Synthetic Biology:   From Bugs to Drugs and Fuels Jay D. Keasling Joint BioEnergy Institute Lawrence Berkeley National Lab...
Building a computer from off-the-shelf parts System Off-the-shelf components Idea Knowledge Base
Building a computer from off-the-shelf parts System Off-the-shelf components Idea Knowledge Base
Building a computer from off-the-shelf parts System Off-the-shelf components Idea Knowledge Base
Building a computer from off-the-shelf parts System Off-the-shelf components Idea Knowledge Base
Building a computer from off-the-shelf parts System Off-the-shelf components Idea Knowledge Base
Building a computer from off-the-shelf parts System Off-the-shelf components Knowledge Base Characterization Idea
Building a computer from off-the-shelf parts System Off-the-shelf components Knowledge Base Characterization Standard  Con...
Building a computer from off-the-shelf parts System Off-the-shelf components Knowledge Base Characterization Standard  Con...
Building a computer from off-the-shelf parts System Off-the-shelf components Knowledge Base Characterization Standard  Con...
Building a computer from off-the-shelf parts System Off-the-shelf components Knowledge Base Characterization Standard  Con...
Styrene
Synthesis of Styrene System Off-the-shelf components Idea Knowledge Base
Synthesis of Styrene System Off-the-shelf components Idea Knowledge Base
Synthesis of Styrene System Off-the-shelf components Idea Knowledge Base
Synthesis of Styrene System Off-the-shelf components Idea Knowledge Base Chemistry
Synthesis of Styrene System Off-the-shelf components Idea Knowledge Base Chemistry Kinetics & Reactor Design
Synthesis of Styrene System Off-the-shelf components Knowledge Base Chemistry Kinetics & Reactor Design Mass Transfer Proc...
Some common features <ul><li>Parts and devices </li></ul><ul><ul><li>Independent </li></ul></ul><ul><ul><li>Characterized ...
Microbial synthesis of artemisinin System Off-the-shelf components Knowledge Base Idea
Malaria <ul><li>Economists have proposed that malaria decreases the GDP of affected countries by as much as 50%. </li></ul...
<ul><li>Most widely-used drugs to treat malaria </li></ul>Quinine-based drugs <ul><li>Plasmodium  in many parts of the wor...
Artemisinin Artemisia annua
A brief history of artemisinin 168 B.C. Recipes For 52 Kinds Of Diseases  found in the Mawangdui Han Dynasty tomb  Hemorr...
Artemisinin is produced in oil sacs on  Artemisia annua  leaves Artemisinin... ...is produced by trichomes... ...found on ...
Current process Artemisinin Plant synthesis Purification Artesunate Artelinate Arteether Artemether Chemical Conversions
Artemisinin-based drugs <ul><li>Price </li></ul><ul><ul><li>$1.00-2.50/treatment </li></ul></ul><ul><ul><li>Fluctuations i...
Microbial synthesis of artemisinin Off-the-shelf components Knowledge Base Idea System Engineer a microorganism to produce...
Semi-synthesis of artemisinin ispH ispG idi isp A dxr dxs ispD DXP Pathway FPP Pyruvate + G3P Microbial synthesis ispF isp...
Constructing an artemisinic acid-producing microbe ispH ispG idi isp A dxr dxs ispD DXP Pathway FPP Pyruvate + G3P Microbi...
Microbial synthesis of artemisinin Knowledge Base Idea System Off-the-shelf components
A chassis for artemisinin biosynthesis Knowledge Base Idea System Off-the-shelf components
Parts needed for artemisinin synthesis Knowledge Base Idea System Off-the-shelf components
How do we get the components we need?
How do we get the components we need? Genome sequencing Genome annotation Components
BioShack? “ Please send me your biological device as you described in your paper.” Reply:  “The device you requested is in...
BioShack? “ It didn’t work as described in your paper.” Reply:  “In my hands, it worked as described in my paper … did you...
Creating an amorphadiene-producing microbe ispH ispG idi isp A dxr dxs ispD DXP Pathway FPP Pyruvate + G3P Microbial synth...
Creating an amorphadiene-producing microbe ispH ispG idi isp A dxr dxs ispD DXP Pathway FPP Pyruvate + G3P Microbial synth...
5- epi- aristolochene synthase  as a model ispH ispG idi isp A dxr dxs ispD DXP Pathway FPP Pyruvate + G3P Microbial synth...
