定向驯化高抗糠醛和对羟基苯甲酸的酵母菌株

Directed domestication of yeast strains with high tolerance to furfural and p-hydroxybenzoic acid

摘要:

在木质纤维素原料预处理过程中不可避免地形成抑制剂，包括糖降解产物(如5-羟甲基糠醛、糠醛)以及木质素降解的酚类化合物(如4-羟基苯甲酸、香兰素)等，这些化合物会降低发酵效率。【目的】提高酵母对纤维素水解液中的抑制物耐受性对工业生物质乙醇高效生产至关重要。【方法】采用浓度较高的糠醛和对羟基苯甲酸对模式菌株W303-1A进行梯度驯化，对比驯化菌株和出发菌株在不同抑制物浓度下的生长曲线及发酵乙醇性能；对驯化后菌株和出发菌株进行高通量基因组重测序，分析其糖代谢途径和耐药性相关的变异点基因，对与耐抑制物有关的变异点进行分析挖掘。【结果】在含有2.0 g/L糠醛的培养基中，F-2菌的乙醇产量为19.40 g/L，比原始菌株高2倍。在含有1.6 g/L糠醛和对羟基苯甲酸的培养基中，B-2菌的最高乙醇产量为20.22 g/L，是原始菌株的7.6倍。通过对出发菌株和驯化后菌株进行高通量基因组重测序发现，糖代谢途径中编码乙醇脱氢酶、果糖-1,6-二磷酸醛缩酶和丙酮酸脱氢酶的基因发生部分突变，而YAP1(参与氧化应激反应和氧化还原稳态的转录激活剂)、PDR5(耐多种化学物质的多效ABC外运载体)和RPN4(锌指蛋白)基因的部分突变对酿酒酵母的耐抑制物具有重要作用。【结论】研究结果为进一步优化和构建模式菌株提供更多的操作靶点。

Abstract:

Inhibitors including sugar degradation products (e.g., 5-hydroxymethylfurfural and furfural) and phenols (e.g., 4-hydroxybenzoic acid and vanillin) from lignin degradation are inevitably formed in the pretreatment process of lignocellulose raw materials, exerting a negative impact on the fermentation efficiency. [Objective] To improve the tolerance of yeast to inhibitors in cellulose hydrolysates and ensure the efficient production of industrial biomass ethanol. [Methods] The model strain W303-1A was domesticated with the inhibitor furfural and p-hydroxybenzoic acid alone or in combination. The growth curves and ethanol fermentation performance of the domesticated strain and the original strain were compared under different inhibitor concentrations. We then conducted high-throughput genome resequencing of both the domesticated and original strains to identify the mutations in genes related to the glucose metabolism and drug resistance, thereby analyzing the variation points related to inhibitor tolerance. [Results] In the medium containing 2.0 g/L furfural, the ethanol yield of F-2 was 19.40 g/L, which was 2 times higher than that of the original strain. In the medium containing 1.6 g/L furfural and p-hydroxybenzoic acid, the highest ethanol yield of B-2 was 20.22 g/L, 7.6 times that of the original strain. Then, high-throughput genome resequencing of the original and domesticated strains revealed several mutations in the genes encoding ethanol dehydrogenase, fructose-1,6-diphosphate aldolase, and pyruvate dehydrogenase in the glucose metabolism pathway. The mutations of YAP1 (transcriptional activator involved in oxidative stress response and REDOX homeostasis), PDR5 (pleiotropic ABC transporter tolerant to multiple chemicals), and RPN4 (zinc finger protein) genes played an important role in the inhibitor tolerance of Saccharomyces cerevisiae. [Conclusion] The findings provide more targets for further optimization and construction of model strains.