Evolutionary Engineering of Cyanobacteria to Enhance the Production of α‑Farnesene from CO₂

Photosynthetic cyanobacteria can fix CO₂ and utilize it as the sole carbon source for cell growth and production of biochemicals. Here, we metabolically engineered Synechococcus elongatus PCC 7942 for an enhanced production of α-farnesene by optimizing the ribosome-binding site (RBS) of the codon-optimized farnesene synthase gene. The production of α-farnesene was found to be enhanced in strains with a low translation initiation rate, resulting in α-farnesene production (0.57 mg/(L day)). Using the RBS variants and random mutations, we performed fluorescence-based analysis of cells grown in 96-well culture plates to screen the α-farnesene-producing strains but could not improve the titers of the RBS-optimized strains. However, evolutionary engineering of the RBS-optimized strains resulted in a twofold increase in α-farnesene production (1.2 mg/(L day)) compared to the previous study. Therefore, combining metabolic and evolutionary engineering might be helpful for enhancing the cellular fitness of cyanobacteria for the production of target chemicals.