Production of a model isoprenoid  in  E. coli Very low production of isoprenoid resulted when using the native gene 0.0001...
Amorphadiene synthase by design ispH ispG idi isp A dxr dxs ispD DXP Pathway FPP Pyruvate + G3P Microbial synthesis Amorph...
Gene synthesis atcttgtgat catcccaaga caaaaccaga gaaaaagacc tgtctgtttt tttaagaagt  ctttatatta ttttttttgt cggagaatct tataagc...
Gene synthesis improves amorphadiene production 142-fold improved production! 0.0001 0.001 0.01 0.1 1 10 100 1000 Construc...
Microbially-derived artemisinin ispH ispG idi isp A dxr dxs ispD Amorphadiene ADS DXP Pathway Synthase FPP Pyruvate + G3P ...
A new pathway    … borrowed from nature ispH ispG idi isp A dxr dxs ispD FPP Pyruvate + G3P ispF ispE PMK MPD MK idi isp A...
Metabolic pathway construction Acetyl-CoA P HMGS tHMGR atoB MevT Mevalonate FPP MBIS P PMK MPD MK idi Mevalonate ispA
Metabolic pathway construction lac I O P P RNAP atoB P HMGS tHMGR atoB
Metabolic pathway construction Connections between genes are not standardized P HMGS tHMGR atoB
BioBricks – Genetic Legos E =  Eco R1 Bg =  Bgl II Ba =  Bam H1 X =  Xho I Restriction site destroyed in the process B0034...
Rapid assembly of metabolic pathways ispA idi MPD PMK MK ispA idi MPD PMK MK ispA idi MPD PMK MK ispA idi MPD PMK MK ispA ...
The yeast mevalonate pathway improves yields ~90-fold 0.0001 0.001 0.01 0.1 1 10 100 1000 Construct Isoprenoid (mg/L)
Growth inhibition by MevT pathway Mevalonate tHMGR AtoB Acetyl-CoA Acetoacetyl -CoA HMG-CoA HMGS atoB HMGS tHMGR atoB HMGS...
Building a computer from off-the-shelf parts System Off-the-shelf components Knowledge Base Characterization Standard  Con...
Systems biology for debugging synthesis System Off-the-shelf components Idea ADS p450 Synthase Hydroxylase Unit CPR PMK MP...
Accumulation of the toxic intermediate HMG-CoA Ac-CoA AcAc-CoA HMG-CoA Mev AtoB HmgS tHmgR PMK MPD MK idi isp A HMGS atoB ...
HMG-CoA inhibits fatty acid production PMK MPD MK idi isp A HMGS atoB tHMGR Amorphadiene ADS Mevalonate pathway (TOP) Meva...
HMG-CoA inhibits fatty acid production PMK MPD MK idi isp A HMGS atoB tHMGR Amorphadiene ADS Mevalonate pathway (TOP) Meva...
Addition of fatty acids to the growth medium restores growth PMK MPD MK idi isp A HMGS atoB tHMGR Amorphadiene ADS Mevalon...
Supplementation with saturated fatty acids improves growth Cell density (OD 600 ) Time post-induction (hrs) Active pathway...
Synthetic scaffolds: another way to solve the problem   Ac-CoA AcAc-CoA HMG-CoA Mev AtoB HmgS tHmgR tHmgR HmgS AtoB
Connecting metabolic pipes with synthetic scaffolds Connecting the enzymes in some way might reduce loss of intermediate t...
Connecting metabolic pipes with synthetic scaffolds Adding additional copies of rate-limiting enzymes might increase pathw...
There are no standards  for connecting enzymes together Ac-CoA AcAc-CoA HMG-CoA Mev AtoB HmgS tHmgR tHmgR HmgS AtoB
Synthetic scaffolds co-localize pathway enzymes and reduce intermediate runoff tHmgR HmgS AtoB AtoB HmgR HmgS
Synthetic scaffolds co-localize pathway enzymes and reduce intermediate runoff Scaffold P tet P BAD Dueber. 2009.  Nat. Bi...
Synthetic scaffolds can control relative enzyme ratios and optimize flux tHmgR HmgS AtoB Dueber. 2009.  Nat. Biotech .   2...
Variation in the number of  HmgS and HmgR on the scaffold Dueber. 2009.  Nat. Biotech .   27:753. AtoB HmgS HmgR HmgR HmgR...
