Dual approach to investigate sustainable isoprene production in genetically modified Synechocystis sp. PCC 6803
Year: 2024
Type: dissertation
Abstract: Sustainable production of otherwise petrochemically sourced products can be achieved in cyanobacteria. These photosynthetic microorganisms can be genetically modified to heterologously produce industrially relevant chemicals such as the terpene isoprene, from sunlight and carbon dioxide. However, this is not yet reality on a large scale. This work was based on investigating culture conditions similar to the ones at the final scale as well as cellular productivity improvements to facilitate the transition from lab-scale proof-of-concept to full industrial capacity. The thesis had two focus areas – investigating the effect of diurnal light dark cycles (LDCs) on isoprene production, and investigation to alleviate bottlenecks in the cyanobacterial isoprene production pathway. The latter aim was focused on 1-deoxy-D-xylulose-5-phosphate synthase (Dxs), a carbon flux bottleneck for the isoprene production, and amino acidresidues involved in its feedback-inhibition by pathway intermediates. For investigation of LDCs, isoprene production by genetically modified cyanobacteria Synechocystis sp. PCC 6803 strains was measured throughout LDCs mimicking the light changes during the day and darkness at night. The results showed that isoprene production correlates to light intensity with highest production at midday and no detectable production in darkness. The second aim focused on reducing the feedback-inhibition of Dxs by investigating Synechocystis sp. PCC 6803 strains expressing different variants of Coleus forskohlii Dxs (CfDXS). Mutant variants contained substitutions of four residues potentially involved in both feedback-inhibition of and monomer interaction in CfDXS – His150, Gln439, Arg440 and Phe477. Mutants were screened by measuring their isoprene production. The results indicated that additional copies of wildtype CfDXS increase isoprene production, while all screened mutants decreased the production. Observations and a brief investigation of the mutants’ effect on culture pigmentation was also done. The results from this thesis give insight into how different conditions and genetic alterations affect isoprene production and can serve as a stepping stone for further studies to elucidate the underlying mechanisms. It also contributes knowledge helpful for the transition to sustainable, large scale isoprene production in genetically modified cyanobacteria.
Author: Beskow, Isa
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W6981280677