Metabolic engineering strategies for constructing methylotrophic cell factories

Abstract

Methanol is a one-carbon compound that has emerged as a promising carbon source for microbial bioproduction due to its abundance and sustainability. Despite the development of synthetic methylotrophic cell factories, challenges, such as the accumulation of toxic intermediates and suboptimal growth rates, have hindered their industrial application. This review summarizes the engineering strategies for methylotrophic cell factory synthesis, including pathway engineering for methanol assimilation optimization, detoxification methods targeting formaldehyde accumulation, and optimization of cell resource utilization. Future challenges and prospects of advancing microbial methanol assimilation in biotechnological applications are also highlighted.

Keywords

Methanol assimilation / Metabolic engineering / Pathway engineering / Adaptive laboratory evolution

References

[1] LiaoJC, MiL, PontrelliS, LuoSS. Fuelling the future: microbial engineering for the production of sustainable biofuels. Nat Rev Microbiol, 2016, 14(5): 288-304

[2] LeeSY, KimHU, ChaeTU, ChoJS, KimJW, ShinJH, KimDI, KoY-S, JangWD, JangY-S. A comprehensive metabolic map for production of bio-based chemicals. Nat Catal, 2019, 2(1): 18-33

[3] ClomburgJM, CrumbleyAM, GonzalezR. Industrial biomanufacturing: the future of chemical production. Science, 2017, 3556320aag0804

[4] CottonCA, ClaassensNJ, Benito-VaquerizoS, Bar-EvenA. Renewable methanol and formate as microbial feedstocks. Curr Opin Biotechnol, 2020, 62: 168-80

[5] WangY, FanL, TuyishimeP, ZhengP, SunJ. Synthetic methylotrophy: A practical solution for methanol-based biomanufacturing. Trends Biotechnol, 2020, 38(6): 650-66

[6] ZhuWL, CuiJY, CuiLY, LiangWF, YangS, ZhangC, XingXH. Bioconversion of methanol to value-added mevalonate by engineered Methylobacterium extorquens AM1 containing an optimized mevalonate pathway. Appl Microbiol Biotechnol, 2016, 100(5): 2171-82

[7] Yang YM, Chen WJ, Yang J, Zhou YM, Hu B, Zhang M, Zhu LP, Wang GY, Yang S. Production of 3-hydroxypropionic acid in engineered Methylobacterium extorquens AM1 and its reassimilation through a reductive route. Microb Cell Fact 2017, 16.

[8] SonntagF, KronerC, LubutaP, PeyraudR, HorstA, BuchhauptM, SchraderJ. Engineering Methylobacterium extorquens for de Novo synthesis of the sesquiterpenoid α-humulene from methanol. Metab Eng, 2015, 32: 82-94

[9] ChistoserdovaL, KalyuzhnayaMG. Current trends in Methylotrophy. Trends Microbiol, 2018, 26(8): 703-14

[10] ZhangWM, ZhangT, WuSH, WuMK, XinFX, DongWL, MaJF, ZhangM, JiangM. Guidance for engineering of synthetic Methylotrophy based on methanol metabolism in Methylotrophy. RSC Adv, 2017, 7(7): 4083-91

[11] JiangW, Hernandez VillamorD, PengH, ChenJ, LiuL, HaritosV, Ledesma-AmaroR. Metabolic engineering strategies to enable microbial utilization of C1 feedstocks. Nat Chem Biol, 2021, 17(8): 845-55

[12] WangY, FanL, TuyishimeP, LiuJ, ZhangK, GaoN, ZhangZ, NiX, FengJ, YuanQ, et al.. Adaptive laboratory evolution enhances methanol tolerance and conversion in engineered Corynebacterium glutamicum. Commun Biol, 2020, 31217

[13] ChenFY, JungHW, TsueiCY, LiaoJC. Converting Escherichia coli to a synthetic Methylotroph growing solely on methanol. Cell, 2020, 182(4): 933-46e914

[14] ZhanC, LiX, LanG, BaidooEEK, YangY, LiuY, SunY, WangS, WangY, WangG, et al.. Reprogramming methanol utilization pathways to convert Saccharomyces cerevisiae to a synthetic Methylotroph. Nat Catal, 2023, 6(5): 435-50

[15] WhitakerWB, SandovalNR, BennettRK, FastAG, PapoutsakisET. Synthetic methylotrophy: engineering the production of biofuels and chemicals based on the biology of aerobic methanol utilization. Curr Opin Biotech, 2015, 33: 165-75

[16] Keller P, Noor E, Meyer F, Reiter MA, Anastassov S, Kiefer P, Vorholt JA. Methanol-dependent Escherichia coli strains with a complete ribulose monophosphate cycle. Nat Commun 2020, 11(1).

