Engineering promoter regulation

Abstract

Systems for easily controlled, conditional induction or repression of gene expression are indispensable tools in fundamental research and industrial-scale biotechnological applications. Both native and rationally designed inducible promoters have been widely used for this purpose. However, inherent regulation modalities or toxic, expensive or inconvenient inducers can impose limitations on their use. Tailored promoters with user-specified regulatory properties would permit sophisticated manipulations of gene expression. Here, we report a generally applicable strategy for the directed evolution of promoter regulation. Specifically, we applied random mutagenesis and a multi-stage flow cytometry screen to isolate mutants of the oxygen-responsive Saccharomyces cerevisiae DAN1 promoter. Two mutants were isolated which were induced under less-stringent anaerobiosis than the wild-type promoter enabling induction of gene expression in yeast fermentations simply by oxygen depletion during cell growth. Moreover, the engineered promoters showed a markedly higher maximal expression than the unmutated DAN1 promoter, under both fastidious anaerobiosis and microaerobisois.

Progress in Metabolic Engineering of Saccharomyces cerevisiae

“…Recently, we proposed that a promoter induced by simply down-regulating the cultivation medium oxygen concentration would represent an inexpensive and manageable tool for conditional gene expression (237). This idea has already been pursued for Escherichia coli heterologous protein production using the Vitreocilla vgb promoter, which is induced by low oxygen (201).…”

“…Given the attenuated mass transfer dynamics at large scales, this step could be time-consuming or its implementation unfeasible under industrial conditions. We therefore attempted to evolve the oxygen sensitivity of the DAN1 promoter (237). The evolved promoters enabled induction of reporter gene expression in yeast fermentation simply by depleting oxygen during cell growth.…”

Towards combinatorial transcriptional engineering

“…Random mutagenesis of the oxygen responsive DAN1 gene promoter in S. cerevisae generated two mutants with decreased anaerobic inducibility. These promoters could be used to regulate gene expression during cell growth and proliferation as transgenes could be expressed upon growth induced oxygen depletion (Nevoigt et al, 2007).…”

GAP Promoter Library for Fine-Tuning of Gene Expression in Pichia pastoris

“…The promoter library permits precise control of gene expression and quantitative assessment that correlates gene expression level with physiologic parameters. Thus, it is a useful toolbox for both basic and applied research in P. pastoris.The creation of a library of engineered promoters of various strengths has enabled the precise control of gene expression in a broad range of activities required for detailed genotypephenotype characterization and the identification of an optimal gene expression level for the desired product yield (1,4,12,20,22,29,35). For example, a library of promoters has been used to assess the influence of phosphoenolpyruvate carboxylase levels on growth yield and deoxyxylulose-P synthase levels on lycopene production and to demonstrate that the optimal expression levels of deoxyxylulose-P synthase are different in a strain preengineered to produce lycopene and in the parental strain (1).…”

“…However, this powerful tool is implemented mainly in bacteria (2,4,12,20,35,36). There are only limited examples in which a promoter library has been created in yeast (1,29,30), and only one promoter library has been created for fine-tuning of gene expression in the methylotrophic yeast Pichia pastoris (15).P. pastoris combines the advantages of eukaryotes, such as efficient protein secretion and posttranslational modifications, with fast growth on economical salt-based media (11).…”