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(12) United States Patent (10) Patent No.: US 8,455,236 B2 Beck Et Al

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(12) United States Patent (10) Patent No.: US 8,455,236 B2 Beck Et Al USOO845.5236B2 (12) United States Patent (10) Patent No.: US 8,455,236 B2 Beck et al. (45) Date of Patent: Jun. 4, 2013 (54) COMPOSITIONS AND METHODS OF PGL WO WO 2009,076676 A2 6, 2009 FOR THE INCREASED PRODUCTION OF WO WO 2009,076676 A3 6, 2009 ISOPRENE WO WO 2009,132220 A9 10/2009 WO WO 2010/OO3OOT A2 1, 2010 WO WO 2010/OO3OOT A3 1, 2010 (75) Inventors: Zachary Q. Beck, Palo Alto, CA (US); WO WO 2010/O14825 A1 2, 2010 Marguerite A. Cervin, Redwood City, WO WO 2010/078457 A2 T 2010 WO WO 2010/078457 A3 T 2010 CA (US); Alex T. Nielsen, Rungsted WO WO 2010, 124146 A2 10/2010 Kyst (DK); Caroline M. Peres, Palo WO WO 2010, 124146 A3 10, 2010 Alto, CA (US) WO WO 2010, 148144 A1 12/2010 (73) Assignee: Danisco US Inc., Palo Alto, CA (US) OTHER PUBLICATIONS (*) Notice: Subject to any disclaimer, the term of this Thomason et al. (J Bacteriol. Dec. 2004; 186(24):8248-53.* Balbas et al. Gene. Jun. 12, 1996; 172(1):65-9.* patent is extended or adjusted under 35 Martin et al. Nat Biotechnol. Jul. 2003:21(7):796-802. Epub Jun. 1, U.S.C. 154(b) by 0 days. 2003.* Sasaki et al. FEBS Lett. Apr. 25, 2005:579(11):2514-8.* (21) Appl. No.: 12/978,324 Anderson, M.S. et al., (1989). “Isopentenyl Diphosphate: Dimethlal lyl Diphosphate Isomerare. An Improved Purification of the Enzyme (22) Filed: Dec. 23, 2010 and Isolation of the Gen From Saccharomyces cerevisia,” J. Biol. Chem. 264(32): 19169-19175. (65) Prior Publication Data Aon, J. et al., (2008) "Suppressing Posttranslational Gluconoylation US 2011/O1595.57 A1 Jun. 30, 2011 of Heterologous Proteins by Metabolic Engineering of Escherichia coli,' Appl. Environ. Microbiol. 74:950-958. Baba, T. et al., (2006) “Construction of Escherichia coli K-12 In Related U.S. Application Data Frame, Single-Gene Knockout Mutants: The Keio Collection.” Mol. Syst. Biol. 2: 2006.0008. (60) Provisional application No. 61/289.959, filed on Dec. Bouvier, F. et al., (2005) “Biogenesis, Moleculars Regulation and 23, 2009. Function of Plant Isoprenoids.” Progress in Lipid Res. 44:357-429. Campbell, E. et al., (1989) “Improved Transformation Efficiency of (51) Int. Cl. Aspregillus niger Using Homologous niaD Gene for Nitrate CI2P 7/42 (2006.01) Reductase. Curr. Genet. 16:53-56. CI2P 5/02 (2006.01) Cherepanov, P.P. et al., (1995) “Gene Disruption in Escherichia coli: CI2N 9/00 (2006.01) Tc' and Km' Cassettes with the Option of Flp-Catalyzed Excision of CI2N L/20 (2006.01) the Antibiotic-Resistance Determinant.” Gene 158(1):9-14. CI2N IS/00 (2006.01) Datsenko, K., et al., (2000) "One-Step Inactivation of Chromosomal C7H 2L/04 (2006.01) Genes in Escherichia coli K-12 Using PCR Products.” Proc. Nat. Acad. Sci. USA 97:6640-6645. (52) U.S. Cl. Dhe-Paganon, S. et al., (1994). “Mechanism of Mevalonate USPC ........ 