CN109913381B

CN109913381B - Method for improving fermentation ethanol yield by regulating cell cycle transcription factor

Info

Publication number: CN109913381B
Application number: CN201910266772.2A
Authority: CN
Inventors: 蒋伶活
Original assignee: Shandong University of Technology
Current assignee: Shandong University of Technology
Priority date: 2019-04-03
Filing date: 2019-04-03
Publication date: 2021-05-04
Anticipated expiration: 2039-04-03
Also published as: CN109913381A

Abstract

The invention discloses a method for improving the yield of fermentation ethanol by regulating cell cycle transcription factors, belonging to the technical field of development of biological energy technology. According to the invention, transcription factor genes MBP1, SWI4 or SWI6 for regulating and controlling the cell cycle are knocked out, so that a new method is provided for improving the production efficiency of ethanol fermentation, and the excellent saccharomyces cerevisiae engineering strain for high-yield ethanol constructed by the invention is fermented for 52 hours, so that the ethanol fermentation yield is improved by 27.3-51.0% compared with a wild strain (WT).

Description

Method for improving fermentation ethanol yield by regulating cell cycle transcription factor

Technical Field

The invention relates to a method for improving the yield of fermentation ethanol by regulating cell cycle transcription factors, belonging to the technical field of development of biological energy technology.

Background

Saccharomyces cerevisiae (Saccharomyces cerevisiae) is the major fermenting microorganism in the current fuel ethanol production process. The technology for producing fuel ethanol by fermentation of saccharomyces cerevisiae by using corn starch as a raw material at home and abroad is very mature. Natural screening and mutation breeding are traditional saccharomyces cerevisiae strain breeding means, but the ethanol fermentation yield of the strain is improved by the natural screening and mutation breeding means to the limit. In recent years, with the rapid development of genetic engineering technology, the possibility is provided for improving the ethanol fermentation level of the saccharomyces cerevisiae strain. Through the modification of transcription factor gene, the expression level of many target genes regulated by the transcription factor gene can be simultaneously changed, so that the character change caused by multiple gene changes can be obtained, and the potential high-yield ethanol character can be obtained, which cannot be achieved through the change of a single gene.

Mbp1 is a DNA binding protein (transcription factor) that forms an MBF complex with Swi6 and constitutes the Mlu1 cell cycle box binding factor. MBF is a sequence-specific transcription factor that regulates gene expression during the G1/S transition of the cell cycle. The genes regulated by MBF are involved in DNA synthesis and DNA repair as well as the G1 cyclin gene. Furthermore, the transcription factor Swi4 can form a similar SBF complex with Swi6, constituting a Swi4/6 cell cycle box binding factor. Genome-wide analysis has found that there are more than 100 target genes in common for MBF and SBF complexes. However, the function of MBF and SBF complex genes for improving ethanol yield has not been reported.

Disclosure of Invention

Through functional genomics technical screening, the invention discovers that 3 transcription factor genes MBP1, SWI4 or SWI6 for regulating and controlling the cell cycle are lacked, and can improve the ethanol yield of saccharomyces cerevisiae fermentation.

The first purpose of the invention is to provide a method for improving the ethanol fermentation yield of saccharomyces cerevisiae, and the method is to knock out the transcription factor genes MBP1, SWI4 or SWI6 for regulating the cell cycle.

In one embodiment of the invention, the nucleotide sequence of the gene MBP1 is shown in NCBI accession No. NM-001180115.1.

In one embodiment of the invention, the nucleotide sequence of the gene SWI4 is shown in NCBI accession No. NM-001179001.1.

In one embodiment of the invention, the nucleotide sequence of the gene SWI6 is shown in NCBI accession No. NM-001182069.1.

In one embodiment of the invention, the Saccharomyces cerevisiae is MBP1 deleted Saccharomyces cerevisiae available from Invitrogen under the number Sc04106576_ s 1.

In one embodiment of the invention, the Saccharomyces cerevisiae is SWI6 deleted Saccharomyces cerevisiae, available from Invitrogen, under the number Sc04147446_ s 1.

In one embodiment of the invention, the Saccharomyces cerevisiae is SWI4 deleted Saccharomyces cerevisiae, available from Invitrogen, under the number Sc04118768_ s 1.

The second purpose of the invention is to provide a method for producing ethanol, which comprises the step of fermenting the strain lacking MBP1, SWI6 or SWI4 in a fermentation medium for 36-60 h.

In one embodiment of the invention, the first 8-10 h of the fermentation is cultured at 160-220 rpm, and then the fermentation is statically cultured.

In one embodiment of the invention, the fermentation medium comprises, in g/L: 100 parts of glucose, 7.5 parts of ammonium sulfate, 3.5 parts of potassium dihydrogen phosphate, 0.75 part of magnesium sulfate heptahydrate, 0.2 part of yeast extract, 0.02 part of histidine, 0.02 part of uracil and 0.1 part of leucine.

In one embodiment of the invention, the strain is activated in YPD medium before fermentation.

The invention also claims the effect of transcription factor genes MBP1, SWI4 and SWI6 for regulating and controlling the cell cycle on improving the ethanol fermentation production efficiency of saccharomyces cerevisiae.

Has the advantages that: the invention provides a new method for improving the production efficiency of ethanol fermentation, and the excellent saccharomyces cerevisiae engineering strain with high ethanol yield, which is constructed by the invention, is fermented for 52 hours, so that the ethanol fermentation yield is improved by 27.3-51.0% compared with a wild strain (WT).

Drawings

FIG. 1 shows the yields (ethanol production/dry weight ratio) of three cell cycle-regulating transcription factor genes MBP1, SWI4 or SWI6 deleted strains and wild-type s.cerevisiae strain (WT) after 32 hours (32h) and 52 hours (52h) of culture in fermentation medium. The asterisks indicate that significant differences were present between each gene-deleted strain and the wild-type strain at the 32 hour and 52 hour time points, respectively.

