Synthesis of Customized Petroleum-replica Fuel Molecules by Targeted Modification of Free Fatty Acid Pools in Escherichia Coli

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 2013 Apr 24

PMID 23610415

Citations 63

Authors

Thomas P Howard

Sabine Middelhaufe

Karen Moore

Christoph Edner

Dagmara M Kolak

George N Taylor

David A Parker

Rob Lee

Nicholas Smirnoff

Stephen J Aves

John Love

Affiliations

Soon will be listed here.

Abstract

Biofuels are the most immediate, practical solution for mitigating dependence on fossil hydrocarbons, but current biofuels (alcohols and biodiesels) require significant downstream processing and are not fully compatible with modern, mass-market internal combustion engines. Rather, the ideal biofuels are structurally and chemically identical to the fossil fuels they seek to replace (i.e., aliphatic n- and iso-alkanes and -alkenes of various chain lengths). Here we report on production of such petroleum-replica hydrocarbons in Escherichia coli. The activity of the fatty acid (FA) reductase complex from Photorhabdus luminescens was coupled with aldehyde decarbonylase from Nostoc punctiforme to use free FAs as substrates for alkane biosynthesis. This combination of genes enabled rational alterations to hydrocarbon chain length (Cn) and the production of branched alkanes through upstream genetic and exogenous manipulations of the FA pool. Genetic components for targeted manipulation of the FA pool included expression of a thioesterase from Cinnamomum camphora (camphor) to alter alkane Cn and expression of the branched-chain α-keto acid dehydrogenase complex and β-keto acyl-acyl carrier protein synthase III from Bacillus subtilis to synthesize branched (iso-) alkanes. Rather than simply reconstituting existing metabolic routes to alkane production found in nature, these results demonstrate the ability to design and implement artificial molecular pathways for the production of renewable, industrially relevant fuel molecules.

Citing Articles

Coordinated conformational changes in P450 decarboxylases enable hydrocarbons production from renewable feedstocks.

Generoso W, Alvarenga A, Simoes I, Miyamoto R, Rodrigues de Melo R, Guilherme E Nat Commun. 2025; 16(1):945.

PMID: 39843428 PMC: 11754895. DOI: 10.1038/s41467-025-56256-4.

Biosynthesis pathways of expanding carbon chains for producing advanced biofuels.

Su H, Lin J Biotechnol Biofuels Bioprod. 2023; 16(1):109.

PMID: 37400889 PMC: 10318755. DOI: 10.1186/s13068-023-02340-0.

Production of Fatty Acids and Derivatives Using Cyanobacteria.

Sattayawat P, Yunus I, Jones P Adv Biochem Eng Biotechnol. 2023; 183:145-169.

PMID: 36764955 DOI: 10.1007/10_2022_213.

Microbial pathways for advanced biofuel production.

Love J Biochem Soc Trans. 2022; 50(2):987-1001.

PMID: 35411379 PMC: 9162456. DOI: 10.1042/BST20210764.

Cellulosic hydrocarbons production by engineering dual synthesis pathways in Corynebacterium glutamicum.

Xu Y, Hua K, Huang Z, Zhou P, Wen J, Jin C Biotechnol Biofuels Bioprod. 2022; 15(1):29.

PMID: 35292099 PMC: 8922798. DOI: 10.1186/s13068-022-02129-7.

References

Choi K, Heath R, Rock C . beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. J Bacteriol. 2000; 182(2):365-70. PMC: 94284. DOI: 10.1128/JB.182.2.365-370.2000. View

Zheng Y, Li L, Liu Q, Yang J, Wang X, Liu W . Optimization of fatty alcohol biosynthesis pathway for selectively enhanced production of C12/14 and C16/18 fatty alcohols in engineered Escherichia coli. Microb Cell Fact. 2012; 11:65. PMC: 3439321. DOI: 10.1186/1475-2859-11-65. View

Kaneda T . Iso- and anteiso-fatty acids in bacteria: biosynthesis, function, and taxonomic significance. Microbiol Rev. 1991; 55(2):288-302. PMC: 372815. DOI: 10.1128/mr.55.2.288-302.1991. View

