Systematic Approaches to Efficiently Produce 2,3-Butanediol in a Marine Cyanobacterium

Cyanobacteria have attracted significant interest as a platform for renewable production of fuel and feedstock chemicals from abundant atmospheric carbon dioxide by way of photosynthesis. While great strides have been made in developing this technology in freshwater cyanobacteria, logistical issues remain in scale-up. Use of the cyanobacterium Synechococcus sp. PCC 7002 (7002) as a chemical production chassis could address a number of these issues given the higher tolerance to salt, light, and heat as well as the fast growth rate of 7002 in comparison to traditional model cyanobacteria such as Synechococcus elongatus PCC 7942 and Synechocystis sp. PCC 6803. However, despite growing interest, the development of genetic engineering tools for 7002 continues to lag behind those available for model cyanobacterial strains. In this work we demonstrate the systematic development of a 7002 production strain for the feedstock chemical 2,3-butanediol (23BD). We expand the range of tools available for use in 7002 by identifying and utilizing new integration sites for homologous recombination, demonstrating the inducibility of theophylline riboswitches, and screening a set of isopropyl β-d-1-thiogalactopyranoside (IPTG) inducible promoters. We then demonstrate improvements of 23BD production with the systematic screening of different conditions including: operon arrangement and copy number, light strength, inducer concentration, cell density at the time of induction, and nutrient concentration. Final production tests yielded titers of 1.6 g/L 23BD after 16 days at a rate of 100 mg/L/day. This work represents great strides in the development of 7002 as an industrially relevant production host.