WO2025174810A2 - Commutateur génétique microbien dépendant de la densité - Google Patents

Commutateur génétique microbien dépendant de la densité

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Publication number
WO2025174810A2
WO2025174810A2 PCT/US2025/015489 US2025015489W WO2025174810A2 WO 2025174810 A2 WO2025174810 A2 WO 2025174810A2 US 2025015489 W US2025015489 W US 2025015489W WO 2025174810 A2 WO2025174810 A2 WO 2025174810A2
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WIPO (PCT)
Prior art keywords
protein
optionally
gene
promoter
genetically engineered
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PCT/US2025/015489
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English (en)
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WO2025174810A3 (fr
Inventor
Gregory Alexander Knauf
Timothy Lyndon Haskett
Tim SCHNABEL
Caroline Kukolj
Tomasz Zajkowski
Elizabeth Leigh Ordeman
Niklaus Hoyt Evitt
James Pearce
Sandipan Samaddar
Daniel Torres NAYLOR
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Switch Bioworks Inc
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Switch Bioworks Inc
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Priority claimed from PCT/US2025/014079 external-priority patent/WO2025166196A1/fr
Application filed by Switch Bioworks Inc filed Critical Switch Bioworks Inc
Publication of WO2025174810A2 publication Critical patent/WO2025174810A2/fr
Publication of WO2025174810A3 publication Critical patent/WO2025174810A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/635—Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H3/00—Processes for modifying phenotypes, e.g. symbiosis with bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora

Definitions

  • Genetically engineered bacterium comprising: (a) one or more heterologous gene expression cassette(s) comprising at least one control element which is operably linked to at least one nucleic acid sequence encoding a first quorum sensing synthase protein (QSSP) able to synthesize a quorum sensing signal molecule Q (QSSM Q) and/or at least one nucleic acid sequence encoding a first quorum sensing regulator protein (QSRP) that can bind said QSSM Q; (b) a heterologous gene expression cassette comprising at least one nucleic acid sequence encoding at least one RNA sequence or protein of interest operably linked to a control element comprising a first quorum sensing (QS) promoter, wherein the first quorum sensing promoter is activated by the first quorum sensing regulator protein (QSRP) and the QSSM Q when the population density of the genetically engineered bacterium exceeds a threshold population
  • QSSP quorum sensing syntha
  • compositions comprising the genetically engineered bacterium and an agriculturally acceptable carrier are also provided.
  • Plant parts or plant propagules which are at least partially coated, imbibed, or mixed with the compositions are also provided. Use of the plant parts or plant propagules which are at least partially coated, imbibed, or mixed with the compositions to grow a crop are also provided.
  • Agricultural systems comprising: (i) at least one of the engineered bacteria; (ii) at least one plant growth medium; and (iii) at least one crop plant, crop plant seed, or crop plant vegetative propagule; wherein the plant growth medium, crop plant, crop seed, and/or crop plant propagule comprise, are at least partially coated with, imbibed with, and/or are mixed with the engineered bacterium or a composition comprising the engineered bacterium and an agriculturally acceptable carrier, are provided.
  • Treated plant seed or plant propagule systems comprising: (i) at least one crop plant seed or crop plant vegetative propagule; and (ii) at least one of the engineered bacteria, wherein the crop plant seed or crop plant propagule are at least partially coated, imbibed, and/or mixed with the engineered bacterium or a composition comprising the engineered bacterium and an agriculturally acceptable carrier, are provided.
  • Methods of producing a bacterial culture comprising: (i) growing the genetically engineered bacterium either: (a) in contact with the quorum quenching compound QQ; or (b) in exposure to a temperature above the threshold temperature; and (ii) harvesting the bacterial culture are provided. Atty. Dkt. No.
  • FIG. 1 shows a gene expression cassette for measuring expression of a green fluorescent protein reporter operably linked to a control element comprising a quorum sensing (QS) promoter. The cognate QSRP transcription factor is expressed constitutively.
  • QS quorum sensing
  • Figure 4 shows population density-dependent induction by strains comprising two heterologous gene expression cassettes: (i) one comprising genes encoding a quorum sensing synthase protein (QSSP) and a quorum sensing regulator protein (QSRP) and (ii) one encoding a GFP reporter protein of interest operably linked to the cognate QS promoters of the QSSP and QSRP in (i).
  • Rows of the figure each contain strains of a specific bacterial host species.
  • Ab Azospirillum brasilense
  • Kr Kosakonia radicincitans
  • Ps Pseudomonas stutzeri.
  • PahlI is the promoter of the AhlRI quorum sensing system
  • PcinI is the promoter of the CinRI quorum sensing system
  • PlasB and PlasI are promoters of the LasRI quorum sensing system.
  • the black “QS” data series came from strains comprising both heterologous gene expression cassettes (i) and (ii) while the gray “WT” data series came from strains comprising only cassette (ii) and missing the cassette encoding the QSSP and QSRP.
  • Figure 5 shows bacterial cell counts for cultures grown in +/- 20 mM lactate (for Ab and Ps) or 20 mM glucose (for Kr), as determined by flow cytometry.
  • Figure 6 shows relative expression in culture (defined as inducible GFP fluorescence / constitutive LSSmScarlett fluorescence) for strains carrying heterologous QS systems grown in the presence/absence of carbon, as determined by flow cytometry.
  • Figure 7 shows bacterial cell counts for bacteria isolated from bulk soil (BS) or from the corn-root rhizoplane (RP), as determined by flow cytometry.
  • BS bulk soil
  • RP corn-root rhizoplane
  • Engineered bacterial strains of Azospirillum brasilense (Ab) and Kosakonia radicincitans (Kr) comprised either the cinRI QS system (CinI QSSP, CinR QSRP, and Atty. Dkt. No. P14610WO00 GFP operably linked to PcinI) or the ahlRI QS system (AhlI QSSP, AhlR QSRP, and GFP operably linked to PahlI).
  • Figure 8 shows relative expression (defined as inducible GFP fluorescence / constitutive LSSmScarlett fluorescence) for bacteria isolated from bulk soil (BS) or from the corn-root rhizoplane (RP), as determined by flow cytometry.
  • Engineered bacterial strains of Azospirillum brasilense (Ab) and Kosakonia radicincitans (Kr) comprised either the cinRI QS system (CinI QSSP, CinR QSRP, and GFP operably linked to PcinI) or the ahlRI QS system (AhlI QSSP, AhlR QSRP, and GFP operably linked to PahlI).
  • Figure 9 shows a system of gene expression cassettes for a quorum sensing system that can be deactivated during fermentation using quorum quenching.
  • Figure 10 shows engineered K. radicincitans soil bacteria that exhibit population density-dependent expression of a GFP protein of interest in a plant growth medium that can be conditionally deactivated by contact with an anhydrotetracycline (aTc) quorum quenching compound in culture.
  • the threshold population density for expression of the protein of interest is about 1 * 10 8 cells/g root.
  • Figure 11 shows a schematic of constructs with different ribosome binding sites for tuning the expression of a QSRP for density-dependent expression from a QS promoter in engineered systems.
  • the construct comprises two gene expression cassettes.
  • a reporter gene (GFP) is operably linked to a quorum sensing promoter (PQS) under control of the ribosome binding site BCD 2.
  • the QSRP-expressing gene (Reg.) is operably linked to an inducible promoter (Ptet) under control of ribosome binding sites of differing strength (here BCD17 and BCD22).
  • Ptet inducible promoter
  • Figure 12 shows the effect of operably linked ribosome binding site on QSRP-mediated expression of a reporter protein of interest.
  • FIG. 13 shows a schematic of constructs with different ribosome binding sites both for tuning the expression of a QSRP and for tuning the expression of a QSSP to alter the rate of AHL production.
  • Each construct comprises three gene expression cassettes.
  • a reporter gene (GFP) is operably linked to a quorum sensing promoter (P QS ) under control of the ribosome binding site BCD 2.
  • the QSRP-expressing gene (Reg.) is operably linked to a repressible promoter (Ptet) under control of ribosome binding sites of differing strength (here BCD17 and BCD22).
  • the QSSP-expressing gene (Synthase) is operably linked to a quorum sensing promoter (P QS ) under control of ribosome binding sites of differing strength (here BCD1 and BCD8). If strains comprise a separate DNA construct expressing the conditional transcriptional repressor rTetR, addition of the quorum quenching compound (in this system anhydrotetracycline) suppresses expression of the QSRP.
  • Figures 14A shows cell density (OD 600 ) and cell density-normalized fluorescence over time in Kosakonia radicincitans grown in liquid culture and engineered with a repressible quorum sensing circuit, wherein the QSSP is operably linked to BCD1 and the QRSP is operably linked to BCD22 (ST2712).
  • QQ+ indicates a condition with a quorum quenching compound to suppress GFP protein of interest expression.
  • QQ- indicates a condition without a quorum quenching compound to suppress GFP protein of interest expression.
  • Figure 14B shows cell density (OD 600 ) and cell density-normalized fluorescence over time in Kosakonia radicincitans grown in liquid culture and engineered with a repressible quorum sensing circuit, wherein the QSSP is operably linked to BCD1 and the QRSP is operably linked to BCD17 (ST2713).
  • QQ+ indicates a condition with a quorum quenching compound to suppress GFP protein of interest expression.
  • QQ- indicates a condition without a quorum quenching compound to suppress GFP protein of interest expression.
  • Figure 14C shows cell density (OD 600 ) and cell density-normalized fluorescence over time in Kosakonia radicincitans grown in liquid culture and engineered with a repressible quorum sensing circuit, wherein the QSSP is operably linked to BCD8 and the QRSP is operably linked to BCD17 (ST2714).
  • QQ+ indicates a condition with a quorum quenching compound to suppress GFP protein of interest expression.
  • QQ- indicates a condition without a quorum quenching compound to suppress GFP protein of interest expression.
  • Figure 14D shows cell density (OD 600 ) and cell density-normalized fluorescence over time in Kosakonia radicincitans grown in liquid culture and engineered with a repressible quorum sensing circuit, wherein the QSSP is operably linked to BCD8 and the QRSP is operably linked to BCD22 (ST2715).
  • QQ+ indicates a condition with a quorum quenching compound to suppress GFP protein of interest expression.
  • QQ- indicates a condition without a quorum quenching compound to suppress GFP protein of interest expression.
  • Figure 15 shows bacterial cell counts for bacteria isolated from bulk soil (BS) or from the corn-root rhizoplane (RP), as determined by flow cytometry.
  • Figure 16 shows relative expression (defined as inducible GFP fluorescence / constitutive LSSmScarlett fluorescence) for bacteria isolated from bulk soil (BS) or from the corn-root rhizoplane (RP), as determined by flow cytometry.
  • P14610WO00 Tet repressor inhibits expression of the AhlR QSRP in the presence of anhydrotetracycline (aTc).
  • aTc anhydrotetracycline
  • the AhlR QSRP, AhlI QSSP, and protein of interest e.g., SfGFP, PhiC rec (e.g., PhiC31 integrase), or a uAT are induced.
  • GEB genetically engineered bacteria
  • QS-P quorum sensing promoters
  • GEB provided herein will typically and advantageously be below the threshold population density which results in expression of the proteins or RNAs of interest following their placement in plant growth media including soil, permitting more rapid and/or more extensive (e.g., higher density) growth in the plant growth media and/or colonization of crop plants grown therein.
  • the desired production of the proteins, RNAs, and/or agriculturally relevant compounds by the GEB occurs once the GEB exceed certain threshold population densities (e.g. about 6 x 10 3 CFU/mL to about 5 x 10 8 CFU/mL).
  • certain threshold population densities e.g. about 6 x 10 3 CFU/mL to about 5 x 10 8 CFU/mL.
  • the genetically engineered bacteria are placed in the plant growth medium (e.g., soil) and then grow to a higher titer until the threshold population density is reached and expression of the protein or RNA of interest which is or causes the production of an agriculturally relevant compound is switched on.
