Synthetic Methylotrophy: Past, Present, and Future

  • Stephanie HeuxEmail author
  • Trygve Brautaset
  • Julia A. Vorholt
  • Volker F. Wendisch
  • Jean Charles Portais


Methane and methanol are regarded as alternative and highly attractive nonfood raw materials for the biotechnology sector. The supply of methane and methanol comes from both fossil and renewable resources, rendering them flexible and sustainable raw materials. Reduced one-carbon (C1) compounds are used by specialized groups of microorganisms, i.e., the methylotrophs, as their sole source of carbon and energy. While progress to engineer and use natural methylotrophs in biotechnology is ongoing, synthetic methylotrophs only recently have gained interest as a parallel approach both in academia and private industry. Synthetic methylotrophy refers to the design and rational engineering of methylotrophy to established non-methylotrophic production hosts for access to methane and methanol as feedstock while maintaining their biotechnological production potential. In this chapter, we will illustrate how combined systems and synthetic biology approaches capitalize on the metabolic versatility and engineered production pathways of industrially well-established microorganisms, such as Escherichia coli, Bacillus subtilis, and Corynebacterium glutamicum, for biotransformation from methane and methanol. Challenges and current prospects for designing and engineering the next generation of synthetic methylotrophs are also discussed.


Methylotrophy Methane Methanol Synthetic biology Industrial host microorganisms 


  1. Ahmad M, Hirz M, Pichler H, Schwab H (2014) Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol 98(12):5301–5317. Scholar
  2. Anthony C (1991) Chapter 4 – Assimilation of carbon by methylotrophs. In: Israel Goldberg, J. Stefan Rokem, Biology of methylotrophs. Butterworth-Heinemann, Oxford, pp 79-109. doi: Scholar
  3. Antonovsky N, Gleizer S, Noor E, Zohar Y, Herz E, Barenholz U, Zelcbuch L, Amram S, Wides A, Tepper N, Davidi D, Bar-On Y, Bareia T, Wernick DG, Shani I, Malitsky S, Jona G, Bar-Even A, Milo R (2016) Sugar synthesis from CO2 in Escherichia coli. Cell 166(1):115–125. Scholar
  4. Arfman N, Watling EM, Clement W, van Oosterwijk RJ, de Vries GE, Harder W, Attwood MM, Dijkhuizen L (1989) Methanol metabolism in thermotolerant methylotrophic Bacillus strains involving a novel catabolic NAD-dependent methanol dehydrogenase as a key enzyme. Arch Microbiol 152(3):280–288CrossRefPubMedGoogle Scholar
  5. Balasubramanian R, Smith SM, Rawat S, Yatsunyk LA, Stemmler TL, Rosenzweig AC (2010) Oxidation of methane by a biological dicopper centre. Nature 465(7294):115–119. Scholar
  6. Bar-Even A (2016) Formate assimilation: the metabolic architecture of natural and synthetic pathways. Biochemistry 55(28):3851–3863. Scholar
  7. Bassham JA, Benson AA, Calvin M (1950) The path of carbon in photosynthesis. J Biol Chem 185(2):781–787PubMedGoogle Scholar
  8. Becker J, Wittmann C (2015) Advanced biotechnology: metabolically engineered cells for the bio-based production of chemicals and fuels, materials, and health-care products. Angew Chem 54(11):3328–3350. Scholar
  9. Bennett RK, Gonzalez JE, Whitaker WB, Antoniewicz MR, Papoutsakis ET (2018) 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 45:75–85. Scholar
  10. Brautaset T, Heggeset M, Marita B, Heux S, Kiefer P, Krog A, Lessmeier L, Muller JE, Portais JC, Potthoff E, Quax WJ, Sibbald M, Vorholt JA, Wendisch VF (2013) Novel methanol dehydrogenase enzymes from Bacillus. WO 2013110797, 1 Aug 2013Google Scholar