Detailed Description

The seed culture medium comprises 2% of glucose, 1% of yeast extract, 2% of peptone and deionized water in terms of m/v, and the pH value is 7.0. The content (g/L) of each component in the fermentation medium is as follows: 100 parts of glucose, 7.5 parts of ammonium sulfate, 3.5 parts of monopotassium phosphate, 0.75 part of magnesium sulfate heptahydrate, 0.2 part of yeast extract, 0.02 part of histidine, 0.02 part of uracil and 0.1 part of leucine, and the pH value is 7.0. Both media were autoclaved (115 ℃, 20 min).

The method for measuring the ethanol content comprises the following steps: detecting the content of ethanol in the fermentation liquid by High Performance Liquid Chromatography (HPLC), and detecting by a differential refraction detector. The chromatographic column used was a BIO-RAD organic acid column, the column temperature was 35 ℃ and the mobile phase was 0.0275% (v/v) dilute sulfuric acid, the flow rate was 0.6 mL/min. The sample amount of each sample was 20. mu.L, and the detection time was 25 min.

The yield of ethanol to thalli is calculated according to the following formula:

wherein y is the ethanol yield, p is the ethanol concentration, and x is the OD value.

Example 1 ethanol fermentation of Saccharomyces cerevisiae

The activated strain was streaked on YPD plates and cultured at 30 ℃ for 2 days. One single colony of each strain was inoculated into 30mL YPD liquid medium and shake-cultured at 30 ℃ and 220r/min for more than 16 hours until saturation. Inoculating the seed bacterial liquid into 100mL of fermentation medium according to the proportion of 10%, performing shake culture at 30 ℃ and 220r/min for 8 hours, and then converting to static culture and fermentation.

At two fermentation time points of 32 hours and 52 hours after inoculation, 1mL of the cell fermentation broth was taken, and the dry weight of the cells was measured. Separately, 1mL of the cell fermentation broth was centrifuged at 12000rpm for 5 minutes, and the supernatant was taken out and used for liquid phase measurement of the ethanol concentration therein. Data were collected and a plot of dry cell weight, ethanol yield was made as shown in FIG. 1. After 52-hour fermentation, compared with wild strains (WT), the alcohol fermentation yield of the saccharomyces cerevisiae strains with single gene deletion of SWI4, SWI6 and MBP1 is respectively improved by 51.0%, 36.4% and 27.3%.

Comparative example 1:

DOT 6-deleted Saccharomyces cerevisiae was purchased from Invitrogen under the number Sc04118417_ s 1.

The same procedure as in example 1 was followed to culture and ferment a DOT6 gene-knocked-out Saccharomyces cerevisiae-deficient strain. The results showed no significant change in the alcoholic fermentation yield compared to the wild type strain (WT) (FIG. 1).

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for improving the ethanol fermentation yield of saccharomyces cerevisiae is characterized in that a transcription factor gene for regulating and controlling the cell cycle is knocked outMBP1，SWI4OrSWI6；

The geneMBP1The nucleotide sequence of (A) is shown as NCBI number: NM-001180115.1;

the geneSWI4The nucleotide sequence of (A) is shown as NCBI number: NM-001179001.1;

the geneSWI6The nucleotide sequence of (1) has the NCBI number of NM-0011820691 is shown in the specification;

the Saccharomyces cerevisiae isMBP1Deleted Saccharomyces cerevisiae, available from Invitrogen, under the Sc04106576_ s 1; or

The Saccharomyces cerevisiae isSWI6Deleted Saccharomyces cerevisiae, purchased from Invitrogen, under the Sc04147446_ s 1; or

The Saccharomyces cerevisiae isSWI4The deleted Saccharomyces cerevisiae, purchased from Invitrogen, was numbered Sc04118768_ s 1.

2. A process for producing ethanol, characterized in that the deletion isMBP1、SWI6OrSWI4The strain is inoculated in a fermentation medium and fermented for 36-60 h;

the geneSWI6The nucleotide sequence of (A) is shown as NCBI number: NM-001182069.1;

absence ofMBP1Saccharomyces cerevisiae (Sc 04106576_ s1, Invitrogen); absence ofSWI6Saccharomyces cerevisiae was purchased from Invitrogen and is numbered Sc04147446_ s 1; absence ofSWI4Saccharomyces cerevisiae (Sc) of (1) was purchased from Invitrogen and is numbered Sc04118768_ s 1.

3. The method of claim 2, wherein the culture is performed at 160 to 220rpm for 8 to 10 hours before the fermentation, and then the culture is performed in a static state.

4. The method according to claim 2, wherein the medium composition for fermentation comprises, in g/L: 80-120 parts of glucose, 6-8 parts of ammonium sulfate, 2-4 parts of monopotassium phosphate, 0.5-1 part of magnesium sulfate heptahydrate, 0.1-0.3 part of yeast extract, 0.01-0.03 part of histidine, 0.01-0.03 part of uracil and 0.05-0.15 part of leucine.

5. A process according to any one of claims 2 to 4, wherein the strain is activated in YPD medium prior to fermentation.

6. The nucleotide sequence of the gene is shown as NCBI numbers of NM-001180115.1, NM-001179001.1 and NM-001182069.1MBP1，SWI4AndSWI6the method has the advantages that the method has the function of improving the ethanol fermentation production efficiency of the saccharomyces cerevisiae; the Saccharomyces cerevisiae was purchased from Invitrogen and is numbered Sc04106576_ s1 or Sc04147446_ s1 or Sc04118768_ s 1.