CN106906152B

CN106906152B - Saccharomyces cerevisiae and application thereof

Info

Publication number: CN106906152B
Application number: CN201710307928.8A
Authority: CN
Inventors: 王颖; 刘贺; 朱家庆; 李炳志; 元英进
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2017-05-04
Filing date: 2017-05-04
Publication date: 2020-06-19
Anticipated expiration: 2037-05-04
Also published as: CN106906152A

Abstract

The invention relates to the field of bioengineering, and in particular relates to saccharomyces cerevisiae and application thereof. The preservation number of the strain is as follows: CGMCC No. 13987. The strain provided by the invention can adapt to the environment containing inhibitors and high-temperature environment, can normally grow in a culture medium containing the inhibitors generated in the production of various cellulose ethanol, can show good growth advantages in cellulose hydrolysate, and realizes efficient ethanol production.

Description

Saccharomyces cerevisiae and application thereof

Technical Field

The invention relates to the field of bioengineering, and in particular relates to saccharomyces cerevisiae and application thereof.

Background

In the cellulosic ethanol production process, acid pretreatment is favored by most institutions due to its low cost and high operability, and is considered to be the most easily industrialized pretreatment method, such as dilute acid pretreatment and steam explosion pretreatment. However, since these chemical pretreatment conditions are generally high temperature and high pressure, some inhibitory substances, such as furans, weak acids, phenols, etc., are generated. These inhibitory substances have a very strong inhibitory effect on the subsequent fermentation microorganisms, and seriously affect the ethanol fermentation yield and rate of the cellulose hydrolysate. Dilute acid pretreatment is considered to be the most promising method for cellulose pretreatment in cellulosic ethanol production processes, but its wide application is limited due to the production of inhibitors and their toxic effects on microorganisms.

Experiments prove that the inhibitors inhibit various fermentation microorganisms, including escherichia coli, zymomonas mobilis, pichia stipitis, saccharomyces cerevisiae and the like, wherein the saccharomyces cerevisiae is one of the strains with the strongest tolerance capability. In addition, the saccharomyces cerevisiae is the most widely used strain in ethanol fermentation, and the advantages of the saccharomyces cerevisiae include: (1) high conversion rate of glucose, (2) high product specificity, (3) robustness to industrial production environment, and the like.

Screening and construction of strains that are tolerant to inhibitors in cellulosic hydrolysates is an important issue in research on cellulosic ethanol production. Early studies showed that these inhibitors primarily inhibit the growth of microorganisms, while the effect on fermentability was much less than the effect on growth. It is desirable to obtain strains of saccharomyces cerevisiae that are tolerant to multiple inhibitors to achieve in situ detoxification and efficient fermentation in cellulosic ethanol production, since the detoxification step significantly increases the cost of cellulosic ethanol production and reduces the market competitiveness of cellulosic ethanol.

Therefore, it is important to provide a strain which can normally grow in a culture medium containing various inhibitors produced in the production of cellulosic ethanol, has good growth advantages and realizes the efficient production of ethanol.

Disclosure of Invention

In view of the above, the present invention provides a saccharomyces cerevisiae and its use. The strain can normally grow in a culture medium containing various inhibitors generated in the production of cellulose ethanol, can show good growth advantages in cellulose hydrolysate, and realizes efficient ethanol production.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides saccharomyces cerevisiae, which has the preservation number as follows: CGMCC No. 13987.

The invention also provides application of the saccharomyces cerevisiae as a xylose utilization strain.

The invention also provides the application of the saccharomyces cerevisiae as a high-temperature resistant strain; the high temperature is not lower than 30 ℃.

In some embodiments of the invention, the elevated temperature is 35 to 45 ℃, preferably 40 ℃.

The invention also provides application of the saccharomyces cerevisiae as a tolerant strain of an inhibitor in ethanol fermentation.

In some embodiments of the invention, the inhibitor is selected from furfural, phenol or acetic acid.

In some embodiments of the invention, the inhibitor contains 1-1.5 g/L of furfural, 0.3-0.7 g/L of phenol and 5-6 g/L of acetic acid.

In some embodiments of the invention, the inhibitor has a furfural content of 1.3g/L, a phenol content of 0.5g/L, and an acetic acid content of 5.3 g/L.

