Alkane Biosynthesis in Bacteria

  • Steven Brown
  • Josh Loh
  • Stephen J. Aves
  • Thomas P. HowardEmail author
Living reference work entry
Part of the Handbook of Hydrocarbon and Lipid Microbiology book series (HHLM)


Biofuels are a commercial reality with ethanol comprising approximately 10% of the US retail fuel market, and biodiesels contributing a little under 5% to the EU retail fuel market. These biofuels are derived from the fermentation of sugars by yeast (ethanol) and from the chemical modification of animal fats and plant oils (biodiesel). However, these biofuel molecules are chemically distinct from the petroleum fuels that they are blended with. Petroleum-based fuels are predominantly composed of alkane and alkene hydrocarbons. These differences impact on fuel properties and infrastructure compatibility resulting in a “blend wall” that – without significant infrastructure realignment and associated costs – limits the use of biofuels. For this reason, there is great interest in biosynthetic routes for alkane and alkene production. Here we will review the known biological routes to alkane/alkene biosynthesis with a focus on bacterial alkane and alkene biosynthetic pathways. Specifically, we will review pathways for which the underlying genetic components have been identified. We will also investigate the development of engineered metabolic pathways that permit the production of alkanes and alkenes that are not naturally synthesized in bacteria (heterologous production) but are suitable for industrial commercial application. Finally, we will highlight some of the challenges facing this research area as it moves from proof-of-principle studies toward industrialization.



J.L. would like to thank the BBSRC for PhD funding (BB/M017036/2).


  1. Akhtar MK, Turner NJ, Jones PR (2013) Carboxylic acid reductase is a versatile enzyme for the conversion of fatty acids into fuels and chemical commodities. Proc Natl Acad Sci U S A 110(1):87PubMedCrossRefGoogle Scholar
  2. Albro PW, Dittmer JC (1969a) The biochemistry of long-chain, nonisoprenoid hydrocarbons. I. Characterization of the hydrocarbons of Sarcina lutea and the isolation of possible intermediates of biosynthesis. Biochemistry 8(1):394PubMedCrossRefGoogle Scholar
  3. Albro PW, Dittmer JC (1969b) The biochemistry of long-chain, nonisoprenoid hydrocarbons. IV. Characteristics of synthesis by a cell-free preparation of Sarcina lutea. Biochemistry 8(8):3317PubMedCrossRefGoogle Scholar
  4. Andre C, Kim SW, Yu X-H, Shanklin J (2013) Fusing catalase to an alkane-producing enzyme maintains enzymatic activity by converting the inhibitory byproduct H2O2 to the cosubstrate O2. Proc Natl Acad Sci U S A 110(8):3191PubMedPubMedCentralCrossRefGoogle Scholar
  5. Aukema KG, Makris TM, Stoian SA, Richman JE et al (2013) Cyanobacterial aldehyde deformylase oxygenation of aldehydes yields n-1 aldehydes and alcohols in addition to alkanes. ACS Catal 3(10):2228PubMedPubMedCentralCrossRefGoogle Scholar
  6. Belcher J, McLean KJ, Matthews S, Woodward LS et al (2014) Structure and biochemical properties of the alkene producing cytochrome P450 OleTJE (CYP152L1) from the Jeotgalicoccus sp. 8456 bacterium. J Biol Chem 289(10):6535PubMedPubMedCentralCrossRefGoogle Scholar
