Minimization of glycerol synthesis in industrial ethanol yeast without influencing its fermentation performance
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.
Section snippets
Yeast strains and media
The genotypes of the microbial strains and plasmids used in the present study are summarized in Table 1. The S. cerevisiae haploid strain ANGA1 (MATa) and ANGA2 (MATα) for genetic manipulation were derived from the polyploid S. cerevisiae CICIMY0086 (http://cicim-cu.sytu.edu.cn/, ethanol producing yeast industrial plants). Strains were routinely grown in a medium composed of 1% yeast extract, 2% Bacto peptone, and 2% glucose (YPD). Solid media contained 2% agar. Incubation conditions were
Expression of GAPN in GPD1 deleted mutant
The genomic DNA analysis showed that the disruption cassette gpd1::PGK1P-gapN-PGK1t-loxp-kanr-loxp was correctly incorporated into the GPD1 locus of the S. cerevisiae chromosome (data not shown). The resultant mutant was named AG1 (gpd1::gapN-kanr). And then, the G418 resistance gene was excised by expression of Cre-recombinase induced by 2% d-galactose. Subsequently, the NADP+-dependent GAPN activities of 10 transformant colonies were investigated. One of the above recombinant strains
Discussion
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). In addition, it is the key precursor used to synthesize the cellular membrane. Thus, we believe completely eliminate its production is not practical, but it can be reduced to a lowest level with keeping its wild type phenotype. Although glycerol formation
Conclusion
In the present study, the predicted higher ethanol yield and lower glycerol yield were achieved by expression of GAPN in GPD1 deleted mutant, but the growth of the strain was severely low during high sugar fermentations. After the genes TPS1 and TPS2 were simultaneously expressed in above recombinant, its desired cellular functions was totally restored and further decrease in glycerol yield was observed. Therefore, minimization of glycerol production to improve ethanol yield without affecting
Acknowledgments
This work was financially supported by the Natural Science Foundation of China (20706024), the “863” Program (2006AA020101, 2007AA10Z359), Innovative Research Team of Jiangsu Province and China Postdoctoral Science Foundation funded project (200801361).
References (49)
- et al.
Osmoregulation in Saccharomyces cerevisiae. Studies on the osmotic induction of glycerol production and glycerol-3-phosphate dehydrogenase (NAD+)
FEBS Lett.
(1991) - et al.
In silico aided metabolic engineering of Saccharomyces cerevisiae for improved bioethanol production
Metab. Eng.
(2006) - et al.
Glass formation in plant anhydrobiotes: survival in the dry state
Cryobiology
(2004) - et al.
A metabolic and genomic study of engineered Saccharomyces cerevisiae strains for high glycerol production
Metab. Eng.
(2007) - et al.
Separation and properties of NAD+-and NADP+-dependent glyceraldehyde-3-phosphate dehydrogenases from Streptococcus mutans
J. Biol. Chem.
(1979) - et al.
Reserve carbohydrates metabolism in the yeast Saccharomyces cerevisiae
FEMS Microbiol. Rev.
(2001) - et al.
Rapid changes of heat and desiccation tolerance correlated with changes of trehalose content in Saccharomyces cerevisiae cells subjected to temperature shifts
FEBS Lett.
(1987) - et al.
Metabolic impact of redox cofactor perturbations in Saccharomyces cerevisiae
Metab. Eng.
(2009) - et al.
Trehalose inhibits ethanol effects on intact yeast cells and liposomes
Biochim. Biophys. Acta
(1994) - et al.
Freezing injury in Saccharomyces cerevisiae: the effect of growth conditions
Cryobiology
(1988)
Mechanisms of intracellular ice formation
Biophys. J.
Optimization of ethanol production in Saccharomyces cerevisiae by metabolic engineering of the ammonium assimilation
Metab. Eng.
Engineering Escherichia coli for the efficient conversion of glycerol to ethanol and co-products
Metab. Eng.
Processes involved in the creation of buffering capacity and in substrate-induced proton extrusion in the yeast Saccharomyces cerevisiae
Biochim. Biophys. Acta
Redox balances in the metabolism of sugars by yeasts
FEMS Microbiol. Rev.
Glucose and sucrose: hazardous fast-food for industrial yeasts?
Trends Biotechnol.
Influence of the nitrogen source on Saccharomyces cerevisiae anaerobic growth and product formation
Appl. Environ. Microbiol.
The role of glycerol in osmotolerance of the yeast Debaryomyces hansenii
J. Gen. Microbiol.
The two isoenzymes for yeast NAD+-dependent glycerol-3-phosphate dehydrogenase encoded by GPD1 and GPD2 have distinct roles in osmoadaption and redox regulation
EMBO J.
Physiological response to anaerobicity of glycerol-3-phosphate dehydrogenase mutants of Saccharomyces cerevisiae
Appl. Environ. Microbiol.
Physiology of osmotolerance in fungi
Adv. Microb. Physiol.
Metabolic control analysis of glycerol synthesis in Saccharomyces cerevisiae
Appl. Environ. Microbiol.
Anhydrobiosis
Annu. Rev. Physiol.
Elimination of glycerol production in anaerobic cultures of a Saccharomyces cerevisiae strain engineered to use acetic acid as an electron acceptor
Appl. Environ. Microbiol.
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