GALpromoter-driven heterologous gene expression inSaccharomyces cerevisiaeΔ strain at anaerobic alcoholic fermentation

Abstract

The removal of Gal80 protein by gene disruption turned into efficient GAL promoter-driven heterologous gene expression under anaerobic alcoholic fermentation of Saccharomyces cerevisiae. Using lipase B from Candida antarctica as a reporter, the relative strength of GAL10 promoter (P(GAL10) ) in Δgal80 mutant that does not require galactose as an inducer was compared to those of ADH1, PDC1, and PGK promoters, which have been known to work well anaerobically in actively fermenting yeast cells under high glucose concentration. P(GAL10) in the Δgal80 mutant showed 0.8-fold (ADH1), fourfold (PDC1), and 50-fold (PGK) in promoter strength.

Stepwise Optimization of Inducible Expression System for the Functional Secretion of Horseradish Peroxidase in Saccharomyces cerevisiae

“…The Optimization of CPS Region in pGal1 Promoter to Roughly Tune HRP Expression. Due to the limited strength of pGal1, 29,30 hybrid promoters that possess CRM Gal1 (the CRM of pGal1) and CPSs from pTEF1, pTDH3, and pGal7 were created to obtain a higher expressional level of HRP. The original CRM Gal1 was used to maintain the regulatory feature induced by galactose and repressed by glucose.…”

“…19 Thus, the common application of a pGal1 promoter for diauxic shift-induced expression provides an alternative solution to synthesize toxic proteins or enzymes. 24,25 Although the knockout of the GAL80 gene allows the thorough release of Gal4p (a transcriptional activator responding to galactose) to improve the expressional strength of pGal1 (Figure 1A) 26,27 and reduction of the medium cost by uncoupling galactose induction, 28 the strength of pGal1 with GAL80 knockout is not high enough; only 0.8fold the strength of pADH1 29 or 0.5-fold the strength of pTDH1. 30 To achieve stronger expression, hybrid promoters were created according to the understanding of the basic components of the promoter, including the core promoter sequence (CPS) and upstream cis-regulatory module (CRM; Figure 1A), 31,32 but the previous research only utilized easy-tofold proteins (such as green fluorescent protein, GFP) to assess the expressional strength, 33−35 which could not reflect the expressional effect on cell growth.…”

“…phaffii . Thus, the common application of a pGal1 promoter for diauxic shift-induced expression provides an alternative solution to synthesize toxic proteins or enzymes. , Although the knockout of the GAL80 gene allows the thorough release of Gal4p (a transcriptional activator responding to galactose) to improve the expressional strength of pGal1 (Figure A) , and reduction of the medium cost by uncoupling galactose induction, the strength of pGal1 with GAL80 knockout is not high enough; only 0.8-fold the strength of pADH1 or 0.5-fold the strength of pTDH1 …”

Advanced Strategies for Production of Natural Products in Yeast

“…The carbon source sensor is extensively used for the construction of dynamic pathway, such as GAL and HXT systems. The promoters of galactose metabolism genes, such as GAL1p and GAL10p, are activated by galactose and repressed by glucose in S. cerevisiae (Ahn et al, 2013). With deletion of GAL80, these GAL promoters are constitutively transcribed at low glucose level and can be used for the construction of biosynthetic pathways responding to glucose level, which can divide fermentation process into cell growth and product biosynthesis phases.…”

Positive Feedback Genetic Circuit Incorporating a Constitutively Active Mutant Gal3 into Yeast GAL Induction System

“…The GAL expression system, which uses the GAL1 – GAL10 divergent promoters, is one of the most frequently used induction techniques, and enables strictly regulated induction in S. cerevisiae . − Both the GAL1 and GAL10 promoters trigger potent transcriptional activation in the presence of galactose, whereas expression from these promoters is robustly suppressed in the presence of glucose. , However, the inducer, galactose, is a relatively expensive sugar compared to other carbon sources such as glucose and glycerol. This cost factor affects for industrial applications, limiting the commercial use of the GAL expression system.…”