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METABOLIC ENGINEERING: Microorganisms as tools in chemistry

METABOLIC ENGINEERING: Microorganisms as tools in chemistry. Taz Cheema January 27, 2011. http://scienceblogs.com/oscillator/2010/09/bacterial_lightbulb.php. WHAT IS METABOLIC ENGINEERING?.

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METABOLIC ENGINEERING: Microorganisms as tools in chemistry

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  1. METABOLIC ENGINEERING:Microorganisms as tools in chemistry TazCheema January 27, 2011 http://scienceblogs.com/oscillator/2010/09/bacterial_lightbulb.php

  2. WHAT IS METABOLIC ENGINEERING? Metabolic engineering is the improvement of cellular activities by manipulation of enzymatic, transport, and regulatory functions of the cell with the use of recombinant DNA technology. Dr. James E. Bailey 1944-2001 Bailey, J., Toward a science of metabolic engineering, Science, 252, 1668, 1991

  3. WHAT IS METABOLIC ENGINEERING? Compound Enzyme http://www.google.com/images http://blogs.nature.com/nm/spoonful/2008/03/

  4. ENGINEERING BIOSYNTHETIC ENZYMES Gene cluster Sorangiumcellulosum Steptomycescoelicolor • Engineer entire biosynthetic cluster into an organism more suitable to laboratory manipulation • Done only if: 1) natural product expression low or 2) natural product difficult to culture and ferment • Engineering biosynthetic enzymes in an organism more desirable than to manipulate each producer organism individually Science, 2000, 287, 640-642 Org.Biomol.Chem.. 2003, 1, 1-4

  5. EPOTHILONE • New chemotherapeutic agent • Arrests cell cycle by binding to microtubules and inhibits depolymerisation • Active in Taxol resistant tumours and multidrug resistant cancer types • Currently analogues are in clinical trials

  6. METABOLIC PATHWAY A • Introduce new pathways using rDNA • Modify existing pathways • Enzymes expressed that define overall conversion process Enzyme 1 B Enzyme 2 Krebs cycle C Enzyme 3 D Enzyme 4 NEW Enzyme 5 E Enzyme 6 1.Stephanopoulos, G (1999) Metabolic Engineering 1:1-11 2.Genetic and metabolic engineering. EJB(1998), Vol.1 No.3

  7. SORBITOL PRODUCTION LDH • Lactobacillus plantarum (lactic acid bacterium) • Hols, P., Applied and Environmental microbiology 2007, 73, 6.. 1864–1872 • Stephanopoulos,G.; Chemical Engineering Science 2002, 57, 2596-2602

  8. SORBITOL PRODUCTION LDH • Sorbitol production in Lactobacillus plantarum (lactic acid bacterium) • Enzymes expressed that define overall conversion process along with totality of cellular functions • Hols, P., Applied and Environmental microbiology 2007, 73, 6.. 1864–1872 • Stephanopoulos,G.; Chemical Engineering Science 2002, 57, 2596-2602

  9. GENES AND THEIR ENZYMES Gene 1 Gene 2 Gene 3 Gene 4 Gene 5 Gene 6 DNA ENZYME 1 ENZYME 1 ENZYME 2 ENZYME 4 ENZYME 3 ENZYME 6 Y X ENZYME 5 Wild Type Stephanopoulos,G.; Chemical Engineering Science 2002, 57, 2596-2602

  10. GENES AND THEIR ENZYMES Gene 1 Gene 2 Gene 3 Gene 4 Gene 5 Gene 6 DNA ENZYME 1 ENZYME 1 ENZYME 2 ENZYME 4 ENZYME 3 ENZYME 6 Y X ENZYME 5 Chemical Engineering Science 2002, 57, 2596-2602

  11. GENES AND THEIR ENZYMES Gene 1 Gene 2 Gene 3 Gene 4 Gene 5 Gene 6 Gene 7 DNA ENZYME 1 ENZYME 1 ENZYME 2 ENZYME 4 ENZYME 7 ENZYME 3 ENZYME 6 Y X ENZYME 5 Stephanopoulos,G.; Chemical Engineering Science 2002, 57, 2596-2602

