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Saccharomyces cerevisiae strain and method for selecting saccharomyces cerevisiae strains expressing activated xylose translocator

A technology of Saccharomyces cerevisiae strain and xylose transporter, which is applied in microorganism-based methods, biochemical equipment and methods, fungi, etc., can solve the problems of large sedimentation coefficient, difficulty in molecular entry and exit, and different xylose transport mechanisms. Achieve the effect of avoiding false positives, simple operation steps, and less demanding operators and equipment

Active Publication Date: 2012-06-13
SHANDONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows researchers to study how certain enzymes work with sugar molecules called XYLs or other sugars like glucose that help cells grow better at producing specific chemical compounds from those sugars. By introducing an organism carrying genes involved in these processes, scientists have been able to predict which products will perform best when added together on top of each others' receptors. Overall this innovation provides technical benefits over existing methods where only one type of substances produced during cell growth has ever happened.

Problems solved by technology

Luteum Crosseaeceae plays key roles during biomass conversion processes like saccharification/liquefaction, polysaccharides extractions, enzyme synthesis, protein secretion, and other technical problem addressed in this patents text relates to developing methods for detecting xylenecarboxymeters involved in these biochemistry reactions without causing errors caused by competitive interactions between them.

Method used

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  • Saccharomyces cerevisiae strain and method for selecting saccharomyces cerevisiae strains expressing activated xylose translocator
  • Saccharomyces cerevisiae strain and method for selecting saccharomyces cerevisiae strains expressing activated xylose translocator
  • Saccharomyces cerevisiae strain and method for selecting saccharomyces cerevisiae strains expressing activated xylose translocator

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Example 1: Construction of strain BSW2ABP expressing xylosidase and assay of xylosidase enzyme activity

[0044] Methods as below:

[0045] (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) is used as the recipient strain, and the strain is classified as Saccharomyces cerevisiae, and has been deposited in the "General Microorganism Center of China Committee for the Collection of Microbial Cultures" on November 17, 2011, with a preservation number of CGMCC No.5465 . Its mycological characteristics are: CEN.PK102-3A derivative, MATa, leu2-3, ura3-52, pYX242-PGKt-TEFpTPIp-xynB-PGKt, the yeast strain is a unicellular eukaryotic microorganism, oval, immobile, solid Or under the culture condition of the fully synthetic auxotrophic culture medium (SC-Leu, this culture medium is existing in the prior art) of liquid, b...

Embodiment 2

[0054] Example 2: Determination of the relationship between incubation time and transport rate measurements

[0055] Methods as below:

[0056] (1) Select the bacterium liquid cultivated for 15 hours in the embodiment 1 step (4), centrifuge (3000r min -1 , 5min) resuspended with 50mmol / L PBS solution of pH 6.5 after removing the original culture medium;

[0057] (2) Determination of the relationship between the incubation time and the measured value of the transport rate: Take an appropriate amount of the complete cell suspension in step (1) with a 96-well plate or EP tube, add pNPX with a final concentration of 6 mmol / L, and then place it at 30 ℃, 200r·min -1 In a shaker, incubate for 10min, 20min, 30min, 40min, 50min and 60min respectively, centrifuge to take 100μl of the supernatant and add it to a 96-well plate, then add an equal volume of 0.4mol / L Na 2 CO 3 Aqueous solution, after mixing, use a microplate reader at OD 405nm The light absorbance of p-nitrophenol (pNP)...

Embodiment 3

[0058] Example 3: Determination of the extracellular and extracellular content of pNP under the condition of complete cell determination

[0059] Methods as below:

[0060] (1) Select the bacterium liquid cultivated for 15 hours in the embodiment 1 step (4), centrifuge (3000r min -1 , 5min) resuspended with 50mmol / L PBS solution of pH 6.5 after removing the original culture medium;

[0061] (2) Determination of the extracellular content of pNP in the state of intact cells: use a 96-well plate or EP tube to take an appropriate amount of the complete cell suspension in step (1), add pNPX with a final concentration of 6mmol / L, and then place it at 30 ℃, 200r·min -1 Incubate on a shaker for 10min and 30min respectively; centrifuge to take 100μl of the supernatant and add it to a 96-well plate, then add an equal volume of 0.4mol / L Na 2 CO 3 Aqueous solution, after mixing, use a microplate reader at OD 405nm The light absorbance of p-nitrophenol (pNP) produced after incubation ...

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Abstract

The invention discloses a saccharomyces cerevisiae strain expressing beta-xylosidase in cells, which is BSW2AB. The classification is named saccharomyces cerevisiae and filed in 'China General Microbiological Culture Collection Center' with the filed number CGMCC No.5465 on November 17, 2011. The saccharomyces cerevisiae strain is capable of expressing activated beta-xylosidase in cells. The invention provides a method for selecting saccharomyces cerevisiae strainsexpressing activated xylose translocator, and the reaction substrate for selecting the translocator is xylose analogue, namely p-nitrophenyl-beta-D-xyloside. The invention further provides a recombination saccharomyces cerevisiae strain BSW2ABT which has high xylose transfer capability and is obtained by the above method. The classification is named saccharomyces cerevisiae and filed in 'China General Microbiological Culture Collection Center' with the filed number CGMCC No.5464 on November 17, 2011.

Description

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Claims

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Application Information

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Owner SHANDONG UNIV
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