Xylose reductase mutant, genetically engineered bacteria and application in producing xylitol

A technology of genetically engineered bacteria and reductase, which is applied in the field of genetic engineering and biology, can solve the problems of reduced catalytic activity and inactivity, and achieve the effects of simplifying separation steps, reducing production costs and improving purity

Active Publication Date: 2016-06-15
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] After arabinose enters the cells, the production of arabinitol can be reduced from the following two aspects: 1) The expression of xylose reductase should not be too much in excess of the enzyme that metabolizes arabinose, otherwise it will block the arabinitol production. Sugars and enzymes that metabolize arabinose cause arabinose to be reduced but not metabolized, so controlling the expression of xylose reductase is crucial to the production of high-purity xylitol; 2) Improve the selectivity of xylose reductase , so that its catalytic activity on arabinose is reduced or even inactive, so that even if a large amount of xylose reductase is expressed, it will not cause the production of arabinitol. more difficult

Method used

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  • Xylose reductase mutant, genetically engineered bacteria and application in producing xylitol
  • Xylose reductase mutant, genetically engineered bacteria and application in producing xylitol
  • Xylose reductase mutant, genetically engineered bacteria and application in producing xylitol

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Embodiment 1

[0028] Embodiment 1 Xylose reductase specific transformation

[0029] Due to the poor specificity of natural xylose reductase, it can catalyze the reduction of arabinose into arabinitol; in the invention patent application document with application number 201510196843.8, XR with a selectivity increased by 50 times (after 5 point mutations) was used , but the arabinose was almost completely converted into arabinitol during fermentation in the fermenter. Therefore, the selectivity of xylose reductase needs to be improved; in order to further reduce the catalytic activity of xylose reductase on arabinose, we performed site-directed mutations on xylose reductase whose selectivity has been increased by 50 times.

[0030] Using the plasmid pTrc99arbs-xr6600 as a template, using the large primer whole plasmid PCR method, using the primers poit-M1-F and poitM1-R to carry out site-directed mutation, the introduced mutation is KSN271-273RTT, and it was transformed into HK401 to obtain s...

Embodiment 2

[0044] Embodiment 2 contains the test of different mutant enzyme genetically engineered bacteria to produce xylitol ability

[0045] Inoculate genetic engineering bacteria HK442, HK452, HK412, HK462 in 45mL improved M9 medium (containing 4~6gNa in 1L medium) by 2%. 2 HPO 4 , 2~5gKH 2 PO 4 , 1~2gNH 4 Cl, 1~5gNaCl, 1~5mmolMgSO 4 , 1~5mmol CaCl 2 , 2~10g yeast extract), cultured at 30℃ to OD 600 0.6 to 1, add an appropriate amount of inducer IPTG and add xylose to the fermentation broth to a final concentration of 20g / L, add glucose to a final concentration of 10g / L, and add arabinose to a final concentration of 5g / L. Continue to cultivate, investigate the fermentation characteristics of each engineering bacteria, the results are as follows: image 3 As shown, there is no significant difference in the production efficiency of xylitol among the genetically engineered bacteria induced by 0.1 mMIPTG.

Embodiment 3

[0046] The construction of embodiment 3 integrative strain IS5-M

[0047] Using the plasmid pRC43 as a template, using the method of full-body PCR with large primers, the primers poit-M1-F and poitM1-R were used for site-directed mutation to obtain the plasmid pRC43M. Using the genome multi-copy integration method in the invention patent application document with application number 201510196843.8, the xylose reductase mutant gene xr-8m was integrated into the Escherichia coli genome to obtain strain IS5-M.

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Abstract

The invention discloses a xylose reductase mutant, genetically engineered bacteria and application in producing xylitol. The amino acid sequence of the xylose reductase mutant is shown in the SEQ ID No.1. Mutation of eight amino acid sites is carried out on xylose reductase, the activity of the xylose reductase mutant for catalyzing arabinose into arabitol is lowered, the selectivity of the xylose reductase mutant for converting xylitol is improved, and the by-product arabitol in the bioconversion product is not detected. The separation step of xylitol bioconversion production downstream can be simplified, the production cost is reduced, and the purity of the xylitol product is improved.

Description

technical field [0001] The invention relates to the fields of genetic engineering and biotechnology, in particular to a xylose reductase mutant, a genetically engineered bacterium and an application in producing xylitol. Background technique [0002] Corn cob hemicellulose hydrolyzate is currently the main raw material for domestic production of xylitol, which contains glucose, arabinose and other components in addition to the main component xylose. In the industrial production of xylitol, when nickel catalyzes hydrogenation, it has no selectivity for various sugars in the hemicellulose hydrolyzate, and when it catalyzes the reduction of xylose to produce xylitol, it will also catalyze other sugars to generate corresponding sugars Alcohol affects the product quality of xylitol. Therefore, before performing chemical catalysis, it is necessary to separate and purify the xylose in the hemicellulose hydrolyzate to remove the miscellaneous sugars in the hydrolyzate as much as po...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N1/21C12P7/18C12R1/19
CPCC12N9/0006C12P7/18C12Y101/01307
Inventor 吴绵斌苏卜利张哲林建平杨立荣
Owner ZHEJIANG UNIV
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