[17] Müller JEN, Meyer F, Litsanov B, Kiefer P, Potthoff E, Heux S, Quax WJ, Wendisch VF, Brautaset T, Portais JC et al. Engineering Escherichia coli for methanol conversion. Metab Eng 2015, 28:190–201.

[18] BogoradIW, ChenCT, TheisenMK, WuTY, SchlenzAR, LamAT, LiaoJC. Building carbon-carbon bonds using a biocatalytic methanol condensation cycle. Proc Natl Acad Sci U S A, 2014, 111(45): 15928-33

[19] SiegelJB, SmithAL, PoustS, WargackiAJ, Bar-EvenA, LouwC, ShenBW, EibenCB, TranHM, NoorE, et al.. Computational protein design enables a novel one-carbon assimilation pathway. Proc Natl Acad Sci U S A, 2015, 112(12): 3704-9

[20] ChouA, ClomburgJM, QianS, GonzalezR. 2-Hydroxyacyl-CoA lyase catalyzes acyloin condensation for one-carbon bioconversion. Nat Chem Biol, 2019, 15(9): 900-6

[21] Lu XY, Liu YW, Yang YQ, Wang SS, Wang Q, Wang XY, Yan ZH, Cheng J, Liu C, Yang X et al. Constructing a synthetic pathway for acetylcoenzyme A from one-carbon through enzyme design. Nat Commun 2019, 10.

[22] Meng X, Hu G, Li X, Gao C, Song W, Wei W, Wu J, Liu L. A synthetic Methylotroph achieves accelerated cell growth by alleviating transcription-replication conflicts. Nat Commun 2025, 16(1).

[23] KimS, LindnerSN, AslanS, YishaiO, WenkS, SchannK, Bar-EvenA. Growth of E. coli on formate and methanol via the reductive glycine pathway. Nat Chem Biol, 2020, 16(5): 538-45

[24] GaoJQ, LiYX, YuW, ZhouYJJ. Rescuing yeast from cell death enables overproduction of fatty acids from sole methanol. Nat Metab, 2022, 47932

[25] TuyishimeP, WangY, FanLW, ZhangQQ, LiQG, ZhengP, SunJB, MaYH. Engineering Corynebacterium glutamicum for methanol-dependent growth and glutamate production. Metab Eng, 2018, 49: 220-31

[26] de la GonzalezJ, MachensF, MesserschmidtK, Bar-EvenA. Core catalysis of the reductive glycine pathway demonstrated in yeast. ACS Synth Biol, 2019, 8(5): 911-7

[27] Wang G, Olofsson-Dolk M, Hansson FG, Donati S, Li X, Chang H, Cheng J, Dahlin J, Borodina I: Engineering yeast Yarrowia lipolytica for methanol assimilation. ACS Synth Biol 2021.

[28] DaiZ, GuH, ZhangS, XinF, ZhangW, DongW, MaJ, JiaH, JiangM. Metabolic construction strategies for direct methanol utilization in Saccharomyces cerevisiae. Bioresour Technol, 2017, 245(Pt B): 1407-12

[29] ChenC-T, ChenFYH, BogoradIW, WuT-Y, ZhangR, LeeAS, LiaoJC. Synthetic methanol auxotrophy of Escherichia coli for methanol-dependent growth and production. Metab Eng, 2018, 49: 257-66

[30] Yu H, Liao JC. A modified Serine cycle in Escherichia coli coverts methanol and CO2 to two-carbon compounds. Nat Commun 2018, 9(1).

