US009353390B2 (12) United States Patent (10) Patent No.: US 9,353,390 B2 Yan et al. (45) Date of Patent: May 31, 2016 (54) GENETICALLY ENGINEERED MICROBES National Center for Biotechnology Information, National Library of AND METHODS FOR PRODUCING Medicine, National Institutes of Health, GenBank Locus 4-HYDROXYCOUMARIN NP 252920. Accession No. NP 252920, "isochorismate-pyruvate lyase IPseudomonas aeruginosa PAO1),” online). Bethesda, MD (71) Applicant: University of Georgia Research retrieved on Aug. 3, 2015. Retrieved from the Internet: <URL: Foundation, Inc., Athens, GA (US) http://www.ncbi.nlm.nih.gov/protein/NP 252920. 1; 3 pgs. National Center for Biotechnology Information, National Library of (72) Inventors: Yajun Yan, Athens, GA (US); Yuheng Medicine, National Institutes of Health, GenBank Locus Lin, Marietta, GA (US) NP 415125. Accession No. NP 415125. "isochorismate synthase 1 Escherichia coli str. K-12 substr. MG 1655, online. Bethesda, (73) Assignee: University of Georgia Research MD retrieved on Aug. 3, 2015). Retrieved from the Internet: <URL: http://www.ncbi.nlm.nih.gov/protein/NP 415125.1.>; 3 pgs. Foundation, Inc., Athens, GA (US) Ajikumar et al. “Isoprenoid Pathway Optimization for Taxol Precur (*) Notice: Subject to any disclaimer, the term of this sor Overproduction in Escherichia coli" Science, Oct. 1, 2010; 330(6000):70-4. patent is extended or adjusted under 35 Anthony et al. “Optimization of the mevalonate-based isoprenoid U.S.C. 154(b) by 0 days. biosynthetic pathway in Escherichia coli for production of the anti malarial drug precursor amorpha-4,11-diene' Metab. Eng. Jan. 2009; (21) Appl. No.: 14/304,105 11(1): 13-9. Epub Aug. 12, 2008. Atsumietal. “Non-fermentative pathways for synthesis of branched (22) Filed: Jun. 13, 2014 chain higher alcohols as biofuels' Nature, Jan. 3, 2008; (65) Prior Publication Data 451(7174):86-9. 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CPC ............... CI2P 17/06 (2013.01); C07D 31 1/46 Chen et al. “Biosynthesis of salicylic acid in plants' Plant Signal Behav. 2009; 4(6):493-6. Epub Jun. 12, 2009. (2013.01); C07D 311/56 (2013.01); C12N Cohen et al. "Venous thromboembolism (VTE) in Europe. The num 15/52 (2013.01) ber of VTE events and associated morbidity and mortality” Thromb. (58) Field of Classification Search Haemost. 2007: 98(4):756-64. None Daruwala et al., “Menaquinone (vitamin K2) biosynthesis: See application file for complete search history. overexpression, purification, and characterization of a new isochorismate synthase from Escherichia coli'J. Bacteriol. 1997; (56) References Cited 179(10), 3133-8. Dhamankar et al. “Microbial chemical factories: recent advances in U.S. PATENT DOCUMENTS pathway engineering for synthesis of value added chemicals' Curr: Opin. Struct. Biol. Aug. 2011; 21(4):488-94. 4,703,004 A 10/1987 Hopp et al. 4,782,137 A 1 1/1988 Hopp et al. (Continued) 5,594,115 A 1/1997 Sharma 5,935,824 A 8/1999 Sgarlato Primary Examiner — Nashaat Nashed OTHER PUBLICATIONS (74) Attorney, Agent, or Firm — Mueting, Raasch & Gebhardt, PA. National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, GenBank Locus BAC78380, (57) ABSTRACT Accession No. BAC78380, “putative salicyl-AMP ligase Provided herein are methods for the biosynthesis of 4-hy Streptomyces sp. WA46).” online. Bethesda, MD retrieved on droxycoumarin. In one embodiment, provided herein are Aug. 3, 2015). Retrieved from the Internet: <URL: http://www.ncbi. genetically engineered microbes that include a metabolic nlm.nih.gov/protein/BAC78380.1 >; 2 pgs. pathway for the production of 4-hydroxycoumarin. 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