Construction of high version thermo-tolerance <italic>Saccharomyces cerevisiae</italic> by random assembly of thermo-tolerance genetic circuits

Abstract

<p indent="0mm">Existing green biomanufacturing systems require controlled fermentation at room temperature, consuming large amount of cooling water and energy. To solve this problem, reprogramming the <italic>Saccharomyces cerevisiae</italic> 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 <italic>Thermoanaerobacter tengcongensis</italic> MB4, <italic>Thermus thermophiles</italic> HB8, and <italic>Bacillus subtilis</italic> were selected as the core functional elements. The constitutive promoters (14) and terminators (10) with different intensities of <italic>S</italic>. <italic>cerevisiae</italic> 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 high-throughput 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 heat-resistant strains was established.