Progress of research on construction and optimization of recombinant Saccharomyces cerevisiae strains for cellulosic ethanol production

doi:10.16085/j.issn.1000-6613.2017-1977

Abstract

Abstract: The increasing challenges on gradual depletion of fossil fuel and global warming are driving the development of alternative fuels and utilization of bioenergy. Production of fuel ethanol from lignocellulosic feedstocks including a variety of agricultural and forestry residues has received increasing interests in recent years. Saccharomyces cerevisiae is the most commonly used microbial organism for ethanol production. However, it cannot efficiently assimilate xylose, which is the most abundant pentose in lignocellulosic hydrolysates. Therefore, construction and optimization of xylose utilizing S. cerevisiae is of great importance for improving economic cellulosic ethanol production. The construction and optimization of recombinant S. cerevisiae strains by integrating xylose utilization pathway, and the effect of xylose transport on xylose assimilation were summarized. Furthermore, the current status of cellulosic ethanol production using the recombinant S. cerevisiae strains was discussed, and further prospects on improvement of production efficiency of cellulosic ethanol was provided. Currently, the yield of ethanol from xylose by the recombinant strains has been improved significantly. However, the regulatory mechanisms of xylose metabolism remain unclear, which limits further optimization of production efficiency. In addition, the evaluation of fermentation performance of the recombinant strains using various lignocellulosic hydrolysates is limited. Future studies will be focused on the regulation of xylose metabolism in the recombinant strains, and the influence of various inhibitors in the hydrolysates on fermentation performance will be emphasized. Strain optimization will be further explored in combination with different fermentation processes, and economic production of celluosic ethanol using the efficient recombinant stain is expected to be achieved.

Key words: biofuel, lignocellulosic biomass, ethanol fermentation, Saccharomyces cerevisiae, metabolic engineering

摘要： 寻找化石能源的替代品以及开发和利用生物能源已引起国内外研究者的广泛关注。提高酿酒酵母利用来源广泛、贮存丰富的农林废弃物等木质纤维素原料生产燃料乙醇的效率是生物能源的重要研究内容，但是，重组酿酒酵母木糖发酵性能低是限制纤维素乙醇经济性的关键问题。本文总结了酿酒酵母中木糖代谢途径的构建和优化以及木糖转运对木糖利用的影响，分析了重组酵母利用纤维素水解液进行乙醇发酵的研究现状，并对进一步提高重组酿酒酵母纤维素乙醇生产效率的研究趋势进行了展望。目前国内外已经构建了可有效利用木糖产乙醇的重组酵母，但对其木糖代谢机制的研究还尚未深入，限制了重组菌株的定向改造。此外，目前缺少在纤维素生物质水解液发酵实际应用过程中对重组菌株的评价。因此，加强重组酵母菌株对木糖利用相关代谢调控机理的分析，注重多种抑制物对菌株发酵性能的影响，结合真实底物纤维素乙醇发酵过程进行重组菌株的构建和优化，从而进一步提高纤维素乙醇生产的经济性，是未来菌株构建的重要研究方向。

关键词: 生物燃料, 木质纤维素类生物质, 乙醇发酵, 酿酒酵母, 代谢工程

CLC Number:

Q939.97

TANG Ruiqi, XIONG Liang, CHENG Cheng, ZHAO Xinqing, BAI Fengwu. Progress of research on construction and optimization of recombinant Saccharomyces cerevisiae strains for cellulosic ethanol production[J]. Chemical Industry and Engineering Progress, 2018, 37(08): 3119-3128.

唐瑞琪, 熊亮, 程诚, 赵心清, 白凤武. 纤维素乙醇生产重组酿酒酵母菌株的构建与优化研究进展[J]. 化工进展, 2018, 37(08): 3119-3128.

