Revitalizing the ethanologenic bacterium Zymomonas mobilis for sugar reduction in high-sugar-content fruits and commercial products

Abstract

The excessive consumption of sugars can cause health issues. Different strategies have been developed to reduce sugars in the diets. However, sugars in fruits and commercial products may be difficult to reduce, limiting their usage among certain populations of people. Zymomonas mobilis is a generally recognized as safe (GRAS) probiotic bacterium with the capability to produce levan-type prebiotics, and thrives in high-sugar environments with unique characteristics to be developed for lignocellulosic biofuel and biochemical production. In this study, the sugar reduction capabilities of Z. mobilis ZM4 were examined using two fruits of pear and persimmon and three high-sugar-content commercial products of two pear pastes (PPs) and one Chinese traditional wine (CTW). Our results demonstrated that Z. mobilis ZM4 can utilize sugars in fruits with about 20 g/L ethanol and less than 5 g/L sorbitol produced within 22 h using pears, and about 45 g/L ethanol and 30 g/L sorbitol produced within 34 h using persimmons. When PPs made from pears were used, Z. mobilis can utilize nearly all glucose (ca. 60 g/L) and most fructose (110 g/L) within 100 h with 40 ~ 60 g/L ethanol and more than 20 g/L sorbitol produced resulting in a final sorbitol concentration above 80 g/L. In the high-sugar-content alcoholic Chinese traditional wine, which contains mostly glucose and ethanol, Z. mobilis can reduce nearly all sugars with about 30 g/L ethanol produced, resulting in a final ethanol above 90 g/L. The ethanol yield and percentage yield of Z. mobilis in 50 ~ 60% CTW were 0.44 ~ 0.50 g/g and 86 ~ 97%, respectively, which are close to its theoretical yields—especially in 60% CTW. Although the ethanol yield and percentage yield in PPs were lower than those in CTW, they were similar to those in fruits of pears and persimmons with an ethanol yield around 0.30 ~ 0.37 g/g and ethanol percentage yield around 60 ~ 72%, which could be due to the formation of sorbitol and/or levan in the presence of both glucose and fructose. Our study also compared the fermentation performance of the classical ethanologenic yeast Saccharomyces cerevisiae BY4743 to Z. mobilis, with results suggesting that Z. mobilis ZM4 had better performance than that of yeast S. cerevisiae BY4743 given a higher sugar conversion rate and ethanol yield for sugar reduction. This work thus laid a foundation for utilizing the advantages of Z. mobilis in the food industry to reduce sugar concentrations or potentially produce alcoholic prebiotic beverages.

Keywords

Zymomonas mobilis / Saccharomyces cerevisiae / Fruits / Chinese wine / Fermentation / Sugar reduction

References

[1] Apovian C, Gokce N. Obesity and cardiovascular disease. Circulation, 2012, 125(9): 1178-1182.

[2] Aziz MG. Production and application of glucose-fructose oxidoreductase for conversion of pineapple juice sugars. Afr J Microbiol Res, 2011, 5(28): 5046-50525.

[3] Bantle JP. Dietary fructose and metabolic syndrome and diabetes. J Nutr, 2009, 9(6): 1263S-1268S.

[4] Berenguer M, Vegara S, Barrajón E, Saura D, Valero M, Martí N. Physicochemical characterization of pomegranate wines fermented with three different Saccharomyces cerevisiae yeast strains. Food Chem, 2016, 190: 848-855.

[5] Brenac L, Baidoo EEK, Keasling JD, Budin I. Distinct functional roles for hopanoid composition in the chemical tolerance of Zymomonas mobilis. Mol Microbiol, 2019, 112(5): 1564-1575.

[6] Delli Bovi AP, Di Michele L, Laino G, Vajro P. Obesity and obesity related diseases, sugar consumption and bad oral health: a fatal epidemic mixtures: the pediatric and odontologist point of view. Transl Med UniSa, 2017, 16: 11-16.

