1. Morten Sommer, MIT/Harvard DOE GtL Center Novozyme 30-Jun-2006 CAD for Synthetic Microbial Biofuels

2. Our DOE GtL Center goals & strengths 1. Basic enabling technologies: omics, models, genome synthesis, evolution, sequencing 2. Fermentative production of alcohols & biodiesel. 3. Improving photosynthetic and conversion efficiencies. 4. Harnessing new insights from ecosystems.

3. Genome & Metabolome Computer Aided Design (CAD) 4.7 Mbp new genetic codes new amino acids 7*7 * 4.7 Mbp mini-ecosystems biosensors, bioenergy, high secretors, DNA & metabolic isolation Top Design Utility, safety & scalability CAD-PAM Synthesis (chip & error correction) Combinatorics Evolution Sequence

4. How? 10 Mbp of oligos / $1000 chip 8K Atactic/Xeotron/Invitrogen Photo-Generated Acid Sheng, Zhou, Gulari, Gao (Houston) 12K Combimatrix Electrolytic 44K Agilent Ink-jet standard reagents 380K Nimblegen Photolabile 5'protection Tian et al. Nature. 432:1050; Carr & Jacobson 2004 NAR; Smith & Modrich 1997 PNAS ~1000X lower oligo costs (= 2 E.coli genomes or 20 Mycoplasmas /chip) Amplify pools of 50mers using flanking universal PCR primers and three paths to 10X error correction Digital Micromirror Array

5. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Martin VJ, et al. Nat. Biotech 2003 Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Ro DK, et al. Nature. 2006 8

6. Programmable ligand-controlled riboregulators to monitor metabolites. Bayer & Smolke 2005 Nature Biotech. ON OFF

7. Smart therapeutics example: Environmentally controlled invasion of cancer cells by engineered bacteria. Anderson et al. J Mol Biol. 2006 Optical imaging: bacteria, viruses, and mammalian cells encoding light- emitting proteins reveal the locations of primary tumors & metastases in animals. Yu, et al. Anal. Bioanal. Chem. 2003. accumulate in tumors at ratios in excess of 1000:1 compared with normal tissues. http://www.vionpharm.com/tapet_virulence.htmlhttp://www.vionpharm.com/tapet_virulence.html Metabolic constraints Regulated Capsule TonB, DapD & new genetic codes for safety

8. LPS - Capsule + Dap - for safety 7 DapD

9. rE.coli: new in vivo genetic codes TTT F 30362TCT S 11495TAT Y 21999TGT C 7048 TTC22516TCC11720TAC16601TGC8816 TTA L 18932TCA9783TAA STOP 2703TGA STOP 1256 TTG18602TCG12166TAG326TGGW20683 CTT L 15002CCT P 9559CAT H 17613CGT R 28382 CTC15077CCC7485CAC13227CGC29898 CTA5314CCA11471CAA Q 20888CGA4859 CTG71553CCG31515CAG39188CGG7399 ATT I 41309ACT T 12198AAT N 24159AGT S 11970 ATC34178ACC31796AAC29385AGC21862 ATA 5967ACA9670AAA K 45687AGA R 2896 ATGM37915ACG19624AAG14029AGG1692 GTT V 24858GCT A 20762GAT D 43719GGT G 33622 GTC20753GCC34695GAC25918GGC40285 GTA14822GCA27418GAA E 53641GGA10893 GTG35918GCG45741GAG24254GGG15090 Freeing 4 tRNAs, 7 codons: UAG, UUR, AGY, AGR e.g. PEG-pAcPhe-hGH (Ambrx, Schultz) high serum stability Isaacs Church Forster Carr Jacobson Jahnz Schultz 1 2 3 4

10. Competition & cooperation Cooperation between two auxotrophs –Overall fitness depends on secretion –Over-production, increase of export Competition among each sub-population –The fastest growing one wins –Increase of uptake Coupling between evolution of import and export properties? –Amplified genes –Transporter & pore genes

11. Cross-feeding symbiotic systems: aphids & Buchnera obligate mutualism nutritional interactions: amino acids and vitamine established 200-250 million years ago close relative of E. coli with tiny genome (618~641kb) Aphids Internal view of the aphid. (by T. Sasaki) Bacteriocyte (Photo by T. Fukatsu) Buchnera (Photo by M. Morioka) http://buchnera.gsc.riken.go.jp

12. Shigenobu et al. Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp.APS. Nature 407, 81-86 (2000).

14. ODE based simulation of population dynamics of cross-feeding ∆Trp-∆Tyr Questions: When mixed in minimum medium, how do the cell population and the amino acid concentrations change over time? What happens when the strains evolve? –improve on amino acid imports –improve on amino acid synthesis and/or exports

15. Governing ODE system density of ∆Trp (gBM/ml) density of ∆Tyr (gBM/ml) conc. of Trp (mmol/ml) conc. of Tyr (mmol/ml) growth rate constant of ∆Trp ([(mmol/ml Trp)-hr] -1 ) growth rate constant of ∆Tyr ([(mmol/ml Tyr)-hr] -1 ) Tyr excretion rate constant of ∆Trp (mmol/gBM-hr) Trp excretion rate constant of ∆Tyr (mmol/gBM-hr) =0.05 Trp requirement of ∆Trp (mmol/gBM) =0.13 Tyr requirement of ∆Tyr (mmol/gBM) Initial conditions:

