US010316323B2 (12 ) United States Patent (10 ) Patent No. : US 10 ,316 , 323 B2 South et al. ( 45 ) Date of Patent: Jun . 11, 2019 ( 54 ) MICROORGANISMS ENGINEERED TO USE OTHER PUBLICATIONS UNCONVENTIONAL SOURCES OF NITROGEN Seffernick , et al. (2001 ) , Journal J . Bacteriol. 183 ( 8 ) , 2405 - 2410 ( 2001 ) . * Boundy -Mills et al ., “ The atzB Gene of Pseudomonas sp . Strain (71 ) Applicant: Novogy , Inc. , Cambridge, MA (US ) ADP Encodes the Second Enzyme of a Novel Atrazine Degradation Pathway ,” Applied and Environmental Microbiology , 1997 , 63 ( 3 ): 916 ( 72 ) Inventors : Colin South , Lexington , MA (US ) ; 923 . Arthur J Shaw , IV , Belmont, MA (US ) Cameron et al. , “ New Family of Biuret Hydrolases Involved in S - Triazine Ring Metabolism ,” ACS Catal, 2011, 1 : 1075 - 1082. (73 ) Assignee : Novogy , Inc ., Cambridge , MA (US ) Cheng et al. , “ Allophanate Hydrolase , Not Urease , Function in Bacterial Cyanuric Acid Metabolism ,” Appl. Environ . Microb. , ( * ) Notice : Subject to any disclaimer, the term of this 2005, 71 ( 8 ) :4437 -4445 . patent is extended or adjusted under 35 Copley, “ Evolution of Efficient Pathways for Degradation of U . S . C . 154 ( b ) by 0 days . Anthropogenic Chemicals ,” Nat. Chem . Biol. , 2009, 5 ( 8 ) :559 -566 . De Souza et al ., “ The atzABC Genes encoding Atrazine Catabolism ( 21 ) Appl. No. : 15 /679 , 312 Are Located on a Self - Transmissible Plasmid in Pseudomonas sp . Strain ADP, ” Appl. Environ . Microb , 1998 , 64 (6 ) :2323 -2326 . (22 ) Filed : Aug. 17 , 2017 Dodge et al ., “ Plasmid Localization and Organization of Melamine Degradation Genes in Rhodococcus sp . Strain Mel. ” Applied and (65 ) Prior Publication Data Environmental Microbiology , 78 : 1397 - 1403 . Eaton et al . , “ Cloning and analysis of s - Triazine catabolic genes US 2018 /0051294 A1 Feb . 22 , 2018 from Pseudomonas sp . Strain NRRLB - 12227, ” Journal of Bacte riology , 1991 173 : 1215 - 1222 . Eaton et al. , “ Cloning and comparison of the DNA encoding Related U . S . Application Data ammelide aminohydrolase and cyanuric acid amidohydrolase from three s - Triazine -degrading bacterial strains, ” Journal of Bacteriol (62 ) Division of application No . 14 / 759, 878, filed as ogy , 1991, 173 : 1363 - 1366 . application No. PCT/ US2014 /010332 on Jan . 6 , El- Sayed et al ., “ Biodegradation of melamine formaldehyde by 2014 , now Pat . No. 9 , 765 ,348 . Micrococcus sp . Strain MF - 1 isolated from aminoplastic wastewater effluent, " International Biodeterioration & Biodegradation , 2006 , 57 :75 - 81 . (60 ) Provisional application No .61 /782 ,351 , filed on Mar . Fruchey et al ., “ On the Origins of Cyanuric Acid Hydrolase : 14 , 2013 , provisional application No . 61/ 748, 901 , Purification , Substrates, and Prevalence of AtzD from Pseudomonas filed on Jan . 4 , 2013 . sp . Strain ADP ,” Appl. Environ . Microb . , 2003 , 69 (6 ): 3653 - 3657 . International Search Report and Written Opinion issued in Interna (51 ) Int . CI. tional Patent Application No. PCT /US2014 / 10332 , dated Jun . 