Study On The Breeding Of FK506 Producing Srain And The Regulation Of Metabolic

In this study, by using random mutagenesis and directed breeding of Streptomyces tsukubaensis No.9993 which produces FK506, a high producing strain was screened. Meanwhile, the production of FK506 was improved by optimizing the regulation of metabolic and fementation process.To screen over-producing strain, mutagenesis approaches such as physical, chemical, complex methods and orientational breeding were applied. Compared with genetic stability and the yield of mentioned methods, UV mutagenesis is the the best method in the study. The mutant production reached up to 150 μg/mL, a 23.6% improvement of FK506.In order to enhance the FK506 production, amino acid, soybean oil, rare earth elements and precursor analogies were added into the fermentation medium of FK506. The result showed that the enrichment of Ala and ornithine in fermentation medium caused to improve the yield of FK506 by 11.6% and 32.1%, respectively. The content of fatty acid in the medium was determined to 0.0296 g in a fermentation medium of 25 mL, which meets the minimum demand of growth. That is the reason why the addition of soybean oil would inhibit the production of FK506. For three kinds of rare earth elements, only LaCl3 can cause a 35.6% increase in the production of FK506. In precursor analogies, ethyl picopelinate hyochdrloride and salicyl alcohol boost the output of FK506:20.24% and 52.2%, respectively.In the biosynthesis of FK506, chorismatase (FkbO) is encoded by fkbO, which is the rate-limiting enzyme in the first step of FK506 biosynthesis. The NRPS FkbP is responsible for extending the set linear polyketide intermediate, then generated 9-deoxo-31-O-demethyl-FK506 (preFK506) through intramolecular lactonization for further tailoring. Utilzing RT-PCR analysed the expression of fkbO and fkbP, it indicated that LaCl3 has a great effect on fkbP, and salicyl alcohol caused a remarkable improvement on fkbO and fkbP.