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46 PROCEEDINGS OF INTERNATIONAL SCIENTIFIC CONFERENCE ON APPLIED BIOTECHNOLOGYselected and screened for double crossover mutant containing catalytically inactive M8DH domain.2.3. Fermentation and isolation of productsFor fermentation of herboxidiene and its derivatives from S. chromofuscus, seed culture was prepared using ISP2 medium as described earlier. After 36 h of seed culture 4% v/v of the seed culture was transferred to the fermenter containing herboxidiene production medium [12]. The fermentation was carried out till the end of 7 days and the culture broth was extracted with double volume of ethyl acetate without removal of mycelial pellets. The ethyl acetate fraction was recovered and dried under reduced pressure using rotary evaporator. Then, the crude extract was reconstituted in 1ml of methanol. Thus, obtained extracts were analyzed by TLC using chloroform: methanol: acetic acid in the ration of 90:9:1 (v/v/v). HPLC analysis was done using a reverse phase C18 column (4.6 %u00d7 250 mm, 50 %u03bcm; KANTO Reagents, Japan) with a solvent system consisting of H2O (0.05% trifluoroacetic acid) and 100% acetonitrile with a flow rate of 1 ml/min using binary gradient method. For purification of the extracts preparative HPLC, with a C18 column [YMC-Pack ODS-AQ (250 x 20 mm I.D., 10 %u00b5m)] connected to a UV detector (236 nm) under the binary condition of H2O and 100% acetonitrile at the flow rate of 10 mL/min. The exact masses of the derivatives were analyzed by HR-QTOF ESI/MS [ACQUITY (UPLC, Waters, Milford, MA, USA)-SYNAPT G2-S (Waters)] in the positive ion mode.3. RESULTS3.1. Identification 8 DH of active siteThe forth module of erythromycin and curacin (2 and 5 module) PKS has well studied DH domain and the active site