  12. BIO SYNTHESIS OF BUTANETRIOL D-xylose D-1,2,4-butanetriol L-arabinose L-1,2,4-butanetriol Frost, J. et al; J. Am. Chem. Soc. 2003, 125, 12998

  13. WHY BUTANETRIOL? H2SO4 , HNO3 D,L-1,2,4-butanetriol trinitrate D,L-1,2,4-butanetriol • Less shock sensitive • More thermally stable • Less volatile than nitro-glycerine • Limited availability of 1,2,4-butanetriol D,L-1,2,3-Trinitroxy propane http://www.allbestwallpapers.com/military_wallpapers.html Frost, J. et al; J. Am. Chem. Soc. 2003, 125, 12998

  14. CURRENT SYNTHESIS OF BUTANETRIOL H+, MeOH Malic acid Dimethylmalate Metal cat. (Ru) H2O 5000 psi H2 160 oC NaBH4 MeOH/ THF 8 % + NaB(OCH3)4 D,L-1,2,4-butanetriol 2.5 % 74 % 1 ton 6 tons 2 % 11 % 2.5 % Frost, J. et al; J. Am. Chem. Soc. 2003, 125, 12998

  15. BIO SYNTHESIS OF BUTANETRIOL Pseudomonas fragi ATCC 4973 a D-Xylose (100g/L) D-Xylonic acid (77g/L) a. D-xylonatedehydrogenase (P. fragi) 70% c Pseudomonas putida 3,4-dihydroxybutanal 3-deoxy-D-glycero-pentulosonic acid c. benzoylformatedecarboxylase(P. putida) Frost, J. et al; J. Am. Chem. Soc. 2003, 125, 12998

  16. BIO SYNTHESIS OF BUTANETRIOL Pseudomonas fragi Pseudomonas putida D-xylose D-1,2,4-butanetriol L-arabinose E. coli DH5α/pWN6.186A L-1,2,4-butanetriol Frost, J. et al; J. Am. Chem. Soc. 2003, 125, 12998

  17. BIO SYNTHESIS OF BUTANETRIOL E. coli DH5α/pWN6.186A a D-Xylonic acid D-Xylose 100 g/L b c d D-1,2,4-butanetriol 3,4-dihydroxybutanal D-3-deoxy-glycero-pentulosonic acid 12 g/L ( 25%) a. D-xylonatedehydrogenase (P. fragi); b. D-xylonatedehydratase (E. coli); c. benzoylformatedecarboxylase(P. putida); d. alcohol dehydrogenase (E. coli) Frost, J. et al; J. Am. Chem. Soc. 2003, 125, 12998

  18. RECOMBINANT DNA Plasmid DNA DNA to be inserted Sticky Ends Sticky Ends DNA ligase • Isolate plasmid DNA • Select DNA • Cut with restriction enzymes • Piece together fragments Recombinant DNA 1. Pray, L.(2008) Recomninant DNA technology and transgenic animals. Nature Education 1 (1) 2. http://www.istockphoto.com/stock-photo-1626198-bacteria.php

  19. RECOMBINANT DNA Metabolite from fermentation Copies of protein isolated • Transform DNA into cell of interest • Cells grow and divide producing copies • Isolate protein/metabolite of interest • Isolate copies of rDNA Copies of gene isolated and transferred to other organism 1. Pray, L.(2008) Recomninant DNA technology and transgenic animals. Nature Education 1 (1) 2. http://www.istockphoto.com/stock-photo-1626198-bacteria.php

  20. CELLULAR METABOLISM IS COMPLEX Cofactors, Vitamins and other substances Complex Carbohydrates • Metabolic network describes which products are made from which substrates • Highly interconnected pathways • Alterations in one path have direct/indirect affects on other pathways • Pathways analysed through Metabolic Flux Analysis (MFA) Nucleotide Complex Lipids Carbohydrates Other amino acids Lipids Amino acids Energy • Lee et al.; Tissue Engineering 1999, 5, 347 • http://microbialgenomics.energy.gov/MicrobialCellProject/thrusts2.shtml

