增强酿酒酵母对木质纤维素水解液抑制剂耐受性的研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2024-0847

摘要/Abstract

摘要：

利用酿酒酵母进行木质纤维素类生物质发酵，从而实现经济和社会可持续发展，具有良好的应用前景，但该类生物质水解液中的羧酸、呋喃醛和多酚对酿酒酵母产生了较强抑制作用，这些抑制剂对细胞膜、线粒体和核糖体等细胞器造成了严重的损伤，影响了酵母对水解液中可发酵糖的利用。本文系统综述了这三类抑制剂对酿酒酵母产生胁迫的信号通路与代谢途径，例如，弱酸会诱导细胞内酸化，呋喃醛和酚类化合物会使胞内氧化还原酶活性下降并抑制翻译进程。基于上述胁迫机理，本文深入探讨了诱变育种、实验室适应性进化、细胞自絮凝和共培养等正向遗传学方法。这些方法借助表型突变结合多组学分析可以挖掘关键基因，从而构建高效菌株。同时，阐述了反向遗传学方法，即利用已知功能基因进行基因组转化，以及基于CRISPR-Cas系统开展菌株定向培育。其中，酿酒酵母过表达目标基因、运用CRISPR-Cas9进行基因敲除以及利用CRISPRa/CRISPRi进行基因调控等方法，有效调节了关键基因的表达，进而提升了酿酒酵母的耐受性能。在此基础上，高通量基因文库与多途径基因调控相结合的综合策略可以大规模地用于关键基因筛选和多维度的基因调控。未来，可利用基因表达和代谢通量联合分析等策略实时监控与耐受性相关的关键酶的活性以及关键代谢通路的碳通量变化，从而更好地应对多重抑制剂对酿酒酵母产生的协同作用，为酿酒酵母高效利用木质纤维素发酵提供参考与建议。

关键词: 酿酒酵母, 木质纤维素水解液, 抑制剂, 耐受性, 响应机制, 遗传改造

Abstract:

Using Saccharomyces cerevisiae to ferment lignocellulosic biomass, thus realizing sustainable economic and social development, and has a good application prospect. However, the carboxylic acid, furfural and polyphenols from it have a strong inhibitory effect on S. cerevisiae. These inhibitors cause severe damages to organelles such as cell membranes, mitochondria and ribosomes, which affects the utilization of fermentable sugars in the hydrolysate by S. cerevisiae. This paper systematically reviews the signal pathways and metabolic pathways by which these three types of inhibitors exert stress on S. cerevisiae. For example, weak acids can induce intracellular acidification, and furfural and phenolic compounds can reduce the activity of intracellular redox enzymes and inhibit the translation process. Based on the above stress mechanisms, this paper deeply explores positive genetic methods, such as mutagenesis breeding, laboratory adaptive evolution, cell self-flocculation, and co-culture. These methods are used to mine key genes by phenotypic mutation combined with multi-omics analysis, so as to construct efficient strains. Meanwhile, reverse genetic methods are also elaborated, that is, genomic transformation using known functional genes and strain-directed breeding based on the CRISPR-Cas system. Among them, methods such as overexpressing the target gene in S. cerevisiae, gene knockout using CRISPR-Cas9, and gene regulation using CRISPRa/CRISPRi effectively regulate the expressions of key genes, thereby enhancing the tolerance performance of S. cerevisiae. On this basis, a comprehensive strategy combining high-throughput gene library with multi-pathway gene regulation can be used for key gene screening and multi-dimensional gene regulation on a large scale. In the future, strategies such as the combined analysis of gene expression and metabolic flux can be used to monitor the activities of key enzymes and the carbon flux changes in key metabolic pathways related to tolerance in real time, so as to better cope with the synergistic effects of multiple inhibitors on S. cerevisiae, and provide references and suggestions for S. cerevisiae to efficiently utilize lignocellulose for fermentation.

Key words: Saccharomyces cerevisiae, lignocellulosic hydrolysate, inhibitors, tolerance, response mechanism, genetic modification

贾承霖, 郭晓鹏, 陆栋, 张苗苗. 增强酿酒酵母对木质纤维素水解液抑制剂耐受性的研究进展[J]. 生物技术通报, 2025, 41(4): 61-75.

