Abstract
Conversion of xylose to xylitol by recombinant Saccharomyces cerevisiae expressing the XYL1 gene, encoding xylose reductase, was investigated by using different cosubstrates as generators of reduced cofactors. The effect of a pulse addition of the cosubstrate on xylose conversion in cosubstrate-limited fed-batch cultivation was studied. Glucose, mannose, and fructose, which are transported with high affinity by the same transport system as is xylose, inhibited xylose conversion by 99, 77, and 78%, respectively, reflecting competitive inhibition of xylose transport. Pulse addition of maltose, which is transported by a specific transport system, did not inhibit xylose conversion. Pulse addition of galactose, which is also transported by a specific transporter, inhibited xylose conversion by 51%, in accordance with noncompetitive inhibition between the galactose and glucose/ xylose transport systems. Pulse addition of ethanol inhibited xylose conversion by 15%, explained by inhibition of xylose transport through interference with the hydrophobic regions of the cell membrane. The xylitol yields on the different cosubstrates varied widely. Galactose gave the highest xylitol yield, 5.6 times higher than that for glucose. The difference in redox metabolism of glucose and galactose was suggested to enhance the availability of reduced cofactors for xylose reduction with galactose. The differences in xylitol yield observed between some of the other sugars may also reflect differences in redox metabolism. With all cosubstrates, the xylitol yield was higher under cosubstrate limitation than with cosubstrate excess.
Full Text
The Full Text of this article is available as a PDF (300.6 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bicho Paul A., Runnals P. Lynn, Cunningham J. Douglas, Lee Hung. Induction of Xylose Reductase and Xylitol Dehydrogenase Activities in Pachysolen tannophilus and Pichia stipitis on Mixed Sugars. Appl Environ Microbiol. 1988 Jan;54(1):50–54. doi: 10.1128/aem.54.1.50-54.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bisson L. F., Fraenkel D. G. Involvement of kinases in glucose and fructose uptake by Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1983 Mar;80(6):1730–1734. doi: 10.1073/pnas.80.6.1730. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
- Busturia A., Lagunas R. Catabolite inactivation of the glucose transport system in Saccharomyces cerevisiae. J Gen Microbiol. 1986 Feb;132(2):379–385. doi: 10.1099/00221287-132-2-379. [DOI] [PubMed] [Google Scholar]
- Cartwright C. P., Li Y., Zhu Y. S., Kang Y. S., Tipper D. J. Use of beta-lactamase as a secreted reporter of promoter function in yeast. Yeast. 1994 Apr;10(4):497–508. doi: 10.1002/yea.320100409. [DOI] [PubMed] [Google Scholar]
- Chambers A., Packham E. A., Graham I. R. Control of glycolytic gene expression in the budding yeast (Saccharomyces cerevisiae). Curr Genet. 1995 Dec;29(1):1–9. doi: 10.1007/BF00313187. [DOI] [PubMed] [Google Scholar]
