TY - GEN
T1 - Protein Engineering to Improve the Function of BicA
T2 - 2021 AIChE Annual Meeting
AU - Parrish, Sydney
AU - Bu, Guanhong
AU - Jones, Christopher
AU - Nielsen, David
AU - Nannenga, Brent
N1 - Publisher Copyright:
© 2021 American Institute of Chemical Engineers. All rights reserved.
PY - 2021
Y1 - 2021
N2 - Cyanobacteria play a large and essential role in the global carbon cycle, collectively contributing to nearly 30% of netfixation of inorganic carbon (Ci). These photoautotrophic microbes also represent programmable chasses forbioproduction of various sustainable fuels and chemicals directly from solar energy and carbon dioxide (CO2). As aprerequisite to their photosynthetic metabolism, however, they must first be able to efficiently uptake dissolved Ci, which under physiological conditions, exists predominantly in the form of bicarbonate (HCO3-). With the ultimategoals of better understanding and improving its function, we are interested in both uncovering structure-functionrelationships 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 widelyresearched solute carrier (SLC26/SulP) family of transporters, insights gained from past studies of related homologshave been applied to construct and study the behaviors of a preliminary subset of BicA structural mutants (denoted asBicA∗). In particular, mutations known to alter the function of various prokaryotic and eukaryotic SLC26/SulPtransporters have been mapped to BicA and the resulting phenotypic changes evaluated. To aid in characterization,we have developed a series of Ci-uptake deficient mutants of Syn7002 to serve as facile genetic screens in whichalterations 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 growthcompared to wild-type BicA. During exponential growth in high-CO2(Air containing 0.5%-1.0% CO2), this fastergrowing mutant strain displays a doubling time approximately 1.4- to 1.6-times faster than the wild-type control. Furtherbiochemical 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 todevelop a broader understanding of the structural features governing function and activity of BicA. To support thisobjective, 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-basedmethod has enabled purified protein yields of 6.5 ± 1.0 mg per liter of culture. Ultimately, these efforts will facilitatestructure 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.
AB - Cyanobacteria play a large and essential role in the global carbon cycle, collectively contributing to nearly 30% of netfixation of inorganic carbon (Ci). These photoautotrophic microbes also represent programmable chasses forbioproduction of various sustainable fuels and chemicals directly from solar energy and carbon dioxide (CO2). As aprerequisite to their photosynthetic metabolism, however, they must first be able to efficiently uptake dissolved Ci, which under physiological conditions, exists predominantly in the form of bicarbonate (HCO3-). With the ultimategoals of better understanding and improving its function, we are interested in both uncovering structure-functionrelationships 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 widelyresearched solute carrier (SLC26/SulP) family of transporters, insights gained from past studies of related homologshave been applied to construct and study the behaviors of a preliminary subset of BicA structural mutants (denoted asBicA∗). In particular, mutations known to alter the function of various prokaryotic and eukaryotic SLC26/SulPtransporters have been mapped to BicA and the resulting phenotypic changes evaluated. To aid in characterization,we have developed a series of Ci-uptake deficient mutants of Syn7002 to serve as facile genetic screens in whichalterations 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 growthcompared to wild-type BicA. During exponential growth in high-CO2(Air containing 0.5%-1.0% CO2), this fastergrowing mutant strain displays a doubling time approximately 1.4- to 1.6-times faster than the wild-type control. Furtherbiochemical 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 todevelop a broader understanding of the structural features governing function and activity of BicA. To support thisobjective, 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-basedmethod has enabled purified protein yields of 6.5 ± 1.0 mg per liter of culture. Ultimately, these efforts will facilitatestructure 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.
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M3 - Conference contribution
AN - SCOPUS:85136169093
T3 - AIChE Annual Meeting, Conference Proceedings
BT - 2021 AIChE Annual Meeting
PB - American Institute of Chemical Engineers
Y2 - 15 November 2021 through 19 November 2021
ER -