Tools and Strategies for Engineering Bacillus methanolicus: A Versatile Thermophilic Platform for Sustainable Bioproduction from Methanol and Alternative Feedstocks

Abstract

Bacillus methanolicus MGA3 is a methylotrophic bacterium with a high potential as a production host in the bioeconomy, particularly with methanol as a feedstock. This review presents the recent acceleration in strain engineering technologies through advances in transformation efficiency, the development of CRISPR/Cas9-based genome editing, and the application of genome-scale models (GSMs) for strain design. The generation of novel genetic tools broadens the biotechnological potential of this thermophilic methylotroph. B. methanolicus is a facultative methylotroph and apart from methanol it can grow on mannitol, arabitol and glucose, and was engineered for starch and xylose utilisation. Here, the central carbon metabolism of B. methanolicus for various native and non-native carbon sources is described, with an emphasis on methanol metabolism. With its expanding product portfolio, B. methanolicus demonstrates its potential as a microbial cell factory for the production of tricarboxylic acid(TCA) cycle and ribulose monophosphate (RuMP) cycle intermediates and their derivatives. Beyond small chemicals, B. methanolicus is both a valuable source of novel thermostable proteins and a host for the production of heterologous proteins, enabled by advances in genetic tools and cultivation methods. Continued progress in understanding its physiology and refining its genetic toolbox will be decisive in transforming B. methanolicus from a promising candidate into a fully established industrial workhorse for sustainable methanol-based biomanufacturing.

Keywords

Methanol / Mannitol / Seaweed hydrolysate / Riboflavin / Amino acids / Genome-scale metabolic model / CRISPR/Cas9

Author Contributions

Conceptualization,. L.F.B. and M.I.; Writing—Original Draft Preparation, L.F.B., M.K.F., H.Z., T.B., V.F.W., M.I.; Writing—Review & Editing, L.F.B., M.K.F., H.Z., T.B., V.F.W., M.I.; Visualization, L.F.B., M.K.F.; Funding Acquisition, L.F.B., T.B., V.F.W., M.I.

Ethics Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Funding

Luciana Fernandes Brito and Markus Klitgaard Friis were funded by the Novo Nordisk Foundation, with grants number NNF24OC0094177 and NNF23OC0086520, respectively. Haowen Zhu gratefully acknowledges support of his PhD studies in Bielefeld, Germany, by scholarship 202206880008 from the China Scholarship Council (CSC).

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References

[1] Arfman N, Dijkhuizen L, Kirchhof G, Ludwig W, Schleifer KH, Bulygina ES, et al. a new species of thermotolerant, methanol-utilizing, endospore-forming bacteria. Int. J. Syst. Evol. Microbiol. 1992, 42, 439-445.

[2] Schendel FJ, Bremmon CE, Flickinger MC, Guettler M, Hanson RS. L-Lysine production at 50 degrees C by mutants of a newly isolated and characterized methylotrophic Bacillus sp. Appl. Environ. Microbiol. 1990, 56, 963-970.

[3] Brautaset T, Jakobsen ØM, Flickinger MC, Valla S, Ellingsen TE. Plasmid-dependent methylotrophy in thermotolerant Bacillus methanolicus. J. Bacteriol. 2004, 186, 1229-1238.

[4] Heggeset TMB, Krog A, Balzer S, Wentzel A, Ellingsen TE, Brautaset T. Genome sequence of thermotolerant Bacillus methanolicus: Features and regulation related to methylotrophy and production of L-lysine and L-glutamate from methanol. Appl. Environ. Microbiol. 2012, 78, 5170-5181.

[5] Irla M, Nærdal I, Virant D, Brautaset T, Busche T, Goranovič D, et al. Systems-level analysis provides insights on methanol-based production of L-glutamate and its decarboxylation product γ-aminobutyric acid by Bacillus methanolicus. Metab. Eng. 2025, 91, 389-404.

[6] Haupka C, Brito LF, Busche T, Wibberg D, Wendisch VF. Genomic and transcriptomic investigation of the physiological response of the methylotroph Bacillus methanolicus to 5-aminovalerate. Front. Microbiol. 2021, 12, 664598.

[7] Irla M, Heggeset TMB, Nærdal I, Paul L, Haugen T, Le SB, et al. Genome-based genetic tool development for Bacillus methanolicus: Theta- and rolling circle-replicating plasmids for inducible gene expression and application to methanol-based cadaverine production. Front. Microbiol. 2016, 7, 1481.

