Effect of Trehalose and Glycerol on the Resistance of Recombinant Saccharomyces cerevisiae Strains to Desiccation, Freeze-Thaw and Osmotic Stresses

M.V. Semkiv; O.T. Ternavska; K.V.  Dmytruk; A.A. Sybirny

doi:10.15407/scine14.06.073

Authors

M.V. Semkiv Institute of Cell Biology, the NAS of Ukraine
O.T. Ternavska Institute of Cell Biology, the NAS of Ukraine
K.V. Dmytruk Institute of Cell Biology, the NAS of Ukraine
A.A. Sybirny Institute of Cell Biology, the NAS of Ukraine

DOI:

https://doi.org/10.15407/scine14.06.073

Keywords:

baker’s yeasts, desiccation, freeze-thaw, glycerol, osmotolerance, trehalose

Abstract

Introduction. Baker's yeast Saccharomyces cerevisiae has been used for manufacturing bakery products, food and feed supplements, alcoholic fermentation etc. In biotechnological processes, yeast cells are exposed to stress factors (high concentration of sugars and ethanol, high temperature, desiccation or freezing etc.), which negatively affects their viability. Yeasts possess certain stress protection systems, including increased accumulation of disaccharide trehalose and glycerol synthesis.
Problem Statement. The strengthening of yeast protective systems by increasing glycerol or trehalose concentrations can help to get increased stress robustness of the S. cerevisiae strains.
Purpose. To construct the recombinant strains of S. cerevisiae with increased trehalose accumulation or glycerol production and to estimate the obtained recombinant strains resistance to a range of stress factors.
Materials and Methods. S. сerevisiae transformation has been performed using Li-Ac-PEG method. Alcoholic fermentation has been carried out at a temperature of 30 °C with stirring at a rate of 120 rpm.
Results. The recombinant strains of S. cerevisiae with enhanced glycerol production (up to 19 g/L) have been constructed based on BY4742. The industrial ethanol-producing strain Y-563 has been used as parental one for construction of recombinant strains with up to 3.3-fold increase in the intracellular trehalose level. The resistance of obtained recombinant strains to different stress factors has been evaluated. BY/TPI25/gpd1gpp2f/fps1 strain with the highest glycerol production has been established to have the highest osmotolerance. The BY/TPI25/gpd1gpp2f, 563/TSL1, 563/TPS1/2 and 563/TPS1/2/TSL1 strains have shown higher viability after freeze-thaw as compared with the corresponding parental strains, but not higher resistance to desiccation. The recombinant strain 563/TPS1/2/TSL1 with a high trehalose content have been established to have higher activity during fermentation of sugar in sweet dough and to longer keep stable at 35°С as compared with the initial strain Y-563.
Conclusion. Constructed recombinant strains of S. cerevisiae with higher osmotolerance or freeze-thaw resistance can be implemented in industrial processes accompanied with these types of stresses. Baker’s yeast made of high trehalose-containing biomass will have prolonged shelf life.

References

Ahmadpour, D., Geijer, C., Tamas, M. J., Lindkvist-Petersson, K., Hohmann, S. (2014). Yeast reveals unexpected roles and regulatory features of aquaporins and aquaglyceroporins. Biochim. Biophys. Acta, 1840(5), 1482-1491.

https://doi.org/10.1016/j.bbagen.2013.09.027

Albertyn, J., Hohmann, S., Thevelein, J. M., Prior, B. A. (1994). GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway. Mol. Cell Biol., 14(6), 4135-4144.

https://doi.org/10.1128/MCB.14.6.4135

Ando, A., Nakamura, T., Murata, Y., Takagi, H., Shima, J. (2007). Identification and classification of genes required for tolerance to freeze-thaw stress revealed by genome-wide screening of Saccharomyces cerevisiae deletion strains. FEMS Yeast Res., 7(2), 244-253.

https://doi.org/10.1111/j.1567-1364.2006.00162.x

Attfield, P. V. (1997). Stress tolerance: the key to effective strains of industrial baker's yeast. Nat. Biotechnol., 15(13), 1351-1357.

https://doi.org/10.1038/nbt1297-1351

Blomberg, A., Adler, L. (1989). Roles of glycerol and glycerol-3-phosphate dehydrogenase (NAD+) in acquired osmotolerance of Saccharomyces cerevisiae. J. Bacteriol., 171(2), 1087-1092.

https://doi.org/10.1128/JB.171.2.1087-1092.1989

Blomberg, A., Adler, L. (1992). Physiology of osmotolerance in fungi. Adv. Microb. Physiol., 33, 145-212.

https://doi.org/10.1016/S0065-2911(08)60217-9

Brown, A. D. (1978). Compatible solutes and extreme water stress in eukaryotic micro-organisms. Adv. Microb. Physiol., 17, 181-242.

https://doi.org/10.1016/S0065-2911(08)60058-2

Byun, S., Lee, E., Lee, K. W. (2017). Therapeutic Implications of Autophagy Inducers in Immunological Disorders, Infection, and Cancer. Int. J. Mol. Sci., 18(9), E1959.

