1111111111111111111i11111n11111uu1111111111u~ (12) United States Patent (io) Patent No.: US 10,131,585 B2 Newman et al. (45) Date of Patent: Nov. 20, 2018 (54) HOPANOIDS PRODUCING BACTERIA AND Witkowski et al., (Biochemistry 38:11643-11650, 1999.* RELATED BIOFERTILIZERS, Kisselev L., (Structure, 2002, vol. 10: 8-9.* COMPOSITIONS, METHODS AND SYSTEMS Komanieck et al. JBC 2014, 289, pp. 35644-35655.* Poonia et al. Intl. j. of Eng and Man res 2011 pp. 36-38.* Battacharjee et al.( App Microbiol Biotech 2008, pp. 199-209.* (71) Applicant: CALIFORNIA INSTITUTE OF Bravo et al.: Fur J. Biochem 2001,268, 1323-1331.* TECHNOLOGY, Pasadena, CA (US) Casadaban, M.J. et al., "Analysis of Gene-Control Signals by DNA-Fusion and Cloning in Escherichia-coli", Journal of Molecu- (72) Inventors: Dianne K. Newman, Pasadena, CA lar Biology, 138(2), pp. 179-207, (1980). (US); Gargi Kulkarni, Pasadena, CA Ehrhardt, D.W. et al., "Depolarization of Alfalfa Root Hair Mem- (US); Brittany Jo Belin, Pasadena, CA brane Potential by Rhizobium meliloti Nod Factors", 256(5059), pp. 998-1000, (1992). (US); Eric Geraud, Marseilles (FR); Kannenberg, E.L. et al., "The Occurrence of Hopanoid Lipids in Antonio Molinaro, Napoli (IT); Alba Bradyrhizobium Bacteria", FEMS Microbiology Letters, 127(3), Silipo, Napoli (IT) pp. 255-261, (1995). Leontein, K. et al., "Assignment of Absolute-Configuration of (73) Assignee: CALIFORNIA INSTITUTE OF Sugars by g.l.c of Their Acetylated Glycosides Formed from Chiral TECHNOLOGY, Pasadena, CA (US) Alcohols", Carbohydrate Research, 62(2), pp. 359-362, (1978), 5 pages. Markowitz, V.M. et al., "Comparative Genome Analysis in the (*) Notice: Subject to any disclaimer, the term of this Integrated Microbial Genomes (IMG) System", Methods Mol. patent is extended or adjusted under 35 Biol., 395, pp. 35-56, (2007), 23 pages. U.S.C. 154(b) by 0 days. Oldroyd, G.E. et al., "The Rules of Engagement in the Legume- Rhizobial Symbiosis", Annu Rev Genet, 45, pp. 119-144,(2011), 28 (21) Appl. No.: 15/298,172 pages. Pearson, W.R. "Searching Protein Sequence Libraries: Comparison (22) Filed: Oct. 19, 2016 of the Sensitivity and Selectivity of the Smith-Waterman and FASTA Algorithms", Genomics, 11(3), pp. 635-650. 16 pages, (1991). (65) Prior Publication Data Regensburger, B.et al.,"RNA-Polymerase from Rhizobiumjaponieum", US 2017/0107160 Al Apr. 20, 2017 Archives of Microbiology, 135(2), pp. 103-109, (1983), 9 pages. Schafer, A. et al., "Small Mobilizable Multi-Purpose Cloning Vec- tors Derived from the Escherichia coli Plasmids pK18 and pK19: Related U.S. Application Data Selection of Defined Deletions in the Chromosome of Corynebacterium (60) Provisional application No. 62/243,418, filed on Oct. glutamicum", Gene, 145(1), pp. 69-73, (1994). Simon, R. et al., "A Broad Host Range Mobilization System for in 19, 2015. vivo Genetic-Engineering: Transposon Mutagenesis in Gram- Negative Bacteria", Bio-Technology, 1(9), pp. 784-791,(1983), 10 (51) Int. Cl. pages. AOIN 43/40 (2006.01) Talbot, H.M. et al., "Structural Characterisation of Unsaturated C12N 1/00 (2006.01) Bacterial Hopanoids by Atmospheric Pressure Chemical Ionisation C12N 15/52 (2006.01) Liquid Chromatography/Ion Trap Mass Spectrometry", Rapid Com- C12N 15/82 (2006.01) munications in Mass Spectrometry, 21, pp. 1613-1622. 