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(12) Patent Application Publication (10) Pub. No.: US 2015/0037853 A1 Fischer Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2015/0037853 A1 Fischer Et Al US 2015 0037853A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0037853 A1 Fischer et al. (43) Pub. Date: Feb. 5, 2015 (54) METHODS AND SYSTEMIS FOR Publication Classification CHEMOAUTOTROPHC PRODUCTION OF ORGANIC COMPOUNDS (51) Int. Cl. CI2P 7/40 (2006.01) (71) Applicant: Ginkgo BioWorks, Inc., Boston, MA CI2P 7/16 (2006.01) (US) CI2N 15/70 (2006.01) (52) U.S. Cl. (72) Inventors: Curt R. Fischer, Cambridge, MA (US); CPC. CI2P 7/40 (2013.01): CI2N 15/70 (2013.01); Austin J. Che, Cambridge, MA (US); CI2P 7/16 (2013.01) Reshma P. Shetty, Boston, MA (US); USPC ....................... 435/136: 435/252.33:435/160 Jason R. Kelly, Cambridge, MA (US) (21) Appl. No.: 14/354,354 (57) ABSTRACT (22) PCT Fled: Oct. 30, 2012 The present disclosure identifies pathways, mechanisms, sys tems and methods to confer chemoautotrophic production of (86) PCT NO.: PCT/US12A62S4O carbon-based products of interest, such as Sugars, alcohols, chemicals, amino acids, polymers, fatty acids and their S371 (c)(1), derivatives, hydrocarbons, isoprenoids, and intermediates (2) Date: Apr. 25, 2014 thereof, in organisms such that these organisms efficiently convertinorganic carbon to organic carbon-based products of Related U.S. Application Data interest using inorganic energy, Such as formate, and in par (63) Continuation of application No. 13/285,919, filed on ticular the use of organisms for the commercial production of Oct. 31, 2011, now Pat. No. 8,349,587. various carbon-based products of interest. Patent Application Publication Feb. 5, 2015 Sheet 1 of 29 US 2015/0037853 A1 Forfate Patent Application Publication Feb. 5, 2015 Sheet 2 of 29 US 2015/0037853 A1 Sissy:3: Figure 2 Patent Application Publication Feb. 5, 2015 Sheet 3 of 29 US 2015/0037853 A1 yruvate O 9 Phosphoenolpyruvate Acetyl-CoA co2 11 b Oxaloacetate Citryl-CoA 2 a s itrate 7b Fumarate co, locatea Oxalosuccinate Succinate 2-oxostant Hco, t CO2 Succinyl-CoA 6 Figure 3 Patent Application Publication Feb. 5, 2015 Sheet 4 of 29 US 2015/0037853 A1 Figure 4 Patent Application Publication Feb. 5, 2015 Sheet 5 of 29 US 2015/0037853 A1 glyoxylate mayl-CoA -2 acetyl-CoA pyruvate Oa malate 1 – HCO 1OC 9 2 malonyl-CoA citramalyl-CoA fumarate 7 2 t 8 23-hydroxypropionate mesaconyl-C4-CoA Succinate 3 1st 2 propionyl-CoA mesaconyl-C1-CoA succinyl-CoA 6 methylmalonyl-CoA 4 Ob methylmalyl-CoA HCO glyoxylate Figure 5 Patent Application Publication Feb. 5, 2015 Sheet 6 of 29 US 2015/0037853 A1 Figure 6 Patent Application Publication Feb. 5, 2015 Sheet 7 of 29 US 2015/0037853 A1 to 1 ner - Hexose 6-P FructOSe 6-P Fructose 1,6-bis-P Dihydroxyacetone-P - 4 6 5 Glyceraldehyde 3-P Erythrose 4-P Fructose 6-P FructOSe 6-P Xylulose 5-P Figure 7 Patent Application Publication Feb. 5, 2015 Sheet 8 of 29 US 2015/0037853 A1 Figure 8 Patent Application Publication Feb. 5, 2015 Sheet 9 of 29 US 