  • bacteria which have the protein or RNA of interest under the control of another promoter which is not quorum sensing will express the protein, RNA, and/or compound before and/or shortly after placement in the plant growth media and/or in association with plants and will not grow to the higher titers achieved by the genetically engineered bacteria provided herein.
  • a constitutive promoter e.g., a constitutive promoter
  • Higher titers of the engineered bacteria provided herein can thus provide for higher titers of the desired proteins, RNAs, and/or agriculturally relevant compounds in the plant growth media and/or in association with plants in comparison to bacteria lacking the quorum sensing promoter-controlled genes.
  • QS-P regulated genes encoding the RNAs and proteins of interest are activated on plant roots and in the rhizosphere (millimeters from plant roots) where cell density is high, but not in bulk soil (far from plant roots) where cell density is comparatively low.
  • Another aspect of the methods and GEB provided herein are genetic systems which can selectively inhibit expression of the proteins or RNAs of interest when the GEB are grown to densities above the threshold population density in fermenters or bioreactors. Such selective inhibition of expression the RNAs and proteins of interest can permit more rapid and economical Atty. Dkt. No. P14610WO00 production of high-titer GEB cultures used to prepare compositions for agricultural use.
  • the density-dependent genetic circuit can provide for desired expression of the RNA or protein of interest when the GEB are in plant growth media and/or associated with a plant and for selective inhibition of undesired expression of the RNA or protein of interest when the GEB are in a fermenter or bioreactor.
  • Quorum sensing promoter-controlled gene expression systems provided herein are especially useful for the production of ammonia by bacteria which can act as bio-fertilizers. Constitutively active ammonia release can significantly inhibit bacteria growth as protein synthesis is effectively shut down.
  • bacteria provided herein are engineered to grow first to a high density in soil before the ammonia release mechanism is activated.
  • quorum sensing gene expression systems allow a desirable delay in ammonia production.
  • the terms “about” or “approximately” indicate values slightly above or below the cited values, e.g., plus or minus 0.1% to 10% of the cited value.
  • the phrase “agriculturally relevant compound” refers to a compound that provides for plant growth, altered plant morphology (e.g., increased branching/surface area of root systems), plant nutrition, plant growth regulation, or plant protection from abiotic (e.g., drought, salinity, cold, heat, or excess water stress) or biotic stress (e.g., plant pests including bacterial, fungal, insect, nematode, and viral plant pests).
  • agriculturally useful bacteria or acronym “AUB” refers to bacteria which can grow in plant growth media (e.g., soil) and/or which can colonize crop plants.
  • the phrase “constitutive promoter” refers to a promoter, which is active under most growth and/or stationary phase conditions (e.g., in biofilms) in a given organism. Constitutive promoters include the promoters of SEQ ID NO: 390-400 and variants thereof having at least 90%, 95% or 99% sequence identity thereto.
  • the phrase “control element” refers to a promoter, a 5’ untranslated region (5’ UTR), a ribosome binding site, an enhancer, an insulator, a silencer, or a terminator.
  • Control elements comprising a promoter, 5’ UTR, an enhancer, an insulator, and/or a silencer can contain Atty. Dkt. No. P14610WO00 transcriptional repressor binding sites, transcriptional activator binding sites, ribozymes, protein recognition sites, and/or sites for chemical modification of nucleobases.
  • the term “gene,” as used herein, refers to a hereditary unit consisting of a sequence of DNA located on a chromosome, plasmid, or other extra-chromosomal element that contains the genetic instruction for a particular characteristic or trait in an organism.
  • the term “gene” thus includes a nucleic acid (for example, DNA or RNA) sequence that comprises coding and/or non-coding sequences necessary for the production of an RNA, a polypeptide, or a precursor of the RNA or protein.
  • a functional polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence as long as the desired activity or functional properties (e.g., enzymatic activity, DNA-binding activity, transcriptional activation, transcriptional repression, pesticidal activity, ligand binding, and/or signal transduction) of the polypeptide are retained.
  • heterologous is used herein to refer to any polynucleotide (e.g., DNA molecule) that has been introduced into a microorganism (e.g., a bacterium) where the polynucleotide is not sourced from the microorganism and/or has been inserted into a new location (e.g., in a distinct DNA sequence in the chromosome, plasmid, or other extrachromosomal element) in the microorganism (e.g., the bacterium).
  • a microorganism e.g., a bacterium
  • Non-limiting examples of heterologous DNA molecules that can be introduced into a microorganism include a non-naturally occurring (e.g., synthetic and/or recombinant) DNA molecule, a DNA molecule found in another microorganism (e.g., an intergeneric transfer of a DNA molecule comprising DNA from an organism of a different taxonomic genus), a DNA molecule found in another species (e.g., an intrageneric transfer of a DNA molecule comprising DNA from an organism of the same taxonomic genus), a DNA molecule found in a different location in the same species, and/or a DNA molecule found in the same strain or isolate of a species, where the DNA molecule has been inserted at a new location.
  • a non-naturally occurring (e.g., synthetic and/or recombinant) DNA molecule e.g., synthetic and/or recombinant) DNA molecule
  • a DNA molecule found in another microorganism
  • phosphate-sensitive promoter refers to a promoter which is activated (e.g., up-regulated) or repressed (e.g., down-regulated) when phosphate concentration changes. In certain embodiments, a phosphate-sensitive promoter is activated or repressed when phosphate concentration crosses a threshold value. In certain embodiments, a phosphate-sensitive promoter is up-regulated or down-regulated proportionally in response to a change in phosphate concentration.
  • a phosphate-sensitive promoter is activated when phosphate concentrations decrease below a threshold value of about 50 ⁇ M, 40 ⁇ M, 30 ⁇ M, 20 ⁇ M, or 10 ⁇ M to about 1 ⁇ M. In certain embodiments, a phosphate-sensitive promoter will be active at a concentration of phosphate from 0 ⁇ M to about 1 ⁇ M, 2 ⁇ M, 5 ⁇ M, 7 ⁇ M, or 10 ⁇ M. [0049] As used herein, the phrase “quorum sensing promoter” refers to a promoter which is activated (e.g., up-regulated) or repressed (e.g., down-regulated) when cell density changes.
  • a quorum sensing promoter is activated or repressed when cell density crosses a threshold value. In certain embodiments, a quorum sensing promoter is activated when cell densities increase above a threshold value of about 6 x 10 3 CFU/mL to about 5 x 10 8 CFU/mL.
  • the term “refactored” refers to a gene, gene cluster, or operon that has been restructured.
  • restructuring may include changing a DNA coding sequence to a DNA sequence divergent from the wild-type gene while still encoding the same polypeptide. In some embodiments, restructuring may include computationally scanning genes to identify control elements, removing them, and optionally replacing them with different control elements.
  • refactored refers to a gene, gene cluster, or operon wherein the naturally-occurring promoter has been modified to contain a new promoter which exhibits different regulatory characteristics.
  • refactored gene clusters include refactored nif and/or fix gene clusters which allow nitrogenase to be expressed without transcriptional down-regulation by fixed nitrogen.
  • fix gene clusters which allow nitrogenase to be expressed without transcriptional down-regulation by fixed nitrogen.
  • refactoring methods and refactored nif gene clusters include those disclosed in US Patent No, 11479516, incorporated herein by reference in its entirety.
  • segment of a 5’ UTR refers to one or more nucleotides of DNA encoding a 5’ UTR (5’ untranslated region) of a transcript and/or RNA comprising one or more nucleotides of a 5’ UTR.
  • the segment of a 5’ UTR will comprise at least the first nucleotide of the 5’ UTR but can also comprise at least a ribosome binding site in a 5’ UTR or the entire 5’ UTR.
  • transcriptional activator refers to proteins or ribonucleoprotein (RNP) complexes capable of activating expression of a particular target gene.
  • Transcription factors can thus include: (i) transcriptional repressors that decrease the rate of transcription of one or more particular target gene(s) upon binding to a specific DNA sequence, (ii) said transcriptional activators defined above, (iii) proteins or ribonucleoprotein (RNP) complexes that stabilize the rate of transcription, (iv) proteins or ribonucleoprotein (RNP) complexes that selectively accomplish a first function selected from a group comprising (i), (ii), or (iii) under a first set of physical and/or chemical conditions and a different function selected from (i), (ii), or (iii) under a different set of physical and/or chemical conditions, and (v) proteins or ribonucleoprotein (RNP) complexes that selectively accomplish a first function selected from a group comprising (i), (ii), or (iii) upon interaction with a first protein binding partner or first specific DNA sequence and a different function selected from (i), (ii), or (
  • one or more bacterial genes are down-regulated to cause the production of the agriculturally relevant compounds.
  • bacterial genes which can be downregulated to produce ammonia include amtB, glnA, glnB, glnK, glnZ, nifL, and/or draT genes.
  • Target amtB, glnA, glnB, glnK, glnZ, nifL, and/or draT gene sequences which can be down-regulated include those set forth in Table 5, in the sequence listing, and in US Patent Application No. US20210315212, which is incorporated herein by reference in its entirety.
  • Target amtB, glnA, glnB, glnK, glnZ, nifL, or draT gene sequences which can be down-regulated further include sequences having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity across the entire length of the sequences set forth in Table 1, in Table 5, in the sequence listing, and in US Patent Application No. US20210315212.
  • Examples of such combinations include down-regulation of GlnA (glutamine synthetase) by quorum sensing promoter- controlled expression of a modified glnE gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity.
  • GlnA glutamine synthetase
  • uAT unidirectional adenylyltransferase
  • Examples of bacterial genes which can be up-regulated to produce ammonia include nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity (also referred to herein as GlnE-uAT), one or more nif cluster gene(s), and/or one or more fix cluster gene(s).
  • uAT unidirectional adenylyltransferase
  • Target nifA, ntrC, glnR, glutaminase encoding genes, GlnE-uAT genes, nif cluster gene(s), and/or fix cluster gene sequences which can be up-regulated include those set forth in Table 3, in the sequence listing, and in US Patent Application No. US20210315212, which is incorporated herein by reference in its entirety.
  • Target nifA, ntrC, glnR, glutaminase encoding genes, GlnE-uAT genes, nif cluster gene(s), and/or fix cluster genes which can be up-regulated further include sequences having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity across the entire length of the sequences set forth in Table 3, in the sequence listing, and in US Patent Application No. US20210315212.
  • P14610WO00 uAT genes, nif cluster gene(s), and/or fix cluster gene sequences which can be up-regulated also include sequences which encode NifA, NtrC, GlnR, glutaminase, GlnE-uAT, nif cluster, and/or fix cluster proteins having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity across the entire length of NifA, NtrC, GlnR, glutaminase, GlnE-uAT, nif cluster, and/or fix cluster proteins encoded by genes set forth in Table 3, in the sequence listing, and/or in US Patent Application No.
  • one or more bacterial genes are up-regulated to cause the production of phosphate from insoluble forms of phosphate (e.g., present in plant growth media).
  • Target phytase-, acid phosphatase- e.g., an acpA, aphA, phoC, napA, napD, or napE gene
  • GAD-, GDH-, or pyrroloquinoline (PQQ) synthase-encoding genes which can be up-regulated further include sequences having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity across the entire length of the sequences set forth in Table 3, in the sequence listing, and in US Patent Application No. US20210345618.
  • Target phytase-, acid phosphatase- e.g., an acpA, aphA, phoC, napA, napD, or napE gene
  • GAD-, GDH-, or pyrroloquinoline (PQQ) synthase-encoding genes which can be up-regulated also include sequences which encode phytase, acid phosphatase (e.g., an AcpA, AphA, PhoC, NapA, NapD, or NapE protein), GAD, GDH, or pyrroloquinoline (PQQ) synthase proteins having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity across the entire length of phytase, acid phosphatase (e.g., an AcpA, AphA, PhoC, NapA, NapD, or NapE protein), GAD, GDH, or pyrroloquinoline (PQQ) synthas
  • one or more genes set forth in Table 3 can be up-regulated by one or more of the quorum sensing systems or genes set forth in Table 4. Particular embodiments of the systems for up- regulation of genes set forth in Table 3 are also disclosed within the following numbered embodiments 1- 131.
