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(12) United States Patent (10) Patent No.: US 8,951,752 B2 Subramanian Et Al

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(12) United States Patent (10) Patent No.: US 8,951,752 B2 Subramanian Et Al US008951752B2 (12) United States Patent (10) Patent No.: US 8,951,752 B2 Subramanian et al. (45) Date of Patent: Feb. 10, 2015 (54) N-DEMETHYASE GENES AND USES (58) Field of Classification Search THEREOF USPC .............. 435/18, 252.3, 320.1, 195; 530/350; 536/23.2 (71) Applicant: University of Iowa Research See application file for complete search history. Foundation, Iowa City, IA (US) (56) References Cited (72) Inventors: Venkiteswaran Subramanian, Coralville, IA (US); Michael Tai-Man U.S. PATENT DOCUMENTS Louie, Longmont, CO (US); Ryan Summers, Iowa City, IA (US) 5,550,041 A * 8/1996 Koide et al. ................... 435/119 (73) Assignee: University of Iowa Research FOREIGN PATENT DOCUMENTS Foundation, Iowa City, IA (US) DE 4316882 A1 11F1993 WO WO-2013059507 A1 4/2013 (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 OTHER PUBLICATIONS U.S.C. 154(b) by 0 days. “International Application Serial No. PCT/US2012/060894, Interna Appl. No.: 14/344,037 tional Search Report mailed Mar. 28, 2013', 7 pgs. (21) “International Application Serial No. PCT/US2012/060894, Invita PCT Fled: Oct. 18, 2012 tion to Pay Additional Fees and Partial Search Report mailed Dec. 19. (22) 2012”, 6 pgs. “International Application Serial No. PCT/US2012/060894, Written (86) PCT NO.: PCT/US2O12/060894 Opinion mailed Mar. 28, 2013”.9 pgs. S371 (c)(1), Dash, et al., “Catabolic pathways and Botechnological applications (2) Date: Mar. 10, 2014 of microbial caffeine degradation'. Biotechnology Letters, vol. 28 (Jan. 1, 2006), 1993-2002. PCT Pub. No.: WO2013/059507 Summers, Ryan M. et al., “Characterization of a broad-specificity (87) non-haem iron N-demethylase from Pseudomonas putida CBB5 PCT Pub. Date: Apr. 25, 2013 capable of utilizing several purine alkaloids as sole carbon and nitro gen source', Microbiology (Reading), vol. 157, No. Part 2, (Feb. (65) Prior Publication Data 2011), 583-592. Summers, Ryan M. et al., “Novel, Highly Specific N-Demethylases US 2014/O227729 A1 Aug. 14, 2014 Enable Bacteria to Live on Caffeine and Related Purine Alkaloids', Journal of Bacteriology, vol. 194, No. 8 (Apr. 2012), 2041-2049. Related U.S. Application Data Yu, Chi Li, et al., “Two Distinct Pathway for Metabolism of Theophylline and Caffeinea are Coexpressed in Pseudomonasputida (60) Provisional application No. 61/549,339, filed on Oct. CBB”, Journal of Bacteriology, vol. 191, No. 14. (Jul. 2009), 4624 20, 2011. 4632. “International Application Serial No. PCT/US2012/060894, Interna (51) Int. C. tional Preliminary Report on Patentability mailed May 1, 2014'. 10 CI2O I/34 (2006.01) pg.S. CI2N IS/00 (2006.01) CI2N L/20 (2006.01) * cited by examiner CI2N 9/14 (2006.01) C07K L/00 (2006.01) Primary Examiner — Maryam Monshipouri C7H 2L/04 (2006.01) (74) Attorney, Agent, or Firm — Schwegman Lundberg & CI2N 9/02 (2006.01) Woessner, P.A. CI2P 17/18 (2006.01) CI2O I/26 (2006.01) (57) ABSTRACT (52) U.S. C. The invention provides isolated nucleic acid encoding one or CPC ............. CI2N 9/0071 (2013.01); C12Y 1 14/13 more gene products that allow for degradation of caffeine and (2013.01); C12Y 114/13128 (2013.01); CI2N related structures, and for preparation of intermediates in that 9/0073 (2013.01); CI2P 17/182 (2013.01); catabolic pathway. Further provided are methods of using one CI2O I/26 (2013.01) or more of those gene products. USPC ....... 435/18; 435/320.1; 435/252.3; 435/195; 530/350,536/23.2 19 Claims, 20 Drawing Sheets U.S. Patent Feb. 10, 2015 Sheet 1 of 20 US 8,951,752 B2 4 20 40 Time (min) FIG, 1 U.S. Patent Feb. 10, 2015 Sheet 2 of 20 US 8,951,752 B2 eouec OScy wg+cz1 VZ'9IAI ?-- . 09 A8 U.S. Patent Feb. 10, 2015 Sheet 3 of 20 US 8,951,752 B2 U.S. Patent Feb. 10, 2015 Sheet 4 of 20 US 8,951,752 B2 8 O S 8 Time (min) FIG, 4 8 400 O & S 8 CO Tire (min) FIG, .5 U.S. Patent Feb. 10, 2015 Sheet 5 of 20 US 8,951,752 B2 „Hz+ U.S. Patent Feb. 10, 2015 Sheet 6 of 20 US 8,951,752 B2 U.S. Patent Feb. 10, 2015 Sheet 7 of 20 US 8,951,752 B2 OZ(9IAI U.S. Patent Feb. 10, 2015 Sheet 8 of 20 US 8,951,752 B2 it treestraatia U.S. Patent Feb. 10, 2015 Sheet 9 of 20 US 8,951,752 B2 U.S. Patent Feb. 10, 2015 Sheet 10 of 20 US 8,951,752 B2 __)~~~~ 24 U.S. Patent US 8,951,752 B2 U.S. Patent Feb. 10, 2015 Sheet 13 of 20 US 8,951,752 B2 XXXXXXXXXXXXXXXXX sww.sixxxswwM S S S sS. S S. SS -xxxx w w y & s & s& S S $ S S & S Sssssssssssssssssssssssssssssssssss sississsssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssss s Wavelergh (ne FIG, 8B S. S s S S royeesepoyeeseS & w , s X. 4xx XXXS sy s SSS S&S s xx S& & SSSX s: www.ss S. &S. s XS SS. S S.roodoicolodolo S. X to S: Wavelenger FIG, 8C U.S. Patent Feb. 10, 2015 Sheet 14 of 20 US 8,951,752 B2 St. O 2 s Time (min) FIG 9A O 3. Time (min) FIG 9B U.S. Patent Feb. 10, 2015 Sheet 15 of 20 US 8,951,752 B2 U.S. Patent US 8,951,752 B2 G/OUupN U.S. Patent US 8,951,752 B2 U.S. Patent Feb. 10, 2015 Sheet 20 of 20 US 8,951,752 B2 (?nZ)quen??ud?SO 'suuun?00??J???Mpº?ndSeMJOO-SIH US 8,951,752 B2 1. 