Brewing yeast strain capable of metabolizing xylose

A Saccharomyces cerevisiae strain and technology of Saccharomyces cerevisiae, applied in fungi, microorganism-based methods, microorganisms, etc., can solve the problems of cumbersome pathways, insufficient xylose conversion efficiency, low efficiency, etc., and achieve the effect of short metabolic pathways

Inactive Publication Date: 2012-07-25
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The path is cumbersome and inefficient
First, there are limitations in the establishment of the two pathways for the conversion of xylose to xylulose in Saccharomyces cerevisiae
① The pathway catalyzed by xylose reductase (XR) and xylitol dehydrogenase (XDH) is uncoupled due to the uncoupling of XR and XDH cofactors, resulting in redox imbalance when the strain introduced into this pathway metabolizes xylose, resulting in side effects The problem of product xylitol accumulation has not been completely solved; ② Although the xylose isomerase (XI) pathway can avoid the problem of redox imbalance, there are only a few xylose isomerases in Saccharomyces cerevisiae Active expression is achieved in the medium, and the enzyme activity is not high at the growth temperature of Saccharomyces cerevisiae (about 30°C), resulting in insufficient conversion efficiency of xylose during fermentation
Secondly, xylulose needs

Method used

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  • Brewing yeast strain capable of metabolizing xylose
  • Brewing yeast strain capable of metabolizing xylose
  • Brewing yeast strain capable of metabolizing xylose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: Construction of recombinant plasmid

[0026] Methods as below:

[0027] (1) Artificially synthesize its DNA according to the sequence of the xylose dehydrogenase gene (Cc-xylB) of Caulobacter crescentus; and use this DNA as a template, and use the nucleotide sequence of XF (XF as SEQ ID No. 2): 5'-GTACGGATCCATGTCCTCAGCCATCTATCC-3' and XR (the nucleotide sequence of XR is shown in SEQ ID No.3): 5'-TGACCTGCAGTCAACGCCAGCCGGCGTCG-3' is a primer to amplify the Cc-xylB fragment; The amplified fragment was restricted by restriction endonucleases BamHI and PstI, and ligated between the TEF1p promoter and PGK1t terminator of the episomal plasmid pJFE3 (use restriction endonucleases BamHI and PstI to restrict pJFE3 enzyme digestion), forming plasmid pJFE3-xylB ( image 3 ).

[0028] The above pJFE3

[0029] (2) Using Escherichia coli (Escherichia coli) chromosome as a template, GF (the nucleotide sequence of GF is shown in SEQ ID No.4): 5'-CAGGTCTAGAAGGAGAAACTC...

Embodiment 2

[0033] Embodiment 2: Construction of recombinant bacterial strain

[0034] (1) Inoculate the CEN.PK102-5B strain in YPD medium and culture overnight at 30°C;

[0035] (2) Transfer the CEN.PK102-5B culture medium cultured overnight to 50ml of fresh YPD, so that the initial OD 600 equal to 0.25, continue to culture at 30°C for about 4 hours to make the culture medium OD 600 equal to 0.7 to 1.0;

[0036] (3) Transform the plasmid pJFE3-yjhG-yjhH-xylB into the CEN.PK102-5B strain with lithium acetate transformation method, wherein, 50% polyethylenediene (PEG) 3350 is used as a protective agent, and single-stranded protist DNA is used for Vector for plasmid transformation.

[0037] (4) The transformed bacterial suspension was coated on a fully synthetic auxotrophic medium SC-Ura plate added with 20 g / L glucose, and transformants were picked.

[0038] (5) The xylose utilization performance of the transformants was tested in SC-Ura medium with 20 g / L xylose as the sole carbon sou...

Embodiment 3

[0040] Embodiment 3: Xylose fermentation of BSHH02B bacterial strain

[0041] Methods as below:

[0042] (1) Strain activation: Pick a single colony of the BSHH02B strain and inoculate it in a fully synthetic auxotrophic medium SC-Ura supplemented with 20g / L glucose, and cultivate it at 30°C and 200rpm for 24 hours; the above-mentioned culture was inoculated with 10% volume The amount was transferred to the fully synthetic auxotrophic medium SC-Ura (20g / L glucose as carbon source), and cultured at 30°C and 200rpm for 12 hours.

[0043] (2) Inoculation: the above activated bacterial solution was used as the strain, rinsed with sterile water and the initial OD 600 =0.1 was inoculated in a 1L fermentor containing 800ml of fully synthetic auxotrophic culture medium SC-Ura (20g / L xylose as carbon source); at 30°C, pH 5.0, ventilation of 0.5L / min, Fermentation was carried out under the condition of stirring speed.

[0044] (3) Determination of growth curve: after inoculation, ta...

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Abstract

The invention discloses a brewing yeast strain capable of metabolizing xylose. The strain is a brewing yeast (Saccharomyces cerevisiae) BSHH02B, and is preserved in the China General Microbiological Culture Collection Center (CGMCC) on Feb. 8th, 2012, wherein the preservation number is CGMCC No. 5747. The strain carries a unique xylose metabolizing way, i.e. a molecular xylose generates a molecular pyruvic acid and a molecular glycolaldehyde through two mesostate, i.e. xylonic acid and 3-deoxyl-D-glycerol-ketopentose acid. By the xylose metabolizing way, the brewing yeast BSHH02B can quickly grow by using the xylose under an aerobic condition, and can be applied to the production of chemical products by using xylose-containing raw materials, wherein the chemical products include ethanol, glycol, carboxylic acid and the like.

Description

technical field [0001] The invention relates to a strain of Saccharomyces cerevisiae engineered strain, in particular to a strain of Saccharomyces cerevisiae engineered strain which has built an exogenous xylose metabolism pathway and can rapidly metabolize xylose. Background technique [0002] The development and stockpiling of alternative petroleum-based fuel and chemical production technologies are necessary for social development and national security. The huge annual output of lignocellulosic raw materials can provide a material basis for the production of large-scale bio-based related products, so it has attracted much attention. The main components of available sugars in lignocellulose are glucose and xylose. Since glucose is an easy-to-use sugar, the conversion of xylose, which can be as high as 30% but is not easy to use, is the key to fully utilizing raw materials and is also the key to reducing bio-based sugars. The basis of chemical production costs. At present...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12R1/865
Inventor 沈煜鲍晓明霍文严刘怀伟
Owner SHANDONG UNIV
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