Reducing glucoamylase usage for commercial-scale ethanol production from starch using glucoamylase expressing Saccharomyces cerevisiae

Abstract

The development of yeast that converts raw corn or cassava starch to ethanol without adding the exogenous α-amylase and/or glucoamylase would reduce the overall ethanol production cost. In this study, two copies of codon-optimized Saccharomycopsis fibuligera glucoamylase genes were integrated into the genome of the industrial Saccharomyces cerevisiae strain CCTCC M94055, and the resulting strain CIBTS1522 showed comparable basic growth characters with the parental strain. We systemically evaluated the fermentation performance of the CIBTS1522 strain using the raw corn or cassava starch at small and commercial-scale, and observed that a reduction of at least 40% of the dose of glucoamylase was possible when using the CIBTS1522 yeast under real ethanol production condition. Next, we measured the effect of the nitrogen source, the phosphorous source, metal ions, and industrial microbial enzymes on the strain’s cell wet weight and ethanol content, the nitrogen source and acid protease showed a positive effect on these parameters. Finally, orthogonal tests for some other factors including urea, acid protease, inoculum size, and glucoamylase addition were conducted to further optimize the ethanol production. Taken together, the CIBTS1522 strain was identified as an ideal candidate for the bioethanol industry and a better fermentation performance could be achieved by modifying the industrial culture media and condition.

Keywords

Starch bioethanol production / Glucoamylase producing Saccharomyces cerevisiae strain / Raw corn starch fermentation / Raw cassava starch fermentation / Ethanol production optimization

References

[1] Altıntaş MM, Ülgen KÖ, Kırdar B, Önsan Zİ, Oliver SG. Optimal substrate feeding policy for fed-batch cultures of S. cerevisiae expressing bifunctional fusion protein displaying amylolytic activities. Enzyme Microb Technol, 2003, 33(2): 262-269.

[2] Birch RM, Walker GM. Influence of magnesium ions on heat shock and ethanol stress responses of Saccharomyces cerevisiae. Enzyme Microb Technol, 2000, 26(9–10): 678-687.

[3] Blagbrough IS, Bayoumi SA, Rowan MG, Beeching JR. Cassava: an appraisal of its phytochemistry and its biotechnological prospects. Phytochemistry, 2010, 71(17–18): 1940-1951.

[4] Casey E, Mosier NS, Adamec J, Stockdale Z, Ho N, Sedlak M. Effect of salts on the Co-fermentation of glucose and xylose by a genetically engineered strain of Saccharomyces cerevisiae. Biotechnol Biofuels, 2013, 6(1): 83.

[5] Chandel AK, Garlapati VK, Singh AK, Antunes FAF, da Silva SS. The path forward for lignocellulose biorefineries: bottlenecks, solutions, and perspective on commercialization. Bioresour Technol, 2018, 264: 370-381.

[6] Chen JP, Wu KW, Fukuda H. Bioethanol production from uncooked raw starch by immobilized surface-engineered yeast cells. Appl Biochem Biotechnol, 2008, 145(1–3): 59-67.

[7] Chi Z, Chi Z, Liu G, Wang F, Ju L, Zhang T. Saccharomycopsis fibuligera and its applications in biotechnology. Biotechnol Adv, 2009, 27(4): 423-431.

[8] Cripwell RA, Rose SH, Favaro L, van Zyl WH. Construction of industrial Saccharomyces cerevisiae strains for the efficient consolidated bioprocessing of raw starch. Biotechnol Biofuels, 2019, 12: 201.

[9] Cripwell RA, Rose SH, Viljoen-Bloom M, van Zyl WH (2019b) Improved raw starch amylase production by Saccharomyces cerevisiae using codon optimisation strategies. FEMS Yeast Res 19(2). https://doi.org/10.1093/femsyr/foy127.

