Date Published: May 21, 2018
Publisher: Springer Berlin Heidelberg
Author(s): Daniel Pereira de Paiva, Tiago Benoliel Rocha, Marciano Regis Rubini, André Moraes Nicola, Viviane Castelo Branco Reis, Fernando Araripe Gonçalves Torres, Lidia Maria Pepe de Moraes.
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.
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). However, the choice of a yeast strain more adapted to the fermentation process is not the only concern that should be considered. The majority of metabolic pathways studies focuses, for example, on the choice of promoters for heterologous expression cassette construction via the selection of appropriate promoters for its strength under different growth conditions (Partow et al. 2010; Peng et al. 2015; Sun et al. 2012), in improving the strength through construction of hybrid promoters (Blazeck et al. 2012) and in optimizing metabolic pathway using combinatorial metabolic libraries (Carquet et al. 2015; Du et al. 2012). But no study to date takes into account the particular genetics of the host strain that will receive the promoters.
Saccharomyces cerevisiae is the most important micro-organism employed in the production of bioethanol. This is due to several characteristics such as high ethanol productivity, tolerance to production stresses and resistance to fermentation by-products (Zaldivar et al. 2002; Zheng et al. 2013). 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. The study conducted by (Della-Bianca and Gombert 2013) compared the physiology of the main strains used in the production of fuel ethanol in Brazil with some well-known laboratory strains, evaluating their tolerance in relation to the classic stress factors and the specific ones faced in the industrial process of fermentation.