[7] Demiray E, Karatay SE, Donmez G. Improvement of bioethanol production from pomegranate peels via acidic pretreatment and enzymatic hydrolysis. Environ Sci Pollut Res Int, 2019, 26(28): 29366-29378.

[8] Duarte-Delgado D, Narvaez-Cuenca CE, Restrepo-Sanchez LP, Kushalappa A, Mosquera-Vasquez T. Development and validation of a liquid chromatographic method to quantify sucrose, glucose, and fructose in tubers of Solanum tuberosum Group Phureja. J Chromatogr B Analyt Technol Biomed Life Sci, 2015, 975: 18-23.

[9] Febbraio MA, Karin M. "Sweet death": Fructose as a metabolic toxin that targets the gut-liver axis. Cell Metab, 2021, 12: 23.

[10] Felczak MM, Bowers RM, Woyke T, TerAvest MA. Zymomonas diversity and potential for biofuel production. Biotechnol Biofuels, 2021, 14(1): 112.

[11] Günan Yücel H, Aksu Z. Ethanol fermentation characteristics of Pichia stipitis yeast from sugar beet pulp hydrolysate: use of new detoxification methods. Fuel, 2015, 158: 793-799.

[12] Hidalgo C, Mateo E, Mas A, Torija MJ. Identification of yeast and acetic acid bacteria isolated from the fermentation and acetification of persimmon (Diospyros kaki). Food Microbiol, 2012, 30(1): 98-104.

[13] Hwang KA, Hwang YJ, Hwang IG, Song J, Cho SM. Cholesterol-lowering effect of astringent persimmon fruits (Diospyros kaki Thunb.) extracts. Food Sci Biotechnol, 2017, 26(1): 229-235.

[14] Idise O, Odum E. Studies of wine produced from banana (Musa sapientum). Int J Biotechnol Mol Biol Res, 2011, 2(12): 209-214.

[15] Jacobson TB, Adamczyk PA, Stevenson DM, Regner M, Ralph J, Reed JL, Amador-Noguez D. ²H and ¹³C metabolic flux analysis elucidates in vivo thermodynamics of the ED pathway in Zymomonas mobilis. Metab Eng, 2019, 54: 301-316.

[16] Johnson RJ, Perez-Pozo SE, Lillo JL, Grases F, Schold JD, Kuwabara M, Sato Y, Hernando AA, Garcia G, Jensen T, Rivard C, Sanchez-Lozada LG, Roncal C, Lanaspa MA. Fructose increases risk for kidney stones: potential role in metabolic syndrome and heat stress. BMC Nephrol, 2018, 19(1): 315.

[17] Jonas R, Silveira M. Sorbitol can be produced not only chemically but also biotechnologically. Appl Biochem Biotechnol, 2004, 118(1–3): 321-336.

[18] Jones-Burrage SE, Kremer TA, McKinlay JB. Cell aggregation and aerobic respiration are important for Zymomonas mobilis ZM4 Survival in an aerobic minimal medium. Appl Environ Microbiol, 2019, 85(10): e00193-e219.

[19] Kohn JC, Azar J, Seta F, Reinhart-King CA. High-fat, high-sugar diet-induced subendothelial matrix stiffening is mitigated by exercise. Cardiovasc Eng Technol, 2017, 9(1): 84-93.

[20] Liu C, Dong H, Zhong J, Ryu DD, Bao J. Sorbitol production using recombinant Zymomonas mobilis strain. J Biotechnol, 2010, 148(2–3): 105-112.

[21] Liu Y, Ghosh IN, Martien J, Zhang Y, Amador-Noguez D, Landick R. Regulated redirection of central carbon flux enhances anaerobic production of bioproducts in Zymomonas mobilis. Metab Eng, 2020, 61: 261-274.

[22] Loos H, Kramer R, Sahm H, Sprenger GA. Sorbitol promotes growth of Zymomonas mobilis in environments with high concentrations of sugar: evidence for a physiological function of glucose-fructose oxidoreductase in osmoprotection. J Bacteriol, 1994, 176(24): 7688-7693.

