Study on Tacrolimus Production using Streptomyces sp.

Abstract

An immunosuppressant tacrolimus-producing strain of Streptomyces sp. TST8 which was isolated and developed by TS corporation in Korea was developed using sequential adaptation of media containing tacrolimus ranging from 600 mg/l to 1600 mg/l. The tacrolimus sequential adaptation was supposed to have the effect of selecting the cells with tacrolimus resistance and reduce product inhibition of the tacrolimus-producing strain. The developed strains produced more tacrolimus than the original strain. In particular, the TST10 adapted in the medium containing 1000 mg/l of tacrolimus produced 972 mg/l of tacrolimus in the final titer after 7 days of cultivation in a 5-l jar fermentor, which is higher than any other reported strain. Because the sequential adaptation was limited by the solubility of tacrolimus in water, the final tacrolimus titer of TST11 adapted in the medium containing 1600 mg/l of tacrolimus was lower than that of TST10. The strain TST8 was permeabilized by ethanol addition during production culture to secrete produced tacrolimus, lower product inhibition, and increase tacrolimus productivity. Tacrolimus production with ethanol addition was about 40% more than that without ethanol addition. Tacrolimus in supernatant and sediment of the cultured broth was quantified respectively to verify tacrolimus secretion and some tacrolimus was found in the supernatant of the broth with ethanol addition. The produced tacrolimus was purified in a process comprising extraction, pre-adsorption using HP20 resin, adsorption using CG161M resin, and crystallization. Purification using the adsorption resin CG161M was optimized by correlating tacrolimus yield with analogue load. One-step adsorption or two-step adsorption using CG161M could be applied selectively to the purification process, according to the analogue load of the input solution. A correlation between the analogue load and the first adsorption yield in the two-step adsorption was determined. Yields according to the analogue loads in the one-step adsorption and the second adsorption of the two-step adsorption were also found. As a result, the purification yields can be predicted by input conditions (analogue load). The purification strategy can be modified to achieve specific goals of purity, yield, and cost.