References

[1] KIM S R, HA S J, WEI N, et al. Simultaneous co-fermentation of mixed sugars:a promising strategy for producing cellulosic ethanol[J]. Trends in Biotechnology, 2012, 30(5):274-282.
[2] 张强, 郭元, 韩德明. 酿酒酵母乙醇耐受性的研究进展[J]. 化工进展, 2014, 33(1):187-192. ZHANG Q, GUO Y, HAN D M. Research progress in the ethanol tolerance of yeast[J]. Chemical Industry and Engineering Progress, 2014, 33(1):187-192.
[3] 张艳, 卢文玉. 酿酒酵母细胞表达异源萜类化合物的研究进展[J]. 化工进展, 2014, 33(5):1265-1270. ZHANG Y, LU W Y. Progress of heterologous expression of terpenes in Saccharomyces cerevisiae[J]. Chemical Industry and Engineering Progress, 2014, 33(5):1265-1270.
[4] KIM S R, PARK Y C, JIN Y S, et al. Strain engineering of Saccharomyces cerevisiae for enhanced xylose metabolism[J]. Biotechnology Advances, 2013, 31(6):851-861.
[5] JO J H, PARK Y C, JIN Y S, et al. Construction of efficient xylose-fermenting Saccharomyces cerevisiae through a synthetic isozyme system of xylose reductase from Scheffersomyces stipitis[J]. Bioresource Technology, 2017, 241:7.
[6] ZHANG X Y, WANG J Y, ZHANG W W, et al. Optimizing the coordinated transcription of central xylose-metabolism genes in Saccharomyces cerevisiae[J]. Applied Microbiology and Biotechnology, 2018, 102:1-11.
[7] KIM S R, HA S J, KONG I I, et al. High expression of XYL2 coding for xylitol dehydrogenase is necessary for efficient xylose fermentation by engineered Saccharomyces cerevisiae[J]. Metabolic Engineering, 2012, 14(4):336-343.
[8] CADETE R M, DE LAS HERAS A M, SANDSTROM A G, et al. Exploring xylose metabolism in Spathaspora species:Xyl 1.2 from Spathaspora passalidarum as the key for efficient anaerobic xylose fermentation in metabolic engineered Saccharomyces cerevisiae[J]. Biotechnology for Biofuels, 2016, 9:1.
[9] WEI N, QUARTERMAN J, KIM S R, et al. Enhanced biofuel production through coupled acetic acid and xylose consumption by engineered yeast[J]. Nature Communications, 2013, 4:2580.
[10] ZHANG G C, KONG I I, WEI N, et al. Optimization of an acetate reduction pathway for producing cellulosic ethanol by engineered yeast[J]. Biotechnology and Bioengineering, 2016, 113(12):2587-2596.
[11] VAN MARIS A J, WINKLER A A, KUYPER M, et al. Development of efficient xylose fermentation in Saccharomyces cerevisiae:xylose isomerase as a key component[M]. Berlin, Heidelberg:Springer, 2007:179-204.
[12] KUYPER M, HARHANGI H R, STAVE A K, et al. High-level functional expression of a fungal xylose isomerase:the key to efficient ethanolic fermentation of xylose by Saccharomyces cerevisiae?[J]. FEMS Yeast Research, 2003, 4(1):69-78.
[13] KUYPER M, AARON A, VAN DIJKEN J P, et al. Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation:a proof of principle[J]. FEMS Yeast Research, 2004, 4(6):655-664.
[14] KUYPER M, HARTOG M M, TOIRKENS M J, et al. Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation[J]. FEMS Yeast Research, 2005, 5(4-5):399-409.
[15] KUYPER M, TOIRKENS M J, DIDERICH J A, et al. Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain[J]. FEMS Yeast Research, 2005, 5(10):925-934.
[16] ZHOU H, CHENG J S, WANG B L, et al. Xylose isomerase overexpression along with engineering of the pentose phosphate pathway and evolutionary engineering enable rapid xylose utilization and ethanol production by Saccharomyces cerevisiae[J]. Metabolic Engineering, 2012, 14(6):611-622.
[17] DOS SANTOS L V, CARAZZOLLE M F, NAGAMATSU S T, et al. Unraveling the genetic basis of xylose consumption in engineered Saccharomyces cerevisiae strains[J]. Scientific Reports, 2016, 6:38676.
[18] DEMEKE M M, FOULQUIE-MORENO M R, DUMORTIER F, et al. Rapid evolution of recombinant Saccharomyces cerevisiae for xylose fermentation through formation of extra-chromosomal circular DNA[J]. PLoS Genetics, 2015, 11(3):e1005010.
