Enhanced Furfural Tolerance in Zymomonas mobilis by the Overexpression of Antioxidant Genes

doi:10.13560/j.cnki.biotech.bull.1985.2019-0040

Abstract

Abstract: Furfural is a major inhibitor in lignocellulosic hydrolysate,which induces the generation of reactive oxygen species（ROS）. Exploration of relationships between antioxidant genes and furfural tolerance contributes to enhanced tolerance performance of strains. ;By overexpressing endogenous antioxidant genes NADH oxidase（ZMO1885）,NADP⁺ reductase（ZMO1753）,and glutathione reductase（ZMO1211）in Zymomonas mobilis,tolerance performance,ROS,and NADH/NAD⁺were measured. The results suggested that overexpression of ZM4 ZMO1885 reduced the intracellular ROS level by 52.1%,increased the biomass by 38.9%,and improved furfural tolerance of ZM4/ZMO1885 with no significant interference of cellular NADH/NAD⁺ in the medium containing 2 g/L furfural,compared to the control. However,recombinant strain ZM4/ZMO1753 and ZM4/ZMO1211 did not demonstrate significantly elevated furfural tolerance. In the simulated lignocellulosic hydrolysate,the biomass of ZM4/ZMO1885 was 46.9% higher than that of the control strain,and the sugar consumption rate increased by 110.3% with the increased ethanol productivity by 195.2%. Collectively,the overexpression of antioxidant genes affects furfural tolerance performance,among which ZMO1885 overexpression improves furfural tolerance through promoting ROS elimination and intracellular furfural conversion without redox level perturbation.

Key words: furfural, reactive oxygen species, NADH oxidase, glutathione reductase, NADP⁺ reductase, tolerance

WEN Yuan, XIA Juan, QI Liang-hua, LIU Xiao-wei, LIU Chen-guang, BAI Feng-wu. Enhanced Furfural Tolerance in Zymomonas mobilis by the Overexpression of Antioxidant Genes[J]. Biotechnology Bulletin, 2019, 35(8): 85-94.

