Improved succinic acid production through the reconstruction of methanol dissimilation in Escherichia coli

Abstract

Synthetic biology has boosted the rapid development on using non-methylotrophy as chassis for value added chemicals production from one-carbon feedstocks, such as methanol and formic acid. The one-carbon dissimilation pathway can provide more NADH than monosaccharides including glucose, which is conducive for reductive chemicals production, such as succinic acid. In this study, the one-carbon dissimilation pathway was introduced in E. coli Suc260 to enhance the succinic acid production capability. Through the rational construction of methanol dissimilation pathway, the succinic acid yield was increased from 0.91 to 0.95 g/g with methanol and sodium formate as auxiliary substrates in anaerobic fed-batch fermentation. Furthermore, the metabolic flux of by-product pyruvate was redirected to succinic acid together with the CO₂ fixation. Finally, through the immobilization on a specially designed glycosylated membrane, E. coli cells are more resistant to adverse environments, and the final yield of succinic acid was improved to 0.98 g/g. This study proved the feasibility of endowing producers with methanol dissimilation pathway to enhance the production of reductive metabolites.

Keywords

Methanol dissimilation / C1-substrates utilization / Succinic acid fermentation / CO₂ fixation / Synthetic biology

References

[1] Ahn JH, Bang J, Kim WJ, Lee SY. Formic acid as a secondary substrate for succinic acid production by metabolically engineered Mannheimia succiniciproducens. Biotechnol Bioeng, 2017, 114(12): 2837-2847.

[2] Balzer GJ, Thakker C, Bennett GN, San KY. Metabolic engineering of Escherichia coli to minimize byproduct formate and improving succinate productivity through increasing NADH availability by heterologous expression of NAD(+)-dependent formate dehydrogenase. Metab Eng, 2013, 20: 1-8.

[3] Bang J, Lee SY. Assimilation of formic acid and CO2 by engineered Escherichia coli equipped with reconstructed one-carbon assimilation pathways. Proc Natl Acad Sci USA, 2018, 115(40): E9271-E9279.

[4] Berrios-Rivera SJ, Bennett GN, San KY. The effect of increasing NADH availability on the redistribution of metabolic fluxes in Escherichia coli chemostat cultures. Metab Eng, 2002, 4(3): 230-237.

[5] Berrios-Rivera SJ, Bennett GN, San KY. Metabolic engineering of Escherichia coli: increase of NADH availability by overexpressing an NAD(+)-dependent formate dehydrogenase. Metab Eng, 2002, 4(3): 217-229.

[6] Bradfield MFA, Mohagheghi A, Salvachua D, Smith H, Black BA, Dowe N, Beckham GT, Nicol W. Continuous succinic acid production by Actinobacillus succinogenes on xylose-enriched hydrolysate. Biotechnol Biofuels, 2015, 8: 1-17.

[7] Chen KQ, Jiang M, Wei P, Yao JM, Wu H. Succinic acid production from acid hydrolysate of corn fiber by Actinobacillus succinogenes. Appl Biochem Biotechnol, 2010, 160(2): 477-485.

[8] Chen KQ, Zhang H, Miao YL, Jiang M, Chen JY. Succinic acid production from enzymatic hydrolysate of sake lees using Actinobacillus succinogenes 130Z. Enzyme Microb Technol, 2010, 47(5): 236-240.

[9] Chen PC, Tao ST, Zheng P. Efficient and repeated production of succinic acid by turning sugarcane bagasse into sugar and support. Bioresour Technol, 2016, 211: 406-413.

[10] Cheng KK, Zhao XB, Zeng J, Zhang JA. Biotechnological production of succinic acid: current state and perspectives. Biofuels Bioprod Biorefin, 2012, 6(3): 302-318.

[11] Dai ZX, Gu HL, Zhang SJ, Xin FX, Zhang WM, Dong WL, Ma JF, Jia HH, Jiang M. Metabolic construction strategies for direct methanol utilization in Saccharomyces cerevisiae. Bioresour Technol, 2017, 245: 1407-1412.

[12] Dai ZX, Guo F, Zhang SJ, Zhang WM, Yang Q, Dong WL, Jiang M, Ma JF, Xin FX. Bio-based succinic acid: an overview of strain development, substrate utilization, and downstream purification. Biofuels Bioprod Biorefin, 2020, 14(5): 965-985.

[13] Dessie W, Zhu JR, Xin FX, Zhang WM, Jiang YM, Wu H, Ma JF, Jiang M. Bio-succinic acid production from coffee husk treated with thermochemical and fungal hydrolysis. Bioprocess Biosyst Eng, 2018, 41(10): 1461-1470.

[14] Eshinimaev BT, Khmelenina VN, Sakharovskii VG, Suzina NE, Trotsenko YA. Physiological, biochemical, and cytological characteristics of a haloalkalitolerant methanotroph grown on methanol. Microbiology, 2002, 71(5): 512-518.

