Abstract
Yeasts are excellent hosts for the production of recombinant proteins. Candida glycerinogenes WL2002-5, an osmotolerant yeast with extremely high glycerol productivity, provides an attractive eukaryotic expression platform. The integrative vectors PURGAP-gfp and PURGPD-gfp harbouring phleomycin-resistance coding sequence and GFP coding sequence with PCgGAP, PCgGPD promoter, respectively, were constructed. The recombinant plasmid PURPpGAP-gfp with the promoter PPpGAP based on the sequence of Pichia pastoris GAPDH gene and the plasmid PURScGAP-gfp with the promoter PScGAP from Saccharomyces cerevisiae were constructed. After transformation, the copy number of gfp gene, which determined using fluorescent quantitative real-time polymerase chain reaction (FQ-RTPCR) in genome of C. glycerinogenes is 1. Expressions of gfp at different levels were conducted using different promoters by osmotic stress containing NaCl or glucose for the recombinant strains. In this study, C. glycerinogenes WL2002-5, expressing xylitol dehydrogenase (XYL2) gene from Pichia stipitis, has the ability to produce glycerol from xylose entered into pentose phosphate pathway. Two recombinant strains of PURGAPX, PURGPDX with XYL2 overexpression were constructed to ferment a mixture of glucose and xylose simultaneously in batch fermentation. Compared to C. glycerinogenes WL2002-5 strain, glycerol production from xylose in strains PURGAPX, PURGPDX were increased by 95.9 and 121.1 %, respectively.
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References
Abdel NA, El-Moghaz (2010) Comparative study of salt tolerance in Saccharomyces cerevisiae and Pichia pastoris yeast strains. Adv Biores 1:169–176
Bisson LF, Neigeborn L, Carlson M, Fraenkel DG (1987) The SNF3 gene is required for high-affinity glucose transport in Saccharomyces cerevisiae. J Bacteriol 169:1656–1662
Blomberg A, Adler L (1992) Physiology of osmotolerance in fungi. Adv Microb Physiol 33:145–212
Bouabe H, Fassler R, Heesemann J (2008) Improvement of reporter activity by IRES-mediated polycistronic reporter system. Nucleic Acids Res 36:e28
Busturia A, Lagunas R (1986) Catabolite inactivation of the glucose transport system in Saccharomyces cerevisiae. J Gen Microbiol 132:379–385
Chen XZ, Fang HY, Rao ZM, Shen W, Zhuge B, Wang ZX, Zhuge J (2008) Cloning and characterization of a NAD+-dependent glycerol-3-phosphate dehydrogenase gene from Candida glycerinogenes, an industrial glycerol producer. FEMS Yeast Res 8:725–734
Cirillo VP (1968) Relationship between sugar structure and competition for the sugar transport system in baker’s yeast. J Bacteriol 95:603–611
Dhanalakshmi C, Michael DM, James S, James N, Aimen FS, Geoffrey PM, Leslie JF (2006) Multicistronic lentiviral vectors containing the FMDV 2A cleavage factor demonstrate robust expression of encoded genes at limiting MOI. Virol J 3:14
Eliasson A, Christensson C, Wahlbom CF, Hahn HB (2000) Anaerobic xylose fermentation by recombinant Saccharomyces cerevisiae carrying XYL1, XYL2, and XKS1 in mineral medium chemostat cultures. Appl Environ Microbiol 66:3381–3386
Garber RC, Turgeon BG, Selker EU, Yoder OC (1988) Organization of ribosomal RNA genes in the fungus Cochliobolus heterostrophus. Curr Genet 14:573–582
Girio FM, Roseiro JC, Sa-Machado P, Duarte-Reis AR, Amaral-Collaqo MT (1994) Effect of oxygen transfer rate on levels of key enzymes of xylose metabolism in Debaryomyces hansenii. Enzyme Microb Technol 16:1074–1078
