For simultaneous conversion of glucose and xylose to ethanol from mixed sugars, ethanologenic glucose/xylose selective E. coli strains were developed by genes deletion and metabolic engineering and their cooperative ethanol fermentation performance was evaluated by shaking flasks fermentation. Strain B0013-2010, a glucose-selective strain, was developed by deleting xylA with blocking of formation of acetate, lactate, formate and succinate by deleting pta-ackA, ldhA, pflB and frdA in E. coli B0013-1031 (pta-ackA, ldhA, pflB, frdA). Strain B0013-2011H, a xylose-selective strain, was obtained by deleting ptsG, glk and manZ and by restoring xylH with a reverse mutation. Two ethanologenic strains, B0013-2010PA and B0013-2011HPA, were developed by transforming pEtac-PA (bearing pdc and adhB from Zymomonas mobilis) into B0013-2010 and B0013-2011H. Ethanol fermentation on glucose and xylose mixture with B0013-2010PA and B0013-2011HPA in cooperative manner was conducted and ethanol production rate was 1.01 g/(L·h), with glucose consumption rate of 2.02 g/(L·h) and xylose of 1.05 g/(L·h). In conclusion, the co-fermentation with sugar-selective strains significantly improved simultaneous utilization of glucose and xylose for ethanol fermentation.
[1] BANERJEE S, MUDLIAR S, SEN R, et al. Commercializing lignocellulosic bioethanol: technology bottlenecks and possible remedies [J]. Biofuels Bioprod Bioref, 2010, 4(1): 77-93.
[2] SANCHEZ R G, KARHUMAA K, FONSECA C, et al. Improved xylose and arabinose utilization by an industrial recombinant Saccharomyces cerevisiae strain using evolutionary engineering [J]. Biotechnol Biofuels, 2010, 3(1):13.
[3] JEFFRIES T W. Engineering yeasts for xylose metabolism[J]. Curr Opin Biotechnol, 2006, 17(3): 320-326.
[4] HO N WY, CHEN Z, BRAINARD A P. Genetically engineered Saccharomyces yeast capable of effective cofermentation of glucose and xylose[J]. Appl Environ Microbio, 1998, 64(5): 1 852-1 859.
[5] MARIS AJ AV, WINKLER A A, KUYPER M, et al. Development of efficient xylose fermentation in Saccharomyces cerevisiae: xylose isomerase as a key component [J]. Adv Biochem Engin Biotechnol, 2007, 108: 179-204.
[6] ZHANG M, EDDY C, DEANDA K, et al. Metabolic engineering of a pentose metabolism pathway in ethanologenic Zymomonas mobilis [J]. Science, 1995, 267(5 195): 240-244.
[7] OHTA K, BEALL D S, MEJIA J P, et al. Metabolic engineering of Klebsiella oxytoca M5A1 for ethanol production from xylose and glucose [J]. Appl Environ Microbiol, 1991, 57(10):2 810-2 815.
[8] INGRAM L O, CONWAY T, CLARK D P, et al. Genetic engineering of ethanol production in Escherichia coli [J]. Appl Environ Microbiol, 1987, 53(10): 2 420-2 425.
[9] NICHOLS N N, DIEN B S, BOTHAST R J. Use of catabolite repression mutants for fermentation of sugar mixtures to ethanol[J]. Appl Microbiol Biotechnol, 2001, 56(1-2):120-125.
[10] YOMANO L P, YORK S W, SHANMUGAM K T, et al. Deletion of methylglyoxal synthase gene (mgsA) increased sugar co-metabolism in ethanol-producing Escherichia coli [J]. Biotechnol Lett, 2009, 31(9):1 389-1 398.
[11] HERNáNDEZ-MONTALVO V, VALLE F, BOLIVAR F, et al. Characterization of sugar mixtures utilization by an Escherichia coli mutant devoid of the phosphotransferase system[J]. Appl Microbiol Biotechnol, 2001, 57(1-2):186-191.
