College of Chemistry Advisory Board Meeting Presentation, 10/24/2011

1. Biomolecular Engineering------Exploitation of Enzymes toward Synthesis of Pharmaceuticals and Biofuels Wenjun Zhang Department of Chemical and Biomolecular Engineering University of California, Berkeley College of Chemistry Advisory Board Meeting Presentation, 10/24/2011

2. Natural Products Pharmaceuticals physostigmine erythromycin lovastatin doxorubicin vancomycin penicillin artemisinin paclitaxel Biofuels

3. Research Themes Genome Mining Discovery Predict New Candidate Natural Products Combinatorial Biosynthesis Biotransformation Pharmaceuticals and Biofuels Enzyme and Pathway Characterization Enzyme and Pathway Engineering Prerequisite

4. Research Approach genome analysis

5. Example 1: Engineered Biosynthesis of Bacterial Aromatic Polyketides ? Streptomyces rimosus

6. Oxytetracycline Biosynthetic Pathway 0 2 4 6 8 10 12 14 16 18 20 22 24 kB otrB oxyB oxyD oxyF oxyH oxyJ oxyL oxyN oxyP oxyR oxyT otrA oxyE oxyG oxyK oxyM oxyO oxyS oxyA oxyC oxyI oxyQ Resistance Minimal PKS Initiation Module KR Cyclase Tailoring Unknown Zhang et al, AEM 2006; Zhang et al, JNP 2006; Zhang et al, JBC 2007; Zhang et al, JACS 2008

7. Combinatorial Biosynthesis: modification of natural product biosynthetic pathways toward the production of “unnatural” natural products. 1 Combined Pathway 2 3

8. Engineered Biosynthesis 1 (yield: ~10 mg/L) Zhang et al, JACS 2008

9. Engineered Biosynthesis 2

10. Engineered Biosynthesis 2 pannorin A C9-C14 and C7-C16 cyclase from the tetracenomycin pathway pannorin analog (yield: ~50 mg/L) Zhang et al, JACS 2007

11. E. coli as a Host for Bacterial Aromatic Polyketide Production Bacterial Aromatic Polyketides Actinomycetes E. coli Macrolides Terpenoids Alkaloids Cyclic Peptides

12. (SAT) (SAT) KS KS MAT MAT PT PT ACP ACP TE/CYC TE/CYC Bacterial and Fungal Aromatic Polyketides Biosynthesis Fungal type I PKS Bacterial type II PKS Malonyl-CoA Malonyl-CoA Backbone Assembly Tailoring No crosstalk Bacterial Aromatic Polyketides Fungal Aromatic Polyketides

13. KS MAT ACP Biosynthesis of Bacterial Aromatic Polyketides in Escherichia coli Bacterial type II PKS Fungal type I PKS Malonyl-CoA Malonyl-CoA Bacterial Aromatic Polyketides Fungal Aromatic Polyketides Zhang et al, PNAS 2008

14. Example 2: Biosynthesis of Uridyl Peptide Antibiotics inhibitors liposidomycins caprazamycins substrate pacidamycins mureidomycins napsamycins sansanmycins capuramycin muraymycins tunicamycins streptoviridins corynetoxins UDP-MurNAc -pentapeptide

15. Pacidamycin Biosynthesis Streptomycescoeruleorubidus ? pacidamycins

16. Pacidamycin Scaffold Assembly Summary Streptomyces coeruleorubidus NRRL 18370 2 kb H A B C D E F G I J K L M N O P Q R S T U V W X Zhang et al, PNAS 2010; Zhang et al, Biochemistry 2010; Zhang et al, JACS 2011; Zhang et al, Biochemistry 2011; Zhang et al, PNAS 2011

17. NRPS Assembly Line

18. In vitro Total Synthesis of >50 Pacidamycin Analogs AA1 = alanyl or H AA2 = m-Tyr or Ala AA5 = Phe or Trp or m-Tyr R = CH3 or H

19. Ongoing Projects sparsomycin physostigmine undecene 4-methylquinazoline antimycin paclitaxel uridyl peptide antibiotics asukamycin

20. Acknowledgements ZheRui Moriah M. Sandy Funding: Berkeley Startup, EBI Berkeley Zhang Lab Christopher T. Walsh Heidi Imker Steven Malcolmson Brian Ames Yi Tang Jixun Zhan Suzanne M. Ma Yanran Li HMS Walsh Lab UCLA Tang Lab