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Saccharomyces cerevisiae bacterial strain with high xylose transport capacity and application thereof

A technology of Saccharomyces cerevisiae strains and Saccharomyces cerevisiae, which is applied in the application field of recombinant Saccharomyces cerevisiae, can solve problems such as plasma membrane incompatibility, correct expression of transport proteins, difficulty in membrane positioning, and differences in chemical composition, and achieve broad application prospects Effect

Active Publication Date: 2012-07-11
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the cell membrane skeletons of bacteria and fungi are both lipid bilayer structures, they contain different appendages, such as sterols, membrane proteins, etc., resulting in differences in the chemical composition of the two, which in turn leads to incompatibility between the plasma membranes In addition, there are differences in the inner membrane system and membrane protein co-regulators of bacterial and fungal cells, resulting in the difficulty of correct expression, membrane localization and full function of bacterial-derived transporters in Saccharomyces cerevisiae strains
So far, there is still a lack of reports on the functional expression of bacterial xylose transporters in Saccharomyces cerevisiae

Method used

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  • Saccharomyces cerevisiae bacterial strain with high xylose transport capacity and application thereof
  • Saccharomyces cerevisiae bacterial strain with high xylose transport capacity and application thereof
  • Saccharomyces cerevisiae bacterial strain with high xylose transport capacity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: Construction of araE Saccharomyces cerevisiae expression vector

[0029] Methods as below:

[0030] (1) Vector selection: pJFE3 (Yeplac195, TEFp-PGKt, Ura - , Amp + ) empty plasmid;

[0031] (2) Expression vector construction: use upstream and downstream primers AraE3up: 5'-ATCGC GGATCC ATGGCAGGGCACATCATCCGCTCAG-3' and AraEdown-His:5'-ACGC GTC GAC TTAGTGGTGGTGGTGGTGGTGAGGCTAAGGAGTGTTTAAGAGC-3'Clone the transporter gene araE from the DNA of Corynebacterium glutamicum ATCC 31831, and construct the Saccharomyces cerevisiae episomal shuttle plasmid on the plasmid in (1) using BamH I and Sal I restriction sites pJFE3-araE, see appendix figure 1 ;

Embodiment 2

[0032] Embodiment 2: Construction of the bacterial strain BSW2ABE expressing AraE transporter

[0033] Methods as below:

[0034] (1) Strain selection: select the constructed laboratory strain BSW2AB (CEN.PK102-3A derivative, MATa, leu2-3, ura3-52, pYX242-PGKt-TEFp TPIp-xynB) with intracellular heterologous expression of xylosidase -PGKt) as the recipient strain, which contains the constructed pYX242-PGKt-TEFp TPIp-xynB-PGKt heterologous expression vector, see figure 2 , the strain was deposited in the General Microorganism Center of China Microbiological Culture Collection Management Committee on November 17, 2011, and the preservation number is CGMCC No.5465;

[0035] (2) Strain construction: the plasmid pJFE3-araE constructed in Example 1 was transformed into the BSW2AB strain by the lithium acetate transformation method, and the correct transformant was screened with the fully synthetic auxotrophic medium SC-Leu-Ura added with 2% glucose;

[0036] (3) Bacterial strain v...

Embodiment 3

[0037] Example 3: Determination of the pNPX Transport Ability of the Bacterial Strain BSW2ABE

[0038] Methods as below:

[0039] (1) Routine strain selection: select the laboratory strain BSW2ABP (CEN.PK102-3A derivative, MATa, leu2-3, ura3- 52, pYX242-PGKt-TEFp TPIp-xynB-PGKt, pJFE3) as a conventional strain;

[0040] (2) Bacterial strain culture: the single bacterium colony of bacterial strain BSW2ABP and BSW2ABE is activated twice with the fully synthetic auxotrophic culture medium SC-Leu-Ura that adds 2% glucose, transfers 40ml to 100ml small Erlenmeyer flask, initial 600nm light absorption (expressed as OD 600 or OD 600nm ) was connected to 0.5, and the culture conditions were 30°C, 200r·min -1 ;

[0041] (3) Preparation of complete cell suspension: take the bacterium liquid cultivated in step (2) for 10 hours, centrifuge (3000r min -1 , 5min) resuspended with 50mmol / L PBS solution of pH 6.5 after removing the original culture medium;

[0042] (4) Determination of...

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Abstract

The invention discloses a saccharomyces cerevisiae bacterial strain with high xylose transport capacity. The bacterial strain is BSW2ABE, is named as saccharomyces cerevisiae in a classified manner and is preserved at China General Microbiological Center Culture Collection Center on December, 27th, 2011, and the preservation number of the bacterial strain is CGMCC No. 5658. The bacterial strain expresses a transport protein gene araE cloned from corynebacterium glutamicum and also expresses the detection on the saccharomyces cerevisiae bacterial strain containing transport protein by utilizing a method for screening and expressing the saccharomyces cerevisiae bacterial strain containing the active xylose transport protein (refer to an invention patent application with an application date of December, 15th , 2011 and an application number of 201110420908.4 submitted by a same applicant); and compared with an initial bacterial strain, the xylose transport speed of the saccharomyces cerevisiae bacterial strain is improved by 1.9 times. The invention also relates to an application of the bacterial strain with improved xylose transport capacity in the construction of recombinant saccharymyces cerevisiae of metabolizable xylose.

Description

technical field [0001] The invention relates to a recombinant strain of Saccharomyces cerevisiae with high xylose transport ability. The bacterial strain expresses the transporter gene araE cloned from Corynebacterium glutamicum, and the expression of the transporter gene araE improves the xylose resistance of the Saccharomyces cerevisiae strain. Transshipment capacity. The present invention also relates to the application of the strain with high xylose transport ability in constructing recombinant brewer's yeast capable of metabolizing xylose. Background technique [0002] At present, the problem of energy shortage has been widely concerned by countries all over the world. It is of great significance to the sustainable development of society in the future to replace non-renewable fossil resources with renewable resources to produce energy substances. In my country, lignocellulosic raw materials, mainly agricultural wastes, are renewable biomass resources with a huge annual...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/31C12N15/63C12R1/865
Inventor 鲍晓明沈煜汪城墙
Owner SHANDONG UNIV
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