https://doi.org/10.3390/ijms18091959

Crowe, J. H. (2007). Trehalose as a "chemical chaperone": fact and fantasy. Adv. Exp. Med. Biol., 594, 143-158.

https://doi.org/10.1007/978-0-387-39975-1_13

Eriksson, P., Andre, L., Ansell, R., Blomberg, A., Adler, L. (1995). Cloning and characterization of GPD2, a second gene encoding sn-glycerol 3-phosphate dehydrogenase (NAD+) in Saccharomyces cerevisiae, and its comparison with GPD1. Mol. Microbiol., 17(1), 95-107.

https://doi.org/10.1111/j.1365-2958.1995.mmi_17010095.x

Francois, J., Parrou, J. L. (2001). Reserve carbohydrates metabolism in the yeast Saccharomyces cerevisiae. FEMS Microbiol. Rev., 25(1), 125-145.

https://doi.org/10.1111/j.1574-6976.2001.tb00574.x

Gonchar, M. V., Maidan, M. M., Pavlishko, H. M., Sibirny, A. A. (2001). A new oxidase-peroxidase kit for ethanol assays in alcoholic beverages. Food Technol. Biotechnol., 39, 37-42.

Grant, W. D. (2004). Life at low water activity. Philos. Trans. R. Soc. Lond. B. Biol. Sci., 359, 1249-1266; discussion 1266-1247.

https://doi.org/10.1098/rstb.2004.1502

Guo, Z. P., Zhang, L., Ding, Z. Y., Shi, G. Y. (2011). Minimization of glycerol synthesis in industrial ethanol yeast without influencing its fermentation performance. Metab. Eng., 13(1), 49-59.

https://doi.org/10.1016/j.ymben.2010.11.003

Hohmann, S. (2002). Osmotic stress signaling and osmoadaptation in yeasts. Microbiol. Mol. Biol. Rev., 66(2), 300-372.

https://doi.org/10.1128/MMBR.66.2.300-372.2002

Ishchuk, O. P., Voronovsky, A. Y., Abbas, C. A., Sibirny, A. A. (2009). Construction of Hansenula polymorpha strains with improved thermotolerance. Biotechnol. Bioeng., 104(5), 911-919.

https://doi.org/10.1002/bit.22457

Jung, Y. J., Park, H. D. (2005). Antisense-mediated inhibition of acid trehalase (ATH1) gene expression promotes ethanol fermentation and tolerance in Saccharomyces cerevisiae. Biotechnol. Lett., 27(23-24), 1855-1859.

https://doi.org/10.1007/s10529-005-3910-3

Kawai, S., Hashimoto, W., Murata, K. (2010). Transformation of Saccharomyces cerevisiae and other fungi: methods and possible underlying mechanism. Bioeng. Bugs., 1(6), 395-403.

https://doi.org/10.4161/bbug.1.6.13257

Kim, J., Alizadeh, P., Harding, T., Hefner-Gravink, A., Klionsky, D. J. (1996). Disruption of the yeast ATH1 gene confers better survival after dehydration, freezing, and ethanol shock: potential commercial applications. Appl. Environ. Microbiol., 62(5), 1563-1569.

https://doi.org/10.1128/AEM.62.5.1563-1569.1996

Larsson, K., Ansell, R., Eriksson, P., Adler, L. (1993). A gene encoding sn-glycerol 3-phosphate dehydrogenase (NAD+) complements an osmosensitive mutant of Saccharomyces cerevisiae. Mol. Microbiol., 10(5), 1101-1111.

https://doi.org/10.1111/j.1365-2958.1993.tb00980.x

Lee, Y. J., Jeschke, G. R., Roelants, F. M., Thorner, J., Turk, B. E. (2012). Reciprocal phosphorylation of yeast glycerol-3-phosphate dehydrogenases in adaptation to distinct types of stress. Mol. Cell Biol., 32(22), 4705-4717.

https://doi.org/10.1128/MCB.00897-12

Londesborough, J., Varimo, K. (1984). Characterization of two trehalases in baker's yeast. Biochem. J., 219(2), 511-518.

https://doi.org/10.1042/bj2190511

Luyten, K., Albertyn, J., Skibbe, W. F., Prior, B. A., Ramos, J., Thevelein, J. M., Hohmann, S. (1995). Fps1, a yeast member of the MIP family of channel proteins, is a facilitator for glycerol uptake and efflux and is inactive under osmotic stress. EMBO J., 14(7), 1360-1371.

https://doi.org/10.1002/j.1460-2075.1995.tb07122.x

Luzhetskyi, T., Semkiv, M., Dmytruk, K., Sibirny, A. (2015). Improving Thermotolerance of Saccharomyces cerevisiae Industrial Yeast Strain via Derepression of Genes of Trehalose Synthesis. In A. Sibirny, D. Fedorovych, M. Gonchar & D. Grabek-Lejko (Eds.), Living Organisms and Bioanalytical Approaches for Detoxification and Monitoring of Toxic Compounds: Monograph. (pp. 259-268). Rzeszow: University of Rzeszow.

