Construction of an Efficient Nicotinate Dehydrogenase Expression System in Comamonas testosteroni CNB-2 with Multi-level
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Construction of an Efficient Nicotinate Dehydrogenase Expression System in Comamonas testosteroni CNB-2 with Multi-level N-Terminal Engineering Zhen-Hua Lu 1 & Li-Rong Yang 1 & Jian-Ping Wu 1 Received: 19 February 2020 / Accepted: 22 May 2020/ # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Nicotinate dehydrogenase (NDHase) is a membrane protein with three subunits (ndhS, ndhL, and ndhM), which is difficult to express in a functional form using common hosts such as Escherichia coli, Bacillus subtilis, or Pichia pastoris. Comamonas testosteroni is a suitable microbial chassis for expressing multi-subunit membrane proteins. However, the expression of NDHase in C. testosteroni is extremely low. We have developed a systematic approach to create an efficient protein expression system in C. testosteroni CNB-2 using multi-level Nterminal engineering. We selected a strong promoter for the Mmp1 system that enables control of transcriptional strength in unconventional bacteria. This enhanced the expression of a green fluorescent reporter protein threefold. Following modification of the N-terminal Shine–Dalgarno sequence and rearrangement of amino acid sequence in the starting area of the gene encoding NDHase, enzyme activity increased from 90.6 to 165 U/L. These optimized N-terminal Shine–Dalgarno and amino acid sequences were used to enhance the expression of ndhL subunit and improve the balance expression of three subunits of NDHase, resulting in enzyme activity of 192 U/L that far surpasses the previously reported level. These results highlight a promising strategy for the development of other heterologous expression systems for challenging proteins using unconventional bacteria. Keywords N-terminal engineering . Promoter engineering . SD sequence modification . SKIKlike strategy . Balanced expression . Nicotinate dehydrogenase
Introduction Recombinant proteins expressed in host cells have a wide range of applications in industries, including biological manufacturing, medicine, and life sciences [1, 2]. At
* Jian-Ping Wu [email protected]
1
Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
Applied Biochemistry and Biotechnology
present, there is a variety of efficient recombinant expression systems available, such as Escherichia coli, Bacillus subtilis, and Pichia pastoris [1, 3, 4]. These systems have particular advantages for enzymes with few subunits and simple structures [5]. However, these effective and conventional expression systems often produce proteins in the form of inclusion bodies or inactive proteins, while some are unable to express multi-subunit proteins with complex functional structures. Nicotinate dehydrogenase (NDHase, EC1.17.1.5), a multi-subunit membrane protein, is an example of a protein that is difficult to express in conventional expression systems [6, 7]. Many researchers have tried to express NDHase in E. coli, but most of the protein is in the form of inclusion bodies in this system. Unti
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