Screening The Key Proteins Of Caffeine Metabolism In Paraburkholderia Caffeinilytica CF1 By Gene Engineering Method

The biodegradation method for tea waste yielded during the tea production is attracting widespread attention because of its advantages of environmental protection,high efficiency and convenience.A comprehensive study on caffeine biodegradation has conducted from the aspects of screening strains,degradation pathways and mechanisms.In this study,Paraburkholderia caffeinilytica CF1 capable of growing on caffeine as sole source of carbon,nitrogen and energy was served as original strain due to the tolerance to high concentration of caffeine(20 g/L).Based on the characteristic of metabolizing high concentration caffeine,bioinformatics analysis was used to explore its tolerance mechanism.During studying the genome of strain CF1,another strain named as Caballeronia jiangsuensis MP-1 which share the same N-demethylase gene cluster with strain CF1 was found.However,strain MP-1cannot use caffeine as the sole source of carbon,nitrogen and energy for growth.By comparing the two gene clusters of caffeine degradation existed in strain CF1 and MP-1,the four genes cdnKJIH possible related to high concentration caffeine metabolism in P.caffeinilytica CF1 was found.The four genes cdnKJIH were proved to be induced by caffeine by qRT-PCR.Then cdnK and cdnH were cloned and expressed in E.coli Rostta-gamiB(DE3)-pLysS.The soluble CdnK and CdnH were got by inclusion body refolding method and verified by Western blot.