[31] YangX, YuanQ, LuoH, LiF, MaoY, ZhaoX, DuJ, LiP, JuX, ZhengY, et al.. Systematic design and in vitro validation of novel one-carbon assimilation pathways. Metab Eng, 2019, 56: 142-53

[32] LuX, LiuY, YangY, WangS, WangQ, WangX, YanZ, ChengJ, LiuC, YangX, et al.. Constructing a synthetic pathway for acetyl-coenzyme A from one-carbon through enzyme design. Nat Commun, 2019, 1011378

[33] 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).

[34] Wu T, Gómez-Coronado PA, Kubis A, Lindner SN, Marlière P, Erb TJ, Bar-Even A, He H. Engineering a synthetic energy-efficient formaldehyde assimilation cycle in Escherichia coli. Nat Commun 2023, 14(1).

[35] WhitakerWB, JonesJA, BennettRK, GonzalezJE, VernacchioVR, CollinsSM, PalmerMA, SchmidtS, AntoniewiczMR, KoffasMA, et al.. Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli. Metab Eng, 2017, 39: 49-59

[36] WoolstonBM, KingJR, ReiterM, Van HoveB, StephanopoulosG. Improving formaldehyde consumption drives methanol assimilation in engineered E. coli. Nat Commun, 2018, 912387

[37] WitthoffS, MühlrothA, MarienhagenJ, BottM. C1 metabolism in Corynebacterium glutamicum: an endogenous pathway for oxidation of methanol to carbon dioxide. Appl Environ Microb, 2013, 79(22): 6974-83

[38] RothTB, WoolstonBM, StephanopoulosG, LiuDR. Phage-assisted evolution of Bacillus methanolicus methanol dehydrogenase 2. ACS Synth Biol, 2019, 8(4): 796-806

[39] PriceJV, ChenL, WhitakerWB, PapoutsakisE, ChenW. Scaffoldless engineered enzyme assembly for enhanced methanol utilization. Proc Natl Acad Sci U S A, 2016, 113(45): 12691-6

[40] FanLW, WangY, TuyishimeP, GaoN, LiQG, ZhengP, SunJB, MaYH. Engineering artificial fusion proteins for enhanced methanol bioconversion. ChemBioChem, 2018, 19(23): 2465-71

[41] Jia M, Shao L, Jiang J, Jiang W, Xin F, Zhang W, Jiang Y, Jiang M. Mitigating toxic formaldehyde to promote efficient utilization of C1 resources. Crit Rev Biotechnol 2024:1–13.

[42] KawanishilM, MatsudaT, YagiT. Genotoxicity of formaldehyde: molecular basis of DNA damage and mutation. Front Environ Sci, 2014, 236

[43] ZhouYJ, KerkhovenEJ, NielsenJ. Barriers and opportunities in bio-based production of hydrocarbons. Nat Energy, 2018, 3(11): 925-35

[44] YuR, LaiYQ, HartwellHJ, MoellerBC, Doyle-EiseleM, KrackoD, BodnarWM, StarrTB, SwenbergJA. Formation, accumulation, and hydrolysis of endogenous and exogenous formaldehyde-induced DNA damage. Toxicol Sci, 2015, 146(1): 170-82

[45] ZhangM, YuanXJ, ZhangC, ZhuLP, MoXH, ChenWJ, YangS. Bioconversion of methanol into valueadded chemicals in native and synthetic Methylotrophs. Curr Issues Mol Biol, 2019, 33: 225-36

[46] BennettRK, GonzalezJE, WhitakerWB, AntoniewiczMR, PapoutsakisET. Expression of heterologous non-oxidative Pentose phosphate pathway from Bacillus methanolicus and phosphoglucose isomerase deletion improves methanol assimilation and metabolite production by a synthetic Escherichia coli Methylotroph. Metab Eng, 2018, 45: 75-85

[47] WuG, YanQ, JonesJA, TangYJ, FongSS, KoffasMAG. Metabolic burden: cornerstones in synthetic biology and metabolic engineering applications. Trends Biotechnol, 2016, 34(8): 652-64

[48] Meyer F, Keller P, Hartl J, Gröninger OG, Kiefer P, Vorholt JA. Methanol-essential growth of Escherichia coli. Nat Commun 2018, 9.