  • the agriculturally relevant compound is not cellulose and/or genes that are upregulated in the numbered embodiments do not encode a protein involved in the synthesis and/or secretion of cellulose.
  • Such proteins involved in the synthesis and/or secretion of cellulose include bcs operon gene(s), a cmcAx gene, a ccpAx gene, a bglAx gene, pgm gene, galU gene, cdg operon, and/or dgc gene set forth in US Patent Application Publication No. US20230034438.
  • Non-limiting summary for gene up-regulation mechanisms at cell densities which exceed a threshold density System Non-limiting materials
  • target genes encoding an RNA or protein of interest can be upregulated or down regulated by an SSR- or integrase- controlled promoter switch where a control element comprising a promoter can be inverted.
  • inversion of the promoter by the SSR- or integrase- can result in the down-regulation of the RNA sequence or protein of interest by disrupting operable linkage of the promoter to DNA encoding the RNA or protein.
  • inversion of the promoter by the SSR- or integrase- can result in the up-regulation of the RNA sequence or protein of interest by operably linking the promoter to DNA encoding the RNA or protein (e.g., an SSR- or integrase- controlled promoter switch).
  • Control elements used in the promoter switch can comprise a constitutive promoter, an inducible promoter, or quorum sensing promoter and at least a segment of a 5’ UTR.
  • an SSR- or integrase-controlled promoter switch can be designed to express a first gene before expression of the SSR or integrase and a second gene after expression of the SSR or integrase by the QS-P.
  • the first gene can be selected from genes targeted for down-regulation in Table 1 and the second gene can be selected from a gene targeted for up-regulation in Table 3.
  • the first gene can be a wild-type or even improved copy of a gene targeted for down- regulation in Table 1 and the second gene can be a wild-type copy of that same first gene from Table 1 which is expressed at levels lower than the wild-type or first gene, a mutated variant of that same first gene from Table 1 with reduced enzymatic or biological activity, or a mutated variant of that same first gene from Table 1 which is also expressed at levels lower than the wild-type or first gene.
  • Reductions in expression of the second gene encoding a wild-type or GS variant can be achieved in a variety of ways including use of weak ribosome binding sites in the 5’ UTR, use of non-preferred codons, Atty. Dkt. No. P14610WO00 substitution of the ATG start codon with a GTG start codon, mutations that result in decrease protein or mRNA and/or protein stability, and combinations thereof. Certain methods of reducing expression of a glnA gene disclosed in US20200331820 can be adapted for use in embodiments disclosed herein.
  • GEB comprising this promoter switch will express wild-type GlnE protein prior to QS-P promoter-mediated activation of the SSR or integrase, promoting growth of the GEB in plant growth media.
  • the promoter in the promoter switch is inverted and operably linked to the GlnE-uAT gene, resulting in the adenylation and inactivation of GS and ammonia production.
  • Sources of GlnE-uAT genes are set forth in Table 3.
  • the SSR- or integrase- controlled promoter switch is placed between a first gene comprising a nifL gene and a second gene comprising a nifA gene, where the promoter in the SSR- or integrase- controlled promoter switch is operably linked to the first nifL gene prior to SSR- or integrase-mediated promoter inversion resulting from QS-P promoter activation of the SSR or integrase.
  • GEB comprising this promoter switch will express wild-type NifL protein prior to QS- P promoter-mediated activation of the SSR or integrase, promoting growth of the GEB in plant growth media.
  • a gene encoding the RNA or protein of interest or which regulates expression of the gene encoding the RNA or protein of interest is at the end of the cascade.
  • a QS-P promoter that drives expression of the first repressor or transcriptional activator, respectively.
  • a repressor cascade can further comprise a gene encoding a transcriptional activator which is controlled by a repressor in the cascade (e.g., the terminal repressor in the cascade which regulates ).
  • a transcriptional activator cascade can further comprise a gene encoding a repressor which is controlled by a transcriptional activator in the cascade (e.g., the Atty. Dkt. No. P14610WO00 terminal repressor in the cascade).
  • a transcriptional activator in the cascade e.g., the Atty. Dkt. No. P14610WO00 terminal repressor in the cascade.
  • each repressor represses the subsequent repressor in the cascade.
  • each transcriptional activator activates the subsequent transcriptional activator in the cascade.
  • control elements comprising QS-P promoters and optionally 5’ UTR segments can be used in the GEB, methods, and systems provided herein. Desirable characteristics of such control elements include activation of expression of operably linked RNAs or proteins in agriculturally useful bacteria in response to population densities which exceed threshold population densities in the plant growth medium.
  • control elements comprising the QS-P are used in bacteria of the genus or the species from which they were derived in whole or in part. Atty. Dkt. No. P14610WO00 [0075]
  • control elements comprising QS-P promoters and optionally 5’ UTR segments will comprise elements which can be bound by an AhlR, CCiR, CinR, CviR, EsaR D91G, EsaR, LasR, LuxR, or TraR QSRP.
  • AUB include bacteria in the taxonomic genera of Acetobacter, Acidothermus, Acinetobacter, Agrobacterium, Aromatoleum, Arthrobacter, Azoarcus, Azorhizobium, Azospirillum, Azotobacter, Bacillus, Bifidobacterium, Bradyrhizobium, Burkholderia, Chromobacterium, Conexibacter, Curtobacterium, Ensifer, Enterobacter, Erwinia, Escherichia, Flavobacterium, Frankia, Gaiella, Gluconacetobacter, Gluconobacter, Herbaspirillum, Klebsiella, Kosakonia, Lactobacillus, Lactococcus, Lysinibacillus, Maritimibacter, Mesorhizobium, Methylobacterium, Nitrosocosmicus, Nitrososphaera, Paenarthrobacter, Paenibacillus, Pantoea, Pediococcus, Peribacillus,
  • Such AUB QS-P sequences can comprise those set forth in SEQ ID NO: 356-364, 373, and 374 and variants thereof comprising 1, 2, or 3 nucleotide substitutions.
  • Such AUB QS-P promoters can comprise DNA molecules having at least 85%, 90%, 95%, 98%, or 99% sequence identity across the entire length of any one of SEQ ID NO: 356-364, 373, and 374.
  • Such QS-P promoters can also include those which are operably linked to a gene encoding a protein having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to a QSSP protein (e.g., an AhlI, CciI, CinI, CviI, EsaI, LuxI, TraI, or LasI protein) set forth in Table 5 and the sequence listing.
  • a QSSP protein e.g., an AhlI, CciI, CinI, CviI, EsaI, LuxI, TraI, or LasI protein
  • control elements comprising the AUB sequences and AUB QS-P promoters provided herein can activate expression of operably linked RNAs or gene-encoding proteins of interest in agriculturally useful bacteria including Gluconacetobacter sp., Azorhizobium sp., Azospirillum sp., Herbaspirillum sp., Kosakonia sp., Paenibacillus sp., and Pseudomonas sp. in response to increases in cell density above a threshold population density.
  • control elements comprising the AUB sequences and AUB QS-P promoters provided herein can activate expression of operably linked RNAs or proteins in a Gluconacetobacter diazotrophicus, Azorhizobium caulinodans, Azospirillum brasilense, Herbaspirillum seropedicae, Kosakonia radicincitans, Paenibacillus azotofixans, and Pseudomonas stutzeri isolate, strain, or derivative thereof.
  • a control element Atty. Dkt. No.
  • Gluconacetobacter sp. e.g., the corresponding AUB sequences and AUB QS-P promoters set forth in Table 5
  • Azorhizobium sp. Azospirillum sp.
  • Herbaspirillum sp. Kosakonia sp.
  • Paenibacillus sp. or Pseudomonas sp.
  • a control element comprising an AUB sequences or AUB QS-P promoter derived in whole or in part from Gluconacetobacter diazotrophicus, Azorhizobium caulinodans, Azospirillum brasilense, Herbaspirillum seropedicae, Kosakonia radicincitans, Paenibacillus azotofixans, or Pseudomonas stutzeri (e.g., the corresponding AUB sequences and AUB QS-P promoters set forth in Table 5) are respectively used in a GEB obtained from a Gluconacetobacter diazotrophicus, Azorhizobium caulinodans, Azospirillum brasilense, Herbaspirillum seropedicae, Kosakonia radicincitans, Paenibacillus azotofixans, or Pseudomonas stutzeri isolate, strain, or derivative thereof.
  • the AUB provided herein comprising the control element comprising an AUB sequences or AUB QS-P promoter which is operably linked to a nucleic acid sequence encoding the RNA or protein of interest can further comprise one or more heterologous gene expression cassettes encoding a quorum sensing synthase protein (QSSP) and/or a quorum sensing regulator protein (QSRP), where the QSSP can produce a quorum sensing signal molecule (QSSM, e.g., an AHL) which can activate the QSRP and the QS-P.
  • QSSP quorum sensing synthase protein
  • QSRP quorum sensing regulator protein
  • Combinations of QSSP, QSRP, and QS-P which can be used together in the agriculturally useful bacteria include: i.
  • an AhlI protein, an AhlR protein, and an ahlI QS-P respectively; ii. a CinI protein, CinR protein, and a cinI QS-P, respectively; iii. a CciI protein, a CciR protein, and a cciI QS-P, respectively; iv. a CviI protein, a CviR protein, and a cviI QS-P, respectively; v. an EsaI protein, an EsaR D91G protein, and an esaR repressable QS-P, respectively; vi. an EsaI protein, an EsaR protein, and an esaI QS-P, respectively; vii.
  • the combinations can be used either in an AUB of the same genus or species from which they were derived (e.g., in a Pseudomonas sp., a Burkholderia sp., a Rhizobium sp., a or a Pantoea sp.).
  • the heterologous gene expression cassette comprising the control elements comprising the sequences and QS-P promoters (e.g., the corresponding AUB sequences and AUB QS-P promoters set forth in Table 5) is integrated at a location in the chromosome of the genetically engineered bacterium which does not comprise the location of an endogenous quorum sensing promoter in the unmodified agriculturally useful bacterium.
  • a control element comprising a QS-P promoter e.g., QS-P promoter in Table 5
  • a control element comprising a cinI_V1 or cinI_V2 QS-P promoter is integrated in the genome of the GEB at a location other than the location of the endogenous (e.g., wild-type) cinI_V1 or cinI_V2 QS-P promoter in the GEB.
  • Methods for inserting control elements comprising QS-P promoters at sites distinct from the endogenous gene include Tn7 transposon mediated insertion (McKenzie and Craig, N.L., 2006, doi: 10.1186/1471-2180-6-39).
  • Methods of producing preparations of the genetically engineered bacterium (GEB) for use in treating plant growth media, plants, plant parts, plant propagules, and/or for formulating a composition comprising the GEB are provided herein.
  • the methods can comprising growing the GEB in a bacterial growth medium comprising a carbon and nitrogen source and harvesting the GEB from the media.
  • Conditions for growing the GEB include axenic growth in continuous stirred tank reactors, batch fermentation reactors, and the like.
  • the GEB are grown either (a) in contact with the quorum quenching compound QQ; or (b) in exposure to a temperature above the threshold temperature to suppress expression of the RNA or protein of interest encoded by the gene which is operably linked to the quorum sensing promoter.
  • Such contact with the quorum quenching compound QQ or in exposure to a temperature above the threshold temperature result in production of one or more deactivators of the quorum sensing system which comprises the quorum sensing synthase protein (QSSP) able to synthesize a quorum sensing signal molecule, the quorum sensing regulator protein (QSRP), and quorum sensing promoter in the GEB.
  • QSSP quorum sensing synthase protein
  • QSRP quorum sensing regulator protein
  • the quorum quenching compound QQ is inorganic phosphate or soluble phosphate, wherein the control element operably linked to the gene encoding the first QSRP comprises a phosphate-sensitive promoter.
  • the control element operably linked to the gene encoding the first QSRP comprises a phosphate-sensitive promoter.
