1. Introduction: Polyketide and Nonribosomal Peptide Natural Products Doxorubicin -Antitumor agent Cyclosporin -Immunosuppressant Yersiniabactin -Siderophore Erythromycin -Antibiotic Enniatin -Antibiotic Epothilone -Antitumor agent OH

2. Gene Expression and Active Enzyme Requirement DEBS1+TE, DEBS2, or DEBS3 -T7 promoter -lac operator BL21(DE3) E. coli strain SDS-PAGE In vitro reaction Analytical TLC DEBS1+TE DEBS2 DEBS3 Standard -Enzyme Activity (Temperature Dependent) sfp KDa 212 158 97.2 66.4 DEBS1+TE DEBS2 DEBS3 -Gene Expression

3. In vivo Polyketide Production BAP1 ATACPKSATKRACPKSATKRACPTE 14 C-Propionate Propionyl-CoA (2S)-Methylmalonyl-CoA DEBS1+TE PrpE PCC DEBS1+TE Triketide Lactone O OH O Standard Complete Negative Culture Control -DEBS1+TE-Triketide Lactone Example 37°C Induce/Feed 22°C

4. In vivo Polyketide Production BAP1 Propionate Propionyl-CoA (2S)-Methylmalonyl-CoA Full DEBS PrpE PCC -DEBS-6dEB Example DEBS2 DEBS3 PCC DEBS1 6dEB

5. Extending Production Capabilities -Using the yersiniabactin synthetase (YBTS) system OH O - O COO - H 3 N + CH 2 CH 2 + S Adenosin e CH 3 H 2 NCOOH SH Salicylate Cysteine S-adenosylmethionine S OH O S O H O S N S N S OH O S N S N OH Yersiniabactin, Ybt YbtE (60 kDa) HMWP2 (230 kDa) HMWP1 (350 kDa) YbtU (41 kDa) NRPS PKS Malonyl-CoA HMWP2 YbtU YbtE HMWP1 BAPint Salicylate S O H O S O H O S N S N S OH O S N S N OH S OH O S N S N N S S OH O S N S N N S OH

6. Extending Production Capabilities HMWP1 HMWP2 YbtE YbtU 22  C -Gene Expression -LC-MS Analysis Fe 3+

7. Summary -Complex polyketide biosynthesis +E. coli polyketide production -Gene transfer -Gene expression -Post-translational modification and protein folding -Substrate requirement +First complex natural product generated through E. coli -Process development/improvement +Fed-batch bioreactor process +Production improved ~200 fold -New production capabilities +Both polyketide and nonribosomal peptide products