10 pages, (2007). C05F 11/08 (2006.01) Talbot, H.M. et al., "Characteristic Fragmentation of C12N 1/20 (2006.01) Bacteriohopanepolyols During Atmospheric Pressure Chemical Ionisa- C12N 1114 (2006.01) tion Liquid Chromatography/Ion Trap Mass Spectrometry", Rapid AOIN 63100 (2006.01) Communications in Mass Spectrometry, 17, pp. 2788-2796. 9 (52) U.S. Cl. pages, (2003). CPC ................ C05F 11/08 (2013.01); C12N 1/20 Westphal, O. et al., `Bacterial Lipopolysaccharide-Extraction with (2013.01); AOIN 63100 (2013.01); C12N 1114 Phenol-Water and Further Applications of the Procedure', Methods (2013.01); C12N 15152 (2013.01); C12N 15182 Carbohydr Chem, 43, pp. 83-91, (1965). (2013.01); C12Y 402/01084 (2013.01) * cited by examiner (58) Field of Classification Search CPC . C12N 1/20; C05F 11/00; C05F 11/08; GO1N Primary Examiner Robert B Mondesi 33/92 Assistant Examiner Mohammad Y Meah USPC ............ 435/252.2, 320.1, 157, 243; 504/101 (74) Attorney, Agent, or Firm Steinfl + Bruno LLP See application file for complete search history. (57) ABSTRACT (56) References Cited Hopanoids, hopanoids-producing nitrogen-fixing bacteria, U.S. PATENT DOCUMENTS and related formulations, systems and methods are described herein. In particular, hopanoids alone or in combination with 9,187,381 B1 * 11/2015 Lanz ...................... AOIN 63/02 hopanoid-producing nitrogen-fixing bacteria can be used as biofertilizer to stimulate plant growth and yield with OTHER PUBLICATIONS enhanced tolerance to diverse stresses found in plant-mi- crobe symbiotic microenvironments. Devos et al., (Proteins: Structure, Function and Genetics, 2000, vol. 41: 98-107).* 26 Claims, 14 Drawing Sheets Whisstock et al., (Quarterly Reviews of Biophysics 2003, vol. 36 (3): 307-340.* Specification includes a Sequence Listing. U.S. Patent Nov. 20, 2018 Sheet 1 of 14 US 10,131,585 B2 A} off I R ON H R R R VO, Hop-97(21)-ene (II) Hop42(29)-ene Hopan-21-ene (V) Hopan-22-o1 Tetrahymanol (VII) (IV, Diploptene) (VI, Diplopterol) H ON N ON ON r i„ 1 R...... a;.. Hz R '. 6N OHL Fl, O H BHP-608 (Vill) Aminobacteriohopanetriol (a) Bacteriohopanetetrol(b) Adenosylhopane(d) R,= H.,CH, B} Na~a:: €3Etttl~i FIG. 1 U.S. Patent Nov. 20, 2018 Sheet 2 of 14 US 10,131,585 B2 A) _ of OH OH 20 1 ~•._~~r OH~352 Hopanoid Totrahymanol Rs H. or CH,: R,= OH or NH, B) ~S)4~~r`r$~GXXhQ,~ r4~~ re~a ,~> >=<I 4llJDlL'lDi> < 1 L—><'-", I I Shc EE 't Hpnp ~ll T-methyldiplopcene HprtH Rffi, adenosyl hopane -~E OH OH 100.: -IV WT VEI 50 I /V V) J Vili X100 • ` o 'I I AhpnP; d q + R gs100, tori_; p --, c~ Ah nH I 50 16 18 20 22 24 26 T28 30 32 34 36 38 40 Time (min) FIG. 2 U.S. Patent Nov. 20, 2018 Sheet 3 of 14 US 10,131,585 B2 Root Nodule Nodule cross-section Plant Cells Symbiosome 3~ i. ------- Nodule Cortical Cells Infected Zone Plant Nucleus Bacteroids Plant Cell Walt Bacteroid Plant Cell Wall Peribacteroid Plant Membrane FIG.3 U.S. Patent Nov. 20, 2018 Sheet 4 of 14 US 10,131,585 B2 Penta-acylated Hexa-acylated Lipid A Lipid A 2622.84 1001 12128-36 WT Hepta-acylated Lipid A 3135.22 so + Hopanoid + Hopanoid A--------------y Traces of 2'°' hopanoid 10-.~_. .w , 36:36.67 Hem- acylated Lipid A 100 2607.56 AhpnA .wy 4)50-Hepta-acylated Lipid A Penta-acylated 3121.09 Lipid A + Hopanoid + Hopanoid o — Traces of 2128.96 211 hopanoid I 10 1 ... ~.ILI.,...i_... ,....aJ III 3636.11 2000 2360 2720 Hexa-acylated Lipid A 100-1 12609.53 AhpnH Penta-acylated Lipid A trace t212 80 10 LiIr~YY.l..,i 2000 2360 2720 30 Mass (mlz) FIG.4 U.S. Patent Nov. 20, 2018 Sheet 5 of 14 US 10,131,585 B2 OA ~nrUe1 r. yn<». 0.38 0.38 0.34 0.32 0,3 WT AhpnP AhpnH FIG. 5 U.S. Patent Nov. 20, 2018 Sheet 6 of 14 US 10,131,585 B2 FIG.6 U.S. Patent Nov. 20, 2018 Sheet 7 of 14 US 10,131,585 B2 Q)Aerobic -30-C B)Aerobic - 37°C 1.5 1.5 LC X: ` :• 1 Cl 0.5 0.5 ... 0 S0 100 ISO 200 p $0 1DO 150 200 F) G) = 5 1 E+ly N 4 i 0 3< **> 1E+4+ 2' s! * QlE+2i --- SDS H2O, HC1 11H2O LNCR336 •WT C_ AhpnP `; xAhpnH FIG.7 U.S. Patent Nov. 20, 2018 Sheet 8 of 14 US 10,131,585 B2 B r G AhpnH AhpnP FIG.8 U.S. Patent Nov. 20, 2018 Sheet 9 of 14 US 10,131,585 B2 B Q ~ FIG.9 U.S. Patent Nov. 20, 2018 Sheet 10 of 14 US 10,131,585 B2 m 1 n 1 9 d.p.i. 14 d.p.i. 21 d.p.i. c B m a 0.8 .c 0.6 0 40.4 U Q 0.2 a 9 d.pJ. 14 d.pJ. 21 d.p.i. WT 'OhpnH ❑.AhpnP FIG. l.0 U.S. Patent Nov. 20, 2018 Sheet 11 of 14 US 10,131,585 B2 1 hpnP she hpnN ~Y M FIG. 11 U.S. Patent Nov. 20, 2018 Sheet 12 of 14 US 10,131,585 B2 FIG. 12 U.R. Patent &nv 20 2018 Sheet 13 of 14 US 10,131,383 G2 a.. > w z .....: .. .. .. :+ ~ \ ©~'~.yy#< «_ma+: . :.. ; w , l 2. ''y :. .. ~4,4 13 FIG. 13 U.S. Patent Nov. 20, 2018 Sheet 14 of 14 US 10,131,585 B2 Fl vMSX 6111% ~3. r,„ ..... ............................................. ............. C. LO m Q 4 8 8 10 13 16 19 22 25 28 31 34 37 40 dpi ~ {1 A { C. ,C G A ...... fiiiii;•iiii. ..:... WT -hmm wl -"~i WT -hpnm FIG. 14 US 10,131,585 B2 2 HOPANOIDS PRODUCING BACTERIA AND method comprises providing one or more candidate nitrogen RELATED BIOFERTILIZERS, fixing rhizobia strains. The method further comprises detect- COMPOSITIONS, METHODS AND SYSTEMS ing among the one or more candidate nitrogen fixing rhizo- bia strains, at least one rhizobia strain capable of producing CROSS REFERENCE TO RELATED 5 C35 hopanoids. The method further comprises providing the APPLICATIONS at least one rhizobia strain capable of producing C35 hopanoids in a form suitable for administration to a legu- The present application claims priority to U.S.