Abstract
Fuel ethanol is an environmentally friendly alternative liquid fuel to the widely used petroleum derived transportation liquid fuels. Since 2007, worldwide fuel ethanol production has increased. Currently ethanol is primarily produced from carbohydrates such as sucrose and starch by fermentation using the yeast Saccharomyces cerevisiae. In this work, new approaches for the selection of S. cerevisiae strains with increased ethanol production from hydrolyzed corn meal are described. An industrial production strain of Saccharomyces cerevisiae AS400 was subjected to positive selection of mutants resistant to toxic concentrations of oxythiamine, trehalose, 3-bromopyruvate, glyoxylic acid, and glucosamine. The selected mutants are characterized by 5-8% increase in ethanol yield (g g-1 of consumed glucose) as compared to the parental industrial ethanol-producing strain. A three-step selection approach that consisted of the use of glyoxylic acid, glucosamine and bromopyruvate resulted in a 12% increase in ethanol yield during fermentation on industrial media. These results indicate that the selected strains are promising candidates for industrial ethanol production.
References
[1] Dashko S., Zhou N., Compagno C., Piškur J., Why, when, and how did yeast evolve alcoholic fermentation?, FEMS Yeast Res., 2004, 14(6), 826-832. 10.1111/1567-1364.12161Search in Google Scholar PubMed PubMed Central
[2] Piškur J., Langkjaer R.B., Yeast genome sequencing: the power of comparative genomics, Mol. Microbiol., 2004, 53, 381–389. 10.1111/j.1365-2958.2004.04182.xSearch in Google Scholar PubMed
[3] Gombert A.K., van Maris A.J., Improving conversion yield of fermentable sugars into fuel ethanol in 1st generation yeast-based production processes, Curr. Opin. Biotechnol., 2015, 7, 33C, 81-86. 10.1016/j.copbio.2014.12.012Search in Google Scholar PubMed
[4] Nissen T.L., Kielland-Brandt M.C., Nielsen J., Villadsen J., Optimization of ethanol production in Saccharomyces cerevisiae by metabolic engineering of the ammonium assimilation, Metab. Eng., 2000, 2, 69-77. 10.1006/mben.1999.0140Search in Google Scholar PubMed
[5] Bro C., Regenberg B., Forster J., Nielsen J., In silico aided metabolic engineering of Saccharomyces cerevisiae for improved bioethanol production, Metab. Eng. 2006, 8, 102-111. Search in Google Scholar
[6] Guo Z., Zhang L., Ding Z., Shi G., Minimization of glycerol synthesis in industrial ethanol yeast without influencing its fermentation performance, Metab. Eng., 2011, 13, 49-59. 10.1016/j.ymben.2010.11.003Search in Google Scholar PubMed
[7] Zhang L., Tang Y., Guo Z., Ding Z., Shi G., Improving the ethanol yield by reducing glycerol formation using cofactor regulation in Saccharomyces cerevisiae, Biotechnol. Lett., 2011, 33, 1375-1380. 10.1007/s10529-011-0588-6Search in Google Scholar PubMed
[8] de Kok S., Kozak B.U., Pronk J.T., Van Maris A., Energy coupling in Saccharomyces cerevisiae: selected opportunities formetabolic engineering, FEMS Yeast Res., 2012, 12, 387-397. 10.1111/j.1567-1364.2012.00799.xSearch in Google Scholar PubMed
[9] Navas M.A., Cerdan S., Gancedo J.M., Futile cycles in Saccharomyces cerevisiae strains expressing the gluconeogenic enzymes during growth on glucose, Proc. Nat. Acad. Sci. USA, 1993, 90, 1290-1294. 10.1073/pnas.90.4.1290Search in Google Scholar PubMed PubMed Central
[10] Dmytruk K.V., Semkiv M.V., Sibirny A.A., Ethanol yield and reduction of biomass accumulation in the recombinant strain of Saccharomyces cerevisiae overexpressing ATPase, WO/2010/151866. 2010. Search in Google Scholar
[11] Semkiv M.V., Dmytruk K.V., Abbas C.A., Sibirny A.A., Increased ethanol accumulation from glucose via reduction of ATP level in a recombinant strain of Saccharomyces cerevisiae overexpressing alkaline phosphatase, BMC Biotechnol., 2014, 14, 42. 10.1186/1472-6750-14-42Search in Google Scholar PubMed PubMed Central
[12] Grossmann M., Kiessling F., Singer J., Schoeman H., Schröder M.B., von Wallbrunn C., Genetically modified wine yeasts and risk assessment studies covering different steps within the wine making process, Ann. Microbiol., 2011, 61, 103–115. 10.1007/s13213-010-0088-2Search in Google Scholar
[13] Chambers P.J., Bellon J.R., Schmidt S.A., Varela C., Pretorius I.S., Non-genetic engineering approaches to isolating and generating novel yeast for industrial applications, In: Kunze G., Satyanarayana T. (eds), Yeast biotechnology: Diversity and applications, Springer Science+Business Media, 433–457, 2009. Search in Google Scholar
[14] Cakar Z.P., Seker U.O., Tamerler C., Sonderegger M., Sauer U., Evolutionary engineering of multiple-stress resistant Saccharomyces cerevisiae, FEMS Yeast Res., 2005, 5, 569–578. 10.1016/j.femsyr.2004.10.010Search in Google Scholar PubMed
