Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering

Biotechnology for Biofuels

Mekonnen M Demeke³  Heiko Dietz²  Yingying Li³  María R Foulquié-Moreno³  Sarma Mutturi⁴  Sylvie Deprez¹  Tom Den Abt³  Beatriz M Bonini³  Gunnar Liden⁴  Françoise Dumortier³  Alex Verplaetse¹  Eckhard Boles²  Johan M Thevelein³ 

[1] Laboratory of Enzyme, Fermentation and Brewing Technology, KAHO Sint-Lieven University College, KU Leuven Association, Gebroeders De Smetstraat 1, 9000, Ghent, Flanders, Belgium

[2] Institute of Molecular Biosciences, Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany

[3] Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, B-3001 Leuven, Heverlee, Flanders, Belgium

[4] Department of Chemical Engineering, Lund University, P.O. Box 124, 22100 Lund, Sweden

关键词: Evolutionary engineering;    Saccharomyces cerevisiae;    Inhibitor tolerance;    D-xylose isomerase;    D-xylose fermentation;    Lignocellulose;    Bioethanol;   

Others  :  798012
DOI  :  10.1186/1754-6834-6-89

 received in 2013-03-06, accepted in 2013-06-12,  发布年份 2013

【 摘 要 】

Background

The production of bioethanol from lignocellulose hydrolysates requires a robust, D-xylose-fermenting and inhibitor-tolerant microorganism as catalyst. The purpose of the present work was to develop such a strain from a prime industrial yeast strain, Ethanol Red, used for bioethanol production.

Results

An expression cassette containing 13 genes including Clostridium phytofermentans XylA, encoding D-xylose isomerase (XI), and enzymes of the pentose phosphate pathway was inserted in two copies in the genome of Ethanol Red. Subsequent EMS mutagenesis, genome shuffling and selection in D-xylose-enriched lignocellulose hydrolysate, followed by multiple rounds of evolutionary engineering in complex medium with D-xylose, gradually established efficient D-xylose fermentation. The best-performing strain, GS1.11-26, showed a maximum specific D-xylose consumption rate of 1.1 g/g DW/h in synthetic medium, with complete attenuation of 35 g/L D-xylose in about 17 h. In separate hydrolysis and fermentation of lignocellulose hydrolysates of Arundo donax (giant reed), spruce and a wheat straw/hay mixture, the maximum specific D-xylose consumption rate was 0.36, 0.23 and 1.1 g/g DW inoculum/h, and the final ethanol titer was 4.2, 3.9 and 5.8% (v/v), respectively. In simultaneous saccharification and fermentation of Arundo hydrolysate, GS1.11-26 produced 32% more ethanol than the parent strain Ethanol Red, due to efficient D-xylose utilization. The high D-xylose fermentation capacity was stable after extended growth in glucose. Cell extracts of strain GS1.11-26 displayed 17-fold higher XI activity compared to the parent strain, but overexpression of XI alone was not enough to establish D-xylose fermentation. The high D-xylose consumption rate was due to synergistic interaction between the high XI activity and one or more mutations in the genome. The GS1.11-26 had a partial respiratory defect causing a reduced aerobic growth rate.

Conclusions

An industrial yeast strain for bioethanol production with lignocellulose hydrolysates has been developed in the genetic background of a strain widely used for commercial bioethanol production. The strain uses glucose and D-xylose with high consumption rates and partial cofermentation in various lignocellulose hydrolysates with very high ethanol yield. The GS1.11-26 strain shows highly promising potential for further development of an all-round robust yeast strain for efficient fermentation of various lignocellulose hydrolysates.

