Construction of an Efficient Nicotinate Dehydrogenase Expression System in Comamonas testosteroni CNB-2 with Multi-level N-Terminal Engineering.
Appl Biochem Biotechnol
; 192(3): 923-934, 2020 Nov.
Article
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| MEDLINE
| ID: mdl-32617841
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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1
Banco de datos:
MEDLINE
Asunto principal:
Ingeniería Genética
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Comamonas testosteroni
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Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH
Idioma:
En
Revista:
Appl Biochem Biotechnol
Año:
2020
Tipo del documento:
Article
País de afiliación:
China