  7. Beller HR, Goh EB, Keasling JD (2010) Genes involved in long-chain alkene biosynthesis in Micrococcus luteus. Appl Environ Microbiol 76(4):1212PubMedCrossRefGoogle Scholar
  8. Bernard A, Domergue F, Pascal S, Jetter R et al (2012) Reconstitution of plant alkane biosynthesis in yeast demonstrates that Arabidopsis ECERIFERUM1 and ECERIFERUM3 are core components of a very-long-chain alkane synthesis complex. Plant Cell 24(7):3106PubMedPubMedCentralCrossRefGoogle Scholar
  9. Bonnett SA, Papireddy K, Higgins S, del Cardayre S et al (2011) Functional characterization of an NADPH dependent 2-alkyl-3-ketoalkanoic acid reductase involved in olefin biosynthesis in Stenotrophomonas maltophilia. Biochemistry 50(44):9633PubMedCrossRefGoogle Scholar
  10. Bourdenx B, Bernard A, Domergue F, Pascal S et al (2011) Overexpression of Arabidopsis ECERIFERUM1 promotes wax very-long-chain alkane biosynthesis and influences plant response to biotic and abiotic stresses. Plant Physiol 156(1):29PubMedPubMedCentralCrossRefGoogle Scholar
  11. Buijs NA, Zhou YJ, Siewers V, Nielsen J (2015) Long-chain alkane production by the yeast Saccharomyces cerevisiae. Biotechnol Bioeng 112(6):1275PubMedCrossRefGoogle Scholar
  12. Cao YX, Xiao WH, Zhang JL, Xie ZX et al (2016) Heterologous biosynthesis and manipulation of alkanes in Escherichia coli. Metab Eng 38:19PubMedCrossRefGoogle Scholar
  13. Cheesbrough TM, Kolattukudy PE (1988) Microsomal preparation from an animal tissue catalyzes release of carbon monoxide from a fatty aldehyde to generate an alkane. J Biol Chem 263(6):2738PubMedGoogle Scholar
  14. Choi YJ, Lee SY (2013) Microbial production of short-chain alkanes. Nature 502(7472):571PubMedCrossRefGoogle Scholar
  15. Choi K-H, Heath RJ, Rock CO (2000) β-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. J Bacteriol 182(2):365PubMedPubMedCentralCrossRefGoogle Scholar
  16. Christenson JK, Jensen MR, Goblirsch BR, Mohamed F et al (2017a) Active multienzyme assemblies for long-chain olefinic hydrocarbon biosynthesis. J Bacteriol 199(9):e00890–16Google Scholar
  17. Christenson JK, Richman JE, Jensen MR, Neufeld JY et al (2017b) Beta-lactone synthetase found in the olefin biosynthesis pathway. Biochemistry 56(2):348PubMedPubMedCentralCrossRefGoogle Scholar
  18. Crepin L, Lombard E, Guillouet SE (2016) Metabolic engineering of Cupriavidus necator for heterotrophic and autotrophic alka(e)ne production. Metab Eng 37:92PubMedCrossRefGoogle Scholar
  19. Das D, Eser BE, Han J, Sciore A et al (2011) Oxygen-independent decarbonylation of aldehydes by cyanobacterial aldehyde decarbonylase: a new reaction of diiron enzymes. Angew Chem Int Ed Engl 50(31):7148PubMedPubMedCentralCrossRefGoogle Scholar
  20. Dennis M, Kolattukudy PE (1992) A cobalt-porphyrin enzyme converts a fatty aldehyde to a hydrocarbon and CO. Proc Natl Acad Sci U S A 89(12):5306PubMedPubMedCentralCrossRefGoogle Scholar
  21. Dunlop MJ, Dossani ZY, Szmidt HL, Chu HC et al (2011) Engineering microbial biofuel tolerance and export using efflux pumps. Mol Syst Biol 7:487PubMedPubMedCentralCrossRefGoogle Scholar
  22. Eser BE, Das D, Han J, Jones PR et al (2011) Oxygen-independent alkane formation by non-heme iron-dependent cyanobacterial aldehyde decarbonylase: investigation of kinetics and requirement for an external electron donor. Biochemistry 50(49):10743PubMedPubMedCentralCrossRefGoogle Scholar