[15] Gao H, Wang J, Wu H, Xin F, Zhang W, Jiang M, Fang Y. Biofilm-integrated glycosylated membrane for biosuccinic acid production. ACS Appl Bio Mater, 2021, 4(10): 7517-7523.

[16] Gokarn RR, Eiteman MA, Altman E. Expression of pyruvate carboxylase enhances succinate production in Escherichia coli without affecting glucose uptake. Biotechnol Lett, 1998, 20(8): 795-798.

[17] Gonzalez JE, Bennett RK, Papoutsakis ET, Antoniewicz MR. Methanol assimilation in Escherichia coli is improved by co-utilization of threonine and deletion of leucine-responsive regulatory protein. Metab Eng, 2018, 45: 67-74.

[18] Gross R, Hauer B, Otto K, Schmid A. Microbial biofilms: new catalysts for maximizing productivity of long-term biotransformations. Biotechnol Bioeng, 2007, 98(6): 1123-1134.

[19] Guarnieri MT, Chou YC, Salvachua D, Mohagheghi A, John PCS, Peterson DJ, Bomble YJ, Beckham GT. Metabolic engineering of Actinobacillus succinogenes provides insights into succinic acid biosynthesis. Appl Environ Microbiol, 2017, 83(17

[20] Guo F, Dai Z, Peng W, Zhang S, Zhou J, Ma J, Dong W, Xin F, Zhang W, Jiang M. Metabolic engineering of Pichia pastoris for malic acid production from methanol. Biotechnol Bioeng, 2021, 118(1): 357-371.

[21] Irla M, Nærdal I, Brautaset T, Wendisch VF. Methanol-based γ-aminobutyric acid (GABA) production by genetically engineered Bacillus methanolicus strains. Ind Crops Prod, 2016, 106: 12-20.

[22] Isikgor FH, Becer CR. Lignocellulosic biomass: a sustainable platform for the production of bio-based chemicals and polymers. Polym Chem, 2015, 6(25): 4497-4559.

[23] Jian X, Li N, Zhang C, Hua Q. In silico profiling of cell growth and succinate production in Escherichia coli NZN111. Bioresour Bioprocess, 2016, 3(1): 48.

[24] Jiang W, Villamor DH, Peng HD, Chen J, Liu L, Haritos V, Ledesma-Amaro R. Metabolic engineering strategies to enable microbial utilization of C1 feedstocks. Nat Chem Biol, 2021, 17(8): 845-855.

[25] Jiang YJ, Liu YS, Zhang XY, Gao H, Mou L, Wu MD, Zhang WM, Xin FX, Jiang M. Biofilm application in the microbial biochemicals production process. Biotechnol Adv, 2021, 48.

[26] Lee SJ, Song H, Lee SY. Genome-based metabolic engineering of Mannheimia succiniciproducens for succinic acid production. Appl Environ Microbiol, 2006, 72(3): 1939-1948.

[27] Lee JW, Yi J, Kim TY, Choi S, Ahn JH, Song H, Lee MH, Lee SY. Homo-succinic acid production by metabolically engineered Mannheimia succiniciproducens. Metab Eng, 2016, 38: 409-417.

[28] Leonardo MR, Dailly Y, Clark DP. Role of NAD in regulating the adhE gene of Escherichia coli. J Bacteriol, 1996, 178(20): 6013-6018.

[29] Li YK, Li MJ, Zhang X, Yang P, Liang QF, Qi QS. A novel whole-phase succinate fermentation strategy with high volumetric productivity in engineered Escherichia coli. Bioresour Technol, 2013, 149: 333-340.

[30] Li YJ, Huang B, Wu H, Li ZM, Ye Q, Zhang YHP. Production of succinate from acetate by metabolically engineered Escherichia coli. ACS Synth Biol, 2016, 5(11): 1299-1307.

[31] Li J, Li Y, Cui Z, Liang Q, Qi Q. Enhancement of succinate yield by manipulating NADH/NAD+ ratio and ATP generation. Appl Microbiol Biotechnol, 2017, 101(8): 3153-3161.

[32] Li Q, Huang B, He QF, Lu JX, Li X, Li ZM, Wu H, Ye Q. Production of succinate from simply purified crude glycerol by engineered Escherichia coli using two-stage fermentation. Bioresour Bioprocess, 2018, 5: 1-10.

[33] Liang LY, Liu RM, Wang GM, Gou DM, Ma JF, Chen KQ, Jiang M, Wei P, Ouyang PK. Regulation of NAD(H) pool and NADH/NAD(+) ratio by overexpression of nicotinic acid phosphoribosyltransferase for succinic acid production in Escherichia coli NZN111. Enzyme Microb Technol, 2012, 51(5): 286-293.

