IP Library › Granted Patent US 12,268,212
Granted Patent B2
US 12,268,212 · App. 18/540,318 · Granted Apr 8, 2025

Agricultural compositions comprising remodeled nitrogen fixing microbes

Inventors: Mark Reisinger (Berkeley, CA); Ernest Sanders (Berkeley, CA); Richard Broglie (Berkeley, CA); Karsten Temme (Berkeley, CA)
Assignee: Pivot Bio, Inc.
A01N63/20
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Quick Facts
Patent No.
US 12,268,212
App. No.
18/540,318
Granted
Apr 8, 2025
Kind
B2
Abstract

The present disclosure provides non-intergeneric remodeled microbes that are able to fix atmospheric nitrogen and deliver such to plants in a targeted, efficient, and environmentally sustainable manner. The utilization of the taught microbial products will enable farmers to realize more productive and predictable crop yields without the nutrient degradation, leaching, or toxic runoff associated with traditional synthetically derived nitrogen fertilizer. The remodeled microbes taught herein are able to be combined with leading agricultural chemistry and elite germplasm. Furthermore, the disclosure provides seed treatments comprising remodeled microbes.

Claims (34)

1. A composition, comprising:

a) a plurality of non-intergeneric remodeled bacteria; and

b) at least one herbicide;

wherein the non-intergeneric remodeled bacteria produce fixed N of at least about 1×10 −17 mmol N per bacterial cell per hour;

wherein the non-intergeneric remodeled bacteria have an average colonization ability per unit of plant root tissue of at least about 1.0×10 4 bacterial cells per gram of fresh weight of plant root tissue; and

wherein the at least one herbicide belongs to a chemical family selected from the group consisting of: cyclohexanediones, aryloxyphenoxypropionates, phenylpyrazolins, imidazolinones, sulfonylureas, triazopyrimidine, sulfonylaminocarbonyltriazolinones, dinitroanilines, benzamide, arylpicolinate, phenoxyacetic acids, benzoic acids, pyridines, triazines, triazinone, phenyl-carbamates, uracils, benzothiadiazoles, nitriles, phenylureas, thiocarbamates, organophosphorus, isoxazolidinone, diphenylether, N-phenylphthalimide, aryl triazolinone, pyrazoles, pyrimidinedione, acetamides, benzofuranes, semicarbazone, bipyridiliums, isoxazole, pyrazolone, and triketone; or

wherein the at least one herbicide is selected from the group consisting of: 2,4-D, 2,4-DB, acetochlor, acifluorfen, alachlor, ametryn, atrazine, aminopyralid, benefin, bensulfuron, bensulide, bentazon, bicyclopyrone, bromacil, bromoxynil, butylate, carfentrazone, chlorimuron, chlorsulfuron, clethodim, clomazone, clopyralid, cloransulam-methyl, cycloate, dimethyl tetrachloroterephthalate (DCPA), desmedipham, dicamba, dichlobenil, diclofop, diclosulam, diflufenzopyr, dimethenamid, diquat, diuron disodium methylarsonate (DSMA), endothall, S-ethyl dipropyl carbamothioate (EPTC), ethalfluralin, ethofumesate, fenoxaprop, fluazifop, flucarbazone, flufenacet, flumetsulam, flumiclorac, flumioxazin, fluometuron, fluroxypyr, fomesafen, foramsulfuron, glufosinate, glyphosate, halosulfuron, hexazinone, imazamethabenz, imazamox, imazapic, imazaquin, imazethapyr, isoxaflutole, lactofen, linuron, 2-methyl-4-chlorophenoxyacetic acid (MCPA), 4-(4-chloro-2-methylphenoxy) butanoic acid (MCPB), mesotrione, metribuzin, indaziflam, metsulfuron, molinate, monosodium methanearsonate (MSMA), napropamide, naptalam, nicosulfuron, norflurazon, oryzalin, oxadiazon, oxyfluorfen, paraquat, pelargonic acid, pendimethalin, phenmedipham, picloram, primisulfuron, prodiamine, prometryn, pronamide, propanil, prosulfuron, pyrazon, pyrithioac, quinclorac, quizalofop, rimsulfuron, S-metolachlor, sethoxydim, siduron, simazine, sulfentrazone, sulfometuron, sulfosulfuron, tebuthiuron, tembotrione, terbacil, thiazopyr, thifensulfuron, thiobencarb, topramezone, tralkoxydim, triallate, triasulfuron, tribenuron, triclopyr, trifluralin, and triflusulfuron.

