(413b) Protein Engineering to Improve the Function of BicA: A Constitutively Expressed, High-Flux Bicarbonate Transporter in Synechococcus sp. PCC 7002

Cyanobacteria play a large and essential role in the global carbon cycle, collectively contributing to nearly 30% of net fixation of inorganic carbon (C_i). These photoautotrophic microbes also represent programmable chasses for bioproduction of various sustainable fuels and chemicals directly from solar energy and carbon dioxide (CO₂). As a prerequisite to their photosynthetic metabolism, however, they must first be able to efficiently uptake dissolved C_i, which under physiological conditions, exists predominantly in the form of bicarbonate (HCO₃^-). With the ultimate goals of better understanding and improving its function, we are interested in both uncovering structure-function relationships as well as applying protein engineering to the high-flux HCO₃^- transporter BicA from Synechococcus sp. PCC 7002 (Syn7002). Although a structure for BicA does not currently exist, as a member of the more widely researched solute carrier (SLC26/SulP) family of transporters, insights gained from past studies of related homologs have been applied to construct and study the behaviors of a preliminary subset of BicA structural mutants (denoted as BicA*). In particular, mutations known to alter the function of various prokaryotic and eukaryotic SLC26/SulP transporters have been mapped to BicA and the resulting phenotypic changes evaluated. To aid in characterization, we have developed a series of C_i-uptake deficient mutants of Syn7002 to serve as facile genetic screens in which alterations in transporter function then correlate directly with cell growth. Using this platform, a number of BicA* mutants imparting significant growth differences have been identified, including one enabling significantly faster growth compared to wild-type BicA. During exponential growth in high-CO₂ (Air containing 0.5%-1.0% CO₂), this faster growing mutant strain displays a doubling time approximately 1.4- to 1.6-times faster than the wild-type control. Further biochemical characterization is now being performed to characterize specific changes in transporter flux and/or affinity. Meanwhile, comprehensive, high-resolution structure determination efforts are also being applied in parallel in order to develop a broader understanding of the structural features governing function and activity of BicA. To support this objective, we have developed novel methods to improve expression and purification of BicA from Synechocystis sp. PCC 6803 in E. coli and are developing enhanced protocols for BicA expression/purification from native, cyanobacterial hosts using a newly developed T7-RNA polymerase expression system. To date, our E. coli-based method has enabled purified protein yields of 6.5 ± 1.0 mg per liter of culture. Ultimately, these efforts will facilitate structure determination efforts and advance our overall understanding regarding the molecular basis underlying HCO₃^- transport function by BicA, while also enhancing our ability to engineer faster growing cyanobacterial cell factories.