Molecular evolutionary engineering of xylose isomerase to improve its catalytic activity and performance of micro-aerobic glucose/xylose co-fermentation in Saccharomyces cerevisiae
Tóm tắt
Expression of d-xylose isomerase having high catalytic activity in Saccharomyces cerevisiae (S. cerevisiae) is a prerequisite for efficient and economical production of bioethanol from cellulosic biomass. Although previous studies demonstrated functional expression of several xylose isomerases (XI) in S. cerevisiae, identification of XIs having higher catalytic activity is needed. Here, we report a new strategy to improve xylose fermentation in the S. cerevisiae strain IR-2 that involves an evolutionary engineering to select top-performing XIs from eight previously reported XIs derived from various species. Eight XI genes shown to have good expression in S. cerevisiae were introduced into the strain IR-2 having a deletion of GRE3 and XKS1 overexpression that allows use of d-xylose as a carbon source. Each transformant was evaluated under aerobic and micro-aerobic culture conditions. The strain expressing XI from Lachnoclostridium phytofermentans ISDg (LpXI) had the highest d-xylose consumption rate after 72 h of micro-aerobic fermentation on d-glucose and d-xylose mixed medium. To enhance LpXI catalytic activity, we performed random mutagenesis using error-prone polymerase chain reaction (PCR), which yielded two LpXI candidates, SS82 and SS92, that showed markedly improved fermentation performance. The LpXI genes in these clones carried either T63I or V162A/N303T point mutations. The SS120 strain expressing LpXI with the double mutation of T63I/V162A assimilated nearly 85 g/L d-glucose and 35 g/L d-xylose to produce 53.3 g/L ethanol in 72 h with an ethanol yield of approximately 0.44 (g/g-input sugars). An in vitro enzyme assay showed that, compared to wild-type, the LpXI double mutant in SS120 had a considerably higher Vmax (0.107 µmol/mg protein/min) and lower Km (37.1 mM). This study demonstrated that LpXI has the highest d-xylose consumption rate among the XIs expressed in IR-2 under micro-aerobic co-fermentation conditions. A combination of novel mutations (T63I and V162A) significantly improved the enzymatic activity of LpXI, indicating that LpXI-T63I/V162A would be a potential construct for highly efficient production of cellulosic ethanol.
Tài liệu tham khảo
Robak K, Balcerek M. Review of second generation bioethanol production from residual biomass. Food Technol Biotechnol. 2018;56:174–87.
Lee J. Biological conversion of lignocellulosic biomass to ethanol. J Biotechnol. 1997;56:1–24.
Hendriks ATWM, Zeeman G. Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresour Technol. 2009;100:10–8.
Hou J, Qiu C, Shen Y, Li H, Bao X. Engineering of Saccharomyces cerevisiae for the efficient co-utilization of glucose and xylose. FEMS Yeast Res. 2017;17:fox034.
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 K m for NADPH increases ethanol production from xylose in recombinant Saccharomyces cerevisiae. Biotechnol Bioeng. 2006;93:665–73.
Watanabe S, Saleh AA, Pack SP, Annaluru N, Kodaki T, Makino K. Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein engineered NADP+-dependent xylitol dehydrogenase. J Biotechnol. 2007;130:316–9.
Karhumaa K, Fromanger R, Hahn-Hägerdal B, Gorwa-Grauslund MF. High activity of xylose reductase and xylitol dehydrogenase improves xylose fermentation by recombinant Saccharomyces cerevisiae. Appl Microbiol Biotechnol. 2007;73:1039–46.
Matsushika A, Sawayama S. Efficient bioethanol production from xylose by recombinant Saccharomyces cerevisiae requires high activity of xylose reductase and moderate xylulokinase activity. J Biosci Bioeng. 2008;106:306–9.
Zeng WY, Tang YQ, Gou M, Sun ZY, Xia ZY, Kida K. Comparative transcriptomes reveal novel evolutionary strategies adopted by Saccharomyces cerevisiae with improved xylose utilization capability. Appl Microbiol Biotechnol. 2017;101:1753–67.
