dc.description.abstract | To fulfil global energy demand and to mitigate economical, geopolitical and
ecological challenges associated with fossil fuel utilisation, the energy sector is moving
towards greater use of sustainable and environmentally friendly energy sources,
including biofuels. The ideal transport biofuel would be hydrocarbons that are identical
to fossil petroleum. However, to date characterised hydrocarbon biosynthetic pathways
include a decarbonylation or decarboxylation reaction, which involves the loss of one
carbon resulting in odd-numbered carbon chain hydrocarbons. This carbon loss
decreases carbon efficiency for alkane production, which reduces microbial fuel
economic competitiveness. Therefore, it is key that new pathways for alkane production
are identified.
The sulphate-reducing bacteria genus Desulfovibrio was previously reported to
synthesise even-numbered carbon chain alkanes, which suggests an alternative route
for alkane production without carbon loss. This investigation aimed to verify Desulfovibrio
alkane biosynthesis and characterise the possible synthetic pathway. Ten Desulfovibrio
strains, representing seven species, were screened for alkane synthesis using
isotopically labelled growth media. The ability to produce alkanes within the Desulfovibrio
genus was confirmed and was shown to be strain-specific under a set of culture
conditions. The biogenic alkanes detected were octadecane (C18), nonadecane (C19)
and eicosane (C20), with a predominance of even-numbered carbon chain alkanes.
Fatty acid analysis of Desulfovibrio strains showed an alkane biosynthetic pathway was
unlikely to involve a decarbonylation or decarboxylation step. A novel hypothesis was
therefore proposed that alkane biosynthesis by Desulfovibrio follows a metabolic route,
which has not previously been characterised, involving a series of reduction reactions
from the fatty acid pool.
The characterisation of the putative Desulfovibrio hydrogenation pathway for
alkane biosynthesis was undertaken via a target-directed genome mining approach. The
genomic DNA of nine Desulfovibrio spp. was purified, sequenced, de novo assembled
and annotated. Seven of these nine genomes are unpublished to date. No homologs of
previously characterised alkane biosynthetic enzymes from bacteria were in silico
identified in the genomes and proteomes of alkane producing Desulfovibrio spp.,
suggesting that Desulfovibrio alkane biosynthetic pathway is likely to be catalysed by
currently uncharacterised enzymes.
The 16S rRNA-based phylogeny of Desulfovibrio spp. supported the hypothesis
that the Desulfovibrio alkane biosynthetic pathway was acquired by a common ancestral
strain via horizontal gene transfer. The ability of Desulfovibrio to produce alkanes was
therefore hypothesised to be due to the presence of recruited genes encoding enzymes
involved in alkane synthesis. A comparative genomic analysis intersecting six-alkane
producing and four non-alkane producing Desulfovibrio genomes resulted in the in silico
identification of 33 hypothetical proteins considered with high confidence to be exclusive
to alkane producing Desulfovibrio strains. A novel hypothetical Desulfovibrio alkane
biosynthetic pathway was proposed involving a V-type ATPase, an uncharacterised
protein, named as a putative reductase in this investigation, and a putative
methyltransferase, which were predicted to be exclusive to alkane producing
Desulfovibrio spp. The inorganic phosphates resulting from the ATP hydrolysis catalysed
by the V-type ATPase would be involved in a reaction with fatty alcohols to form alkyl
phosphates, which are putative activated intermediates required for the hydrogenation
route from fatty alcohols to alkanes. The putative reductase and the methyltransferase,
predicted to share similar structural features with known alkane-binding proteins, would
subsequently reduce alkyl phosphates to alkanes and to iso-alkanes respectively.
Empirical investigation of the candidate molecular basis function in Desulfovibrio alkane
biosynthesis was undertaken. The Desulfovibrio alkane biosynthetic pathway remains to
be fully characterised. | en_GB |