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Therapeutics from Microbes: Pathways and Specific Examples

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Presentation on theme: "Therapeutics from Microbes: Pathways and Specific Examples"— Presentation transcript:

1 Therapeutics from Microbes: Pathways and Specific Examples
Blaine Pfeifer Department of Chemical & Biological Engineering Tufts University Medford, MA 02155, U.S.A. FMM Industry Day February 23, 2011

2 Motivation I – Range of therapeutics and activities
Penicillium chrysogenum Artemisia annua Salinospora tropica Penicillin Artemisinin Lomaiviticin Antibiotic Antimalarial Anticancer 23Feb11

3 Motivation II – Market value and emerging drug resistance
NCE’s between 1981 and 2006 34% were natural products or semi-synthetic derivatives Global antibacterial market generated $42 billion in sales in 2009 ~4% annual growth over last five years MRSA, VRSA, other antibiotic resistant microbes in community and hospital environments Now, MRSA kills more U.S. citizens each year than AIDS Infectious Diseases Society of America “10 by ‘20” initiative Pollack A. New York Times Nov 10. Clin Infect Dis Apr 15;50(8): 23Feb11 Hasmad B. Nat Rev Drug Discov Sep;9(9):675-6. Newman DJ, Cragg GM. J Nat Prod Mar;70(3):

4 Challenges I – Intractable natural sources
Penicillium chrysogenum Artemisia annua Salinospora tropica Artemisinin Antimalarial Lomaiviticin Anticancer Penicillin Antibiotic 23Feb11

5 Challenges II – Molecular complexity
Penicillium chrysogenum Artemisia annua Salinospora tropica Antimalarial Anticancer Antibiotic Penicillin Artemisinin Lomaiviticin 23Feb11

6 Approach I – Heterologous Biosynthesis
Identification & Isolation Heterologous Expression GeneA GeneA GeneB GeneC GeneB Escherichia coli GeneC Scientific Motivation: Complex, slow-growing organisms Complex chemistry Vast knowledge Discovery Metagenomics Genome sequencing and bioinformatics High-throughput and activity screening Reconstitution Microbiology Molecular biology Biochemistry Development Metabolic engineering Bioprocess engineering Protein engineering Eventual Outcome: Therapeutic access Less expensive drugs Widespread use Challenges: Uncompetitive production titers 23Feb11

7 Approach II – Metabolic and process optimization
23Feb11

8 Examples – Erythromycin and Taxol
Saccharopolyspora erythraea Polyketide Propionyl-CoA 6 × (2S)-Methymalonyl-CoA 6-Deoxyerythronolide B Erythromycin Carbon Source(s) 3 × Isoprenoid IPP Taxus brevifolia DMAPP Taxadiene Taxol 23Feb11

9 Erythromycin – Complete heterologous biosynthesis
23Feb11 Zhang H, et al. Chem Biol Nov;17(11): Pfeifer BA, et al. Science Mar;291(5509):

10 Taxol – Isoprenoid pathway optimization
IPP GGPPS TXS 5α Pyruvate Glyceraldehyde 3-phosphate 1-deoxy-D-xylulose-5-phosphate (DXP) 2C-methyl-D-erythritol-4-phosphate (MEP) CDP-ME MEC HMBPP CDP-MEP DMAPP DXS DXR IspD IspE IspF IspG IspH IDI Plasmid, promoter, strain combinations 23Feb11 Ajikumar PK, et al. Science Oct 1;330(6000):70-4.

11 Opportunities I – Access Nature’s potential
25 predicted polyketide, nonribosomal peptide, and terpene gene clusters No products derived from these clusters (besides erythromycin) were indentified on 50 different types of solid and liquid media 23Feb11 Boakes S, et al. J Mol Microbiol Biotechnol. 2004;8(2):73-80. Oliynyk M, et al. Nat Biotechnol Apr;25(4):

12 Opportunities II – Manipulate biosynthesis
DEBS1 DEBS2 DEBS3 Tailoring Enzymes Propionate Sfp PrpE PCC DEBS1 Propionate DEBS2 DEBS3 Tailoring Enzymes Sfp PrpE PCC Benzoate 23Feb11 Zhang H, et al In preparation.

13 Opportunities III – Production speed and economy
Process Time: ~100 years ~Weeks ~Weeks ~Weeks ~Days ~Days 100 year old tree → 3 kg bark → 300 mg Taxol → 1 dose! T. brevifolia Extraction T. canadensis Cell-Culture A. thaliana Heterologous Tomato Heterologous S. cerevisiae Heterologous E. coli Heterologous (Horwitz 1994 Nature) (Ketchum, et al B&B) (Besumbes, et al B&B) (Kovacs, et al Transgenic Res) (Engels, et al Metab Eng) (Ajikumar, et al Science) 23Feb11

14 Opportunities IV – High(er) throughput process optimization
-Media optimization through 96-well format LB Medium: -Yeast Extract -NaCl Tryptone Defined Medium: Potassium Phosphates Ammonium Sulfate Glycerol Magnesium Sulfate Vitamins Trace Metals HPLC-ELSD Analysis 96-well format Plackett-Burman Screening: Tryptone Trace Metals 6dEB (mg/L) LB Medium: -Yeast Extract -NaCl -Tryptone Enhanced Medium: Yeast Extract Tryptone Glycerol NaCl Vitamins Trace Metals Defined Medium: Potassium Phosphates Ammonium Sulfate Glycerol Magnesium Sulfate Vitamins -Trace Metals 23Feb11 Pistorino M, Pfeifer BA. Biotechnol Prog Sep-Oct;25(5):

15 Acknowledgements Students: Haoran Zhang Brett Boghigian Jiequn Wu
Karin Skalina Yong Wang Funding/Collaborators: NIH (AI074224; GM085323) NSF ( ; ) Milheim Foundation Greg Stephanopoulos (MIT) 23Feb11

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