RESUMO
Tanshinones are the bioactive components of the Chinese medicinal herb Salvia miltiorrhiza, while its biosynthetic pathway remains to be characterized. Rapid identification and characterization of the genes correlated to tanshinones biosynthesis is very important. As one of the intermediates of tanshinones biosynthesis, the ferruginol content is relative low in both root and engineered bacteria. It is urgent to construct an efficient system for conversion of miltiradiene to ferruginol to obtain large amount of ferruginol as the substrates for further identifying other downstream genes involved in tanshinones biosynthesis. In this study, we constructed the whole-cell yeast biocatalysts co-expressing miltiradiene oxidase CYP76AH1 and cytochrome P450 reductases (SmCPR1) from Salvia miltiorrhiza, and then characterized it with RT-PCR. After permeabilization, the yeast whole-cell could catalyze turnover of miltiradiene to ferruginol efficiently through single-step biotransformation with a conversion efficiency up to 69.9%. The yeast whole-cell biocatalyst described here not only provide an efficient platform for producing ferruginol in recombinant yeast but also an alternative strategy for identifying other CYP genes involved in tanshinones biosynthesis.
RESUMO
Sepiolite--an inexpensive, resourceful, fibrous yet inoffensive mineral--made DNA transformation rapid, simple and efficient but the mechanism for DNA transformation was still unclear. Through RNA competition test, we proposed the different transforming mechanisms from the previous report. Meanwhile, we optimized the transforming method and could transfer a colony stored at 4 degrees C for a month with plasmid through sepiolite fibers. The cells could be transformed well without competent cells preparation or incubation process. In sum, this was a novel potential transforming method, which could be explored further if the chemical method and electroporation could not be used.