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SECTION I: MOLECULAR GENETIC ENGINEERING AND BIOCHEMICAL TECHNOLOGY 47amino acids are identified [7,8]. Moreover, the loss of function mutation of erythromycin PKS DH domain was successfully done [13]. Based on the identified active site amino acids, from tylosin, erythromycin and pikromycin PKS, the HxxxGxxxxP motif and DxxxQ/H motif were identified in herboxidiene PKS by multiple sequence alignment (Fig. 3). Histidine residue and aspartic acid residue were identified in the active site of DH domain and replaced with the residue aspartic acid and asparagine respectively as described in previous studies.Figure 3. Identification of active site residue of M8DH domain of herboxidiene PKS by multiple sequence alignment with M5DH of tylosin PKS, M4DH of erythromycin PKS, M4DH of pikromycin PKS and M7 and M8 DH of herboxidiene PKS3.2. Confirmation of recombinantsThe recombinant plasmid was transformed into S. chromofuscus ATCC 49982 after that the single crossover mutants of S. chromofuscus %u0394M8DH was selected by PCR of apramycin resistant gene from genomic DNA which was present on the vector back bone (Fig. 4). The desired double crossover mutant was confirmed by loss of apramycin resistance gene in the vector backbone followed by 6 generations of colony transfer in 37%u00b0C.