(164n) Exploring Structure-Function Relationships Governing Activity of the Cyanobacterial Bicarbonate Transporter Sbta

Cyanobacteria play a large and essential role in the global carbon cycle, collectively contributing to nearly 30% of net primary inorganic carbon (C_i) fixation. In addition, they also represent programmable platforms for the sustainable bioproduction of fuels and chemicals directly from sunlight and carbon dioxide (CO₂). Photosynthetic carbon fixation, however, depends on the ability to cyanobacteria to efficiently uptake dissolved C_i which, under physiological conditions, exists mainly in the form of bicarbonate (HCO₃^-). SbtA is one of two main HCO₃^- transporters in cyanobacteria, and it is highly conserved across most species. That said, past reports have shown that individual SbtA homologs can display diverse flux vs. affinity behaviors, suggesting that small sequence differences can impart diverse changes in function. Accordingly, our group is interested in enhancing our structure-function understanding of SbtA and using these insights to engineer improved HCO₃^- transporters. To this end, several approaches are being applied in parallel, including i) structural modeling to identify features of putative importance to controlling HCO₃^- uptake rates among SbtA homologs, ii) structure determination of purified SbtA expressed in both Synechococcus sp. PCC 7002 and Escherichia coli, and iii) functional characterization of SbtA variants in C_i-uptake deficient PCC 7002 mutants as well as E. coli strains lacking carbonic anhydrase (CA).

Recently, the structure of SbtA from Synechocystis sp. PCC 6803 (expressed in E. coli) was reported and has been used to develop Robetta models of SbtA’s from PCC 7002, PCC 6803, and Cyanobium sp. PCC 7001; three homologs displaying vastly different kinetics. We are still using these models to learn about inherent structure-function relationships and generate new hypotheses regarding HCO₃^- transport behaviors displayed by different SbtA homologs.

In support of attaining higher resolution structures, we have developed a platform for sbtA expression and purification from heterologous E. coli that yields sufficient levels of SbtA for single particle cryo-EM. Furthermore, we are working to advance our structural objectives by developing protocols for sbtA expression and purification from native, cyanobacterial hosts using a newly developed T7-RNA polymerase expression system that has been shown to enable very high protein production (up to two thirds of total cellular protein content in PCC 7002).

Finally, we have developed a series of Ci-uptake deficient mutants of PCC 7002 to serve as facile genetic screens in which alterations in transporter function then correlate directly with cell growth. Meanwhile, to provide an additional, heterotrophic platform for SbtA functional screening, we have also developed a CA-free strain of E. coli that is also optimized for succinate fermentation; thereby providing a strong C_i sink. Ultimately, these collectively efforts will further our understanding of key structural features that influence transport activity (i.e., flux and/or affinity) by SbtA, as well as enhance our ability to engineer faster growing and more productive cell factories.