The invention provides application of saccharomyces cerevisiae as a tolerant strain of an inhibitor in high-temperature and ethanol fermentation, and the saccharomyces cerevisiae can simultaneously tolerate the inhibitor in high-temperature and ethanol fermentation. The high temperature is not lower than 30 ℃, preferably 35-45 ℃, and more preferably 40 ℃. The inhibitor is selected from furfural, phenol or acetic acid. The content of furfural in the inhibitor is 1-1.5 g/L, the content of phenol is 0.3-0.7 g/L, and the content of acetic acid is 5-6 g/L. In some embodiments of the invention, the inhibitor has a furfural content of 1.3g/L, a phenol content of 0.5g/L, and an acetic acid content of 5.3g/L

The invention also provides application of the saccharomyces cerevisiae in producing cellulosic ethanol.

The invention also provides application of the saccharomyces cerevisiae in producing ethanol.

The invention also provides a method for producing ethanol by fermentation, and the saccharomyces cerevisiae is inoculated in fermentation raw materials for fermentation to prepare the ethanol.

In some embodiments of the invention, the seed medium or fermentation medium of Saccharomyces cerevisiae comprises: peptone, 15-25 g/L; 5-15 g/L of yeast powder; 15-25 g/L of xylose; more specifically, the method comprises the following steps: peptone, 20 g/L; 10g/L of yeast powder; xylose, 20 g/L; sterilizing at 121 deg.C for 20min under high temperature and high pressure; xylose is added after being separately killed.

In some embodiments, the seed medium further comprises 30-50 g/L glucose. In some embodiments, the seed medium further comprises 40g/L glucose.

The condition of the saccharomyces cerevisiae expansion culture is as follows: culturing at 25-35 ℃ and 200-250 rpm for 20-30 h; more specifically, culturing at 30 ℃ and 220rpm for 24 h;

the fermentation production conditions of the saccharomyces cerevisiae are as follows: culturing at 25-45 deg.C and 100-200 rpm for 70-80 h, more specifically at 30 deg.C and 150rpm for 72h or at 40 deg.C and 150rpm for 72 h.

The invention provides saccharomyces cerevisiae, which has the preservation number as follows: CGMCC No. 13987. The experimental results show that: when the strain provided by the invention is fermented at 30 ℃ in a fermentation medium containing the inhibitor, the strain begins to enter a stable period after 72 hours, namely OD₆₀₀Is 6.435 and the residual amount of sugar consumption was 1.269 g/l. While the original strain was still in log phase, OD₆₀₀Is 3.94, and the residual amount of xylose is 7.18g/l, the sugar consumption is higher and the growth is faster than that of the original strain. Therefore, the E7-12 strain shows obvious growth advantages under the condition of containing the inhibitor, can adapt to the environment containing the inhibitor compared with the original strain, and shows good xylose utilization advantages.

The strain E7-12 provided by the invention enters a stationary phase after 15h when fermented at 30 ℃ under the condition of mixed sugar without inhibitor, and OD₆₀₀Is 14.025, and glucose remains 4.50337g/L, less than 5g/L at 9 h. While the original strain was still in log phase, OD, at 15h₆₀₀Was 8.43 and glucose was 16.06737g/L remaining at 9 h. Both are nearly identical in xylose utilization. Therefore, the E7-12 strain can show obvious growth advantages and can show good glucose and xylose utilization advantages under the condition of mixed sugar without inhibitors.

The strain E7-12 provided by the invention enters a stationary phase after 15h when fermented at 40 ℃ under the condition of mixed sugar without inhibitor, and OD₆₀₀Is 9.765, 0.04969g/L and less than 1g/L of glucose are remained after 9h, and 1g/L of xylose is remained after 24 h. While the original strain had an OD of 12h₆₀₀Is 3.77, and glucose remains 19.49938g/L after 9h, and after 21h, glucose is not consumed any more, the remaining amount is 8.97926g/L, the xylose content of the original strain is not consumed any more after 18h, the remaining amount is 14.77384 g/L. Therefore, the E7-12 strain can show obvious growth advantages under the high-temperature mixed sugar condition, and can show good glucose and xylose utilization advantages;

the strain E7-12 provided by the invention enters a stationary phase after 48 hours when fermented at 40 ℃ under the condition of mixed sugar containing an inhibitor, and the OD₆₀₀14.025, and the residual amount of xylose after the glucose is consumed for 24 hours is 4.6797g/L and less than 5g/L after the xylose content is 72 hours. While the original strain had an OD of 48h₆₀₀Is 8.43, and glucose is glucose at 24hThe residual glucose content is 6.29134g/L, the xylose content of the original strain is 72h, the xylose content is 12.40712g/L, and the residual xylose content is far higher than E7. Therefore, the E7-12 strain can show obvious growth advantages and can show good glucose and xylose utilization advantages under the high-temperature mixed sugar condition containing the inhibitor.