[23] Martien JI, Hebert AS, Stevenson DM, Regner MR, Khana DB, Coon JJ, Amador-Noguez D. Systems-level analysis of oxygen exposure in Zymomonas mobilis: implications for isoprenoid production. mSystems, 2019, 4(1): e00284.

[24] Millis N. A study of the cider-sickness Bacillus - a New Variety of Zymomonas anaerobia. J Gen Microbiol, 1956, 15(3): 521-528.

[25] Musatti A, Mapelli C, Rollini M, Foschino R, Picozzi C. Can Zymomonas mobilis substitute Saccharomyces cerevisiae in cereal dough leavening?. Foods, 2018, 7(4): 61.

[26] Nilambari D, Jadhav B. Technology development for ethanol production from the wild fruits of Mimusops Hexandra. Res J Biotechnol, 2010, 5(3): 63-67.

[27] Ong WK, Courtney DK, Pan S, Andrade RB, Kiley PJ, Pfleger BF, Reed JL. Model-driven analysis of mutant fitness experiments improves genome-scale metabolic models of Zymomonas mobilis ZM4. PLoS Comput Biol, 2020, 16(8): e1008137.

[28] Park S, Baratti J. Comparison of ethanol production by Zymomonas mobilis from sugar beet substrates. Appl Microbiol Biotechnol, 1991, 1991(35): 283-291.

[29] Parker C, Peekhaus N, Zhang X, Conway T. Kinetics of sugar transport and phosphorylation influence glucose and fructose cometabolism by Zymomonas mobilis. Appl Environ Microbiol, 1997, 63(9): 3519-3525.

[30] Rice T, Zannini E, E, K.A., Coffey, A.. A review of polyols-biotechnological production, food applications, regulation, labeling and health effects. Crit Rev Food Sci Nutr, 2020, 60(12): 2034-2051.

[31] Roda A, Lucini L, Torchio F, Dordoni R, De Faveri DM, Lambri M. Metabolite profiling and volatiles of pineapple wine and vinegar obtained from pineapple waste. Food Chem, 2017, 229: 734-742.

[32] Seo JS, Chong H, Park HS, Yoon KO, Jung C, Kim JJ, Hong JH, Kim H, Kim JH, Kil JI, Park CJ, Oh HM, Lee JS, Jin SJ, Um HW, Lee HJ, Oh SJ, Kim JY, Kang HL, Lee SY, Lee KJ, Kang HS. The genome sequence of the ethanologenic bacterium Zymomonas mobilis ZM4. Nat Biotechnol, 2005, 23(1): 63-68.

[33] Shen W, Zhang J, Geng B, Qiu M, Hu M, Yang Q, Bao W, Xiao Y, Zheng Y, Peng W, Zhang G, Ma L, Yang S. Establishment and application of a CRISPR-Cas12a assisted genome-editing system in Zymomonas mobilis. Microb Cell Fact, 2019, 18(1): 162.

[34] Silbir S, Dagbagli S, Yegin S, Baysal T, Goksungur Y. Levan production by Zymomonas mobilis in batch and continuous fermentation systems. Carbohydr Polym, 2014, 99: 454-461.

[35] Silveira M, Jonas R. The biotechnological production of sorbitol. Appl Microbiol Biotechnol, 2002, 59(4–5): 400-408.

[36] Stoneman HR, Wrobel RL, Place M, Graham M, Krause DJ, De Chiara M, Liti G, Schacherer J, Landick R, Gasch AP, Sato TK, Hittinger CT. CRISpy-pop: a web tool for designing CRISPR/Cas9-driven genetic modifications in diverse populations. G3 (bethesda), 2020, 10(11): 4287-4294.

[37] Tastan O, Sozgen G, Baysal T, Kaplan Turkoz B. Production of prebiotic 6-kestose using Zymomonas mobilis levansucrase in carob molasses and its effect on 5-HMF levels during storage. Food Chem, 2019, 297: 124897.