2. The composition of claim 1 , wherein a member of the plurality of non-intergeneric remodeled bacteria comprises at least one genetic variation introduced into a gene selected from the group consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nifQ, a gene associated with biosynthesis of a nitrogenase enzyme, and combinations thereof.

3. The composition of claim 1 , formulated as a seed treatment.

4. The composition of claim 1 , formulated as an in-furrow treatment.

5. The composition of claim 1 , in combination with a corn seed.

6. The composition of claim 1 , in combination with a genetically modified corn seed, wherein said genetically modified corn seed comprises an herbicide tolerance trait, an insect resistance trait, or both.

7. The composition of claim 1 , wherein the non-intergeneric remodeled bacteria are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen.

8. The composition of claim 1 , wherein a member of the plurality of non-intergeneric remodeled bacteria comprises at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of a nitrogen fixation or assimilation genetic regulatory network.

9. The composition of claim 1 , wherein a member of the plurality of non-intergeneric remodeled bacteria comprises an introduced control sequence operably linked to at least one gene of a nitrogen fixation or assimilation genetic regulatory network.

10. The composition of claim 1 , wherein a member of the plurality of non-intergeneric remodeled bacteria comprises a heterologous promoter operably linked to at least one gene of a nitrogen fixation or assimilation genetic regulatory network.

11. The composition of claim 1 , wherein a member of the plurality of non-intergeneric remodeled bacteria comprises at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of a nitrogen fixation or assimilation genetic regulatory network that results in one or more of: increased expression or activity of NifA or glutaminase; decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GinK, DraT, AmtB; decreased adenylyl-removing activity of GlnE; or decreased uridylyl-removing activity of GlnD.

12. The composition of claim 1 , wherein a member of the plurality of non-intergeneric remodeled bacteria comprises at least one genetic modification to a niff gene, glnE gene, or glnD gene.

13. The composition of claim 1 , wherein the plurality of non-intergeneric remodeled bacteria comprise at least two different species of bacteria.

14. The composition of claim 1 , wherein the plurality of non-intergeneric remodeled bacteria comprise at least two different strains of the same species of bacteria.

15. The composition of claim 1 , wherein the plurality of non-intergeneric remodeled bacteria comprise bacteria selected from the genera of: Rahnella, Klebsiella, Achromobacter, Achromobacter, Microbacterium, Kluyvera, Kosakonia, Enterobacter, Azospirillum , and combinations thereof.

16. The composition of claim 1 , wherein the plurality of non-intergeneric remodeled bacteria comprise bacteria from the genera of Klebsiella and Kosakonia.

17. The composition of claim 1 , wherein the plurality of non-intergeneric remodeled bacteria comprise Klebsiella variicola and Kosakonia sacchari.

18. The composition of claim 1 , wherein the non-intergeneric remodeled bacteria have an average colonization ability per unit of plant root tissue of at least about 1.0×10 7 bacterial cells per gram of fresh weight of plant root tissue.

19. The composition of claim 1 , wherein the at least one herbicide is selected from the group consisting of: 2,4-D, 2,4-DB, acetochlor, acifluorfen, alachlor, ametryn, atrazine, aminopyralid, benefin, bensulfuron, bensulide, bentazon, bicyclopyrone, bromacil, bromoxynil, butylate, carfentrazone, chlorimuron, chlorsulfuron, clethodim, clomazone, clopyralid, cloransulam-methyl, cycloate, dimethyl tetrachloroterephthalate (DCPA), desmedipham, dicamba, dichlobenil, diclofop, diclosulam, diflufenzopyr, dimethenamid, diquat, diuron, disodium methylarsonate (DSMA), endothall, S-ethyl dipropyl carbamothioate (EPTC), ethalfluralin, ethofumesate, fenoxaprop, fluazifop, flucarbazone, flufenacet, flumetsulam, flumiclorac, flumioxazin, fluometuron, fluroxypyr, fomesafen, foramsulfuron, glufosinate, glyphosate, halosulfuron, hexazinone, imazamethabenz, imazamox, imazapic, imazaquin, imazethapyr, isoxaflutole, lactofen, linuron, 2-methyl-4-chlorophenoxyacetic acid (MCPA), 4-(4-chloro-2-methylphenoxy) butanoic acid (MCPB), mesotrione, metribuzin, indaziflam, metsulfuron, molinate, monosodium methanearsonate (MSMA), napropamide, naptalam, nicosulfuron, norflurazon, oryzalin, oxadiazon, oxyfluorfen, paraquat, pelargonic acid, pendimethalin, phenmedipham, picloram, primisulfuron, prodiamine, prometryn, pronamide, propanil, prosulfuron, pyrazon, pyrithioac, quinclorac, quizalofop, rimsulfuron, S-metolachlor, sethoxydim, siduron, simazine, sulfentrazone, sulfometuron, sulfosulfuron, tebuthiuron, tembotrione, terbacil, thiazopyr, thifensulfuron, thiobencarb, topramezone, tralkoxydim, triallate, triasulfuron, tribenuron, triclopyr, trifluralin, and triflusulfuron.