In conclusion, the strain provided by the invention can adapt to the environment containing inhibitors and high-temperature environment, can normally grow in a culture medium containing the inhibitors generated in the production of various cellulose ethanol, can show good growth advantages in cellulose hydrolysate, and can realize efficient ethanol production.

Biological preservation Instructions

Biomaterial E7-12, taxonomic nomenclature: saccharomyces cerevisiae, was deposited at the general microbiological center of the China Committee for culture Collection of microorganisms 6.4.2017 at the institute of microbiology, China academy of sciences, No. 3, West Lu 1, North Chen, south China, Beijing, with the accession number of CGMCC No. 13987.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the verification that the constructed strain is a diploid strain;

FIG. 2 shows xylose consumption and ethanol production curves of E7-12 strain with the original S strain in YPX mixed inhibitors on shake flasks;

FIG. 3 shows growth curves of E7-12 strain with the original S strain under YPX mixed inhibitor on shake flasks;

FIG. 4 shows the glucose consumption curves of E7-12 strain and original S strain at 30 ℃ in a mixed sugar medium;

FIG. 5 shows the ethanol production curves of E7-12 strain and original S strain at 30 ℃ in a mixed sugar medium;

FIG. 6 shows the growth curves of E7-12 strain and the original S strain in a mixed sugar medium at 30 ℃;

FIG. 7 shows the sugar consumption curves of E7-12 strain and the original S strain in a mixed sugar medium at a high temperature of 40 ℃;

FIG. 8 shows ethanol production curves of E7-12 strain and original S strain in a mixed sugar medium at a high temperature of 40 ℃;

FIG. 9 shows the growth curves of E7-12 strain and the original S strain in a mixed sugar medium at a high temperature of 40 ℃;

FIG. 10 shows the sugar consumption curves of the E7-12 strain and the original S strain in a medium containing a sugar-mixing inhibitor at a high temperature of 40 ℃;

FIG. 11 shows ethanol production curves for E7-12 strain and the original S strain in a medium of a carbohydrate-mixing inhibitor at a high temperature of 40 ℃;

FIG. 12 shows the growth curves of E7-12 strain and the original S strain in a medium containing a mixed sugar inhibitor at a high temperature of 40 ℃.

Detailed Description

The invention discloses saccharomyces cerevisiae and application thereof, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

A strain of Saccharomyces cerevisiae tolerant to multiple inhibitors is named as Saccharomyces cerevisiae SyBE-SC02100032, E7-12 for short.

The new strain E7-12 of the Saccharomyces cerevisiae, which can tolerate various inhibitors and high temperature, is obtained by carrying out spore separation on industrial Saccharomyces cerevisiae diploid (Saccharomyces cerevisiae) to obtain MATa 10#, and carrying out ultraviolet mutagenesis to obtain the strain 12# with improved tolerance of the inhibitors. Then, the strain is crossed with xylose utilization strain E7 to construct diploid xylose utilization strain.

The ultraviolet mutagenesis strategy was: diluting the culture solution (YEPD culture medium: glucose 20g/L, yeast powder 10g/L, peptone 20g/L) of strain No. 10, spreading on the screening culture medium containing inhibitor (YEPD culture medium added with inhibitor), irradiating the spread plate at a distance of 56cm below a 15W ultraviolet lamp for 7min, and placing in a 30 ℃ incubator for 2 days in the dark.

The conditions for strain hybridization were: the selected 12# colonies were inoculated into 5ml YPD liquid medium under conditions of 30 ℃ at 220rpm for 8 hours. Xylose utilization strain E75 ml YPX liquid medium, the culture conditions were 30 ℃,220rpm,8 h. The 12# colony and E7 were then transferred to fresh YPD liquid medium at OD 0.5:0.5, 30 ℃,220rpm,6 h. Then 100ul of the cells were spread on YNBX solid plates (6.7 g/L yeast nitrogen source, 20g/L xylose), colonies with large morphology were picked and verified to be diploid and haploid by yeast colony PCR, and the results are shown in FIG. 1. E7-12 is a diploid strain.