  23. Frias JA, Richman JE, Wackett LP (2009) C29 olefinic hydrocarbons biosynthesized by Arthrobacter species. Appl Environ Microbiol 75(6):1774PubMedPubMedCentralCrossRefGoogle Scholar
  24. Frias JA, Richman JE, Erickson JS, Wackett LP (2011) Purification and characterization of OleA from Xanthomonas campestris and demonstration of a non-decarboxylative Claisen condensation reaction. J Biol Chem 286(13):10930PubMedPubMedCentralCrossRefGoogle Scholar
  25. Fukuda H, Fujii T, Sukita E, Tazaki M et al (1994) Reconstitution of the isobutene-forming reaction catalyzed by cytochrome P450 and P450 reductase from Rhodotorula minuta: decarboxylation with the formation of isobutene. Biochem Biophys Res Commun 201(2):516PubMedCrossRefGoogle Scholar
  26. Gianoulis TA, Griffin MA, Spakowicz DJ, Dunican BF et al (2012) Genomic analysis of the hydrocarbon-producing, cellulolytic, endophytic fungus Ascocoryne sarcoides. PLoS Genet 8(3):e1002558PubMedPubMedCentralCrossRefGoogle Scholar
  27. Griffin MA, Spakowicz DJ, Gianoulis TA, Strobel SA (2010) Volatile organic compound production by organisms in the genus Ascocoryne and a re-evaluation of myco-diesel production by NRRL 50072. Microbiology 156(Pt 12):3814PubMedCrossRefGoogle Scholar
  28. Grossi V, Raphel D, Aubert C, Rontani J-F (2000) The effect of growth temperature on the long-chain alkenes composition in the marine coccolithophorid Emiliania huxleyi. Phytochemistry 54(4):393PubMedCrossRefGoogle Scholar
  29. Harger M, Zheng L, Moon A, Ager C et al (2013) Expanding the product profile of a microbial alkane biosynthetic pathway. ACS Synth Biol 2(1):59PubMedCrossRefGoogle Scholar
  30. Hong KK, Nielsen J (2012) Metabolic engineering of Saccharomyces cerevisiae: a key cell factory platform for future biorefineries. Cell Mol Life Sci 69(16):2671PubMedCrossRefGoogle Scholar
  31. Howard RW (1982) Chemical ecology and biochemistry of insect hydrocarbons. Annu Rev Entomol 27:149CrossRefGoogle Scholar
  32. Howard RW, McDaniel CA, Nelson DR, Blomquist GJ et al (1982) Cuticular hydrocarbons of Reticulitermes virginicus (banks) (Isoptera, Rhinotermitidae) and their role as potential species-recognition and caste-recognition cues. J Chem Ecol 8(9):1227PubMedCrossRefGoogle Scholar
  33. Howard TP, Middelhaufe S, Moore K, Edner C et al (2013) Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli. Proc Natl Acad Sci U S A 110(19):7636PubMedPubMedCentralCrossRefGoogle Scholar
  34. Jimenez-Diaz L, Caballero A, Perez-Hernandez N, Segura A (2017) Microbial alkane production for jet fuel industry: motivation, state of the art and perspectives. Microb Biotechnol 10(1):103PubMedCrossRefGoogle Scholar
  35. Kallio P, Pasztor A, Thiel K, Akhtar MK et al (2014) An engineered pathway for the biosynthesis of renewable propane. Nat Commun 5:4731PubMedPubMedCentralCrossRefGoogle Scholar
  36. Kancharla P, Bonnett SA, Reynolds KA (2016) Stenotrophomonas maltophilia OleC-catalyzed ATP-dependent formation of long-chain Z-olefins from 2-Alkyl-3-hydroxyalkanoic acids. Chembiochem 17(15):1426PubMedCrossRefGoogle Scholar
  37. Kang MK, Zhou YJ, Buijs NA, Nielsen J (2017) Functional screening of aldehyde decarbonylases for long-chain alkane production by Saccharomyces cerevisiae. Microb Cell Factories 16(1):74CrossRefGoogle Scholar
