US 20170 107160A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0107160 A1 NEWMAN et al. (43) Pub. Date: Apr. 20, 2017 (54) HOPANOIDS PRODUCING BACTERIA AND Publication Classification RELATED BIOFERTILIZERS, (51) Int. Cl. COMPOSITIONS, METHODS AND SYSTEMS C05F II/08 (2006.01) (71) Applicant: CALIFORNLA INSTITUTE OF CI2N L/20 (2006.01) TECHNOLOGY, Pasadena, CA (US) GOIN 33/92 (2006.01) (52) U.S. Cl. (72) Inventors: Dianne K. NEWMAN, PASADENA, CPC .............. C05F II/08 (2013.01); G0IN 33/92 CA (US); Gargi KULKARNI, (2013.01); C12N 1/20 (2013.01); G0IN PASADENA, CA (US); Brittany Jo 2405/00 (2013.01) BELIN, PASADENA, CA (US) (57) ABSTRACT (21) Appl. No.: 15/298,172 Hopanoids, hopanoids-producing nitrogen-fixing bacteria, and related formulations, systems and methods are described (22) Filed: Oct. 19, 2016 herein. In particular, hopanoids alone or in combination with hopanoid-producing nitrogen-fixing bacteria can be used as Related U.S. Application Data biofertilizer to stimulate plant growth and yield with (60) Provisional application No. 62/243,418, filed on Oct. enhanced tolerance to diverse stresses found in plant-mi 19, 2015. crobe Symbiotic microenvironments. Patent Application Publication Apr. 20, 2017. Sheet 1 of 14 US 2017/O107160 A1 A) f d C. top-2-ene top-22.9}-ere apart-2-ene (W) Bopan-22-ol Tetraymanov W. Dokopee) W, biplopteros) --- R, ii, a GH oh s, ri o e-P-508 vil Afinobacteriohopatrio a Bacteriahopaetetra (b. Adenosylhopane (d) R, s - orch, B) sit: A lioid Fitiparoi F.G. Patent Application Publication Apr. 20, 2017. Sheet 2 of 14 US 2017/O107160 A1 A. oH ch o: X-------3s 28 ^-NY23 s R. t 2 oH R, g2 hiopanoid Tetrahyrnatiot R, x - or c-, R. so- or x-, B) s &S& C &yss& S & 9 & & & S. 8 O C-:)) I} { {KXCIC3) {:KI IX} 38s --------~~~~scale?e : Stic f f a. diploptee C5r ci, th& QC, 2-methyidipogteineC Hipnh 8, adenosylhopane rSC 5 O 100. 5 0. oil. Tr: --------------------- ---- 18 8 20 22 24 28 28 33 33 34 36 38 40 Time (min) FG. 2 Patent Application Publication Apr. 20, 2017. Sheet 3 of 14 US 2017/O107160 A1 Root Nodule Nodule cross-section Pant Ces Symbiosome Noctule Cotics Ces facedone Pas Nicles Bacteris Pat Ce:Wai Batteroic at Cea Peribacteroid Parterbare F.G. 3 Patent Application Publication Apr. 20, 2017. Sheet 4 of 14 US 2017/O107160 A1 Penta-acylated hexa-acylated Lipid A 100- 2622.84. W epta-acylated tipid A 335,22 SO. # topanoid + Hopanoid --------------- races of 2-hopanoid O. 36365? s 2000 236 2720 3O8O o 3800 hexa-acylated lipid A OO 287.58 AhpnP 50 Hepta-acylated Lipid A Penta-acylated 329 lipid A + Hopanoid # opanoid -es- -- Traces of i 228.98 2 hopanoid 1 O 3838. 2000 2360 2720 3800 Hexa-acylated Lipid A OO 289.3 Ahpnh 50 Penta-acylated 21yeLipid A trace O 2000 2360 2720 3O80 3440 3800 Mass (mfz) F.G. 4 Patent Application Publication Apr. 20, 2017. Sheet 5 of 14 US 2017/O107160 A1 0, 4. :: 25C 40: 38. 36. 34. 32. 0.3 : Patent Application Publication Apr. 20, 2017. Sheet 6 of 14 US 2017/O107160 A1 F.G. 6 Patent Application Publication Apr. 20, 2017. Sheet 7 of 14 US 2017/O107160 A1 A) Aerobic - 30°C B) Aerobic - 37°C s s 38 s 0. Time (h (WT: Ahpn?& Ahpnh Cotri NCR335 Iwi Aliprap 8Ahpnh FIG. 7 Patent Application Publication Apr. 20, 2017. Sheet 8 of 14 US 2017/O107160 A1 - a. - s 8 & 3. - g s 2 s -gs E al F.G. 8 Patent Application Publication Apr. 20, 2017. Sheet 9 of 14 US 2017/0107160 A1 0. 2 - ). 8 F.G. 9 Patent Application Publication Apr. 20, 2017. Sheet 10 of 14 US 2017/0107160 A1 O 2 21 d.p.i. FG, 10 Patent Application Publication Apr. 20, 2017. Sheet 11 of 14 US 2017/O107160 A1 hpnP shc hpni ey. FIG. Patent Application Publication Apr. 20, 2017. Sheet 12 of 14 US 2017/0107160 A1 2. Š- C. 3 wildtype 20 dpi : 8 -hpah & 3 : " : S : : 3 R.; 1. C.. ... : b. c. s. s S. s s S. s c 2 WT Alphi Patent Application Publication Apr. 20, 2017. Sheet 13 of 14 US 2017/0107160 A1 Exp: 38c an as: F.G. 13 Patent Application Publication Apr. 20, 2017. Sheet 14 of 14 US 2017/O107160 A1 3. 8 S. 8 33 8 -ip: F.G. 4 US 2017/01 07160 A1 Apr. 20, 2017 HOPANOIDS PRODUCING BACTERIA AND fixing rhizobia strains. The method further comprises detect RELATED BIOFERTILIZERS, ing among the one or more candidate nitrogen fixing rhizo COMPOSITIONS, METHODS AND SYSTEMS bia Strains, at least one rhizobia Strain capable of producing Cs hopanoids. The method further comprises providing the CROSS REFERENCE TO RELATED at least one rhizobia Strain capable of producing Cs APPLICATIONS hopanoids in a form Suitable for administration to a legu 0001. The present application claims priority to U.S. minous plant or seed or soil Surrounding a leguminous plant Provisional Application No. 62/243,418, entitled “Using or seed, thus providing a biofertilizer for the leguminous Hopanoids to Improve Stress Resistance in Biological Nitro plant. In some embodiments, the method further comprises gen Fixation filed on Oct. 19, 2015, with docket number providing the at least one rhizobia Strain capable of produc CIT-7327-P which is incorporated herein by reference in its ing Cs hopanoids in combination with carrier allowing entirety. increased stability, viability and/or effectiveness of the nitro gen-fixing bacteria gas exchance of the one or more nitro STATEMENT OF GOVERNMENT GRANT gen-fixing rhizobia. 