Characterization of a membrane enzymatic complex for heterologous production of poly-γ-glutamate inE. coli

Abstract

ABSTRACTPoly-γ-glutamic acid (PGA) produced by manyBacillusspecies is a polymer with many distinct and desirable characteristics. However, the multi-subunit enzymatic complex responsible for its synthesis, PGA Synthetase (PGS), has not been well characterized yet, in native nor in recombinant contexts. Elucidating structural and functional properties are crucial for future engineering efforts aimed at altering the catalytic properties of this enzyme. This study focuses on expressing the enzyme heterologously in theEscherichia colimembrane and characterizing localization, orientation, and activity of this heterooligomeric enzyme complex. InE. coli, we were able to produce high molecular weight PGA polymers with minimal degradation at titers of approximately 13 mg/L in deep-well microtiter batch cultures. Using fusion proteins, we observed, for the first time, the association and orientation of the different subunits with the inner cell membrane. These results elucidate provide fundamental structural information on this poorly studied enzyme complex and will aid future fundamental studies and engineering efforts.HIGHLIGHTSSuccessfully expressed active poly-γ-glutamate synthetase (PGS) inE. coli.Confirmed PGS localization at inner membrane ofE. coli.Elucidated topology of PGS components inE. colimembrane.Culture and expression in microplates might allow future screening of a high number of samples.Faster production of poly-γ-glutamate inE. colisupernatant compared toB. subtilis.