Elucidation of alkane metabolism in the filamentous fungi Ascocoryne sarcoides

Abstract

Ascocoryne sarcoides has been reported to produce a variety of secondary metabolites such as linear and cyclic alkanes that are suitable for biofuel applications. Alkanes and alkenes are important as they are fully compatible with current fuel infrastructure. The genetic and biochemical basis for the biosynthesis of linear alkanes in fungi is not known and routes for cyclic alkane biosynthesis in any domain remains to be established. In this thesis, A. sarcoides was able to grow robustly in chemically-defined media in which linear, but not cyclic, alkanes are the sole carbon source, providing evidence for fungal degradation of alkanes. To establish alkane metabolic pathways in A. sarcoides, the genome and metabolome of six publicly available A. sarcoides isolates were examined. The genomes of all six isolates were sequenced, assembled and annotated. For each isolate, over 10, 000 gene products were identified by combining expression data with Hidden Markov machine learning. Each genome and predicted proteome achieved over 90% complete annotation against BUSCO’s database, considered the threshold for a high-quality dataset. No homology to known alkane producing genes were detected in any A. sarcoides isolates. By integrating annotations, pathway mapping and gene ontology with comparative analysis, hypothetical pathways for alkane degradation (via ALK-like P450), linear alkane biosynthesis (via fdc1-mediated fatty acid decarboxylation/decarbonylation) and cyclic alkane biosynthesis (via lipid lyase route) are proposed. These findings provide candidate genes for downstream heterologous expression and have the potential to increase the available toolkit for advanced biofuel applications. Solvent extraction and stir bar sorptive methods coupled to GC/MS were used to screen for biogenic hydrocarbon metabolites. The solvent extraction method did not identified the presence of biogenic alkanes. Moreover, results from SBSE were inconclusive in establishing A. sarcoides as an alkane producer due to exogenic alkane contamination and will require further method development.