24 , C12P 7 / 48 ( 2006 .01 ) 2014 . C12N 15 / 81 ( 2006 .01 ) Kamo et al. , “ Limited distribution of natural cyanamide in higher plants : Occurrence in Vicia villosa subsp . varia , V . cracca , and C12N 1 / 20 ( 2006 .01 ) Robinia pseudo - acacia ,” Phytochemistry , 2008 , 68 : 1166 - 1172 . C12N 9 / 14 ( 2006 .01 ) Kamo et al ., “ Quantification of Cyanamide Contents in Herbaceous C12N 9 / 88 ( 2006 .01 ) Plants , ” Bioscience , Biotechnology , and Biochemistry , 2006 , 70 :2310 C12P 3 / 00 ( 2006 .01 ) 2312 . C12N 15 / 70 ( 2006 .01 ) (Continued ) (52 ) U . S . CI. CPC . .. .. .. C12N 15 /815 (2013 .01 ) ; C12N 1 /20 ( 2013 .01 ) ; C12N 9 / 14 ( 2013 .01 ) ; C12N 9 /88 Primary Examiner — Tekchand Saidha ( 2013 .01 ) ; C12N 15 / 70 ( 2013 .01 ) ; C12N (74 ) Attorney , Agent, or Firm — Norton Rose Fulbright 15 /81 ( 2013 . 01 ) ; C12P 3700 (2013 .01 ) ; C12Y US LLP 308 /01 ( 2013. 01 ) ; C12Y 402/ 01069 ( 2013 .01 ) ( 58 ) Field of Classification Search CPC .. .. .. C12N 15 /815 ; C12N 1 / 20 ; C12N 9 / 14 (57 ) ABSTRACT USPC . .. .. .. .. .. .. 435/ 18 Disclosed are genetically engineered organisms, such as See application file for complete search history . yeast and bacteria , that have the ability to metabolize atypical nitrogen sources, such as melamine and cyanamide . ( 56 ) References Cited Fermentation methods using the genetically engineered U . S . PATENT DOCUMENTS organisms are also described . The methods of the invention are robust processes for the industrial bioproduction of a 2011/ 0008809 AL 1 / 2011 Krebs variety of compounds, including commodities, fine chemi 2011/ 0127108 A1 6 /2011 Teichert 2011/ 0129566 Al 6 /2011 Van Vuuren et al. cals , and pharmaceuticals . FOREIGN PATENT DOCUMENTS 7 Claims, 29 Drawing Sheets WO WO 2003 / 040379 5 / 2003 Specification includes a Sequence Listing. US 10 ,316 ,323 B2 Page 2 ( 56 ) References Cited Shapir et al ., “ Purification and Characterization of Allophanate Hydrolase ( AtzF ) from Pseudomonas sp . Strain ADP, ” J . Bacteriol, 2005, 187 ( 11) :3731 -3738 . OTHER PUBLICATIONS Shapir et al. , “ Purification and Characterization of TrzF : Biuret Karns , “ Gene Sequence and Properties of an s - Triazine Ring Hydrolysis by Allophanate Hydrolase Supports Growth ,” Applied Cleavage Enzyme from Pseudomonas sp . Strain NRRLB - 12227, " and Environmental Microbiology , 2006 , 72 : 2491 - 2495 . 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Bacteriol, 2007 , 189 ( 3 ) :674 682 . * cited by examiner atent Jun . 11 , 2019 Sheet 1 of 29 US 10 , 316 , 323 B2 NN melarine (EC 3 . 5 . 2 . 15 ) carboxybiuret (EC 3 . 5 . 4 . - ) CHON ( spontaneous ) ht 3rmeline biuret ( EC 3 .5 . 4 . 3 ) 41 (EC 3 . 5 . 1 .84 ) NON ammelide MOOM allophante ( EC 3 . 5 . 99 . 4 ) (EC 3 . 5 . 1 .54 ) cyanuric acid 2 N3 + 2 CC , atent Jun . 11 , 2019 Sheet 2 of 29 US 10 , 316 , 323 B2 Figure 2 Compound Hydrazine 88 % 5 - Aminotetrazole CHIN 82 % Tetrazole 80 % Melamine CHON 67 % Cyanamide CH?N 67 % 2 - Cyanoguanidine CHANA Sodium azide NaN ; 65 % Carbohydrazide CHENO 62 % 1 , 2 , 3 - Triazole CH3N3 1 , 2 , 4 - Triazole C2H3N : 1, 3 -Diaminoguanidine HCL Ammeline CzH : N50 55 % 1 , 3 , 5 - triazine Aminoacetonitrile CHAN : Cyanoethylhydrazine C ; H ; N ; Azodicarbonamide Biurea CH .NO2 47 % Formamidoxime CHNO 47 % 1 , 2 -Dimethylhydrazine CH3N2 1 , 1 - Dimethylhydrazine CN3 $ 7 % ethylhydrazine C2H / N2 47 % Ethylenediamine CHsN : 47 % Sodium dicyanamide C2N Na 47 % Guanidine carbonate CHEN3 * 112 Methylamine CHN 45 % Aizunelide CH4N402 Hydroxylamine 42 % Malononitrile 42 % Biuret Ch? Nga 41% Diethyltriamine CH3N3 U . S . Patent Jun . 11, 2019 Sheet 3 of 29 US 10 , 316 , 323 B2 Hexamethylenetetramine CH2N4