JIA Cheng-lin, GUO Xiao-peng, LU Dong, ZHANG Miao-miao. Advances in Improving the Tolerance of Saccharomyces cerevisiae to Lignocellulose Hydrolysate Inhibitors[J]. Biotechnology Bulletin, 2025, 41(4): 61-75.

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关键基因 Key gene	编码产物 Encoded product	调控方法 Regulation method	抑制剂 Inhibitor	调控结果 Regulatory results	参考文献 References
CAR1	精氨酸酶	过表达	乙酸	+	［8］
PDR18	ATP结合盒转运蛋白	过表达	乙酸	+	［17］
ERG6	甾醇C-24甲基转移酶	敲除	有机酸	-	［18］
HAA1	转录因子Haa1p	引入点突变	有机酸	+	［19-20］
PPR1	锌指转录因子	过表达	乙酸	+	［22］
ACS2	乙酰辅酶A合成酶	过表达	乙酸	+	［23］
FDH1	甲酸脱氢酶	过表达	甲酸	+	［24］
SET5	组蛋白H4甲基转移酶	过表达	甲酸	+	［25］
GRE2	NADPH依赖性醛还原酶	过表达	糠醛	+	［32-34］
IDH1	异柠檬酸脱氢酶	过表达	糠醛	+	［33-34］
GSH1等	抗氧化功能的蛋白质	过表达	呋喃醛	+	［35-36］
RDS1	醛还原酶	过表达	呋喃醛	+	［35-36］
AAD3	芳醇脱氢酶	过表达	呋喃醛	+	［37］
SIW14	肌醇磷酸酶	敲除	HMF	-	［38］
GRE2	NADPH依赖性醛还原酶	过表达	香草醛	+	［46］
YRR1	锌指转录因子	敲除	香草醛	+	［47］
TMA17	分子伴侣Tma17	过表达	酚类化合物	+	［49］
ADE1	参与IMP和AMP合成的氧化还原酶	过表达	抑制剂混合物	+	［51-52］
MCR1	线粒体还原酶	过表达	云杉水解液	+	［53］
ZWF1	NADPH依赖性葡萄糖-6-磷酸脱氢酶	过表达	小麦秸秆水解液	+	［56］
THI2	硫胺素生物合成基因转录激活因子	敲除	抑制剂混合物	-	［58］
VMS1	肽基-tRNA水解酶	敲除	抑制剂混合物	-	［59］
YOS9	内质网质控凝集素	敲除	抑制剂混合物	-	［59］
AST2	醛还原酶	全基因组转化	HMF	+	［80］
AADH	乙酰化乙醛脱氢酶	异源转化	乙酸	+	［81］
CBH1等	纤维素酶	基因整合	乙酸	+	［86］
MET5	硫氨酸还原酶	敲除	乙酸和乳酸	+	［87］
SIZ1	泛素修饰蛋白连接酶	敲除	乙酸和乳酸	+	［87］
SPT15	通用转录因子	单碱基替换	乙醇	+	［89］
SSK2	促分裂原活化蛋白激酶	CRISPRi	糠醛	+	［92］
RPT5	19S碱基组装亚基	CRISPRi	乙酸	+	［93-94］
PRN9	26S蛋白酶体调节亚基	CRISPRi	乙酸	+	［93-94］
SIZ1	泛素修饰蛋白连接酶	CRISPRi	糠醛	+	［96］
NAT1	N-末端乙酰转移酶亚基	CRISPRa	糠醛	+	［96］
PDR1	负调控耐药转录因子	CRISPRi	糠醛	+	［96］
YAP1	亮氨酸拉链转录因子	过表达	云杉水解液	+	［97］
HAA1	转录因子Haa1p	过表达	云杉水解液	+	［97］
GRE3	醛糖还原酶	敲除	玉米秸秆水解液	+	［100］
PHO13	碱性磷酸酶	敲除	玉米秸秆水解液	+	［100］
TAL1	参与PPP途径	过表达	玉米秸秆水解液	+	［100］
SUT1	木糖转运蛋白	基因整合	蓝桉木质素磺酸液	+	［101］
XYL3	D-木酮糖激酶	基因整合	蓝桉木质素磺酸液	+	［101］
PsTAL1	转醛醇酶	基因整合	蓝桉木质素磺酸液	+	［101］

