Abstract
Polymorphism is well known in Saccharomyces cerevisiae strains used for different industrial applications, however little is known about its effects on promoter efficiency. In order to test this, five different promoters derived from an industrial and a laboratory (S288c) strain were used to drive the expression of eGFP reporter gene in both cells. The ADH1 promoter (PADH1) in particular, which showed more polymorphism among the promoters analyzed, also exhibited the highest differences in intracellular fluorescence production. This was further confirmed by Northern blot analysis. The same behavior was also observed when the gene coding for secreted α-amylase from Cryptococcus flavus was placed under the control of either PADH1. These results underline the importance of the careful choice of the source of the promoter to be used in industrial yeast strains for heterologous expression.
Highlights
It has been previously described that S. cerevisiae strains present different patterns of gene expression according to environmental stress factors (James et al 2003; Kvitek et al 2008)
In order to determine if commonly used promoters in synthetic biology show different expression patterns, we analyzed and compared five promoters (PCYC1, PTEF1, PPGK1, PPGI1 and PADH1) selected from S. cerevisiae strains JPU and S288C, for the heterologous expression of the eGFP gene and the α-amylase gene (AMY1) from C. flavus (Galdino et al 2008b)
A study of the resistance to different types of stress carried out in yeast strains used for wine production (Ivorra et al 1999) was used as a basis for understanding the physiology of ethanol-producing strains isolated from Brazilian sugarcane mills
Summary
It has been previously described that S. cerevisiae strains present different patterns of gene expression according to environmental stress factors (James et al 2003; Kvitek et al 2008). The Brazilian fermentation process is an adaptation of the Melle-Boinot process where cells are intensively recycled through a process of centrifugation and washing in diluted sulfuric acid resulting in high cell densities (Babrzadeh et al 2012; Basso et al 2008; Wheals et al 1999). This process occurs in non-sterile conditions making it susceptible to contamination and genetically and physiologically adapted strains tend to dominate (da Silva-Filho et al 2005a; Zaldivar et al 2002; Zheng et al 2013). In order to determine if commonly used promoters in synthetic biology show different expression patterns, we analyzed and compared five promoters (PCYC1, PTEF1, PPGK1, PPGI1 and PADH1) selected from S. cerevisiae strains JPU (industrial) and S288C (laboratory), for the heterologous expression of the eGFP gene and the α-amylase gene (AMY1) from C. flavus (Galdino et al 2008b).
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