0009. According to a third aspect, a method to provide a 0002 This invention was made with government support biofertilizer for a leguminous plant, is described. The from NASA (NNX12AD93G), the National Science Foun method comprises genetically engineering a nitrogen fixing dation (1224158) and the Howard Hughes Medical Institute rhizobia Strain incapable of producing Cls hopanoids to (HHMI). The government has certain rights in the invention. introduce Cs synthesis genes thus providing a genetically engineered nitrogen fixing rhizobia strains capable of pro FIELD ducing Cls hopanoids. The method further comprises pro 0003. The present disclosure relates to hopanoids, viding the genetically engineered nitrogen fixing rhizobia hopanoids producing bacteria and related biofertilizers, strains in a form suitable for administration to a leguminous microorganisms, compositions, methods and systems com plant or seed, and/or a soil Surrounding leguminous plant or prising hopanoid-producing microorganisms and related for seed thus providing the biofertilizer for the leguminous mulations, and uses as biofertilizer in agriculture industry. plant. 0010. According to a fourth aspect, a biofertilizer com BACKGROUND position for a leguminous plant is described. The biofertil 0004. The exploitation of beneficial microbes as a biofer izer composition comprises one or more biofertilizers essen tilizer has become of paramount importance in agriculture tially consisting of one or more nitrogen-fixing rhizobia sector for their potential role in food safety and sustainable capable of producing Cls hopanoids and an acceptable crop production. vehicle. In some embodiments, the biofertilizer composition 0005. There remains however a challenge for developing can further comprise one or more Cs hopanoids. In the eco-friendly and economically feasible alternatives to biofertilizer composition the one or more biofertilizers and chemical fertilizers, which can improve soil fertility, health the vehicle are formulated for administration to a legumi of the environment, and agricultural productivity particu nous plant and/or for administration to a leguminous seed. In Some embodiments the vehicle comprises at least one carrier larly under different soil conditions. allowing increased stability, viability and/or effectiveness of the nitrogen-fixing bacteria gas exchance of the one or more SUMMARY nitrogen-fixing rhizobia. 0006 Provided herein are hopanoid-producing bacteria, 0011. According to a fifth aspect, a biofertilizer compo and related biofertilizers, microorganisms, formulations, and sition is described. The biofertilizer composition comprises methods which can comprise one or more particular types of one or more nitrogen-fixing rhizobia capable of producing hopanoids and/or one or more hopanoid-producing nitrogen Cs hopanoids and one or more Cs hopanoids. In some fixing bacteria. In several embodiments, the microorgan embodiments the composition can be formulated for admin isms, and related biofertilizers, compositions methods and istration to a leguminous plant, a leguminous seed and/or systems described herein can improve stress-resistance dur soil Surrounding the leguminous plant or leguminous seed. ing the progression of plant-microbe Symbiosis and in In some embodiments, the biofertilizer composition further particular of a legume-microbe Symbiosis. comprises one or more carriers allowing increased stability, 0007 According to a first aspect, a biofertilizer for a viability and/or effectiveness of the nitrogen-fixing bacteria leguminous plant is described. The biofertilizer essentially gas exchance of the one or more nitrogen-fixing rhizobia. consists of one or more nitrogen-fixing rhizobia capable of producing Cs hopanoids. In the biofertilizer, the one or 0012. According to a sixth aspect, a genetically modified more nitrogen-fixing rhizobia capable of producing Cs nitrogen-fixing rhizobium is described. The genetically hopanoids are in a form suitable for administration to one or modified nitrogen-fixing rhizobium is a rhizobium naturally more leguminous plant or seed, and/or for administration to incapable of producing Cs hopanoids and genetically engi a Soil Surrounding the one or more leguminous plant or seed. neered to produce Cls hopanoids. In some embodiments, the biofertilizer is in combination 0013. According to a seventh aspect, described herein is with carrier allowing increased stability, viability and/or a leguminous seed coated and/or inoculated with a biofer effectiveness of the nitrogen-fixing bacteria gas exchance of tilizer, and/or biofertilizer composition herein described, the one or more nitrogen-fixing rhizobia.