[15] Zeyl C., The number of mutations selected during adaptation in a laboratory population of Saccharomyces cerevisiae, Genetics, 2005, 169, 1825–1831. 10.1534/genetics.104.027102Search in Google Scholar PubMed PubMed Central
[16] Kuyper M., Toirkens M.J., Diderich J.A., Winkler A.A., van Dijken J.P., Pronk J.T., Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain, FEMS Yeast Res., 2005, 5, 925–934. 10.1016/j.femsyr.2005.04.004Search in Google Scholar PubMed
[17] Higgins V.J., Bell P.J.L., Dawes I.W., Attfield P.V., Generation of a novel Saccharomyces cerevisiae strain that exhibits strong maltose utilization and hyperosmotic resistance using nonrecombinant techniques, Appl. Environ. Microbiol., 2001, 67, 4346–4348. 10.1128/AEM.67.9.4346-4348.2001Search in Google Scholar PubMed PubMed Central
[18] Stanley D., Fraser S., Chambers P.J., Rogers P., Stanley G.A., Generation and characterisation of stable ethanol-tolerant mutants of Saccharomyces cerevisiae, J. Ind. Microbiol. Biot., 2010, 37, 139–149. 10.1007/s10295-009-0655-3Search in Google Scholar PubMed
[19] Cadiere A., Ortiz-Julien A., Camarasa C., Dequin S., Evolutionary engineered Saccharomyces cerevisiae wine yeast strains with increased in vivo flux through the pentose phosphate pathway, Metab. Eng., 2011, 13, 263–271. 10.1016/j.ymben.2011.01.008Search in Google Scholar PubMed
[20] Lawford H.G., Rousseau J.D., Corn steep liquor as a cost-effective nutrition adjunct in high-performance Zymomonas ethanol fermentations, Applied Biochemistry and Biotechnology, Spring 1997, 63-65, 1, 287-304. 10.1007/BF02920431Search in Google Scholar PubMed
[21] Christianson D.D., Cavins J.F., Wall J.S., Steep liquor constituents, identification and determination of nonprotein nitrogenous substances in corn steep liquor., J. Agric. Food Chem., 1965, 13(3), 277–280. 10.1021/jf60139a023Search in Google Scholar
[22] Lin Y., Tanaka S., Ethanol fermentation from biomass resources: current state and prospects, Appl. Microbiol. Biotechnol., 2006, 69, 627-642. 10.1007/s00253-005-0229-xSearch in Google Scholar PubMed
[23] Gonchar M.V., Sensitive method for quantitative determination of hydrogen peroxide and oxidase substrates in biological samples, Ukr. Biokhim. Zh., 1998, 70, 157–163. Search in Google Scholar
[24] Gonchar M.V., Maidan M.M., Pavlishko H.M., Sibirny A.A., A new oxidase-peroxidase kit for ethanol assays in alcoholic beverages, Food. Technol. Biotechnol. 2001, 39, 37–42. Search in Google Scholar
[25] Tylicki A., Czerniecki J., Dobrzyn P., Matanowska A., Olechno A., Strumilo S., Modification of thiamine pyrophosphate dependent enzyme activity by oxythiamine in Saccharomyces cerevisiae cells, Can. J. Microbiol., 2005, 51(10), 833-839. 10.1139/w05-072Search in Google Scholar PubMed
[26] Kaino T., Takagi H., Gene expression profiles and intracellular contents of stress protectants in Saccharomyces cerevisiae under ethanol and sorbitol stresses, Appl. Microbiol. Biotechnol., 2008, 79, 273–283. 10.1007/s00253-008-1431-4Search in Google Scholar
[27] Lippert K., Galinski E.A., Trueper H.G., Biosynthesis and function of trehalose in Ectothiorhodospira halochloris, Antonie Leeuwenhoek 1993, 63, 85-91. 10.1007/BF00871735Search in Google Scholar
[28] Ko Y.H., Pedersen P.L., Geschwind J.F., Glucose catabolism in the rabbit VX2 tumor model for liver cancer: characterization and targeting hexokinase, Cancer Lett., 2001, 173, 83–91. 10.1016/S0304-3835(01)00667-XSearch in Google Scholar
[29] Geschwind J.F., Georgiades C.S., Ko Y.H., Pedersen P.L., Recently elucidated energy catabolism pathways provide opportunities for novel treatments in hepatocellular carcinoma, Expert Rev. Anticancer Ther., 2004, 4, 449–457. 10.1586/14737140.4.3.449Search in Google Scholar
[30] Kurylenko O.O., Ruchala J., Hryniv O.B., Abbas C.A, Dmytruk K.V., Sibirny A.A., Metabolic engineering and classical selection of the methylotrophic thermotolerant yeast Hansenula polymorpha for improvement of high-temperature xylose alcoholic fermentation, Microb. Cell. Fact., 2014, 13, 122. 10.1186/s12934-014-0122-3Search in Google Scholar
[31] Uhlemann H., Schellenberger A., Glyoxylic acid as an active site marker of yeast pyruvate decarboxylase, FEBS Lett., 1976, 63, 37-39. 10.1016/0014-5793(76)80189-5Search in Google Scholar
[32] Bekesi J.G., Molnar Z., Richard J., Winzler inhibitory effect of d-glucosamine and other sugar analogs on the viability and transplantability of ascites tumor cells, Cancer Res., 1969, 29, 353–359. Search in Google Scholar
© 2016 Kostyantyn V. Dmytruk et al.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.