[10] Cripwell RA, Favaro L, Viljoen-Bloom M, van Zyl WH. Consolidated bioprocessing of raw starch to ethanol by Saccharomyces cerevisiae: achievements and challenges. Biotechnol Adv, 2020, 42: 107579.

[11] Diao L, Liu Y, Qian F, Yang J, Jiang Y, Yang S. Construction of fast xylose-fermenting yeast based on industrial ethanol-producing diploid Saccharomyces cerevisiae by rational design and adaptive evolution. BMC Biotechnol, 2013, 13(1): 110.

[12] Doran-Peterson J, Jangid A, Brandon SK, DeCrescenzo-Henriksen E, Dien B, Ingram LO. Simultaneous saccharification and fermentation and partial saccharification and co-fermentation of lignocellulosic biomass for ethanol production. Methods Mol Biol, 2009, 581: 263-280.

[13] Duhan JS, Kumar A, Tanwar SK. Bioethanol production from starchy part of tuberous plant (potato) using Saccharomyces cerevisiae MTCC-170. Afr J Microbiol Res, 2013, 7(46): 5253-5260.

[14] Favaro L, Jooste T, Basaglia M, Rose SH, Saayman M, Gorgens JF, Casella S, van Zyl WH. Codon-optimized glucoamylase sGAI of Aspergillus awamori improves starch utilization in an industrial yeast. Appl Microbiol Biotechnol, 2012, 95(4): 957-968.

[15] Gibson BR, Lawrence SJ, Leclaire JP, Powell CD, Smart KA. Yeast responses to stresses associated with industrial brewery handling. FEMS Microbiol Rev, 2007, 31(5): 535-569.

[16] Gorgens JF, Bressler DC, van Rensburg E. Engineering Saccharomyces cerevisiae for direct conversion of raw, uncooked or granular starch to ethanol. Crit Rev Biotechnol, 2015, 35(3): 369-391.

[17] Guillaume A, Thorigné A, Carré Y, Vinh J, Levavasseur L (2019) Contribution of proteases and cellulases produced by solid-state fermentation to the improvement of corn ethanol production. Bioresour Bioprocess 6(1). doi:https://doi.org/10.1186/s40643-019-0241-0

[18] Hahn-Hagerdal B, Galbe M, Gorwa-Grauslund MF, Liden G, Zacchi G. Bio-ethanol—the fuel of tomorrow from the residues of today. Trends Biotechnol, 2006, 24(12): 549-556.

[19] Harris PV, Xu F, Kreel NE, Kang C, Fukuyama S. New enzyme insights drive advances in commercial ethanol production. Curr Opin Chem Biol, 2014, 19: 162-170.

[20] Innis MA, Holland MJ, McCabe PC, Cole GE, Wittman VP, Tal R, Watt KW, Gelfand DH, Holland JP, Meade JH. Expression, glycosylation, and secretion of an Aspergillus Glucoamylase by Saccharomyces cerevisiae. Science, 1985, 228(4695): 21-26.

[21] Jansen MLA, Bracher JM, Papapetridis I, Verhoeven MD, de Bruijn H, de Waal PP, van Maris AJA, Klaassen P, Pronk JT (2017) Saccharomyces cerevisiae strains for second-generation ethanol production: from academic exploration to industrial implementation. FEMS Yeast Res 17(5). https://doi.org/10.1093/femsyr/fox044.

[22] Johnston DB, McAloon AJ. Protease increases fermentation rate and ethanol yield in dry-grind ethanol production. Bioresour Technol, 2014, 154: 18-25.

[23] Karim AS, Curran KA, Alper HS. Characterization of plasmid burden and copy number in Saccharomyces cerevisiae for optimization of metabolic engineering applications. FEMS Yeast Res, 2013, 13(1): 107-116.

[24] Kim JH, Kim HR, Lim MH, Ko HM, Chin JE, Lee HB, Kim IC, Bai S. Construction of a direct starch-fermenting industrial strain of Saccharomyces cerevisiae producing glucoamylase, alpha-amylase and debranching enzyme. Biotechnol Lett, 2010, 32(5): 713-719.