  • a temperature above the threshold temperature is used to suppress expression of the recombinase which is operably linked to the quorum sensing promoter
  • temperature induced de-repression of the deactivator or temperature induced de-activation of the first QSSP and/or the first QSRP can be used.
  • QS systems are deactivated by quorum quenching enzymes (QEs) that degrade AHL quorum sensing signal molecules (QSSMs).
  • QEs span several classes of QSSM- degrading enzymes, including AHL acylase enzymes (SEQ ID NO: 320-322, and 323), alpha-beta hydrolase fold lactonase proteins (SEQ ID NO: 324, 325, and 326), metallo-beta-lactamase-like lactonase proteins (SEQ ID NO: 327-330, and 331), phosphotriesterase-like lactonase proteins (SEQ ID NO: 332-335, and 336), and variants of the QEs having at least 70%, 80%, 85%, 90%, 95%, 98%, and 99% sequence identity to SEQ ID NO: 320 to 335, or 336).
  • AHL acylase enzymes SEQ ID NO: 320-322, and 323
  • alpha-beta hydrolase fold lactonase proteins SEQ ID NO: 324, 325, and 326
  • the adhesive agents can comprise one or more waxes (e.g., carnauba wax, beeswax, or Chinese wax, shellac wax, spermaceti wax, candelilla wax, castor wax, ouricury wax, or rice bran wax), a polysaccharide (e.g., starch, dextrins, maltodextrins, alginate, and chitosans), a fat, oil, a protein (e.g., gelatin and zeins), gum ambles, and/or a shellac.
  • waxes e.g., carnauba wax, beeswax, or Chinese wax, shellac wax, spermaceti wax, candelilla wax, castor wax, ouricury wax, or rice bran wax
  • a polysaccharide e.g., starch, dextrins, maltodextrins, alginate, and chitosans
  • P14610WO00 adhesive agents can comprise one or more polymers or copolymers including polyvinyl acetates, polyvinyl acetate copolymers, ethylene vinyl acetate (EVA) copolymers, polyvinyl alcohols, polyvinyl alcohol copolymers, celluloses (e.g., ethylcelluloses, methylcelluloses, hydroxymethylcelluloses, hydroxypropylcelluloses, and carboxymethylcelluloses), polyvinylpyrolidones, vinyl chloride, vinylidene chloride copolymers, calcium lignosulfonates, acrylic copolymers, polyvinylacrylates, polyethylene oxide, acylamide polymers and copolymers, polyhydroxyethyl acrylate, methylacrylamide monomers, and/or polychloroprene.
  • EVA ethylene vinyl acetate
  • the LuxI protein comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 354; or (ix) the TraI protein comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 355. [00105] 15.
  • the at least one agriculturally relevant compound is selected from the group consisting of a. at least one fertilizer or plant nutrient, optionally wherein the fertilizer or plant nutrient is selected from the group consisting of ammonia, ammonium, bioavailable carbon, calcium, iron, nitrate, nitrite, nitrogen, potassium, phosphate, sulfur, urea, zinc, a combination thereof, and a mixture thereof; b. at least one pesticide, optionally wherein the pesticide is an RNA- and/or protein-based fungicide, insecticide, nematicide, antibacterial agent, and/or antiviral agent; c.
  • the first target gene(s) is/are glnA, amtB, glnB, glnK, glnZ, nifL, and/or draT and the fertilizer is ammonia; or (u) a transcriptional activator protein, wherein the transcriptional activator protein binds a natural or synthetic DNA motif in a promoter of any one or more first target gene(s) and increases expression of any one or more of the first target gene(s), optionally wherein the transcriptional activator protein comprises the tet responsive element-binding tTA transcription factor or a transcriptional activator domain fused to a DNA targeting protein, optionally wherein the DNA targeting protein is a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator- like effector (TALE), or any variant thereof, optionally wherein the transcriptional activator domain
  • TALE transcription activator-like effector
  • the plant nutrient is zinc or potassium and the protein of interest is a gluconate dehydrogenase (GAD) enzyme;
  • GAD gluconate dehydrogenase
  • the plant nutrient is iron and the protein of interest is a siderophore biosynthetic and transport proteins optionally selected from a dhbACDEBF gene cluster; a non-ribosomal peptide synthetase (NRPS), polyketide synthase (PKS), and NRPS-independent siderophore synthetase (NIS); and major facilitator superfamily (MFS) transporters (ymfE), TonB, ExbD, and/or ExbB; (iii) the phytohormone is auxin and the
  • the integrase is a serine integrase
  • the serine integrase is a phage PhiC31 serine integrase, IntS, IntM, IntG – ICEMcSym 1271, Yd–L - ICEBs, or I–t - ICE SXT/R39 integrase
  • the SSRRS are attB and attP sites recognized respectively by the PhiC31, IntS, IntM, IntG, YdcL, or Sxt/R39 integrase.
  • the genetically engineered bacterium of embodiment 42 wherein the genetically engineered bacterium lacks (ii) and (iii), and the first target gene comprises: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and/or one or more fix cluster gene(s), and the agriculturally relevant compound is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein
  • the genetically engineered bacterium of embodiment 42 wherein the genetically engineered bacterium further comprises (ii) and (iii), and the first target gene comprises: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and/or one or more fix cluster gene(s) and the agriculturally relevant compound is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein
  • Dkt. No. P14610WO00 comprise(s) one or more internal integrative SSRRS and retain(s) activity of said second target gene(s), promoter(s), and/or 5’ UTR(s) and wherein the genetically engineered bacterium further comprises an integrative element comprising an SSRRS; optionally wherein the internal integrative SSRRS is/are an attB site(s), the integrative element comprises an SSRRS comprising an attP site; and/or optionally wherein the second target gene(s) is/are glnA, amtB, glnB, glnK, glnZ, nifL, and/or draT and the fertilizer is ammonia; (n) a site-specific recombinase (SSR) or integrase protein, wherein a control element comprising a promoter and/or at least a segment of a 5’ UTR: (i) is flanked by site-specific recombinase
  • any one or more second target gene(s) of the genetically engineered bacterium comprise(s) one or more in-frame insertion(s) comprising DNA encoding the PSRS in the protein coding region of the second target gene(s), wherein the target protein product comprising the one or more in-frame insertion(s) has activity, and wherein cleavage of the target protein product(s) by the protease deactivates the target protein product(s), optionally wherein the location of the one or more in-frame insertion is given by Table 6, optionally wherein the second target gene is under the control of a constitutive promoter, and/or optionally wherein the second target gene(s) is/are glnA, amtB, glnB, glnK, glnZ, nifL, and/or draT and the fertilizer is ammonia
  • PSRS protease specific recognition sequence
  • the genetically engineered bacterium of embodiment 52 wherein the target gene is a glnA, amtB, glnB, glnK, glnZ, nifL, and/or draT gene and the agriculturally relevant compound is ammonia.
  • the protein of interest is a transcriptional activator protein and wherein the genetically engineered bacterium further comprises: (i) a second control element comprising a promoter which is activated by the transcriptional activator protein and operably linked to a gene encoding a repressor protein; and Atty. Dkt. No.
  • P14610WO00 (ii) a third control element comprising a promoter which is repressed by the repressor and operably linked to a target gene.
  • the target gene is a glnA, amtB, glnB, glnK, glnZ, nifL, and/or draT gene and the agriculturally relevant compound is ammonia.
  • the target gene is a glnA, amtB, glnB, glnK, glnZ, nifL, and/or draT gene and the agriculturally relevant compound is ammonia.
  • the genetically engineered bacterium of embodiment 32 wherein the glnA, amtB, glnB, glnK, glnZ, nifL, and/or draT gene in (n), (o), (p), or (q) is under the control of a heterologous constitutive promoter.
  • 57. The method of embodiment 48, wherein the glnA, amtB, glnB, glnK, glnZ, nifL, and/or draT gene in (q), (r), (s), or (t) is under the control of a heterologous constitutive promoter.
  • the genetically engineered bacterium of embodiment 31, wherein the plant nutrient is phosphate and the protein of interest operably linked to the quorum sensing promoter comprises: a. a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase; b. an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof; c.
  • a protein which stimulates organic acid release from the bacterium optionally wherein the protein comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), a pyrroloquinoline (PQQ) synthase including pqqFABCDEG, or any combination of GAD, GDH, and PQQ; or, d. any combination of proteins of a, b, or c. [00149] 59.
  • GAD gluconate dehydrogenase
  • GDH glucose dehydrogenase
  • PQQ pyrroloquinoline
  • the genetically engineered bacterium of any one of embodiments 1 to 58 wherein: (i) the genetically engineered bacterium comprises the heterologous gene expression cassettes in a bacterium originally isolated from a plant growth medium or a plant; and (ii) wherein the bacterium originally isolated from the plant growth medium or the plant lacks the heterologous gene expression cassettes.
  • the bacteria are selected from the group of gram-negative bacteria.
  • Rhodococcus Rhodoplanes, Rhodopseudomonas, Rhodospirillum, Serratia, Solirubrobacter, Sphingobacterium, Sphingomonas, Stenotrophomonas, Streptomyces, Stutzerimonas, Variovorax, Xanthobacter, and Yoonia, optionally wherein the bacteria are selected from at least one of the taxonomic genera selected from the group consisting of Herbaspirillum, Azospirillum, Kosakonia, or Pseudomonas. [00153] 63.
  • compositions comprising the genetically engineered bacterium of any one of any one of embodiments 1 to 62 and an agriculturally acceptable carrier.
  • the composition further comprises: (i) an agriculturally acceptable adjuvant, optionally wherein the adjuvant comprises an adhesive agent, a desiccant, and/or a dispersant; (ii) a fungicide, an insecticide, a nematicide, a rodenticide, and/or a bacteriocide; and/or (iii) a fertilizer, optionally wherein the fertilizer comprises nitrogen, phosphorous, potassium, calcium, sulfur, magnesium, boron, chloride, manganese, iron, zinc, copper, molybdenum, and/or selenium.
  • the plant part of embodiment 67 wherein the part is a leaf, stem, root, or seed.
  • 69 The plant propagule of embodiment 67, wherein the propagule comprises a cutting, tuber, or stolon.
  • 70 Use of the plant part or plant propagule of embodiment 67 to grow a crop.
  • 71 The use of embodiment 70, wherein fertilizer input is reduced in comparison to a crop grown from a plant part or plant propagule which has not been treated.
  • An agricultural system comprising: (i) at least one engineered bacterium of any one of embodiments 1 to 62; (ii) at least one plant growth medium; and (iii) at least one crop plant, crop plant seed, or crop plant vegetative propagule; wherein the plant growth medium, crop plant, crop seed, and/or crop plant propagule comprise, are at least partially coated, imbibed, and/or are mixed with the engineered bacterium or a composition comprising the engineered bacterium and an agriculturally acceptable carrier. [00163] 73.
  • the system of embodiment 72, wherein the crop plant, seed, or vegetative propagule is an alfalfa, apple, banana, barley, bean, buckwheat, cabbage, cassava, chili, clover, coffee, corn, cotton, cowpea, cucumber, fonio, garlic, herb, lettuce, maize, melon, millet, nut, oat, oilseed rape, olive, onion, orange, Atty. Dkt. No.
  • the plant growth medium comprises soil and/or water, optionally wherein the soil and/or water is non-axenic.
  • 75 The system of embodiment 72, 73, or 74, wherein the vegetative propagule comprises a cutting, tuber, or stolon.
  • 76 The system of embodiment 72, 73, or 74, wherein the vegetative propagule comprises a cutting, tuber, or stolon.
  • a treated plant seed or plant propagule system comprising: (i) at least one crop plant seed or crop plant vegetative propagule; and (ii) at least one engineered bacterium of any one of embodiments 1 to 62, wherein the crop plant seed or crop plant propagule are at least partially coated, imbibed, and/or mixed with the engineered bacterium or a composition comprising the engineered bacterium and an agriculturally acceptable carrier. [00167] 77.
