Special Focus Topic: Synthetic Biology Title: The Next Phase of Biology - Synthetic Biology for Synthetic Professor Jay Keasling, Joint Bioenergy Institute, Berkeley, CA

1. Synthetic Biology: From Bugs to Drugs and Fuels Jay D. Keasling Joint BioEnergy Institute Lawrence Berkeley National Laboratory University of California, Berkeley

2. Building a computer from off-the-shelf parts System Off-the-shelf components Idea Knowledge Base

3. Building a computer from off-the-shelf parts System Off-the-shelf components Idea Knowledge Base

4. Building a computer from off-the-shelf parts System Off-the-shelf components Idea Knowledge Base

5. Building a computer from off-the-shelf parts System Off-the-shelf components Idea Knowledge Base

6. Building a computer from off-the-shelf parts System Off-the-shelf components Idea Knowledge Base

7. Building a computer from off-the-shelf parts System Off-the-shelf components Knowledge Base Characterization Idea

8. Building a computer from off-the-shelf parts System Off-the-shelf components Knowledge Base Characterization Standard Connections Idea

9. Building a computer from off-the-shelf parts System Off-the-shelf components Knowledge Base Characterization Standard Connections Independent Devices Idea

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. 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. Styrene

13. Synthesis of Styrene System Off-the-shelf components Idea Knowledge Base

14. Synthesis of Styrene System Off-the-shelf components Idea Knowledge Base

15. Synthesis of Styrene System Off-the-shelf components Idea Knowledge Base

16. Synthesis of Styrene System Off-the-shelf components Idea Knowledge Base Chemistry

17. Synthesis of Styrene System Off-the-shelf components Idea Knowledge Base Chemistry Kinetics & Reactor Design

18. Synthesis of Styrene System Off-the-shelf components Knowledge Base Chemistry Kinetics & Reactor Design Mass Transfer Process Design Standard Connections Idea

20. Microbial synthesis of artemisinin System Off-the-shelf components Knowledge Base Idea

23. Artemisinin Artemisia annua

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. Artemisinin is produced in oil sacs on Artemisia annua leaves Artemisinin... ...is produced by trichomes... ...found on Artemisia annua leaves...

26. Current process Artemisinin Plant synthesis Purification Artesunate Artelinate Arteether Artemether Chemical Conversions

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. 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. 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. Microbial synthesis of artemisinin Knowledge Base Idea System Off-the-shelf components

32. A chassis for artemisinin biosynthesis Knowledge Base Idea System Off-the-shelf components

33. Parts needed for artemisinin synthesis Knowledge Base Idea System Off-the-shelf components

34. How do we get the components we need?

35. How do we get the components we need? Genome sequencing Genome annotation Components

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. 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. 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. 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. 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. 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. 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

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. 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. 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. Metabolic pathway construction Acetyl-CoA P HMGS tHMGR atoB MevT Mevalonate FPP MBIS P PMK MPD MK idi Mevalonate ispA

48. Metabolic pathway construction lac I O P P RNAP atoB P HMGS tHMGR atoB

49. Metabolic pathway construction Connections between genes are not standardized P HMGS tHMGR atoB

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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. Synthetic scaffolds: another way to solve the problem Ac-CoA AcAc-CoA HMG-CoA Mev AtoB HmgS tHmgR tHmgR HmgS AtoB

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. 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. There are no standards for connecting enzymes together Ac-CoA AcAc-CoA HMG-CoA Mev AtoB HmgS tHmgR tHmgR HmgS AtoB

65. Synthetic scaffolds co-localize pathway enzymes and reduce intermediate runoff tHmgR HmgS AtoB AtoB HmgR HmgS

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. 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. 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. Synthetic scaffolds have a dramatic effect on the mevalonate pathway Dueber. 2009. Nat. Biotech . 27:753. HmgS HmgR AtoB n = 1

70. Synthetic scaffolds can control relative enzyme ratios and optimize flux tHmgR tHmgR AtoB HmgS HmgR HmgR HmgS HmgS HmgS AtoB

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. 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. 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. 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. 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. Alignment of known terpene hydroxylases

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. 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. Research, Development & Delivery Keasling Laboratory Amyris Biotechnologies Sanofi-Aventis

80. Production of advanced biofuels System Off-the-shelf components Idea Knowledge Base

81. Production of advanced biofuels Off-the-shelf components Idea Knowledge Base System

84. Production of advanced biofuels Off-the-shelf components Idea Knowledge Base System

85. Production of advanced biofuels Idea Knowledge Base System Off-the-shelf components lmnS gerS pptS waxS fnsS

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