Synthetic scaffolds have a dramatic effect on the mevalonate pathway Dueber. 2009.  Nat. Biotech .   27:753. HmgS HmgR Ato...
Synthetic scaffolds can control relative enzyme ratios and optimize flux tHmgR tHmgR AtoB HmgS HmgR HmgR HmgS HmgS HmgS AtoB
Component optimization and debugging yields another 50 fold 0.0001 0.001 0.01 0.1 1 10 100 1000 Construct Isoprenoid (mg/L)
Fermentation optimization pushes yields beyond 25 g/L! Fermentation and further microbe optimization done by  Amyris . 0.0...
Identify final enzyme in pathway (P450/AMO) and transplant into  E. coli PMK MPD MK idi isp A HMGS atoB tHMGR Amorphadiene...
Proposed artemisinin biosynthetic pathway Cytochrome P450  monooxygenase Alcohol  dehydrogenase Aldehyde  dehydrogenase FP...
Lettuce, chicory, and sunflower produce isoprenoids like artemisinin Amorphadiene ( Artemisia annua )   Artemisinin H H O ...
Alignment of known terpene hydroxylases
P450 candidate produces artemisinic acid FPP Amorphadiene Artemisinic acid C H 2 O H O H C H O O C 121 248 93 188 79 105 2...
Completing the biosynthetic pathway in  E. coli p450 Amorphadiene Artemisinic Acid 1 2 3 >25 g/L Current titer in E. coli ...
Research, Development &  Delivery Keasling  Laboratory Amyris  Biotechnologies Sanofi-Aventis
Production of advanced biofuels System Off-the-shelf components Idea Knowledge Base
Production of advanced biofuels Off-the-shelf components Idea Knowledge Base System
Biofuels beyond ethanol <ul><li>does not have the full fuel value of gasoline </li></ul><ul><li>requires energy-intensive ...
Microbial synthesis of biofuels <ul><li>Isopentanol & isopentenol </li></ul><ul><li>Low miscibility with water </li></ul><...
Production of advanced biofuels Off-the-shelf components Idea Knowledge Base System
Production of advanced biofuels Idea Knowledge Base System Off-the-shelf components lmnS gerS pptS waxS fnsS
Acknowledgements Funding Department of Energy National Science Foundation Office of Naval Research University of Californi...
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09 CeoMeeting- Final Talk_Jay Keasling

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Special Focus Topic: Synthetic Biology

Title: The Next Phase of Biology - Synthetic Biology for Synthetic

Professor Jay Keasling, Joint Bioenergy Institute, Berkeley, CA

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  • Ancient Chinese medical texts dating from around 150 B.C. suggest the use of sweet Annie for people with hemorrhoids .[1] Other writings from 340 A.D. are the first known to mention sweet Annie as a treatment for people with fevers.[2] It has been used ever since for a variety of infections in Traditional Chinese Medicine . 1. Foster S, Yue CX. Herbal Emissaries: Bringing Chinese Herbs to the West. Rochester, VT: Healing Arts Press, 1992, 322. 2. Foster S, Yue CX. Herbal Emissaries: Bringing Chinese Herbs to the West. Rochester, VT: Healing Arts Press, 1992, 322.
  • One way you can improve expression of plant genes (and in fact genes from many different organisms) in a microbial host is to resynthesize the gene, making it look like a microbial gene even though it encodes the plant enzyme. The way we do this is to run the amino acid sequence through a computer program that exchanges all of the rare codons in the gene (at least the codons that are rare for the host (E. coli)) for codons that are used frequently by E. coli.
  • This new gene improved production of amorphadiene 142 fold (not shown to scale in the graph). In essence, we eliminated a bottleneck and opened up the metabolic pipes at the end of the process.
  • So, we decided that the best approach was to bring an entirely new metabolic pathway into the cell. The new pathway that we added is the mevalonate pathway from yeast that is responsible for cholesterol biosynthesis. Since no other growth factors for cell growth were produced by this pathway, we would not have to worry about it disrupting growth. The pathway functioned beautifully and improved production another 90 fold.
  • So, we decided that the best approach was to bring an entirely new metabolic pathway into the cell. The new pathway that we added is the mevalonate pathway from yeast that is responsible for cholesterol biosynthesis. Since no other growth factors for cell growth were produced by this pathway, we would not have to worry about it disrupting growth. The pathway functioned beautifully and improved production another 90 fold.