[19] HOU J, SHEN Y, JIAO C L, et al. Characterization and evolution of xylose isomerase screened from the bovine rumen metagenome in Saccharomyces cerevisiae[J]. Journal of Bioscience and Bioengineering, 2016, 121(2):160-165.
[20] KO J K, UM Y, LEE S M. Effect of manganese ions on ethanol fermentation by xylose isomerase expressing Saccharomyces cerevisiae under acetic acid stress[J]. Bioresource Technology, 2016, 222:422-430.
[21] VERHOEVEN M D, LEE M, KAMOEN L, et al. Mutations in PMR1 stimulate xylose isomerase activity and anaerobic growth on xylose of engineered Saccharomyces cerevisiae by influencing manganese homeostasis[J]. Scientific Reports, 2017, 7:46155.
[22] HOU J, JIAO C L, PENG B Y, et al. Mutation of a regulator Ask10p improves xylose isomerase activity through up-regulation of molecular chaperones in Saccharomyces cerevisiae[J]. Metabolic Engineering, 2016, 38:241-250.
[23] THOMIK T, WITTIG I, CHOE J Y. An artificial transport metabolon facilitates improved substrate utilization in yeast[J]. Nature Chemical Biology, 2017, 13(11):1158-1163.
[24] BRAT D, BOLES E, WIEDEMANN B. Functional expression of a bacterial xylose isomerase in Saccharomyces cerevisiae[J]. Applied and Environmental Microbiology, 2009, 75(8):2304-2311.
[25] MADHAVAN A, TAMALAMPUDI S, USHIDA K, et al. Xylose isomerase from polycentric fungus Orpinomyces:gene sequencing, cloning, and expression in Saccharomyces cerevisiae for bioconversion of xylose to ethanol[J]. Applied Microbiology and Biotechnology, 2009, 82(6):1067-1078.
[26] MERT M J, ROSE S H, LA GRANGE D C, et al. Quantitative metabolomics of a xylose-utilizing Saccharomyces cerevisiae strain expressing the Bacteroides thetaiotaomicron xylose isomerase on glucose and xylose[J]. Journal of Industrial Microbiology & Biotechnology, 2017, 44(10):1459-1470.
[27] AELING K A, SALMON K A, LAPLAZA J M, et al. Co-fermentation of xylose and cellobiose by an engineered Saccharomyces cerevisiae[J]. Journal of Industrial Microbiology & Biotechnology, 2012, 39(11):1597-1604.
[28] HECTOR R E, DIEN B S, COTTA M A, et al. Growth and fermentation of D-xylose by Saccharomyces cerevisiae expressing a novel D-xylose isomerase originating from the bacterium Prevotella ruminicola TC2-24[J]. Biotechnology for Biofuels, 2013, 6:84.
[29] PENG B Y, HUANG S C, LIU T T, et al. Bacterial xylose isomerases from the mammal gut Bacteroidetes cluster function in Saccharomyces cerevisiae for effective xylose fermentation[J]. Microbial Cell Factories, 2015, 14:70.
[30] KATAHIRA S, MURAMOTO N, MORIYA S, et al. Screening and evolution of a novel protist xylose isomerase from the termite Reticulitermes speratus for efficient xylose fermentation in Saccharomyces cerevisiae[J]. Biotechnology for Biofuels, 2017, 10:203.
[31] SARTHY A, MCCONAUGHY B, LOBO Z, et al. Expression of the Escherichia coli xylose isomerase gene in Saccharomyces cerevisiae[J]. Applied and Environmental Microbiology, 1987, 53(9):1996-2000.
[32] XIA P F, ZHANG G C, LIU J J, et al. GroE chaperonins assisted functional expression of bacterial enzymes in Saccharomyces cerevisiae[J]. Biotechnology and Bioengineering, 2016, 113(10):2149-2155.
[33] LI H X, SHEN Y, WU M L, et al. Engineering a wild-type diploid Saccharomyces cerevisiae strain for second-generation bioethanol production[J]. Bioresour Bioprocess, 2016, 3(1):51.
[34] KO J K, UM Y, WOO H M, et al. Ethanol production from lignocellulosic hydrolysates using engineered Saccharomyces cerevisiae harboring xylose isomerase-based pathway[J]. Bioresource Technology, 2016, 209:290-296.
[35] LEE S M, JELLISON T, ALPER H S. Systematic and evolutionary engineering of a xylose isomerase-based pathway in Saccharomyces cerevisiae for efficient conversion yields[J]. Biotechnology and Biofuels, 2014, 7:122.
