Application of New Synthetic Biology Tools for Organic Acid Production in Cyanobacteria

Cyanobacteria are an interesting platform for metabolic engineering due to their ability to directly convert sunlight and CO₂ to fuels and chemicals. In order to harness this intrinsic ability, engineering tools and strategies must be developed to control gene expression and direct carbon flux to products of interest. The cyanobacterium Synechococcus sp. PCC 7002 was used as a host platform due to the fast growth rate and ability to grow under a variety of conditions. In order to efficiently test a variety of constructs, we have developed a novel counter selection system based on acrylic acid sensitivity to quickly construct strains. This development has allowed us to design, build, and modify new promoters and induction systems for controlling gene expression. These tools were used to express heterologous pathways for the production of the organic acids lactate and 3-hydroxypropionate (3HP). Lactate was produced by expressing a lactate dehydrogenase from Bacillus subtilis in an inducible operon with a soluble transhydrogenase. 3HP was produced by expression of a malonyl-CoA reductase and malonate semialdehyde reductase derived from archaea. Expression was tuned via inducer titration to maximize product production. Additional strains with gene deletions and over expression constructs were built to increase carbon flux to these products. Gene expression and carbon flux modification strategies developed in these systems could be used to improve production of other cyanobacterial chemical production strains.