20. The composition of claim 1 , wherein the at least one herbicide belongs to a chemical family selected consisting from the group of: cyclohexanediones, aryloxyphenoxypropionates, phenylpyrazolins, imidazolinones, sulfonylureas, triazopyrimidine, sulfonylaminocarbonyltriazolinones, dinitroanilines, benzamide, arylpicolinate, phenoxyacetic acids, benzoic acids, pyridines, triazines, triazinone, phenyl-carbamates, uracils, benzothiadiazoles, nitriles, phenylureas, thiocarbamates, organophosphorus, isoxazolidinone, diphenylether, N-phenylphthalimide, aryl triazolinone, pyrazoles, pyrimidinedione, acetamides, benzofuranes, semicarbazone, bipyridiliums, isoxazole, pyrazolone, and triketone.

21. The composition of claim 1 , wherein the at least one herbicide is selected from the group consisting of: 2,4-D, 2,4-DB, acetochlor, acifluorfen, atrazine, bentazon, bicyclopyrone, bromoxynil, carfentrazone, chlorimuron, clethodim, clomazone, clopyralid, cloransulam-methyl, desmedipham, dicamba, diflufenzopyr, dimethenamid-p, diquat, S-ethyl dipropyl carbamothioate (EPTC), ethalfluralin, ethofumesate, fenoxaprop, florasulam, fluazifop, flufenacet, flumetsulam, flumiclorac, flumioxazin, fluroxypyr, fluthiacet-methyl, fomesafen, glufosinate, glyphosate, halauxifen, halosulfuron, hexazinone, imazamox, imazethapyr, iodosulfuron, isoxaflutole, lactofen, linuron, mcpa, mesosulfuron, mesotrione, metolachlor, metribuzin, nicosulfuron, paraquat, pendimethalin, phenmedipham, pinoxaden, primisulfuron, pronamide, propoxycarbazone, prosulfuron, pyraflufen-ethyl, pyrasulfotole, pyroxasulfone, pyroxsulam, quizalofop, rimsulfuron, saflufenacil, sethoxydim, simazine, s-metolachlor, sulfentrazone, tembotrione, terbacil, thiencarbazone-methyl, thifensulfuron, topramezone, tribenuron, trifluralin, triflusulfuron, and combinations thereof.

22. The composition of claim 1 , wherein the at least one herbicide is selected from the group consisting of: thiencarbazone-methyl, isoxaflutole, tembotrione, glufosinate, iodosulfuron, and combinations thereof.

23. The composition of claim 1 , wherein the at least one herbicide is glufosinate.

24. The composition of claim 1 , wherein the at least one herbicide is glyphosate.

25. A method, comprising: applying the composition of claim 1 to a plant or a field that will comprise a plant.

26. A method of making the composition of claim 1 , comprising: combining the a) plurality of non-intergeneric remodeled bacteria and the b) at least one herbicide.

27. A method of making the composition of claim 1 , comprising: combining in a field the a) plurality of non-intergeneric remodeled bacteria and the b) at least one herbicide.

28. A method of making the composition of claim 1 , comprising: combining in an agricultural delivery system the a) plurality of non-intergeneric remodeled bacteria and the b) at least one herbicide.

