Sezai TÜRKEL, Elif ARIK, Sinem GÜZELVARDAR

The effect of hperosmotic stress and nitrogen starvation growth and $ Beta$-galactosidase synthesis in kluyveromyces lactis and kluyveromyces marxianus

Kluyveromyces lactis ve Kluyveromyces marxianus β-galaktozidaz enzimi üretiminde yaygın olarak kullanılan endüstriyel mayalardır. β-galaktozidaz’ın biyosentezi glukoz baskılaması ile kontrol edilir. Bu çalışmada β-galaktozidaz biyozentezi derepresyonunun yüksek ozmotik stress tarafından inhibe edildiği gösterildi. K. lactis ve K. marxianus’ta β-galactozidaz aktivitelerinin bu maya türleri NaCl, KCl veya sukroz tarafından oluşturulan yüksek ozmotik strese uğradıklarında β-galaktozidaz aktivitelerinin yaklaşık olarak baskılanmış seviyede olduğu bulundu. K. marxianus’da β-galaktozidaz biyosentezinin ozmotik strese K. lactis’den daha hassas olduğu görülmektedir. Buna ek olarak, azot açlığının K. lactis’de β-galaktozidaz biyosentezinde önemli seviyede azalmaya neden olurken, K. marxianus’da 2 kat artışa yol açtığı gösterildi. Bu çalışmanın sonuçları yüksek seviyede sukroz’un değil NaCl’nin K. lactis ve K. marxianus’da üremeyi inhibe ettiğini gösterdi.

Yüksek ozmotik stres ve azot açlığının kluyveromyces lactis ve kluyveromyces marxianus'ta üreme hızı ve $ Beta$-galaktozidaz sentezine etkileri

Abstract: Kluyveromyces lactis and Kluyveromyces marxianus are industrial yeasts widely used in the production of the β-galactosidase enzyme. Biosynthesis of β-galactosidase is controlled by glucose repression. In this study it was demonstrated that the derepression of β-galactosidase biosynthesis in these yeast strains is inhibited by high osmotic stress. It was found that the β-galactosidase activity of K. lactis and K. marxianus remained approximately at the repressed level when these yeast cells were subjected to NaCl-, KCl-, or sucrose-induced high osmotic stress. Derepression of β-galactosidase biosynthesis seems to be more sensitive to high osmotic stress in K. marxianus than in K. lactis. In addition, it was shown that nitrogen starvation resulted in a significant decrease in the level of β-galactosidase biosynthesis in K. lactis, while nitrogen starvation led to a 2-fold increase in β-galactosidase biosynthesis in K. marxianus. Results of this study indicated that high levels of NaCl, but not sucrose, inhibited the growth of K. lactis and K. marxianus.

