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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
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Natural Products Pharmaceuticals erythromycin doxorubicin lovastatin
physostigmine vancomycin penicillin artemisinin paclitaxel Biofuels
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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
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Research Approach genome analysis
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Example 1: Engineered Biosynthesis of Bacterial Aromatic Polyketides
? Streptomyces rimosus
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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
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Combinatorial Biosynthesis: modification of natural product biosynthetic pathways toward the production of “unnatural” natural products. 1 Combined Pathway 2 3
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Engineered Biosynthesis 1
(yield: ~10 mg/L) Zhang et al, JACS 2008
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Engineered Biosynthesis 2
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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
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E. coli as a Host for Bacterial Aromatic Polyketide Production
Bacterial Aromatic Polyketides Actinomycetes E. coli Macrolides Cyclic Peptides Terpenoids Alkaloids
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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
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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
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Example 2: Biosynthesis of Uridyl Peptide Antibiotics
inhibitors liposidomycins caprazamycins substrate pacidamycins mureidomycins napsamycins sansanmycins capuramycin muraymycins UDP-MurNAc -pentapeptide tunicamycins streptoviridins corynetoxins
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Pacidamycin Biosynthesis
Streptomyces coeruleorubidus ? pacidamycins
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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
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NRPS Assembly Line
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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
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Ongoing Projects sparsomycin physostigmine undecene
4-methylquinazoline antimycin uridyl peptide antibiotics paclitaxel asukamycin
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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
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