[8] Nærdal I, Netzer R, Irla M, Krog A, Heggeset TMB, Wendisch VF, et al. L-Lysine production by Bacillus methanolicus: Genome-based mutational analysis and L-lysine secretion engineering. J. Biotechnol. 2017, 244, 25-33.

[9] Liu P, Yuan Q, Yang X, Wang Q, Chang T, Bi Y, et al. A synthetic biology toolkit for interrogating plasmid-dependent methylotrophy and enhancing methanol-based biosynthesis of Bacillus methanolicus. bioRxiv 2025. doi:10.1101/2025.05.06.652373.

[10] Jakobsen ØM, Benichou A, Flickinger MC, Valla S, Ellingsen TE, Brautaset T. Upregulated transcription of plasmid and chromosomal ribulose monophosphate pathway genes is critical for methanol assimilation rate and methanol tolerance in the methylotrophic bacterium Bacillus methanolicus. J. Bacteriol. 2006, 188, 3063-3072.

[11] Brautaset T, Jakobsen ØM, Degnes KF, Netzer R, Nærdal I, Krog A, et al. Bacillus methanolicus pyruvate carboxylase and homoserine dehydrogenase I and II and their roles for L-lysine production from methanol at 50 °C. Appl. Microbiol. Biotechnol. 2010, 87, 951-964.

[12] Drejer EB, Chan DTC, Haupka C, Wendisch VF, Brautaset T, Irla M.Methanol-based acetoin production by genetically engineered Bacillus methanolicus. Green. Chem. 2020, 22, 788-802.

[13] Brito LF, Irla M, Nærdal I, Le SB, Delépine B, Heux S, et al. Evaluation of heterologous biosynthetic pathways for methanol-based 5-aminovalerate production by thermophilic Bacillus methanolicus. Front. Bioeng. Biotechnol. 2021, 9, 686319.

[14] Irla M, Hakvåg S, Brautaset T. Developing a riboswitch-mediated regulatory system for metabolic flux control in thermophilic Bacillus methanolicus. Int. J. Mol. Sci. 2021, 22, 4686.

[15] Li B, Yang Z, Li Z, Zhang Y, Zhang L, Wang W. Enabling genetic manipulation and robustness of Bacillus methanolicus for methanol-based bio-manufacturing. Metab. Eng. 2025, 89, 121-134.

[16] Schultenkämper K, Brito LF, Wendisch VF. Impact of CRISPR interference on strain development in biotechnology. Biotechnol. Appl. Biochem. 2020, 67, 7-21.

[17] Schultenkämper K, Gütle DD, López MG, Keller LB, Zhang L, Einsle O, et al. Interrogating the role of the two distinct fructose-bisphosphate aldolases of Bacillus methanolicus by site-directed mutagenesis of key amino acids and gene repression by CRISPR interference. Front. Microbiol. 2021, 12, 669220.

[18] Schultenkämper K, Brito LF, López MG, Brautaset T, Wendisch VF. Establishment and application of CRISPR interference to affect sporulation, hydrogen peroxide detoxification, and mannitol catabolism in the methylotrophic thermophile Bacillus methanolicus. Appl. Microbiol. Biotechnol. 2019, 103, 5879-5889.

[19] Rütering M, Cress BF, Schilling M, Rühmann B, Koffas MAG, Sieber V, et al. Tailor-made exopolysaccharides—CRISPR-Cas9 mediated genome editing in Paenibacillus polymyxa. Synth. Biol. 2017, 2, ysx007.

[20] Khider MLK, Irla M, López MG, Konjetzko T, Meliawati M, Schmid J, et al. CRISPR-Cas-driven HDR-/NHEJ-genome editing in Bacillus methanolicus. Front. Microbiol. 2025, submitted.

[21] Mougiakos I, Mohanraju P, Bosma EF, Vrouwe V, Finger Bou M, Naduthodi MIS, et al. Characterizing a thermostable Cas9 for bacterial genome editing and silencing. Nat. Commun. 2017, 8, 1647.

[22] Styles MQ, Nesbitt EA, Hoffmann TD, Queen J, Ortenzi MV, Leak DJ. The heterologous production of terpenes by the thermophile Parageobacillus thermoglucosidasius in a consolidated bioprocess using waste bread. Metab. Eng. 2021, 65, 146-155.