[12] KARIMOVA G, LADANT D, ULLMANN A. Relief of catabolite repression in a cAMP-independent catabolite gene activator mutant of Escherichia coli [J]. Res Microbiol, 2004, 155(2):76-79.
[13] FU N, PEIRIS P. Co-fermentation of a mixture of glucose and xylose to ethanol by Zymomonas mobilis and Pachysolen tannophilus [J]. World J Microbiol Biotechnol, 2008, 24(7): 1 091-1 097.
[14] FU N, PEIRIS P, MARKHAM J, et al. A novel co-culture process with Zymomonas mobilis and Pichia stipitis for efficient ethanol production on glucose/xylose mixtures [J]. Enzyme Microb Technol, 2009, 45(3): 210-217.
[15] EITEMAN M A, LEE S A, ALTMAN E. A co-fermentation strategy to consume sugar mixtures effectively [J]. J Biol Eng, 2008, 2(1):1-8.
[16] EITEMAN M A, LEE S A, ALTMAN R, et al. A Substrate-selective co-fermentation strategy with Escherichia coli produces lactate by simultaneously consuming xylose and glucose [J]. Biotechnol Bioeng, 2009, 102(3): 822-827.
[17] TIAN X,ALTMAN E,EITEMAN M A. Succinate production from xylose-glucose mixtures using a consortium of engineered Escherichia coli [J]. Eng Life Sci, 2015, 15(1): 65-72.
[18] 孙金凤,徐敏,张峰,等. 利用木糖和葡萄糖合成乙醇的新型重组大肠杆菌的研究[J]. 微生物学报, 2004, 31(5):600-604.
[19] 曹剑磊,周丽,张梁,等. 产琥珀酸重组大肠杆菌的构建和发酵性能[J]. 应用与环境生物学报, 2010,16 (6):851-857.
[20] ZHOU L, ZUO Z R, CHEN X Z, et al. Evaluation of genetic manipulation strategies on D-lactate production by Escherichia coli[J]. Curr Microbiol, 2011, 62(3):981-989.
[21] 孙金凤,田康明,沈微,等. 大肠杆菌不同菌株木糖代谢差异性的遗传本质[J].食品与发酵工业,2017, 43(10): 68-73.
[22] 周丽,牛丹丹,李宁,等. 基于Red重组系统和Xer重组系统的大肠杆菌多基因删除方法[J]. 微生物学通报, 2010, 37(6):23-28.
[23] DATSENKO K A, WANNER B L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products [J]. Proc Natl Acad Sci USA, 2000, 97(12):6 640-6 645.
[24] JOSEPH S, RUSSELL D W. Molecular cloning: A laboratory manual [M].2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001.
[25] ZHOU L, NIU D D, TIAN K M, et al. Genetically switched D-lactate production in Escherichia coli[J]. Metab Eng, 2012, 14(5):560-568.
[26] KHANKAL R, CHIN J W, CIRINO P C. Role of xylose transporters in xylitol production from engineered Escherichia coli [J]. J Biotechnol, 2008, 134(3): 246-252.
[27] SONG S, PARK C. Organization and regulation of the D-xylose operons in Escherichia coli K-12: XylR acts as a transcriptional activator [J]. J Bacteriol, 1997, 179(22):7 025-7 032.
[28] GOSSET G. Improvement of Escherichia coli production strains by modification of the phosphoenolpyruvate:sugar phosphotransferase system[J]. Microb Cell Fact, 2005, 4(1):14.
[29] CURTIS S J, EPSTEIN W. Phosphorylation of D-glucose in Escherichia coli mutants defective in glucosephosphotransferase, mannosephosphotransferase, and glucokinase [J]. J Bacteriol, 1975, 122(3): 1 189-1 199.
[30] DEUTSCHER J, FRANCKE C, POSTMA P W. How phosphotransferase system-related protein phosphorylation regulates carbohydrate metabolism in bacteria [J]. Microbiol Mol Biol Rev, 2006, 70(4): 939-1 031.
[31] HUBER F, ERNI B. Membrane topology of the mannose transporter of Escherichia coli K12 [J]. Eur J Biochem, 1996, 239(3):810-817.