  11. Brautaset T, Jakobsen MO, Flickinger MC, Valla S, Ellingsen TE (2004) Plasmid-dependent methylotrophy in thermotolerant Bacillus methanolicus. J Bacteriol 186(5):1229–1238CrossRefPubMedPubMedCentralGoogle Scholar
  12. Burgard AP, Osterhout RE, Van Dien SJ, Tracewell CA, Pharkya P, Andrae S (2014) Microorganisms and methods for enhancing the availability of reducing equivalents in the presence of methanol, and for producing 1,4-butanediol related thereto. US 20140058056 A1, 27 Feb 2014Google Scholar
  13. Bystrykh LV, Vonck J, van Bruggen EF, van Beeumen J, Samyn B, Govorukhina NI, Arfman N, Duine JA, Dijkhuizen L (1993) Electron microscopic analysis and structural characterization of novel NADP(H)-containing methanol: N,N′-dimethyl-4-nitrosoaniline oxidoreductases from the gram-positive methylotrophic bacteria Amycolatopsis methanolica and Mycobacterium gastri MB19. J Bacteriol 175(6):1814–1822CrossRefPubMedPubMedCentralGoogle Scholar
  14. Carbonell P, Currin A, Jervis AJ, Rattray NJ, Swainston N, Yan C, Takano E, Breitling R (2016) Bioinformatics for the synthetic biology of natural products: integrating across the design-build-test cycle. Nat Prod Rep 33(8):925–932. Scholar
  15. Chistoserdova L (2011) Modularity of methylotrophy, revisited. Environ Microbiol 13(10):2603–2622. Scholar
  16. Chung BK, Selvarasu S, Andrea C, Ryu J, Lee H, Ahn J, Lee H, Lee DY (2010) Genome-scale metabolic reconstruction and in silico analysis of methylotrophic yeast Pichia pastoris for strain improvement. Microb Cell Fact 9:50. Scholar
  17. Clomburg JM, Crumbley AM, Gonzalez R (2017) Industrial biomanufacturing: the future of chemical production. Science 355(6320). Scholar
  18. Coleman WJ, Vidanes GM, Cottarel G, Muley S, Kamimura R, Javan AF, Sun J, Groban ES (2014) Biological conversion of multi-carbon compounds from methane. US 20140273128 A1, 18 Sep 2014Google Scholar
  19. Conrado RJ, Gonzalez R (2014) Chemistry. Envisioning the bioconversion of methane to liquid fuels. Science 343(6171):621–623. Scholar
  20. Dai Z, Gu H, Zhang S, Xin F, Zhang W, Dong W, Ma J, Jia H, Jiang M (2017) Metabolic construction strategies for direct methanol utilization in Saccharomyces cerevisiae. Bioresour Technol 245:1407–1412. Scholar
  21. Dedysh SN, Naumoff DG, Vorobev AV, Kyrpides N, Woyke T, Shapiro N, Crombie AT, Murrell JC, Kalyuzhnaya MG, Smirnova AV, Dunfield PF (2015) Draft genome sequence of Methyloferula stellata AR4, an obligate methanotroph possessing only a soluble methane monooxygenase. Genome Announc 3(2). Scholar
  22. Ekeroth L, Villadsen J (1991) Chapter 9 – Single cell protein production from C1 compounds. In: Israel Goldberg, J. Stefan Rokem. Biology of methylotrophs. Butterworth-Heinemann: Oxford, pp 205-231. doi: Scholar
  23. Erb TJ, Berg IA, Brecht V, Muller M, Fuchs G, Alber BE (2007) Synthesis of C5-dicarboxylic acids from C2-units involving crotonyl-CoA carboxylase/reductase: the ethylmalonyl-CoA pathway. Proc Natl Acad Sci USA 104(25):10631–10636. Scholar
  24. Erb TJ, Jones PR, Bar-Even A (2017) Synthetic metabolism: metabolic engineering meets enzyme design. Curr Opin Chem Biol 37:56–62. Scholar
  25. Ghosh M, Avezoux A, Anthony C, Harlos K, Blake CC (1994) X-ray structure of PQQ-dependent methanol dehydrogenase. Exs 71:251–260PubMedGoogle Scholar
  26. Gilman A, Laurens LM, Puri AW, Chu F, Pienkos PT, Lidstrom ME (2015) Bioreactor performance parameters for an industrially-promising methanotroph Methylomicrobium buryatense 5GB1. Microb Cell Fact 14:182. Scholar
  27. Gou Z, Xing XH, Luo M, Jiang H, Han B, Wu H, Wang L, Zhang F (2006) Functional expression of the particulate methane mono-oxygenase gene in recombinant Rhodococcus erythropolis. FEMS Microbiol Lett 263(2):136–141. Scholar