  • the crop plant, seed, or vegetative propagule is an alfalfa, apple, banana, barley, bean, buckwheat, cabbage, cassava, chili, clover, coffee, corn, cotton, cowpea, cucumber, fonio, garlic, herb, lettuce, maize, melon, millet, nut, oat, oilseed rape, olive, onion, orange, sunflower, pea, Phaseolus bean, plantain, potato, quinoa, rice, rye, safflower, sorghum, soybean, sugar beet, sugar cane, sunflower, tangerine, tobacco, tomato, triticale, turnip, wheat, or yam plant, seed, or vegetative propagule.
  • any one of embodiments 79 to 86 further comprising combining the harvested bacterial culture with an agriculturally acceptable carrier and optionally (i) an agriculturally acceptable adjuvant, optionally wherein the adjuvant comprises an adhesive agent, a desiccant, and/or a dispersant; (ii) a fungicide, an insecticide, a nematicide, a rodenticide, and/or a bacteriocide; and/or (iii) a fertilizer, optionally wherein the fertilizer comprises nitrogen, phosphorous, potassium, calcium, sulfur, magnesium, boron, chloride, manganese, iron, zinc, copper, molybdenum, and/or selenium, thereby forming a composition. Atty. Dkt.
  • a method of providing at least one agriculturally relevant compound to a plant comprising placing at least one genetically engineered bacterium of any one of embodiments 1 to 62 into a plant growth medium, wherein said at least one RNA sequence or protein of interest is or causes the production of said at least one agriculturally relevant compound when the population density of the genetically engineered bacterium exceeds a threshold population density in the plant growth medium and activates expression of said at least one RNA sequence or protein of interest.
  • the plant growth medium comprises soil and/or water, optionally wherein the soil and/or water is non-axenic.
  • the method of embodiment 91, wherein the placing is prior to, during, and/or after depositing a seed in the plant growth medium.
  • 94 The method of embodiment 91, wherein the placing is prior to, during, and/or after depositing a vegetative propagule in the plant growth medium.
  • 95 The method of embodiment 91, wherein the placing comprises depositing a seed which is at least partially coated with the genetically engineered bacterium in the plant growth medium or depositing both the seed and a composition comprising the genetically engineered bacterium in the plant growth medium.
  • 96 96.
  • the method of embodiment 91, wherein the placing comprises depositing the seed in furrow and contacting the seed in the furrow with a composition comprising the genetically engineered bacterium.
  • the placing of the genetically engineered bacterium in the plant growth medium is prior to, during, and/or after establishment of a plant in the plant growth medium.
  • P14610WO00 (iv) with a seed in the form of bio-priming where the seed is imbibed with an aqueous composition comprising the genetically engineered bacterium before planting; and/or (v) with a root dip transplant whereby a seedling root system is dipped in an aqueous composition comprising the genetically engineered bacterium. [00189] 99.
  • the at least one agriculturally relevant compound is selected from the group consisting of: (a) at least one fertilizer or plant nutrient, optionally wherein the fertilizer or plant nutrient is selected from the group consisting of ammonia, ammonium, bioavailable carbon, calcium, iron, nitrate, nitrite, nitrogen, potassium, phosphate, sulfur, urea, zinc, a combination thereof, and a mixture thereof; (b) at least one pesticide, optionally wherein the pesticide is an RNA- and/or protein-based fungicide, insecticide, nematicide, antibacterial agent, and/or antiviral agent; (c) at least one phytohormone or plant growth regulator, optionally wherein the phytohormone or plant growth regulator is an auxin, a cytokinin, a gibberellin, abscisic acid, a brassinosteroid, jasmonic acid, a polyamine, a strigolactone, trehalose, and
  • RNA sequence or protein of interest encoded by the heterologous gene expression cassette and operably linked to the control element comprises: (a) a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity and the fertilizer is ammonia; (b) a NifA protein, wherein the genetically engineered bacterium optionally comprises a loss-of- function mutation in any one or more of the amtB, draT, glnA, glnB, glnK, glnR, glnZ, or nifL genes, wherein the genetically engineered bacterium optionally comprises one or more heterologous genes from a wild- type or refactored nif or fix gene cluster and the fertilizer is ammonia; (c) a GlnR protein and the fertilizer is ammonia; (d) a glutaminase
  • first target gene(s) is/are a glnA, amtB, glnB, glnK, glnZ, nifL, and/or draT gene and the fertilizer is ammonia; (g) a non-coding synthetic small RNA (sRNA); optionally wherein the non-coding synthetic small RNA (sRNA) binds a natural or synthetic DNA and/or RNA motif in the promoter, 5’ UTR, and/or coding region of any one or more first target gene(s) of the genetically engineered bacterium, optionally wherein the non-coding synthetic small RNA (sRNA) comprises a guide RNA that additionally binds an RNA- guided DNA or RNA endonuclease or variant thereof and/or optionally wherein the first target gene(s) is/are a glnA, amtB, glnB, glnK, glnZ, nifL, and/
  • the first target gene(s) is/are glnA, amtB, glnB, glnK, glnZ, nifL, and/or draT and the fertilizer is ammonia; (l) a site-specific recombinase (SSR) or integrase protein, wherein a control element comprising a promoter and at least a segment of a 5’ UTR: (i) is flanked by site-specific recombinase recognition sites (SSRRS) in an inverted configuration; and (ii) is operably linked to one or more first target gene(s) of the genetically engineered bacterium upon inversion by the SSR or integrase; optionally wherein the control element which is operably linked to said first target gene(s) upon inversion by the SSR or integrase comprises a constitutive promoter, an inducible promoter, or
  • any one or more first target gene(s) of the genetically engineered bacterium comprise(s) an in-frame insertion(s) of DNA encoding the PSRS at the N-terminus of the protein coding region of the gene followed by a -Leu, - Phe, -Trp, or -Tyr residue and wherein cleavage of PSRS from the N-terminus of the protein(s) encoded by the gene(s) by the protease results in a protein comprising an N-terminal
  • the first target gene(s) is/are glnA, amtB, glnB, glnK, glnZ, nifL, and/or draT and the fertilizer is ammonia; or (u) a transcriptional activator protein, wherein the transcriptional activator protein binds a natural or synthetic DNA motif in a promoter of any one or more first target gene(s) and increases expression of any one or more of the first target gene(s), optionally wherein the transcriptional activator protein comprises the tet responsive element-binding tTA transcription factor or a transcriptional activator domain fused to a DNA targeting protein, wherein the DNA targeting protein is optionally a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator- like effector (TALE), or any variant thereof, wherein the transcriptional activator domain is optional
  • integrase is a serine integrase
  • serine integrase is a phage PhiC31 serine integrase, IntS, IntM, IntG – ICEMcSym 1271, YdcL - ICEBs, or Int - ICE SXT/R39 integrase
  • the SSRRS are attB and attP sites recognized respectively by the PhiC31, IntS, IntM, IntG, YdcL, or Sxt/R39 integrase.
  • one or more SSR is a yeast flippase (FLP) recombinase and the SSRRS are FRT sites; or (ii) one or more SSR is a Cre-recombinase and the SSRRS are loxP sites. [00193] 103.
  • FLP yeast flippase
  • the PSRS comprises a tobacco etch virus (TEV) protease and the PSRS comprises the peptide EXXYXQ- (S/G) or ENLYFQ-(S/G/A/M/C/H), wherein X is any amino acid and the TEV protease cleaves between the Q and the S, G, A, M, C, or H residues;
  • b. comprises a tobacco vein mottling virus (TVMV) protease and the PSRS comprises the peptide ETVRFQ-(G/S), wherein the TVMV protease cleaves between the Q and S or G residues;
  • SMMV sunflower mild mosaic virus
  • PSRS comprises the peptide EEIHLQ-(S/G), wherein the SMMV protease cleaves between the Q and S or G residues
  • d. comprises a turnip mosaic virus (TrMV) protease and the PSRS comprises the peptide VXHQ or VRHQ-S, wherein X is any amino acid and the TrMV protease cleaves C-terminal to the Q residue
  • TrMV turnip mosaic virus
  • HCV hepatitis C virus
  • PSRS comprises the peptide (D/E)XXXXC(A/S), wherein X is any amino acid and the HCV protease cleaves between the C and the A or S residues
  • h. comprises an enterokinase and the PSRS comprises the peptide DDDDK, wherein the enterokinase cleaves C-terminal to the K residue
  • i. comprises a Factor Xa protease and the PSRS comprises the peptide I(D/E)GR, wherein the Factor Xa protease cleaves C-terminal to the R residue
  • j a hepatitis C virus
  • the plant growth regulator is a volatile organic compound and the proteins are Glyceraldehyde- 3-Phosphate Dehydrogenase (GAPDH) and 2,3-butanediol Dehydrogenase (BDH);
  • GPDH Glyceraldehyde- 3-Phosphate Dehydrogenase
  • BDH 2,3-butanediol Dehydrogenase
  • the agriculturally relevant compound is trehalose and the protein(s) of interest is OtsA, OtsB, and TreS or TreS
  • the carbon containing compound is calcium carbonate and the protein of interest is a beta- carbonic anhydrase or alpha-carbonic anhydrase.
  • the protein of interest comprises a site-specific recombinase (SSR) or integrase protein
  • a control element comprising a promoter and/or at least a segment of a 5’ UTR: (i) is flanked by site-specific recombinase recognition sites (SSRRS) in an inverted configuration; (ii) is operably linked to one or more first target gene(s) of the genetically engineered bacterium; and (iii) is operably linked to one or more second target gene(s) of the genetically engineered bacterium upon inversion by the SSR or integrase; and wherein: (a) the first target gene is a glnA gene encoding a wild-type glutamine synthetase (GS) or variant thereof with improved catalytic activity in comparison to wild-type GS and the second target gene is a glnA gene encoding a wild-type GS with reduced levels of expression in comparison to
  • SSR site-specific
  • 107 The method of embodiment 106, wherein the control element which is operably linked to said target genes comprises a constitutive promoter, an inducible promoter, or quorum sensing promoter.
  • 108 The method of embodiment 106 or 107, wherein the integrase is a serine integrase, optionally wherein the serine integrase is a phage PhiC31 serine integrase, IntS, IntM, IntG – ICEMcSym 1271, YdcL - ICEBs, or Int - ICE SXT/R39 integrase, and the SSRRS are attB and attP sites recognized respectively by the PhiC31, IntS, IntM, IntG, YdcL, or Sxt/R39 integrase.
  • P14610WO00 optionally a third control element comprising a promoter which is repressed by the second repressor protein and operably linked to a gene encoding a third repressor protein; (iii) optionally a fourth control element comprising a promoter which is repressed by the third repressor protein and operably linked to a gene encoding a fourth repressor protein; and (iv) a control element comprising a promoter which is repressed by the second, third, or fourth repressor protein and which is operably linked to a first target gene,optionally wherein the first, second, third, and/or fourth repressor protein(s) comprise(s) the lambda repressor (cI), the tet repressor (TetR), the lac repressor (LacI), a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-
  • the genetically engineered bacterium lacks (ii) and (iii), and the first target gene comprises: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and/or one or more fix cluster gene(s), and the agriculturally relevant compound is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphata
  • the genetically engineered bacterium further comprises (ii) and (iii), and the first target gene comprises: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and/or one or more fix cluster gene(s) and the agriculturally relevant compound is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatas
  • the genetically engineered bacterium comprises an even number of repressors and the first target gene comprises: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity, one or more nif cluster gene(s), and/or one or more fix cluster gene(s) and the agriculturally relevant compound is ammonia; or (b) a gene encoding a phytase enzyme, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase, a gene encoding an acid phosphatase enzyme, optionally wherein the acid phosphatase is encoded by an a
  • 116 The method of embodiment 110, wherein the genetically engineered bacterium comprises an even number of repressors and the first target gene encodes an RNA sequence or protein comprising: a. a GlnE protein lacking an adenylyl removing domain which exhibits unidirectional adenylyltransferase (uAT) activity and the fertilizer is ammonia; b.