Sarthy AV, McConaughy BL, Lobo Z, Sundstrom JA, Furlong CE, Hall BD. Expression of the Escherichia coli xylose isomerase gene in Saccharomyces cerevisiae. Appl Environ Microbiol. 1987;53:1996–2000.
Amore R, Wilhelm M, Hollenberg CP. The fermentation of xylose—an analysis of the expression of Bacillus and Actinoplanes xylose isomerase genes in yeast. Appl Microbiol Biotechnol. 1989;30:351–7.
Moes CJ, Pretorius IS, van Zyl WH. Cloning and expression of the Clostridium thermosulfurogenes d-xylose isomerase gene (xylA) in Saccharomyces cerevisiae. Biotechnol Lett. 1996;18:269–74.
Walfridsson M, Bao X, Anderlund M, Lilius G, Bülow L, Hahn-Hägerdal 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;62:4648–51.
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.
Madhavan A, Tamalampudi S, Srivastava A, Fukuda H, Bisaria VS, Kondo A. Alcoholic fermentation of xylose and mixed sugars using recombinant Saccharomyces cerevisiae engineered for xylose utilization. Appl Microbiol Biotechnol. 2009;82:1037–47.
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;82:1067–78.
Tanino T, Hotta A, Ito T, Ishii J, Yamada R, Hasunuma T, Ogino C, Ohmura N, Ohshima T, Kondo A. Construction of a xylose-metabolizing yeast by genome integration of xylose isomerase gene and investigation of the effect of xylitol on fermentation. Appl Microbiol Biotechnol. 2010;88:1215–21.
Brat D, Boles E, Wiedemann B. Functional expression of a bacterial xylose isomerase in Saccharomyces cerevisiae. Appl Environ Microbiol. 2009;75:2304–11.
Demeke MM, Dietz H, Li Y, Foulquié-Moreno MR, Mutturi S, Deprez S, Den Abt T, Bonini BM, Liden G, Dumortier F, Verplaetse A, Boles E, Thevelein JM. Development of a d-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering. Biotechnol Biofuels. 2013;6:89.
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;39:1597–604.
Hector RE, Dien BS, Cotta MA, Mertens JA. Growth and fermentation of d-xylose by Saccharomyces cerevisiae expressing a novel d-xylose isomerase originating from the bacterium Prevotella ruminicola TC2-24. Biotechnol Biofuels. 2013;6:84.
de Figueiredo Vilela L, de Mello VM, Reis VC, Bon EP, Gonçalves Torres FA, Neves BC, Eleutherio EC. Functional expression of Burkholderia cenocepacia xylose isomerase in yeast increases ethanol production from a glucose–xylose blend. Bioresour Technol. 2013;128:792–6.
de Figueiredo Vilela L, de Araujo VPG, de Sousa Paredes R, da Silva Bon EP, Torres FAG, Neves BC, Eleutherio ECA. Enhanced xylose fermentation and ethanol production by engineered Saccharomyces cerevisiae strain. AMB Express. 2015;5:1–7.
Harcus D, Dignard D, Lépine G, Askew C, Raymond M, Whiteway M, Wu C. Comparative xylose metabolism among the ascomycetes C. albicans, S. stipitis and S. cerevisiae. PLoS ONE. 2013;8:e80733.
Waltman MJ, Yang ZK, Langan P, Graham DE, Kovalevsky A. Engineering acidic Streptomyces rubiginosus d-xylose isomerase by rational enzyme design. Protein Eng Des Sel. 2014;27:59–64.
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;78:5708–16.
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;180:134–41.
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.
Xia PF, Zhang GC, Liu JJ, Kwak S, Tsai CS, Kong II, Sung BH, Sohn JH, Wang SG, Jin YS. GroE chaperonins assisted functional expression of bacterial enzymes in Saccharomyces cerevisiae. Biotechnol Bioeng. 2016;113:2149–55.
