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

• Published in: Metabolic engineering communications Vol. 11; p. e00144
• Main Authors: Motta Nascimento, Bruno, Nair, Nikhil U.
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.12.2020; Elsevier
• Subjects: Biopolymer; cell membranes; enzymes; Escherichia coli; Heterologous; Localization; Membrane; molecular weight; Poly-gamma-glutamate; Special issue on Model chassis cells edited by Long Liu and Rodrigo Ledesma- Amaro; Synthetase; Synthetase; Membrane; Poly-gamma-glutamate; Localization; Heterologous; Biopolymer
• ISSN: 2214-0301; 2214-0301
• DOI: 10.1016/j.mec.2020.e00144

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. •Successfully expressed active poly-γ-glutamate synthetase (PGS) in E. coli.•Confirmed PGS localization at inner membrane of E. coli.•Elucidated topology of PGS components in E. coli membrane.•Culture and expression in microplates might allow future screening of a high number of samples.•Faster production of poly-γ-glutamate in E. coli supernatant compared to B. subtilis.