外源添加乙酸对酿酒酵母(Saccharomyces cerevisiae)产2,3-丁二醇影响初探

doi:10.11924/j.issn.1000-6850.casb20190500171

中国农学通报 ›› 2020, Vol. 36 ›› Issue (21): 104-112.doi: 10.11924/j.issn.1000-6850.casb20190500171

所属专题：生物技术

外源添加乙酸对酿酒酵母(Saccharomyces cerevisiae)产2,3-丁二醇影响初探

杨智宇¹^,², 佟天奇¹^,², 刘磊¹^,², 平文祥¹^,², 葛菁萍¹^,²()

¹ 微生物黑龙江省高校重点实验室/黑龙江大学生命科学学院,哈尔滨 150080
² 黑龙江大学农业微生物技术教育部工程研究中心,哈尔滨 150500

收稿日期:2019-05-21 修回日期:2019-06-30 出版日期:2020-07-25 发布日期:2020-07-21
通讯作者: 葛菁萍
作者简介:杨智宇,男,1994年出生,黑龙江齐齐哈尔人,硕士研究生,研究方向：生物学。通信地址：150080 黑龙江省哈尔滨市南岗区学府路74号,Tel：0451-86609016,E-mail：yangzhiyu0606@163.com。
基金资助:
国家自然科学基金“从2,3-丁二醇代谢角度构建工程微生物群体及其生态学机制研究”(31570492);黑龙江省教育厅重点项目“利用肺炎克雷伯氏菌发酵生产2,3-丁二醇及机理探讨”(HDJCCX-2016Z05)

Acetic Acid Addition: Effects on the Production of 2,3-butanediol by Saccharomyces cerevisiae

Yang Zhiyu¹^,², Tong Tianqi¹^,², Liu Lei¹^,², Ping Wenxiang¹^,², Ge Jingping¹^,²()

¹ Key Laboratory of Microbiology of Heilongjiang Province, Life Science College, Heilongjiang University, Harbin 150080
² Agricultural Microorganisms Technology Education Engineering Research Center, Harbin 150500

Received:2019-05-21 Revised:2019-06-30 Online:2020-07-25 Published:2020-07-21
Contact: Ge Jingping

摘要/Abstract

摘要：

为验证乙酸作为信号分子的作用,本研究分别将酿酒酵母(Saccharomyces cerevisiae)W141上清液（对照组）、W141-07 (△aldh6)上清液及1.5 g/L乙酸添加至对数生长期的W141发酵液中,检测2,3-BD产量、乙酰乳酸合成酶(ILV2)及2,3-丁二醇脱氢酶(BDH1)酶活。结果表明：当添加1.5 g/L乙酸时,2,3-BD产量、ILV2和BDH1酶活性均达到最高,分别为3.01±0.04 g/L、1.41±0.03 U/mg和0.12±0.002 U/mg,且较其余两组相比差异极显著(P<0.01)。同时,测定3组条件下ilv2(24 h)和bdh1(60 h)基因的表达情况时发现,添加W141-07上清液后,ilv2和bdh1基因的表达量分别下调了31.6%和25.0%;而添加1.5 g/L乙酸后,ilv2和bdh1的表达量均发生上调,分别是对照组的4.38及1.24倍。表明乙酸可作为信号分子驱动相关基因的表达,进而提高2,3-BD产量。

关键词: 2,3-丁二醇, 酿酒酵母, 乙酸, 荧光定量PCR, 2,3-丁二醇脱氢酶, α-乙酰乳酸合成酶

Abstract:

To verify the role of acetic acid as a signal molecule, the supernatant of Saccharomyces cerevisiae W141 (the control group) and W141-07 (△aldh6), as well as 1.5 g/L acetic acid were added into S. cerevisiae W141 fermentation broth at the logarithmic phase. The content of 2,3- butanediol(2,3-BD), the activity of acetolactate synthase (ILV2) and 2,3-BD dehydrogenase (BDH1) were detected. The results showed that under the condition of 1.5 g/L acetic acid, the content of 2,3-BD, the ILV2 and BDH1 enzyme activities reached the highest value, which were 3.01±0.04 g/L, 1.41±0.03 U/mg and 0.12±0.002 U/mg, respectively. The acetic acid addition group was significantly higher than those of supernatant addition groups (P<0.01). At the same time, the expressions of ilv2 (24 h) and bdh1 (60 h) genes under the three conditions were determined. After adding W141-07 supernatant, the expression levels of ilv2 and bdh1 genes were down-regulated by 31.6% and 25.0%, respectively. After adding acetic acid with a final concentration 1.5 g/L, the expression levels of ilv2 and bdh1 were up-regulated, which were 4.38 and 1.24 times that of the control, respectively. These results indicate that acetic acid can act as a signal molecule to drive the expression of related genes, thus to improve 2,3-BD production.

