Page 50 - Demo

54 PROCEEDINGS OF INTERNATIONAL SCIENTIFIC CONFERENCE ON APPLIED BIOTECHNOLOGY4. DISCUSSIONIt is worth noting down that the herboxidiene polyketide has 19 carbon chain and 17 carbon intermediates is transferred to last module of herboxidiene PKS. The malonyl extension unit supplied by AT domain is condensed and added to 17 carbon intermediates to give 19 carbon chained herboxidiene polyketide. Before the chain release from TE domain by hydrolysis process, the added malonyl unit is tailored by KR and DH domain to give enoyl function to the added carbon chain between 2 and 3 position. Thus, it is speculated that the THP ring formation happens in herboxidiene biosynthesis pathway by spontaneous process with the ring closure reaction between C-7 hydroxyl and 3 (2-3 enoyl) functional groups [4]. As reducing functions of a modular polyketide synthase is not absolutely necessary for the activity of PKS protein but exist just for tailoring the added extension unit through different degree of reduction, disfunctioning of one of a reducing domain can be used as a tool to introduce variation to the polyketide chain. The dehydratase domain of erythromycin PKS is well studied and has been reported that the DH domain consist of the active site motif HXXXGXXXXP and DXXX(Q/H). The purpose of this experiment is to unravel if the activity of DH domain of herboxidiene PKS depend on residues histidine and aspartic acid. Special attention was given while designing the mutation construct for replacing the original version of module 8 DH to avoid the codon shift to get over the possible polar effect that might occur and to keep the codon usages to optimum level as that of native HerD protein. The dehydration step catalyzed by DH domain involves deprotonation of extender unit %u03b1-carbon by the histidine and removal of %u03b2-hydroxyl group by aspartic acid [15]. During herboxidiene biosynthesis, the malonyl unit added to the