Meynial Salles, I., Forchhammer, N., Croux, C., Girbal, L., Soucaille, P. (2007). Evolution of a Saccharomyces cerevisiae metabolic pathway in Escherichia coli. Metab. Eng., 9(2), 152-159.

https://doi.org/10.1016/j.ymben.2006.09.002

Norbeck, J., Pahlman, A. K., Akhtar, N., Blomberg, A., Adler, L. (1996). Purification and characterization of two isoenzymes of DL-glycerol-3-phosphatase from Saccharomyces cerevisiae. Identification of the corresponding GPP1 and GPP2 genes and evidence for osmotic regulation of Gpp2p expression by the osmosensing mitogen-activated protein kinase signal transduction pathway. J. Biol. Chem., 271(23), 13875-13881.

https://doi.org/10.1074/jbc.271.23.13875

Nwaka, S., Mechler, B., Holzer, H. (1996). Deletion of the ATH1 gene in Saccharomyces cerevisiae prevents growth on trehalose. FEBS Lett., 386(2-3), 235-238.

https://doi.org/10.1016/0014-5793(96)00450-4

Oliveira, A. P., Ludwig, C., Picotti, P., Kogadeeva, M., Aebersold, R., Sauer, U. (2012). Regulation of yeast central metabolism by enzyme phosphorylation. Mol. Syst. Biol., 8, 623.

https://doi.org/10.1038/msb.2012.55

Overkamp, K. M., Bakker, B. M., Kotter, P., Luttik, M. A., Van Dijken, J. P., Pronk, J. T. (2002). Metabolic engineering of glycerol production in Saccharomyces cerevisiae. Appl. Environ. Microbiol., 68(6), 2814-2821.

https://doi.org/10.1128/AEM.68.6.2814-2821.2002

Pahlman, A. K., Granath, K., Ansell, R., Hohmann, S., Adler, L. (2001). The yeast glycerol 3-phosphatases Gpp1p and Gpp2p are required for glycerol biosynthesis and differentially involved in the cellular responses to osmotic, anaerobic, and oxidative stress. J. Biol. Chem., 276(5), 3555-3563.

https://doi.org/10.1074/jbc.M007164200

Pettersson, N., Filipsson, C., Becit, E., Brive, L., Hohmann, S. (2005). Aquaporins in yeasts and filamentous fungi. Biol. Cell., 97(7), 487-500.

https://doi.org/10.1042/BC20040144

Randez-Gil, F., Sanz, P., Prieto, J. A. (1999). Engineering baker's yeast: room for improvement. Trends. Biotechnol., 17(6), 237-244.

https://doi.org/10.1016/S0167-7799(99)01318-9

Remize, F., Barnavon, L., Dequin, S. (2001). Glycerol export and glycerol-3-phosphate dehydrogenase, but not glycerol phosphatase, are rate limiting for glycerol production in Saccharomyces cerevisiae. Metab. Eng., 3(4), 301-312.

https://doi.org/10.1006/mben.2001.0197

Semkiv, M. V., Dmytruk, K. V., Abbas, C. A., Sibirny, A. A. (2017). Metabolic engineering for high glycerol production by the anaerobic cultures of Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol., 101(11), 4403-4416.

https://doi.org/10.1007/s00253-017-8202-z

Tamas, M. J., Luyten, K., Sutherland, F. C., Hernandez, A., Albertyn, J., Valadi, H., Li, H., Prior, B. A., Kilian, S. G., Ramos, J., Gustafsson, L., Thevelein, J. M., Hohmann, S. (1999). Fps1p controls the accumulation and release of the compatible solute glycerol in yeast osmoregulation. Mol. Microbiol., 31(4), 1087-1104.

https://doi.org/10.1046/j.1365-2958.1999.01248.x

Tapia, H., Koshland, D. E. (2014). Trehalose is a versatile and long-lived chaperone for desiccation tolerance. Curr. Biol., 24(23), 2758-2766.

https://doi.org/10.1016/j.cub.2014.10.005

ter Linde, J. J., Liang, H., Davis, R. W., Steensma, H. Y., van Dijken, J. P., Pronk, J. T. (1999). Genome-wide transcriptional analysis of aerobic and anaerobic chemostat cultures of Saccharomyces cerevisiae. J. Bacteriol., 181(24), 7409-7413.

https://doi.org/10.1128/JB.181.24.7409-7413.1999

Wiemken, A. (1990). Trehalose in yeast, stress protectant rather than reserve carbohydrate. Antonie Van Leeuwenhoek, 58(3), 209-217.

https://doi.org/10.1007/BF00548935

Yancey, P. H., Clark, M. E., Hand, S. C., Bowlus, R. D., Somero, G. N. (1982). Living with water stress: evolution of osmolyte systems. Science, 217(4566), 1214-1222.

https://doi.org/10.1126/science.7112124