Quantitative insight into the metabolism of isoprene-producing Synechocystis sp. PCC 6803 using steady state 13C-MFA

Abstract

Cyanobacteria are photosynthetic bacteria, widely studied for the conversion of atmospheric carbon dioxide to useful platform chemicals. Isoprene is one such industrially important chemical, primarily used for production of synthetic rubber and biofuels. Synechocystis sp. PCC 6803, a genetically amenable cyanobacterium, produces isoprene on heterologous expression of isoprene synthase gene, albeit in very low quantities. Rationalized metabolic engineering to re-route the carbon flux for enhanced isoprene production requires in-dept knowledge of the metabolic flux distribution in the cell. Hence, in the present study, we undertook steady state ¹³C-metabolic flux analysis of glucose-tolerant wild-type (GTN) and isoprene-producing recombinant (ISP) Synechocystis sp. to understand and compare the carbon flux distribution in the two strains. The R-values for amino acids, flux analysis predictions and gene expression profiles emphasized predominance of Calvin cycle and glycogen metabolism in GTN. Alternatively, flux analysis predicted higher activity of the anaplerotic pathway through phosphoenolpyruvate carboxylase and malic enzyme in ISP. The striking difference in the Calvin cycle, glycogen metabolism and anaplerotic pathway activity in GTN and ISP suggested a possible role of energy molecules (ATP and NADPH) in regulating the carbon flux distribution in GTN and ISP. This claim was further supported by the transcript level of selected genes of the electron transport chain. This study provides the first quantitative insight into the carbon flux distribution of isoprene-producing cyanobacterium, information critical for developing Synechocystis sp. as a single cell factory for isoprenoid/terpenoid production.

Keywords: Cyanobacterium; Flux analysis; MEP pathway; Metabolic model; qRT-PCR.