  21. METABOLIC FLUX ANALYSIS (MFA) Flow (Flux)) Substrate Product http://www.google.com/images

  22. METABOLIC FLUX ANALYSIS (MFA) Substrate Product Flow Mr. Wong http://www.google.com/images

  23. METABOLIC FLUX ANALYSIS (MFA) exchange flux Internal flux Internal metabolite concentration Substrates /products • Genomic data not sufficient to describe flux distribution • Exchange flux easy to determine (uptake vs secretion) • Internal fluxes hardest to determine • Lee et al.; Tissue Engineering 1999, 5, 347 • Stephanopoulos,G.; Chemical Engineering Science 2002, 57, 2596-2602 • Tanis, J.; Metabolic engineering flux balance analysis, 2006

  24. METABOLIC CELL FACTORY D,L-1,2,4-butanetriol (12g/L) Frost, J. et al; J. Am. Chem. Soc. 2003, 125, 12998 Indigo J.Indu. Microbiology & Biotchnology 2002,28,127 Riboflavin (B2) Appl.Micro.Bio.2003,53,509 Artemisinin ACS.Chem.Bio, 2007,3, 64 http://pi.lilly.com/us/humulin-n-ppi.pdf Polyhydroxyalkanoates (PHAs) Microbiology and molecular bio. 1999, 61, 21 Codeine Curr.Opion.Plant Bio. 2005, 8, 280 Morphine Nature Chem. Bio. 2010, 6,251

  25. METABOLIC CELL FACTORY Algae Biofuel J.R.Soc.Interface, 2010, 7, 703 Atrazine Gallivan,P.; Nature Chem.Bio, 2010, 6, 464 Doxorubicin J.Bacteriol. 1999 Lomovskaya et.al. 181, 1, 305 Bioluminescent E.coli http://2010.igem.org/Team:Cambridge Bioremediation of Oil spills Technology in Society 2010, 32, 331 Taxol MacMillan,D.;Synlett 2007, 10, 1477

  26. METABOLIC CELL FACTORY Oleic acid (Canola/Soybean oil) Plant Physiology, 2001, 125, 160 Vitamin E Plant Cell, 2003, 15, 3007 Priming crops with transgenic plants Curr. Opin. Biotech, 2008,19, 181 Insect resistant Plants Plant Physiology, 2008, 146, 881 Vitamin A Science, 2000, 287, 303 Dhurrin (cyanogenicglucoside) PNAS, 2005, 102, 1779

  27. METABOLIC CELL FACTORY Taxol MacMillan,D.;Synlett 2007, 10, 1477 Priming crops with transgenic plants Curr. Opin. Biotech, 2008,19, 181 Vitamin A Science, 2000, 287, 303

  28. TAXOL • Potent anticancer agent • Isolated in 1966 from bark of Pacific yew tree • Binds to microtubules and inhibits depolymerisation into tubulin • 10 kg of dried bark = 1 g of Taxol • Major supply crisis • Challenging chemical synthesis • 10-deacetylbaccatin III (10-DAB) (Leaves of European yew) 1g / 1kg Pacific yew tree • MacMillan,D.;Synlett 2007, 10, 1477 • Nature 1979, 277, 665 • http://itech.dickinson.edu/chemistry/?tag=michael-dalton

  29. TAXOL VIA SYNTHETIC CHEMISTRY 51 chemical steps 37 chemical steps Nicolaou (1994) Wender (1997) Isopyromucic acid Verbenone 47chemical steps 41 chemical steps Holton (1994) Kuwajima (1998) Propionaldehyde Patchoulene oxide 47 chemical steps 38 chemical steps Danishefsky (1995) Mukaiuama (1999) Serine Wieland Miescherketone • 6 independent total synthesis of Taxol achieved • Wender’s synthesis shortest and most efficient; overall yield 0.4% MacMillan,D.; Synlett 2007, 10, 1477

  30. TAXOL VIA SYNTHETIC CHEMISTRY KOtBu O3 85% Prenyl Bromide 79% h (450 W) 85% Verbenone 500 g 1.LDA/ Ethyl propiolate 2. TMSCl 89% 1.Me2CuLi 2. AcOH 97% 95% LiAlH4 KHMDS N-(phenylsulfonyl)phenyloxazirdine Wender,P.; J. Am. Chem. Soc. 1997, 119, 2755