2015/0037853 A1 Sedoheptulose 1.7-bis-P-Sedoheptulose 7-P Ribose 5-P Erythrose 4-P Xylulose 5-P Fructose 6-P le. Ribose 5-P 13 Fructose 1,6-bis-P Ribulose 1,5-bis-P Dihydroxyacetone 3-P- --Glyceraldehyde 3-P- -Glycerate 1,3-bis-P- -Glycerate 3-P Figure 9 Patent Application Publication Feb. 5, 2015 Sheet 10 of 29 US 2015/0037853 A1 Figure 10 Patent Application Publication Feb. 5, 2015 Sheet 11 of 29 US 2015/0037853 A1 Succinate 3-hydroxypropionate reductases dehydratase 2 reductases 4-hydroxybutyrate acrylic acid 1,3-propanediol reductases eStefaSe etransaminase eSterase 1,4-butanediolates Y-butyrolactone acrylamide acrylate esters tetrahydrofuran Figure 11 Patent Application Publication Feb. 5, 2015 Sheet 12 of 29 US 2015/0037853 A1 glutamate ... itaconic aci : synthase 8 3 reductases s 5s, Y poly(glutamic acid) tSE C c cy 8 5 w s s w h g C s . t S w s 2-Me-1,4-butanediol glutaric acid o w dehydratase 2-Me-1,4-butanediamine esterases 3-Me-tetrahydrofuran t ->Y-valerolactone v 1,5-pentanediol Figure 12 Patent Application Publication Feb. 5, 2015 Sheet 13 of 29 US 2015/0037853 A1 D-alpha-glucose-6-P 1. beta-D-fructose-6-P 2. D-mannose-6-P 3. D-mannose-1-P 4. GDP-mannose 5. GDP-L-galactose 6. L-galactose-1-P 7. L-galactose Figure 13 Patent Application Publication Feb. 5, 2015 Sheet 14 of 29 US 2015/0037853 A1 yruvate CO2 tomato- 3 5 acetyl-CoA 4 acetaldehyde ethanol Figure 14 Patent Application Publication Feb. 5, 2015 Sheet 15 of 29 US 2015/0037853 A1 glyceraldehyde 3-P pyruvate 1-deoxy-D-xylulose 5-P (DOXP) 2. 2-C-methylerythritol 4-P (MEP) 3. 4-diphophocytidyl-2-C-methylerythritol (CDP-ME) 4. 4-diphophocytidyl-2-C-methylerythritol 2-P (CDP-MEP) 5. 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate (MEcPP) 6. (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) isopenty pyrophosphate (IPP) dimethylallyl pyrophosphate (DMAPP) Figure 15 Patent Application Publication Feb. 5, 2015 Sheet 16 of 29 US 2015/0037853 A1 acetyl-CoA 1-acetoacetyl-CoA 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) 3. mevalonic acid 4. mevalonate-5-P 5. mevalonate-5-pyrophosphate 6 —-coa isopenty pyrophosphate (IPP) 7. dimethylallyl pyrophosphate (DMAPP) Figure 16 Patent Application Publication Feb. 5, 2015 Sheet 17 of 29 US 2015/0037853 A1 dihydroxyacetone-P 1. sn-glycerol-3-P 2. glycerol 3. 3-hydroxypropanal 4. 1,3-propanediol Figure 17 Patent Application Publication Feb. 5, 2015 Sheet 18 of 29 US 2015/0037853 A1 2 acetyl-CoA acetoacetyl-CoA 2 3-hydroxybutyryl-CoA 3 t (addition onto a growing chain) polyhydroxybutyrate Figure 18 Patent Application Publication Feb. 5, 2015 Sheet 19 of 29 US 2015/0037853 A1 L-glutamate Xaloacetate L-aspartate 2-oxoglutarate L-aspartyl-4-P 3. L-aspartate semialdehyde 4. (S)-2,3-dihydrodipicolinate 5. (S)-2,3,4,5-tetrahydrodipicolinate 6. N-succinyl-2-amino-6-ketopimelate 7. N-succinyl-L,L-2,6-diaminopimelate 8. L,L-diaminopimelate 9. meso-diaminopimelate 0. L-lysine Figure 19 Patent Application Publication Feb. 5, 2015 Sheet 20 of 29 US 2015/0037853 A1 3-hydroxypropionyl-CoA 1. propionyl-CoA 2 acetyl-CoA 3-ketovaleryl-CoA 3. (R)-3-hydroxyvaleryl-CoA 4. 