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Introduction: Polyketide and Nonribosomal Peptide Natural Products

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

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Introduction: Polyketide and Nonribosomal Peptide Natural Products

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

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

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

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

  5. S S O O H H O O S S N N S S OH O O H H O O S S N N O O H H S S N N S S S S N N S S N N S S O O N N S S O O H H O O S S N N H H O O S H - O H N C O O H O H O 2 YbtE HMWP1 BAPint - C O O + H N 3 HMWP2 YbtU C H 2 C H 2 + S e A d e n o s i n C H 3 Salicylate Extending Production Capabilities -Using the yersiniabactin synthetase (YBTS) system HMWP2 (230 kDa) HMWP1 (350 kDa) NRPS NRPS PKS YbtE (60 kDa) NRPS YbtU (41 kDa) Salicylate Cysteine Yersiniabactin, Ybt S-adenosylmethionine Malonyl-CoA

  6. YbtE YbtU HMWP1 22C HMWP2 Extending Production Capabilities -Gene Expression -LC-MS Analysis Fe3+

  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

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