过表达tRNA基因tL(CAA)K提高酿酒酵母乙酸耐受性

Overexpression of a leucine transfer RNA gene tL(CAA)K improves the acetic acid tolerance of Saccharomyces cerevisiae

摘要:

乙酸是木质纤维素类生物质水解液中的常见毒性抑制物，选育乙酸耐受性好的酿酒酵母菌株，有利于高效利用木质纤维素类生物质，发酵生产生物燃料和生物基化学品。目前对酿酒酵母抗逆性的研究多集中在转录水平，但对转运RNA (Transfer RNA，tRNA) 在耐受性中的作用研究较少。在对酿酒酵母抗逆性研究过程中发现，一些转运RNA基因在耐受性好的酿酒酵母菌株中转录明显上调。本文深入分析了精氨酸tRNA基因tR(ACG)D和亮氨酸tRNA基因tL(CAA)K过表达对酿酒酵母耐受木质纤维素水解液的影响。结果表明，在4.2 g/L乙酸胁迫条件下进行乙醇发酵时，过表达tL(CAA)K的菌株生长和发酵性能均优于对照酵母菌株，乙醇生产强度比对照菌株提高了29.41%，但过表达tR(ACG)D基因的菌株生长和代谢能力较对照菌株明显降低，体现了不同tRNA的不同调控作用。进一步分析发现，过表达tL(CAA)K的重组酵母菌株乙酸耐受性调控相关基因HAA1、MSN2和MSN4等胁迫耐受性相关转录因子编码基因的转录水平上调。本文的研究为选育高效利用木质纤维素资源进行生物炼制的酵母菌株提供了新的改造策略，也为进一步揭示酿酒酵母tRNA基因表达调控对抗逆性的影响提供了基础。

Abstract:

Acetic acid is a common inhibitor present in lignocellulosic hydrolysate. Development of acetic acid tolerant strains may improve the production of biofuels and bio-based chemicals using lignocellulosic biomass as raw materials. Current studies on stress tolerance of yeast Saccharomyces cerevisiae have mainly focused on transcription control, but the role of transfer RNA (tRNA) was rarely investigated. We found that some tRNA genes showed elevated transcription levels in a stress tolerant yeast strain. In this study, we further investigated the effects of overexpressing an arginine transfer RNA gene tR(ACG)D and a leucine transfer RNA gene tL(CAA)K on cell growth and ethanol production of S. cerevisiae BY4741 under acetic acid stress. The tL(CAA)K overexpression strain showed a better growth and a 29.41% higher ethanol productivity than that of the control strain. However, overexpression of tR(ACG)D showed negative influence on cell growth and ethanol production. Further studies revealed that the transcriptional levels of HAA1, MSN2, and MSN4, which encode transcription regulators related to stress tolerance, were up-regulated in tL(CAA)K overexpressed strain. This study provides an alternative strategy to develop robust yeast strains for cellulosic biorefinery, and also provides a basis for investigating how yeast stress tolerance is regulated by tRNA genes.