The strain can show good growth advantage in cellulose hydrolysate when normally growing in a culture medium containing various inhibitors generated in the production of cellulose ethanol.

The inhibitor is furfural, phenol and acetic acid.

The content of the inhibitor furfural is 1.3g/L, the content of phenol is 0.5g/L, and the content of acetic acid is 5.3 g/L.

The strain provided by the invention can adapt to the environment containing inhibitors and high-temperature environment, can normally grow in a culture medium containing the inhibitors generated in the production of various cellulose ethanol, can show good growth advantages in cellulose hydrolysate, and realizes efficient ethanol production. Compared with the original strain, the strain provided by the invention has the advantages that the growth advantage and the xylose utilization rate are remarkably improved (P is less than 0.01), and the yield of ethanol is remarkably improved (P is less than 0.01).

The saccharomyces cerevisiae and the raw materials and reagents used in the application thereof provided by the invention are all available in the market.

The invention is further illustrated by the following examples:

example 1 construction of E7-12 Strain

The ultraviolet mutagenesis strategy was: diluting the culture solution (YPD medium: glucose 20g/L, yeast powder 10g/L, peptone 20g/L) of strain No. 10, spreading on screening medium (YPD medium added with inhibitor) containing inhibitor, irradiating the spread plate 56cm away from 15W ultraviolet lamp for 7min, and culturing in 30 deg.C incubator in dark for 2 days.

The conditions for strain hybridization were: the selected 12# colony was inoculated into 5ml YPD liquid medium under conditions of 30 ℃ at 220rpm for 8 hours. Xylose utilization strain E75 ml YPX liquid medium, the culture conditions were 30 ℃,220rpm,8 h. E7-12 and E7 were then transferred to fresh YPD broth at OD 0.5:0.5, 30 ℃ at 220rpm, for 6 h. Then 100ul of the cells were spread on YNBX solid plates (6.7 g/L yeast nitrogen source, 20g/L xylose), colonies with large morphology were picked and verified to be diploid and haploid by yeast colony PCR, and the results are shown in FIG. 1. E7-12 is a diploid strain.

Example 2 comparison of the growth of E7-12 Strain with the original Strain on Shake flasks

1. Test materials: strain E7-12 the laboratory screened and constructed strain; the xylose utilization strain S.cerevisiae CGMCC NO.6634 is constructed at the early stage of the original strain E7 laboratory.

2. The test method comprises the following steps:

seed culture medium:

e7-12 strain: peptone, 20 g/L; 10g/L of yeast powder; xylose, 20 g/L. Sterilizing at 121 deg.C for 20min under high temperature and high pressure. Xylose was separately sterilized and then mixed. Original strain: peptone, 20 g/L; 10g/L of yeast powder; xylose, 20 g/L. Sterilizing at 121 deg.C for 20min under high temperature and high pressure. Xylose was separately sterilized and then mixed.

Fermentation medium:

peptone, 20 g/L; 10g/L of yeast powder; xylose, 20 g/L. Sterilizing at 121 deg.C for 20min under high temperature and high pressure. Xylose was separately sterilized and then mixed. Adding inhibitor before fermentation to make the final concentration reach furfural 0.39g/L, phenol 0.15g/L and acetic acid 1.59 g/L.

Respectively inoculating E7-12 strain and original strain S.cerevisiae CGMCC NO.6634 in 50mL seed culture medium, culturing at 30 deg.C and 220rpm for 24 hr, and determining initial thallus concentration OD₆₀₀The cells were inoculated in a 250mL conical flask containing 100mL of the fermentation medium at 1.0, cultured at 30 ℃ and 150rpm, and the cell growth curve was measured using a type 722 spectrophotometer.

