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bioRxiv · 10.1101/2020.07.17.209205

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

Motta Nascimento, B.·Nair, N. U.
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Abstract

Poly-{gamma}-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 elucidate provide fundamental structural information on this poorly studied enzyme complex and will aid future fundamental studies and engineering efforts. HIGHLIGHTSO_LISuccessfully expressed active poly-{gamma}-glutamate synthetase (PGS) in E. coli. C_LIO_LIConfirmed PGS localization at inner membrane of E. coli. C_LIO_LIElucidated topology of PGS components in E. coli membrane. C_LIO_LICulture and expression in microplates might allow future screening of a high number of samples. C_LIO_LIFaster production of poly-{gamma}-glutamate in E. coli supernatant compared to B. subtilis. C_LI

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Motta Nascimento, B., Nair, N. U.. 2020-07-17. Characterization of a membrane enzymatic complex for heterologous production of poly-γ-glutamate in E. coli. https://doi.org/10.1101/2020.07.17.209205

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