435/252.3; 435/146; 435/167; 435/183; Pyrophosphate Decarboxylase: Evidence for a Carbocationic Tran 435/320.1; 536/23.2 sition State.” Biochemistry 33(45): 13355-13362. (58) Field of Classification Search EcoGene Accession No. EG13231, located at <www.ecogene.org>, None last visited on Jun. 25, 2012, 1 page. See application file for complete search history. GenBank Accession No. AAG06570.1, located at <http://www.ncbi. nlm.nih.gov), last visited on Jun. 25, 2012, 2 pages. (56) References Cited GenBank Accession No. AE004091, located at < http://www.ncbi. nlm.nih.gov), last visited on Jun. 25, 2012, 908 pages. GenBank Accession No. NC 001140, located at <http://www.ncbi. U.S. PATENT DOCUMENTS nlm.nih.gov), last visited on Jun. 25, 2012, 204 pages. 7,132,527 B2 11/2006 Payne et al. GenBank Accession No. U00096, located at <http://www.ncbi.nlm. 2009,0203102 A1 8, 2009 Cervin et al. nih.gov), last visited on Jun. 25, 2012, 352 pages. 2010.0003716 A1 1/2010 Cervin et al. 2010, 004.8964 A1 2/2010 Calabria et al. Grawert, T. et al., (2004). “IspH Protein of Escherichia coli: Studies 2010, OO86978 A1 4/2010 Becket al. on Iron-Sulfur Cluster Implementation and Catalysis,” Journal 2010.0113846 A1 5, 2010 McAuliffe et al. American Chemistry Society, 126(40): 12847-12855. 2010.016737O A1 7, 2010 Chotani et al. 2010.01673.71 A1 7, 2010 Chotani et al. (Continued) 2010, O184178 A1 7, 2010 Becket al. 2010.0196977 A1 8, 2010 Chotani et al. Primary Examiner — Christian Fronda FOREIGN PATENT DOCUMENTS (74) Attorney, Agent, or Firm — Morrison & Foerster LLP EP 1510 583 A1 3, 2005 (57) ABSTRACT WO WO96,35796 A1 11, 1996 WO WO 98.02550 A2 1, 1998 Provided herein are improved compositions and methods for WO WO 98.02550 A3 1, 1998 the increased production of isoprene. Also provided herein WO WO 2004/003175 A2 1, 2004 are improved compositions and methods for the increased WO WO 2004/003175 A3 1, 2004 WO WO 2004/033646 A2 4/2004 production of heterologous polypeptides capable of biologi WO WO 2004/033646 A3 4/2004 cal activity. WO WO 2005/080583 A2 9, 2005 WO WO 2005/080583 A3 9, 2005 15 Claims, 66 Drawing Sheets US 8,455,236 B2 Page 2 OTHER PUBLICATIONS Self, W.T. et al., (2001). “Molybdate Transport.” Res Microbiol. Grunden, A.M. et al., (1997). “Molybdate Transport and Regulation 152:311-321. in Bacteria, Arch Microbiol. 168:345-354. Sharkey, T. et al., (2005). “Evolution of the Isoprene Biosynthetic Hedl, M. et al., (2002). “Enterococcus faecalis Acetoacetyl-Coen Pathway in Kudza.” Plant Physiology 137:700-712. Zyme A Thiolase/3-Hydroxy-3-Methyglutary-Coenzyme A Silver, G. et al., (1995). “Characterization of Aspen Isoprene Reductase, A Dual-Function Protein of Isopentenyl Diphosphate Synthase. An Enzyme Responsible for Leaf Isoprene Emission to the Biosynthesis,” J. Bacteriol. 184(8):2116-2122. Atmosphere.” JBC 270(22): 13010-13016. Hoeffler, J-F. et al., (2002). “Isoprenoid Biosynthesis via the Sinha, A. et al., (1992). “Induction of Specific Enzymes of the Oxi Methylerythritol Phosphate Pathway. Mechanistic Investigations of the 1-Deoxy-D-Xylulose 5-Phosphate D Reductiosimerase.” Eur: J. dative Pentose Phosphate Pathway by Glucono-6-Lactone in Sac Biochen. 269:4446-4457. charomyces cerevisiae.” J. Gen. Microbiol. 