  • This is what is known of the artemisinin biosynthetic pathway. It begins with farnesyl diphosphate, a 15-carbon precursor that is found in every organism. Besides being the precursor to artemisinin, farnesyl diphosphate is also the precursor to cholesterol.
  • The goals of the Fuels Synthesis Division are to develop organisms that can efficiently produce, in high concentration, existing and next generation biofuels from the sugars of cellulose depolymerization and organisms that can withstand high concentrations of the fuels. Early deliverables from the Fuels Synthesis Division will be pathways for production of next generation biofuels enabled by our strength in synthetic biology. Understanding the toxicity and stress encountered by organisms in response to fuels, enabled by functional genomics methods developed through the Genomics:GTL programs and informatics to integrate these methods, will allow us to engineer organisms that are better able to withstand high concentrations of the fuels. Using mathematical models of metabolism and gene regulation we will engineer microorganisms to efficient convert sugars into biofuels. Our long-term goal is to develop microorganisms capable of depolymerizing cellulose into sugars and converting the sugars into biofuels in a single pot.
  • 09 CeoMeeting- Final Talk_Jay Keasling

    1. 1. Synthetic Biology: From Bugs to Drugs and Fuels Jay D. Keasling Joint BioEnergy Institute Lawrence Berkeley National Laboratory University of California, Berkeley
    2. 2. Building a computer from off-the-shelf parts System Off-the-shelf components Idea Knowledge Base
    3. 3. Building a computer from off-the-shelf parts System Off-the-shelf components Idea Knowledge Base
    4. 4. Building a computer from off-the-shelf parts System Off-the-shelf components Idea Knowledge Base
    5. 5. Building a computer from off-the-shelf parts System Off-the-shelf components Idea Knowledge Base
    6. 6. Building a computer from off-the-shelf parts System Off-the-shelf components Idea Knowledge Base
    7. 7. Building a computer from off-the-shelf parts System Off-the-shelf components Knowledge Base Characterization Idea
    8. 8. Building a computer from off-the-shelf parts System Off-the-shelf components Knowledge Base Characterization Standard Connections Idea
    9. 9. Building a computer from off-the-shelf parts System Off-the-shelf components Knowledge Base Characterization Standard Connections Independent Devices Idea
    10. 10. Building a computer from off-the-shelf parts System Off-the-shelf components Knowledge Base Characterization Standard Connections Independent Devices Models, Design, & Debugging Idea
    11. 11. Building a computer from off-the-shelf parts System Off-the-shelf components Knowledge Base Characterization Standard Connections Independent Devices Models, Design, & Debugging Silicon Wafer Processing Idea
    12. 12. Styrene
    13. 13. Synthesis of Styrene System Off-the-shelf components Idea Knowledge Base
    14. 14. Synthesis of Styrene System Off-the-shelf components Idea Knowledge Base
    15. 15. Synthesis of Styrene System Off-the-shelf components Idea Knowledge Base
    16. 16. Synthesis of Styrene System Off-the-shelf components Idea Knowledge Base Chemistry
    17. 17. Synthesis of Styrene System Off-the-shelf components Idea Knowledge Base Chemistry Kinetics & Reactor Design
    18. 18. Synthesis of Styrene System Off-the-shelf components Knowledge Base Chemistry Kinetics & Reactor Design Mass Transfer Process Design Standard Connections Idea
    19. 19. Some common features <ul><li>Parts and devices </li></ul><ul><ul><li>Independent </li></ul></ul><ul><ul><li>Characterized </li></ul></ul><ul><li>Standards </li></ul><ul><ul><li>Connections </li></ul></ul><ul><ul><li>Characterization </li></ul></ul><ul><li>Design & Fabrication </li></ul>
    20. 20. Microbial synthesis of artemisinin System Off-the-shelf components Knowledge Base Idea
    21. 21. Malaria <ul><li>Economists have proposed that malaria decreases the GDP of affected countries by as much as 50%. </li></ul><ul><li>1-3 million people die of malaria every year </li></ul><ul><ul><li>90% are children </li></ul></ul><ul><li>300-500 million people infected </li></ul>Source: Roll Back Malaria World Malaria Report 2005
    22. 22. <ul><li>Most widely-used drugs to treat malaria </li></ul>Quinine-based drugs <ul><li>Plasmodium in many parts of the world is largely resistant to chloroquine </li></ul>Source: Roll Back Malaria World Malaria Report 2005
    23. 23. Artemisinin Artemisia annua
    24. 24. A brief history of artemisinin 168 B.C. Recipes For 52 Kinds Of Diseases found in the Mawangdui Han Dynasty tomb  Hemorrhoids 340 A.D. Zhou Hou Bei Ji Fang (Handbook of Prescriptions for Emergency Treatments)  Fevers (malaria) 1972 Active ingredient (artemisinin) isolated
    25. 25. Artemisinin is produced in oil sacs on Artemisia annua leaves Artemisinin... ...is produced by trichomes... ...found on Artemisia annua leaves...