References

[1] Azapagic A, Perdan S, Clift R, et. al. Sustainable development in practice:case studies for engineers and scientists[M]. New York:John Wiley & Sons, 2004.
[2] Morales M, Quintero J, Conejeros R, et. al. Life cycle assessment of lignocellulosic bioethanol:environmental impacts and energy balance[J]. Renewable and Sustainable Energy Reviews, 2015, 42:1349-1361.
[3] 曹莲莹, 李凯, 李凡, 等. 木质纤维素乙醇关键技术研究进展[J]. 生物产业技术, 2018(4):25-32.
[4] Matsufuji Y, Fujimura S, Ito T, et al.Acetaldehyde tolerance in Saccharomyces cerevisiae involves the pentose phosphate pathway and oleic acid biosynthesis[J]. Yeast, 2010, 25(11):825-833.
[5] Wang S, Sun X, Yuan Q.Strategies for enhancing microbial tolerance to inhibitors for biofuel production:A review[J]. Bioresource Technology, 2018, 258:302-309.
[6] Allen SA, Clark W, Mccaffery JM, et al.Furfural induces reactive oxygen species accumulation and cellular damage in Saccharomyces cerevisiae[J]. Biotechnology for Biofuels, 2010, 3(1):2-11.
[7] Yang S, Fei Q, Zhang Y, et al.Zymomonas mobilis as a model system for production of biofuels and biochemical[J]. Microbial Biotechnology, 2016, 9(6):699-717.
[8] Franden MA, Pilath HM, Mohagheghi A, et al.Inhibition of growth of Zymomonas mobilis model compounds found in lignocellulosic hydrolysates[J]. Biotechnology for Biofuels, 2013, 6(1):99-113.
[9] Huang S, Xue T, Wang Z, et al.Furfural-tolerant Zymomonas mobilis derived from error-prone PCR-based whole genome shuffling and their tolerant mechanism[J]. Applied Microbiology and Biotechnology, 2018, 102(7):3337-3347.
[10] Yi X, Gu H, Gao Q, et al.Transcriptome analysis of Zymomonas mobilis ZM4 reveals mechanisms of tolerance and detoxification of phenolic aldehyde inhibitors from lignocellulose pretreatment[J]. Biotechnology for Biofuels, 2015, 8(1):153-167.
[11] Wang L, Chong H, Jiang R.Comparison of alkyl hydroperoxide reductase and two water-forming NADH oxidases from Bacillus cereus ATCC 14579[J]. Applied Microbiology and Biotechnology, 2012, 96(5):1265-1273.
[12] André Jänsch, Freiding S, Jürgen Behr, et al. Contribution of the NADH-oxidase(Nox)to the aerobic life of Lactobacillus sanfranciscensis DSM20451T[J]. Food Microbiology, 2011, 28;(1):29-37.
[13] Higuchi M, Yamamoto Y, Kamio Y.Molecular biology of oxygen tolerance in lactic acid bacteria:Functions of NADH oxidases and Dpr in oxidative stress[J]. Journal of Bioscience and Bioengineering, 2000, 90(5):484-493.
[14] Xu S, Zhou J, Qin Y, et al.Water-forming NADH oxidase protects Torulopsis glabrata against hyperosmotic stress[J]. Yeast, 2010, 27(4):207-216.
[15] Shi X, Zou Y, Chen Y, et al.Overexpression of a water-forming NADH oxidase improves the metabolism and stress tolerance of Saccharomyces cerevisiae in aerobic fermentation[J]. Frontiers in Microbiology, 2016, 7:1427-1439.
[16] Minard KI, McAlister-Henn L. Antioxidant function of cytosolic sources of NADPH in yeast[J]. Free Radical Biology and Medicine, 2001, 31(6):832-843.
[17] Couto N, Wood J, Barber J.The role of glutathione reductase and related enzymes on cellular redox homoeostasis network[J]. Free Radical Biology and Medicine, 2016, 95:27-42.
[18] Achary VMM, Reddy CS, Pandey P, et al.Glutathione reductase a unique enzyme:molecular cloning, expression and biochemical characterization from the stress adapted C 4 plant, Pennisetum glaucum(L.)R. Br[J]. Molecular Biology Reports, 2015, 42(5):947-962.
[19] Zhang MM, Zhao XQ, 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.
[20] Dong H, Fan L, Luo Z, et al.Improvement of ethanol productivity and energy efficiency by degradation of inhibitors using recombinant Zymomonas mobilis(pHW20a-fdh)[J]. Biotechnol Bioeng, 2013, 110:2395-2404.
[21] Rutkis R, Strazdina I, Balodite E, et al.The low energy-coupling respiration in Zymomonas mobilis accelerates flux in the Entner-Doudoroff pathway[J]. PLoS One, 2016, 11(4):e0153866.
[22] Schorsch M, Kramer M, Goss T, et al.A unique ferredoxin acts as a player in the low-iron response of photosynthetic organisms[J]. Proceedings of the National Academy of Sciences, 2018, 115(51):E12111-E12120.
[23] Schuller JM, Birrell JA, Tanaka H, et al.Structural adaptations of photosynthetic complex I enable ferredoxin-dependent electron transfer[J]. Science, 2019, 363(6424):257-260.
[24] Arcinas AJ, Maiocco SJ, Elliott SJ, et al.Ferredoxins as interchangeable redox components in support of MiaB, a radical S-Adenosylmethionine Methylthiotransferase[J]. Protein Science, 2019, 28(1):267-282.
[25] Juhnke H, Krems B, Kötter P, et al.Mutants that show increased sensitivity to hydrogen peroxide reveal an important role for the pentose phosphate pathway in protection of yeast against oxidative stress[J]. Molecular and General Genetics, 1996, 252(4):456-464.
[26] Gorsich SW, Dien BS, Nichols NN, et al.Tolerance to furfural-induced stress is associated with pentose phosphate pathway genes ZWF1, GND1, RPE1, and TKL1 in Saccharomyces cerevisiae[J]. Applied Microbiology and Biotechnology, 2006, 71(3):339-349.
[27] Kim D, Hahn JS.Roles of the Yap1 transcription factor and antioxidants in Saccharomyces cerevisiae’s tolerance to furfural and 5-hydroxymethylfurfural, which function as thiol-reactive electrophiles generating oxidative stress[J]. Applied and Environmental Microbiology, 2013, 79(16):5069-5077.
[28] Liu CG, Xue C, Lin YH, et al.Redox potential control and applications in microaerobic and anaerobic fermentations[J]. Biotechnology Advances, 2013, 31(2):257-265.
[29] 郝学密, 杜斌, 刘黎阳, 等. ORP对酿酒酵母在木质纤维素水解液抑制物中发酵的影响[J]. 化工学报, 2015, 66(3):1066-1071.
[30] Shi XC, Zou YN, Chen Y, et al.A water-forming NADH oxidase regulates metabolism in anaerobic fermentation[J]. Biotechnology for Biofuels, 2016, 9(1):103-114.
[31] Heux S, Cachon R, Dequin S.Cofactor engineering in Saccharomyces cerevisiae:expression of a H₂O-forming NADH oxidase and impact on redox metabolism[J]. Metabolic Engineering, 2006, 8(4):303-314.
[32] Ji XJ, Xia ZF, Fu NH, et al.Cofactor engineering through heterologous expression of an NADH oxidase and its impact on metabolic flux redistribution in Klebsiella pneumoniae[J]. Biotechnology for Biofuels, 2013, 6(1):7-15.
[33] Wang X, Gao Q, Bao J. Enhancement of furan aldehydes conversion in Zymomonas mobilis by elevating dehydrogenase activity and cofactor regeneration[J]. Biotechnology for Biofuels, 2017, 10;(1):24-32.