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

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

Combinatorial Biosynthesis 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

Research Approach genome analysis

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

Oxytetracycline Biosynthetic Pathway 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 oxyA oxyC oxyE oxyG oxyI oxyK oxyM oxyO oxyQ oxyS 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

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

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

Engineered Biosynthesis 2

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

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

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

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

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

Pacidamycin Biosynthesis Streptomyces coeruleorubidus ? pacidamycins

Pacidamycin Scaffold Assembly Summary Streptomyces coeruleorubidus NRRL 18370 2 kb A B C D E F G H 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

NRPS Assembly Line

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

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

Acknowledgements Zhe Rui Funding: Moriah M. Sandy 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