Delebecque C, Lindner A, Silver P, Aldaye F . Organization of intracellular reactions with rationally designed RNA assemblies. Science. 2011; 333(6041):470-4. DOI: 10.1126/science.1206938. View

Berdyshev E . Mass spectrometry of fatty aldehydes. Biochim Biophys Acta. 2011; 1811(11):680-93. PMC: 3205333. DOI: 10.1016/j.bbalip.2011.08.018. View

Handke P, Lynch S, Gill R . Application and engineering of fatty acid biosynthesis in Escherichia coli for advanced fuels and chemicals. Metab Eng. 2010; 13(1):28-37. DOI: 10.1016/j.ymben.2010.10.007. View

Perera M, Qin W, Yandeau-Nelson M, Fan L, Dixon P, Nikolau B . Biological origins of normal-chain hydrocarbons: a pathway model based on cuticular wax analyses of maize silks. Plant J. 2010; 64(4):618-32. DOI: 10.1111/j.1365-313X.2010.04355.x. View

Dellomonaco C, Clomburg J, Miller E, Gonzalez R . Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals. Nature. 2011; 476(7360):355-9. DOI: 10.1038/nature10333. View

Burger B, Reiter B, Borzyk O, Du Plessis M . Avian exocrine secretions. I. Chemical characterization of the volatile fraction of the uropygial secretion of the green woodhoopoe, Phoeniculus purpureus. J Chem Ecol. 2004; 30(8):1603-11. DOI: 10.1023/b:joec.0000042071.65335.f3. View

10.

Peralta-Yahya P, Zhang F, del Cardayre S, Keasling J . Microbial engineering for the production of advanced biofuels. Nature. 2012; 488(7411):320-8. DOI: 10.1038/nature11478. View

11.

Ro D, Paradise E, Ouellet M, Fisher K, Newman K, Ndungu J . Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature. 2006; 440(7086):940-3. DOI: 10.1038/nature04640. View

12.

Akhtar M, Turner N, Jones P . 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. 2012; 110(1):87-92. PMC: 3538209. DOI: 10.1073/pnas.1216516110. View

13.

Westfall P, Pitera D, Lenihan J, Eng D, Woolard F, Regentin R . Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin. Proc Natl Acad Sci U S A. 2012; 109(3):E111-8. PMC: 3271868. DOI: 10.1073/pnas.1110740109. View

14.

Steen E, Kang Y, Bokinsky G, Hu Z, Schirmer A, McClure A . Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature. 2010; 463(7280):559-62. DOI: 10.1038/nature08721. View

15.

Meighen E . Molecular biology of bacterial bioluminescence. Microbiol Rev. 1991; 55(1):123-42. PMC: 372803. DOI: 10.1128/mr.55.1.123-142.1991. View

16.

Folch J, Lees M, SLOANE STANLEY G . A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957; 226(1):497-509. View

17.

Zhang F, Carothers J, Keasling J . Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids. Nat Biotechnol. 2012; 30(4):354-9. DOI: 10.1038/nbt.2149. View

18.

Bokinsky G, Peralta-Yahya P, George A, Holmes B, Steen E, Dietrich J . Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli. Proc Natl Acad Sci U S A. 2011; 108(50):19949-54. PMC: 3250153. DOI: 10.1073/pnas.1106958108. View

19.

Yuan L, Voelker T, Hawkins D . Modification of the substrate specificity of an acyl-acyl carrier protein thioesterase by protein engineering. Proc Natl Acad Sci U S A. 1995; 92(23):10639-43. PMC: 40667. DOI: 10.1073/pnas.92.23.10639. View

20.

Koma D, Yamanaka H, Moriyoshi K, Ohmoto T, Sakai K . Production of aromatic compounds by metabolically engineered Escherichia coli with an expanded shikimate pathway. Appl Environ Microbiol. 2012; 78(17):6203-16. PMC: 3416637. DOI: 10.1128/AEM.01148-12. View