Temer B, Dos Santos LV, Negri VA, Galhardo JP, Magalhães PHM, José J, Marschalk C, Corrêa TLR, Carazzolle MF, Pereira GAG. Conversion of an inactive xylose isomerase into a functional enzyme by co-expression of GroEL-GroES chaperonins in Saccharomyces cerevisiae. BMC Biotechnol. 2017;17:71.
Zhang GC, Liu JJ, Kong II, Kwak S, Jin YS. Combining C6 and C5 sugar metabolism for enhancing microbial bioconversion. Curr Opin Chem Biol. 2015;29:49–57.
Moysés DN, Reis VC, de Almeida JR, de Moraes LM, Torres FA. Xylose Fermentation by Saccharomyces cerevisiae: challenges and prospects. Int J Mol Sci. 2016;17:207.
Rose M. Molecular and biochemical characterization of the hexokinase from the starch-utilizing yeast Schwanniomyces occidentalis. Curr Genet. 1995;27:330–8.
Demeke MM, Foulquié-Moreno MR, Dumortier F, Thevelein JM. Rapid evolution of recombinant Saccharomyces cerevisiae for xylose fermentation through formation of extra-chromosomal circular DNA. PLoS Genet. 2015;11:e1005010.
Boles E, Schulte F, Miosga T, Freidel K, Schlüter 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;179:2987–93.
Kobayashi Y, Sahara T, Suzuki T, Kamachi S, Matsushika A, Hoshino T, Ohgiya S, Kamagata Y, Fujimori KE. Genetic improvement of xylose metabolism by enhancing the expression of pentose phosphate pathway genes in Saccharomyces cerevisiae IR-2 for high-temperature ethanol production. J Ind Microbiol Biotechnol. 2017;44:879–91.
Kobayashi Y, Sahara T, Ohgiya S, Kamagata Y, Fujimori KE. Systematic optimization of gene expression of pentose phosphate pathway enhances ethanol production from a glucose/xylose mixed medium in a recombinant Saccharomyces cerevisiae. AMB Express. 2018;8:139.
Sedlak M, Ho NW. Characterization of the effectiveness of hexose transporters for transporting xylose during glucose and xylose co-fermentation by a recombinant Saccharomyces yeast. Yeast. 2004;21:671–84.
Farwick A, Bruder S, Schadeweg V, Oreb M, Boles E. Engineering of yeast hexose transporters to transport d-xylose without inhibition by d-glucose. Proc Natl Acad Sci USA. 2014;111:5159–64.
Li YC, Mitsumasu K, Gou ZX, Gou M, Tang YQ, Li GY, Wu XL, Akamatsu T, Taguchi H, Kida K. Xylose fermentation efficiency and inhibitor tolerance of the recombinant industrial Saccharomyces cerevisiae strain NAPX37. Appl Microbiol Biotechnol. 2016;100:1531–42.
Kuriyama H, Seiko Y, Murakami T, Kobayashi H. Continuous ethanol fermentation with cell recycling using flocculating yeast. J Ferment Technol. 1985;63:159–65.
Sahara T, Fujimori KE, Nezuo M, Tsukahara M, Tochigi Y, Ohgiya S, Kamagata Y. Draft genome sequence of Saccharomyces cerevisiae IR-2, a useful industrial strain for highly efficient production of bioethanol. Genome Announc. 2014;2:e01160-13.
Güldener 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;24:2519–24.
Akashi H. Translational selection and yeast proteome evolution. Genetics. 2003;164:1291–303.
Ohgiya S, Sahara T. Protein production at low temperature by yeast Saccharomyces cerevisiae. Biosci Ind. 2007;65:130–1.
Matsushika A, Watanabe S, Kodaki T, Makino K, Inoue H, Murakami K, Takimura O, Sawayama S. Expression of protein engineered NADP+-dependent xylitol dehydrogenase increase ethanol production from xylose in recombinant Saccharomyces cerevisiae. Appl Microbiol Biotechnol. 2008;81:243–55.