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.

烟酸脱氢酶（NDHase）是具有三个亚基（ndhS，ndhL和ndhM）的膜蛋白，使用常见宿主（例如大肠杆菌，枯草芽孢杆菌或巴斯德毕赤酵母）很难以功能形式表达。睾丸Comomonas睾丸激素是表达多亚基膜蛋白的合适微生物底盘。但是，NDHase在睾丸梭菌中的表达极低。我们已经开发了一种系统的方法来创建C. testosteroni中有效的蛋白质表达系统CNB-2使用多层N端工程。我们为Mmp1系统选择了一个强大的启动子，该启动子可以控制非常规细菌的转录强度。这将绿色荧光报道蛋白的表达提高了三倍。在修饰NDHase编码基因的起始区域，对N端Shine-Dalgarno序列进行了修饰，并对氨基酸序列进行了重排后，酶的活性从90.6提高至165 U / L。这些优化的N端Shine–Dalgarno和氨基酸序列用于增强ndhL亚基的表达并改善NDHase的三个亚基的平衡表达，从而使192 U / L的酶活性远远超过先前报道的水平。