3-pentenoyl-CoA 5. 4-hydroxypentanoyl-CoA a Cetate acetyl-CoA 4-hydroxypentanoate 7. Y-valerolactone Figure 20 Patent Application Publication Feb. 5, 2015 Sheet 21 of 29 US 2015/0037853 A1 Negative contro, NADP+ Negative control, NAD+ 4. * Pasrid 2:30, NADP+ * * Plasmid 2438, NA+ 12 * Passniid 2:29, NAD+ * * Plasmid 2428, NAD+ Positive contris, A+ 1 : rt c. 0.8 E 0.6 w t c. O.4 O.2 O O 50 OO 150 200 250 300 350 4OO 450 500 550 600 650 700 750 Time (s) Figure 21 Patent Application Publication Feb. 5, 2015 Sheet 22 of 29 US 2015/0037853 A1 Negative control & wePlasmid 4768 Time (min) Figure 22 Patent Application Publication Feb. 5, 2015 Sheet 23 of 29 US 2015/0037853 A1 14 **** Negative control in Rasic A986 12 . :: Plasmid 4986 to AP * Plasmid 4988, 3d CoAS “Plasmid 4988, to NADPH ^plasmid 4986, a 3-HFA E O.8 O.4 O2 is O O 2CO 4 OO 6OC 80 OOO 1200 400 1600 18OO 2COO Time (s) Figure 23 Patent Application Publication Feb. 5, 2015 Sheet 24 of 29 US 2015/0037853 A1 Stain 2:2 Stair 24, 2-fold ::itic WStrait 242, 4-fold citation ŠStrait 32 & Stair 392 2.500E-03 ; y - 3.48E {4x + 1.28 E-03 2.000E-03 Rs. 9.4.8 1,500E-03 y 59E-Cax + 7.19.83 1.000E-03 5.000E-04 - y - 7, 1 E-35x - 3.58E-04: (.000E+00 y is 400E-35x - 3.163-34 -5.000E-04 »oxw x: : 8,83E-6x S.E.C. -1990E-93 icoscycross OS 5 S. Time (mins) Figure 24 Patent Application Publication Feb. 5, 2015 Sheet 25 of 29 US 2015/0037853 A1 9 8 Standards 7 -MM Fit Formate, mM Figure 25 Patent Application Publication Feb. 5, 2015 Sheet 26 of 29 US 2015/0037853 A1 GltA AspC xaloacetate-Ho-Citraten n Aspartate Malate SOCitrate AspA FumEB an Fumarate 2-oxoglutarate & FrCABCD CO2 Succinateco S Figure 26 Patent Application Publication Feb. 5, 2015 Sheet 27 of 29 US 2015/0037853 A1 s . i res fit 88.3 F-833i a. Patent Application Publication Feb. 5, 2015 Sheet 28 of 29 US 2015/0037853 A1 & 33i: initiatig 8&i Ekx. forcea (miringfiri. E85. Ex_2e)x & imagigFrisis: Ew-hr- Existiev. W firrig .Ji is 38 Figure 28 Patent Application Publication Feb. 5, 2015 Sheet 29 of 29 US 2015/0037853 A1 , Sys,sy. is Y.is Ss sS S. &St. a. 8 & 2 x 18 8 Maximum isooctanol productivity, gll hr Figure 29 US 2015/003 7853 A1 Feb. 5, 2015 METHODS AND SYSTEMIS FOR (twenty minutes), reflective of low total productivities. In CHEMOAUTOTROPHC PRODUCTION OF addition, techniques for genetic manipulation (homologous ORGANIC COMPOUNDS recombination, transformation or transfection of nucleic acid molecules, and recombinant gene expression) are inefficient, STATEMENT REGARDING GOVERNMENT time-consuming, laborious or non-existent. LICENSE RIGHTS 0006. Accordingly, the ability to endow an otherwise het 0001. This invention was made with government support erotrophic organism with chemoautotrophic capability would under contract number DE-AR0000091 awarded by U.S. significantly enable more energy- and carbon-efficient pro Department of Energy, Office of ARPA-E. The government duction of carbon-based products of interest.
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