[34] Liu YQ, Bai CX, Xu Q, Yu JH, Zhou XS, Zhang YX, Cai MH. Improved methanol-derived lovastatin production through enhancement of the biosynthetic pathway and intracellular lovastatin efflux in methylotrophic yeast. Bioresour Bioprocess, 2018, 5(1): 22.

[35] McKinlay JB, Vieille C. C-13-metabolic flux analysis of Actinobacillus succinogenes fermentative metabolism at different NaHCO₃ and H-2 concentrations. Metab Eng, 2008, 10(1): 55-68.

[36] Minh DP, Besson M, Pinel C, Fuertes P, Petitjean C. Aqueous-phase hydrogenation of biomass-based succinic acid to 1,4-butanediol over supported bimetallic catalysts. Top Catal, 2010, 53(15–18): 1270-1273.

[37] Mokhtari-Hosseini ZB, Vasheghani-Farahani E, Shojaosadati SA, Karimzadeh R, Heidarzadeh-Vazifekhoran A. Effect of feed composition on PHB production from methanol by HCDC of Methylobacterium extorquens (DSMZ 1340). J Chem Technol Biotechnol, 2009, 84(8): 1136-1139.

[38] Muller JEN, Meyer F, Litsanov B, Kiefer P, Potthoff E, Heux S, Quax WJ, Wendisch VF, Brautaset T, Portais JC, Vorholt JA. Engineering Escherichia coli for methanol conversion. Metab Eng, 2015, 28: 190-201.

[39] Naerdal I, Pfeifenschneider J, Brautaset T, Wendisch VF. Methanol-based cadaverine production by genetically engineered Bacillus methanolicus strains. Microb Biotechnol, 2015, 8(2): 342-350.

[40] Nash T. Citation classic—the colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Curr Contents Life Sci, 1981, 14: 14.

[41] Sanchez AM, Bennett GN, San KY. Efficient succinic acid production from glucose through overexpression of pyruvate carboxylase in an Escherichia coli alcohol dehydrogenase and lactate dehydrogenase mutant. Biotechnol Progr, 2005, 21(2): 358-365.

[42] Sehested J. Industrial and scientific directions of methanol catalyst development. J Catal, 2019, 371: 368-375.

[43] Sonntag F, Buchhaupt M, Schrader J. Thioesterases for ethylmalonyl-CoA pathway derived dicarboxylic acid production in Methylobacterium extorquens AM1. Appl Microbiol Biotechnol, 2014, 98(10): 4533-4544.

[44] Wang D, Li QA, Song ZY, Zhou W, Su ZG, Xing JM. High cell density fermentation via a metabolically engineered Escherichia coli for the enhanced production of succinic acid. J Chem Technol Biotechnol, 2011, 86(4): 512-518.

[45] Wang X, Wang Y, Liu J, Li Q, Zhang Z, Zheng P, Lu F, Sun J. Biological conversion of methanol by evolved Escherichia coli carrying a linear methanol assimilation pathway. Bioresour Bioprocess, 2017, 4(1): 41.

[46] Wang Y, Fan LW, Tuyishirne P, Zheng P, Sun JB. Synthetic methylotrophy: a practical solution for methanol-based biomanufacturing. Trends Biotechnol, 2020, 38(6): 650-666.

[47] Wegner GH. Emerging applications of the methylotrophic yeasts. FEMS Microbiol Lett, 1990, 87(3–4): 279-283.

[48] Werpy TA, Frye JG, Holladay JE. Succinic acid—a model building block for chemical production from renewable resources, 2006, Richland: Pacific Northwest National Lab (PNNL).

[49] Whitaker WB, Jones JA, Bennett RK, Gonzalez JE, Vernacchio VR, Collins SM, Palmer MA, Schmidt S, Antoniewicz MR, Koffas MA, Papoutsakis ET. Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli. Metab Eng, 2017, 39: 49-59.

[50] Yu J, Li ZM, Ye Q, Yang Y, Chen SL. Development of succinic acid production from corncob hydrolysate by Actinobacillus succinogenes. J Ind Microbiol Biotechnol, 2010, 37(10): 1033-1040.

[51] Zeikus JG, Jain MK, Elankovan P. Biotechnology of succinic acid production and markets for derived industrial products. Appl Microbiol Biotechnol, 1999, 51(5): 545-552.

[52] Zhang W, Zhang T, Song M, Dai Z, Zhang S, Xin F, Dong W, Ma J, Jiang M. Metabolic engineering of escherichia coli for high yield production of succinic acid driven by methanol. ACS Synth Biol, 2018, 7(12): 2803-2811.

[53] Zhang WM, Zhu JR, Zhu XG, Song M, Zhang T, Xin FX, Dong WL, Ma JF, Jiang M. Expression of global regulator IrrE for improved succinate production under high salt stress by Escherichia coli. Bioresour Technol, 2018, 254: 151-156.

Funding

National Key R&D Program of China(2018YFA0901500)

National Natural Science Foundation of China(22078151)

Natural Science Foundation of Jiangsu Province(BK20200683)