降解呕吐毒素的微生物/功能基因的筛选及其降解机制的研究

Vomitoxin (Deoxynivalenol，DON) is the major mycotoxin produced by Fusarium fungi in grains. DON is stable at high temperature and have an immunosuppressive effect on the health of animals and humans, so that food and feed contaminated with DON pose a health risk to humans and livestock. Consequently, detoxification of deoxynivalenol has particular merit. Physical and chemical approaches for removal and detoxification of DON often result in nutritional changes and had limit effects. Biological decontamination of DON by microbial cultures have been observed and enzymatic detoxification have been investigated. However, genes involved the transformation reactions is undiscovered, and insight into transformation mechanisms of the organisms is a prerequisite. In the study, DON-transformating microbes will be first screened from chicken and pig intestinal microbiota using gut chemostat system, and the genes encoding DON-detoxification-enzyme will be screened by constructed Fosmid library. The project also aims to understand the enzymatic mechanism of DON-detoxification through analysis genes and regulations involved in detoxification reactions and identification the metabolite by mass spectroscopy. The project might highly improve the screening efficiency for DON-transformating microbes due to cooperative effects of chemostat system and metagemomics, and can likely provide foresight insights on detoxification of the trichothecenes, stimulate the development of technologies for elimination of mycotoxins from food and feed chains.

呕吐毒素（DON）是镰孢菌产生的、普遍存在于谷物中的一种霉菌毒素，其结构稳定、毒性较强，对人体健康和畜牧业的发展都有很大的危害。目前，物理法和化学法处理DON的效果不理想，生物法处理DON虽有效果好、绿色环保的优点，但是人们对DON降解基因及生物降解机制的认识仍不够明确。因此，筛选高效降解DON的微生物菌株、挖掘其降解基因、解析其降解机制是一项有重要意义的工作。本项目以筛选可降解DON的功能微生物和功能基因为目的，利用动物肠道体外模型富集可降解DON的肠道菌群，筛选可降解DON的菌株；再通过构建Fosmid文库、结合功能元基因组学方法筛选降解/转化呕DON的基因或基因簇并验证其功能。通过基因结构组成分析DON的代谢通路，再结合质谱技术解析DON代谢产物，以揭示DON的生物转化机制。通过本项目的开展，为DON降解菌的高效筛选提供新思路和新方法，并为DON的生物降解提供理论依据和工作基础。

呕吐毒素（DON）和玉米赤霉烯酮都是由镰刀菌产生的，普遍存在于合谷类作物中的霉菌毒素，对人体健康和畜牧业的发展都有很大的影响。本项目从自然环境包括土壤和动物肠道菌群中分离筛选可降解DON和ZEA的微生物，结果表明：（1）不同培养基对肠道菌群降解DON的影响较大，鸡盲肠菌群CC在VL、BMM和L10培养基中对DON的降解率均超过了90%，而鸡大肠菌群SL和猪肠道菌群P在BMM和L10培养基中对DON的降解率也超过了80%，说明BMM和L10培养基适合筛选DON降解菌。同样的体外发酵条件下，鸡肠道菌群对DON的降解能力要高于猪的肠道菌群，说明鸡体内存在更多的能降解DON的微生物。（2）从土壤中分离筛选了可降解DON的微生物，共筛选到5株对DON有较强降解效果的菌株，经鉴定均为芽孢杆菌属，其中3株菌131、374和DL2对DON的降解率最好，均在50%以上。LC-MS结果表明，菌株374和DL2可能将DON转化为其同分异构体，推测为3-epi-DON。菌株374和DL2对DON的降解速率也有所不同，374的降解速度要大于DL2，在起始浓度为200μg/ml的DON菌株374能在80h以内降解完，而DL2需要120h的时间。（3）挖掘到2个能够对DON发生生物转化的功能基因，并对其编码酶进行了表达及功能验证，发现功能酶TRI可将DON转化为3A-DON，其Km值为25μM，Kcat值为101s-1。另外我们寻找到两个环氧水解酶能降解DON，初步研究结果表明，来源于宏基因组的CLEH酶对DON的降解效率为98.5%，另一个来源于Rhodococcus erythropolis的酶LEH对DON的降解率为12.3%，这两个酶的具体作用机理及条件正在进一步深入研究过程中。（4）筛选到两株在24h内可完全降解玉米赤霉烯酮ZEA的菌株716和816，两株菌均属于枯草芽孢杆菌，初步试验结果为716的作用部位可能为菌体和胞外分泌物，816可能是胞外分泌物的作用。