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.