Assignments (2)
SECURITY INTEREST Recorded Dec 22, 2025
From: PIVOT BIO, INC.
To: HSBC BANK USA, N.A.
Reel/Frame 073285/0020 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Dec 15, 2023
From: REISINGER, MARK; SANDERS, ERNEST; BROGLIE, RICHARD; TEMME, KARSTEN
To: PIVOT BIO, INC.
Reel/Frame 065881/0613 →
Continuity (4)
Continuation 17254175
Provisional Application 62808693 · Feb 21, 2019
Provisional Application 62690621 · Jun 27, 2018
Related Publication 20240196903A1 · Jun 20, 2024
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GenBank: AAA86265.1. CryIA(a), partial [Bacillus thuringiensis serovar kurstaki]. Jan. 30, 1996. [cited by applicant]
GenBank: AAD46139.1. insecticidal crystal protein [Bacillus thuringiensis]. Aug. 1, 1999. [cited by applicant]
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GenBank: AAD55382.1. 135 kDa insecticidal protein [Bacillus thuringiensis serovar kurstaki]. Sep. 16, 1999. [cited by applicant]
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GenBank: AAP40639.1. Cry1Aa [Bacillus thuringiensis]. Dec. 1, 2004. [cited by applicant]
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GenBank: AAE33526.1. Sequence 7 from U.S. Pat. No. 5,973,231. Aug. 31, 2000. [cited by applicant]
GenBank: ACU57499.1. pesticidal crystal protein [Bacillus thuringiensis]. Feb. 3, 2010. [cited by applicant]
GenBank: GQ483512.1. Bacillus thuringiensis strain G7-1 Cry56Aa-like protein gene, complete cds. Jun. 1, 2011. [cited by applicant]
GenBank: JX025567.1. Bacillus thuringiensis strain HS18-1 Cry56Aa1-like protein gene, complete cds. May 7, 2016. [cited by applicant]
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GenBank: JN790647.1. Bacillus thuringiensis strain Bm59-2 cry4-like protein gene, complete cds. Sep. 26, 2015. [cited by applicant]
GenBank: ACR43758.2. cry4 delta-toxin-like protein (plasmid) [Bacillus thuringiensis]. Jul. 24, 2016. [cited by applicant]
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GenBank: EA057254.1. Sequence 628 from U.S. Pat. No. 7166424. Feb. 7, 2007. [cited by applicant]
GenBank: EEM99278.1. Pesticidial crystal protein cry 15Aa [Bacillus thuringiensis IBL 4222]. Apr. 30, 2009. [cited by applicant]
GenBank: GU810818.1. Bacillus thuringiensis serovar malayensis strain 4AV1 crystal protein gene, complete cds. Apr. 3, 2010. [cited by applicant]
GenBank: EA057253.1. Sequence 626 from U.S. Pat. No. 7166424. Feb. 7, 2007. [cited by applicant]
GenBank: EEM99279.1. Pesticidial crystal protein cry 15Aa [Bacillus thuringiensis IBL 4222]. Apr. 30, 2009. [cited by applicant]
GenBank: HM035087.1. Bacillus thuringiensis strain Sbt009 pesticidal crystal protein Cry0092 (cry0092) gene, complete cds. Dec. 31, 2013. [cited by applicant]
GenBank: HM132125.1. Bacillus thuringiensis strain HD868(E5) Cry7-like protein gene, partial cds. Jul. 25, 2016. [cited by applicant]
GenBank: EEM19308.1. hypothetical protein bthur0001_55730 [Bacillus thuringiensis serovar tochigiensis BGSC 4Y1]. Apr. 30, 2009. [cited by applicant]
GenBank: HM485581.1. Bacillus thuringiensis strain Sbt021 Cry0211 gene, complete cds. Dec. 31, 2019. [cited by applicant]
GenBank: HM461686.1. [cited by applicant]
GenBank: BAJ05397.1. crystal protein [Bacillus thuringiensis]. Jul. 10, 2014. [cited by applicant]
GenBank: HM485582.1. Bacillus thuringiensis strain Sbt009 Cry0094 gene, complete cds. Dec. 31, 2019. [cited by applicant]
GenBank: HQ113114.1. Bacillus thuringiensis Cry20-like protein (cry20) gene, complete cds. Dec. 30, 2012. [cited by applicant]
GenBank: HQ401006.1. Bacillus thuringiensis strain BtMC28 Cry-like protein (cry) gene, complete cds. Nov. 1, 2013. [cited by applicant]