【 授权许可】

   
2013 Demeke et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Zaldivar J, Nielsen J, Olsson L: Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration. Appl Microbiol Biotechnol 2001, 56:17-34.
• [2]Lau MW, Gunawan C, Balan V, Dale BE: Comparing the fermentation performance of Escherichia coli KO11, Saccharomyces cerevisiae 424A(LNH-ST) and Zymomonas mobilis AX101 for cellulosic ethanol production. Biotechnol Biofuels 2010, 3:11. BioMed Central Full Text
• [3]Ebringerová A, Hromádková Z, Heinze T: Hemicellulose. In Polysaccharides I. Volume 186. Edited by Heinze T. Berlin. Heidelberg: Springer-Verlag; 2013:1-67.
• [4]Hahn-Hagerdal 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]Weber C, Farwick A, Benisch F, Brat D, Dietz H, Subtil T, Boles E: Trends and challenges in the microbial production of lignocellulosic bioalcohol fuels. Appl Microbiol Biotechnol 2010, 87:1303-1315.
• [6]Almeida JR, Modig T, Petersson A, Hahn-Hagerdal B, Liden G, Gorwa-Grauslund MF: Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae. J Chem Technol Biotechnol 2007, 82:340-349.
• [7]Miller EN, Jarboe LR, Turner PC, Pharkya P, Yomano LP, York SW, Nunn D, Shanmugam KT, Ingram LO: Furfural inhibits growth by limiting sulfur assimilation in ethanologenic Escherichia coli strain LY180. Appl Environ Microbiol 2009, 75:6132-6141.
• [8]Huang CF, Lin TH, Guo GL, Hwang WS: Enhanced ethanol production by fermentation of rice straw hydrolysate without detoxification using a newly adapted strain of Pichia stipitis. Bioresour Technol 2009, 100:3914-3920.
• [9]Hahn-Hagerdal B, Karhumaa K, Jeppsson M, Gorwa-Grauslund MF: Metabolic engineering for pentose utilization in Saccharomyces cerevisiae. Adv Biochem Eng Biotechnol 2007, 108:147-177.
• [10]Nevoigt E: Progress in metabolic engineering of Saccharomyces cerevisiae. Microbiol Mol Biol Rev 2008, 72:379-412.
• [11]Jeppsson M, Bengtsson O, Franke K, Lee H, Hahn-Hagerdal B, Gorwa-Grauslund MF: The expression of a Pichia stipitis xylose reductase mutant with higher K(M) for NADPH increases ethanol production from xylose in recombinant Saccharomyces cerevisiae. Biotechnol Bioeng 2006, 93:665-673.
• [12]Johansson B, Christensson C, Hobley T, Hahn-Hagerdal B: Xylulokinase overexpression in two strains of Saccharomyces cerevisiae also expressing xylose reductase and xylitol dehydrogenase and its effect on fermentation of xylose and lignocellulosic hydrolysate. Appl Environ Microbiol 2001, 67:4249-4255.
• [13]Johansson B, Hahn-Hagerdal B: The non-oxidative pentose phosphate pathway controls the fermentation rate of xylulose but not of xylose in Saccharomyces cerevisiae TMB3001. FEMS Yeast Res 2002, 2:277-282.
• [14]Bengtsson O, Hahn-Hagerdal 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
• [15]Runquist D, Hahn-Hagerdal B, Bettiga M: Increased ethanol productivity in xylose-utilizing Saccharomyces cerevisiae via a randomly mutagenized xylose reductase. Appl Environ Microbiol 2010, 76:7796-7802.
• [16]Olofsson K, Runquist D, Hahn-Hagerdal B, Liden G: A mutated xylose reductase increases bioethanol production more than a glucose/xylose facilitator in simultaneous fermentation and co-fermentation of wheat straw. AMB Express 2011, 1:4. BioMed Central Full Text
• [17]Peng B, Shen Y, Li X, Chen X, Hou J, Bao X: Improvement of xylose fermentation in respiratory-deficient xylose-fermenting Saccharomyces cerevisiae. Metab Eng 2012, 14:9-18.
• [18]Walfridsson M, Bao X, Anderlund M, Lilius G, Bulow L, Hahn-Hagerdal B: Ethanolic fermentation of xylose with Saccharomyces cerevisiae harboring the Thermus thermophilus xylA gene, which expresses an active xylose (glucose) isomerase. Appl Environ Microbiol 1996, 62:4648-4651.
