Yiu

1. Metabolic Engineering and Systems Biotechnology Laboratory Ka - Yiu San

2. Metabolic engineering recombinant DNA technology/ genetic engineering

3. Recombined plasmid Restriction cleavage mRNA Gene of interest Translation Restriction sites Ligation Transcription Protein Restriction cleavage Transformation Cloning vector Host cell Recombinant proteins by microorganisms

4. Recombinantproteins by microorganisms Some early products Year Products Disease Company 1982 Humulin Type 1 diabetes Genentech, Inc. (synthetic insulin) 1985 Protropin Growth hormone Genentech, Inc. Deficiency

5. Metabolic engineering is referred to as the directed improvement of cellular properties through the modification of specific biochemical reactions or the introduction of new ones, with the use of recombinant DNA technology What is Metabolic Engineering?

6. Three major projects • Pathway and co-factor engineering • Quantitative systems biotechnology • (genomics, transcriptomics, proteomics and metabolomics) • Plant metabolic engineering

7. Metabolic evolution Molecular Biology Techniques PCR, RE digest, ligation, transformation, knockout, etc. Insert/Knockout Genes/pathways Potential strains Metabolic Engineering/systems biotechnology Cycle Gene Targets Identified Shake Flask/Bioreactor Experiments Modeling Analysis metabolomics proteomics transcriptomics

8. Pathway design and manipulation

9. Succinic acid

10. Top Value Added Chemicals from Biomass (2004 DOE report )

11. Top Value Added Chemicals from Biomass (2004 DOE report )

12. Glucose NADH limitation Max theoretical Yield 1 mole/mole PEP Pyruvate Biomass Glucose-6-P ATP ADP Succinate Fructose 1,6-diP NAD+ NADH Glycerate-1,3-diP ADP CO2 ATP PEP OAA Pepc ADP ATP 2NADH Ldh Pyruvate Lactate 2NAD+ NADH NAD+ Formate CoA CO2 Pta Acetyl-P H2 Acetyl- CoA ADP Ack 2NADH ATP 2NAD+ Ethanol Acetate Simplified central anaerobic pathway in E. coli

13. succinate E. coli Wild Type Problems: low yield; mixed acid fermentation

14. Glucose Glucose PEP PEP Pyruvate Pyruvate Glucose-6P Biomass Glucose-6-P ATP ATP ADP ADP Fructose 1,6-diP Fructose 1,6-diP NAD+ NAD+ NADH NADH Glycerate-1,3-diP ADP Glycerate 1,3-diP CO2 ATP ADP PEP OAA Pepc ATP ADP CO2 PEP ATP ADP Ldh CO2 Pyk Pyruvate Lactate ATP NADH NAD+ 2NADH OAA Pyc Pyruvate CO2 NADH 2NAD+ Formate CO2 CoA Citrate H2 NAD+ Malate Pfl H2 aceB Formate Acetyl-CoA aceA ADP Pta Ack Isocitrate Acetyl-P Acetyl- CoA Succinate glyoxylate Fumarate Acetyl-CoA ATP NADH 2NADH aceA Acetate NAD+ 2NAD+ Glyoxylate pathway (no NADH requirements) Fermentative pathway Ethanol Succinate NADH limitation Max theoretical Yield 1 mole/mole Experimental yield= 1.6 mol/mol Simplified central anaerobic pathway in E. coli Resulting pathway design

15. Glucose PEP Pyruvate Glucose-6P ATP ADP Fructose 1,6-diP Dihydroxyacetone-P Glyceraldehyde-3 P Pi + NAD+ NADH Glycerate 1,3-diP ADP ATP CO2 PEP ADP CO2 Pyk ATP OAA PYC Pyruvate CO 2 NADH Citrate NAD+ Malate aceB PFL Formate CoA - Acetyl H 2 aceA Isocitrate glyoxylate Fumarate Acetyl - CoA NADH aceA NAD+ Succinate Resulting pathway design Glyoxylate pathway (no NADH requirements) Fermentative pathway

16. E. coli Wild Type SBS550MG (pHL413) succinate Succinic acid produced from 20g/L (~110 moles) of glucose succinate

17. Glucose PEP ptsG Pyruvate G6P G3P CO2 PEP Pyruvate CO2 CO2 Acetyl-CoA OAA Citrate CO2 Acetyl-CoA Malate Isocitrate 2-ketoglutarate icd aceA Glyoxylate aceB CO2 Fumarate Succinyl-CoA Succinate Succinate Succinate Three routes engineered for succinate production Strain SBS552 Genes inactivated: sdhAB ackA-pta poxB iclR ptsG ldhA adhE (anaerobic & aerobic) (anaerobic) (aerobic)

18. Cofactor manipulations

19. Cofactor manipulations • NAD+/NADH; NADP+/NADPH • Coenzyme A ( CoA )

20. Examples • esters • chiral compounds • terpenoids – lycopene • polyketides • Cancer (adriamycin) • Infection disease (tetracyclines, erythromycin) • Cardiovascular (mevacor, lovastatin) • Immunosuppression (rapamycin, tacrolimus)

21. Enzymes + Cofactors Substrate Products Importance of cofactor manipulation

22. 136.0% (2.4) 136.7% (2.4) Example: lycopene production Lycopene production is limited by NADPH availability WT mutant

23. Plant metabolic engineering

24. vinblastine vincristine Metabolic Engineering of the Terpenoid and Indole Pathways in Catharanthus roseus hairy roots Catharanthus roseus (Madagascar Periwinkle) • Produce a wide range of secondary metabolites • Ajmalicine and Serpentine – hypertension • Vinblastine and Vincristine – anticancer drugs used to treat lymphomas and leukemia

25. Terpenoid Indole Alkaloid Pathway Pyruvate + G3P DXS 1-Deoxy-D-Xylulose-5-Phosphate Chorismate Mevalonate AS 2-C-Methyl-D-erythitol-4-phosphate Anthranilate DMAPP IPP IndolePathway GPP Tryptophan Terpenoid Pathway Geraniol TDC G10H Tryptamine 10-Hydroxygeraniol Loganin STR Secologanin Strictosidine Catharanthine Ajmalicine Tabersonine Serpentine Vindoline Vinblastine Vincristine Lochnericine Hörhammericine

26. Clone Generation Adapt to Liquid Media (24 weeks) Plasmid Construction in E. coli Agrobacterium Transgene Ri Infection (6 weeks) Selection Media (6 weeks) Sterile Grown Plants (5 weeks)

27. Discovery of new compounds • Isolated and verified the structures of 11 new phenolic compounds • Structure very similar to existing agents that exhibit anti-fungal, anti-cancer properties • Currently testing new compounds for biological activities

28. Produce useful compounds that exhibit desirable biological functions

29. Collaborators: Dr. George N. Bennett Department of Biochemistry and Cell Biology Rice University Dr. Jacqueline V. Shanks Department of Chemical Engineering Iowa State University Dr. Sue Gibson Department of Plant Biology University of Minnesota Dr. Steve Cox Department of Computational and Applied Mathematics Rice University Dr. Ill-Min Chung KonKuk University, Korea

30. Questions ? ???