[23] Irla M, Brito LF, Langlo J, Wohlers C, Benninghaus L, Heid C, et al. Development of a markerless tool for targeted chromosome modification in the thermophilic and methylotrophic bacterium Bacillus methanolicus. Microb. Cell Factories

[24] Brito LF, Arampu A, Pérez-García F, Kaya FEA, Sayar NA, Akbulut BS, et al. Model-based engineering of Bacillus methanolicus towards de novo polyamine bioproduction from methanol. New Biotechnol. 2025, 89, 91-104.

[25] Bozdag A, Komives C, Flickinger MC. Growth of Bacillus methanolicus in 2 M methanol at 50 °C: The effect of high methanol concentration on gene regulation of enzymes involved in formaldehyde detoxification by the ribulose monophosphate pathway. J. Ind. Microbiol. Biotechnol. 2015, 42, 1027-1038.

[26] Schoberth SM, Chapman BE, Kuchel PW, Wittig RM, Grotendorst J, Jansen P, et al. Ethanol transport in Zymomonas mobilis measured by using in vivo nuclear magnetic resonance spin transfer. J. Bacteriol. 1996, 178, 1756-1761.

[27] Murooka Y, Harada T. Active transport of alcohol in Corynebacterium acetophilum. J. Bacteriol. 1974, 118, 149-154.

[28] Anthony C.The Biochemistry of Methylotrophs; Academic Press Inc.: London, UK, 1982.

[29] Bellion E, Kent ME, Aud JC, Alikhan MY, Bolbot JA. Uptake of methylamine and methanol by Pseudomonas sp. strain AM1. J. Bacteriol. 1983, 154, 1168-1173.

[30] Arfman N, Watling EM, Clement W, van Oosterwijk RJ, de Vries GE, Harder W, et al. Methanol metabolism in thermotolerant methylotrophic Bacillus strains involving a novel catabolic NAD-dependent methanol dehydrogenase as a key enzyme. Arch. Microbiol. 1989, 152, 280-288.

[31] Krog A, Heggeset TMB, Müller JEN, Kupper CE, Schneider O, Vorholt JA, et al. Methylotrophic Bacillus methanolicus encodes two chromosomal and one plasmid born NAD+ dependent methanol dehydrogenase paralogs with different catalytic and biochemical properties. PLoS ONE 2013, 8, e59188.

[32] Krüsemann JL, Rainaldi V, Cotton CA, Claassens NJ, Lindner SN. The cofactor challenge in synthetic methylotrophy: Bioengineering and industrial applications. Curr. Opin. Biotechnol. 2023, 82, 102953.

[33] Le TK, Lee YJ, Han GH, Yeom SJ. Methanol dehydrogenases as a key biocatalysts for synthetic methylotrophy. Front. Bioeng. Biotechnol. 2021, 9, 787791.

[34] Müller JEN, Meyer F, Litsanov B, Kiefer P, Potthoff E, Heux S, et al. Engineering Escherichia coli for methanol conversion. Metab. Eng. 2015, 28, 190-201.

[35] Klein VJ, Irla M, Gil López M, Brautaset T, Fernandes Brito L. Unravelling formaldehyde metabolism in bacteria: Road towards synthetic methylotrophy. Microorganisms 2022, 10, 220.

[36] Murrell JC, Dalton H. Methane and Methanol Utilizers; Springer: Boston, MA, USA, 1992.

[37] Müller JEN, Litsanov B, Bortfeld-Miller M, Trachsel C, Grossmann J, Brautaset T, et al. Proteomic analysis of the thermophilic methylotroph Bacillus methanolicus MGA3. Proteomics 2014, 14, 725-737.

[38] Price JV, Chen L, Whitaker WB, Papoutsakis E, Chen W.Scaffoldless engineered enzyme assembly for enhanced methanol utilization. Proc. Natl. Acad. Sci. USA 2016, 113, 12691-12696.

[39] Fan L, Wang Y, Tuyishime P, Gao N, Li Q, Zheng P, et al. Engineering artificial fusion proteins for enhanced methanol bioconversion. Chembiochem Eur. J. Chem. Biol. 2018, 19, 2465-2471.

[40] Stolzenberger J, Lindner SN, Wendisch VF. The methylotrophic Bacillus methanolicus MGA3 possesses two distinct fructose 1,6-bisphosphate aldolases. Microbiol. Read. Engl. 2013, 159, 1770-1781.

[41] Pfeifenschneider J, Markert B, Stolzenberger J, Brautaset T, Wendisch VF. Transaldolase in Bacillus methanolicus: Biochemical characterization and biological role in ribulose monophosphate cycle. BMC Microbiol. 2020, 20, 63.

