Cyanobacteria are valuable organisms for studying the physiology of photosynthesis and carbon fixation, as well as metabolic engineering for the sustainable production of fuels and chemicals. This talk will describe our ongoing efforts to use Synechococcus sp. PCC 7002, a fast growing, halotolerant, and naturally transformable cyanobacteria as a platform for producing commodity chemicals from sunlight and carbon dioxide.  The talk will describe a novel counter selection method based on organic acid toxicity as well as new gene expression tools (promoters, RBS, post-transcriptional regulation) that facilitate metabolic engineering in PCC 7002. In Begemann et al., (PLoS One, 2013) the organic acids acrylate, 3-hydroxypropionate, and propionate were shown to be inhibitory towards PCC 7002 and other cyanobacteria at low concentrations. Inhibition was overcome by a loss of function mutation in the gene acsA, which was annotated as an acetyl-CoA ligase. Loss of AcsA function was used as a basis for an acrylate counter selection method that facilitates scarless mutagenesis of the chromosome. To overcome the lack of known PCC 7002 promoters, a series of promoter libraries were developed and tested. The expression of fluorescent proteins generated by each promoter was compared in E. coli and PCC 7002 and found to have little correlation. Inducible promoters were engineered to enable controlled expression of metabolic genes and the Ribosome Binding Site Calculator was evaluated as a tool for engineering translation rates. The combined toolset was used to engineer production of organic acids in PCC 7002. A challenge with photosynthetic chemical production is the cost associated with photobioreactors. The talk will include a brief discussion of combining wastewater remediation with chemical production to meet economic targets.

Engineering cyanobacteria for use as chemical factories

247th National Meeting (2014)

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