    26. 26. Current process Artemisinin Plant synthesis Purification Artesunate Artelinate Arteether Artemether Chemical Conversions
    27. 27. Artemisinin-based drugs <ul><li>Price </li></ul><ul><ul><li>$1.00-2.50/treatment </li></ul></ul><ul><ul><li>Fluctuations in artemisinin price </li></ul></ul><ul><li>Source </li></ul><ul><ul><li>Extraction from plant </li></ul></ul><ul><ul><li>Quality of active ingredient </li></ul></ul><ul><li>Use </li></ul><ul><ul><li>Monotherapies </li></ul></ul><ul><ul><li>Resistance </li></ul></ul>
    28. 28. Microbial synthesis of artemisinin Off-the-shelf components Knowledge Base Idea System Engineer a microorganism to produce artemisinin from an inexpensive, renewable resource.
    29. 29. Semi-synthesis of artemisinin ispH ispG idi isp A dxr dxs ispD DXP Pathway FPP Pyruvate + G3P Microbial synthesis ispF ispE Central metabolism Amorphadiene Artemisinic Acid ADS p450 Synthase Hydroxylase Unit CPR OPP Artemisinic Acid Purification OPP OPP Artemisinin Chemical Conversions Artesunate Artelinate Arteether Artemether
    30. 30. Constructing an artemisinic acid-producing microbe ispH ispG idi isp A dxr dxs ispD DXP Pathway FPP Pyruvate + G3P Microbial synthesis ispF ispE Central metabolism OPP Amorphadiene Artemisinic Acid ADS p450 Synthase Hydroxylase Unit CPR OPP OPP
    31. 31. Microbial synthesis of artemisinin Knowledge Base Idea System Off-the-shelf components
    32. 32. A chassis for artemisinin biosynthesis Knowledge Base Idea System Off-the-shelf components
    33. 33. Parts needed for artemisinin synthesis Knowledge Base Idea System Off-the-shelf components
    34. 34. How do we get the components we need?
    35. 35. How do we get the components we need? Genome sequencing Genome annotation Components
    36. 36. BioShack? “ Please send me your biological device as you described in your paper.” Reply: “The device you requested is in this package … please use it as described in our paper.”
    37. 37. BioShack? “ It didn’t work as described in your paper.” Reply: “In my hands, it worked as described in my paper … did you try X and Y and Z?”
    38. 38. Creating an amorphadiene-producing microbe ispH ispG idi isp A dxr dxs ispD DXP Pathway FPP Pyruvate + G3P Microbial synthesis Amorphadiene ADS Synthase ispF ispE Central metabolism ADS amorphadiene FPP OPP OPP OPP
    39. 39. Creating an amorphadiene-producing microbe ispH ispG idi isp A dxr dxs ispD DXP Pathway FPP Pyruvate + G3P Microbial synthesis Amorphadiene ADS Synthase ispF ispE Central metabolism EAS amorphadiene FPP “ Please send me your amorphadiene synthase as you described in your paper.” Reply: “No … you are my competitor.” OPP OPP OPP
    40. 40. 5- epi- aristolochene synthase as a model ispH ispG idi isp A dxr dxs ispD DXP Pathway FPP Pyruvate + G3P Microbial synthesis ispF ispE Central metabolism 5-epi-aristolochene EAS Synthase Tobacco EAS 5- epi- aristolochene FPP OPP OPP OPP
    41. 41. Production of a model isoprenoid in E. coli Very low production of isoprenoid resulted when using the native gene 0.0001 0.001 0.01 0.1 1 10 100 1000 Construct Isoprenoid (mg/L)
    42. 42. Amorphadiene synthase by design ispH ispG idi isp A dxr dxs ispD DXP Pathway FPP Pyruvate + G3P Microbial synthesis Amorphadiene ADS Synthase ispF ispE Central metabolism ADS amorphadiene FPP OPP OPP OPP