[36] 左颀, 张明明, 程诚, 等. 不同宿主来源的重组酿酒酵母混合糖代谢比较[J]. 微生物学通报, 2014, 41(7):1270-1277. ZUO Q, ZHANG M M, CHENG C, et al. Comparison of glucose/xylose cofermentation in recombinant Saccharomyces cerevisiae strains using different hosts[J]. Microbiology China, 2014, 41(7):1270-1277.
[37] 程诚, 熊亮, 李勇昊, 等. 混合糖发酵重组酿酒酵母的菌株构建和菊芋秸秆同步糖化发酵研究[J]. 微生物学通报, 2016, 43(7):1411-1418. CHENG C, XIONG L, LI Y H, et al. Construction of mixed-sugar fermenting recombinant Saccharomyces cerevisiae and ethanol production from Jerusalem artichoke stalk by simultaneous saccharification and fermentation[J]. Microbiology China, 2016, 43(7):1411-1418.
[38] LI Y C, LI G Y, GOU M, et al. Functional expression of xylose isomerase in flocculating industrial Saccharomyces cerevisiae strain for bioethanol production[J]. Journal of Bioscience and Bioengineering, 2016, 121(6):7.
[39] QI X, ZHA J, LIU G G, et al. Heterologous xylose isomerase pathway and evolutionary engineering improve xylose utilization in Saccharomyces cerevisiae[J]. Frontiers in Microbiology, 2015, 6:1165.
[40] LI Y C, ZENG W Y, GUO M, et al. Transcriptome changes in adaptive evolution of xylose-fermenting industrial Saccharomyces cerevisiae strains with δ-integration of different xylA genes[J]. Applied Microbiology and Biotechnology, 2017, 101(20):7741-7753.
[41] DU J, LI S J, ZHAO H M. Discovery and characterization of novel D-xylose-specific transporters from Neurospora crassa and Pichia stipitis[J]. Molecular Biosystems, 2010, 6(11):2150-2156.
[42] MOYSES D N, REIS V C, DE ALMEIDA J R, et al. Xylose fermentation by Saccharomyces cerevisiae:challenges and prospects[J]. International Journal of Molecular Sciences, 2016, 17(3):207.
[43] FARWICK A, BRUDER S, SCHADEWEG V, et al. Engineering of yeast hexose transporters to transport D-xylose without inhibition by D-glucose[J]. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(14):5159-5164.
[44] REIDER APEL A, OUELLET M, SZMIDT-MIDDLETON H, et al. Evolved hexose transporter enhances xylose uptake and glucose/xylose co-utilization in Saccharomyces cerevisiae[J]. Scientific Reports, 2016, 6:19512.
[45] WANG M, LI S J, ZHAO H M. Design and engineering of intracellular-metabolite-sensing/regulation gene circuits in Saccharomyces cerevisiae[J]. Biotechnology and Bioengineering, 2016, 113(1):206-215.
[46] WANG M, YU C Z, ZHAO H. Identification of an important motif that controls the activity and specificity of sugar transporters[J]. Biotechnology and Bioengineering, 2016, 113(7):1460-1467.
[47] NIJLAND J G, VOS E, SHIN H Y, et al. Improving pentose fermentation by preventing ubiquitination of hexose transporters in Saccharomyces cerevisiae[J]. Biotechnology for Biofuels, 2016, 9:158.
[48] HECTOR R E, QURESHI N, HUGHES S R, et al. Expression of a heterologous xylose transporter in a Saccharomyces cerevisiae strain engineered to utilize xylose improves aerobic xylose consumption[J]. Applied Microbiology and Biotechnology, 2008, 80(4):675-684.
[49] SLOOTHAAK J, TAMAYO-RAMOS J A, ODONI D I, et al. Identification and functional characterization of novel xylose transporters from the cell factories Aspergillus niger and Trichoderma reesei[J]. Biotechnology for Biofuels, 2016, 9:148.
[50] WANG M, YU C Z, ZHAO H M. Directed evolution of xylose specific transporters to facilitate glucose-xylose co-utilization[J]. Biotechnology and Bioengineering, 2016, 113(3):484-491.
[51] YOUNG E M, TONG A, BUI H, et al. Rewiring yeast sugar transporter preference through modifying a conserved protein motif[J]. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(1):131-136.
[52] LI H, SCHMITZ O, ALPER H S. Enabling glucose/xylose co-transport in yeast through the directed evolution of a sugar transporter[J]. Applied Microbiology and Biotechnology, 2016, 100(23):10215-10223.
[53] YOUNG E M, COMER A D, HUANG H, et al. A molecular transporter engineering approach to improving xylose catabolism in Saccharomyces cerevisiae[J]. Metabolic Engineering, 2012, 14(4):401-411.