  • the genetically engineered bacterium optionally comprises a loss-of- function mutation in any one or more of the amtB, draT, glnA, glnB, glnK, glnR, glnZ, or nifL genes, wherein the genetically engineered bacterium optionally comprises one or more heterologous genes from a wild-type or refactored nif or fix gene cluster and the fertilizer is ammonia; c. a GlnR protein and the fertilizer is ammonia; d. a glutaminase enzyme and the fertilizer is ammonia; e.
  • a protein product of a refactored nif or fix gene cluster and the fertilizer is ammonia; f. a phytase enzyme and the agriculturally relevant compound is phosphate, optionally wherein the phytase enzyme comprises a cysteine phytase, a histidine acid phytase, or a beta-propeller phytase; g. an acid phosphatase enzyme and the agriculturally relevant compound is phosphate, optionally wherein the acid phosphatase enzyme is encoded by an acpA, aphA, phoC, napA, napD, or napE gene or variant thereof; h.
  • a protein which stimulates organic acid release from the bacterium and the agriculturally relevant compound is phosphate, optionally wherein the protein which stimulates organic acid release comprises a gluconate dehydrogenase (GAD), a glucose dehydrogenase (GDH), or a pyrroloquinoline (PQQ) synthase; Atty. Dkt. No. P14610WO00 i.
  • GAD gluconate dehydrogenase
  • GDH glucose dehydrogenase
  • PQQ pyrroloquinoline
  • a repressor protein wherein the repressor protein binds a natural or synthetic DNA motif in the promoter of any one or more second target gene(s) of the genetically engineered bacterium and inhibits expression of any one or more of the protein product(s) of the second target gene(s) and/or wherein the repressor protein optionally comprises the lambda repressor (cI), the tet repressor (TetR), the lac repressor (LacI), a catalytically inactive RNA-guided DNA binding protein, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), or any variant thereof and/or optionally wherein the second target gene(s) is/are a glnA, amtB, glnB, glnK, glnZ, nifL, and/or draT gene and the fertilizer is ammonia; j.
  • the repressor protein optional
  • non-coding synthetic small RNA binds a natural or synthetic DNA and/or RNA motif in the promoter, 5’ UTR, and/or coding region of any one or more second target gene(s) of the genetically engineered bacterium
  • the non-coding synthetic small RNA comprises a guide RNA that additionally binds an RNA- guided DNA endonuclease, an RNA-guided RNA endonuclease, or variant thereof and/or optionally wherein the second target gene(s) is/are a glnA, amtB, glnB, glnK, glnZ, nifL, and/or draT gene and the fertilizer is ammonia; k.
  • SSR site-specific recombinase
  • integrase protein wherein any one or more second target gene(s) of the genetically engineered bacterium and/or a promoter operatively linked thereto are flanked by site-specific recombinase recognition sites (SSRRS) in a direct configuration, optionally wherein the SSRRS comprise attL and attR sites and the genetically engineered bacterium comprises a gene encoding a recombinase directionality factor (RDF) and/or optionally wherein the second target gene(s) is/are a glnA, amtB, glnB, segment of glnE encoding an adenylyl-removing domain of a glutamine synthetase adenylyltransferase, glnK, glnZ, nifL, and/or draT gene and the fertilizer is ammonia; l.
  • SSRRS site-specific
  • any one or more second target gene(s), the promoter(s) thereof, and/or the 5’ UTR(s) thereof of the genetically engineered bacterium comprise(s) one or more internal synthetic SSRRS and wherein the gene(s), promoter(s), the 5’ UTR(s), and/or a segment(s) thereof is/are excised or inactivated after a recombination event; optionally wherein the genetically engineered bacterium further comprises a plasmid comprising an SSRRS; optionally wherein the genetically engineered bacterium further comprises one or more genes encoding a recombinase directionality factor (RDF); and/or optionally wherein the second target gene(s) is/are a glnA, amtB, glnB, glnE, glnK, glnZ, nifL, and/or draT
  • any one or more second target gene(s), the promoter(s) thereof, and/or the 5’ UTR(s) thereof of the genetically engineered bacterium comprise(s) one or more internal integrative SSRRS and retain(s) activity of said second target gene(s), promoter(s), and/or 5’ UTR(s) and wherein the genetically engineered bacterium further comprises an integrative element comprising an SSRRS; optionally wherein the internal integrative SSRRS is/are an attB Atty. Dkt. No.
  • the integrative element comprises an SSRRS comprising an attP site; and/or optionally wherein the second target gene(s) is/are glnA, amtB, glnB, glnK, glnZ, nifL, and/or draT and the fertilizer is ammonia; n.
  • SSR site-specific recombinase
  • integrase protein wherein a control element comprising a promoter and/or at least a segment of a 5’ UTR: (i) is flanked by site-specific recombinase recognition sites (SSRRS) in an inverted configuration; and (ii) is operably linked to one or more second target gene(s) of the genetically engineered bacterium; optionally wherein the control element which is operably linked to said second target gene(s) comprises a constitutive promoter, an inducible promoter, or quorum sensing promoter; and/or optionally wherein the second target gene(s) is/are glnA, amtB, glnB, glnK, glnZ, nifL, and/or draT and the fertilizer is ammonia; o.
  • SSR site-specific recombinase
  • integrase protein wherein a control element comprising a promoter and at least a segment of a 5’ UTR: (i) is flanked by site-specific recombinase recognition sites (SSRRS) in an inverted configuration; and (ii) is operably linked to one or more second target gene(s) of the genetically engineered bacterium upon inversion by the SSR or integrase; optionally wherein the control element which is operably linked to said second target gene(s) upon inversion by the SSR or integrase comprises a constitutive promoter, an inducible promoter, or quorum sensing promoter; and/or optionally wherein the second target gene(s) is/are: (a) nifA, ntrC, glnR, a gene encoding a glutaminase enzyme, a gene encoding a GlnE protein lacking an adenylyl
  • any one or more second target gene(s) of the genetically engineered bacterium comprise(s) one or more specific DNA sequence(s) recognized by the site-specific DNA endonuclease, optionally wherein the site-specific DNA endonuclease comprises an RNA-guided DNA endonuclease, a protein comprising a DNA-binding zinc finger domain, a transcription activator-like effector (TALE), a meganuclease, a homing endonuclease, or a restriction endonuclease, and/or optionally wherein the second target gene(s) is/are glnA, amtB, glnB, glnK, glnZ, nifL, and/or draT and the fertilizer is ammonia; q.
  • TALE transcription activator-like effector
  • any one or more second target gene(s) of the genetically engineered bacterium comprise(s) an in-frame insertion(s) of DNA encoding the PSRS at the N-terminus of the protein coding region of the gene(s) followed by a -Leu, -Phe, -Trp, or -Tyr residue and wherein cleavage of PSRS from the N-terminus of the protein(s) encoded by the gene(s) by the protease results in a protein comprising an N-terminal -Leu, -Phe, -Trp, or -Tyr residue which is degraded by native ClpS and ClpAP, optionally wherein the second target gene(s) is/are glnA, amtB, glnB, glnK, glnZ, nifL, and/or draT and the fertilizer is am
  • PSRS protease specific recognition sequence
  • Cells say “Intermediate” if the fluorescent reporter (GFP) protein of interest was induced 2 to 3-fold upon addition of an AHL QSSM inducer in a certain engineered strain.
  • Cells say “On” if the fluorescent reporter (GFP) protein of interest was expressed constantly in the presence and absence of an AHL QSSM inducer in a certain engineered strain.
  • Cells say “Non-functional” if the fluorescent reporter (GFP) Atty. Dkt. No. P14610WO00 protein of interest was not expressed in the presence or absence of an AHL QSSM inducer in a certain engineered strain. “NT” indicates not tested.
  • the quorum sensing promoter PahlI (SEQ ID NO: 356), PcinI (SEQ ID NO: 359), PlasI (SEQ ID NO: 373), or PlasB (SEQ ID NO: 374) was operably linked to a gene encoding a Superfolder GFP (sfGFP) protein of interest on a broad host range reporter plasmid.
  • sfGFP Superfolder GFP
  • Rows are organized by different QS systems (QSSP, QSRP, QS promoter triads). Columns are organized by different agriculturally relevant bacterial strains. Cells express a bacterial population density range in cells/mL that represents the threshold population density for each QS system-strain combination. Cells say “On” if the fluorescent reporter (GFP) protein of interest was expressed Atty. Dkt. No. P14610WO00 independently of bacterial population density. Cells say “Non-functional” if the fluorescent reporter (GFP) protein of interest was not significantly expressed at a tested bacterial population density. QSSP + QSRP + QS Promoter A. brasilense K. radicincitans P.
  • RP rhizosphere and rhizoplane
  • Root fresh weight was recorded for normalization of bacterial population density.
  • 5 experimental replicates for each experimental condition without planted seeds were sampled by flushing sample bottles with 20 mL of 1% KCl and vortexing, denoted the “bulk soil” (BS) fraction.
  • BS bulk soil
  • RP and BS fractions were cleared of residual sand and other particles by centrifugation for 30 sec at 1000 x g, and 100 ⁇ L aliquots of the resulting supernatants were analyzed by flow cytometry.
  • the bacterial population density in sand where corn seeds were not planted was about 1000-fold lower than the bacterial population density observed on corn roots (measured as cells g -1 root) (FIGURE 7).
  • Ab and Kr demonstrated population density-dependent induction of the GFP protein of interest on plant roots Atty. Dkt. No. P14610WO00 with the AhlRI and CinRI QS systems respectively (FIGURE 8).
  • the threshold population density at which each QS system caused each bacterial strain to produce the reporter protein of interest is given in TABLE 10. [00242] Table 10. Threshold population density at which various agriculturally relevant bacteria comprising various quorum sensing systems begin expressing a protein of interest in a plant growth medium.
  • Rows are organized by different QS systems (QSSP, QSRP, QS promoter triads). Columns are organized by different agriculturally relevant bacterial strains. Cells express a bacterial population density range in cells/g root or cells/g soil that represents the threshold population density for each QS system-strain combination. Cells say “On” if the fluorescent reporter (GFP) protein of interest was expressed independently of bacterial population density. “NT” indicates not tested. QSSP + QSRP + QS Promoter A. brasilense K. radicincitans .
  • RNA sequence or protein of interest under control of a QS system first colonize and grow on crop roots before beginning energy-intensive production of agriculturally relevant compounds such as fixed nitrogen.
  • bacterial population density as a trigger for production of agriculturally relevant compounds facilitates sufficient bacterial growth because it requires a threshold population density of microbes to be reached before any product formation.
  • microbes are manufactured in a fermentor at a higher population density (e.g. greater than about 1 * 10 10 cells/mL) than the threshold population density at which energy-intensive product formation is initiated by the QS system.
  • QS systems are deactivated by quorum quenching enzymes (QEs) that degrade AHL quorum sensing signal molecules (QSSMs). Expression of said QEs reduces the intracellular concentration of QSSMs, the concentration of QSSM-bound QSRPs, and the abundance of activated QS promoters.
  • QEs quorum quenching enzymes
  • QSSMs AHL quorum sensing signal molecules
  • QEs span several classes of QSSM-degrading enzymes, including AHL acylase enzymes (SEQ ID NO: 320- 322, and 323), alpha-beta hydrolase fold lactonase proteins (SEQ ID NO: 324, 325, and 326), metallo- beta-lactamase-like lactonase proteins (SEQ ID NO: 327-330, and 331), and phosphotriesterase-like lactonase proteins (SEQ ID NO: 332-335, and 336).
  • AHL acylase enzymes SEQ ID NO: 320- 322, and 323
  • alpha-beta hydrolase fold lactonase proteins SEQ ID NO: 324, 325, and 326
  • metallo- beta-lactamase-like lactonase proteins SEQ ID NO: 327-330, and 331
  • phosphotriesterase-like lactonase proteins SEQ ID NO: 332-335, and 336
  • the first “QS circuit” gene expression cassette comprised control elements operably linked to a gene encoding the CinI QSSP (SEQ ID NO: 351) and a gene encoding the CinR QSRP (SEQ ID NO: 342) as well as the PcinI quorum sensing promoter (SEQ ID NO: 359) operably linked to a sequence encoding a GFP reporter protein of interest.