    43. 43. Gene synthesis atcttgtgat catcccaaga caaaaccaga gaaaaagacc tgtctgtttt tttaagaagt ctttatatta ttttttttgt cggagaatct tataagcatg gcttcaggag gatcaaagtc ggcagctttc atgcttctga tgctgaatct tggtctctat ttcgtcatca ccatcatcgc ttcttgggct gttaatcacg gcatcgagag aactcgcgag tctggtaact aacaaagata acaactgatt aagtaacaat taatccaacg ttagaaaatg tcatcatcaa tcttcttttt gtggtatttt gcagcgtcga cactgtcact tccggcgaag atattcccga tatacttccc ggtggggaac atggcgaccg gttttttcgt aatattcacg ttaatcgccg gcgtcgtcgg atcttgtgat catcccaaga caaaaccaga gaaaaagacc tgtctgtttt tttaagaagt ctttatatta ttttttttgt cggagaatct tataagcatg gcttcaggag gatcaaagtc ggcagctttc atgcttctga tgctgaatct tggtctctat ttcgtcatca ccatcatcgc ttcttgggct gttaatcacg gcatcgagag aactcgcgag tctggtaact aacaaagata acaactgatt aagtaacaat taatccaacg ttagaaaatg tcatcatcaa tcttcttttt gtggtatttt gcagcgtcga cactgtcact tccggcgaag atattcccga tatacttccc ggtggggaac atggcgaccg gttttttcgt aatattcacg ttaatcgccg gcgtcgtcgg <ul><li>Remove rare codons </li></ul><ul><li>Insert restriction sites </li></ul>Plant Sequence Microbial Sequence
    44. 44. Gene synthesis improves amorphadiene production 142-fold improved production! 0.0001 0.001 0.01 0.1 1 10 100 1000 Construct Isoprenoid (mg/L)
    45. 45. Microbially-derived artemisinin ispH ispG idi isp A dxr dxs ispD Amorphadiene ADS DXP Pathway Synthase FPP Pyruvate + G3P ispF ispE Central metabolism Limitations in the native biosynthetic pathways OPP OPP OPP
    46. 46. A new pathway … borrowed from nature ispH ispG idi isp A dxr dxs ispD FPP Pyruvate + G3P ispF ispE PMK MPD MK idi isp A HMGS atoB tHMGR Amorphadiene ADS FPP Mevalonate Acetyl- CoA The mevalonate pathway is responsible for cholesterol production. OPP
    47. 47. Metabolic pathway construction Acetyl-CoA P HMGS tHMGR atoB MevT Mevalonate FPP MBIS P PMK MPD MK idi Mevalonate ispA
    48. 48. Metabolic pathway construction lac I O P P RNAP atoB P HMGS tHMGR atoB
    49. 49. Metabolic pathway construction Connections between genes are not standardized P HMGS tHMGR atoB
    50. 50. BioBricks – Genetic Legos E = Eco R1 Bg = Bgl II Ba = Bam H1 X = Xho I Restriction site destroyed in the process B0034 AMP E Bg Ba X C0010 AMP E Bg Ba X Cut with E & Ba E Bg Ba B0034 Cut with E & Bg Mix & Ligate C0010 AMP Ba X B0034 E Bg C0010 AMP Ba X E Bg
    51. 51. Rapid assembly of metabolic pathways ispA idi MPD PMK MK ispA idi MPD PMK MK ispA idi MPD PMK MK ispA idi MPD PMK MK ispA idi MPD PMK MK Best producer Assemble using biobricks
    52. 52. The yeast mevalonate pathway improves yields ~90-fold 0.0001 0.001 0.01 0.1 1 10 100 1000 Construct Isoprenoid (mg/L)
    53. 53. Growth inhibition by MevT pathway Mevalonate tHMGR AtoB Acetyl-CoA Acetoacetyl -CoA HMG-CoA HMGS atoB HMGS tHMGR atoB HMGS tHMGR atoB HMGS tHMGR atoB HMGS tHMGR
    54. 54. Building a computer from off-the-shelf parts System Off-the-shelf components Knowledge Base Characterization Standard Connections Independent Devices Models, Design, & Debugging Idea
    55. 55. Systems biology for debugging synthesis System Off-the-shelf components Idea ADS p450 Synthase Hydroxylase Unit CPR PMK MPD MK idi isp A HMGS atoB tHMGR Mevalonate pathway (TOP) Mevalonate pathway (BOTTOM) FPP Mevalonate Knowledge Base Debugging Metabolomics Proteomics Transcriptomics 0 5 10 15 20 25 Time [min] Asp Phe Glu Pro Ile Leu Lys Arg Val His Met
