Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA; Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA.
Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA; Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA; Department of Bioenergy Science and Technology, Chonnam National University, Gwangju, 500-757, Korea.
J Biotechnol. 2018 Jun 10;275:53-59. doi: 10.1016/j.jbiotec.2018.04.008. Epub 2018 Apr 13.
To efficiently ferment intermediate cellodextrins released during cellulose hydrolysis, Saccharomyces cerevisiae has been engineered by introduction of a heterologous cellodextrin utilizing pathway consisting of a cellodextrin transporter and either an intracellular β-glucosidase or a cellobiose phosphorylase. Among two types of cellodextrin transporters, the passive facilitator CDT-2 has not enabled better cellobiose fermentation than the active transporter CDT-1, which suggests that the CDT-2 might be engineered to provide energetic benefits over the active transporter in cellobiose fermentation. We attempted to improve cellobiose transporting activity of CDT-2 through laboratory evolution. Nine rounds of a serial subculture of S. cerevisiae expressing CDT-2 and cellobiose phosphorylase on cellobiose led to the isolation of an evolved strain capable of fermenting cellobiose to ethanol 10-fold faster than the original strain. After sequence analysis of the isolated CDT-2, a single point mutation on CDT-2 (N306I) was revealed to be responsible for enhanced cellobiose fermentation. Also, the engineered strain expressing the mutant CDT-2 with cellobiose phosphorylase showed a higher ethanol yield than the engineered strain expressing CDT-1 and intracellular β-glucosidase under anaerobic conditions, suggesting that CDT-2 coupled with cellobiose phosphorylase may be better choices for efficient production of cellulosic ethanol with the engineered yeast.
为了有效地发酵纤维素水解过程中释放的中间纤维二糖，通过引入一个由纤维二糖转运蛋白和胞内β-葡萄糖苷酶或纤维二糖磷酸酶组成的异源纤维二糖利用途径，对酿酒酵母进行了工程改造。在两种类型的纤维二糖转运蛋白中，被动促进剂 CDT-2 并不能比主动转运蛋白 CDT-1 更有效地发酵纤维二糖，这表明 CDT-2 可能经过工程改造，在纤维二糖发酵中提供比主动转运蛋白更有利的能量效益。我们试图通过实验室进化来提高 CDT-2 对纤维二糖的转运活性。在纤维二糖上表达 CDT-2 和纤维二糖磷酸酶的酿酒酵母连续传代培养九轮，导致分离出的一个进化菌株能够将纤维二糖发酵为乙醇的速度比原始菌株快 10 倍。对分离出的 CDT-2 进行序列分析后，发现 CDT-2 上的单个点突变（N306I）是导致增强纤维二糖发酵的原因。此外，在厌氧条件下，表达突变型 CDT-2 和纤维二糖磷酸酶的工程菌株比表达 CDT-1 和胞内β-葡萄糖苷酶的工程菌株表现出更高的乙醇产量，这表明 CDT-2 与纤维二糖磷酸酶的偶联可能是利用工程酵母高效生产纤维素乙醇的更好选择。
