Fig. 3.

Construction sketch of Ivermectins polyketide synthetase IveA1 by domain swapping. AT, acyltransferase; ACP, acyl carrier protein; KS, beta-ketoacyl synthase; DH, dehydratase; ER, enoylreductase; KR, ketoreductase. A, the first round of PCR targeting. Meilingmycin PKS module 2 DH2-ER2-KR2 encoding DNA was amplified by PCR and fused with Spectinomycin resistant gene aadA. Then, the DH2-ER2-KR2-aadA DNA fragment was employed to substitute the Avermectin PKS module 2 DH2-KR2 by PCR targeting, and the aadA gene was subsequently released by DNA recombination. B, the second round of PCR targeting. The MEI KR2-ACP2 linker DNA fragment and AVE ACP2 encoding DNA fragment were amplified by PCR, and then fused with aadA gene. The linker-ACP2-aadA DNA fragment was employed to substitute the scar-ACP2 region by PCR targeting, and the aadA gene was subsequently released to generate a scarless IveA1 encoding gene. Avermectin PKS domains are shown in red, Meilingmycin PKS domains are shown in magenta.