[38] Tatli M, Hebert AS, Coon JJ, Amador-Noguez D. Genome wide phosphoproteome analysis of Zymomonas mobilis under anaerobic, aerobic, and N₂-fixing conditions. Front Microbiol, 2019, 10: 1986.

[39] Taylor SR, Ramsamooj S, Liang RJ, Katti A, Pozovskiy R, Vasan N, Hwang SK, Nahiyaan N, Francoeur NJ, Schatoff EM, Johnson JL, Shah MA, Dannenberg AJ, Sebra RP, Dow LE, Cantley LC, Rhee KY, Goncalves MD. Dietary fructose improves intestinal cell survival and nutrient absorption. Nature, 2021, 597(7875): 263-267.

[40] Todhanakasem T, Wu B, Simeon S. Perspectives and new directions for bioprocess optimization using Zymomonas mobilis in the ethanol production. World J Microbiol Biotechnol, 2020, 36(8): 112.

[41] Vera JM, Ghosh IN, Zhang Y, Hebert AS, Coon JJ, Landick R. Genome-scale transcription-translation mapping reveals features of Zymomonas mobilis transcription units and promoters. mSystems, 2020, 5(4): e00250.

[42] Wang X, He Q, Yang Y, Wang J, Haning K, Hu Y, Wu B, He M, Zhang Y, Bao J, Contreras LM, Yang S. Advances and prospects in metabolic engineering of Zymomonas mobilis. Metab Eng, 2018, 50: 57-73.

[43] Xia J, Yang Y, Liu CG, Yang S, Bai FW. Engineering Zymomonas mobilis for robust cellulosic ethanol production. Trends Biotechnol, 2019, 37(9): 960-972.

[44] Yang S, Fei Q, Zhang Y, Contreras LM, Utturkar SM, Brown SD, Himmel ME, Zhang M. Zymomonas mobilis as a model system for production of biofuels and biochemicals. Microb Biotechnol, 2016, 9(6): 699-717.

[45] Yang H, Sun J, Tian T, Gu H, Li X, Cai G, Lu J. Physicochemical characterization and quality of Dangshan pear wines fermented with different Saccharomyces cerevisiae. J Food Biochem, 2019, 43(8): e12891.

[46] Yang Q, Yang Y, Tang Y, Wang X, Chen Y, Shen W, Zhan Y, Gao J, Wu B, He M, Chen S, Yang S. Development and characterization of acidic-pH-tolerant mutants of Zymomonas mobilis through adaptation and next-generation sequencing-based genome resequencing and RNA-Seq. Biotechnol Biofuels, 2020, 13: 144.

[47] Yang Y, Geng B, Song H, He Q, He M, Bao J, Bai FW, Yang S. Progress and perspectives on developing Zymomonas mobilis as a chassis cell. Synth Biol J, 2021, 2(1): 59-90.

[48] Zheng Y, Han J, Wang B, Hu X, Li R, Shen W, Ma X, Ma L, Yi L, Yang S, Peng W. Characterization and repurposing of the endogenous Type I-F CRISPR-Cas system of Zymomonas mobilis for genome engineering. Nucleic Acids Res, 2019, 47(21): 11461-11475.

[49] Zhu JC, Niu YW, Feng T, Liu SJ, Cheng HX, Xu N, Yu HY, Xiao ZB. Evaluation of the formation of volatiles and sensory characteristics of persimmon (Diospyros kaki Lf) fruit wines using different commercial yeast strains of Saccharomyces cerevisiae. Nat Prod Res, 2014, 28(21): 1887-1893.

[50] Zou B, Wu J, Yu Y, Xiao G, Xu Y. Evolution of the antioxidant capacity and phenolic contents of persimmon during fermentation. Food Sci Biotechnol, 2017, 26(3): 563-571.

Funding

National Natural Science Foundation of China(21978071)