SOD1, a New Kluyveromyces lactis Helper Gene for Heterologous Protein Secretion

Abstract

Bottlenecks in protein expression and secretion often limit the development of industrial processes. By manipulating chaperone and foldase levels, improvements in yeast secretion were found for a number of proteins. Recently, sustained endoplasmic reticulum stress, occurring due to recombinant protein production, was reported to cause oxidative stress in yeast. Saccharomyces cerevisiae cells are able to trigger an adaptive response to oxidative-stress conditions, resulting in the upregulation of both primary and secondary antioxidant defenses. SOD1 encodes for a superoxide dismutase that catalyzes the dismutation of superoxide anions (O 2 ؊ ) into oxygen and hydrogen peroxide. It is a Cu 2؉ /Zn 2؉ metalloenzyme and represents an important antioxidant defense in nearly all aerobic and aerotolerant organisms. We found that overexpression of the Kluyveromyces lactis SOD1 (KlSOD1) gene was able to increase the production of two different heterologous proteins, human serum albumin (HSA) and glucoamylase from Arxula adeninivorans. In addition, KlSOD1 overexpression led to a significant decrease in the amount of reactive oxygen species (ROS) that originated during protein production. The yield of HSA also increased when K. lactis cells were grown in the presence of the antioxidant agent ascorbic acid and decreased when cells were challenged with menadione, a ROS generator compound. Moreover, we observed that, in high-osmolarity medium, cells overexpressing KlSOD1 showed higher growth rates than control cells. Our results thus further support the notion that the production of some heterologous proteins may be improved by manipulating genes involved in general stress responses.

Rational strain engineering interventions to enhance cellulase secretion by Saccharomyces cerevisiae

“…Many of the published secretion enhancing gene modifications were proposed to affect the protein production and secretory pathway in a direct manner, by assisting in product folding or secretion vesicle fusion . Less obvious mechanisms by which modifications could modulate secretion, included the general maintenance of cellular health under environmental and general secretory pathway stress conditions …”

Identification of genes that enhance cellulase protein production in yeast

“…Deletion of the CYM1 gene, which encodes a lysine-specific metalloprotease, enhanced human holecystokinin protein secretion (Jonson et al, 2004). On the other hand, overexpression of the SOD1 gene, which encodes a superoxide dismutase, the PDI1 gene, which encodes a protein disulfide isomerase, or the RPP0 gene, which encodes a ribosomal protein, also enhanced heterologous protein production (Raimondi et al, 2008;Robinson et al, 1994;Wentz and Shusta, 2008). In addition, mutant strains that exhibited enhanced endoglucanase II activity from T. reesei have been isolated, but the genes responsible for the enhanced cellulase protein production were not identified (Aho et al, 1996).…”

Assembling a cellulase cocktail and a cellodextrin transporter into a yeast host for CBP ethanol production

“…However, it still needs further improvement before industry application. For example, elevation of the enzyme production could be achieved by improving the enzyme secretion ability with better secretion leader sequence [28] or by reforming the protein folding with related chaperon gene co-expression [29,30]. Furthermore, the enzyme activity can be further improved by increasing the thermostability by addition of specific ions in the growth media [31].…”