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1. Rajoka MI, Khan S, Shahid R. Kinetics and regulation studies of the production of beta-galactosidase from Kluyveromyces marxianus grown on different substrates. Food Technol Biotechnol 41: 315-320, 2003.
2. Siso M. Beta-galactosidase production by Kluyveromyces lactis on milk whey. Batch versus Fed-batch cultures. Process Biochem 29: 565-568, 1994.
3. Rubio-Texeira M. Endless versatility in the biotechnological applications of Kluyveromyces LAC genes. Biotechnology Adv 24: 212-225, 2006.
4. Adam AC, Rubio-Texeira M, Polaina J. Lactose: The milk sugar from a biotechnological perspective. Crit Rev Food Sci Nutr 44: 553-557, 2004.
5. Lee Y-J, Kim CS, Oh D-K. Lactulose production by β-galactosidase in permeabilized cells of Kluyveromyces lactis. Appl Microbiol Biotechnol 64: 787-793, 2004.
6. Sheetz R, Dickson RC. LAC4 is the structural gene for β- galactosidase in Kluyveromyces lactis. Genetics 98: 729-745, 1981.
7. Zachariae W, Kuger P, Breunig KD. Glucose repression of lactose/galactose metabolism in Kluyveromyces lactis is determined by the concentration of the transcriptional activator LA1C9 (K1GAL4). Nucl Acids Res 21: 69-77, 1993.
8. Breunig KD. Glucose repression of LAC gene expression in yeast is mediated by the transcriptional activator LAC9. Mol Gen Genet 216: 422-427, 1989.
9. van Ooyen AJJ, Dekker P, Huang M et al. Heterologous protein production in the yeast Kluyveromyces lactis. FEMS Yeast Res 6: 381-392, 2006.
10. Attfield PV, Kletsas S. Hyperosmotic stress response by strains of bakers’ yeasts in high sugar concentration medium. Lett Applied Microbiol 31: 323-327, 2000.
11. Myers DK, Lawlor DTM, Attfield PV. Influence of invertase and glycerol synthesis and retention on fermentation of media with a high sugar concentration by Saccharomyces cerevisiae. Appl Environ Microbiol 63: 145-150, 1997.
12. Pinheiro R, Belo I, Mota M. Growth and β-galactosidase activity in cultures of Kluyveromyces marxianus under increased air pressure. Lett Applied Microbiol 37: 438-442, 2003.
13. Furlan SA, Schneider ALS, Merkle R et al. Optimization of pH, temperature and inoculum ratio for the production of b-Dgalactosidase by Kluyveromyces marxianus using lactose-free medium. Acta Biotechnol 21: 57-64, 2001.
14. Dickson RC, Markin JS. Physiological studies of β-galactosidase induction in Kluyveromyces lactis. J Bacteriol 142: 777-785,1980.
15. Türkel S, Turgut T, Kayakent N. Effect of osmotic stress on the derepression of invertase synthesis in nonconventional yeasts. Lett Applied Microbiol 42: 78-82, 2006.
16. Aon JC, Cortassa S. Involvement of nitrogen metabolism in the triggering of ethanol fermentation in aerobic chemostat cultures of Saccharomyces cerevisiae. Metabolic Eng 3: 250-264, 2001.
17. Silveira MCF, Oliveira EMM, Carjaval E et al. Nitrogen regulation of Saccharomyces cerevisiae invertase. Role of the URE2 gene. Appl Biochem Biotechnol 84-86: 247-254, 2000.
18. Cooper TG. Transmitting the signal of excess nitrogen in Saccharomyces cerevisiae from the Tor proteins to the GATA factors: connecting the dots. FEMS Microbiol Rev 26: 223-238,2002.
19. Hohmann S. Osmotic stress signaling and osmoadaptation in yeasts. Microbiol Mol Biol Rev 66: 300-372, 2002.
20. Siderius M, Kolen CPAM, van Heerikhuizen H et al. Candidate osmosensors from Candida utilis and Kluyveromyces lactis: structural and functional homology to the Sho1p putative osmosensor from Saccharomyces cerevisiae. Biochim Biophys Acta 1517: 143-147, 2000.
21. Rep M, Krantz M, Thevelein JM et al. The transcriptional response of Saccharomyces cerevisiae to osmotic shock. Hot1p and Msn2p/Msn4p are required for the induction of subsets of high osmolarity glycerol pathway-dependent genes. J Biol Chem 275: 8290-8300, 2000.
22. Springael J-Y, Penninckx MJ. Nitrogen-source regulation of yeast g-glutamyl transpeptidase synthesis involves the regulatory network including the GATA zinc-finger factors Gln3, Nil/Gat1 and Gzf3. Biochem J 371: 589-595, 2003.
23. Flores MV, Voget CE, Ertola RJJ. Permeabilization of yeast cells (Kluyveromyces lactis) with organic solvents. Enzyme Microb Technol 16: 340-346, 1994.
24. Guarente L. Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol 101: 181-191, 1983.
25. Bussereau F, Casaregola S, Lafay J-F et al. The Kluyveromyces lactis repertoire of transcriptional regulators. FEMS Yeast Res 6: 325-335, 2006.
26. Proft M, Struhl K. MAP kinase-mediated stress relief that precedes and regulates the timing of transcriptional induction. Cell 118: 351-361, 2004.
27. ter Schure EG, van Riel NAW, Verrips CT. The role of ammonia metabolism in nitrogen catabolite repression in Saccharomyces cerevisiae. FEMS Microbiol Rev 24: 67-83, 2000.
28. Magasanik B, Kaiser CA. Nitrogen regulation in Saccharomyces cerevisiae. Gene 290: 1-18, 2002.
29. Beltran G, Novo M, Rozes N et al. Nitrogen catabolite repression in Saccharomyces cerevisiae during wine fermentations. FEMS Yeast Res 4: 625-632, 2004.
30. Haro R, Garciadeblas B, Rodriguez-Navarro A. A novel P-type ATPase from yeast involved in sodium transport. FEBS Lett 291: 189-191, 1991.
31. Wieland J, Nitsche AM, Strayle J et al. The PMR2 gene cluster encodes functionally distinct isoforms of a putative Na+ pump in the plasma membrane. EMBO J 14: 3870-3882, 1995.
32. Garciadeblas B, Rubio F, Quintero FJ et al. Differential expression of two genes encoding isoforms of the ATPase involved in sodium efflux in Saccharomyces cerevisiae. Mol Gen Genet 236: 363-368, 1993.