Multi-omics and in vitro characterisation of cyanobacteria biochemistry

Abstract

Cyanobacteria have been one of the most important organisms in the Earth’s biogeochemical cycles. Subject to millions of years of evolution, it is a diverse phylum of photoautotrophs which is responsible for fixing 20-30% of global carbon dioxide. This thesis is an attempt to explore the composition and function of some fast-growing biotechnologically important strains of cyanobacteria in differential growth conditions (ambient, 0.5% or 3% CO2 in low or high light) using high throughput endometabolomics and proteomic analysis along with in vitro assays, to capture the holistic snapshot of metabolism and resource allocation. We focus on proteins related to carbon fixation and light harvesting along with metabolites like sugar phosphates, amino acids and organic acids families. Photosynthetic efficiency with respect to light harvesting and carbon fixation was assessed using cell-free cyanobacterial lysates. The different in vitro assays developed here, expand our understanding of the strains and their potential by decoupling metabolism from cell growth and viability constraints. Cyanobacterial lysates show accumulation of 3-phosphoglycerate when supplemented with RuBP as substrate; and glucovanillin via a cascade of enzymatic steps which is then used to understand rates of carbon fixation and gluconeogenesis. Light harvesting capabilities of the cell-free extracts are measured by using the ATP dependent conversion of glucose to glucose-6-phosphate using hexokinase. Together, the compositional and functional datasets will highlight how environmental cues and differential regulation impact CO2 fixation, light harvesting, and metabolic flux toward precursor molecules of interest to guide subsequent engineering and biosynthesis efforts.