Controlling promoter strength and regulation in Saccharomyces cerevisiae using synthetic hybrid promoters

Abstract

A dynamic range of well-controlled constitutive and tunable promoters are essential for metabolic engineering and synthetic biology applications in all host organisms. Here, we apply a synthetic hybrid promoter approach for the creation of strong promoter libraries in the model yeast, Saccharomyces cerevisiae. Synthetic hybrid promoters are composed of two modular components-the enhancer element, consisting of tandem repeats or combinations of upstream activation sequences (UAS), and the core promoter element. We demonstrate the utility of this approach with three main case studies. First, we establish a dynamic range of constitutive promoters and in doing so expand transcriptional capacity of the strongest constitutive yeast promoter, P(GPD) , by 2.5-fold in terms of mRNA levels. Second, we demonstrate the capacity to impart synthetic regulation through a hybrid promoter approach by adding galactose activation and removing glucose repression. Third, we establish a collection of galactose-inducible hybrid promoters that span a nearly 50-fold dynamic range of galactose-induced expression levels and increase the transcriptional capacity of the Gal1 promoter by 15%. These results demonstrate that promoters in S. cerevisiae, and potentially all yeast, are enhancer limited and a synthetic hybrid promoter approach can expand, enhance, and control promoter activity.

Modular optimization of multi-gene pathways for fatty acids production in E. coli

“…Engineering mutant and hybrid promoters with modified RNA polymerase, activator or repressor-binding activities has been widely used in metabolic engineering for transcriptional finetuning and pathway optimization 17,27,28 . However, the genetic recalcitrance of the bacteriophage T7 promoter has restricted our ability to manipulate the consensus promoter core sequence ( À 35 and À 10 region) and the lacI repressor binding region (lacO) 29 .…”

Synthetic Biology and Metabolic Engineering Approaches and Its Impact on Non-Conventional Yeast and Biofuel Production

“…Saccharomyces cerevisiae have been considered as a model system for studying cell and molecular biology and is a wellstudied organism (Blazeck et al, 2012;Blount et al, 2012a,b;Bao et al, 2015). Many synthetic biology and metabolic engineering efforts have been already established in S. cerevisiae for improving biofuel production (Tsai et al, 2015).…”

“…However, recently a large number of genome sequences of non-conventional yeasts are available (e.g., Y. lipolytica) and have boosted both basic and applied research to understand the genotypic and phenotypic features and further development of metabolic engineering tools for biofuel production. Most of the promoter engineering work has been concentrated on upstream regulatory sequences (Hartner et al, 2008;Xuan et al, 2009), engineering core promoter elements (Blazeck et al, 2012), and/or by creating random mutations in core promoter elements (Berg et al, 2013), and this will lead to tight and tuneable control over a metabolic regulatory pathways (Teo and Chang, 2014).…”

“…The constitutive pGAP (glyceraldehyde-3-phosphate dehydrogenase promoter) served as a scaffold, and this resulted in the development of a series of promoters with a range of activity from 0.01-to 19.6-fold of wild-type pGAP activity (Qin et al, 2011). Blazeck et al (2012) reported that the combination of core promoter elements with synthetic upstream activator sequence could increase the expression level.…”

Advances in Metabolic Engineering of Saccharomyces cerevisiae for the Production of Industrially and Clinically Important Chemicals

“…Kanamasa et al isolated cis-aconitic acid decarboxylase (CAD), which is the key enzyme in the conversion of cis-aconitate to itaconic acid in A. terreus, and its heterologous expression in S. cerevisiae showed the possibility of itaconic acid production in yeast [20]. Blazeck et al utilized a synthetic hybrid promoter carrying an enhancer and a core promoter module to optimize CAD expression in S. cerevisiae [19,21]. A genome-wide metabolic model of the yeast was used to identify gene deletion targets to further increase the itaconic acid titer.…”