• [19]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.
• [20]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.
• [21]Bettiga M, Hahn-Hagerdal B, Gorwa-Grauslund MF: Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains. Biotechnol Biofuels 2008, 1:16. BioMed Central Full Text
• [22]Chang Q, Griest TA, Harter TM, Petrash JM: Functional studies of aldo-keto reductases in Saccharomyces cerevisiae. Biochim Biophys Acta 2007, 1773:321-329.
• [23]Traff KL, Jonsson LJ, Hahn-Hagerdal B: Putative xylose and arabinose reductases in Saccharomyces cerevisiae. Yeast 2002, 19:1233-1241.
• [24]Toivari MH, Salusjarvi L, Ruohonen L, Penttila M: Endogenous xylose pathway in Saccharomyces cerevisiae. Appl Environ Microbiol 2004, 70:3681-3686.
• [25]Traff KL, Otero Cordero RR, Van Zyl WH, Hahn-Hagerdal B: Deletion of the GRE3 aldose reductase gene and its influence on xylose metabolism in recombinant strains of Saccharomyces cerevisiae expressing the xylA and XKS1 genes. Appl Environ Microbiol 2001, 67:5668-5674.
• [26]Aguilera J, Prieto JA: The Saccharomyces cerevisiae aldose reductase is implied in the metabolism of methylglyoxal in response to stress conditions. Curr Genet 2001, 39:273-283.
• [27]Brat D, Boles E, Wiedemann B: Functional expression of a bacterial xylose isomerase in Saccharomyces cerevisiae. Appl Environ Microbiol 2009, 75:2304-2311.
• [28]Kuyper M, Toirkens MJ, Diderich JA, Winkler AA, Van Dijken JP, Pronk JT: Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain. FEMS Yeast Res 2005, 5:925-934.
• [29]Wisselink HW, Toirkens MJ, Wu Q, Pronk JT, Van Maris AJ: Novel evolutionary engineering approach for accelerated utilization of glucose, xylose, and arabinose mixtures by engineered Saccharomyces cerevisiae strains. Appl Environ Microbiol 2009, 75:907-914.
• [30]Karhumaa K, Wiedemann B, Hahn-Hagerdal B, Boles E, Gorwa-Grauslund MF: Co-utilization of L-arabinose and D-xylose by laboratory and industrial Saccharomyces cerevisiae strains. Microb Cell Fact 2006, 5:18. BioMed Central Full Text
• [31]Van Maris AJ, Winkler AA, Kuyper M, De Laat WT, Van Dijken JP, Pronk JT: Development of efficient xylose fermentation in Saccharomyces cerevisiae: xylose isomerase as a key component. Adv Biochem Eng Biotechnol 2007, 108:179-204.
• [32]Boles E, Schulte F, Miosga T, Freidel K, Schluter E, Zimmermann FK, Hollenberg CP, Heinisch JJ: Characterization of a glucose-repressed pyruvate kinase (Pyk2p) in Saccharomyces cerevisiae that is catalytically insensitive to fructose-1,6-bisphosphate. J Bacteriol 1997, 179:2987-2993.
• [33]Wiedemann B, Boles E: Codon-optimized bacterial genes improve L-arabinose fermentation in recombinant Saccharomyces cerevisiae. Appl Environ Microbiol 2008, 74:2043-2050.
• [34]Hamacher T, Becker J, Gardonyi M, Hahn-Hagerdal B, Boles E: Characterization of the xylose-transporting properties of yeast hexose transporters and their influence on xylose utilization. Microbiology 2002, 148:2783-2788.
• [35]Subtil T, Boles E: Competition between pentoses and glucose during uptake and catabolism in recombinant Saccharomyces cerevisiae. Biotechnol Biofuels 2012, 5:14. BioMed Central Full Text
• [36]Wisselink HW, Toirkens MJ, Del Rosario Franco Berriel M, Winkler AA, Van Dijken JP, Pronk JT, Van Maris AJ: Engineering of Saccharomyces cerevisiae for efficient anaerobic alcoholic fermentation of L-arabinose. Appl Environ Microbiol 2007, 73:4881-4891.
• [37]Wahlbom CF, Van Zyl WH, Jonsson LJ, Hahn-Hagerdal B, Otero RR: Generation of the improved recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3400 by random mutagenesis and physiological comparison with Pichia stipitis CBS 6054. FEMS Yeast Res 2003, 3:319-326.
