Study On The Biosynthesis Related Genes Of Tacrolimus And Genetic Manipulation On The Producing Strain

Tacrolimus, a 23-membered macrolide compound produced by streptomycete strain, is clinic widely used as an important immunosuppressant in organ transplantation and autoimmune disease in many countries. Low titer of the fermentation broth and producing chemical similar byproducts, such as FK520 and dihydrogen-FK506, are bottlenecks for FK506 industry production. This study focused on the FK506 biosynthesis related genes and attempted improving fermentation yield of FK506 producing strain by genetic manipulation.FK506 producing strain Streptomyces sp. TL01 (0311-20) was used as original strain. FK506 biosynthesis genes fkbM encoding 31-O-demethyl-FK506 methyltransferase, regulatory gene fkbN and tcs7, primary metabolism related genes zwf2, bccA2, gltD and extraneous source gene vgb encoding Vitreoscilla hemoglobin (VHb) were systematically studied. Above-mentioned genes were cloned and verified by PCR and sequencing analysis, respectively. Suitable shuttle plasmids carrying the target gene were constructed and introduced into the FK506 producing strain Streptomyces sp. TL01 by performing E.coli-Streptomyces conjugation. Engineering strains of homologous exchange were screened by growing on the plates containing different selection antibiotics and verified by PCR.Flask fermentation was performed and the broth was extracted and analysed by HPLC. The results indicated that engineering strain carrying 1 additional copy of fkbN gene reduced 32% production of FK506, however strain Streptomyces sp. Tdfn produced no FK506; engineering strain carrying 1 additional copy of tcs7 gene reduced 54% production of FK506, however strain Streptomyces sp. Tdt7 improved 6% production of FK506, the result is not significant; engineering strain carrying 1 additional copy of zwf2 gene improved 13% production of FK506; engineering strain carrying 1 additional copy of bccA2 gene reduced 9% production of FK506; Streptomyces sp. Tdgd improved 135% production of FK506; engineering strain carrying 1 additional copy of fkbM gene improved 13% production of FK506; As for vgb gene, engineering strain with the extraneous source gene named Vitreoscilla hemoglobin brought more biomass than the original strain.In the fermentation of producing FK506, fkbN may acted as negative regulatory but necessary gene while tcs7 acted as negative regulatory gene. Among the 3 primary metabolism related genes, zwf2 and bccA2 showed very light effect on the production of FK506, but knockout gltD gene improved the production of FK506 significantly. As for the biosynthesis genes fkbM, no distinct improvement was observed by introducing 1 additional copy of them. As for vgb gene, engineering strain with the extraneous source gene brought more biomass than the original strain, which means Vitreoscilla hemoglobin could carry more oxygen and supply for the mycelium growing during fermentation.This study revealed the effect of fkbN, tcs7, zwf2, bccA2, gltD, fkbM and vgb genes on the fermentation of FK506 producing strain tentatively, which lay the foundation for further breeding high producing strain by genetic manipulation.