Growth and fermentation of D-xylose by Saccharomyces cerevisiae expressing a novel D-xylose isomerase originating from the bacterium Prevotella ruminicola TC2-24

Biotechnology for Biofuels

Ronald E Hector¹  Bruce S Dien¹  Michael A Cotta¹  Jeffrey A Mertens¹ 

[1] Bioenergy Research Unit, United States Department of Agriculture, Agricultural Research Service, National Center for Agricultural Utilization Research, Peoria, IL 61604, USA

关键词: Lignocellulose;    Ethanol;    D-xylose isomerase;    Bacteroides;    Prevotella ruminicola;    Saccharomyces cerevisiae;   

Others  :  798020
DOI  :  10.1186/1754-6834-6-84

 received in 2013-04-03, accepted in 2013-05-22,  发布年份 2013

【 摘 要 】

Background

Saccharomyces cerevisiae strains expressing D-xylose isomerase (XI) produce some of the highest reported ethanol yields from D-xylose. Unfortunately, most bacterial XIs that have been expressed in S. cerevisiae are either not functional, require additional strain modification, or have low affinity for D-xylose. This study analyzed several XIs from rumen and intestinal microorganisms to identify enzymes with improved properties for engineering S. cerevisiae for D-xylose fermentation.

Results

Four XIs originating from rumen and intestinal bacteria were isolated and expressed in a S. cerevisiae CEN.PK2-1C parental strain primed for D-xylose metabolism by over expression of its native D-xylulokinase. Three of the XIs were functional in S. cerevisiae, based on the strain’s ability to grow in D-xylose medium. The most promising strain, expressing the XI mined from Prevotella ruminicola TC2-24, was further adapted for aerobic and fermentative growth by serial transfers of D-xylose cultures under aerobic, and followed by microaerobic conditions. The evolved strain had a specific growth rate of 0.23 h^-1 on D-xylose medium, which is comparable to the best reported results for analogous S. cerevisiae strains including those expressing the Piromyces sp. E2 XI. When used to ferment D-xylose, the adapted strain produced 13.6 g/L ethanol in 91 h with a metabolic yield of 83% of theoretical. From analysis of the P. ruminicola XI, it was determined the enzyme possessed a V_max of 0.81 μmole/min/mg protein and a K_m of 34 mM.

Conclusion

This study identifies a new xylose isomerase from the rumen bacterium Prevotella ruminicola TC2-24 that has one of the highest affinities and specific activities compared to other bacterial and fungal D-xylose isomerases expressed in yeast. When expressed in S. cerevisiae and used to ferment D-xylose, very high ethanol yield was obtained. This new XI should be a promising resource for constructing other D-xylose fermenting strains, including industrial yeast genetic backgrounds.