3. And (3) test results:

as shown in FIG. 2 (data shown in tables 1 and 2) and FIG. 3 (data shown in Table 3), the strain E7-12 started to enter stationary phase after 72 hours had passed, and OD was determined₆₀₀Is 6.435 and the residual amount of sugar consumption was 1.269 g/l. While the original strain was still in log phase, OD₆₀₀Is 3.94, and the residual amount of xylose is 7.18g/l, the sugar consumption is higher and the growth is faster than that of the original strain. Therefore, the E7-12 strain shows obvious growth advantages under the condition of containing the inhibitor, can adapt to the environment containing the inhibitor compared with the original strain, and shows good xylose utilization advantages.

TABLE 1

TABLE 2

TABLE 3

Time (h)	E7 (mean value)	E7 (variance)	E7-12 (mean)	E7-12 (variance)
					0	1	0	1	0
12	1.3	0.016831162	1.5	0.008412893
					24	1.96	0.038667054	2.38	0.019185696
36	2.545	0.009934202	3.475	0.141245216
					48	2.925	0.04850987	4.79	0.108942932
60	3.41	0.007488281	6.005	0.067346149
					72	3.945	0.034033243	6.435	0.032194771
84	4.2	0.009014966	6.555	0

4. And (4) conclusion:

in the presence of inhibitors (at the concentrations described above), the original strain showed a weak growth and fermentation capacity, while E7-12 was able to adapt to the inhibitor environment, achieving good growth and xylose utilization advantages in the inhibitor environment.

Example 3 Mixed sugar fermentation growth of E7-12 strain was compared to the original strain on shake flasks

2. The test method comprises the following steps:

seed culture medium:

e7-12 strain: peptone, 20 g/L; 10g/L of yeast powder; glucose, 40 g/L; xylose, 20 g/L. Sterilizing at 121 deg.C for 20min under high temperature and high pressure. Xylose was separately sterilized and then mixed. Original strain: peptone, 20 g/L; 10g/L of yeast powder; glucose, 40g/L, xylose, 20 g/L. Sterilizing at 121 deg.C for 20min under high temperature and high pressure. Xylose was separately sterilized and then mixed.

Fermentation medium:

peptone, 20 g/L; 10g/L of yeast powder; xylose, 20 g/L. Sterilizing at 121 deg.C for 20min under high temperature and high pressure. Xylose was separately sterilized and then mixed.

The E7-12 strain and the original strain S.cerevisiae CGMCC NO.6634 were respectively inoculated into 100mL of seed culture medium, cultured at 30 ℃ and 220rpm for 24h, inoculated into a 250mL conical flask with 100mL of fermentation medium at the initial cell concentration OD600 of 1.0, cultured at 30 ℃ and 150rpm, and the cell growth curve was measured using a type 722 spectrophotometer.

3. And (3) test results:

as shown in FIG. 4 (data in tables 4 and 5), FIG. 5 (data in tables 6 and 7), and FIG. 6 (data in tables 8 and 9), the strain E7-12 started to enter the stationary phase after 15h, and its OD was adjusted to OD₆₀₀Is 14.025, and glucose remains 4.50337g/L, less than 5g/L at 9 h. While the original strain was still in log phase, OD, at 15h₆₀₀Was 8.43 and glucose was 16.06737g/L remaining at 9 h. Both are nearly identical in xylose utilization. Therefore, the E7-12 strain can show obvious growth advantages and can show good glucose and xylose utilization advantages under the condition of mixed sugar without inhibitors.

TABLE 4

E7	Mean value of	Variance (variance)	Mean value of	Variance (variance)
					Time(h)	glucose		xylose
0	40.00036	0.07885	20.00005	0.08383
					3	37.01875	0.04055	19.40058	0.26693
6	29.46476	0.24524	18.5254	0.02064
					9	16.06737	0.20963	17.4517	0.00754
12	0.22162	0.01139	13.58468	0.02608
					15	0	0	10.33839	0.0642
18			7.0758	0.05989
					21			4.52437	0.01049
24			2.39534	0.00995
					27			1.82073	0.06826
30			1.04652	0.01551
					33			0.59829	0.02485
36			0.33385	0.06594
					48			0.04558	0

TABLE 5

E7-12	Mean value of	Variance (variance)	Mean value of	Variance (variance)
					Time(h)	glucose		xylose
0	40.00036	0.07885	20.028	0.08383
					3	36.74827	0.20512	18.5829	0.29558
6	24.98851	0.07073	17.56887	0.2873
					9	4.50337	0.47775	15.1358	0.03214
12	0	0	10.32205	0.01666
					15	0	0	8.50197	0.08507
18			6.85626	0.13938
					21			5.56897	0.12485
24			4.1368	0.10421
					27			3.73997	0.0944
30			2.84232	0.07098
					33			2.25037	0.09746
36			1.74438	0.08013
					48			0.68494	0.0252