[54] ZHAO X Q, ZI L H, BAI F W, et al. Bioethanol from lignocellulosic biomass[M]. Berlin, Heidelberg:Springer, 2012:25-51.
[55] ZHU J Q, QIN L, LI B Z, et al. Simultaneous saccharification and co-fermentation of aqueous ammonia pretreated corn stover with an engineered Saccharomyces cerevisiae SyBE005[J]. Bioresource Technology, 2014, 169:9-18.
[56] MAHBOUBI A, YLITERVO P, DOYEN W, et al. Continuous bioethanol fermentation from wheat straw hydrolysate with high suspended solid content using an immersed flat sheet membrane bioreactor[J]. Bioresource Technology, 2017, 241:296-308.
[57] QIN L, LI X, LIU L, et al. Dual effect of soluble materials in pretreated lignocellulose on simultaneous saccharification and co-fermentation process for the bioethanol production[J]. Bioresource Technology, 2017, 224:342-348.
[58] 李洪兴, 张笑然, 沈煜, 等. 纤维素乙醇生物加工过程中的抑制物对酿酒酵母的影响及应对措施[J]. 生物工程学报, 2009, 25(9):1321-1328. LI H X, ZHANG X R, SHEN Y, et al. Inhibitors and their effects on Saccharomyces cerevisiae and relevant countermeasures in bioprocess of ethanol production from lignocellulose:a review[J]. Chinese Journal of Biotechnology, 2009, 25(9):1321-1328.
[59] 刘贺, 朱家庆, 纵秋瑾, 等. 生物质转化工程酿酒酵母的研究进展[J]. 生物技术通报, 2017, 33(1):93-98. LIU H, ZHU J Q, ZONG Q J, et al. The development of engineered Saccharomyces cerevisiae for biomass conversion[J]. Biotechnology Bulletin, 2017, 33(1):93-98.
[60] ZHANG M M, ZHAO X Q, CHENG C, et al. Improved growth and ethanol fermentation of Saccharomyces cerevisiae in the presence of acetic acid by overexpression of SET5 and PPR1[J]. Biotechnology Journal, 2015, 10(12):1903-1911.
[61] ZHANG M M, ZHANG K Y, MEHMOOD M A, et al. Deletion of acetate transporter gene ADY2 improved tolerance of Saccharomyces cerevisiae against multiple stresses and enhanced ethanol production in the presence of acetic acid[J]. Bioresource Technology, 2017, 245:1461-1468.
[62] DEMEKE M M, DIETZ H, LI Y, et al. Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering[J]. Biotechnology for Biofuels, 2013, 6(1):89.
[63] ZHU J Q, QIN L, LI W C, et al. Simultaneous saccharification and co-fermentation of dry diluted acid pretreated corn stover at high dry matter loading:overcoming the inhibitors by non-tolerant yeast[J]. Bioresource Technology, 2015, 198:39-46.
[64] PARREIRAS L S, BREUER R J, NARASIMHAN R A, et al. Engineering and two-stage evolution of a lignocellulosic hydrolysate-tolerant Saccharomyces cerevisiae strain for anaerobic fermentation of xylose from AFEX pretreated corn stover[J]. PLoS One, 2014, 9(9):e107499.
[65] WANG R F, UNREAN P, FRANZEN C J. Model-based optimization and scale-up of multi-feed simultaneous saccharification and co-fermentation of steam pre-treated lignocellulose enables high gravity ethanol production[J]. Biotechnology for Biofuels, 2016, 9(88):13.
[66] LIU Z H, CHEN H Z. Simultaneous saccharification and co-fermentation for improving the xylose utilization of steam exploded corn stover at high solid loading[J]. Bioresource Technology, 2016, 201:15-26.
[67] LEE Y G, JIN Y S, CHA Y L, et al. Bioethanol production from cellulosic hydrolysates by engineered industrial Saccharomyces cerevisiae[J]. Bioresource Technology, 2017, 228:355-361.
[68] JIN M J, LIU Y P, DA COSTA SOUSA L, et al. Development of rapid bioconversion with integrated recycle technology for ethanol production from extractive ammonia pretreated corn stover[J]. Biotechnology and Bioengineering, 2017, 114(8):1713-1720.
[69] LIU G, ZHANG Q, LI H X, et al. Dry biorefining maximizes the potentials of simultaneous saccharification and co-fermentation for cellulosic ethanol production[J]. Biotechnology and Bioengineering, 2018, 115(1):60-69.
[70] 吕永坤, 堵国成, 陈坚, 等. 合成生物学技术研究进展[J]. 生物技术通报, 2015, 31(4):134-148. LÜ Y K, DU G C, CHEN J, et al. Advances in synthetic biology[J]. Biotechnology Bulletin, 2015, 31(4):134-148.