Minimization of glycerol synthesis in industrial ethanol yeast without influencing its fermentation performance

Abstract

To synthesize glycerol, a major by-product during anaerobic production of ethanol, the yeast Saccharomyces cerevisiae would consume up to 4% of the sugar feedstock in typical industrial ethanol processes. The present study was dedicated to decreasing the glycerol production mostly in industrial ethanol producing yeast without affecting its desirable fermentation properties including high osmotic and ethanol tolerance, natural robustness in industrial processes. In the present study, the GPD1 gene, encoding NAD⁺-dependent glycerol-3-phosphate dehydrogenase in an industrial ethanol producing strain of S. cerevisiae, was deleted. Simultaneously, a non-phosphorylating NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Bacillus cereus was expressed in the mutant deletion of GPD1. Although the resultant strain AG1A (gpd1△ P_PGK-gapN) exhibited a 48.7±0.3% (relative to the amount of substrate consumed) lower glycerol yield and a 7.6±0.1% (relative to the amount of substrate consumed) higher ethanol yield compared to the wild-type strain, it was sensitive to osmotic stress and failed to ferment on 25% glucose. However, when trehalose synthesis genes TPS1 and TPS2 were over-expressed in the above recombinant strain AG1A, its high osmotic stress tolerance was not only restored but also improved. In addition, this new recombinant yeast strain displayed further reduced glycerol yield, indistinguishable maximum specific growth rate (μ_max) and fermentation ability compared to the wild type in anaerobic batch fermentations. This study provides a promising strategy to improve ethanol yields by minimization of glycerol production.

Introduction

Ethanol is one of the most important products originating from the biotechnological industry with respect to both value and amount. It has been estimated that up to 4% of the sugar feedstock is converted into glycerol, a major by-product produced by Saccharomyces cerevisiae, in typical industrial ethanol processes (Nissen et al., 2000a). Glycerol synthesis plays significant physiological roles in metabolism of the yeast including osmoregulation and maintaining intracellular redox balance under anaerobic conditions (Blomberg and Adler, 1992, Van Dijken and Scheffers, 1986). It is produced from dihydroxyacetone phosphate (DHAP) in two sequential steps catalyzed by glycerol-3-phosphate dehydrogenase (GPD) and by glycerol-3-phosphate phosphatase (GPP). The first step of glycerol formation, catalyzed by GPD encoded for by two highly homologous isogenes GPD1 and GPD2, is rate-controlling (Cronwright et al., 2002). In S. cerevisiae, glycerol serves as a compatible solute at high extracellular osmolarity protecting the cell against lysis (Larsson et al., 1993), which is extremely important in current very high gravity (VHG) fermentations. On the other hand, a surplus of NADH, formed in synthesis of biomass and secondary fermentation products, e.g. acetic, pyruvic and succinic acid, was re-oxidized to NAD⁺ by glycerol production during anaerobic conditions, since ethanol formation from glucose is a redox-neutral process (Albers et al., 1996). However, formation of glycerol represents an unwanted loss of carbon source if the aim is to produce maximum amounts of ethanol. To improve the efficiency of substrate utilization by reduction in glycerol formation is a prospective way to make ethanol an economically feasible bio-fuel. Therefore, great attempts have been made to decrease the glycerol yield, e.g. by deleting the GPD1 and GPD2 genes (Nissen et al., 2000b, Valadi et al., 1998), or by regulating redox balance in ammonia metabolism by over-expression of the GLT1 and GLN1 genes, encoding glutamate synthase and glutamine synthase, respectively (Nissen et al., 2000a, Valenzuela et al., 1998). Guadalupe Medina et al. (2010) recently reported a valuable metabolic engineering strategy to completely eliminate glycerol production by engineering S. cerevisiae such that it can re-oxidize NADH by the reduction of acetic acid to ethanol via NADH-dependent reactions. Unfortunately, consistent with earlier studies (Björkqvist et al., 1997, Nissen et al., 2000a), the maximum specific growth rate and product formation were severely lowered in such strains. Besides, the osmotic stress sensitivity of the engineered yeast makes it impractical to use in industrial fermentations with high initial sugar concentrations. Another possible metabolic engineering strategy for reducing glycerol production and redirecting the flux of carbon from glycerol flux towards ethanol is to substitute the NAD⁺-reducing reactions in biomass formation by NADP⁺-reducing reactions (Bro et al., 2006). Regulation of redox cofactors are emerging to be attractive targets to induce widespread changes in metabolism due to their pivotal role in coupling catabolism with anabolism and energy generation (Hou et al., 2009). In previous study by Bro et al. (2006), a non-phosphorylating NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Streptococcus mutants was over-expressed to replace the NAD⁺-dependent glyceraldehyde-3-phosphate dehydrogenase of S. cerevisiae and hence, decrease the formation of NADH during the glycolysis. Consequently, the glycerol yield was reduced by 40% while the ethanol yield increased 3% without affecting the maximum specific growth rate. In the present work, in order to further improve ethanol yield, GAPN from Bacillus cereus was over-expressed in S. cerevisiae concomitant with deletion of GPD1.

On the other hand, it is well known that trehalose functions as one of the major stress protectants and that the synthesis of trehalose is induced by many stress conditions at the transcriptional level in S. cerevisiae, as is glycerol (Kaino and Takagi, 2008). Owing to the abilities of these compounds to prevent the influx of excess salts into the cell or irreversible cell dehydration, the osmotic imbalance after hyper-osmotic shock can be rapidly restored (Shima and Takagi, 2009). Thus, to eliminate the adverse influences of deletion of GPD1 on desirable fermentation properties including high osmotic and ethanol tolerance, natural robustness of the yeast in industrial processes, we then introduced the genes TPS1 and TPS2, encoding trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, respectively, into the recombinant strain deletion of GPD1 and simultaneous expression of GAPN. Growth and product formation of the engineered strains, as well as the reference strain used as a control, were then compared when 5% and 25% glucose were provided as carbon sources. The strategy used in the present study was shown in Fig. 1. We are dedicated to exploring the most promising way to improve ethanol yield by minimization of glycerol production.