Fine-tuning the expression of pathway gene in yeast using a regulatory library formed by fusing a synthetic minimal promoter with different Kozak variants

Microbial Cell Factories

Liping Xu¹  Zhubo Dai²  Xueli Zhang²  Pingping Liu²  Feiyu Fan² 

[1] School of Life Science, University of Science and Technology of China, No. 96, JinZhai Road, Baohe District, 230026, Hefei, Anhui, People’s Republic of China;Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, 300308, Tianjin, People’s Republic of China;Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, 300308, Tianjin, People’s Republic of China;Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, 300308, Tianjin, People’s Republic of China;Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, 300308, Tianjin, People’s Republic of China;National Innovation Center for Synthetic Biotechnology, 300308, Tianjin, People’s Republic of China;

关键词: Artificial minimal promoters;    Kozak sequence;    Chimeric promoter library;    Saccharomyces cerevisiae;    Pathway engineering;   

DOI  :  10.1186/s12934-021-01641-z

来源: Springer

【 摘 要 】

BackgroundTailoring gene expression to balance metabolic fluxes is critical for the overproduction of metabolites in yeast hosts, and its implementation requires coordinated regulation at both transcriptional and translational levels. Although synthetic minimal yeast promoters have shown many advantages compared to natural promoters, their transcriptional strength is still limited, which restricts their applications in pathway engineering.ResultsIn this work, we sought to expand the application scope of synthetic minimal yeast promoters by enhancing the corresponding translation levels using specific Kozak sequence variants. Firstly, we chose the reported UASF-E-C-Core1 minimal promoter as a library template and determined its Kozak motif (K0). Next, we randomly mutated the K0 to generate a chimeric promoter library, which was able to drive green fluorescent protein (GFP) expression with translational strengths spanning a 500-fold range. A total of 14 chimeric promoters showed at least two-fold differences in GFP expression strength compared to the K0 control. The best one named K528 even showed 8.5- and 3.3-fold increases in fluorescence intensity compared with UASF-E-C-Core1 and the strong native constitutive promoter PTDH3, respectively. Subsequently, we chose three representative strong chimeric promoters (K540, K536, and K528) from this library to regulate pathway gene expression. In conjunction with the tHMG1 gene for squalene production, the K528 variant produced the best squalene titer of 32.1 mg/L in shake flasks, which represents a more than 10-fold increase compared to the parental K0 control (3.1 mg/L).ConclusionsAll these results demonstrate that this chimeric promoter library developed in this study is an effective tool for pathway engineering in yeast.

【 授权许可】

CC BY