TABLE 6

E7	Mean value of	Variance (variance)
			Time(h)	ethanol
0	0	0
			3	2.2827	0.01726
6	5.21084	0.03678
			9	11.06869	0.04655
12	20.11675	0.00707
			15	22.03596	0.02914
18	23.62298	0.01256
			21	25.23214	0.15042
24	25.90255	0.07077
			27	25.90026	0.1033
30	26.15018	0.40677
			33	26.73918	0.39304
36	26.48896	0.14568
			48	26.53717	0.18103

TABLE 7

E7-12	Mean value of	Variance (variance)
			Time(h)	ethanol
0	0	0
			3	2.32451	0.00538
6	7.26813	0.01568
			9	17.28687	0.23149
12	22.00298	0.0722
			15	22.93606	0.04381
18	23.48922	0.15677
			21	24.31802	0.13729
24	24.65172	0.09119
			27	24.66244	5.26461E-4
30	24.98204	0.05058
			33	25.23967	0.06934
36	25.62597	0.0132
			48	26.33749	0.11492

TABLE 8

E7	Mean value of	Variance (variance)
			Time(h)	OD600
0	1	0
			3	1.93	0.05
6	3.115	0.025
			9	5.18	0.02
12	7.83	0.05
			15	8.43	0
18	8.82	0.12
			21	9.27	0.18
24	9.72	0.42
			27	9.66	0.12
36	10.185	0.615

TABLE 9

E7-12	Mean value of	Variance (variance)
			Time(h)	OD600
0	1	0
			3	2.67	0.01
6	5.71	0.17
			9	11	0.2
12	12.53	0.15
			15	14.025	0.195
18	13.965	0.225
			21	14.19	0.15
24	14.415	0.135
			27	14.835	0.525
36	14.475	0.105

4. And (4) conclusion:

in the absence of inhibitors, E7-12 was able to exhibit significant growth advantages and exhibited good glucose and xylose utilization advantages.

Example 4 Mixed sugar fermentation growth of E7-12 strain was compared to the original strain on shake flasks

2. The test method comprises the following steps:

seed culture medium:

e7-12 strain: peptone, 20 g/L; 10g/L of yeast powder; glucose, 40g/L, xylose, 20 g/L. Sterilizing at 121 deg.C for 20min under high temperature and high pressure. Xylose was separately sterilized and then mixed. Original strain: peptone, 20 g/L; 10g/L of yeast powder; glucose, 40g/L, xylose, 20 g/L. Sterilizing at 121 deg.C for 20min under high temperature and high pressure. Xylose was separately sterilized and then mixed.

Fermentation medium:

The E7-12 strain and the original strain S.cerevisiae CGMCC NO.6634 were respectively inoculated into 100mL of seed culture medium, cultured at 30 ℃ and 220rpm for 24h, inoculated into a 250mL conical flask with 100mL of fermentation medium at the initial cell concentration OD600 of 1.0, cultured at 40 ℃ and 150rpm, and the cell growth curve was measured using a type 722 spectrophotometer.

3. And (3) test results:

as shown in FIG. 7 (data shown in tables 10 and 11), FIG. 8 (data shown in tables 12 and 13) and FIG. 9 (data shown in tables 14 and 15), the strain E7-12 started to enter stationary phase after 15h, OD₆₀₀Is 9.765, 0.04969g/L and less than 1g/L of glucose are remained after 9h, and 1g/L of xylose is remained after 24 h. While the original strain had an OD of 12h₆₀₀Is 3.77, and glucose remains 19.49938g/L after 9h, and after 21h, glucose is not consumed any more, the remaining amount is 8.97926g/L, the xylose content of the original strain is not consumed any more after 18h, the remaining amount is 14.77384 g/L. Therefore, the E7-12 strain can show obvious growth advantages under high-temperature mixed sugar conditions, and can show good glucose and xylose utilization advantages.

