Method for improving sucrose fermentation performance of Saccharomyces cerevisiae and application thereof

A technology of Saccharomyces cerevisiae and sucrose, which is applied in the field of genetic engineering, can solve problems that have not been reported, and achieve the effects of increased ethanol yield, efficient fermentation of sucrose to produce bioethanol and rare biochemical products, and increased sucrose consumption rate

Inactive Publication Date: 2017-10-20
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, studies on the involvement of sucrose transporters in sucrose metabolism have not been reported, nor has the study on the sucrose transporter gene sut1 improving sucrose fermentation performance in Saccharomyces cerevisiae

Method used

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  • Method for improving sucrose fermentation performance of Saccharomyces cerevisiae and application thereof
  • Method for improving sucrose fermentation performance of Saccharomyces cerevisiae and application thereof
  • Method for improving sucrose fermentation performance of Saccharomyces cerevisiae and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 : Construction of expression vector pRS6K-sut1 containing sucrose transporter gene sut1

[0028] The target gene sut1 was obtained by PCR technology, with a size of 1551bp (PCR upstream primer: 5'-aaaacaggatccccc AAAA ATGGAGAATGGTACAAAAAAG-3', downstream primer: 5'-gaattcctgcagcccTTATTTTAATGGAAAGCCCCATG-3', the lowercase part is the sequence homologous to the vector, and the underlined part is the Kozak sequence. The PCR reaction program was: 98°C pre-denaturation for 3 minutes, 98°C for 10s, 53°C for 15s, 72°C for 2min, 30 cycles, and finally 72°C extension for 10 minutes), and then the linearized expression vector pRS426K was digested with restriction endonuclease SmalⅠ, The size is 6636bp.

[0029] Finally, using TaRaKa's HD Cloning Kit connects two DNA fragments, transforms Escherichia coli JM109 competent cells, screens positive transformants on LA-resistant plates containing 100 mg / mL ampicillin, and then sends the positive transformants to Huada ...

Embodiment 2

[0030] Example 2 : Construction of recombinant yeast strain SC1-ST expressing sut1 gene heterologously

[0031] Saccharomyces cerevisiae S.cerevisiae INVSC1 was used as the starting strain, and the expression plasmid pRS6K-sut1 containing the sucrose transporter gene and the empty plasmid pRS426K were transformed with lithium acetate (Gietz and Schiestl, 2007, Quick and easy yeast transformation using the LiAc / SS carrier DNA / PEG method, Nat.Protoc., 2(1):35-37) were introduced into S.cerevisiae INVSC1 respectively, and positive transformants were screened on the screening plate.

[0032] The screening plate medium is uracil (URA) auxotroph: YNB yeast basic nitrogen source 6.7g / L, glucose 20g / L, histidine 30mg / L, leucine 60mg / L, tryptophan 30mg / L, The culture temperature is 30°C, and the culture time is 3-5 days. Select transformants from the selection plate, and obtain positive recombinant strains after colony PCR verification (PCR reaction program: 98°C for 10 minutes, 98...

Embodiment 3

[0034] Example 3 : Detection of sucrose fermentation performance of strain SC1-ST under the condition of sucrose as the only carbon source

[0035] (1) Streak the Saccharomyces cerevisiae strain SC1-ST on the YPD solid plate (YPD solid medium composition: peptone 20g / L, yeast powder 10g / L, glucose 20g / L, agar powder 15g / L), and place it in 30 Cultivate in a constant temperature incubator at ℃ for 3 days, select a single colony and inoculate it in the seed medium for overnight culture (seed medium composition: peptone 20g / L, yeast powder 10g / L, glucose 20g / L), and continuously transfer and activate the culture for 2 Second-rate.

[0036] (2) Inoculate the above-mentioned activated bacterial strain SC1-ST in 100mL sucrose fermentation medium (fermentation medium components: peptone 20g / L, yeast powder 10g / L, sucrose 10% and 20%), initial cell OD 600 The value is 0.5, cultured at 30° C. and 100 rpm, and samples are taken regularly for detection. Each experiment was done in 3 ...

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Abstract

The invention specifically relates to a method for improving the output of ethanol through rapid fermentation of sucrose and application thereof, belonging to the field of genetic engineering technology. According to the invention, a recombinant Saccharomyces cerevisiae strain SC1-ST for heterogenous expression of a sucrose transport protein gene sut1, and the strain SC1-ST can rapidly ferment sucrose to produce bioethanol in a medium with sucrose as a carbon source. Compared with fermentation of sucrose with wild bacterial strains, the method for fermenting sucrose with the SC1-ST strain in the invention can substantially improve the sucrose fermentation performance of Saccharomyces cerevisiae and allows biomass output, ethanol yield and sucrose consumption to be substantially increased. Thus, it is proved that the method for improving the sucrose fermentation performance of Saccharomyces cerevisiae via sucrose transport protein has great industrial application potential in high-efficiency fermentation of sucrose for production of bioethanol and rare biochemical products.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a method for improving the sucrose fermentation performance of Saccharomyces cerevisiae and an application thereof. Background technique [0002] Since the beginning of the 21st century, with the depletion of fossil energy and the deterioration of the ecological environment, the discourses on energy crisis and ecological crisis that have emerged in recent years have attracted the attention of countries all over the world. In response to these current problems, countries around the world have successively proposed the development and implementation of renewable energy strategies, and biomass energy, as a sustainable renewable energy source, is an ideal substitute for fossil fuels. [0003] Microorganisms can use a variety of biomass raw materials in nature, such as fiber, starch and sugar, as carbon sources to produce biomass energy through fermentation. Ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/08C12P7/06C12N1/19C12R1/865
CPCC07K14/415C12P7/06C12P7/08Y02E50/10
Inventor 张志凯杜丽琴庞浩汤宏赤林丽华兰青黄日波韦宇拓
Owner GUANGXI UNIV
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