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Construction of an Efficient Nicotinate Dehydrogenase Expression System in Comamonas testosteroni CNB-2 with
Zhen-Hua Lu1, Li-Rong Yang1, Jian-Ping Wu2
1Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, China.
Applied Biochemistry and Biotechnology
|July 4, 2020
Summary
Researchers optimized Nicotinate dehydrogenase (NDHase) expression in Comamonas testosteroni using N-terminal engineering. This strategy significantly boosted enzyme activity, offering a new method for expressing difficult proteins.
Area of Science:
- Microbiology
- Protein Expression
- Biotechnology
Background:
- Nicotinate dehydrogenase (NDHase), a three-subunit membrane protein, is challenging to express functionally in common hosts like E. coli.
- Comamonas testosteroni is a promising chassis for multi-subunit membrane proteins, but NDHase expression is very low.
Purpose of the Study:
- To develop an efficient heterologous protein expression system for NDHase in C. testosteroni.
- To enhance NDHase expression and activity through multi-level N-terminal engineering.
Main Methods:
- Engineered N-terminal sequences, including Shine-Dalgarno sequences and amino acid arrangements, for NDHase subunits (ndhS, ndhL, ndhM).
- Utilized the Mmp1 promoter system for controlled transcriptional strength in C. testosteroni.
- Optimized expression of the ndhL subunit and balanced expression of all three subunits.
Main Results:
- Achieved a threefold increase in green fluorescent reporter protein expression using the Mmp1 system.
- Increased NDHase enzyme activity from 90.6 to 165 U/L through N-terminal modifications.
- Further enhanced NDHase activity to 192 U/L by optimizing ndhL expression and subunit balance.
Conclusions:
- Multi-level N-terminal engineering in C. testosteroni is an effective strategy for enhancing heterologous expression of challenging membrane proteins like NDHase.
- The developed system significantly surpasses previously reported NDHase expression levels.
- This approach provides a promising foundation for expressing other difficult proteins in unconventional bacterial hosts.
