Abstract
To produce an industrial strain of Saccharomyces cerevisiae that metabolizes xylose, we constructed a rDNA integration vector and YIp integration vector, containing the xylose-utilizing genes, XYL1 and XYL2, which encode xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis, and XKS1, which encodes xylulokinase (XK) from S. cerevisiae, with the G418 resistance gene KanMX as a dominant selectable marker. The rDNA results in integration of multiple copies of the target genes. The industrial stain of S. cerevisiae NAN-27 was transformed with the two integration vectors to produce two recombinant strains, S. cerevisiae NAN-127 and NAN-123. Upon transformation, multiple copies of the xylose-utilizing genes were integrated into the genome rDNA locus of S. cerevisiae. Strain NAN-127 consumed twice as much xylose and produced 39% more ethanol than the parent strain, while NAN-123 consumed 10% more xylose and produced 10% more ethanol than the parent strain over 94 h.
Similar content being viewed by others
References
Blomqvist K, Suihko ML, Knowles J, Penttilä M (1991) Chromosomal integration and expression of two bacterial α-acetolactate decarboxylase genes in brewer's yeast. Appl. Environ. Microbiol. 57: 2796–2803.
Eliasson A, Christensson C, Wahlbom CF, Hahn-Hägerdal B (2000) Anaerobic xylose fermentation by recombinant Saccharomyces cerevisiae carrying XYL1, XYL2 and XKS1 in mineral medium chemostat cultures. Appl. Environ. Microbiol. 66: 3381–3386.
Gietz RD, Schiestl RH, Willems AR, Woods RA (1995) Studies on the transformation of intact yeast cells by the LiAc/SSDNA/PEG procedure. Yeast 11: 355–360.
Lopes TS, Klootwijk J, Veenstra AE, van der Aar PC, van Heerikhuizen H, Raue HA, Planta RJ (1989) High-copynumber integration into the ribosomal DNA of Saccharomyces cerevisiae: a new vector for high-level expression. Gene 79: 199–206.
Mao H, Qu YB, Gao PJ, Li W (1996) Improvement of xylose fermentation by intergeneric protoplast fusion of Pichia stipitis and Saccharomyces cerevisiae. Chin. J. Biotechnol. 12: 157–162.
Meinander NQ, Boels I, Hahn-Hägerdal B (1999) Fermentation of xylose/glucose mixtures by metabolically engineered Saccharomyces cerevisiae strains expressing XYL1 and XYL2 from Pichia stipitis with and without overexpression of TAL1. Bioresour. Technol. 68: 79–87.
Mellor J, Dobson MJ, Roberts NA, Tuite MF, Emtage JS, White S, Lowe PA, Patel T, Kingsman AJ, Kingsman SM (1983) Efficient synthesis of enzymatically active calf chymosin in Saccharomyces cerevisiae. Gene 24: 1–14.
Rizzi M, Harwart K, Erleman P, Bui-Thanh NA, Dellweg H (1989) Purification and properties of the NAD+-dependent xylitol dehydrogenase from yeast Pichia stipitis. J. Ferment. Bioeng. 67: 20–24.
Shamanna DK, Sanderson KE (1979) Uptake and catabolism of Dxylose in Salmonella typhimurium LT2. J. Bacteriol. 139: 64–70.
Smiley KL, Bolen PL (1982) Demonstration of D-xylose reductase and D-xylitol dehydrogenase in Pachysolen tannophilus. Biotechnol. Lett. 4: 607–610.
van Zyl C, Prior BA, Kilian SG, Brandt EV (1993) Role of D-ribose as a cometabolite in D-xylose metabolism by Saccharomyces cerevisiae. Appl. Environ. Microbiol. 59: 1487–1494.
Wach A, Brachat A, Pohlmann R, Philippsen P (1994) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10: 1793–1808.
Walfridsson M, Bao X, Anderlund M, Lilius G, Bülow L, Hahn-Hägerdal B (1996) Ethanolic fermentation of xylose with Saccharomyces cerevisiae harboring the Thermus thermophilus xylA gene which expresses an active xylose (glucose) isomerase. Appl. Environ. Microbiol. 62: 4648–4651.
Walker ME, Gardner JM, Vystavelova A, McBryde C, de Barros Lopes M, Jiranek V (2003) Application of the reuseable, Kan MX selectable marker to industrial yeast: construction and evaluation of heterothallic wine strains of Saccharomyces cerevisiae, possessing minimal foreign DNA sequences. FEMS Yeast Res. 4: 339–347.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Wang, Y., Shi, WL., Liu, XY. et al. Establishment of a xylose metabolic pathway in an industrial strain of Saccharomyces cerevisiae . Biotechnology Letters 26, 885–890 (2004). https://doi.org/10.1023/B:bile.0000025897.21106.92
Issue Date:
DOI: https://doi.org/10.1023/B:bile.0000025897.21106.92