关键基因 Key gene	编码产物 Encoded product	调控方法 Regulation method	抑制剂 Inhibitor	调控结果 Regulatory results	参考文献 References
CAR1	精氨酸酶	过表达	乙酸	+	［8］
PDR18	ATP结合盒转运蛋白	过表达	乙酸	+	［17］
ERG6	甾醇C-24甲基转移酶	敲除	有机酸	-	［18］
HAA1	转录因子Haa1p	引入点突变	有机酸	+	［19-20］
PPR1	锌指转录因子	过表达	乙酸	+	［22］
ACS2	乙酰辅酶A合成酶	过表达	乙酸	+	［23］
FDH1	甲酸脱氢酶	过表达	甲酸	+	［24］
SET5	组蛋白H4甲基转移酶	过表达	甲酸	+	［25］
GRE2	NADPH依赖性醛还原酶	过表达	糠醛	+	［32-34］
IDH1	异柠檬酸脱氢酶	过表达	糠醛	+	［33-34］
GSH1等	抗氧化功能的蛋白质	过表达	呋喃醛	+	［35-36］
RDS1	醛还原酶	过表达	呋喃醛	+	［35-36］
AAD3	芳醇脱氢酶	过表达	呋喃醛	+	［37］
SIW14	肌醇磷酸酶	敲除	HMF	-	［38］
GRE2	NADPH依赖性醛还原酶	过表达	香草醛	+	［46］
YRR1	锌指转录因子	敲除	香草醛	+	［47］
TMA17	分子伴侣Tma17	过表达	酚类化合物	+	［49］
ADE1	参与IMP和AMP合成的氧化还原酶	过表达	抑制剂混合物	+	［51-52］
MCR1	线粒体还原酶	过表达	云杉水解液	+	［53］
ZWF1	NADPH依赖性葡萄糖-6-磷酸脱氢酶	过表达	小麦秸秆水解液	+	［56］
THI2	硫胺素生物合成基因转录激活因子	敲除	抑制剂混合物	-	［58］
VMS1	肽基-tRNA水解酶	敲除	抑制剂混合物	-	［59］
YOS9	内质网质控凝集素	敲除	抑制剂混合物	-	［59］
AST2	醛还原酶	全基因组转化	HMF	+	［80］
AADH	乙酰化乙醛脱氢酶	异源转化	乙酸	+	［81］
CBH1等	纤维素酶	基因整合	乙酸	+	［86］
MET5	硫氨酸还原酶	敲除	乙酸和乳酸	+	［87］
SIZ1	泛素修饰蛋白连接酶	敲除	乙酸和乳酸	+	［87］
SPT15	通用转录因子	单碱基替换	乙醇	+	［89］
SSK2	促分裂原活化蛋白激酶	CRISPRi	糠醛	+	［92］
RPT5	19S碱基组装亚基	CRISPRi	乙酸	+	［93-94］
PRN9	26S蛋白酶体调节亚基	CRISPRi	乙酸	+	［93-94］
SIZ1	泛素修饰蛋白连接酶	CRISPRi	糠醛	+	［96］
NAT1	N-末端乙酰转移酶亚基	CRISPRa	糠醛	+	［96］
PDR1	负调控耐药转录因子	CRISPRi	糠醛	+	［96］
YAP1	亮氨酸拉链转录因子	过表达	云杉水解液	+	［97］
HAA1	转录因子Haa1p	过表达	云杉水解液	+	［97］
GRE3	醛糖还原酶	敲除	玉米秸秆水解液	+	［100］
PHO13	碱性磷酸酶	敲除	玉米秸秆水解液	+	［100］
TAL1	参与PPP途径	过表达	玉米秸秆水解液	+	［100］
SUT1	木糖转运蛋白	基因整合	蓝桉木质素磺酸液	+	［101］
XYL3	D-木酮糖激酶	基因整合	蓝桉木质素磺酸液	+	［101］
PsTAL1	转醛醇酶	基因整合	蓝桉木质素磺酸液	+	［101］

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