[49] GaneshI, VidhyaS, EomGT, HongSH. Construction of Methanol-Sensing Escherichia coli by the introduction of a Paracoccus denitrificans MxaY-Based chimeric Two-Component system. J Microbiol Biotechnol, 2017, 27(6): 1106-11

[50] WoolstonBM, RothT, KohaleI, LiuDR, StephanopoulosG. Development of a formaldehyde biosensor with application to synthetic Methylotrophy. Biotechnol Bioeng, 2018, 115(1): 206-15

[51] RohlhillJ, Gerald HarJR, AntoniewiczMR, PapoutsakisET. Improving synthetic Methylotrophy via dynamic formaldehyde regulation of Pentose phosphate pathway genes and redox perturbation. Metab Eng, 2020, 57: 247-55

[52] OerlemansR, TimmermansS, van HestJ. Artificial organelles: towards adding or restoring intracellular activity. ChemBioChem, 2021, 22: 2051-78

[53] Grewal PS, Samson JA, Baker JJ, Choi B, Dueber JE. Peroxisome compartmentalization of a toxic enzyme improves alkaloid production. Nat Chem Biol 2020.

[54] Schmid-DannertC, Lopez-GallegoF. Advances and opportunities for the design of self-sufficient and spatially organized cell-free biocatalytic systems. Curr Opin Chem Biol, 2019, 49: 97-104

[55] TranPHN, LeeTS. Harnessing organelle engineering to facilitate biofuels and biochemicals production in yeast. J Microbiol, 2025, 633e2501006

[56] HammerSK, AvalosJL. Harnessing yeast organelles for metabolic engineering. Nat Chem Biol, 2017, 13(8): 823-32

[57] DeLoacheWC, RussZN, DueberJE. Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways. Nat Commun, 2016, 711152

[58] LiM, SunW, WangX, ChenK, FengY, TanZ. A eukaryote-featured membrane phospholipid enhances bacterial formaldehyde tolerance and assimilation of one-carbon feedstocks. ACS Synth Biol, 2024, 13(12): 4074-84

[59] Godoy-Gallardo M, Labay C, Jansman MM, Ek PK, Hosta-Rigau L. Intracellular microreactors as artificial organelles to conduct multiple enzymatic reactions simultaneously. Adv Healthc Mater 2017, 6(4).

[60] Zhou P, Liu H, Meng X, Zuo HY, Qi MY, Guo L, Gao C, Song W, Wu J, Chen XL et al. Engineered artificial membraneless organelles in Saccharomyces cerevisiae to enhance chemical production. Angew Chem Int Ed 2023, 62(14).

[61] de JongH, GeiselmannJ, RopersD. Resource reallocation in bacteria by reengineering the gene expression machinery. Trends Microbiol, 2017, 25(6): 480-93

[62] Montano LopezJ, DuranL, AvalosJL. Physiological limitations and opportunities in microbial metabolic engineering. Nat Rev Microbiol, 2021, 20: 35-48

[63] GaoC, XuP, YeC, ChenX, LiuL. Genetic circuit-assisted smart microbial engineering. Trends Microbiol, 2019, 27(12): 1011-24

[64] Leão-HelderAN, KrikkenAM, van der KleiIJ, KielJAKW, VeenhuisM. Transcriptional down-regulation of peroxisome numbers affects selective peroxisome degradation in Hansenula polymorpha. J Biol Chem, 2003, 278(42): 40749-56

[65] GonzalezJE, BennettRK, PapoutsakisET, AntoniewiczMR. 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

[66] Lee J-Y, Sung BH, Oh S-H, Kwon KK, Lee H, Kim H, Lee D-H, Yeom S-J, Lee S-G. C1 compound biosensors: design, functional study, and applications. Int J Mol Sci 2019, 20(9).