  31. TAXOL VIA SYNTHETIC CHEMISTRY 1. O2, P(OEt)3 KOtBu 2. NaBH4 91% 1.TBSCl 2. PPTS 91% 1.m-CPBA 2. DABCO 3.TIPSCl 85% H2, Crabtree’s cat. 2. TMSCl 3. Triphosgene 98% PCC • 14 synthetic steps (AB Ring) • All C, O in correct pattern Wender,P.; J. Am. Chem. Soc. 1997, 119, 2755

  32. TAXOL VIA SYNTHETIC CHEMISTRY KHMDS 91% 1N HCl, NaI 94% 1. 2. , Et3N Eschenmoser’s salt 97% TESCl 92% 1. ZnCl2 89% 2. BOMCl 3. NH4F; PhLi 93% 4. Ac2O 79% 1. 80% 2. O3 86% Wender,P.; J. Am. Chem. Soc. 1997, 119, 2755

  33. TAXOL VIA SYNTHETIC CHEMISTRY 1.HCl, NaI 2. MsCl 3. LiBr 67% 1. 72% (11:1) 2. TrocCl 1.OsO4 2.Triphosgene 3. KCN 92% 1.TASF 2. PhLi 1.∆ 2. Ac2O 89% 10-deacetylbaccatin III (46%) + Tetrahedron 1992, 48, 6985 Baccatin III (33%) Taxol - 37 total steps(0.4%) Wender,P.; J. Am. Chem. Soc. 1997, 119, 2755

  34. TAXOL VIA SYNTHETIC CHEMISTRY 20 synthetic steps 14 synthetic steps Baccatin III Aldehyde (A-B ring) 3 steps • 500 g Verbenone starting material used • Overall yield of 0.4% (2 g) • “Stop and go” synthesis not practical for industrial scale Taxol - 37 total steps Wender,P.; J. Am. Chem. Soc. 1997, 119, 2755

  35. TAXOL VIA SYNTHETIC BIOLOGY Species: Taxus media • Plant cells perceive environmental changes, generate biological response • Accumulation of taxol is thought to be a biological response • 1996 Yukimune produced Baccatin III content with 100 µM Methyl Jasmonate addition to Taxus media cell culture • Methyl Jasmonate induces biological response in plants • Taxol = 110.3mg/L/2 weeks, Baccatin III = 25.2 mg/L/2 weeks • Issues: >100 µM M. Jasmonate decrease product production by 15 fold Yukimune,Y., Nat. Biotech. 1996, 14, 1129

  36. TAXOL VIA SYNTHETIC BIOLOGY Endophytic Fungi: Fusariummairei • Taxuschinesis and fungis in 20 L co-bioreactor. • Taxol production = 25.63 mg/L/15 days with fungis vs. 0.68 mg/L/15days no fungi • Issues: 1. No co-biorecactor for industrial use available. • 2. Biomass of fungi decreasses with inoculation time Cheng, L.;Appl. MicrobioBiotechnol (2009) 83, 233

  37. TAXOL VIA SYNTHETIC BIOLOGY • November 2010 Ajikumar & Stephanopoulos optimized overproduction of taxadiene • Taxadiene, first committed Taxol intermediate • Used a multivariate-modular approach • Used modified CYP450 • Produced 1g/L = 15, 000 fold increase in an engineered E. Coli strain CYP450 Taxadiene Taxadien-5α-ol Escherichia coli Ajikumar,P. et al. Science 330, 70 (2010)

  38. TAXOL VIA SYNTHETIC BIOLOGY Taxadiene 5αhydroxylase 1 CoA-acylation 2. Sidechain CYP450 Taxadien-5α-ol Baccatin III Taxol • Removed bottlenecks • Created two operons 1: (dxs-idi-ispDF), 2. (G-T) • 300 mg/litertaxadiene • Engineered CYP450 of E.coli Ajikumar,P. et al. Science 330, 70 (2010)

  39. TAXOL: SYNTHETIC CHEM OR BIO? Wender,P.; J. Am. Chem. Soc. 1997, 119, 2755 Ajikumar,P. et al. Science 330, 70 (2010) • 14 synthetic steps to AB ring • 20 synthetic steps from AB ring to Baccatin III • 37 total synthetic steps from Verbenone to Taxol • Overall yield of 0.4% • Time to synthesize: months? Years? • Multivariate-modular pathway engineering • Able to produce 300 mg/L of Taxadiene in 48 hours (30 Kg/ 100 K L) • Taxadiene to Taxadiene-5α-ol via CYP450 • Further enzymatic reactions required