16. density of ∆Trp (gBM/ml) density of ∆Tyr (gBM/ml) conc. of Trp (mmol/ml) conc. of Tyr (mmol/ml) growth rate constant of ∆Trp ([(mmol/ml Trp)-hr] -1 ) growth rate constant of ∆Tyr ([(mmol/ml Tyr)-hr] -1 ) Tyr excretion rate constant of ∆Trp (mmol/gBM-hr) Trp excretion rate constant of ∆Tyr (mmol/gBM-hr) =0.05 Trp requirement of ∆Trp (mmol/gBM) =0.13 Tyr requirement of ∆Tyr (mmol/gBM) “Steady-state” solution: Variables: Parameters:

17. Invasion of advantageous mutants

18. Fong SS, Burgard AP, Herring CD, Knight EM, Blattner FR, Maranas CD, Palsson BO. In silico design and adaptive evolution of Escherichia coli for production of lactic acid. Biotechnol Bioeng. 2005 91(5):643-8. Rozen DE, Schneider D, Lenski RE Long-term experimental evolution in Escherichia coli. XIII. Phylogenetic history of a balanced polymorphism. J Mol Evol. 2005 61(2):171-80 Andries K, et al. (J&J) A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis. Science. 2005 307:223-7. Shendure et al. Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome Science 2005 309:1728 (Select for secretion & ‘altruism’). Intelligent Design & Metabolic Evolution

19. ‘Next Generation’ Technology Development Multi-molecule Our role Affymetrix Software 454 LifeSci Paired ends, emulsion Solexa/Lynx Multiplexing & polony AB/APG Seq by Ligation (SbL) Complete Genomics SbL Gorfinkel Polony to Capillary Single molecules Helicos Biosci SAB, cleavable fluors Pacific Biosci Advisor KPCB Agilent Nanopores Visigen Biotech AB

20. HPLC autosampler (96 wells) syringe pump Polony Sequencing Equipment HMS/AB/APG microscope with xyz controls flow-cell temperature control

21.  trp/  tyrA pair of genomes shows the best co-growth Reppas, Lin & Church ; Shendure et al. Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome(2005) Science 309:1728 Second Passage First Passage Synthetic combinatorics & evolution of 7*7* 4.7 Mbp genomes

22. Consensus error rate Total errors (E.coli) (Human) 1E-4 Bermuda/Hapmap 500 600,000 4E-5 454 @40X 200 240,000 3E-7 Polony-SbL @6X 0 1800 1E-8 Goal for 2006 0 60 Goal of genotyping & resequencing  Discovery of variants E.g. cancer somatic mutations ~1E-6 (or lab evolved cells) Why low error rates? Also, effectively reduce (sub)genome target size by enrichment for exons or common SNPs to reduce cost & # false positives.

23. PositionType GeneLocation ABI Confirm Comments 986,334 T > GompFPromoter-10 Only in evolved strain 985,797 T > GompFGlu > Ala Only in evolved strain 931,960 ▲ 8 bplrpframeshift Only in evolved strain 3,957,960 C > TppiC5' UTR MG1655 heterogeneity -3274 T > CcIGlu > Glu  red heterogeneity -9846 T > CORF61Lys > Gly  red heterogeneity Mutation Discovery in Engineered/Evolved E.coli Shendure, Porreca, et al. (2005) Science 309:1728

24. Glu-117 → Ala (in the pore) Charged residue known to affect pore size and selectivity Promoter mutation at position (-12) Makes -10 box more consensus-like -12 -11 -10 -9 -8 -7 -6 A AAGAT C AAGAT Can increase import & export capability simultaneously ompF - non-specific transport channel

25. Sequence monitoring of evolution (optimize small molecule synthesis/transport) Sequence trp - Reppas, Lin & Church

26. 3 independent lines of Trp/Tyr co-culture frozen. OmpF: 42R-> G, L, C, 113 D->V, 117 E->A Promoter: -12A->C, -35 C->A Lrp: 1bp deletion, 9bp deletion, 8bp deletion, IS2 insertion, R->L in DBD. Heterogeneity within each time-point reflecting colony heterogeneity. Co-evolution of mutual biosensors sequenced across time & within each time-point

27. Prochlorococcus 40ºN - 40ºS Ocean chl a (Aug 1997 –Sept 2000) Provided by the SeaWiFS Project, NASA

28.  -Glc-1P ADP-Glc  -1,4-glucosyl-glucan glycogen Central Carbon Metabol. glgC glgX glgA glgB glgP Zinser et al. unpubl. Light regulated Prochlorococcus metabolism

29. Photosynthetic Genes in Phage Podovirus P-SSP7 46 kb PCHLIPsFdD1 12kb 24kb PCHLIPsFdD1 12kb 24kb ~500bp HLIPsD1D2 6.4kb2.8kb ~500bp Myovirus P-SSM4 181 kb HLIPsD1D2 6.4kb2.8kb Lindell, Sullivan, Chisholm et al. 2004 HLIPD1 Myovirus P-SSM2 255 kb

30. RNA Responses to Phage MED4-0682 (60 aa Conserved URF) Phage SSP7 psbA MED4 host psbA Lindell,Sullivan, Zinser, Chisholm Lindell, Sullivan, Zinser, Chisholm

31. Our DOE GtL Center goals & strengths 1. Basic enabling technologies: omics, models, genome synthesis, evolution, sequencing 2. Fermentative production of alcohols & biodiesel. 3. Improving photosynthetic and conversion efficiencies. 4. Harnessing new insights from ecosystems.

32. Morten Sommer, MIT/Harvard DOE GtL Center Novozyme 30-Jun-2006 CAD for Synthetic Microbial Biofuels

33. .