  38. Kato A, Takatani N, Use K, Uesaka K et al (2015) Identification of a cyanobacterial RND-type efflux system involved in export of free fatty acids. Plant Cell Physiol 56(12):2467PubMedCrossRefGoogle Scholar
  39. Lennen RM, Politz MG, Kruziki MA, Pfleger BF (2012) Identification of transport proteins involved in free fatty acid efflux in Escherichia coli. J Bacteriol 195:135–144PubMedCrossRefGoogle Scholar
  40. Li N, Norgaard H, Warui DM, Booker SJ et al (2011) Conversion of fatty aldehydes to alka(e)nes and formate by a cyanobacterial aldehyde decarbonylase: cryptic redox by an unusual dimetal oxygenase. J Am Chem Soc 133(16):6158PubMedPubMedCentralCrossRefGoogle Scholar
  41. Li N, Chang WC, Warui DM, Booker SJ et al (2012) Evidence for only oxygenative cleavage of aldehydes to alk(a/e)nes and formate by cyanobacterial aldehyde decarbonylases. Biochemistry 51(40):7908PubMedCrossRefGoogle Scholar
  42. Liu Y, Wang C, Yan J, Zhang W et al (2014) Hydrogen peroxide-independent production of a-alkenes by OleTJE P450 fatty acid decarboxylase. Biotechnol Biofuels 7(1):1CrossRefGoogle Scholar
  43. Matthews S, Tee KL, Rattray NJ, McLean KJ et al (2017) Production of alkenes and novel secondary products by P450 OleTJE using novel H2 O2 -generating fusion protein systems. FEBS Lett 591(5):737PubMedCrossRefGoogle Scholar
  44. McInnes AG, Walter JA, Wright JL (1980) Biosynthesis of hydrocarbons by algae: decarboxylation of stearic acid to N-heptadecane in Anacystis nidulans determined by 13 C-and 2 H-labeling and 13 C nuclear magnetic resonance. Lipids 15(9):609PubMedCrossRefGoogle Scholar
  45. Mehrer CR, Hernández Lozada NJ, Lai R-Y, Pfleger BF (2016) Production of fatty acids and derivatives by metabolic engineering of bacteria. In: Sang Yup Lee (ed) Consequences of microbial interactions with hydrocarbons, oils, and lipids: production of fuels and chemicals. Springer, p 1.
  46. Meighen EA (1998) Enzymes and genes from the lux operons of bioluminescent bacteria. Annu Rev Microbiol 42:151CrossRefGoogle Scholar
  47. Mendez-Perez D, Begemann MB, Pfleger BF (2011) Modular synthase-encoding gene involved in alpha-olefin biosynthesis in Synechococcus sp. strain PCC 7002. Appl Environ Microbiol 77(12):4264PubMedPubMedCentralCrossRefGoogle Scholar
  48. Mendez-Perez D, Herman NA, Pfleger BF (2014) A desaturase gene involved in the formation of 1,14-nonadecadiene in Synechococcus sp. strain PCC 7002. Appl Environ Microbiol 80(19):6073PubMedPubMedCentralCrossRefGoogle Scholar
  49. Mukhopadhyay A, Redding AM, Rutherford BJ, Keasling JD (2008) Importance of systems biology in engineering microbes for biofuel production. Curr Opin Biotechnol 19(3):228PubMedCrossRefGoogle Scholar
  50. Nichols D, Nichols PD, McKMeekin TA (1995) A new n-C31:9 polyene hydrocarbon from Antarctic bacteria. FEMS Microbiol Lett 125(2–3):281CrossRefGoogle Scholar
  51. Oku H, Kaneda T (1998) Biosynthesis of branched-chain fatty acids in Bacillus subtilis. J Biol Chem 263(34):18386Google Scholar
  52. Paddon CJ, Westfall PJ, Pitera DJ, Benjamin K et al (2013) High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 496(7446):1CrossRefGoogle Scholar
  53. Patel KG, Welch M, Gustafsson C (2016) Leveraging gene synthesis, advanced cloning techniques, and machine learning for metabolic pathway engineering. In: Van Dien S (ed) Metabolic engineering for bioprocess commercialization. Springer International Publishing Switzerland, p 53.
