Characterization of a membrane enzymatic complex for heterologous production of poly-γ-glutamate in E. coli
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. El...
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| Published in | Metabolic engineering communications Vol. 11; p. e00144 |
|---|---|
| Main Authors | , |
| Format | Journal Article |
| Language | English |
| Published |
Elsevier B.V
01.12.2020
Elsevier |
| Subjects | |
| Online Access | Get full text |
| ISSN | 2214-0301 2214-0301 |
| DOI | 10.1016/j.mec.2020.e00144 |
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| Abstract | 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. |
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| AbstractList | 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. 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.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. 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. 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. 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. |
| ArticleNumber | e00144 |
| Author | Motta Nascimento, Bruno Nair, Nikhil U. |
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| Keywords | Synthetase Membrane Poly-gamma-glutamate Localization Heterologous Biopolymer |
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| Notes | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Website: https://sites.tufts.edu/nairlab. Present/permanent address: Science and Technology Center #276, 4 Colby St., Medford, MA 02155. |
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| Snippet | Poly-γ-glutamic acid (PGA) produced by many Bacillus species is a polymer with many distinct and desirable characteristics. However, the multi-subunit... Poly-γ-glutamic acid (PGA) produced by many Bacillus species is a polymer with many distinct and desirable characteristics. However, the multi-subunit... |
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| SubjectTerms | 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 |
| Title | Characterization of a membrane enzymatic complex for heterologous production of poly-γ-glutamate in E. coli |
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