Watch 10

E7	Mean value of	Variance (variance)	Mean value of	Variance (variance)
					Time(h)	glucose		xylose
0	40.00036	0.07885	20.00005	0.08383
					6	27.33518	0.40368	17.99691	0.03412
9	19.49938	0.38575	16.92407	0.08779
					12	12.94972	0.31574	15.76895	0.02473
15	10.69749	0.18942	15.13612	0.03842
					18	9.45085	0.20277	14.77384	0.06973
21	8.97926	0.01471	14.6636	0.02117
					24	8.91216	0.32611	14.3851	0.12258
27	9.12391	0.12057	14.67693	0.09588
					30	8.94344	0.10905	14.38811	0.1324
33	8.76286	0.28283	14.27746	0.0727
					36	8.87936	0.20911	14.35644	0.0602
48	8.88251	0.23577	14.40335	0.06348

TABLE 11

E7-12	Mean value of	Variance (variance)	Mean value of	Variance (variance)
					Time(h)	glucose		xylose
0	40.00036	0.07885	20.00005	0.08383
					6	17.78115	0.17833	17.11719	0.00648
9	0.04969	1.00044E-4	12.46175	0.00959
					12			6.80614	0.01635
15			4.33112	0.03444
					18			2.37767	0.04546
21			1.24231	0.04567
					24			0.55312	0.05002
27			0.4155	0.0328
					30			0.29173	9.12091E-4
33			0.20683	0.02779
					36			0.0868	0.00718
48			0.23482	0.00691

TABLE 12

E7	Mean value of	Variance (variance)
			Time(h)	Ethanol
0	0	0
			6	6.22055	0.12067
9	9.80778	0.14451
			12	13.24192	0.1632
15	14.21794	0.09503
			18	14.62005	0.05641
21	14.93453	0.02621
			24	14.72966	0.03117
27	14.98515	0.22243
			30	14.7086	0.25214
33	14.53581	0.07498
			36	14.77603	0.16012
48	14.86519	0.04276

Watch 13

E7-12	Mean value of	Variance (variance)
			Time(h)	Ethanol
0	0	0
			6	11.03823	0.03445
9	20.75095	0.02802
			12	23.84387	0.02738
15	25.13343	0.03546
			18	25.86882	0.06487
21	26.51915	0.07585
			24	26.66777	0.34103
27	26.38968	0.23315
			30	26.61207	0.31106
33	25.90113	0.25488
			36	26.07407	0.02862
48	25.91222	0.06742

TABLE 14

E7	Mean value of	Variance (variance)
			Time(h)	OD600
0	1	0
			6	2.685	0.075
9	3.34	0.1
			12	3.77	0.01
15	3.615	0.105
			18	3.69	0.09
21	3.69	0.12
			24	3.6	0.09
27	3.405	0.015
			36	3.57	0.15
48	3.345	0.015

Watch 15

E7-12	Mean value of	Variance (variance)
			Time(h)	OD600
0	1	0
			6	5.38	0.08
9	8.85	0.07
			12	9.14	0.32
15	9.765	0.255
			18	9.75	0.09
21	9.42	0.09
			24	9.435	0.105
27	9.045	0.015
			36	9.06	0.15
48	9.525	0.105

4. And (4) conclusion:

under high temperature conditions, E7-12 can show significant growth advantages and can exhibit good glucose and xylose utilization advantages.

Example 5 Mixed sugar fermentation growth of E7-12 strain was compared to the original strain on shake flasks

2. The test method comprises the following steps:

seed culture medium:

Fermentation medium:

3. And (3) test results:

as shown in FIG. 10 (data in tables 16 and 17), FIG. 11 (data in tables 18 and 19) and FIG. 12 (data in tables 20 and 21), the strain E7-12 started to enter the stationary phase after 48h, OD₆₀₀14.025, and the residual amount of xylose after the glucose is consumed for 24 hours is 4.6797g/L and less than 5g/L after the xylose content is 72 hours. While the original strain had an OD of 48h₆₀₀Is 8.43, the residual glucose amount in 24 hours of glucose is 6.29134g/L, the xylose content in the original strain is 72 hours, the xylose content is 12.40712g/L, and the residual xylose amount is far higher than E7. Therefore, the E7-12 strain can show obvious growth advantages under high-temperature mixed sugar conditions, and can show good glucose and xylose utilization advantages.