  28. Hadadi N, Hafner J, Shajkofci A, Zisaki A, Hatzimanikatis V (2016) ATLAS of biochemistry: a repository of all possible biochemical reactions for synthetic biology and metabolic engineering studies. ACS Synth Biol 5(10):1155–1166. Scholar
  29. Hakemian AS, Rosenzweig AC (2007) The biochemistry of methane oxidation. Annu Rev Biochem 76:223–241. Scholar
  30. Haynes CA, Gonzalez R (2014) Rethinking biological activation of methane and conversion to liquid fuels. Nat Chem Biol 10(5):331–339. Scholar
  31. Helman N, Clarke E, Greenfield D (2015) Synthetic methanotrophic and methylotrophic microorganisms. WO 2015160848 A1, 22 Oct 2015Google Scholar
  32. Hibi Y, Asai K, Arafuka H, Hamajima M, Iwama T, Kawai K (2011) Molecular structure of La3+-induced methanol dehydrogenase-like protein in Methylobacterium radiotolerans. J Biosci Bioeng 111(5):547–549. Scholar
  33. Irla M, Heggeset TM, Naerdal I, Paul L, Haugen T, Le SB, Brautaset T, Wendisch VF (2016) 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 7:1481. Scholar
  34. Jeffryes JG, Colastani RL, Elbadawi-Sidhu M, Kind T, Niehaus TD, Broadbelt LJ, Hanson AD, Fiehn O, Tyo KE, Henry CS (2015) MINEs: open access databases of computationally predicted enzyme promiscuity products for untargeted metabolomics. J Cheminf 7:44. Scholar
  35. Kalyuzhnaya MG, Puri AW, Lidstrom ME (2015) Metabolic engineering in methanotrophic bacteria. Metab Eng 29:142–152. Scholar
  36. Kolb S (2009) Aerobic methanol-oxidizing bacteria in soil. FEMS Microbiol Lett 300(1):1–10. Scholar
  37. Koopman FW, de Winde JH, Ruijssenaars HJ (2009) C(1) compounds as auxiliary substrate for engineered Pseudomonas putida S12. Appl Microbiol Biotechnol 83(4):705–713. Scholar
  38. Lessmeier L, Pfeifenschneider J, Carnicer M, Heux S, Portais JC, Wendisch VF (2015) Production of carbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as co-substrate. Appl Microbiol Biotechnol.
  39. Lidstrom ME (2006) Aerobic methylotrophic prokaryotes. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E (eds) The prokaryotes, Ecophysiology and biochemistry, vol 2. Springer New York, New York, NY, pp 618–634. Scholar
  40. Lloyd JS, De Marco P, Dalton H, Murrell JC (1999) Heterologous expression of soluble methane monooxygenase genes in methanotrophs containing only particulate methane monooxygenase. Arch Microbiol 171(6):364–370CrossRefPubMedGoogle Scholar
  41. Lynch M (2014) Microorganisms for the conversion of methane and methanol to higher value chemicals and fuels. WO 2014165763 A1, 9 Oct 2014Google Scholar
  42. Matelbs RI, Tannenbaum SE (1968) Single-Cell protein. Econ Bot 22(1):42–50. Scholar
  43. Medema MH, van Raaphorst R, Takano E, Breitling R (2012) Computational tools for the synthetic design of biochemical pathways. Nat Rev Microbiol 10(3):191–202. Scholar
  44. Merkx M, Kopp DA, Sazinsky MH, Blazyk JL, Muller J, Lippard SJ (2001) Dioxygen activation and methane hydroxylation by soluble methane monooxygenase: a tale of two irons and three proteins A list of abbreviations can be found in Section 7. Angew Chem 40(15):2782–2807CrossRefGoogle Scholar
  45. Mes TZD, de Stams AJM, Zeeman G (2003) Chapter 4. Methane production by anaerobic digestion of wastewater and solid wastes. In: Reith JH, Wijffels RH, Barten H (eds) Biomethane and biohydrogen: status and perspectives of biological methane and hydrogen production. Dutch Biological Hydrogen Foundation – NOVEM, The Hague, The Netherlands, pp 58–94Google Scholar
  46. Methanol Institute (1989) Accessed 20 Jan 2016
  47. Muller JE, Meyer F, Litsanov B, Kiefer P, Vorholt JA (2015a) Core pathways operating during methylotrophy of Bacillus methanolicus MGA3 and induction of a bacillithiol-dependent detoxification pathway upon formaldehyde stress. Mol Microbiol 98(6):1089–1100. Scholar
