Sequencing and Analysis of the Diel Transcriptome of Botryococcus braunii
Thesis or dissertation
University of Exeter
This thesis is available for Library use on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement.
Reason for embargo
To allow time for a journal publication.
Microalgae are widely viewed as a potential source of renewable biofuels. Microalgae are highly productive and can be cultured in recycled water on margial or non-agricultural land. Despite their advantages, the industrial scale deployment of microalgae faces numerous challenges including relatively little knowledge of the algae themselves and the comparatively expensive infrastructures required for culture. The green microalga, Botryococcus braunii is particularly interesting because it synthesizes long-chain (C30- C40) hydrocarbons that can be converted to liquid fuel by hydrogenation and catalytic cracking. Moreover, B. braunii is the major fossil present in the Ordovician oil shales and kerogen deposits. Although studied since the 1970s, very little is known regarding critical aspects of B. braunii, notably its molecular biology. In higher plants molecular clocks have been well defined and transcript profiling has revealed a sophisticated network of circadian scheduling of metabolic processes. Characterization of temporal controls over hydrocarbon synthesis is therefore of importance to optimization of biofuel production from B. braunii. In this project B. braunii (Race B, strain Guadeloupe) were cultured in a 12-hour photoperiod and either maintained in that regime or transferred to constant light. Algae were sampled every 4 hours, during a 28-hour time-course and mRNA extracted. mRNA was reverse-transcribed to cDNA and sequenced using a paired-end protocol on an Illumina HiSeq 2000 platform. Over 2 billion sequence reads of 100 bp were generated and assembled de novo, into a complete transcriptome for B. braunii. The transcriptome was comprehensively annotated using global and targeted protocols and differential expression and co-expression analyses were performed. Metabolic pathway analysis confirmed the presence, and photoperiodic regulation of the MEP/DOXP Terpenoid Backbone synthesis pathway. Targeted annotation and expression analysis revealed two predicted B. braunii circadian clock components, which were incorporated into a B. braunii circadian clock model. In non-hierarchical cluster analysis, contigs of the B. braunii transcriptome clustered under four distinct patterns of diel expression. Networks of co- and anti-expressed contigs were elucidated by hierarchical clustering. These results demonstrate the exquisite control over metabolism in B. braunii. Such knowledge is essential for the industrial applications of B. braunii, either directly or through the engineering of selected B. braunii genes or molecular pathways into alternative chassis.
Biotechnology and Biological Sciences Research Council
Plymouth Marine Laboratory
PhD in Biological Sciences