A microbial platform for renewable propane synthesis based on a fermentative butanol pathway

Abstract

BackgroundPropane (C3H8) is a volatile hydrocarbon with highly favourable physicochemical properties as a fuel, in addition to existing global markets and infrastructure for storage, distribution and utilization in a wide range of applications. Consequently, propane is an attractive target product in research aimed at developing new renewable alternatives to complement currently used petroleum-derived fuels. This study focuses on the construction and evaluation of alternative microbial biosynthetic pathways for the production of renewable propane. The new pathways utilize CoA intermediates that are derived from clostridial-like fermentative butanol pathways and are therefore distinct from the first microbial propane pathways recently engineered in Escherichia coli.ResultsWe report the assembly and evaluation of four different synthetic pathways for the production of propane and butanol, designated a) atoB-adhE2 route, b) atoB-TPC7 route, c) nphT7-adhE2 route and d) nphT7-TPC7 route. The highest butanol titres were achieved with the atoB-adhE2 (473 ± 3 mg/L) and atoB-TPC7 (163 ± 2 mg/L) routes. When aldehyde deformylating oxygenase (ADO) was co-expressed with these pathways, the engineered hosts also produced propane. The atoB-TPC7-ADO pathway was the most effective in producing propane (220 ± 3 μg/L). By (i) deleting competing pathways, (ii) including a previously designed ADOA134F variant with an enhanced specificity towards short-chain substrates and (iii) including a ferredoxin-based electron supply system, the propane titre was increased (3.40 ± 0.19 mg/L).ConclusionsThis study expands the metabolic toolbox for renewable propane production and provides new insight and understanding for the development of next-generation biofuel platforms. In developing an alternative CoA-dependent fermentative butanol pathway, which includes an engineered ADO variant (ADOA134F), the study addresses known limitations, including the low bio-availability of butyraldehyde precursors and poor activity of ADO with butyraldehyde.Graphical abstractPropane synthesis derived from a fermentative butanol pathway is enabled by metabolic engineering.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0231-1) contains supplementary material, which is available to authorized users.

Rapid Reduction of the Diferric-Peroxyhemiacetal Intermediate in Aldehyde-Deformylating Oxygenase by a Cyanobacterial Ferredoxin: Evidence for a Free-Radical Mechanism

“…22,62,75 Short-chain aldehydes are also superior substrates for ADO in vitro compared to the natural long-chain substrates, due to their greater solubility. This phenomenon is particularly important because short-chain alkanes have been specifically targeted for their uses as liquefied petroleum gas (e.g., propane and butane) and as "drop-in" gasoline and jet-fuel components (e.g., heptane to undecane).…”

Distributed Biomanufacturing of Liquefied Petroleum Gas

“…coli containing engineered pathways that contain ADO, and 5x greater than E. coli containing CvFAP G462V and fed with butyrate. 6,7,9 In Halomonas, access of light to the biocatalyst was found to be a limiting factor as revealed by the linear dependence of propane titre on light intensity (Figure 5c inset). Optimal light intensity is a balance between competing factors.…”

“…Engineered biological pathways to propane have been developed based on decarbonylation of butyraldehyde incorporating natural or engineered variants of the enzyme aldehyde deformylating oxygenase (ADO). [6][7][8][9][10] The low turnover number of ADO (~ 3-5 h -1 ), however, limits implementation of these pathways in scaled bio-propane production. 6,7,9 The low activity of ADO has stimulated searches for alternative biocatalysts.…”

“…[6][7][8][9][10] The low turnover number of ADO (~ 3-5 h -1 ), however, limits implementation of these pathways in scaled bio-propane production. 6,7,9 The low activity of ADO has stimulated searches for alternative biocatalysts. A novel fatty acid photodecarboxylase (FAP) was described recently that catalyzes blue light-dependent decarboxylation of fatty acids to n-alkanes or n-alkenes (Figure 1).…”

Engineered fatty acid catabolism for fuel and chemical production

“…reported propane production (3.5mg/L) via butylaldehyde, which was generated from butyryl-CoA by acyl-CoA reductase, AdhE2 [49], whereas Sheppard et al used both a thioesterase and a carboxylic acid reductase to generate hexanal from hexanoyl-CoA, which subsequently converted to pentane (1.5mg/L) [31].…”