carried out by fusion PCR to obtain the integrated fragment of LoxP-KanMX4-LoxP-TEF1p.

[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 [25 Yeast Genetic Strain and Plasm...

Embodiment 2

[0048] Example 2: Acquisition of Saccharomyces cerevisiae Cre-LoxP Genetic Operating System Tool Plasmid YEp-CH

[0049] When the Cre enzyme is expressed in the cell 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 homologous LoxP site sequences to be deleted from the DNA. . YEp-CH is a tool plasmid with Cre-LoxP genetic operating system used in Saccharomyces cerevisiae, and its Cre enzyme gene is expressed under galactose-induced conditions. The construction method of the YEp-CH plasmid is obtained by methods such as cloning construction and DNA sequence synthesis known in the art.

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

Embodiment 3

[0052] Example 3: Mutation of the TEC1 gene in situ on the chromosome of Saccharomyces cerevisiae

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

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

[0055] Using plasmid pUG6 (GenBank: AF298793.1) as a template, amplified with primer tec1-5 (SEQ ID NO: 43) and primer tec1-6 (SEQ ID NO: 44) to obtain DNA frag...

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Abstract

The invention discloses a method for improving the xylose utilization capacity of a recombinant saccharomyces cerevisiae strain by utilizing a transcription factor Tec1p mutant or for producing ethanol by utilizing xylose-containing raw materials. The method expresses the transcription factor Tec1p mutant in the recombinant saccharomyces cerevisiae with the xylose utilization capacity so as to improve the xylose utilization capacity of the strain; and the transcription factor Tec1p mutant is named as Tec1pT273M and is formed by mutating threonine at 273rd site of the wild-type transcription factor Tec1p with an amino acid sequence as shown in SEQ ID NO:1 into methionine, and the amino acid sequence of the Tec1pT273M is as shown in SEQ ID NO:2. Experiments prove that when the method is applied to glucose and xylose co-fermentation, the growth of the mutant strain is obviously superior to that of a starting strain BSGX001, and the utilization rate of xylose and the production rate of ethanol are obviously higher than that 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 technical field of bioengineering. Background technique [0002] There are many kinds of lignocellulose resources, and the annual output is huge. Their comprehensive utilization can not only bring economic benefits, but also reduce environmental pollution caused by improper disposal such as incineration. The content of xylose component in lignocellulosic raw materials is as high as about 30%, second only to glucose component, and its comprehensive utilization can significantly improve the utilization rate of raw materials. Saccharomyces cerevisiae (Saccharomyces cerevisiae) is a microorganism widely used in food and industrial production, which has the characteristics of strong robustness, vigorous metabolism and food-grade safety. Through g...

Claims

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Application Information

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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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