Figure 2. Diagram of disorazol A gene cluster engineering.

Firstly, the backbone of plasmid pBeloBAC11-dis (i) was replaced by p15A ori-tps casstte to form p15A-dis (ii) which containing an original MycoMar transposon by Red/ET recombineering. In this way dis gene cluster was driven by P_tet promoter. Then, the interrupted esterase gene orf3’ in pTn-Rec_IE2 plasmid (iii) from transposon mutant So12_EXI_IE-3³⁶ was recovered, repaired and engineered to form the vector p15A-amp-cm-orf2’-orf3’-spect (iv) that contained the whole length of the esterase gene orf3’. Next, linear DNA fragment released by Hind III was integrated into disorazol vector p15A-dis (ii) to get the final construct p15A-dis-est (v) via Red/ET recombinantion. Finally, two types of modified vectors p15A-dis (ii) and p15A-dis-est (v) were electroporated into M. xanthus respectively and kanamycin-resistant colonies were selected for further analysis. Hind III restriction sites used for releasing linear fraction “cm^R-orf2’-orf3’-spect^R” were indicated in ↑. The insertion site of the linear fragment DNA containing orf2’ and orf3’ gene was marked with ↓.