TABLE 16

E7-12	Mean value of	Variance (variance)	Mean value of	Variance (variance)
					Time(h)	glucose		xylose
0	39.3127	0.23443	19.76149	0.15478
					12	21.24351	0.7627	17.46089	0.35866
24	6.29134	0.20216	14.07728	0.98177
					30	4.60741	0.04194	14.53486	0.03295
36	3.74298	0.07377	14.12665	0.01074
					48	2.51748	0.00733	12.86986	0.82538
60	2.22644	0.18916	13.55135	0.0208
					72	1.805	0.05594	12.40712	0.88572
84	1.70819	0.00895	12.27478	0.71121
					96	1.59903	0.09335	12.5136	0.20088
108	1.56237	0.01773	12.33695	0.20471
					120	1.4582	0.02399	12.51914	0.07774

TABLE 17

E7-12	Mean value of	Variance (variance)	Mean value of	Variance (variance)
					Time(h)	glucose		xylose
0	39.3127	0.23443	19.7099	0.15478
					12	1.39383	0.54884	14.68416	0.26051
24	0	0	8.70594	0.11384
					30			7.32768	0.17064
36			6.55903	0.15099
					48			5.58423	0.15267
60			5.07246	0.14283
					72			4.6797	0.12953
84			4.18835	0.04914
					96			4.15422	0.04248
108			4.00282	0.09983
					120			4.02758	0.09646

Watch 18

E7	Mean value of	Variance (variance)
			Time(h)	Ethanol
0	0	0
			12	10.03623	0.06817
24	16.43685	1.0796
			36	17.01976	0.45375
48	17.65743	0.6408
			72	17.91833	1.74738
84	18.09587	1.14428
			96	18.97861	0.22074
108	19.21427	0.20663
			120	19.79732	0.99832

Watch 19

E7-12	Mean value of	Variance (variance)
			Time(h)	Ethanol
0	0	0
			12	19.95399	0.04012
24	22.49729	0.21913
			36	22.87434	0.25952
48	22.99435	0.17787
			72	23.93371	0.29144
84	22.36588	0.77785
			96	23.6333	0.15761
108	22.90605	0.29312
			120	23.52915	0.00884

Watch 20

E7	Mean value of	Variance (variance)
			Time(h)	OD600
0	1	0
			12	1.93	0.05
24	3.115	0.025
			30	5.18	0.02
36	7.83	0.05
			48	8.43	0
60	8.82	0.12
			72	9.27	0.18
96	9.66	0.12
			120	10.575	0.765

TABLE 21

E7-12	Mean value of	Variance (variance)
			Time(h)	OD600
0	1	0
			12	2.67	0.01
24	5.71	0.17
			30	11	0.2
36	12.53	0.15
			48	14.025	0.195
60	13.965	0.225
			72	14.19	0.15
96	14.835	0.525
			120	14.685	0.255

4. And (4) conclusion:

under the condition of high-temperature inhibitor, E7-12 can show obvious growth advantage and can show good glucose and xylose utilization advantage.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. Saccharomyces cerevisiae (Saccharomyces cerevisiae) with the preservation number: CGMCC No. 13987.

2. Use of the saccharomyces cerevisiae as described in claim 1 as xylose utilization strain.

3. Use of the saccharomyces cerevisiae strain according to claim 1 as a high temperature tolerant strain, said high temperature being between 30 ℃ and 40 ℃.

4. Use according to claim 3, wherein the elevated temperature is 35-40 ℃.

5. Use of saccharomyces cerevisiae according to claim 1 as tolerant strain of inhibitors in ethanol fermentation selected from one or more of furfural at 1-1.5 g/L, phenol at 0.3-0.7 g/L or acetic acid at 5-6 g/L.

6. Use of saccharomyces cerevisiae as a tolerant strain of inhibitors in high temperature and ethanol fermentation selected from one or more of furfural at 1-1.5 g/L, phenol at 0.3-0.7 g/L or acetic acid at 5-6 g/L according to claim 1; the high temperature is 30-40 ℃.

7. Use of the saccharomyces cerevisiae according to claim 1 for the production of ethanol.

8. A method for producing ethanol by fermentation, which is characterized in that the saccharomyces cerevisiae of claim 1 is inoculated in a fermentation raw material for fermentation to prepare the ethanol.