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

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Bruno Motta Nascimento, Nikhil U. Nair
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引用次数: 4

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.

大肠杆菌异源生产聚γ-谷氨酸的膜酶复合物的表征
聚γ-谷氨酸(PGA)是由许多芽孢杆菌种产生的一种具有许多独特和理想特性的聚合物。然而,负责其合成的多亚基酶复合物PGA合成酶(PGS)尚未在天然环境和重组环境中得到很好的表征。阐明该酶的结构和功能特性对未来改变其催化性能的工程研究至关重要。本研究主要研究了该酶在大肠杆菌膜上的异种表达,并对其定位、取向和活性进行了表征。在大肠杆菌中,我们能够产生高分子量的PGA聚合物,在深孔微滴批培养中,其滴度约为13 mg/L,降解最小。利用融合蛋白,我们首次观察到不同亚基与内细胞膜的结合和取向。这些结果为这种研究较少的酶复合物提供了基本的结构信息,并将有助于未来的基础研究和工程努力。
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来源期刊
Metabolic Engineering Communications
Metabolic Engineering Communications Medicine-Endocrinology, Diabetes and Metabolism
CiteScore
13.30
自引率
1.90%
发文量
22
审稿时长
18 weeks
期刊介绍: Metabolic Engineering Communications, a companion title to Metabolic Engineering (MBE), is devoted to publishing original research in the areas of metabolic engineering, synthetic biology, computational biology and systems biology for problems related to metabolism and the engineering of metabolism for the production of fuels, chemicals, and pharmaceuticals. The journal will carry articles on the design, construction, and analysis of biological systems ranging from pathway components to biological complexes and genomes (including genomic, analytical and bioinformatics methods) in suitable host cells to allow them to produce novel compounds of industrial and medical interest. Demonstrations of regulatory designs and synthetic circuits that alter the performance of biochemical pathways and cellular processes will also be presented. Metabolic Engineering Communications complements MBE by publishing articles that are either shorter than those published in the full journal, or which describe key elements of larger metabolic engineering efforts.
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