[42] Markert B, Stolzenberger J, Brautaset T, Wendisch VF. Characterization of two transketolases encoded on the chromosome and the plasmid pBM19 of the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus. BMC Microbiol. 2014, 14, 7.

[43] Delépine B, López MG, Carnicer M, Vicente CM, Wendisch VF, Heux S. Charting the metabolic landscape of the facultative methylotroph Bacillus methanolicus. Msystems 2020, 5, e00745-20.

[44] Stolzenberger J, Lindner SN, Persicke M, Brautaset T, Wendisch VF. Characterization of fructose 1,6-bisphosphatase and sedoheptulose 1,7-bisphosphatase from the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus. J. Bacteriol. 2013, 195, 5112-5122.

[45] Balzer Le S, Heggeset TMB, Haugen T, Nærdal I, Brautaset T. 6-Phosphofructokinase and ribulose-5-phosphate 3-epimerase in methylotrophic Bacillus methanolicus ribulose monophosphate cycle. Appl. Microbiol. Biotechnol. 2017, 101, 4185-4200.

[46] López MG, Irla M, Brito LF, Wendisch VF. Characterization of D-arabitol as newly discovered carbon source of Bacillus methanolicus. Front. Microbiol. 2019, 10, 1725.

[47] Irla M, Neshat A, Winkler A, Albersmeier A, Heggeset TMB, Brautaset T, et al. Complete genome sequence of Bacillus methanolicus MGA3, a thermotolerant amino acid producing methylotroph. J. Biotechnol. 2014, 188, 110-111.

[48] Hakvåg S, Nærdal I, Heggeset TMB, Kristiansen KA, Aasen IM, Brautaset T. Production of value-added chemicals by Bacillus methanolicus strains cultivated on mannitol and extracts of seaweed Saccharina latissima at 50 °C. Front. Microbiol. 2020, 11, 680.

[49] Irla M, Drejer EB, Brautaset T, Hakvåg S.Establishment of a functional system for recombinant production of secreted proteins at 50 °C in the thermophilic Bacillus methanolicus. Microb. Cell Factories 2020, 19, 151.

[50] Klein VJ, Brito LF, Perez-Garcia F, Brautaset T, Irla M. Metabolic engineering of thermophilic Bacillus methanolicus for riboflavin overproduction from methanol. Microb. Biotechnol. 2023, 16, 1011-1026.

[51] Bastem GM, Sayar NA, Brito LF, Brautaset T, Virant D, Sariyar Akbulut B. Development of a novel process towards an L-malate biorefinery using methanol as feedstock. Chem. Eng. Res. Des. 2024, 212, 158-167.

[52] Krog A, Heggeset TMB, Ellingsen TE, Brautaset T. Functional characterization of key enzymes involved in L-glutamate synthesis and degradation in the thermotolerant and methylotrophic bacterium Bacillus methanolicus. Appl. Environ. Microbiol. 2013, 79, 5321-5328.

[53] Brito LF, Luciano D, Irla M, Virant D, Courtade G, Brautaset T. Identification of MscS as a key L-glutamate exporter in Bacillus methanolicus. Microb. Biotechnol. 2025, 18, e70252.

[54] Schendel FJ, Dillingham R, Hanson RS, Sano K, Matsui K. Production of Glutamate Using Wild Type Bacillus methanolicus. U.S. Patent 6,083,728, 4 July 2000.

[55] Brautaset T, Jakobsen ØM, Josefsen KD, Flickinger MC, Ellingsen TE. Bacillus methanolicus: A candidate for industrial production of amino acids from methanol at 50 °C. Appl. Microbiol. Biotechnol. 2007, 74, 22-34.

[56] Nærdal I, Pfeifenschneider J, Brautaset T, Wendisch VF. Methanol-based cadaverine production by genetically engineered Bacillus methanolicus strains. Microb. Biotechnol. 2015, 8, 342-350.

[57] Liu Y, Zhang Q, Qi X, Gao H, Wang M, Guan H, et al. Metabolic engineering of Bacillus subtilis for riboflavin production: A review. Microorganisms 2023, 11, 164.

[58] de Boer L, Vrijbloed JW, Grobben G, Dijkhuizen L. Regulation of aromatic amino acid biosynthesis in the ribulose monophosphate cycle methylotroph Nocardia sp. 239. Arch. Microbiol. 1989, 151, 319-325.

[59] Gruenberg M, Irla M, Myllek S, Draths K. Characterization of two 3-deoxy-d-arabino-heptulosonate 7-phosphate synthases from Bacillus methanolicus. Protein Expr. Purif. 2021, 188, 105972.