138:1865-1873. International Search Report mailed on Jun. 15, 2011, for PCT Patent Sprenger, G.A. et al., (1997). “Identification of a Thiamin-Dependent Application No. PCT/US2010/062099, filed Dec. 23, 2010, 5 pages. Synthase in Escherichia coli Required for the Formation of the Koga, et al., (2007). "Biosyntheseis of Ester-Type Polar Lipids in 1-DeoxY-o-Xylulose 5-Phosphate Precursor to Isoprenoids, Archea and Evolutionary Consideration.” Microbiology and Mol. Thiamin, and Pyridoxol.” PNAS 94:12857-12862. Biology Reviews 71(1):97-120. Studier F.W. et al., (2009). “Understanding the Differences Between Kutsche, M. et al., (Apr. 1996). “Promoters Controlling Expression Genome Sequences of Escherichia coli B Strains REL606 and of the Alternative Nitrogenase and the Molybdenum Uptake System BL21 (DE3) and Comparison of the E. coli B and K-12 Genomes.”J. in Rhodobacter capsulatus Are Activated by NtrC, Independent of Mol. Biol. 394(4):653-680. o', and Repressed by Molybdenum.” Journal of Bacteriology Sutherlin, A. et al., (2002). “Enterococcus faecalis 3-Hydrozy-3- 178(7):2010-2017. Methylglutaryl Coenzyme A Synthase. An Enzyme of Isopentenyl Luttgen, H. etal. (2000). "Biosynthesis of Terpenoids:YchB Protein Diphosphate Biosynthesis,” J. Bacteriol. 184(15):4065-4070. Escherichia coli Phosphorylates the 2-Hydrozy Group of Thomason, L.C. et al., (Dec. 2004). “Identification of the Escherichia 4-Diphosphocytidyl-2C-Methyl-o-Erythritol.” PNAS 97(3):1062 coli K-12 ybhF Gene as pgil, Encoding 6-Phosphogluconolactonase.” 1067. J. Bact. 186(24):8248-8253. Miclet, E. et al., (Sep. 14, 2001). “NMR Spectroscopic Analysis of Thomason, L.C., (Jul. 2007). 'E. coli Genome Manipulation by Pl the First Two Steps of the Pentose-Phosphate Pathway Elucidates the Transduction.” Curr: Protocols Mol. Biol. Chapter 1, Unit 1.17. Role of 6-Phosphogluconolactonase.”.J. Biol. Chem. 276(37):34840 Tsay, Y.H. et al., (1991). “Cloning and Characterization of ERG8, An 34846. Essential Gene of Saccharomyces cerevisiae That Encodes Miller, B. etal. (2001). “First Isolation of an Isoprene Synthase Gene From Poplar and Successful Expression of the Gene in Escherichia Phosphomevalonate Kinase.” Mol Cell Biol. 11(2):620-631. coli. Planta 2 13:483-487. UniProtKB/Swiss-Prot Accession No. P38858 (PGL polypeptide), Oulmouden, A. et al., (1991). "Nucleotide Sequence of the ERG 12 located at <http://www.ncbi.nlm.nih.gov), last visited on Jun. 25. Gene of Saccharomyces cerevisiae EncodinO Mevalonate Kinase.” 2012, 3 pages. Curr Genet. 19:9-14. UniProtKB/Swiss-Prot Accession No. P52697 (PGL polypeptide), Rohdich, F. et al., (1999). "Cytidine 5'-Triphosphate-Dependent located at <http://www.ncbi.nlm.nih.gov), last visited on Jun. 25. Biosynthesis of Isoprenoids: YgbP Protein of Escherichia coli Cata 2012, 7 pages. lyzes the Formation of 4-Diphosphocytidyl-D-2-C- Zepeck, F. et al., (2005). "Biosynthesis of Isoprenoids. Purification Methylerythritol.” PNAS 96(21): 11758-11763. and Properties of IspG Protein from Escherichia coli,'.J. Org. Chem. Rohdich, F. et al., (2000). "Biosynthesis of Terpenoids: TO:9168-9174. 4-Diphosphocytidly-2C-Methyl-o-Erythritol Synthase of Arabidopsis thaliana.” PNAS97(12):6451-6456. * cited by examiner U.S. Patent Jun. 4, 2013 Sheet 2 of 66 US 8,455,236 B2 Figure B. 2 CECO-S-CoA acetyl-CoA o o HC-C-Cig SCoA acetoacetyl-CoA Chico-S-CoA CO-S-CoA Chi HC-C-OH G-CoA E66HC 3 y CHOH CH2 HCCOH iseyasi acid 666HC * six CHO-P Chi Hscg3.
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