    56. 56. Accumulation of the toxic intermediate HMG-CoA Ac-CoA AcAc-CoA HMG-CoA Mev AtoB HmgS tHmgR PMK MPD MK idi isp A HMGS atoB tHMGR Amorphadiene ADS FPP Mevalonate Acetyl- CoA OPP
    57. 57. HMG-CoA inhibits fatty acid production PMK MPD MK idi isp A HMGS atoB tHMGR Amorphadiene ADS Mevalonate pathway (TOP) Mevalonate pathway (BOTTOM) Synthase FPP Mevalonate Acetyl- CoA HMG-CoA Fatty acid biosynthetic pathway OPP
    58. 58. HMG-CoA inhibits fatty acid production PMK MPD MK idi isp A HMGS atoB tHMGR Amorphadiene ADS Mevalonate pathway (TOP) Mevalonate pathway (BOTTOM) Synthase FPP Mevalonate Acetyl- CoA HMG-CoA Fatty acid biosynthetic pathway OPP
    59. 59. Addition of fatty acids to the growth medium restores growth PMK MPD MK idi isp A HMGS atoB tHMGR Amorphadiene ADS Mevalonate pathway (TOP) Mevalonate pathway (BOTTOM) Synthase FPP Mevalonate Acetyl- CoA HMG-CoA Fatty acid addition to the growth medium OPP HO O
    60. 60. Supplementation with saturated fatty acids improves growth Cell density (OD 600 ) Time post-induction (hrs) Active pathway No supplement Active pathway 16:0 Supplement Inactive pathway
    61. 61. Synthetic scaffolds: another way to solve the problem Ac-CoA AcAc-CoA HMG-CoA Mev AtoB HmgS tHmgR tHmgR HmgS AtoB
    62. 62. Connecting metabolic pipes with synthetic scaffolds Connecting the enzymes in some way might reduce loss of intermediate to the bulk Ac-CoA AcAc-CoA HMG-CoA Mev AtoB HmgS tHmgR tHmgR HmgS AtoB
    63. 63. Connecting metabolic pipes with synthetic scaffolds Adding additional copies of rate-limiting enzymes might increase pathway flux Ac-CoA AcAc-CoA HMG-CoA Mev AtoB HmgS tHmgR tHmgR HmgS AtoB
    64. 64. There are no standards for connecting enzymes together Ac-CoA AcAc-CoA HMG-CoA Mev AtoB HmgS tHmgR tHmgR HmgS AtoB
    65. 65. Synthetic scaffolds co-localize pathway enzymes and reduce intermediate runoff tHmgR HmgS AtoB AtoB HmgR HmgS
    66. 66. Synthetic scaffolds co-localize pathway enzymes and reduce intermediate runoff Scaffold P tet P BAD Dueber. 2009. Nat. Biotech . 27:753. AtoB HmgS HmgR AtoB HmgR HmgS
    67. 67. Synthetic scaffolds can control relative enzyme ratios and optimize flux tHmgR HmgS AtoB Dueber. 2009. Nat. Biotech . 27:753. AtoB HmgS HmgR HmgR HmgR
    68. 68. Variation in the number of HmgS and HmgR on the scaffold Dueber. 2009. Nat. Biotech . 27:753. AtoB HmgS HmgR HmgR HmgR HmgR AtoB HmgS HmgR HmgR AtoB HmgS HmgR AtoB HmgS HmgR HmgS AtoB HmgS HmgR HmgR HmgS AtoB HmgS HmgS HmgR HmgS HmgS HmgR HmgR HmgR AtoB HmgS HmgS HmgR HmgR HmgR HmgR AtoB HmgS HmgS HmgR HmgS HmgS HmgR AtoB HmgS HmgS HmgS HmgS HmgR HmgR HmgS
    69. 69. Synthetic scaffolds have a dramatic effect on the mevalonate pathway Dueber. 2009. Nat. Biotech . 27:753. HmgS HmgR AtoB n = 1
    70. 70. Synthetic scaffolds can control relative enzyme ratios and optimize flux tHmgR tHmgR AtoB HmgS HmgR HmgR HmgS HmgS HmgS AtoB
    71. 71. Component optimization and debugging yields another 50 fold 0.0001 0.001 0.01 0.1 1 10 100 1000 Construct Isoprenoid (mg/L)
    72. 72. Fermentation optimization pushes yields beyond 25 g/L! Fermentation and further microbe optimization done by Amyris . 0.0001 0.001 0.01 0.1 1 10 100 1000 Construct Isoprenoid (mg/L)