  • the second “QQ circuit” gene expression cassette comprised a gene encoding the QqlM QE (SEQ ID NO: 325) operably linked to the tetracycline-inducible Ptet promoter (SEQ ID NO: 32) as well as a constitutively expressed gene encoding TetR (SEQ ID NO: 27).
  • the CinI QSSP synthesizes an AHL QSSM, which binds the CinR QSRP, activating the PcinI QS promoter from which the GFP protein of interest is expressed in a population density-dependent manner.
  • aTc quorum quenching compound
  • the “QQ circuit” is activated.
  • aTc binds TetR, inducing de- repression of the QE deactivator, degradation of the AHL QSSM, and abrogation of population density- dependent expression of the protein of interest.
  • Variant “QQ circuit” gene expression cassettes comprising a weak BCD22 (SEQ ID NO: 307), medium BCD13 (SEQ ID NO: 298), or strong BCD2 (SEQ ID NO: 288) ribosome binding site operably linked to the gene encoding the QqlM QE were cloned into the reporter plasmid of EXAMPLE 1 and FIGURE 2 comprising PcinI.
  • the resulting plasmids were mobilized into a K. radicincitans strain comprising a chromosomally integrated cinRI “QS circuit” (SEQ ID NO: 318) encoding the CinR QSRP and the CinI QSSP.
  • radicincitans comprising QS and QQ circuits were inoculated into plant growth media, bacterial population density was quantified in bulk soil (BS) and on the rhizoplane (RP) of corn roots, and population density-dependent expression of a GFP reporter protein of interest was assessed by flow cytometry (FIGURE 10).
  • the QQ circuit was not active in the absence of the quorum quenching compound aTc, and the three engineered K. radicincitans strains demonstrated population density-dependent expression of the GFP reporter protein of interest with a threshold population density of about 1 * 10 8 cells per gram root on the rhizoplane. Atty. Dkt. No. P14610WO00 Example 4.
  • the control element operably linked to the QSRP-encoding gene was replaced with an inducible control element comprising a promoter that is inactive in common fermentation conditions but active in common field conditions.
  • an inducible control element comprising a promoter that is inactive in common fermentation conditions but active in common field conditions.
  • promoter is an inducible promoter that can be de-repressed or activated through addition of a chemical to fermentation media that is not present in agricultural fields.
  • promoters examples include the tetracycline-inducible Ptet promoter (SEQ ID NO: 32), the allolactose-inducible lac promoter (SEQ ID NO: 33), and the diacetylphoroglucinol-inducible PphlA promoter (SEQ ID NO: 34).
  • Another type of promoter that is selectively active in a fermenter but not in an agricultural field is a promoter with variable activity based on temperature. Bacterial fermentation is carried out at higher temperatures than the soil temperature in agricultural fields. As such, promoters that bind temperature- sensitive transcription factors permit selective expression of the QSRP at low field temperatures but not at higher fermenter temperatures.
  • cIts2 acts as a transcriptional repressor for the promoter P L (SEQ ID NO: 31) but a transcriptional activator for the promoter P RM (SEQ ID NO: 338).
  • P L the promoter of the promoter P L
  • P RM the transcriptional activator for the promoter P RM
  • cIts2 achieves de-repression of a target gene at higher fermentation temperatures.
  • P RM In combination with P RM , cIts2 achieves de-activation of a target gene at higher temperatures.
  • a third type of promoter that can be selectively active in a fermenter but not in an agricultural field is a phosphate-sensitive promoter (SEQ ID NO: 378-389). Bacteria are fermented with high-phosphate media to expedite growth. In such growth media, the phosphate-sensitive promoter is inactive.
  • phosphate is initially at high concentration due to the application of chemical fertilizer but decreases as crop plants utilize that fertilizer to grow.
  • the phosphate-sensitive promoter is activated, expressing an operably linked target gene.
  • the QS system Atty. Dkt. No. P14610WO00 is inactive in the fermenter (where there is a high concentration of phosphate) but active in the field once there is a low concentration of phosphate.
  • the expression level of the QSRP is tuned so that expression from the QS promoter is density-dependent upon QS-promoter induction.
  • AhlR QSRP SEQ ID NO: 339
  • a reporter construct was generated wherein (i) the chemically inducible Ptet promoter (SEQ ID NO: 32) was operably linked to the gene encoding AhlR in one gene expression cassette and (ii) the PahlI promoter (SEQ ID NO: 356) was operably linked to both the strong ribosome binding site BCD2 (SEQ ID NO: 288) and a green fluorescent protein (GFP) reporter gene in a separate gene expression cassette of the same construct.
  • GFP green fluorescent protein
  • FIGURE 11 A schematic of the two separate constructs for tuning density-dependent AhlR expression are shown as FIGURE 11.
  • the constructs of FIGURE 11 were synthesized and cloned into the mini-Tn7 delivery plasmid pUC18R6K-mini-Tn7T-Gm digested at the SacI restriction site.
  • FIGURE 11 containing BCD22 operably linked to the AhlR-encoding gene demonstrated a greater fold-change of reporter gene expression across a range of AHL concentrations than the construct with BCD17.
  • a greater fold-change of reporter expression across a range of AHL concentrations predicted a greater density-dependent response in a full QS system including a quorum sensing synthase protein (QSSP).
  • QSSP quorum sensing synthase protein
  • sets of DNA constructs analogous to those of FIGURE 11 were designed additionally comprising a third gene expression cassette, wherein the QS promoter PahlI (SEQ ID NO: 356) was operably linked to both a gene encoding the QSSP AhlI (SEQ ID NO: 348) and a ribosome binding site of differing strength in each set of constructs.
  • the first set of constructs comprised the strong ribosome binding site BCD1 (SEQ ID NO: 287).
  • the second set of constructs comprised the weak ribosome binding site BCD8 (SEQ ID NO: 293).
  • the resulting constructs included four permutations: (1) AhlI-encoding gene operably linked to BCD1 with AhlR-encoding gene operably linked to BCD 22; (2) AhlI-encoding gene operably linked to BCD1 with AhlR-encoding gene operably linked to BCD 17; (3) AhlI-encoding gene operably linked to BCD8 with AhlR-encoding gene operably linked to Atty. Dkt. No. P14610WO00 BCD 22; and (4) AhlI-encoding gene operably linked to BCD8 with AhlR-encoding gene operably linked to BCD 17.
  • FIGURE 13 The constructs of FIGURE 13 were synthesized and cloned into the mini-Tn7 delivery plasmid pUC18R6K-mini-Tn7T-Gm digested at the SacI restriction site. These were stably integrated into the chromosome of Kosakonia radicincitans DSM16656 (Kr) to generate four strains to test density-dependent expression of a GFP protein of interest with and without repression of the QSRP. The four strains of Kr were then transformed with a plasmid expressing the anhydrotetracycline (aTc) responsive transcription factor rTetR (SED ID NO: 28) under control of the Pomega2 promoter (SEQ ID NO: 377).
  • aTc anhydrotetracycline
  • the transformed strains were grown in liquid culture under QSRP-repressing conditions where an aTc quorum quenching compound was present (QQ+) and quorum sensing conditions where aTc was absent (QQ-). GFP fluorescence and OD600 were measured using spectroscopy to assess density-dependent induction.
  • the four Kr strains comprising the DNA constructs of FIGURE 13 and the rTetR-expressing plasmid demonstrated density-dependent expression of the GFP protein of interest in the absence of the aTc quorum quenching compound with silencing of GFP expression in the presence of aTc at all tested cell densities (FIGURES 14A, 14B, 14C, 14D).
  • the engineered Kr strains with transcription-mediated quorum sensing deactivation tested in liquid culture were subsequently tested for suppression of density-dependent expression of a GFP protein of interest during fermentation followed by activation of density-dependent expression of GFP by the same bacteria after addition to a plant growth medium containing a plant.
  • the four Kr strains were cultured in the presence of the quorum quenching aTc compound, then inoculated onto non-germinated corn seeds, and isolated for quantification of the GFP protein of interest using flow cytometry according to the method of EXAMPLE 2.
  • the bacterial population density in sand where corn seeds were not planted was about 1000-fold lower than the bacterial population density observed on corn roots (measured as cells g -1 root) (FIGURE 15).
  • the threshold population density at which each QS system caused each bacterial strain to produce the reporter protein of interest is about 1 * 10 5 cells per gram root/soil to about 5 * 10 8 cells per gram root/soil.
  • Intracellular glutamine concentration is the dominant indicator of nitrogen status in many nitrogen- fixing bacteria. Decreasing intracellular glutamine concentration prevents cells from sensing high ammonia levels in the environment. Intracellular glutamine concentration can be decreased by increasing expression or activity of glutaminase, an enzyme that converts glutamine into glutamate. Separately, intracellular Atty. Dkt. No. P14610WO00 glutamine concentration can be lowered by decreasing expression or activity of glutamine synthase (GS), an enzyme encoded by the gene glnA that converts ammonia into glutamine.
  • GS glutamine synthase
  • GS is regulated post-translationally by GS adenylyltransferase (GlnE), a bidirectional enzyme encoded by the glnE gene that catalyzes both the adenylylation and de-adenylylation of GS through activity of its adenylyltransferase (AT) and adenylyl- removing (AR) domains, respectively.
  • GlnE GS adenylyltransferase
  • AT adenylyltransferase
  • AR adenylyl- removing
  • uATs Unidirectional ATases
  • the glnE gene encoding glutamine synthetase adenylyltransferase is identified in soil bacteria such as those of the genera Acetobacter, Acidothermus, Acinetobacter, Agrobacterium, Aromatoleum, Arthrobacter, Azoarcus, Azorhizobium, Azospirillum, Azotobacter, Bacillus, Bifidobacterium, Bradyrhizobium, Burkholderia, Conexibacter, Curtobacterium, Ensifer, Enterobacter, Erwinia, Escherichia, Flavobacterium, Frankia, Gaiella, Gluconacetobacter, Gluconobacter, Herbaspirillum, Klebsiella, Kosakonia, Lactobacillus, Lactococcus, Lysinibacillus, Maritimibacter, Methylobacterium, Nitrosocosmicus, Nitrososphaera, Paenarthrobacter, Paenibac
  • the locations of the adenylyl-transferring (AT) and adenylyl-removing (AR) domains within each ATase are predicted.
  • AT adenylyl-transferring
  • AR adenylyl-removing domains within ATase
  • their locations are predicted through alignment of the N- and C- terminal halves of ATase to each other before Atty. Dkt. No. P14610WO00 designing uAT variants based on this intraprotein homology.
  • the ATase amino acid sequence in one organism is aligned to that of another to identify the AT and AR domains.
  • Ammonia release is measured by growing strains in nitrogen-free liquid minimal media with an atmosphere of less than 3% oxygen, or by growing them in nitrogen-free semi-solid agar where bacteria form a pellicle at an oxygen concentration that permits nitrogenase activity. After incubation under these conditions, ammonia is then quantified in samples of cleared supernatant using an ammonium probe or a colorimetric assay such as the indophenol assay described by Schnabel and Sattely (doi: 10.1128/AEM.00582-21). Example 6.
  • N 2 -fixing catalyst nitrogenase is expressed only under conditions of nitrogen starvation, and the same conditions stimulate upregulation of high-affinity ammonia assimilation by the enzyme glutamine synthetase (GlnA, GS), preventing release of excess ammonia for plants (FIGURE 17).
  • Diazotrophs can be engineered to produce and release ammonia by decoupling their ability to repress nitrogenase under nitrogen replete conditions. De-repression of nitrogenase can be achieved by a number of genetic strategies, and in many bacteria, results in more ammonia produced than can be assimilated, causing diffusion of ammonia from the cell. [00263]
  • a distinct method to stimulate ammonia production and release is to prevent bacteria from assimilating fixed nitrogen derived from N2 into glutamine, the intracellular signal for nitrogen status. This can be accomplished by chemically or genetically inactivating glutamine synthetase by a number of strategies.
