Saccharomyces cerevisiae strain and application thereof in comprehensive utilizing xylose mother liquor and corn cob residues to produce xylitol

A technology of Saccharomyces cerevisiae and xylose mother liquor, which is applied in the field of microorganisms to achieve the effects of reducing production costs, high conversion rate and good tolerance

Active Publication Date: 2017-11-24
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the search found that there is no engineering strain of Saccharomyces cerevisiae and its application for the comprehensive utilization of xylose mother liquor and xylose residue to produce xylitol

Method used

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  • Saccharomyces cerevisiae strain and application thereof in comprehensive utilizing xylose mother liquor and corn cob residues to produce xylitol
  • Saccharomyces cerevisiae strain and application thereof in comprehensive utilizing xylose mother liquor and corn cob residues to produce xylitol
  • Saccharomyces cerevisiae strain and application thereof in comprehensive utilizing xylose mother liquor and corn cob residues to produce xylitol

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1 Cre-LoxP site-specific recombination system and use of the system to remove screening marker genes

[0029] The Cre-LoxP site-specific recombination system consists of two modules. Module 1 is the gene to be integrated on the Saccharomyces cerevisiae chromosome, and the expression cassette of the screening marker gene KanMX4. Among them, there is a LoxP sequence in the upstream and downstream of the KanMX4 expression cassette, and the two LoxP sequences are in the same direction. The LoxP-KanMX4-LoxP cassettes involved in the present invention are all cloned from the plasmid pUG6 (GenBank: AF298793.1). The second module is the episomal plasmid YEp-CH (Chinese invention patent application, application number 201510747241.7), the selection marker of this plasmid in Saccharomyces cerevisiae is the hygromycin resistance gene hygB, and the inducible recombinase expression box GAL1p-Cre-CYC1t .

[0030] The use steps of this system are: (1) Module 1 transforms Sac...

Embodiment 2

[0031] Example 2 Screening the starting strain suitable for fermenting xylose mother liquor to produce xylitol

[0032] The starting strain suitable for fermenting xylose mother liquor to produce xylitol needs to have good growth ability in xylose mother liquor. Wild-type industrial yeast has higher resistance to inhibitory factors. Therefore, several commercially available strains (BSIF, RC212, 6508, GZ-4, GZ-5, NAN-27, etc.) of wild-type industrial yeast were tested, and their After cultivation, the bacteria were collected by centrifugation, and resuspended in sterile water to make the bacterial suspension OD 600 =1. Carry out ten-fold gradient dilution to this bacterium suspension, each gradient gets 4mL and drips on the plate of 20% (v / v) xylose mother liquor, 1% yeast powder, 2% peptone, cultivates 3 days, observes the colony growth situation, The results showed that RC212, 6508, and GZ-5 had higher tolerance to xylose mother liquor than other strains ( figure 1 A). ...

Embodiment 3

[0034] Example 3 Construction of Xylitol Production Strain

[0035] (1) Integrating the xylose reductase gene XYL1 in the δ-sequence region of rDNA and retrotransposon Ty1, so that Saccharomyces cerevisiae has the ability to efficiently reduce xylose to xylitol.

[0036] The XYL1 gene on the recombinant plasmid pYMIKP-XYL1 is under the control of the yeast constitutive strong promoter PGK1p, and the plasmid has rDNA homologous sequences as integration arms. In the integration arm, digest pYMIKP-XYL1 at the HpaI site to linearize it, and then transform Saccharomyces cerevisiae 6508 with the LiAc method. The transformation solution contained 400 μg mL- 1 Transformants were obtained on the YPD plate of G418. Randomly selected transformants were transferred to cells containing 20000 μg mL- 1 The transformant XGH2 with obvious growth advantage was screened on the YPD plate of G418 [Li Min et al., Construction of Industrial Saccharomyces cerevisiae with Stable and High Expression ...

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Abstract

The invention discloses a saccharomyces cerevisiae strain. The strain is named as saccharomyces cerevisiae genetically engineered bacterium X3kZPM, the strain is preserved in China General Microbiological Culture Collection in twenty seventh of June, 2017, and a preservation number is CGMCC No.14362. The invention further discloses application of the saccharomyces cerevisiae in utilizing xylose mother liquor to produce xylitol or comprehensively utilizing the xylose mother liquor and corn cob residues to produce the xylitol. Experiments prove that a sugar alcohol converting rate is high and can reach 100% of a theoretical value when the saccharomyces cerevisiae genetically engineered bacterium disclosed by the invention is used; a concentration of the xylitol in fermentation liquor is obviously improved and reaches 83 to 91g L<-1>; the saccharomyces cerevisiae strain basically has actual industrialization potency and a wide industrialized application prospect.

Description

technical field [0001] The present invention relates to a strain of Saccharomyces cerevisiae and its application, in particular to a strain of Saccharomyces cerevisiae engineering strain and its application in the comprehensive utilization of xylose mother liquor and xylose residue to produce xylitol, which belongs to the field of microbial technology. Background technique [0002] Xylitol is a five-carbon polyol with excellent properties such as low calorie, high sweetness, and negative heat of fusion. It is widely used in food, medicine, industrial manufacturing and other industries, and the market demand is increasing day by day. At present, the main production method in China is to use corncobs rich in xylose as raw materials, hydrolyze with dilute acid, and recover the hydrolyzate rich in xylose. Hydrogenation and reduction into xylitol under high temperature and high pressure environment. In the above process, the part that cannot be hydrolyzed in the hydrolysis link ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12P7/18C12R1/865
CPCC12N9/0006C12P7/18C12Y101/01049C12Y101/01307
Inventor 沈煜鲍晓明何瑶侯进陈丽媛
Owner SHANDONG UNIV
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