  54. Qiu Y, Tittiger C, Wicker-Thomas C, Le Goff G et al (2012) An insect-specific P450 oxidative decarbonylase for cuticular hydrocarbon biosynthesis. Proc Natl Acad Sci U S A 109(37):14858PubMedPubMedCentralCrossRefGoogle Scholar
  55. Reed JR, Vanderwel D, Choi S, Pomonis JG et al (1994) Unusual mechanism of hydrocarbon formation in the housefly: cytochrome P450 converts aldehyde to the sex pheromone component (Z)-9-tricosene and CO2. Proc Natl Acad Sci U S A 91(21):10000PubMedPubMedCentralCrossRefGoogle Scholar
  56. Rude MA, Schirmer A (2009) New microbial fuels: a biotech perspective. Curr Opin Microbiol 12(3):274PubMedCrossRefGoogle Scholar
  57. Rude MA, Baron TS, Brubaker S, Alibhai M et al (2011) Terminal olefin (1-alkene) biosynthesis by a novel P450 fatty acid decarboxylase from Jeotgalicoccus species. Appl Environ Microbiol 77(5):1718PubMedPubMedCentralCrossRefGoogle Scholar
  58. Rui Z, Li X, Zhu X, Liu J et al (2014) Microbial biosynthesis of medium-chain 1-alkenes by a nonheme iron oxidase. Proc Natl Acad Sci U S A 111(51):18237PubMedPubMedCentralCrossRefGoogle Scholar
  59. Rumbold K, van Buijsen HJ, Overkamp KM, van Groenestijn JW et al (2009) Microbial production host selection for converting second-generation feedstocks into bioproducts. Microb Cell Factories 8:64CrossRefGoogle Scholar
  60. Runguphan W, Keasling JD (2014) Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals. Metab Eng 21:103PubMedCrossRefGoogle Scholar
  61. Schirmer A, Rude MA, Li X, Popova E et al (2010) Microbial biosynthesis of alkanes. Science 329(5991):559PubMedCrossRefGoogle Scholar
  62. Schrader J, Bohlmann J (2015) Biotechnology of isoprenoids, vol 148. Advances in biochemical engineering/biotechnology. Springer.
  63. Sheppard MJ, Kunjapur AM, Prather KL (2016) Modular and selective biosynthesis of gasoline-range alkanes. Metab Eng 33:28PubMedCrossRefGoogle Scholar
  64. Singleton C, Howard TP, Smirnoff N (2014) Synthetic metabolons for metabolic engineering. J Exp Bot 65(8):1947PubMedCrossRefGoogle Scholar
  65. Sinha M, Weyda I, Sorensen A, Bruno KS et al (2017) Alkane biosynthesis by Aspergillus carbonarius ITEM 5010 through heterologous expression of Synechococcus elongatus acyl-ACP/CoA reductase and aldehyde deformylating oxygenase genes. AMB Express 7(1):18PubMedPubMedCentralCrossRefGoogle Scholar
  66. Smirnova N, Reynolds KA (2001) Branched-chain fatty acid biosynthesis in Escherichia coli. J Ind Microbiol Biotechnol 27(4):246PubMedCrossRefGoogle Scholar
  67. Song X, Yu H, Zhu K (2016) Improving alkane synthesis in Escherichia coli via metabolic engineering. Appl Microbiol Biotechnol 100(2):757PubMedCrossRefGoogle Scholar
  68. Sorigue D, Legeret B, Cuine S, Morales P et al (2016) Microalgae synthesize hydrocarbons from long-chain fatty acids via a light-dependent pathway. Plant Physiol 171(4):2393PubMedPubMedCentralGoogle Scholar
  69. Spakowicz DJ, Strobel SA (2015) Biosynthesis of hydrocarbons and volatile organic compounds by fungi: bioengineering potential. Appl Microbiol Biotechnol 99(12):4943PubMedPubMedCentralCrossRefGoogle Scholar