- Chambers A., Stanway C., Tsang J. S., Henry Y., Kingsman A. J., Kingsman S. M. ARS binding factor 1 binds adjacent to RAP1 at the UASs of the yeast glycolytic genes PGK and PYK1. Nucleic Acids Res. 1990 Sep 25;18(18):5393–5399. doi: 10.1093/nar/18.18.5393. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chambers A., Tsang J. S., Stanway C., Kingsman A. J., Kingsman S. M. Transcriptional control of the Saccharomyces cerevisiae PGK gene by RAP1. Mol Cell Biol. 1989 Dec;9(12):5516–5524. doi: 10.1128/mcb.9.12.5516. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cirillo V. P. Galactose transport in Saccharomyces cerevisiae. I. Nonmetabolized sugars as substrates and inducers of the galactose transport system. J Bacteriol. 1968 May;95(5):1727–1731. doi: 10.1128/jb.95.5.1727-1731.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Does A. L., Bisson L. F. Comparison of glucose uptake kinetics in different yeasts. J Bacteriol. 1989 Mar;171(3):1303–1308. doi: 10.1128/jb.171.3.1303-1308.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- HARRIS G., THOMPSON C. C. The uptake of nutrients by yeasts. III. The maltose permease of a brewing yeast. Biochim Biophys Acta. 1961 Sep 2;52:176–183. doi: 10.1016/0006-3002(61)90915-5. [DOI] [PubMed] [Google Scholar]
- Hallborn J., Gorwa M. F., Meinander N., Penttilä M., Keränen S., Hahn-Hägerdal B. The influence of cosubstrate and aeration on xylitol formation by recombinant Saccharomyces cerevisiae expressing the XYL1 gene. Appl Microbiol Biotechnol. 1994 Nov;42(2-3):326–333. doi: 10.1007/BF00902737. [DOI] [PubMed] [Google Scholar]
- Hallborn J., Walfridsson M., Airaksinen U., Ojamo H., Hahn-Hägerdal B., Penttilä M., Keräsnen S. Xylitol production by recombinant Saccharomyces cerevisiae. Biotechnology (N Y) 1991 Nov;9(11):1090–1095. doi: 10.1038/nbt1191-1090. [DOI] [PubMed] [Google Scholar]
- Heredia C. F., Sols A., DelaFuente G. Specificity of the constitutive hexose transport in yeast. Eur J Biochem. 1968 Aug;5(3):321–329. doi: 10.1111/j.1432-1033.1968.tb00373.x. [DOI] [PubMed] [Google Scholar]
- Holland M. J., Holland J. P. Isolation and identification of yeast messenger ribonucleic acids coding for enolase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase. Biochemistry. 1978 Nov 14;17(23):4900–4907. doi: 10.1021/bi00616a007. [DOI] [PubMed] [Google Scholar]
- Kotyk A. Properties of the sugar carrier in baker's yeast. II. Specificity of transport. Folia Microbiol (Praha) 1967;12(2):121–131. doi: 10.1007/BF02896872. [DOI] [PubMed] [Google Scholar]
- Kuhn A., van Zyl C., van Tonder A., Prior B. A. Purification and partial characterization of an aldo-keto reductase from Saccharomyces cerevisiae. Appl Environ Microbiol. 1995 Apr;61(4):1580–1585. doi: 10.1128/aem.61.4.1580-1585.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kuo S. C., Cirillo V. P. Galactose transport in Saccharomyces cerevisiae. 3. Characteristics of galactose uptake in transferaseless cells: evidence against transport-associated phosphorylation. J Bacteriol. 1970 Sep;103(3):679–685. doi: 10.1128/jb.103.3.679-685.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kötter P., Amore R., Hollenberg C. P., Ciriacy M. Isolation and characterization of the Pichia stipitis xylitol dehydrogenase gene, XYL2, and construction of a xylose-utilizing Saccharomyces cerevisiae transformant. Curr Genet. 1990 Dec;18(6):493–500. doi: 10.1007/BF00327019. [DOI] [PubMed] [Google Scholar]
- Lagunas R. Energy metabolism of Saccharomyces cerevisiae discrepancy between ATP balance and known metabolic functions. Biochim Biophys Acta. 1976 Sep 13;440(3):661–674. doi: 10.1016/0005-2728(76)90049-9. [DOI] [PubMed] [Google Scholar]
- Meinander N., Zacchi G., Hahn-Hägerdal B. A heterologous reductase affects the redox balance of recombinant Saccharomyces cerevisiae. Microbiology. 1996 Jan;142(Pt 1):165–172. doi: 10.1099/13500872-142-1-165. [DOI] [PubMed] [Google Scholar]
- Mäkinen K. K. Xylitol and oral health. Adv Food Res. 1979;25:137–158. doi: 10.1016/s0065-2628(08)60236-0. [DOI] [PubMed] [Google Scholar]
