Systems metabolic engineering of cyanobacteria for the production of bio-alkenes

The increase in atmospheric carbon dioxide (CO2) levels due to human activities, such as burning fossil fuels and deforestation, has led to concerns about its potential impacts on ecosystems. Managing CO2 emissions and enhancing carbon sequestration are suggested strategies for mitigating these effects of climate change. Photosynthetic microorganisms, such as cyanobacteria, are considered as potent platform for the environmentally-friendly manufacturing of fuels and chemicals, owing to their native ability to transform renewable resources (CO2 and light) into organic material. This thesis focuses on the photobiological production of isobutene, an alkene-type fuel precursor compound, in the unicellular cyanobacterium Synechocystis sp. PCC 6803. First, the heterologous genes encoding α-ketoisocaproate dioxygenase from Rattus norvegicus (RnKICD) and mevalonate-3-kinase from Picrophilus torridus (PtM3K) have been introduced into Synechocystis cells, which resulted in the light-driven bioproduction of isobutene. RnKICD was further recognized as a sole catalyst required for isobutene generation; yet, its promiscuous activity hinders high production capability. Therefore, RnKICD was protein-engineered with the aim to shift its substrate selectivity towards α-ketoisocaproate (KIC), the precursor for isobutene formation. The semi-rationally designed N363A/F336V variant presented a 4-fold enhanced conversion efficiency from KIC into isobutene in vitro, relative to the wildtype variant. In vivo analysis revealed that the best engineered Synechocystis strain, Syn-F336V, showed a 4-fold increase in specific volumetric content, as compared to the base strain. In addition, a comprehensive computational analysis of Synechocystis metabolism was conducted, culminating in the development of a novel machine-learning-based framework. These analyses were devised to gain a deeper insight into the internal metabolic flux, with the ultimate aim of enhancing biomolecule production through global metabolic rewiring. In summary, this thesis demonstrates the usefulness of systems metabolic engineering approach for achieving optimized cyanobacterial strains. The findings presented here contribute to the development of a circular, blue bioeconomy utilizing cyanobacteria as microbial cell factories.