  70. Sugihara S, Hori R, Nakanowatari H, Takada Y et al (2010) Possible biosynthetic pathways for all cis-3,6,9,12,15,19,22, 25,28-hentriacontanonaene in bacteria. Lipids 45(2):167PubMedCrossRefGoogle Scholar
  71. Sukovich DJ, Seffernick JL, Richman JE, Gralnick JA et al (2010a) Widespread head-to-head hydrocarbon biosynthesis in bacteria and role of OleA. Appl Environ Microbiol 76(12):3850PubMedPubMedCentralCrossRefGoogle Scholar
  72. Sukovich DJ, Seffernick JL, Richman JE, Hunt KA et al (2010b) Structure, function, and insights into the biosynthesis of a head-to-head hydrocarbon in Shewanella oneidensis strain MR-1. Appl Environ Microbiol 76(12):3842PubMedPubMedCentralCrossRefGoogle Scholar
  73. Tillman JA, Seybold SJ, Jurenka RA, Blomquist GJ (1999) Insect pheromones – an overview of biosynthesis and endocrine regulation. Insect Biochem Mol 29(6):481CrossRefGoogle Scholar
  74. Vickers CE, Blank LM, Kromer JO (2010) Chassis cells for industrial biochemical production. Nat Chem Biol 6(12):875PubMedCrossRefGoogle Scholar
  75. Warui DM, Li N, Norgaard H, Krebs C et al (2011) Detection of formate, rather than carbon monoxide, as the stoichiometric co-product in conversion of fatty aldehydes to alkanes by a cyanobacterial aldehyde decarbonylase. J Am Chem Soc 133(10):3316PubMedPubMedCentralCrossRefGoogle Scholar
  76. Westfall PJ, Pitera DJ, Lenihan JR, Eng D et al (2012) Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin. Proc Natl Acad Sci U S A 109(3):111CrossRefGoogle Scholar
  77. Winters K, Parker PL, Van Baalen C (1969) Hydrocarbons of blue-green algae: geochemical significance. Science 163(3866):467PubMedCrossRefGoogle Scholar
  78. Xie M, Wang W, Zhang W, Chen L et al (2017) Versatility of hydrocarbon production in cyanobacteria. Appl Microbiol Biotechnol 101(3):905PubMedCrossRefGoogle Scholar
  79. Yan H, Wang Z, Wang F, Tan TW et al (2016) Biosynthesis of chain-specific alkanes by metabolic engineering in Escherichia coli. Eng Life Sci 16(1):53CrossRefGoogle Scholar
  80. Yoshino T, Liang Y, Arai D, Maeda Y et al (2015) Alkane production by the marine cyanobacterium Synechococcus sp. NKBG15041c possessing the alpha-olefin biosynthesis pathway. Appl Microbiol Biotechnol 99(3):1521PubMedCrossRefGoogle Scholar
  81. Zhang, J. J., Lu, X. F., Li, J. J. (2013) Conversion of fatty aldehydes into alk (a/e)nes by in vitro reconstituted cyanobacterial aldehyde-deformylating oxygenase with the cognate electron transfer system. Biotechnology for Biofuels 6:86.
  82. Zhang L, Liang Y, Wu W, Tan X et al (2016) Microbial synthesis of propane by engineering valine pathway and aldehyde-deformylating oxygenase. Biotechnol Biofuels 9:80PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  • Steven Brown
    • 1
  • Josh Loh
    • 2
  • Stephen J. Aves
    • 3
  • Thomas P. Howard
    • 2
    • 4
    Email author
  1. 1.Synthace Ltd.The London Bioscience Innovation CentreLondonUK
  2. 2.School of Natural and Environmental Sciences, Faculty of Science and EngineeringNewcastle UniversityNewcastle-Upon-TyneUK
  3. 3.Biosciences, College of Life and Environmental SciencesUniversity of ExeterExeterUK
  4. 4.The Centre for Synthetic Biology and the BioeconomyNewcastle UniversityNewcastle-Upon-TyneUK

Personalised recommendations