[67] VemuriGN, AltmanE, SangurdekarDP, KhodurskyAB, EitemanMA. Overflow metabolism in Escherichia coli during steady-state growth: transcriptional regulation and effect of the redox ratio. Appl Environ Microb, 2006, 72(5): 3653-61

[68] MüllerJEN, MeyerF, LitsanovB, KieferP, VorholtJA. Core pathways operating during Methylotrophy of Bacillus methanolicus MGA3 and induction of a bacillithiol-dependent detoxification pathway upon formaldehyde stress. Mol Microbiol, 2015, 98(6): 1089-100

[69] WuY, JameelA, XingX-H, ZhangC. Advanced strategies and tools to facilitate and streamline microbial adaptive laboratory evolution. Trends Biotechnol, 2021, 40(1): 38-59

[70] ReiterMA, BradleyT, BüchelLA, KellerP, HegedisE, GasslerT, VorholtJA. A synthetic Methylotrophic Escherichia coli as a chassis for bioproduction from methanol. Nat Catal, 2024, 7(5): 560-73

[71] Bang J, Ahn JH, Lee JA, Hwang CH, Kim GB, Lee J, Lee SY. Synthetic formatotrophs for one-carbon biorefinery. Adv Sci 2021.

[72] ZhangZ, WangY, ZhengP, SunJ. Promoting lignin valorization by coping with toxic C1 byproducts. Trends Biotechnol, 2021, 39(4): 331-5

[73] FabariusJT, WegatV, RothA, SieberV. Synthetic Methylotrophy in yeasts: towards a circular bioeconomy. Trends Biotechnol, 2021, 39(4): 348-58

[74] Zhou P, Gao C, Song W, Wei W, Wu J, Liu L, Chen X. Engineering status of protein for improving microbial cell factories. Biotechnol Adv 2024, 70.

[75] XuN, LiuY, JiangH, LiuJ, MaY. Combining protein and metabolic engineering to construct efficient microbial cell factories. Curr Opin Biotechnol, 2020, 66: 27-35

[76] GarabedianMV, WangW, DabdoubJB, TongM, CaldwellRM, BenmanW, SchusterBS, DeitersA, GoodMC. Designer membraneless organelles sequester native factors for control of cell behavior. Nat Chem Biol, 2021, 17: 998-1007

[77] avalos sKHJL. Harnessing yeast organelles for metabolic engineering. Nat Chem Biol, 2017, 13: 823-32

[78] Wang T, Guan C, Guo J, Liu B, Wu Y, Xie Z, Zhang C, Xing X-H. Pooled CRISPR interference screening enables genome-scale functional genomics study in bacteria with superior performance. Nat Commun 2018, 9(1).

[79] GarstAD, BassaloMC, PinesG, LynchSA, Halweg-EdwardsAL, LiuRM, LiangLY, WangZW, ZeitounR, AlexanderWG, et al.. Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering. Nat Biotechnol, 2017, 35(1): 48-55

[80] NiehL-Y, ChenFYH, JungH-W, SuK-Y, TsueiC-Y, LinC-T, LeeY-Q, LiaoJC. Evolutionary engineering of Methylotrophic E. coli enables fast growth on methanol. Nat Commun, 2024, 1518840

[81] LiB, YangZ, LiZ, ZhangY, ZhangL, WangW. Enabling genetic manipulation and robustness of Bacillus methanolicus for methanol-based bio-manufacturing. Metab Eng, 2025, 89: 121-34

[82] GuoF, LiuK, QiaoYY, ZhengYM, LiuCG, WuY, ZhangZH, JiangWK, JiangYJ, XinFX, et al.. Evolutionary engineering of Saccharomyces cerevisiae: crafting a synthetic Methylotroph via self-reprogramming. Sci Adv, 2024, 1051eadq3484

[83] ZhouP, LiuH, MengX, ZuoH, QiM, GuoL, GaoC, SongW, WuJ, ChenX, et al.. Engineered artificial membraneless organelles in Saccharomyces cerevisiae to enhance chemical production. Angew Chem Int Ed, 2023, 6214e202215778

Funding

Key Research and Development Project of Henan Province(231111310700)

Key Technologies R&D Program of Henan Province (252102311184)

Key Research Projects of Higher Education Institutions in Henan Province(25A416005)

Natural Science Foundation of Henan Province(252300420649)

RIGHTS & PERMISSIONS

Jiangnan University

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