Abstract
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.
Highlights
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
The best minimal constitutive yeast promoter, with the highest transcriptional activity reported to date, is UASF-E-C-Core1, which was constructed by combining a minimal-sized core sequence with tandem synthetic upstream activator sequence (UAS) elements [18]
To test whether this minimal promoter can function in other yeast expression systems, we first used the UASF-E-C-Core1 promoter to drive green fluorescent protein (GFP) expression using the centromeric plasmid pRS313 in the S. cerevisiae BY4742
Summary
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. The endogenous promoters of S. cerevisiae include constitutive promoters (e.g. promoters of the genes encoding glyceraldehyde-3-phosphate dehydrogenase, PTDH3, cytochrome c isoform, P CYC1, translation elongation factor, PTEF1, etc.), which allow continuous transcription under all circumstances, as well as regulated promoters (e.g. the galactose-inducible PGAL1/PGAL2/ PGAL7/PGAL10 and the C u2+-inducible PCUP1), which are active only in response to specific stimuli [9,10,11]. Because these endogenous promoters have different expression strengths, their combination and tuning of the gene copy numbers can be used to fine-tune the transcript abundance of target genes within a wide and dynamic range spanning several orders of magnitude. The activity of the best minimal promoter reported to date is still 30% below the endogenous strong promoter P TDH3 [18]
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