(149g) Biological Synthesis of Chemicals from CO2

Chemical production in photosynthetic organisms is a nascent technology with great promise for renewable chemical production. Cyanobacteria are under investigation as a means to utilize light energy to directly recycle CO₂ into renewable chemical compounds. We have previously engineered production of the chemical feedstock 2,3-butanediol (23BD) from an obligate photoautotrophic cyanobacterium, Synechococcus elongatus PCC 7942. We subsequently optimized 23BD production by varying ribosomal binding site and promoter strength, operon organization, and gene expression at the transcriptional and translational level. The resulting engineered strains exhibited enhanced total carbon fixation and 23BD production under continuous light conditions. We observed a concurrent increase in oxygen evolution due to greater carbon redirection away from metabolism, indicating the possibility of an overall increase in photosynthetic efficiency. However, nearly all cyanobacterial production studies have exclusively used continuous lighting in laboratory conditions when measuring productivity. Natural sunlight is freely available for any photo-dependent chemical platform and may aid the commercial viability of the production. This natural lighting includes periods of darkness where many cyanobacteria naturally stop growing, fixing carbon, and producing biochemicals. To overcome these limitations, the 23BD strain was engineered for enhanced production of 23BD via glucose supplementation. This study achieved comparable 23BD production in diurnal and continuous light conditions. However, the carbon yield was only 40% of the theoretical maximum yield possible from glucose alone. Therefore, we developed a strategy to optimize glucose and CO₂ utilization and to improve 23BD production and yield. First, glucose metabolism was rewired through the oxidative pentose phosphate pathway to overproduce ribulose-5-phosphate (Ru5P). Next, because carbon metabolism in cyanobacteria is precisely controlled in response to environmental changes such as light and carbon availability, cp12, a regulatory gene of the Calvin Benson cycle, was deleted to amplify conversion of Ru5P to ribulose-1,5-bisphosphate, a direct precursor of CO₂ fixation. The engineered strain efficiently uses both CO₂ and glucose, and produces 12.6 g/L of 2,3-butanediol with a rate of 1.1 g/L/d. This represents a significant step towards industrial viability and a robust example of carbon metabolism plasticity.