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Construction of an Efficient Nicotinate Dehydrogenase Expression System in Comamonas testosteroni CNB-2 with Multi-level N-Terminal Engineering

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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 N-terminal 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.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 21676240) and the National Key R&D Program of China (No. 2019YFA09005000). We are grateful for the donation of PMmP1-LacO- sfgfp-p321 plasmid by Professor Guoqiang Chen (Center for Synthetic and Systems Biology, Tsinghua University, China).

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  1. Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, China

    Zhen-Hua Lu, Li-Rong Yang & Jian-Ping Wu

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  1. Zhen-Hua Lu
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  2. Li-Rong Yang
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Correspondence to Jian-Ping Wu.

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Lu, ZH., Yang, LR. & Wu, JP. Construction of an Efficient Nicotinate Dehydrogenase Expression System in Comamonas testosteroni CNB-2 with Multi-level N-Terminal Engineering. Appl Biochem Biotechnol 192, 923–934 (2020). https://doi.org/10.1007/s12010-020-03354-2

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  • DOI: https://doi.org/10.1007/s12010-020-03354-2

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