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Method for promoting microbial cells to transport glucose, xylose and arabinose and application thereof in fermentation of biobased products

A technology of arabinose and cells, applied in biochemical equipment and methods, botany equipment and methods, chemical instruments and methods, etc., can solve problems such as potential changes and unexplained

Active Publication Date: 2015-08-19
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

As early as 1974, it was reported that Neurospora co-transports protons while actively transporting sugar into cells (Slayman et al.1974. Depolarization of the plasma membrane of Neurospora during active transport of glucose: evidence for a proton-dependent cotransport system.Proc Natl Acad Sci USA,71(5):1935-1939.), but at the molecular level, which sugar transporters function to cause the apparent potential change has not yet been elucidated

Method used

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  • Method for promoting microbial cells to transport glucose, xylose and arabinose and application thereof in fermentation of biobased products
  • Method for promoting microbial cells to transport glucose, xylose and arabinose and application thereof in fermentation of biobased products
  • Method for promoting microbial cells to transport glucose, xylose and arabinose and application thereof in fermentation of biobased products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Example 1.GLT-1 is a glucose transporter that enables microorganisms to obtain the ability to transport and utilize glucose

[0127] 1. Construction of GLT-1 gene expression vector

[0128] Using primers GLT-F (sequence: 5'-CGCGGATCCATGGGTCTCTTCTCGAAAAAGTC-3') (SEQ ID NO.: 11) and GLT-R (sequence: 5'-CCGGAATTCCTAAACCTCTCCATGGCTTGAGG-3') (SEQ ID NO.: 12) from vein The coding reading frame of the GLT-1 gene was amplified by PCR from the cDNA of the sporozoite. The PCR reaction system was: 5×phusion HF buffer 10μL, 10mM dNTPs 1μL, GLT-F 2.5μL, GLT-R 2.5μL, cDNA 1μL, Phusion DNA Polymerase 0.5 μL, water 32.5 μL. The PCR reaction conditions were as follows: first 98°C for 30s; then 98°C for 10s, 65°C for 30s, 72°C for 1.5min, 35 cycles; finally 72°C for 10min, 4°C for 10min. After the PCR reaction was completed, the PCR product and plasmid pRS426-PGK1 were combined using restriction enzymes BamHI and EcoRI [the plasmid was constructed according to reference (Galazka, J.M.,...

Embodiment 2

[0145] Example 2.XYT-1 is a xylose transporter that enables microorganisms to obtain the ability to transport xylose

[0146] 1. Construction of XYT-1 gene expression vector

[0147] Using primers XYT-F (sequence: 5'-GGACTAGTATGGTTCTGGGGAAAAAGTCAATC-3') (SEQ ID NO.: 13) and XYT-R (sequence: 5'-CCCAAGCTTCTAAACCCTATGGTTAATAACCTT-3') (SEQ ID NO.: 14) from Grain The coding reading frame of the XYT-1 gene was amplified by PCR from the cDNA of the sporozoite. The PCR reaction system was: 5×phusion HF buffer 10μL, 10mM dNTPs 1μL, XYT-F 2.5μL, XYT-R 2.5μL, cDNA 1μL, Phusion DNA Polymerase 0.5 μL, water 32.5 μL. The PCR reaction conditions were as follows: first 98°C for 30s; then 98°C for 10s, 60°C for 30s, 72°C for 1.5min, 35 cycles; finally 72°C for 10min, 4°C for 10min. After the PCR reaction was completed, the PCR product and the plasmid pRS426-PGK1 were combined using restriction enzymes SpeI and HindIII [the plasmid was constructed according to reference (Galazka, J.M., et al....

Embodiment 3

[0150] Example 3.XAT-1 is a xylose and arabinose transporter, enabling microorganisms to obtain the ability to transport xylose and arabinose

[0151] 1. Construction of XAT-1 gene expression vector

[0152] Using primers XAT-F (sequence: 5'-CGCGGATCCATGAAGCCATTTCTGGGGCTC-3') (SEQ ID NO.: 15) and XAT-R (sequence: 5'-CCCAAGCTTCTACGACTCCCGATTACCTCCAT-3') (SEQ ID NO.: 16) The coding reading frame of the XAT-1 gene was amplified by PCR from the cDNA of the sporozoite. The PCR reaction system was: 5×phusion HF buffer 10μL, 10mM dNTPs 1μL, XAT-F 2.5μL, XAT-R 2.5μL, cDNA 1μL, Phusion DNA Polymerase 0.5 μL, water 32.5 μL. The PCR reaction conditions were as follows: first 98°C for 30s; then 98°C for 10s, 65°C for 30s, 72°C for 1.5min, 35 cycles; finally 72°C for 10min, 4°C for 10min. After the PCR reaction was completed, the PCR product and plasmid pRS426-PGK1 were combined using restriction enzymes BamHI and HindIII [the plasmid was constructed according to reference (Galazka, J.M....

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Abstract

The present invention discloses a method for promoting microbial cells to transport glucose, xylose and arabinose and application thereof in fermentation of biobased products. Five transport proteins provided by the present invention have capabilities of transporting glucose, xylose or arabinose. The method for promoting microbial cells to transport glucose, xylose and arabinose is characterized in that the transportproteins are guided into microbiological strains, the obtained reconstructed microbiological strains obtain or improve the capabilities in transporting glucose, xylose or arabinose, and further take use of glucose, xylose and arabinose to produce fuel ethanol and other biobased fermentation products.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for promoting microbial cells to transport glucose, xylose and arabinose. It mainly involves transport proteins (GLT-1, XYT-1, XAT-1, LAT-1 and MtLAT-1) and coding genes, as well as making them obtain or improve glucose, xylose or arabinose transport ability and their Application in microbial fermentation to produce bio-based products. Background technique [0002] Biomass is the largest renewable resource on earth and the most widely distributed carbohydrate. In the face of energy crisis and resource shortage, the use of renewable biomass to produce bioenergy provides hope for the sustainable development of human beings. Biomass is mainly composed of cellulose, hemicellulose and lignin, and its degradation products mainly include glucose, xylose, arabinose and other monosaccharides and some oligosaccharides. [0003] In order to improve the utilization rate of biomass degr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/37C07K14/38C07K14/39C12N15/31C12N15/81C12N15/80C12P7/06
CPCY02E50/10
Inventor 田朝光李金根蔡鹏丽王邦许晶马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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