Enhanced FK506Production In Streptomyces Tsukubaensis By Comparative Metabolic Profiling Analysis

FK506, a23-membered polyketide macrolide, is produced by industrial fermentationprocesses using various Streptomyces species. The immunosuppressive activity ofFK506is10-100folds potent of cyclosporin A. it has been the most important drugused to prevent rejection of transplanted livers and kidneys and in the treatment ofautoimmune diseases. It is believed that FK506is the super-star drug that will exerthuge influence onto the furture market of autoimmune diseases drugs. However, as asecond metabolite, the low titer and long production period resulted in difficulties inthe rectification and high production cost, which have become the bottlenecks for itsapplication and industrialization.To date, the improvement of FK506titer still relies on mutagenesis and genemanipulation. Traditional mutagenesis method is accompanied with the strongrandomness and blindness. Although some properties of the strain can be changedwithin a relatively short time, it lacks of rational thinking and the certain mutationdirection, which requires a lot of money and manpower; The key enzyme activity canbe rapidly modified with the help of gene manipulation method, however, as a kind ofsecondary metabolites, the overproduction mechanism of FK506is very complex. Soit is not easy to effectively improve the FK506titer by operation on only a certain partof the genes.Towards this end, comparative metabolomics approach was employed to analyzemetabolite concentration differences of Streptomyces tsukubaensis cultivated in twomedia with low and high productivities. Initially,98intracellular metabolites wereidentified and13metabolites involved in5pathways were determined to be directlycorrelated with FK506biosynthesis, including pyruvate, sedoheptulose7-phosphate,shikimate, threonine, valine, erythrose4-phosphate, isoleucine,lactate,malonyl-CoA, methylmalonyl-CoA,proline,and succinyl-CoA. Then in-depthanalysis elucidated that during the main production period (120-168h), theavailability of most precursors, serving as key building blocks of FK506, wassubstantially reduced. This was most pronounced for methylmalonyl-CoA andshikimate, probably responsible for the cease of FK506production in the latefermentation phase (168-192h). Based on such key information, rationally designedfeeding strategy was carried out. Results showed that the FK506yield increased from251mg/L to405mg/L, whereas, by-products FK520and37,38-dihydro-FK506 decreased by31%and39%, respectively, compared with the values of control. Thisnovel method developed by us provided a new way to impove the low-titer biomassproducts.