Xylose utilization yeast and application thereof

A technology of xylose and Saccharomyces cerevisiae, which is applied in genetic engineering and biological fields, can solve the problems of high production cost and inability to produce on a large scale, and achieve the effect of reducing waste, reducing production cost and eliminating inhibition

Active Publication Date: 2020-06-05
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the production of carotenoids through biological fermentation mainly uses glucose as the carbon source, and the production cost is h

Method used

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  • Xylose utilization yeast and application thereof
  • Xylose utilization yeast and application thereof
  • Xylose utilization yeast and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Construction of xylose utilization metabolic pathway in Saccharomyces cerevisiae

[0028] Since Saccharomyces cerevisiae itself cannot metabolize xylose, in order to use xylose to synthesize carotenoids, this example introduces xylose reductase gene XYL1 and xylitol dehydrogenase gene XLY2 from Scheffersomyces stipitis. Use the plasmid pHCas9-gRNA as a template (pHCas9-gRNA contains the guide sequence N 20 ), use the primers gRNA-XK-F / gRNA-R to amplify the functional modules, and construct the genome integration plasmid pHCas9-XK. Using the Saccharomyces cerevisiae genome as a template, primers EFT1-F-2, EFT1-F / EFT1-R, PGK-F / PGK-R, HSP82-F / HSP82-R were used to amplify EFT1, PGK1, and HSP82 promoters respectively; Scheffersomyces stipitis genome was used as a template, and primers XYL1-F / XYL1-R, XYL1-R-2, XYL2-F / XYL2-R, XYL2-R-2 were used to amplify XYL1 (Gene ID: 4839234), XYL2 (Gene ID :4852013) gene. Using overlapping PCR, build the EFT1-XYL1-PGK1-XYL2-HS...

Embodiment 2

[0035] Embodiment 2 pentose phosphate ketolysis (PK) pathway construction

[0036] In the above-mentioned Example 1, after introducing the xylose metabolism pathway into Saccharomyces cerevisiae, although the engineered bacteria can already utilize xylose, the content of the target product is still relatively low. In order to improve the product yield, the pentose phosphate ketolysis (PK) pathway was introduced in this example. Use the plasmid pHCas9-gRNA as a template (pHCas9-gRNA contains the guide sequence N 20 ), use the primers gRNA-720a-F / gRNA-R to amplify the functional modules, and construct the genome integration plasmid pHCas9-720a. Using the Leuconostoc mesenteroides genome as a template, use primers xpk-F / xpk-R, xpk-R-2 to amplify the xPK gene (GenBank: TJY30451.1); using the Clostridium butyricum genome as a template, use primers pta-F / pta-R , pta-R-2, 720-R amplified PTA gene (GenBank: EEP53689.1). Using the Saccharomyces cerevisiae genome as a template, prime...

Embodiment 3

[0041] Example 3 Pho13 gene knockout

[0042] After the above-mentioned Example 2, the xylose utilization rate of the engineered bacteria is still relatively slow. In order to improve the xylose utilization efficiency, the Pho13 gene (Gene ID: 851362) is knocked out in this example. Using the plasmid pHCas9-gRNA as a template, the primers gRNA-Pho13-F / gRNA-R were used to amplify the functional modules to construct the genome-integrated plasmid pHCas9-Pho13. Using the Saccharomyces cerevisiae genome as a template, primers Pho13-UP-F / Pho13-UP-R, Pho13-DOWN-F / Pho13-DOWN-R were used to amplify the upstream and downstream homology arms of the Pho13 gene, and the Pho13 gene knockout was constructed by overlapping PCR. In addition to the module, the strain SC103 constructed in Example 2 was introduced together with pHCas9-Pho13, and the identification primer Pho13-check-F / Pho13-check-R was used for identification to obtain the engineering strain SC104. The obtained recombinant strai...

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Abstract

The invention discloses xylose utilization yeast and an application thereof. The yeast is integration type engineering bacteria containing an xylose reductase gene XYL1m and an xylitol dehydrogenase gene XLY2 which are mutated, the amino acid sequence of xylose reductase contains one point mutation, K at the 271st-site amino acid is mutated into N, the integration type engineering bacteria also contain a phosphoketolase gene xPK and a phosphotransacetylase gene PTA, the Pho13 gene is knocked out, the integration type engineering bacteria also contain hexose transport protein Gal2 which is mutated, N at the 376th-site amino acid for Gal2 is mutated into F, a promoter at the upper stream of the Gal2 gene is replaced with SSA1, and a promoter of an xylulokinase XK gene is replaced with a strong promoter HSP82. A saccharomyces cerevisia platform for synthetizing carotenoids from xylose is constructed, and important support is provided for preparation of carotenoids by economically utilizing a biological fermentation method.

Description

technical field [0001] The invention belongs to the field of genetic engineering and biotechnology, and relates to a xylose-utilizing yeast and its application, in particular to a xylose-utilizing Saccharomyces cerevisiae and its application in carotenoid synthesis. Background technique [0002] Lignocellulosic biomass resources are the most abundant and cheapest renewable resources on the earth; my country is a large agricultural country with extremely rich biomass resources such as agricultural waste. At present, most of these resources have not been fully utilized, and often cause pollution to varying degrees; with the increasing depletion of non-renewable resources such as fossil fuels and the resulting environmental pollution, climate change and other issues, the development of renewable resources The use of biomass resources to produce bio-based chemicals and biofuels is a current research hotspot. Lignocellulose includes cellulose (34-50%), hemicellulose (19-35%) and...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12P33/00C12R1/865
CPCC12N9/0006C12N9/1022C12N9/104C07K14/395C12P33/00C12Y101/01307C12Y101/01009
Inventor 朱红惠苏卜利宋丹丹冯广达
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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