Grace QC, Jiann-Tsyh L (2010) Use of Quantitative Polymerase Chain Reaction for Determining Copy Numbers of Transgenes in Lesquerella fendleri. Am J Agric Biol Sci 5:415–421
Hernandez M, Esteve T, Prat S, Pla M (2004) Development of realtime PCR systems based on SYBR Green I, Amplifluor and TaqMan technologies for specific quantitative detection of the transgenic maize event GA21. J Cereal Sci 39:99–107
Hohmann S (2002) Osmotic stress signaling and osmoadaptation in yeasts. Microbiol Mol Biol Rev 66:300–372
Hohmann S, Krantz M, Nordlander B (2007) Yeast osmoregulation. Methods Enzymol 428:29–45
Jayaram M, Li YY, Broach JR (1983) The yeast plasmid 2μ encodes components required for its high copy propagation. Cell 34:95–104
Kengo S, Daisuke S, Yuri S, Hiroshi T, Ryosuke Y, Tomohisa H, Chiaki O, Akihiko K (2013) Ethanol fermentation by xylose- assimilating Saccharomyces cerevisiae using sugars in a rice straw liquid hydrolysate concentrated by nanofiltration. Bioresour Technol 147:84–88
Kleinzeller A, Kotyk A (1967) Transport of monosaccharides in yeast cells and its relationship to cell. In: Mills AK, Krebs H (eds) Aspects of yeast metabolism. F. A. Davis Co., Philadelphia, pp 33–45
Kotyk A (1967) Properties of the sugar carrier in baker’s yeast. II. Specificity of transport. Folia Microbiol 12:121–131
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lopes TS, Hakkaart GJ, Koerts BL, Raué HA, Planta RJ (1991) Mechanism of high-copy-number integration of pMIRY type vectors into the ribosomal DNA of Saccharomyces cerevisiae. Gene 105:83–90
Lopes TS, Klootwijk J, Veenstra AE, van der Aar PC, van HH, Raúe HA, Planta RJ (1989) High-copy number integration into the ribosomal DNA of Saccharomyces cerevisiae: a new vector for high-level expression. Gene 79:199–206
Lopes TS, de Wijs IJ, Steenhauer SI, Verbakel J, Planta RJ (1996) Factors affecting the mitotic stability of high-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae. Yeast 12:467–477
Maleszka R, Clark-Walker GD (1993) Yeasts have a four-fold variation in ribosomal DNA copy number. Yeast 9:53–58
Mazzola JL, Sirover MA (2003) Subcellular localization of human glyceraldehydes -3-phosphate dehydrogenase is independent of its glycolytic function. Biochim Biophys Acta 1622:50–56
Meiswinkel TM, Gopinath V, Lindner SN, Nampoothiri KM, Wendisch VF (2013) Accelerated pentose utilization by Corynebacterium glutamicum for accel-erated production of lysine, glutamate, ornithine and putrescine. Microb Biotechnol 6:131–140
Melamed D, Pnueli L, Arava Y (2008) Yeast translational response to high salinity: global analysis reveals regulation at multiple levels. RNA 14:1337–1351
Nolleau V, Preziosi-Belloy L, Delgenes JP, Navarro JM (1993) Xylitol production from xylose by two yeast strains: sugar tolerance. Curr Microbial 27:191–197
Petes TD (1979) Yeast ribosomal DNA genes are located on chromosomeXII. Proc Natl Acad Sci USA 76:410–414
Ponchel F (2003) Real-time PCR based on SYBR-Green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions. BMC Biotechnol 3:18
Ralser M, Walmelink MW, Kowald A, Gerisch B, Heeren G, Struys EA, Klipp E, Jakobs C, Breitenbach M, Lehrach H, Krobitsch S (2007) Dynamic rerouting of the carbohydrate flux is key to counteracting oxidative stress. J Biol 6:10
