Biofuels produced by cyanobacteria have the potential to reduce the cost and climate change impacts of biofuel production. Carbon fixation and conversion to fuel may completed together in the cultivation of the cyanobacteria. Organic carbon feedstocks are not required, which reduces competition for agricultural resources. Reported product titers for most molecules of interest produced using cyanobacteria lag behind what has been achieved using yeast and E. coli cultures. In part, this is due to many achievements in the control of gene expression in those organisms. We attempted to increase the product titer of the sesquiterpene, bisabolene, which may be converted to bisabolane, a possible diesel replacement in Synechocystis sp. PCC 6803 (S. 6803). One initial strain expressed the bisabolene synthase gene from the grand fir tree (Abies grandis), codon optimized by GenScript, with transcription driven by the IPTG-inducible Ptic2op promoter. This strain produced 1.1 ± 0.08 mg/L bisabolene after five days of growth in continuous light. To increase the expression of bisabolene synthase, we varied the codon usage of the bisabolene synthase gene, using commercial gene synthesis company codon optimization services as well as the academic license software, EuGene, to design four additions coding sequence. At least three ribosome binding sites (most of which were designed using the RBS Calculator) were tested for each codon usage sequence. The effect on bisabolene production of coexpression of the farnesyl pyrophosphate synthase gene from E. coli, which catalyzes the formation of the precursor for bisabolene, was also tested. Bisabolene titers ranged from un-detected to 7.9 ± 0.6 mg/L. Bisabolene synthase abundance was measured by Western blot and found to be well correlated with titer. To move towards the conditions engineered strains might experience when grown in industrial settings, two strains were also tested in 12:12 light:dark cycles. Similar titers were reached after the same amount of light exposure time for both strains. One engineered strain was also tested in 1.5L photobioreactors exposed to sinusoidally oscillating light (with a 12-hour dark period) with maximum light intensity of 1,600 μmol photons m-2 s-1the bisabolene titer reached 22.2 mg/L after 36 days of growth.

Investigating the effects of codon optimization and ribosome binding sequences on heterologous protein expression in Synechocystis sp. PCC 6803 for improved alpha-bisabolene production

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.