GenBank: JQ821388.1. Bacillus thuringiensis strain BtMC28 Cry69Aa-like protein gene, complete cds. Jan. 1, 2016. [cited by applicant]
GenBank: JN209957.1. Bacillus thuringiensis strain hs18-1 insecticidal Cry-like protein (cry) gene, complete cds. Jul. 1, 2015. [cited by applicant]
GenBank: JN646781.1. Bacillus thuringiensis strain HS18-1 cry-like protein gene, complete cds. Sep. 26, 2015. [cited by applicant]
GenBank: EEL67276.1. 83-kDa crystal protein [Bacillus mycoides]. Jun. 25, 2018. [cited by applicant]
GenBank: JX025568.1. Bacillus thuringiensis strain HS18-1 Cry53Ab1-like protein gene, complete cds. May 7, 2016. [cited by applicant]
GenBank: JX025569.1. Bacillus thuringiensis strain HS18-1 mosquitocidal toxin-like protein gene, complete cds. May 7, 2016. [cited by applicant]
GenBank: X03182.1. Bacillus thuringiensis gene for crystal protein (Mr 28 000). Oct. 23, 2008. [cited by applicant]
GenBank: Z14147.1. B.thuringiensis gene for CytB toxin. Apr. 18, 2005. [cited by applicant]
GenBank: X98793.1. B.thuringiensis cyt1Ab1 gene. Apr. 18, 2005. [cited by applicant]
GenBank: U37196.1. Bacillus thuringiensis delta endotoxin gene, complete cds. Jul. 11, 1996. [cited by applicant]
GenBank: U52043.1. Bacillus thuringiensis plasmid pRX80 CytB toxin homolog (cytB) gene, complete cds. Jul. 26, 2016. [cited by applicant]
GenBank: HQ439788. Bacillus thuringiensis strain PS9-C12 Cry 1A-like protein gene, partial cds Jul. 25, 2016. [cited by applicant]
Genbank: HQ439790. Bacillus thuringiensis strain PS9-D12 Cry1A-like protein gene, partial cds Nov. 4, 2016. [cited by applicant]
GenBank: HQ685121. Bacillus thuringiensis strain LS-R-21 Cry1Aa gene, partial cds Jul. 25, 2016. [cited by applicant]
GenBank: JF340156. Bacillus thuringiensis strain SK-798 plasmid Cry toxin (cry) gene, complete cds Jul. 25, 2016. [cited by applicant]
GenBank: JN651496. Bacillus thuringiensis strain LTS-209 Cry1Aa gene, partial cds Feb. 5, 2015. [cited by applicant]
GenBank: KC158223. Bacillus thuringiensis strain Lip plasmid insecticidal crystal protein (cry1Aa) gene, complete cds Jul. 26, 2016. [cited by applicant]
GenBank: AAA22330. entomocidal protoxin [Bacillus thuringiensis] Apr. 26, 1993. [cited by applicant]
GenBank: AAA22613. insecticidal endotoxin [Bacillus thuringiensis] Apr. 26, 1993. [cited by applicant]
GenBank: AAA22561. crystal protein precursor [Bacillus thuringiensis] Apr. 26, 1993. [cited by applicant]
GenBank: BAA00071. delta-endotoxin [Bacillus thuringiensis serovar kurstaki str. HD-1] Sep. 29, 2007. [cited by applicant]
GenBank: CAA31620. unnamed protein product [Bacillus thuringiensis] Apr. 18, 2005. [cited by applicant]
GenBank: AAA22551. insecticidal protein [ Bacillus thuringiensis] Apr. 26, 1993. [cited by applicant]
GenBank: CAA38701. unnamed protein product [Bacillus thuringiensis] Oct. 23, 2008. [cited by applicant]
GenBank: A29125, Replaced by Q93T21. Cry1Ab16 Nov. 28, 2006. [cited by applicant]
GenBank: AAV83251. Transcriptional regulator, Cro/CI family [Idiomarina loihiensis L2TR] Jan. 31, 2014. [cited by applicant]
GenBank: AAC64003. crystal protein (plasmid) [Bacillus thuringiensis serovar kurstaki] Jul. 25, 2016. [cited by applicant]
GenBank: AAN76494. insecticidal protein P [Bacillus thuringiensis] Dec. 2, 2002. [cited by applicant]
GenBank: AAG16877. delta endotoxin [Bacillus thuringiensis] Oct. 1, 2000. [cited by applicant]
GenBank: AA013302. ze28d06.r1 Soares retina N2b4HR [cited by applicant]
GenBank: AAK55546. Cry1Ab16 [Bacillus thuringiensis] Oct. 16, 2002. [cited by applicant]
GenBank: AAT46415. parasporal crystal protein [Bacillus thuringiensis] Jun. 20, 2004. [cited by applicant]
Cited By (8)
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