耐热基因线路随机组装构建高版本耐热酿酒酵母

Construction of high version thermo-tolerance Saccharomyces cerevisiae by random assembly of thermo-tolerance genetic circuits

针对现有绿色生物制造体系因控制常温发酵需要消耗大量冷却水所带来的能耗问题,本文以工程化思维,借助合成生物学技术进行耐热基因线路随机组装,重编程酿酒酵母细胞工厂提高其耐热能力.本文选择来自腾冲嗜热菌(Thermoanaerobacter tengcongensis)、嗜热栖热菌(Thermus thermophilus)、枯草芽孢杆菌(Bacillus subtilis)等来源的32个耐热基因作为核心功能元件,以酿酒酵母(Saccharomyces cerevisiae)不同强度的组成型启动子(14个)和终止子(10个)作为调控元件,采用Golden Gate组装技术将耐热基因元件和调控元件进行随机组装,基于全自动高通量筛选平台和高通量细胞筛选技术,实现耐热酵母的高通量筛选.从96孔深孔板-1000 mL三角瓶的二级筛选体系,以及合成培养基-真实物料液化醪的二级发酵培养基进行高版本耐热酵母筛选,最终筛选得到工程菌4741-GRX5和4741-ttha0122,42℃培养72 h残留葡萄糖含量分别为对照BY4741的77.4%和74.5%,乙醇产量分别比BY4741提高5.8%和2.5%,建立了全新的耐热菌株筛选平台.

Existing green biomanufacturing systems require controlled fermentation at room temperature,consuming large amount of cooling water and energy.To solve this problem,reprogramming the Saccharomyces cerevisiae cell factory to improve its thermo-tolerance by randomly assembling the thermo-tolerance gene circuits with the aid of engineering and synthetic biology approaches is developed.In this study,32 thermo-tolerance genes from Thermoanaerobacter tengcongensis MB4,Thermus thermophiles HB8,and Bacillus subtilis were selected as the core functional elements.The constitutive promoters(14)and terminators(10)with different intensities of S.cerevisiae were employed as regulatory elements.The thermo-tolerance genes and regulatory elements were randomly assembled by Golden Gate assembly.High-throughput screening of thermo-tolerance yeast was realized based on an automated highthroughput screening platform and high-throughput cell screening.The high version heat-resistant yeast was screened from the secondary screening system of 96-hole deep hole plate-1000 mL triangular flask and the secondary fermentation medium of synthetic medium-real material liquefied mash.Finally,the engineered strains of 4741-GRX5 and 4741-ttha0122 were obtained with residual glucose content of 77.4% and 74.5% of the control(BY4741),respectively.Furthermore,4741-GRX5 and 4741-ttha0122 were found to augment the ethanol yield by 5.8% and 2.5% compared to BY4741,respectively.Therefore,a new screening platform for heatresistant strains was established.