  48. Muller JE, Meyer F, Litsanov B, Kiefer P, Potthoff E, Heux S, Quax WJ, Wendisch VF, Brautaset T, Portais JC, Vorholt JA (2015b) Engineering Escherichia coli for methanol conversion. Metab Eng 28:190–201. Scholar
  49. Murrell JC (2002) Expression of soluble methane monooxygenase genes. Microbiology 148(Pt 11):3329–3330. Scholar
  50. Murrell JC, Gilbert B, McDonald IR (2000) Molecular biology and regulation of methane monooxygenase. Arch Microbiol 173(5–6):325–332CrossRefPubMedGoogle Scholar
  51. Naerdal I, Pfeifenschneider J, Brautaset T, Wendisch VF (2015) Methanol-based cadaverine production by genetically engineered Bacillus methanolicus strains. Microb Biotechnol 8(2):342–350. Scholar
  52. Nguyen AD, Hwang IY, Chan JY, Lee EY (2016) Reconstruction of methanol and formate metabolic pathway in non-native host for biosynthesis of chemicals and biofuels. Biotechnol Bioproc E 21(4):477–482. Scholar
  53. Olah GA (2013) Towards oil independence through renewable methanol chemistry. Angew Chem Int Ed 52(1):104–107. Scholar
  54. Orita I, Sakamoto N, Kato N, Yurimoto H, Sakai Y (2007) Bifunctional enzyme fusion of 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. Appl Microbiol Biotechnol 76(2):439–445. Scholar
  55. Papoutsakis ET, Nicolaou S, Fast A, Falara V, Bennett RK, Whitaker WB, Sandoval NR, Gonzalez J, Antoniewicz M (2015) Synthetic methylotrophy to liquid fuels and chemicals. WO 2015/108777 A1, 23 Jul 2015Google Scholar
  56. Peyraud R, Kiefer P, Christen P, Massou S, Portais JC, Vorholt JA (2009) Demonstration of the ethylmalonyl-CoA pathway by using 13C metabolomics. Proc Natl Acad Sci USA 106(12):4846–4851. Scholar
  57. Pfeifenschneider J, Brautaset T, Wendisch VF (2017) Methanol as carbon substrate in the bio-economy: Metabolic engineering of aerobic methylotrophic bacteria for production of value-added chemicals. Biofuels Bioprod Biorefin 11(4):719–731. Scholar
  58. Planson AG, Carbonell P, Grigoras I, Faulon JL (2012) A retrosynthetic biology approach to therapeutics: from conception to delivery. Curr Opin Biotechnol 23(6):948–956. Scholar
  59. Pol A, Barends TR, Dietl A, Khadem AF, Eygensteyn J, Jetten MS, Op den Camp HJ (2014) Rare earth metals are essential for methanotrophic life in volcanic mudpots. Environ Microbiol 16(1):255–264. Scholar
  60. Price JV, Chen L, Whitaker WB, Papoutsakis E, Chen W (2016) Scaffoldless engineered enzyme assembly for enhanced methanol utilization. Proc Natl Acad Sci USA.
  61. Quayle JR (1972) The metabolism of one-carbon compounds by micro-organisms. In: Rose AH, Tempest DW (eds) Advances in microbial physiology, vol 7. Academic Press, San Diego, CA, pp 119–203. Scholar
  62. Russmayer H, Buchetics M, Gruber C, Valli M, Grillitsch K, Modarres G, Guerrasio R, Klavins K, Neubauer S, Drexler H, Steiger M, Troyer C, Al Chalabi A, Krebiehl G, Sonntag D, Zellnig G, Daum G, Graf AB, Altmann F, Koellensperger G, Hann S, Sauer M, Mattanovich D, Gasser B (2015) Systems-level organization of yeast methylotrophic lifestyle. BMC Biol 13(1):80. Scholar
  63. Schrader J, Schilling M, Holtmann D, Sell D, Filho MV, Marx A, Vorholt JA (2009) Methanol-based industrial biotechnology: current status and future perspectives of methylotrophic bacteria. Trends Biotechnol 27(2):107–115. Scholar
  64. Selvakumar G, Nazim S, Kundu S (2008) Chapter 4: Methylotrophy in bacteria. Concept and significance. In: Saikia R (ed) Microbial biotechnology. New India Publishing, New Delhi, pp 67–85Google Scholar
