(149c) Improving Heterologous Protein Expression in Synechocystis Sp. PCC 6803 for Alpha-Bisabolene Production

Plants use enzymes to generate a massively diverse array of terpenoids, many of which have present applications in industries including pharmaceuticals and food. Expressing such enzymes in bacteria would lead to lower costs of production of these chemicals due to faster growth rates and simpler harvesting. One challenge faced by microbial production of plant terpenoids is the difficulty of expressing enough of the heterologous enzymes that produce the terpenoid of interest to compete with the native pathways. Our objective is to improve cyanobacterial production of bisabolene, a precursor to a potential diesel replacement, bisabolane, to facilitate conversion of sunlight into liquid fuel. This work focused on modulating the important gene expression regulatory elements, ribosome binding sites and codon usage, to improve bisabolene production in the cyanobacteria, Synechocystis sp. PCC6803. A small set of 20 strains of this organism was generated, each with a different combination of ribosome binding site designed using the RBS Calculator and bisabolene synthase codon optimization. The strains were tested for bisabolene production with titers ranging from 0 (non-detect) to 7.9 ± 0.6 mg/L after five days of growth in continuous light. Similar titers were achieved for strains grown in 12hr:12hr light:dark cycles after 10 days. Bisabolene synthase abundance was found to correlate well with bisabolene titer.