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

• Main Authors: Bruno Motta Nascimento, Nikhil U. Nair
• Format: Article
• Language: English
• Published: Elsevier 2020-12-01
• Series: Metabolic Engineering Communications
• Subjects: Heterologous; Poly-gamma-glutamate; Synthetase; Biopolymer; Localization; Membrane
• Online Access: http://www.sciencedirect.com/science/article/pii/S2214030120300444

Poly-γ-glutamic acid (PGA) produced by many Bacillus species 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 the Escherichia coli membrane and characterizing localization, orientation, and activity of this heterooligomeric enzyme complex. In E. 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 provide fundamental structural information on this poorly studied enzyme complex and will aid future fundamental studies and engineering efforts.