[60] Doroshenko VG, Livshits VA, Airich LG, Shmagina IS, Savrasova EA, Ovsienko MV, et al. Metabolic engineering of Escherichia coli for the production of phenylalanine and related compounds. Appl. Biochem. Microbiol. 2015, 51, 733-750.

[61] Wang C, Zada B, Wei G, Kim SW. Metabolic engineering and synthetic biology approaches driving isoprenoid production in Escherichia coli. Bioresour. Technol. 2017, 241, 430-438.

[62] Irla M, Neshat A, Brautaset T, Rückert C, Kalinowski J, Wendisch VF. Transcriptome analysis of thermophilic methylotrophic Bacillus methanolicus MGA3 using RNA-sequencing provides detailed insights into its previously uncharted transcriptional landscape. BMC Genom. 2015, 16, 73.

[63] Carnicer M, Vieira G, Brautaset T, Portais JC, Heux S.Quantitative metabolomics of the thermophilic methylotroph Bacillus methanolicus. Microb. Cell Factories 2016, 15, 92.

[64] Gao B, Zhao N, Deng J, Gu Y, Jia S, Hou Y, et al. Constructing a methanol-dependent Bacillus subtilis by engineering the methanol metabolism. J. Biotechnol. 2022, 343, 128-137.

[65] Hennig G, Haupka C, Brito LF, Rückert C, Cahoreau E, Heux S, et al. Methanol-essential growth of Corynebacterium glutamicum: Adaptive laboratory evolution overcomes limitation due to methanethiol assimilation pathway. Int. J. Mol. Sci. 2020, 21, 3617.

[66] Müller JEN, Heggeset TMB, Wendisch VF, Vorholt JA, Brautaset T. Methylotrophy in the thermophilic Bacillus methanolicus, basic insights and application for commodity production from methanol. Appl. Microbiol. Biotechnol. 2015, 99, 535-551.

[67] Antoniewicz MR. Synthetic methylotrophy: Strategies to assimilate methanol for growth and chemicals production. Curr. Opin. Biotechnol. 2019, 59, 165-174.

[68] Girvan HM, Poddar H, McLean KJ, Nelson DR, Hollywood KA, Levy CW, et al. Structural and catalytic properties of the peroxygenase P450 enzyme CYP152K6 from Bacillus methanolicus. J. Inorg. Biochem. 2018, 188, 18-28.

[69] Zhang SY, Guo ZW, Wu XL, Ou XY, Zong MH, Lou WY. Recombinant expression and characterization of a novel cold-adapted type I pullulanase for efficient amylopectin hydrolysis. J. Biotechnol. 2020, 313, 39-47.

[70] Li L, Zheng Z, Zhao X, Wu F, Zhang J, Yang Z. Production, purification and characterization of a milk clotting enzyme from Bacillus methanolicus LB-1. Food Sci. Biotechnol. 2019, 28, 1107-1116.

[71] Liu J, Ma Y, Zhang M, Lai T, Wang Y, Yang Z. Biosynthesis of lactosucrose by a new source of β-fructofuranosidase from Bacillus methanolicus LB-1. J. Biosci. Bioeng. 2023, 135, 118-126.

[72] Pfeifenschneider J, Brautaset T, Wendisch VF. Methanol as carbon substrate in the bio-economy: Metabolic engineering of aerobic methylotrophic bacteria for production of value-added chemicals. Biofuels Bioprod. Biorefin. 2017, 11, 719-731.

[73] Drejer EB, Hakvåg S, Irla M, Brautaset T. Genetic tools and techniques for recombinant expression in thermophilic Bacillaceae. Microorganisms 2018, 6, 42.

[74] Nilasari D, Dover N, Rech S, Komives C. Expression of recombinant green fluorescent protein in Bacillus methanolicus. Biotechnol. Prog. 2012, 28, 662-668.

[75] Pluschkell SB, Flickinger MC. Dissimilation of [ 13C]methanol by continuous cultures of Bacillus methanolicus MGA 3 at 50 °C studied by 13C NMR and isotope-ratio mass spectrometry. Microbiology 2002, 148, 3223-3233.

[76] Liu WC, Gong T, Wang QH, Liang X, Chen JJ, Zhu P. Scaling-up fermentation of Pichia pastoris to demonstration-scale using new methanol-feeding strategy and increased air pressure instead of pure oxygen supplement. Sci. Rep. 2016, 6, 18439.