  • nifA In Azotobacter vinelandii, overexpression of nifA from a heterologous promoter drives constitutive nitrogenase activity and stimulates ammonia release.
  • intracellular levels of active NifA protein are controlled by two key factors: transcription of the nifLA operon and inhibition of NifA protein activity by protein-protein interaction with the NifL protein. Increasing the transcription level of the nifLA operon leads to a higher intracellular concentration of NifA proteins, which increases expression of nitrogenase, the rate of ammonia production, and resulting ammonia release.
  • PII proteins are global nitrogen response regulators, acting on a suite of nitrogen metabolism proteins.
  • ORS 571 In Rhodobacter capsulatus and Azorhizobium caulinodans ORS 571, deletion of both PII proteins forces the adenylyl transferase (AT) to adenylate the GS protein, leading to de-repressed nitrogenase activity in the presence of ammonia. When both PII genes are deleted, ORS 571 releases ammonia into the growth media when grown under nitrogen fixing conditions. Atty. Dkt. No. P14610WO00 [00271] Separately, nitrogenase is regulated by feedback repression at the transcriptional and post translational level by ADP-ribosylation via the DraT-DraG system.
  • DraT catalyzes the ADP-ribosylation of the nitrogenase Fe protein and shuts off of nitrogenase under nitrogen excessive conditions
  • DraG catalyzes the removal of ADP ribose and reactivation of nitrogenase under nitrogen starvation. Deletion of DraT in a cell where nitrogenase is not regulated by feedback repression at the transcriptional level results in nitrogenase activity that escapes feedback repression.
  • Example 8
  • GS glutamine synthetase activity blocks bacteria from assimilating fixed nitrogen and results in ammonia release into the environment. Transcriptionally repressing glnA in response to increases in bacterial population densities above threshold densities can down-regulate GS and lead to conditional ammonia release at those bacterial population densities.
  • the native glnA in soil bacteria such as those of the genera Acetobacter, Acidothermus, Acinetobacter, Agrobacterium, Aromatoleum, Arthrobacter, Azoarcus, Azorhizobium, Azospirillum, Azotobacter, Bacillus, Bifidobacterium, Bradyrhizobium, Burkholderia, Conexibacter, Curtobacterium, Ensifer, Enterobacter, Erwinia, Escherichia, Flavobacterium, Frankia, Gaiella, Gluconacetobacter, Gluconobacter, Herbaspirillum, Klebsiella, Kosakonia, Lactobacillus, Lactococcus, Lysinibacillus, Maritimibacter, Methylobacterium, Nitrosocosmicus, Nitrososphaera, Paenarthrobacter, Paenibacillus, Panotea, Pedi
  • a protein-coding sequence encoding a repressor protein paired with the repressible promoter, TetR (SEQ ID NO: 27), LacI (SEQ ID NO: 29), or PhlF (SEQ ID NO: 30) respectively, is operably linked to a quorum sensing promoter (SEQ ID NO: e.g., SEQ ID NO: 356-364, 373, and 374) in a heterologous gene expression cassette, which is introduced into a soil bacterium along with compatible QSSP and QSRP (e.g., an AhlI protein, a TetR promoter operably linked to an AhlR protein, with an ahlI promoter operably linked to a PhlF encoding gene) to create a genetically engineered bacterium.
  • QSSP and QSRP e.g., an AhlI protein, a TetR promoter operably linked to an AhlR protein, with an ahlI promoter operably linked to a PhlF en
  • the genetically engineered bacterium is fermented in bacterial growth media such that the bacteria grow to high cell density (e.g., in the presence of doxycycline, tetracycline, and/or anhydrotetracycline to prevent PhlF mediated repression of glnA at high cell densities).
  • the GEB are then introduced into a plant growth medium in an agricultural context. Once the GEB population densities exceed a threshold density in the plant growth medium (e.g., see representative threshold density ranges in Tables 9 and 10 for certain quorum sensing systems in representative bacterial Atty. Dkt. No.
  • Glutamine synthetase is catalytically inactivated by disrupting the structure of its active site through cleavage of the GS peptide backbone.
  • Hydrolysis of the GS peptide backbone can be catalyzed by proteases, a class of enzymes that recognize specific short amino acid sequences and cleave a protein’s peptide backbone a consistent distance from said amino acid recognition sequences, herein referred to as protease-specific recognition sequences (PSRS).
  • proteases a class of enzymes that recognize specific short amino acid sequences and cleave a protein’s peptide backbone a consistent distance from said amino acid recognition sequences, herein referred to as protease-specific recognition sequences (PSRS).
  • PSRS protease-specific recognition sequences
  • a GS protein variant that is controllably cleaved by a protease is engineered by encoding one or more PSRS within the coding sequence of the glnA gene.
  • GS is catalytically inactivated in response to environmental changes.
  • introduction of an environmental stimulus induces expression of a protease.
  • the protease then binds the one or more PSRS in the engineered GS and cleaves the GS backbone, causing a structural change in GS that inactivates it catalytically and causes ammonia accumulation due to GS down- regulation.
  • the GS protein is engineered to comprise one or more PSRS.
  • Multiple methods are used to identify the solvent-facing surface of the GS protein, including visualization of external amino acid residues using crystal structure data, alignment of the amino acid sequence of GS variants without a crystal structure to those with one in order to identify homologous regions of the protein, in silico structure prediction for GS variants without a crystal structure and identification of solvent-facing regions of the protein, and mass- spectrometry based protein labeling methods that identify solvent-facing regions of a protein. Additionally, catalytic deactivation of GS is more complete if protease cleavage compromises the structure of the GS enzyme active site.
  • GS In the Pseudomonas stutzeri GS polypeptide (SEQ ID NO: 230), locations to incorporate one or more PSRS that meet the above criteria include between amino acid residues 98 and 99, 119 and 120, 283 and 284, and/or 298 and 299. These insertion sites are depicted in Table 6. [00278] To achieve inducible deactivation of GS, GS must function normally until an environmental stimulus triggers protease expression, at which point enzymatic activity is minimized.
  • the native glnA gene in a bacterial strain is replaced with an engineered copy of glnA encoding the one or more PSRS so that the engineered GS is expressed by way of the native glnA genetic context of the host strain.
  • the engineered copy of glnA encoding the one or more PSRS is the only glnA allele in the host strain.
  • the gene encoding GS, glnA is an essential gene for many bacterial strains.
  • replacing a single native allele with a single engineered allele is accomplished by inserting certain site-specific recombinase recognition sites (SSRRS) flanking a bacterium’s native glnA allele in the bacterium’s chromosome (including FRT sites and/or loxP sites), encoding the corresponding site-specific recombinase (a yeast flippase (FLP) recombinase if FRT SSRRS or a Cre-recombinase if loxP SSRRS) under inducible control on a plasmid with the glnA allele encoding a GS variant comprising the one or more PSRS, and inducing recombination to swap the engineered glnA allele into the chromosome while excising the native allele.
  • SSRRS site-specific recombinase recognition sites
  • Such a process achieves exchange of the wild-type glnA allele for the engineered glnA allele encoding the GS that comprises the one or more PSRS without ever requiring deletion of the essential glnA gene.
  • a heterologous gene expression cassette comprising a protease-encoding gene (e.g. SEQ ID NO: 54, 55, or 56) operably linked to a quorum sensing Atty. Dkt. No.
  • the genetically engineered bacterium (GEB) is fermented in in contact with the quorum quenching compound QQ and/or in exposure to a temperature above the threshold temperature such that the bacteria grow to high cell density.
  • the GEB are then introduced into a plant growth medium in an agricultural context.

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Abstract

L'invention divulgue des bactéries génétiquement modifiées qui expriment des ARN ou des protéines qui produisent ou représentent des composés utiles en agriculture y compris des engrais et des nutriments végétaux lors de l'augmentation de la densité cellulaire lorsqu'ils sont cultivés dans des milieux de croissance végétale tels que le sol. L'invention divulgue également des bactéries génétiquement modifiées dans lesquelles l'expression d'ARN ou de protéines qui produisent ou représentent des composés utiles en agriculture y compris des engrais et des nutriments végétaux peut être inhibée lorsque des densités cellulaires accrues sont atteintes quand elles sont cultivées dans un fermenteur ou un bioréacteur.
PCT/US2025/015489 2024-02-12 2025-02-12 Commutateur génétique microbien dépendant de la densité Pending WO2025174810A2 (fr)

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Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7098014B2 (en) 2002-01-23 2006-08-29 Lian Hui Zhang Ralstonia ahl-acylase gene
US20110124522A1 (en) 2006-10-27 2011-05-26 Athena Biotechnologies, Inc. Methods of Disrupting Quorum Sensing to Affect Microbial Population Cell Density
US20150133339A1 (en) 2011-12-16 2015-05-14 The Regents Of The University Of California Multiscale platform for coordinating cellular activity using synthetic biology
WO2019032926A1 (fr) 2017-08-09 2019-02-14 Pivot Bio, Inc. Procédés et compositions d'amélioration de microbes modifiés
US20200331820A1 (en) 2017-10-25 2020-10-22 Pivot Bio, Inc. Gene targets for nitrogen fixation targeting for improving plant traits
US20210315212A1 (en) 2018-11-01 2021-10-14 Pivot Bio, Inc. Biofilm compositions with improved stability for nitrogen fixing microbial products
US20210345618A1 (en) 2018-09-21 2021-11-11 Pivot Bio, Inc. Methods and compositions for improving phosphate solubilization
US11479516B2 (en) 2015-10-05 2022-10-25 Massachusetts Institute Of Technology Nitrogen fixation using refactored NIF clusters
US20220411769A1 (en) 2021-06-18 2022-12-29 Sabrina Robin Mueller-Spitz Methods of using bacterial quorum quenching enzymes
US20230034438A1 (en) 2019-12-03 2023-02-02 Crobio Ltd Genetically engineered microorganisms
US20230148607A1 (en) 2020-05-01 2023-05-18 Pivot Bio, Inc. Stable liquid formulations for nitrogen-fixing microorganisms
US20240010576A1 (en) 2015-07-13 2024-01-11 Pivot Bio, Inc. Methods and compositions for improving plant traits

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021221690A1 (fr) * 2020-05-01 2021-11-04 Pivot Bio, Inc. Souches bactériennes modifiées pour une fixation améliorée d'azote

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7098014B2 (en) 2002-01-23 2006-08-29 Lian Hui Zhang Ralstonia ahl-acylase gene
US20110124522A1 (en) 2006-10-27 2011-05-26 Athena Biotechnologies, Inc. Methods of Disrupting Quorum Sensing to Affect Microbial Population Cell Density
US20150133339A1 (en) 2011-12-16 2015-05-14 The Regents Of The University Of California Multiscale platform for coordinating cellular activity using synthetic biology
US20240010576A1 (en) 2015-07-13 2024-01-11 Pivot Bio, Inc. Methods and compositions for improving plant traits
US11479516B2 (en) 2015-10-05 2022-10-25 Massachusetts Institute Of Technology Nitrogen fixation using refactored NIF clusters
WO2019032926A1 (fr) 2017-08-09 2019-02-14 Pivot Bio, Inc. Procédés et compositions d'amélioration de microbes modifiés
US20200331820A1 (en) 2017-10-25 2020-10-22 Pivot Bio, Inc. Gene targets for nitrogen fixation targeting for improving plant traits
US20210345618A1 (en) 2018-09-21 2021-11-11 Pivot Bio, Inc. Methods and compositions for improving phosphate solubilization
US20210315212A1 (en) 2018-11-01 2021-10-14 Pivot Bio, Inc. Biofilm compositions with improved stability for nitrogen fixing microbial products
US20230034438A1 (en) 2019-12-03 2023-02-02 Crobio Ltd Genetically engineered microorganisms
US20230148607A1 (en) 2020-05-01 2023-05-18 Pivot Bio, Inc. Stable liquid formulations for nitrogen-fixing microorganisms
US20220411769A1 (en) 2021-06-18 2022-12-29 Sabrina Robin Mueller-Spitz Methods of using bacterial quorum quenching enzymes

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