Expression of mitochondrial cytochrome c oxidase chaperone gene (COX20) improves tolerance to weak acid and oxidative stress during yeast fermentation

Abstract

Introduction Saccharomyces cerevisiae is the micro-organism of choice for the conversion of fermentable sugars released by the pre-treatment of lignocellulosic material into bioethanol. Pretreatment of lignocellulosic material releases acetic acid and previous work identified a cytochrome oxidase chaperone gene (COX20) which was significantly up-regulated in yeast cells in the presence of acetic acid. Results A cox20 strain was sensitive to the presence of acetic acid compared with the background strain. Overexpressing COX20 using a tetracycline-regulatable expression vector system in a cox20 strain, resulted in tolerance to the presence of acetic acid and tolerance could be ablated with addition of tetracycline. Assays also revealed that overexpression improved tolerance to the presence of hydrogen peroxide-induced oxidative stress. Conclusion This is a study which has utilised tetracycline-regulated protein expression in a fermentation system, which was characterised by improved (or enhanced) tolerance to acetic acid and oxidative stress. Introduction Fossil-based hydrocarbon fuels for generating energy, such as coal and crude oil, are finite resources and at the present rate of human consumption are predicted to be completely depleted by 2050 [1, 2]. An alternative, renewable source of energy is lignocellulosic residue from agricultural, forestry, municipal or industrial processes [3]. Sugars can be released from the lignocellulosic feed stocks using industrial pre-treatment processes, followed by enzymatic digestion and then converted to transportation biofuels, such as bioethanol or biobutanol by microbial fermentation [4]. Saccharomyces cerevisiae is currently used for the production of bioethanol, first generation bioethanol production has involved the conversion of hexose sugars derived from sucrose present in crops such as sugar cane in Brazil and from starch in crops such as maize in the USA [5]. Use of lignocellulosic feed stocks for biofuel production is more challenging, in order to increase fermentation efficiency, the problem of pre-treatment generated inhibitor compounds, and fermentation stresses, also have to be addressed. Pre-treatment of lignocellulose to release constituent sugars results in the release of aromatic and acidic compounds such as acetic acid, formic acid, furfural, hydroxy-methyl furfural (HMF), and vanillin [6] that are detrimental to the growth of S. cerevisiae. In addition, fermentations carried out within bioreactors generates further problems, such as osmotic stress due to high sugar levels, elevated heat and increasing ethanol concentrations [7-9]. Thus, resistance to all these fermentation stresses are desirable phenotypic attributes for improved bioethanol productivity. Using a systems biology approach and a phenotypic microarray assay we previously identified quantitative trait loci (QTLs) associated with response to weak acids [10], recipricol hemizygosity analysis of genes within the loci highlighted the potential role of COX20 in weak acid response [10]. Cytochrome C oxidase activity has been associated with programmed cell death (PCD) in yeast [11], where a loss of function along with addition of acetic acid has been shown to induce PCD [12]. Cytochrome C release has been shown to involve the ATP/ADP carrier as a component of the mitochondrial outer membrane [11] and has been shown to be released in response to reactive oxygen species (ROS) [13]. Yeast strains with altered cytochrome C oxidase activity maybe more tolerant to the inducement of PCD by acetic acid, the importance of cytochrome C oxidase has been reported in work on improving acetate tolerance in E. coli [14]. COX20 encodes for a chaperone that facilitates proteolytic processing of the mitochondrial gene product Cox2p and its assembly into the mitochondrial inner membrane cytochrome C oxidase complex [15]. In the work reported here, COX20 was expressed using tetracycline-regulatable vectors [16] in a co20 strain and response to acetic acid was measured using phenotypic microarrays and during fermentation. Incomplete assembly or mis-functioning cytochrome C oxidases have been associated with elevated levels of oxidative stress in the yeast cell [17] so the effect of oxidative stress on COX20 was also investigated. Material And Methods Yeast strains and growth conditions Yeast strains employed in this work derive from S. cerevisiae BY4741 (w) (Table 1). All strains were grown in YPD [1% (w/v) yeast extract (Oxoid); 2% (w/v) Bacto-peptone (Oxoid); 2% (w/ v) glucose]. Strains were deleted for tryptophan biosynthesis (trp1: URA3) using pAG60 (Euroscarf, Frankfurt, Germany) with a URA3 selectable marker using primers (Table 2). A cox20 null mutant was obtained from Euroscarf (Frankfurt, Germany) and TRP1 was also deleted from this strain as above for wild-type (BY4741).