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

Tailoring 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. In 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 UAS.sub.F-E-C-Core1 minimal promoter as a library template and determined its Kozak motif (K.sub.0). Next, we randomly mutated the K.sub.0 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 K.sub.0 control. The best one named K.sub.528 even showed 8.5- and 3.3-fold increases in fluorescence intensity compared with UAS.sub.F-E-C-Core1 and the strong native constitutive promoter P.sub.TDH3, respectively. Subsequently, we chose three representative strong chimeric promoters (K.sub.540, K.sub.536, and K.sub.528) from this library to regulate pathway gene expression. In conjunction with the tHMG1 gene for squalene production, the K.sub.528 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 K.sub.0 control (3.1 mg/L). All these results demonstrate that this chimeric promoter library developed in this study is an effective tool for pathway engineering in yeast.