Psedomonas sp. FDS-1对伏马毒素B1的降解途径及其降解机理研究

Fumonisin B1 (FB1) is a secondary metabolite produced by Fusarium proliferatum and Fusarium verticillioides, which is very common in corns. FB1 is of high toxicity and poses a serious threat to animal and human health. Degradation of FB1 by microorganism is the most promising approach for FB1 pollution. At present, the microbial resources for degradation of FB1 are very limited, and the degradation mechanism of FB1 has not completely elucidated. Psedomonas sp. FDS-1 is the FB1 degrading bacterium that was isolated in our lab. We have already studied the degradation characteristics of FB1 by strain FDS-1 and its field application effects. In addition, we have constructed the mutant library of FDS-1 by transposon mutation. The structure of degradation products will be analyzed by spectroscopy methods and the FB1 degradation pathway by FDS-1 will be elucidated. The FB1 degradation genes will be cloned by means of genomics, differential protein atlas and modern molecular biology methods. Moreover, the function of the degrading genes will be verified by gene knockout and functional complementation experiments. Then we will clarify the degradation process of FB1 by FDS-1 at the level of genes and enzymes. The FB1 degrading genes will be expressed in E.coli BL21 (DE3) and enzymology characteristics of FB1 degrading enzyme will be studied. The results of this study will elucidate the molecular mechanism of FB1 degradation by bacteria, lay the foundation for the development of FB1 degrading enzyme and genetically modified antitoxin transgenic crops, and provide theoretical and technological foundation for biological control of FB1 pollution.

伏马毒素B1(FB1)是由镰刀菌产生的一种次级代谢产物，在谷物中广泛存在，具有很高的毒性，严重威胁人畜健康。微生物降解是控制FB1污染最有前景的方法，目前关于微生物降解 FB1的资源有限，降解效率不高，解毒机理研究还不完善。Psedomonas sp. FDS-1是本课题组分离的降解菌，目前已经明确了该菌株对FB1的降解特性、田间控毒效果，构建了突变子文库。本研究以FDS-1为研究对象，通过波谱学方法分析降解产物结构，解析FB1降解途径；借助于基因组学、差异蛋白谱以及现代分子生物学相结合的方法克隆FB1降解基因，采用基因敲除和回补的方法验证基因功能，从基因水平和酶学水平阐明FDS-1降解FB1过程；研究FB1降解酶基因的外源表达和酶学特性。本研究结果将能全面解析细菌降解FB1的机理，为FB1降解酶制剂和抗毒素转基因作物的开发奠定基础，为FB1污染的生物控制提供理论和技术支撑。

伏马毒素B1(FB1)是由镰刀菌产生的一种次级代谢产物，在谷物中广泛存在，具有很高的毒性，严重威胁人畜健康。微生物降解是控制FB1污染最有前景的方法，目前关于微生物降解FB1的资源有限，降解效率不高，解毒机理研究还不完善。为此本项目首先为了保证有充足的伏马毒素标品进行降解基因克隆，降解途径解析及降解效果验证实验，利用大孔树脂联合高速逆流色谱开发了一种高效制备高纯度FB1的方法，单次可制备124 mg FB1。在此基础上以前期分离到的FB1降解菌FDS-1和FDS-2为研究对象，进行系统分类鉴定，两株降解菌分别鉴定为Sphingopyxis sp. FDS-1和Exophiala spinifera FDS-2，丰富了伏马毒素降解菌种资源库。利用液相色谱串联质谱等技术，分离了FB1的降解产物并进行结构分析鉴定，推测了降解菌FDS-1和FDS-2 对FB1的降解途径，探究了降解菌对FB1的降解机理，结果表明两株降解菌均能将FB1降解为HFB1，其中FDS-1能继续将HFB1转化为2-keto-HFB1，生成的降解产物的毒性较FB1显著降低。对FDS-1和FDS-2中的降解酶进行了定位，结果表明两株菌的降解酶均位于细胞内；通过对降解菌进行全基因组测序，NCBI进行序列比对分析，从两株降解菌中克隆了FB1水解酶基因fumD和转氨酶基因fumI，在大肠杆菌表达系统中分别进行异源表达，明确了FB1水解酶FD酶和FB1转氨酶FI的酶学特性。将两株菌的FB1水解酶基因分别转化至芽孢杆菌WB800和酵母菌表达系统中制备降解菌剂，并将菌剂应用于玉米酒糟中进行脱毒，结果显示两种菌剂对酒糟中的FB1具有较好的降解效果，降解率分别为70%和67%。本项目研究结果表明降解菌株FDS-1和FDS-2对FB1具有优良的降解效果，制备的降解菌剂对玉米酒糟的脱毒效果明显，脱毒机制明确，安全可靠，可以应用于实际生产，具有良好的应用前景，为FB1污染的生物控制提供理论和技术支撑。