  65. Siegel JB, Smith AL, Poust S, Wargacki AJ, Bar-Even A, Louw C, Shen BW, Eiben CB, Tran HM, Noor E, Gallaher JL, Bale J, Yoshikuni Y, Gelb MH, Keasling JD, Stoddard BL, Lidstrom ME, Baker D (2015) Computational protein design enables a novel one-carbon assimilation pathway. Proc Natl Acad Sci USA 112(12):3704–3709. Scholar
  66. Strong PJ, Xie S, Clarke WP (2015) Methane as a resource: can the methanotrophs add value? Environ Sci Technol 49(7):4001–4018. Scholar
  67. Schwander T, Schada von Borzyskowski L, Burgener S, Cortina NS, Erb TJ (2016) A synthetic pathway for the fixation of carbon dioxide in vitro. Science 354(6314):900–904. Scholar
  68. Tai YS, Zhang K (2015) Enzyme pathways: C1 metabolism redesigned. Nat Chem Biol 11(6):384–386. Scholar
  69. Tani Y (1991) Chapter 11 – Production of useful chemicals by methylotrophs. In: Biology of methylotrophs. Butterworth-Heinemann, Oxford, pp 253–270. Scholar
  70. de la Torre A, Metivier A, Chu F, Laurens LM, Beck DA, Pienkos PT, Lidstrom ME, Kalyuzhnaya MG (2015) Genome-scale metabolic reconstructions and theoretical investigation of methane conversion in Methylomicrobium buryatense strain 5G(B1). Microb Cell Fact 14:188. Scholar
  71. Vieira G, Carnicer M, Portais JC, Heux S (2014) FindPath: a Matlab solution for in silico design of synthetic metabolic pathways. Bioinformatics 30(20):2986–2988. Scholar
  72. Vorholt JA (2002) Cofactor-dependent pathways of formaldehyde oxidation in methylotrophic bacteria. Arch Microbiol 178(4):239–249. Scholar
  73. Wendisch VF, Brito LF, Gil Lopez M, Hennig G, Pfeifenschneider J, Sgobba E, Veldmann KH (2016) The flexible feedstock concept in industrial biotechnology: metabolic engineering of Escherichia coli, Corynebacterium glutamicum, Pseudomonas, Bacillus and yeast strains for access to alternative carbon sources. J Biotechnol 234:139–157. Scholar
  74. West CA, Salmond GP, Dalton H, Murrell JC (1992) Functional expression in Escherichia coli of proteins B and C from soluble methane monooxygenase of Methylococcus capsulatus (Bath). J Gen Microbiol 138(7):1301–1307. Scholar
  75. Whitaker WB, Jones JA, Bennett RK, Gonzalez JE, Vernacchio VR, Collins SM, Palmer MA, Schmidt S, Antoniewicz MR, Koffas MA, Papoutsakis ET (2017) Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli. Metab Eng 39:49–59. Scholar
  76. Whitaker WB, Sandoval NR, Bennett RK, Fast AG, Papoutsakis ET (2015) Synthetic methylotrophy: engineering the production of biofuels and chemicals based on the biology of aerobic methanol utilization. Curr Opin Biotechnol 33:165–175. Scholar
  77. Witthoff S, Schmitz K, Niedenfuhr S, Noh K, Noack S, Bott M, Marienhagen J (2015) Metabolic engineering of Corynebacterium glutamicum for methanol metabolism. Appl Environ Microbiol 81(6):2215–2225. Scholar
  78. Wood TK (2002) Active expression of soluble methane monooxygenase from Methylosinus trichosporium OB3b in heterologous hosts. Microbiology 148(Pt 11):3328–3329. Scholar
  79. Yurimoto H, Kato N, Sakai Y (2009) Genomic organization and biochemistry of the ribulose monophosphate pathway and its application in biotechnology. Appl Microbiol Biotechnol 84(3):407–416. Scholar
  80. Yurimoto H, Oku M, Sakai Y (2011) Yeast methylotrophy: metabolism, gene regulation and peroxisome homeostasis. Int J Microbiol 2011:101298. Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Stephanie Heux
    • 1
    Email author
  • Trygve Brautaset
    • 2
  • Julia A. Vorholt
    • 3
  • Volker F. Wendisch
    • 4
  • Jean Charles Portais
    • 1
  1. 1.LISBP, Université de Toulouse, CNRS, INRA, INSAToulouseFrance
  2. 2.Department of BiotechnologyNorwegian University of Science and Technology (NTNU)TrondheimNorway
  3. 3.ETH ZurichZurichSwitzerland
  4. 4.Genetics of Prokaryotes, Faculty of Biology & CeBiTecBielefeld UniversityBielefeldGermany

Personalised recommendations