假单胞菌NyZ12降解环己胺的分子机理研究

In recent years, an enormously large amount of cyclohexylamine, as an industrial organic pollutant suspected of being a weak carcinogen, has been produced and released into the environment. Microbial degradation is one of the effective ways to eliminate organic pollutants.But little is known about the degradation of cyclohexylamine by microorganisms in the environment since neither complete metabolic pathway nor genes encoding the key enzymes involved in the degradation of cyclohexylamine have been characterized so far.Pseudomonas NyZ12 is a Gram-negative bacterial strain that can utilize cyclohexylamine-HCl as a sole source of carbon, nitrogen, and energy.Preliminary results indicated that this strain completely metabolized cyclohexylamine via cyclohexanone degradative pathway. The gene encoding cyclohexanone monooxygenase has also been cloned with arbitrary oligonucleotide primers designed based on the conserved sequence of cyclohexanone monooxygenases.This study aims to illustrate the metabolic mechanism for cyclohexylamine degradation at both biochemical and genetic levels, including clone of catabolic gene clusters, characterization of relevant enzymes and identification of metabolic intermediates.The key enzyme - cyclohexylamine oxidase catalyzing the initial degradation of cyclohexylamine - will be heterologously expressed, purified and further characterized. Furthermore, gene knockout and the intermediates accumulated in the medium during transformation of cyclohexylamine would be conducted to demonstrate its degradation pathways. The results will provide both a scientific basis for in-depth study of the microbial degradation mechanism of cyclohexylamine and a theoretical and technical basis for the bioremediation of environmental pollution caused by the organic amine contaminants.

环己胺是一种大宗工业有机污染物，且有致癌性。微生物降解是消除有机污染物的有效途径之一。目前对环己胺微生物降解的机理研究还很不充分，完整的代谢途径及参与代谢的关键酶的编码基因尚不清楚。假单胞菌NyZ12是一株可利用环己胺为唯一碳氮源生长的革兰氏阴性菌，初步研究结果表明该菌株通过环己酮途径完全代谢环己胺。我们根据环己酮单加氧酶的保守序列设计简并引物，已克隆了环己酮单加氧酶基因。本研究拟通过底物利用、相关酶系的诱导和降解代谢中间产物鉴定，阐明环己胺的代谢途径，进而通过分子生物学方法获得整个环己胺降解基因簇，并对环己胺代谢途径的关键酶- - 环己胺氧化酶进行表达纯化及酶学特性研究。采用基因敲除、结合中间代谢产物积累分析，从分子生物学水平揭示微生物代谢环己胺的降解途径和机理。研究结果为环己胺的微生物降解机制的深入研究提供科学依据，为有机胺类污染物引起的环境问题的生物修复提供理论和技术基础。

环己胺是一种重要的精细化工中间体, 用于生产石油产品添加剂、金属缓蚀剂、杀菌剂、杀虫剂、橡胶硫化促进剂等，被大量释放到环境中造成严重的环境污染。关于环己胺的微生物降解目前仅停留在菌株的筛选以及代谢途径的鉴定，分子生物学和生化水平的相关报道较少。本研究选取革兰氏阴性菌Pseudomonas plecoglossicida NyZ12为研究对象，首次从生化和分子水平上对环己胺的代谢机理进行研究。完成菌株NyZ12的全基因组测序，递交GenBank (Accession Number: CP010359)。克隆了参与环己胺下游代谢途径的降解基因簇-环己酮降解基因簇RK21_02866-RK21_2870（chnCBADE）, RK21_02866为6-己内酯水解酶基因(chnC)，RK21_02867为环己酮单加氧酶基因(chnB), RK21_02868与环己醇脱氢酶基因（chnA）具有同源性，RK21_02869为6-羟基己酸脱氢酶基因(chnD)，RK21_02870为己醛酸脱氢酶基因(chnE)。基因簇chnCBADE位于基因岛，来源于水平基因转移，与微生物对环境的适应性相关。转录组结合荧光定量PCR结果显示各降解基因表达受底物环己胺诱导上调。将整个降解基因簇克隆后异源表达，利用全细胞生物转化可使环己酮转变成己二酸。基因敲除和互补实验证实RK21_02867 (chnB) 是NyZ12代谢环己胺必须的基因，菌株NyZ12降解环己胺经过唯一的环己酮途径降解的。基于基因组和转录组分析，结合酶学分析和产物鉴定，从五个候选胺氧化酶基因中筛选到一个环己胺氧化酶基因RK21_02631，克隆表达该基因，并对酶学性质进行了研究。对环己胺氧化酶基因RK21_02631敲除，突变体仍然可以在底物上生长，表明在菌株NyZ12中催化环己胺生成环己酮的环己胺氧化酶存在同工酶或者存在其他机制催化环己胺生成环己酮，首次在革兰氏阴性菌中发现存在这种遗传上的冗余现象。本研究在生物化学和分子水平上阐明了环己胺的微生物降解机制，为有机胺类污染物引起的环境问题的生物修复提供理论和技术基础。