Key words: 2,3-butanediol, Saccharomyces cerevisiae, acetic acid, fluorescent quantitative polymerase chain reaction, 2,3-butanediol dehydrogenase, α-acetolactatesynthase

中图分类号:

Q935

杨智宇, 佟天奇, 刘磊, 平文祥, 葛菁萍. 外源添加乙酸对酿酒酵母(Saccharomyces cerevisiae)产2,3-丁二醇影响初探[J]. 中国农学通报, 2020, 36(21): 104-112.

Yang Zhiyu, Tong Tianqi, Liu Lei, Ping Wenxiang, Ge Jingping. Acetic Acid Addition: Effects on the Production of 2,3-butanediol by Saccharomyces cerevisiae[J]. Chinese Agricultural Science Bulletin, 2020, 36(21): 104-112.

图/表 12

引物名称	引物序列(5’-3’)
18S-up	TCACC AGGTC CAGAC ACAAT
18S-down	AGCAG ACAAA TCACT CCACC
qilv2-up	CGTCCAATTCCTCTTGCTTC
qilv2-down	ATGGCAATCCCTGTTCTACG
qbdh1-up	TTTGCTGAACAAGTCGTAGTC
qbdh1-down	CCCAGTTTCTTGGCCATTTC

表1. 引物序列

图1. S. cerevisiae W141菌株对乙酸的耐受性

图2. S. cerevisiae W141的OD600值随发酵时间变化的关系曲线

图3. S. cerevisiae W141的2,3-BD产量随发酵时间变化的关系曲线

图4. 添加上清液及乙酸后对2,3-BD产量及OD600值的影响

图5. ILV2酶活力测定结果

图6. BDH1酶活力测定结果

图7. 菌株ilv2、bdh1和18S-rRNA基因qRT-PCR扩增曲线及熔解曲线 A1：添加S. cerevisiae W141上清液（对照）/添加W141-07上清液/添加1.5 g/L乙酸的ilv2基因扩增曲线;B1：添加S. cerevisiae W141上清液（对照）/添加W141-07上清液/添加1.5 g/L乙酸的ilv2基因熔解曲线;A2：添加S. cerevisiae W141上清液（对照）/添加W141-07上清液/添加1.5 g/L乙酸的bdh1基因扩增曲线;B2：添加S. cerevisiae W141上清液（对照）/添加W141-07上清液/添加1.5 g/L乙酸的bdh1基因熔解曲线

图8. 添加S. cerevisiae W141上清液（对照）/添加W141-07上清液/添加1.5 g/L乙酸的ilv2基因的相对表达量

图9. 添加S. cerevisiae W141上清液（对照）/添加W141-07上清液/添加1.5 g/L乙酸的bdh1基因的相对表达量

时间	组别	CT值		△CT	△△CT	2^-△△^CT
时间	组别	CT-ilv2	CT-18S	△CT	△△CT	2^-△△^CT
24 h	添加S. cerevisiae W141上清液（对照）	23.99±0.32	20.13±0.11	3.77	0	1
	添加S. cerevisiae W141-07上清液	23.76±0.22	19.59±0.16	4.17	0.40	0.76
	添加1.5 g/L乙酸	23.01±0.16	18.97±0.45	4.04	-2.13	4.38

表2. 添加S. cerevisiae W141上清液（对照）/添加W141-07上清液/添加1.5 g/L乙酸的ilv2基因的相对表达量

时间	组别	CT值		△CT	△△CT	2^-△△^CT
时间	组别	CT- bdh1	CT-18S	△CT	△△CT	2^-△△^CT
60 h	添加S. cerevisiae W141上清液（对照）	24.57±0.21	19.03±0.31	5.54	0	1
	添加S. cerevisiae W141-07上清液	24.94±0.32	19.07±0.16	5.87	0.33	0.80
	添加1.5 g/L乙酸	23.82±0.25	19.33±0.41	4.49	-0.31	1.24

表3. 添加S. cerevisiae W141上清液（对照）/添加W141-07上清液/添加1.5 g/L乙酸的bdh1基因的相对表达量

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外源添加乙酸对酿酒酵母(Saccharomyces cerevisiae)产2,3-丁二醇影响初探

Acetic Acid Addition: Effects on the Production of 2,3-butanediol by Saccharomyces cerevisiae

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