微生物降解全氟化合物PFOA的机理研究

Perfluorinated compounds (PFCs) such as perfluorooctane acid (PFOA) and perfluorooctane sulfonate (PFOS) that are characterized by strong surface activity and superior physical and chemical stability, have been widely used in pharmaceutical and other industries. However, with the long-term use of these compounds, it has been found that they are a new type of persistent organic pollutants, and have potentially toxicity to human and animal. Microbial transformation is an effective remediation approach to prevent and clean up PFOA/PFOS contamination. To date, certain strains of wild-type microbes have been reported to have limited capacity to degrade PFOA, Selection of superior strains and determination of the molecular mechanism associated with microbial degradation of PFOA become urgently necessary. In our preliminary experiments, we got a PFOA-decomposing bacterium, Pseudomonas parafulva, isolated from PFCs-contaminated soils, accomplished the complete genome sequencing of this strain and obtained an improved mutant F3-52 using genome shuffling that had high degradation efficiency of PFOA. Based on our preliminary data, we propose to make comparative genomics and comparative transcriptomics investigation on an effective PFOA-degrading mutant F3-52, using wild Pseudomonas parafulva as control, analyze the relationship between genomic constitution varieties, gene express differences and the capacity of degrading PFOA, and capture candidate genes relevant to microbial degradation of PFOA. Further, we will characterize the function of PFOA-degrading related candidate genes by homologous combination knock-out strategy and real time PCR. Meantime, we will collect culture solution of wild strain and mutant F3-52 at regular time, and determine metabolic intermediates of PFOA by LC-MS/MS. Combined with PFOA-degrading related genes function, we could speculate the metabolism pathway about microbial transformation of PFOA. This study will be for the first time to investigate the molecular mechanism associated with microbial degradation of PFOA, determination of PFOA-decomposing related genes and metabolic intermediates of PFOA will enhance understanding microbial metabolism pathway of PFOA, and provide useful information for genetic breeding of PFOA-decomposing microorganisms by metabolism engineering.

全氟辛酸（PFOA）有机化合物具有持久性、生物积累性和毒性，随着广泛应用带来的环境污染问题受到高度关注。微生物降解PFOA效率低，降解机理不清楚，极大限制了其应用。本项目以筛选获得的降解PFOA的类黄色假单胞菌野生株和通过基因组改组技术选育的降解效率高的突变株F3-52为研究对象，开展比较基因组学和比较转录组学研究，探讨两种性状下基因结构组成变化、基因表达差异与降解PFOA能力的关系，筛选降解相关的候选基因，并采用基因敲除技术对候选基因逐一进行功能验证。同时，定时收集两种性状微生物的发酵液，采用LC-MS/MS检测分析降解PFOA的代谢中间产物，结合确证基因功能信息推测微生物降解PFOA的代谢转化路径。本项目的开展可望挖掘出系列与PFOA代谢分解相关的功能基因，初步阐明微生物降解PFOA的代谢途径。项目首次全面解析微生物降解PFOA的机理，将为通过代谢工程改良菌种提高降解效率奠定理论基础。

全氟辛酸（PFOA）有机化合物具有持久性、生物积累性和毒性，随着广泛应用带来的环境污染问题受到高度关注。微生物降解PFOA效率低，降解机理不清楚，极大限制了其应用。课题组前期筛选获得以PFOA为唯一碳源生长的4株细菌，其中类黄色假单胞菌（Pseudomonas parafulva）YAB-1为优势降解菌株，以此为基础开展本研究工作。①开展菌株YAB-1对PFOA胁迫的生理响应和降解特性研究，发现对PFOA胁迫生理响应表现出明显的剂量—效应关系，最优降解条件为初始pH7.0、培养温度30℃、接菌量2%、底物PFOA浓度500 mg/L。此条件下发酵96 h，YAB-1的最大PFOA降解效率为32.4%。②基于基因组改组技术对菌株YAB-1改良，获得9株PFOA耐受力和降解效率均提高的正突变株，其中突变株F3-52降解率为58.6%，且具有稳定遗传。③菌株YAB-1的全基因组测序与生物信息学分析表明，基因组全长为5,119,844 bp，G+C%为61.3%，预测得4,807个CDS，基因经GO、COG、KEGG注释，结合比较基因组学数据，推测编码原儿茶酸脂3, 4-双加氧酶、乙醛还原酶、细胞色素P450单加氧酶等的基因参与了PFOA的降解转化。④菌株YAB-1的转录组测序与分析，发现8个显著差异表达基因，分别编码细胞色素P450单加氧酶、乙醇脱氢酶、乙醛还原酶、ccb-3型细胞色素C氧化酶、TolC家族I型外膜分泌蛋白、F0F1 ATP合成酶、谷氨酸脱羧酶、ABC转运蛋白，推测可能与微生物降解PFOA紧密关联。⑤以PFOA为唯一碳源筛选、分离获得2株降解PFOA的真菌（AF1和AF2），初步鉴定AF1属于木霉菌（Trichoderma sp.）、AF2属于毛霉菌（Mucor sp.）。对AF2进行转录组测序及生物信息学分析，推测编码原儿茶酸脂3, 4-双加氧酶、乙醛还原酶、FDA为辅酶的单加氧酶、细胞色素P450单加氧酶等9个基因参与了PFOA的降解转化。项目获得了降解PFOA性能优良的微生物菌种，为PFOA污染物的微生物修复积累了种质资源。同时，首次从分子水平解析了微生物降解PFOA的机理，为理解微生物降解PFOA的代谢途径及挖掘与鉴定微生物中参与代谢分解PFOA的功能基因奠定了坚实基础。