- Nevado J., Navarro M. A., Heredia C. F. Galactose inhibition of the constitutive transport of hexoses in Saccharomyces cerevisiae. Yeast. 1993 Feb;9(2):111–119. doi: 10.1002/yea.320090202. [DOI] [PubMed] [Google Scholar]
- Nevado J., Navarro M. A., Heredia C. F. Transport of hexoses in yeast. Re-examination of the sugar phosphorylation hypothesis with a new experimental approach. Yeast. 1994 Jan;10(1):59–65. doi: 10.1002/yea.320100106. [DOI] [PubMed] [Google Scholar]
- Postma E., Verduyn C., Kuiper A., Scheffers W. A., van Dijken J. P. Substrate-accelerated death of Saccharomyces cerevisiae CBS 8066 under maltose stress. Yeast. 1990 Mar-Apr;6(2):149–158. doi: 10.1002/yea.320060209. [DOI] [PubMed] [Google Scholar]
- Schiestl R. H., Gietz R. D. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. doi: 10.1007/BF00340712. [DOI] [PubMed] [Google Scholar]
- Seaston A., Inkson C., Eddy A. A. The absorption of protons with specific amino acids and carbohydrates by yeast. Biochem J. 1973 Aug;134(4):1031–1043. doi: 10.1042/bj1341031. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Serrano R., Delafuente G. Regulatory properties of the constitutive hexose transport in Saccharomyces cerevisiae. Mol Cell Biochem. 1974 Dec 20;5(3):161–171. doi: 10.1007/BF01731379. [DOI] [PubMed] [Google Scholar]
- Serrano R. Energy requirements for maltose transport in yeast. Eur J Biochem. 1977 Oct 17;80(1):97–102. doi: 10.1111/j.1432-1033.1977.tb11861.x. [DOI] [PubMed] [Google Scholar]
- Thestrup H. N., Hahn-Hägerdal B. Xylitol formation and reduction equivalent generation during anaerobic xylose conversion with glucose as cosubstrate in recombinant Saccharomyces cerevisiae expressing the xyl1 gene. Appl Environ Microbiol. 1995 May;61(5):2043–2045. doi: 10.1128/aem.61.5.2043-2045.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tuite M. F., Dobson M. J., Roberts N. A., King R. M., Burke D. C., Kingsman S. M., Kingsman A. J. Regulated high efficiency expression of human interferon-alpha in Saccharomyces cerevisiae. EMBO J. 1982;1(5):603–608. doi: 10.1002/j.1460-2075.1982.tb01215.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Verduyn C., Postma E., Scheffers W. A., Van Dijken J. P. Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast. 1992 Jul;8(7):501–517. doi: 10.1002/yea.320080703. [DOI] [PubMed] [Google Scholar]
- Walfridsson M., Hallborn J., Penttilä M., Keränen S., Hahn-Hägerdal B. Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing the TKL1 and TAL1 genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase. Appl Environ Microbiol. 1995 Dec;61(12):4184–4190. doi: 10.1128/aem.61.12.4184-4190.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Walsh M. C., Smits H. P., Scholte M., van Dam K. Affinity of glucose transport in Saccharomyces cerevisiae is modulated during growth on glucose. J Bacteriol. 1994 Feb;176(4):953–958. doi: 10.1128/jb.176.4.953-958.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- van Zyl C., Prior B. A., Kilian S. G., Brandt E. V. Role of D-ribose as a cometabolite in D-xylose metabolism by Saccharomyces cerevisiae. Appl Environ Microbiol. 1993 May;59(5):1487–1494. doi: 10.1128/aem.59.5.1487-1494.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- van Zyl C., Prior B. A., Kilian S. G., Kock J. L. D-xylose utilization by Saccharomyces cerevisiae. J Gen Microbiol. 1989 Nov;135(11):2791–2798. doi: 10.1099/00221287-135-11-2791. [DOI] [PubMed] [Google Scholar]