【 授权许可】

   
2013 Hector et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Bettiga M, Bengtsson O, Hahn-Hägerdal B, Gorwa-Grauslund MF: Arabinose and xylose fermentation by recombinant Saccharomyces cerevisiae expressing a fungal pentose utilization pathway. Microb Cell Fact 2009, 8:40. BioMed Central Full Text
• [2]Bera AK, Sedlak M, Khan A, Ho NWY: Establishment of l-arabinose fermentation in glucose/xylose co-fermenting recombinant Saccharomyces cerevisiae 424A(LNH-ST) by genetic engineering. Appl Microbiol Biotechnol 2010, 87:1803-1811.
• [3]Van-Vleet JH, Jeffries TW: Yeast metabolic engineering for hemicellulosic ethanol production. Curr Opin Biotechnol 2009, 20:300-306.
• [4]Hahn-Hägerdal B, Karhumaa K, Fonseca C, Spencer-Martins I, Gorwa-Grauslund MF: Towards industrial pentose-fermenting yeast strains. Appl Microbiol Biotechnol 2007, 74:937-953.
• [5]Scalcinati G, Otero JM, Van-Vleet JR, Jeffries TW, Olsson L, Nielsen J: Evolutionary engineering of Saccharomyces cerevisiae for efficient aerobic xylose consumption. FEMS Yeast Res 2012, 12:582-597.
• [6]Zhou H, Cheng JS, Wang BL, Fink GR, Stephanopoulos G: Xylose isomerase overexpression along with engineering of the pentose phosphate pathway and evolutionary engineering enable rapid xylose utilization and ethanol production by Saccharomyces cerevisiae. Metab Eng 2012, 14:611-622.
• [7]Sonderegger M, Sauer U: Evolutionary engineering of Saccharomyces cerevisiae for anaerobic growth on xylose. Appl Environ Microb 2003, 69:1990-1998.
• [8]Jeffries TW, Jin YS: Metabolic engineering for improved fermentation of pentoses by yeasts. Appl Microbiol Biotechnol 2004, 63:495-509.
• [9]Ho NWY, Chen Z, Brainard AP: Genetically engineered Saccharomyces yeast capable of effective cofermentation of glucose and xylose. Appl Environ Microb 1998, 64:1852-1859.
• [10]Richard P, Toivari MH, Penttilä M: The role of xylulokinase in Saccharomyces cerevisiae xylulose catabolism. FEMS Microbiol Lett 2000, 190:39-43.
• [11]Harhangi HR, Akhmanova AS, Emmens R, van der-Drift C, De-Laat WT, Van-Dijken JP, Jetten MS, Pronk JT, Op den Camp HJ: Xylose metabolism in the anaerobic fungus Piromyces sp. strain E2 follows the bacterial pathway. Arch Microbiol 2003, 180:134-141.
• [12]Kuyper M, Harhangi HR, Stave AK, Winkler AA, Jetten MS, De-Laat WT, Den-Ridder JJ, Op den Camp HJ, van Dijken JP, Pronk JT: High-level functional expression of a fungal xylose isomerase: the key to efficient ethanolic fermentation of xylose by Saccharomyces cerevisiae? FEMS Yeast Res 2003, 4:69-78.
• [13]Lee SM, Jellison T, Alper HS: Directed evolution of xylose isomerase for improved xylose catabolism and fermentation in the yeast Saccharomyces cerevisiae. Appl Environ Microbiol 2012, 78:5708-5716.
• [14]Brat D, Boles E, Wiedemann B: Functional expression of a bacterial xylose isomerase in Saccharomyces cerevisiae. Appl Environ Microbiol 2009, 75:2304-2311.
• [15]Ma M, Liu Z, Moon J: Genetic engineering of inhibitor-tolerant Saccharomyces cerevisiae for improved xylose utilization in ethanol production. BioEnergy Research 2012, 5:459-469.
• [16]Aeling KA, Salmon KA, Laplaza JM, Li L, Headman JR, Hutagalung AH, Picataggio S: Co-fermentation of xylose and cellobiose by an engineered Saccharomyces cerevisiae. J Ind Microbiol Biotechnol 2012, 39:1597-1604.
• [17]Madhavan A, Tamalampudi S, Ushida K, Kanai D, Katahira S, Srivastava A, Fukuda H, Bisaria VS, Kondo A: Xylose isomerase from polycentric fungus Orpinomyces: gene sequencing, cloning, and expression in Saccharomyces cerevisiae for bioconversion of xylose to ethanol. Appl Microbiol Biotechnol 2009, 82:1067-1078.
• [18]Matte A, Forsberg CW, Gibbins AMV: Enzymes associated with metabolism of xylose and other pentoses by Prevotella (Bacteroides) ruminicola strains, Selenomonas ruminantium D, and Fibrobacter succinogenes S85. Can J Microbiol 1992, 38:370-376.
• [19]Dodd D, Mackie RI, Cann IK: Xylan degradation, a metabolic property shared by rumen and human colonic Bacteroidetes. Mol Microbiol 2011, 79:292-304.
• [20]Avgustin G, Wright F, Flint HJ: Genetic diversity and phylogenetic relationships among strains of Prevotella (Bacteroides) ruminicola from the rumen. Int J Syst Bacteriol 1994, 44:246-255.
