Parreiras Lucas S, Breuer Rebecca J, Avanasi Narasimhan Ragothaman, Higbee Alan J, La Reau Alex, Tremaine Mary, Qin Li, Willis Laura B, Bice Benjamin D, Bonfert Brandi L, Pinhancos Rebeca C, Balloon Allison J, Uppugundla Nirmal, Liu Tongjun, Li Chenlin, Tanjore Deepti, Ong Irene M, Li Haibo, Pohlmann Edward L, Serate Jose, Withers Sydnor T, Simmons Blake A, Hodge David B, Westphall Michael S, Coon Joshua J, Dale Bruce E, Balan Venkatesh, Keating David H, Zhang Yaoping, Landick Robert, Gasch Audrey P, Sato Trey K
DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America; Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
PLoS One. 2014 Sep 15;9(9):e107499. doi: 10.1371/journal.pone.0107499. eCollection 2014.
The inability of the yeast Saccharomyces cerevisiae to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of S. cerevisiae to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting S. cerevisiae strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent S. cerevisiae strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in GRE3, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust S. cerevisiae strain with the ability to ferment xylose anaerobically from ACSH.
酿酒酵母在厌氧条件下无法有效发酵木糖，这是木质纤维素生物燃料经济生产的主要障碍。尽管通过基因工程方法已使酿酒酵母能够在特定的实验室培养基中将木糖高效转化为乙醇，但在无氧条件下，很少有菌株能够发酵木质纤维素水解产物中的木糖。据信，这种有限的木糖转化是由生物质预处理和水解过程中产生的小分子导致的，这些小分子会引起细胞应激并损害新陈代谢。在此，我们描述了一种能够发酵木糖的酿酒酵母菌株的开发过程，该菌株对一系列预处理和水解的木质纤维素具有耐受性，包括氨纤维膨胀（AFEX）预处理的玉米秸秆水解产物（ACSH）。我们通过基因工程构建了一种带有细菌木糖异构酶的抗水解产物酵母菌株，然后应用了需氧和厌氧两个独立阶段的定向进化。新出现的酿酒酵母菌株在严格的厌氧条件下能迅速将实验室培养基和ACSH中的木糖转化为乙醇。代谢组学、遗传学和生化分析表明，在厌氧进化过程中获得的GRE3基因中的一个错义突变，通过减少木糖异构酶抑制剂木糖醇的细胞内产生，有助于提高木糖转化率。这些结果验证了我们的组合方法，该方法利用表型菌株选择、合理工程设计和定向进化来生成一种强大的酿酒酵母菌株，该菌株能够在无氧条件下从ACSH中发酵木糖。