Serrano R, Delafuente G (1974) Regulatory properties of the constitutive hexose transport in Saccharomyces cerevisiae. Mol Cell Biochem 5:161–171
Sirover MA (2005) New nuclear functions of the glycolytic protein, glyceraldehydes- 3-phosphate dehydrogenase in mammalian cells. J Cell Biochem 95:45–52
Suk-Jin H, Soo RK, Heejin K, Jing D, Jamie HDC, Yong-Su J (2013) Continuous co-fermentation of cellobiose and xylose by engineered Saccharomyces cerevisiae. Bioresour Technol 149:525–531
Tetsuya G, Kanako N, Masaharu T, Hiroyuki I, Shinichi Y, Tamotsu H, Akinori M (2013) Ethanol fermentation from xylose by metabolically engineered strains of Kluyveromyces marxianus. J Biosci Bioeng 116:551–554
Udem SA, Warner JR (1972) Ribosomal RNA synthesis in Saccharomyces cerevisiae. J Mol Biol 65:227–242
Vall ZC, Prior BA, Brandt EV (1993) Role of d-ribose as a cometabolite in d-xylose metabolism by Saccharomyces cerevisiae. Appl Environ Micorbiol 59:1487–1494
Vandeska E, Kuzmanova S, Jeffries TW (1995) Xylitol formation and key enzyme activities in Cundidu boidinii under different oxygen transfer rates. J Ferment Bioeng 80:513–516
Van HP, van Dijken JP, Pronk JT (2000) Regulation of fermentative capacity and levels of glycolytic enzymes in chemostat cultures of Saccharomyces cerevisiae. Enzyme Microb Technol 26:724–736
Walfridsson M, Anderlund M, Bao X, Hahn-Hagerdal B (1997) Expression of different levels of enzymes from the Pichia stipitis XYL1 and XYL2 genes in Saccharomyces cerevisiae and its effects on product formation during xylose utilisation. Appl Microbiol Biotechnol 48:218–224
Wang CQ, Shen Y, Hou J, Suo F, Bao XM (2013) An assay for functional xylose transporters in Saccharomyces cerevisiae. Anal Biochem 442:241–248
Wang M, Fu J, Zhang X, Chen T (2012) Metabolic engineering of Bacillus subtilisfor enhanced production of acetoin. Biotechnol Lett 34:1877–1885
Wang ZX, Zhuge J, Fang H, Prior BA (2001) Glycerol production by microbial fermentation: a review. Biotechnol Adv 19:201–223
Waschk D, Klabunde J, Suckow M, Hollenberg CP (2002) Characteristics of the Hansenulapolymorpha genome. In: Gellissen G (ed) Hansenulapolymorpha biology and applications. Wiley, Weinheim, pp 95–104
Yuan JS, Burris J, Stewart NR, Mentewab A, Stewart CN (2007) Statistical tools for transgene copy number estimation based on real-time PCR. BMC Bioinformatics 8: S6
Zhang C, Zhuge B, Zhan XB, Fang HY, Zong H, Zhuge J (2013) Cloning and characterization of a novel NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase gene from Candida glycerinogenes and use of its promoter. Yeast 30:157–163
Zhuge J, Fang HY, Wang ZX, Chen DZ, Jin HR, Gu HL (2001) Glycerol production by a novel osmotolerant yeast Candida glycerinogenes. Appl Microbiol Biotechnol 55:686–692
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This work was funded by China National ‘‘863’’ High-Tech Program (No.2012AA021201, No.2011AA02A207) and supported by the National Natural Science Foundation of China (No. 31270080).
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Zhang, C., Zong, H., Zhuge, B. et al. Integrative expression vectors for overexpression of xylitol dehydrogenase (XYL2) in Osmotolerant yeast, Candida glycerinogenes WL2002-5. J Ind Microbiol Biotechnol 42, 113–124 (2015). https://doi.org/10.1007/s10295-014-1530-4
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DOI: https://doi.org/10.1007/s10295-014-1530-4