• [38]Kuhn A, Van Zyl C, Van Tonder A, Prior BA: Purification and partial characterization of an aldo-keto reductase from Saccharomyces cerevisiae. Appl Environ Microbiol 1995, 61:1580-1585.
• [39]Zhang JG, Liu XY, He XP, Guo XN, Lu Y, Zhang BR: Improvement of acetic acid tolerance and fermentation performance of Saccharomyces cerevisiae by disruption of the FPS1 aquaglyceroporin gene. Biotechnol Lett 2011, 33:277-284.
• [40]Bellido C, Bolado S, Coca M, Lucas S, Gonzalez-Benito G, Garcia-Cubero MT: Effect of inhibitors formed during wheat straw pretreatment on ethanol fermentation by Pichia stipitis. Bioresour Technol 2011, 102:10868-10874.
• [41]Bellissimi E, Van Dijken JP, Pronk JT, Van Maris AJ: Effects of acetic acid on the kinetics of xylose fermentation by an engineered, xylose-isomerase-based Saccharomyces cerevisiae strain. FEMS Yeast Res 2009, 9:358-364.
• [42]Wright J, Bellissimi E, De Hulster E, Wagner A, Pronk JT, Van Maris AJ: Batch and continuous culture-based selection strategies for acetic acid tolerance in xylose-fermenting Saccharomyces cerevisiae. FEMS Yeast Res 2011, 11:299-306.
• [43]Persson P, Larsson S, Jonsson LJ, Nilvebrant NO, Sivik B, Munteanu F, Thorneby L, Gorton L: Supercritical fluid extraction of a lignocellulosic hydrolysate of spruce for detoxification and to facilitate analysis of inhibitors. Biotechnol Bioeng 2002, 79:694-700.
• [44]Ask M, Olofsson K, Felice TD, Ruohonen L, Penttilä M, Lidén G, Olsson L: Challenges in enzymatic hydrolysis and fermentation of pretreated Arundo donax revealed by a comparison between SHF and SSF. Process Biochem 2012, 47:1452-1459.
• [45]Mutturi S, Liden G: Effect of temperature on simultaneous saccharification and fermentation of pretreated spruce and arundo. Ind Eng Chem Res 2013, 52:1244-1251.
• [46]Olofsson K, Rudolf A, Liden G: Designing simultaneous saccharification and fermentation for improved xylose conversion by a recombinant strain of Saccharomyces cerevisiae. J Biotechnol 2008, 134:112-120.
• [47]Liu JJ, Ding WT, Zhang GC, Wang JY: Improving ethanol fermentation performance of Saccharomyces cerevisiae in very high-gravity fermentation through chemical mutagenesis and meiotic recombination. Appl Microbiol Biotechnol 2011, 91:1239-1246.
• [48]Pais TM, Foulquié-Moreno MR, Hubmann G, Duitama J, Swinnen S, Goovaerts A, Yang Y, Dumortier F, Thevelein JM: Comparative polygenic analysis of maximal ethanol accumulation capacity and tolerance to high ethanol levels of cell proliferation in yeast. PLoS Genet 2013, 9(6):e1003548.
• [49]Bellissimi E, Richards C: Yeast propagation. In The alcohol textbook, a reference for the beverage, fuel and industrial alcohol industries. 5th edition. Edited by Ingledew WM, Kelsall DR, Austin GD, Kluhspies C. Nottingham: University Press; 2009:145-159.
• [50]Shen Y, Chen X, Peng B, Chen L, Hou J, Bao X: An efficient xylose-fermenting recombinant Saccharomyces cerevisiae strain obtained through adaptive evolution and its global transcription profile. Appl Microbiol Biotechnol 2012, 96:1079-1091.
• [51]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.
• [52]Chu BC, Lee H: Genetic improvement of Saccharomyces cerevisiae for xylose fermentation. Biotechnol Adv 2007, 25:425-441.
• [53]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.
• [54]Liu E, Hu Y: Construction of a xylose-fermenting Saccharomyces cerevisiae strain by combined approaches of genetic engineering, chemical mutagenesis and evolutionary adaptation. Biochem Eng J 2010, 48:204-210.
• [55]Sonderegger M, Sauer U: Evolutionary engineering of Saccharomyces cerevisiae for anaerobic growth on xylose. Appl Environ Microbiol 2003, 69:1990-1998.