2 N-DEMETHYASE GENES AND USES The holoenzyme included a reductase component with cyto THEREOF chrome reductase activity (CCr) and a soluble, 2 subunit N-demethylase (Ndim) that demethylates caffeine to Xan CROSS-REFERENCE TO RELATED thine, with a native molecular mass of 240,000 Da. The two APPLICATIONS subunits of Ndm, designated as NdmA and NdmB, displayed This application is a U.S. National Stage Filing under 35 apparent Mr of 40 and 35 kDa, respectively. Cer transfers U.S.C. 371 from International Application No. PCT/US2012/ reducing equivalents from NAD(P)H to Ndim, and the NAD 060894, filed on 18 Oct. 2012, and published as WO 2013/ (P)H-dependent reaction only occurs when the reductase 059507 A1 on 25 Apr. 2013, which application claims the component couples with Ndim and Ndm consumes one mol benefit of the filing date of U.S. application Ser. No. 61/549, 10 ecule of oxygen per methyl group that is removed from caf 339, filed on Oct. 20, 2011, which applications and publica feine as formaldehyde. Paraxanthine and 7-methylxanthine tions are incorporated by reference herein. were determined to be substrates with apparent K, and k, BACKGROUND values of 50.4+6.8 uM and 16.2+0.6 min' and 63.8+7.5 uM 15 and 94.8+3.0 min', respectively. Ndimalso displayed activ Caffeine (1,3,7-trimethylxanthine) and related methylxan ity towards caffeine, theobromine, theophylline, and 3-meth thines are widely distributed in many plant species. Caffeine ylxanthine, all of which are growth Substrates for this organ is also a major human dietary ingredient that can be found in ism. Ndm was deduced as a Rieske 2Fe-2S domain common beverages and food products, such as coffee, tea, containing non-heme iron oxygenase based on N-terminal and chocolates. In pharmaceuticals, caffeine is used generally sequencing, UV absorbance spectrum, and iron content. as a cardiac, neurological, and respiratory stimulant, as well N-demethylase genes were isolated from a genomic DNA as a diuretic (Dash et al., 2006). Hence, caffeine and related library of Pseudomonas putida CBB5 using a nested PCR methylxanthines enter soil and water easily through decom approach, and were cloned individually into an expression posed plant materials and other means, such as effluents from vector as C-terminal His-tagged fusion proteins. The gene coffee- and tea-processing facilities. Therefore, it is not Sur 25 sequences of NdmA and NdmB were most similar to the prising that microorganisms capable of degrading caffeine catalytic subunits of other Rieske oxygenases known to have been isolated from various natural environments, withor cleave C O and C N bonds. The E. coli expressed proteins without enrichment procedures (Dash et al., 2006; Maz were purified using a Ni-NTA column. Upon expression of Zafera, 2004). Bacteria use oxidative and N-demethylating the cloned Ndim genes, NdmA-His alone plus partially puri pathways for catabolism of caffeine. Oxidation of caffeine by 30 fied reductase was capable of N-demethylating caffeine to a Rhodococcus sp.-Klebsiella sp. mixed-culture consortium at the C-8 position to form 1,3,7-trimethyluric acid (TMU) theobromine (TB) and was determined to be highly specific has been reported (Madyastha and Sridnar, 1995). An for the N-1 methyl group. In contrast, NdmB-His alone plus 85-kDa, flavin-containing caffeine oxidase was purified from reductase was highly specific for N-3 methyl group, produc this consortium (Madyastha et al., 1996). Also, Mohapatra et 35 ing paraxanthine (PX) from caffeine. NdmA and B, which al. (2006) purified a 65-kDa caffeine oxidase from Alcali were not separable via purification from the wild-type, were genes sp. Strain CF8. Cells of a caffeine-degrading fully resolved by genetic approach. Thus, two positional Pseudomonas putida strain (ATCC 700097) isolated from specific N-demethylases were purified from P.
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