    73. 73. Identify final enzyme in pathway (P450/AMO) and transplant into E. coli PMK MPD MK idi isp A HMGS atoB tHMGR Amorphadiene Artemisinic Acid ADS Mevalonate pathway (TOP) Mevalonate pathway (BOTTOM) p450 Synthase Hydroxyase Unit FPP Mevalonate CPR Acetyl- CoA A. annua p450 CPR Amorphadiene Artemisinic Acid OPP
    74. 74. Proposed artemisinin biosynthetic pathway Cytochrome P450 monooxygenase Alcohol dehydrogenase Aldehyde dehydrogenase FPP Amorphadiene Artemisinic acid Artemisinin C H 2 O H O H C H O O C C H 2 O H O H C H O O C
    75. 75. Lettuce, chicory, and sunflower produce isoprenoids like artemisinin Amorphadiene ( Artemisia annua ) Artemisinin H H O H O H H O O O H H O O Germacrene A (Chicory, sunflower and lettuce) O O O O H HO OH O O O H lettucenin A niveusin A O H O O P450’s involved
    76. 76. Alignment of known terpene hydroxylases
    77. 77. P450 candidate produces artemisinic acid FPP Amorphadiene Artemisinic acid C H 2 O H O H C H O O C 121 248 93 188 79 105 216 136 162 173 145 55 67 201 233 121 93 248 79 188 105 136 216 162 173 145 55 67 201 233 Relative Ion Abundance Peak 1 Yeast product Peak 2 Artemisinic acid m/z
    78. 78. Completing the biosynthetic pathway in E. coli p450 Amorphadiene Artemisinic Acid 1 2 3 >25 g/L Current titer in E. coli (lab scale) > 1 g/L P450/AMO Catalyzes 3 Separate Oxidations
    79. 79. Research, Development & Delivery Keasling Laboratory Amyris Biotechnologies Sanofi-Aventis
    80. 80. Production of advanced biofuels System Off-the-shelf components Idea Knowledge Base
    81. 81. Production of advanced biofuels Off-the-shelf components Idea Knowledge Base System
    82. 82. Biofuels beyond ethanol <ul><li>does not have the full fuel value of gasoline </li></ul><ul><li>requires energy-intensive purification processes </li></ul><ul><li>is toxic at high concentrations </li></ul><ul><li>is not transported using traditional means </li></ul>
    83. 83. Microbial synthesis of biofuels <ul><li>Isopentanol & isopentenol </li></ul><ul><li>Low miscibility with water </li></ul><ul><li>High octane </li></ul><ul><li>Appropriate vapor pressure </li></ul><ul><li>Isoprenoid esters </li></ul><ul><li>Diesel replacements </li></ul><ul><li>Appropriate cetane numbers </li></ul><ul><li>Terpenes </li></ul><ul><li>Jet fuel replacements </li></ul><ul><li>Low freezing point </li></ul>
    84. 84. Production of advanced biofuels Off-the-shelf components Idea Knowledge Base System
    85. 85. Production of advanced biofuels Idea Knowledge Base System Off-the-shelf components lmnS gerS pptS waxS fnsS
    86. 86. Acknowledgements Funding Department of Energy National Science Foundation Office of Naval Research University of California Discovery Grant Bill & Melinda Gates Foundation Keasling lab Jennifer Anthony Michelle Chang Howard Chou John Dueber Connie Kang Lance Kizer Jim Kirby Taek Soon Lee Vincent Martin Karyn Newman Farnaz Nowroozi Mario Ouellet Eric Paradise Chris Petzold Brian Pfleger Doug Pitera Dae-Kyun Ro Christina Smolke Sydnor Withers Gabriel Wu Yasuo Yoshikuni Amyris # Jack Newman Chris Paddon Kinkead Reiling Rika Regentin Neil Renninger # Jay Keasling has a financial interest in Amyris & LS9. Joint Genome Institute

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