Abstract
BackgroundMicroalgae hold promise for yielding a biofuel feedstock that is sustainable, carbon-neutral, distributed, and only minimally disruptive for the production of food and feed by traditional agriculture. Amongst oleaginous eukaryotic algae, the B race of Botryococcus braunii is unique in that it produces large amounts of liquid hydrocarbons of terpenoid origin. These are comparable to fossil crude oil, and are sequestered outside the cells in a communal extracellular polymeric matrix material. Biosynthetic engineering of terpenoid bio-crude production requires identification of genes and reconstruction of metabolic pathways responsible for production of both hydrocarbons and other metabolites of the alga that compete for photosynthetic carbon and energy.ResultsA de novo assembly of 1,334,609 next-generation pyrosequencing reads form the Showa strain of the B race of B. braunii yielded a transcriptomic database of 46,422 contigs with an average length of 756 bp. Contigs were annotated with pathway, ontology, and protein domain identifiers. Manual curation allowed the reconstruction of pathways that produce terpenoid liquid hydrocarbons from primary metabolites, and pathways that divert photosynthetic carbon into tetraterpenoid carotenoids, diterpenoids, and the prenyl chains of meroterpenoid quinones and chlorophyll. Inventories of machine-assembled contigs are also presented for reconstructed pathways for the biosynthesis of competing storage compounds including triacylglycerol and starch. Regeneration of S-adenosylmethionine, and the extracellular localization of the hydrocarbon oils by active transport and possibly autophagy are also investigated.ConclusionsThe construction of an annotated transcriptomic database, publicly available in a web-based data depository and annotation tool, provides a foundation for metabolic pathway and network reconstruction, and facilitates further omics studies in the absence of a genome sequence for the Showa strain of B. braunii, race B. Further, the transcriptome database empowers future biosynthetic engineering approaches for strain improvement and the transfer of desirable traits to heterologous hosts.
Highlights
Microalgae hold promise for yielding a biofuel feedstock that is sustainable, carbon-neutral, distributed, and only minimally disruptive for the production of food and feed by traditional agriculture
A transcriptome sequence database cannot capture untranscribed genomic regions and does not reflect post-transcriptional regulation, it can still serve as a useful tool for gene discovery and metabolic pathway and network reconstruction, and will inform further proteomic and genomic analyses
As a first step to construct such a database, we isolated total RNA from seven time points during the four week culture cycle of the Showa strain of B. braunii, race B. These time points are centered on the early portion of the culture cycle because previous studies have shown that the biosynthesis and accumulation rate of botryococcenes is maximal in this period [35,37,38]
Summary
Microalgae hold promise for yielding a biofuel feedstock that is sustainable, carbon-neutral, distributed, and only minimally disruptive for the production of food and feed by traditional agriculture. The B race of Botryococcus braunii is unique in that it produces large amounts of liquid hydrocarbons of terpenoid origin. These are comparable to fossil crude oil, and are sequestered outside the cells in a communal extracellular polymeric matrix material. Microalgae grow faster, have higher photosynthetic productivity [1,5], and accumulate biofuel feedstocks to a much higher percentage of their total biomass than land plants [6,7] These advantages translate to 15–60 times higher annual oil productivity as compared to soybean, the main US oil crop, grown on the same area of land [2,4,5]
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