  40. PLANT METABOLIC ENGINEERING Herbivore Extreme heat http://www.physorg.com/news157900895.html Pathogen Flooding www.google.com/images

  41. PLANT METABOLIC ENGINEERING • Plant secondary metabolites not essential for basic growth and development • >200, 000 secondary metabolites produced • For plants: pollination & seed dispersion, interactions with other plants, recognition of herbivores, and defence by attracting predators of herbivores • For humans: interesting applications in pharmacology, chemical industry, crops. • Structural complexity makes it very difficult to synthesize chemically in adequate yields Quinine Taxol Vincristine A.G.Fett-Neto (ed.), Plant Secondary Metabolism Engineering, Methods in Mol. Bio. 643

  42. PLANT PATHWAY BOTTLENECKS Can manipulate the Developmental control Organ/tissue specific Abiotic/biotic stresses Circadian rhythms A.G.Fett-Neto (ed.), Plant Secondary Metabolism Engineering, Methods in Mol. Bio. 643

  43. PLANT PATHWAY BOTTLENECKS • Some pathways entirely or partially introduced and expressed in bacteria/ yeast • Modifications done via recombinant DNA • Transfer of pathways between plant species much more difficult but can be done Dhurrin in Arabidopsis plant PNAS, 2005, 102, 1779 1.A.G.Fett-Neto (ed.), Plant Secondary Metabolism Engineering, Methods in Mol. Bio. 643 2.Image: http://en.wikipedia.org/wiki/Arabidopsis_thaliana

  44. PRO-VITAMIN A • Most successful metabolic-engineering done in plants: • Rice milled to remove outer layer which removes several nutrients such as provitamin A • Vitamin A deficiency is a serious public health problem • Symptoms ranging from night blindness to total blindness, diarrhea, respiratory diseases, and measles • Quarter of a million children go blind each year due to provitamin A deficiency Vitamin A • Ye, X; Science, 2000, 287, 303-305 • Science, 2000, 287, 303

  45. PRO-VITAMIN A • Express pathway, in rice endosperm, to vitamin A precursor β-carotene • Entire β-carotene biosynthetic pathway introduced into rice endosperm in single transformation with three vectors www.menurice.com/All_About_Rice 1.Ye, X; Science, 2000, 287, 303-305 2 .A.G.Fett-Neto (ed.), Plant Secondary Metabolism Engineering, Methods in Mol. Bio. 643

  46. PRO-VITAMIN A Immature rice endosperm Geranylgeranyldiphosphate phytoenedesaturase uncolored carotene phytoene z-carotene desaturase bacterial carotene destaurase lycopeneβ-cyclase Ye, X; Science, 2000, 287, 303-305

  47. PRO-VITAMIN A Ye, X; Science, 2000, 287, 303-305 1.6 μg/g Pain, J.; Nature biotech., 2005, 23, 482-487 37 μg/g

  48. PLANT VOLATILES 1. Curr. Opin. Biotech, 2008,19, 181 2. http://healthsystemcio.com

  49. PLANT VOLATILES • In response to herbivore attack plants emit divers volatile blends • > 200 different compounds • Directly intoxicate, repel or deter herbivorous insects, or attract natural predators of herbivors • Potential in agriculture, preserve forest ecosystems, and crop protection • Priming crops by planting few transgenic plants that constantly emit defence volatiles 1. Curr. Opin. Biotech, 2008,19, 181 2. http://healthsystemcio.com

  50. Attracting Bodyguard to Arabidopsis • M.E of Arabidopsis thaliana plants to emit two new isoprenoids • These attracted carnivorous predatory mites that aid the plants’ defence mechanism against herbivorous anthropods (3S)-(E)-nerolidol (E)- DMNT Spider mites http://www.hydro-gardens.com/spidermite.htm Predatory Mites http://www.entomology.wisc.edu/predatory-mites Iris F. Kappers, et al. Science 309, 2070 (2005)

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