• [21]Avgustin G, Wallace RJ, Flint HJ: Phenotypic diversity among ruminal Isolates of Prevotella ruminicola: Proposal of Prevotella brevis sp. nov., Prevotella byantii sp. nov., and Prevotella albensis sp. nov. and redefinition of Prevotella ruminicola. International Journal of Systematic Bacteriology. Int J Syst Bacteriol 1997, 47:284-288.
• [22]Funke M, Diederichs S, Kensy F, Müller C, Büchs J: The baffled microtiter plate: Increased oxygen transfer and improved online monitoring in small scale fermentations. Biotechnol Bioeng 2009, 103:1118-1128.
• [23]Warringer J, Blomberg A: Automated screening in environmental arrays allows analysis of quantitative phenotypic profiles in Saccharomyces cerevisiae. Yeast 2003, 20:53-67.
• [24]Kuyper M, Hartog MM, Toirkens MJ, Almering MJ, Winkler AA, Van-Dijken JP, Pronk JT: Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation. FEMS Yeast Res 2005, 5:399-409.
• [25]Karhumaa K, Garcia Sanchez R, Hahn-Hägerdal B, Gorwa-Grauslund MF: Comparison of the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant Saccharomyces cerevisiae. Microb Cell Fact 2007, 6:5. BioMed Central Full Text
• [26]Parachin NS, Gorwa-Grauslund MF: Isolation of xylose isomerases by sequence- and function-based screening from a soil metagenomic library. Biotechnol Biofuels 2011, 4:9. BioMed Central Full Text
• [27]Hector RE, Dien BS, Cotta MA, Qureshi N: Engineering industrial Saccharomyces cerevisiae strains for xylose fermentation and comparison for switchgrass conversion. J Ind Microbiol Biotechnol 2011, 38:1193-1202.
• [28]Bera AK, Ho NW, Khan A, Sedlak M: A genetic overhaul of Saccharomyces cerevisiae 424A(LNH-ST) to improve xylose fermentation. J Ind Microbiol Biot 2011, 38:617-626.
• [29]Jeppsson M, Bengtsson O, Franke K, Lee H, Hahn-Hägerdal B, Gorwa-Grauslund MF: The expression of a Pichia stipitis xylose reductase mutant with higher Km for NADPH increases ethanol production from xylose in recombinant Saccharomyces cerevisiae. Biotechnol Bioeng 2006, 93:665-673.
• [30]Petschacher B, Nidetzky B: Altering the coenzyme preference of xylose reductase to favor utilization of NADH enhances ethanol yield from xylose in a metabolically engineered strain of Saccharomyces cerevisiae. Microb Cell Fact 2008, 7:9. BioMed Central Full Text
• [31]Bengtsson O, Hahn-Hägerdal B, Gorwa-Grauslund MF: Xylose reductase from Pichia stipitis with altered coenzyme preference improves ethanolic xylose fermentation by recombinant Saccharomyces cerevisiae. Biotechnol Biofuels 2009, 2:9. BioMed Central Full Text
• [32]Krahulec S, Klimacek M, Nidetzky B: Analysis and prediction of the physiological effects of altered coenzyme specificity in xylose reductase and xylitol dehydrogenase during xylose fermentation by Saccharomyces cerevisiae. J Biotechnol 2012, 158:192-202.
• [33]Stambuk BU, Dunn B, Alves SL Jr, Duval EH, Sherlock G: Industrial fuel ethanol yeasts contain adaptive copy number changes in genes involved in vitamin B1 and B6 biosynthesis. Genome Res 2009, 19:2271-2278.
• [34]Dunham MJ, Badrane H, Ferea T, Adams J, Brown PO, Rosenzweig F, Botstein D: Characteristic genome rearrangements in experimental evolution of Saccharomyces cerevisiae. P Natl Acad Sci USA 2002, 99:16144-16149.
• [35]Sambrook J, Russell DW: Molecular Cloning: A Laboratory Manual. 3rd edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2001.
• [36]Gietz D, Woods RA: Transformation of yeasts by the lithium acetate/single-stranded carrier/polyethylene glycol method. Methods Enzymol 2002, 350:87-96.
• [37]Amberg BC, Burke DJ, Strathern JN: Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual. 2005 edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2005.
• [38]Whitehead TR, Cotta MA, Collins MD, Falsen E, Lawson PA: Bacteroides coprosuis sp. nov., isolated from swine-manure storage pits. Int J Syst Evol Microbiol 2005, 55:2515-2518.
• [39]Christianson TW, Sikorski RS, Dante M, Shero JH, Hieter P: Multifunctional yeast high-copy-number shuttle vectors. Gene 1992, 110:119-122.
• [40]Hector RE, Mertens JA, Bowman MJ, Nichols NN, Cotta MA, Hughes SR: Saccharomyces cerevisiae engineered for xylose metabolism requires gluconeogenesis and the oxidative branch of the pentose phosphate pathway for aerobic xylose assimilation. Yeast 2011, 28:645-660.