• [56]Jingping G, Hongbing S, Gang S, Hongzhi L, Wenxiang P: A genome shuffling-generated Saccharomyces cerevisiae isolate that ferments xylose and glucose to produce high levels of ethanol. J Ind Microbiol Biotechnol 2012, 39:777-787.
• [57]Zhang W, Geng A: Improved ethanol production by a xylose-fermenting recombinant yeast strain constructed through a modified genome shuffling method. Biotechnol Biofuels 2012, 5:46. BioMed Central Full Text
• [58]Gong J, Zheng H, Wu Z, Chen T, Zhao X: Genome shuffling: Progress and applications for phenotype improvement. Biotechnol Adv 2009, 27:996-1005.
• [59]Kuyper M, Winkler AA, Van Dijken JP, Pronk JT: Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle. FEMS Yeast Res 2004, 4:655-664.
• [60]Reyes LH, Winkler J, Kao KC: Visualizing evolution in real-time method for strain engineering. Front Microbiol 2012, 3:198.
• [61]Almario MP: Reyes LH. Kao KC: Evolutionary engineering of Saccharomyces cerevisiae for enhanced tolerance to hydrolysates of lignocellulosic biomass. Biotechnol Bioeng; 2013.
• [62]Elena SF, Lenski RE: Evolution experiments with microorganisms: the dynamics and genetic bases of adaptation. Nat Rev Genet 2003, 4:457-469.
• [63]Karhumaa K, Garcia Sanchez R, Hahn-Hagerdal 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
• [64]Schmidt-Dannert C, Arnold FH: Directed evolution of industrial enzymes. Trends Biotechnol 1999, 17:135-136.
• [65]Palmqvist E, Hahn-Hägerdal B: Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition. Bioresour Technol 2000, 74:25-33.
• [66]Hou L: Novel methods of genome shuffling in Saccharomyces cerevisiae. Biotechnol Lett 2009, 31:671-677.
• [67]Huxley C, Green ED, Dunham I: Rapid assessment of S. cerevisiae mating type by PCR. Trends Genet 1990, 6:236.
• [68]Gietz RD, Schiestl RH, Willems AR, Woods RA: Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 1995, 11:355-360.
• [69]Gietz RD, Schiestl RH: High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2007, 2:31-34.
• [70]Hoffman CS, Winston F: A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 1987, 57:267-272.
• [71]Sambrook J, Fritsch EF, Maniatis T: Molecular cloning: a laboratory manual. 2nd edition. New York: Cold Spring Harbor; 1989.
• [72]Dower WJ, Miller JF, Ragsdale CW: High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 1988, 16:6127-6145.
• [73]Guldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH: A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 1996, 24:2519-2524.
• [74]Carter Z, Delneri D: New generation of loxP-mutated deletion cassettes for the genetic manipulation of yeast natural isolates. Yeast 2010, 27:765-775.
• [75]Popolo L, Vanoni M, Alberghina L: Control of the yeast cell cycle by protein synthesis. Exp Cell Res 1982, 142:69-78.
• [76]Kersters-Hilderson H, Callens M, Van Opstal O, Vangrysperre W, De Bruyne CK: Kinetic characterization of D-xylose isomerases by enzymatic assays ising D-sorbitol dehydrogenase. Enzyme Microb Technol 1987, 9:145-148.
• [77]Leitão L, Prista C, Moura TF, Loureiro-Dias MC, Soveral G: Grapevine aquaporins: gating of a tonoplast intrinsic protein (TIP2;1) by cytosolic pH. PLoS One 2012, 7:e33219.
• [78]Swinnen S, Schaerlaekens K, Pais T, Claesen J, Hubmann G, Yang Y, Demeke M, Foulquie-Moreno MR, Goovaerts A, Souvereyns K, et al.: Identification of novel causative genes determining the complex trait of high ethanol tolerance in yeast using pooled-segregant whole-genome sequence analysis. Genome Res 2012, 22:975-984.
• [79]Bertilsson M, Olofsson K, Liden G: Prefermentation improves xylose utilization in simultaneous saccharification and co-fermentation of pretreated spruce. Biotechnol Biofuels 2009, 2:8. BioMed Central Full Text