[25] Kong, II, Turner TL, Kim H, Kim SR, Jin YS (2018) Phenotypic evaluation and characterization of 21 industrial Saccharomyces cerevisiae yeast strains. FEMS Yeast Res 18(1). https://doi.org/10.1093/femsyr/foy001.

[26] Kotaka A, Sahara H, Hata Y, Abe Y, Kondo A, Kato-Murai M, Kuroda K, Ueda M. Efficient and direct fermentation of starch to ethanol by sake yeast strains displaying fungal glucoamylases. Biosci Biotechnol Biochem, 2008, 72(5): 1376-1379.

[27] Liao B, Hill G, Roesler W. Stable expression of barley α-amylase in S. cerevisiae for conversion of starch into bioethanol. Biochem Eng J, 2012, 64: 8-16.

[28] Luangthongkam P, Fang L, Noomhorm A, Lamsal B. Addition of cellulolytic enzymes and phytase for improving ethanol fermentation performance and oil recovery in corn dry grind process. Ind Crop Prod, 2015, 77: 803-808.

[29] Murthy GS, Townsend DE, Meerdink GL, Bargren GL, Tumbleson ME, Singh V. Effect of Aflatoxin B1 on dry-grind ethanol process. Cereal Chem, 2005, 82(3): 302-304.

[30] Nakamura Y, Kobayashi F, Ohnaga M, Sawada T. Alcohol fermentation of starch by a genetic recombinant yeast having glucoamylase activity. Biotechnol Bioeng, 1997, 53(1): 21-25.

[31] Nonato RV, Shishido K. Alpha-factor-directed synthesis of Bacillus stearothermophilus alpha-amylase in Saccharomyces cerevisiae. Biochem Biophys Res Commun, 1988, 152(1): 76-82.

[32] Peplow M. Cellulosic ethanol fights for life. Nature, 2014, 507(7491): 152-153.

[33] Perez-Carrillo E, Serna-Saldivar SO, Chuck-Hernandez C, Cortes-Callejas ML. Addition of protease during starch liquefaction affects free amino nitrogen, fusel alcohols and ethanol production of fermented maize and whole and decorticated sorghum mashes. Biochem Eng J, 2012, 67: 1-9.

[34] Pretorius IS, Lambrechts MG, Marmur J. The glucoamylase multigene family in Saccharomyces cerevisiae var. diastaticus: an overview. Crit Rev Biochem Mol Biol, 1991, 26(1): 53-76.

[35] van Zyl WH, Bloom M, Viktor MJ. Engineering yeasts for raw starch conversion. Appl Microbiol Biotechnol, 2012, 95(6): 1377-1388.

[36] Vidal BC, Rausch KD, Tumbleson ME, Singh V. Protease treatment to improve ethanol fermentation in modified dry grind corn processes. Cereal Chem, 2009, 86(3): 323-328.

[37] Walker GM (1998) Yeast physiology and biotechnology. Wiley

[38] Wang X, Yang J, Yang S, Jiang Y. Unraveling the genetic basis of fast l-arabinose consumption on top of recombinant xylose-fermenting Saccharomyces cerevisiae. Biotechnol Bioeng, 2019, 116(2): 283-293.

[39] Yue GJ, Yu JL, Zhang X, Tan TW. The influence of nitrogen sources on ethanol production by yeast from concentrated sweet sorghum juice. Biomass Bioenerg, 2012, 39: 48-52.

[40] Zhao XQ, Xue C, Ge XM, Yuan WJ, Wang JY, Bai FW. Impact of zinc supplementation on the improvement of ethanol tolerance and yield of self-flocculating yeast in continuous ethanol fermentation. J Biotechnol, 2009, 139(1): 55-60.

Funding

National Natural Science Foundation of China(31921006)