College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310032, China.
Technology Center, China Tobacco Zhejiang Industrial Co., Ltd., Hangzhou, 310009, China.
Appl Biochem Biotechnol. 2021 Sep;193(9):2793-2805. doi: 10.1007/s12010-021-03566-0. Epub 2021 Jun 1.
Nicotine-degrading Pseudomonas sp. JY-Q is a preferred strain utilized in reconstituted tobacco process for tobacco waste treatment. However, its efficiency of nicotine metabolism still requires to be improved via genomic technology such as promoter engineering based on genomic information. Concerning upstream module of nicotine metabolic pathway, we found that two homologous genes of nicotine dehydrogenase (nicA2 and nox) coexisted in strain JY-Q. However, the transcriptional amount of nox was 20-fold higher than that of nicA2. Thus, the nicA2 expression required improvement. Combinatorial displacement was accomplished for two predicted endogenous promoters, named as PnicA2 and Pnox for nicA2 and nox, respectively. The mutant with Pnox as the promoters for both nicA2 and nox exhibited the best nicotine metabolic capacity which increased by 66% compared to the wild type. These results suggested that endogenous promoter replacement is also feasible for function improvement of metabolic modules and strain enhancement of biodegradation capacity to meet real environment demand.
用于处理烟草废物的再造烟叶过程中，尼古丁降解假单胞菌 JY-Q 是一种被广泛应用的优势菌株。然而，其尼古丁代谢效率仍需要通过基因组技术（如启动子工程）进行改进，这些技术是基于基因组信息得到的。在尼古丁代谢途径的上游模块中，我们发现菌株 JY-Q 中存在两个尼古丁脱氢酶（nicA2 和nox）的同源基因。然而，nox 的转录量是 nicA2 的 20 倍。因此，需要改进 nicA2 的表达。我们对两个预测的内源性启动子（分别命名为 PnicA2 和 Pnox）进行了组合替换，以实现 nicA2 和 nox 的表达。与野生型相比，以 Pnox 为 nicA2 和 nox 启动子的突变体表现出最佳的尼古丁代谢能力，提高了 66%。这些结果表明，内源性启动子替换对于代谢模块的功能改进和生物降解能力的增强也是可行的，以满足实际环境的需求。
