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WO2025227114A1 - Procédés et compositions utiles pour modifier génétiquement des cellules végétales - Google Patents

Procédés et compositions utiles pour modifier génétiquement des cellules végétales

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Publication number
WO2025227114A1
WO2025227114A1 PCT/US2025/026509 US2025026509W WO2025227114A1 WO 2025227114 A1 WO2025227114 A1 WO 2025227114A1 US 2025026509 W US2025026509 W US 2025026509W WO 2025227114 A1 WO2025227114 A1 WO 2025227114A1
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Prior art keywords
agrobacterium
library
strains
cells
strain
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English (en)
Inventor
Patrick M. Shih
Simon ALAMOS
Matthew SZARZANOWICZ
Mitchell G. Thompson
Liam KIRKPATRICK
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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Publication of WO2025227114A1 publication Critical patent/WO2025227114A1/fr
Pending legal-status Critical Current
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention is in the field of genetic transformation of eukaryotic cells.
  • BACKGROUND OF THE INVENTION Bacteria in the group Agrobacterium are plant pathogens that can transfer a piece of DNA known as the transfer-DNA (T-DNA) into the plant cell nucleus.
  • T-DNA is part of a tumor-inducing plasmid (pTi) which carries virulence associated vir genes necessary to sense, respond to and infect plant cells.
  • pTi tumor-inducing plasmid
  • the genes encoded within the T-DNA are necessary for tumor formation they are otherwise dispensable for all other steps of pathogenesis, up to and including DNA transfer.
  • a fundamental aspect of any engineering discipline is having a predictive and quantitative model based on first principles that describes the behavior of the system in question, yet, despite recent progress, we still lack a predictive theoretical framework to capture agrobacterium T-DNA transfer in single plant cells.
  • the present invention provides for a composition comprising a first binary vector and second binary vector.
  • Each binary vector has a different origin of replication in Agrobacterium.
  • Suitable origins of replication in Agrobacterium include, but are not limited to, pVS1, BBR1, oriV, repA, OriA, and StaA origins of replication.
  • the first binary vector and the second binary vector have different origin of replication.
  • the first binary vector has a pVS1 origin of replication
  • the second binary vector has a BBR1 origin of replication
  • the composition further comprises a vir helper plasmid.
  • Each binary vector comprises one or more of the following: (1) An origin of replication or ORI for E. coli or OriE, or for another bacterium: a particular element on the plasmid for starting its replication in E. coli, or in another bacterium. This component is useful for allowing maintenance of the vector in E. coli, or in another bacterium. (2) An origin of replication or ORI for Agrobacterium or OriA: a particular site on the plasmid for starting its replication in Agrobacterium.
  • the first binary vector comprises a first gene of interest
  • the second binary vector comprises a second gene of interest.
  • Poly(A) signals an element containing poly-A, important to produce a protein.
  • Reporter a sequence encoding a particular protein with a specific function for monitoring the recombinant protein, such as ⁇ -glucuronidase or GUS, luciferase or LUC, or Green Fluorescent Protein or GFP), or any other fluorescent protein described herein.
  • the first binary vector and the second binary vector comprise a fluorescent protein having a different color from each other.
  • each first binary vector and/or each second binary vector comprises: (1) an origin of replication or ORI for another bacterium, (2) an origin of replication or ORI for Agrobacterium, (3) a multiple cloning sites (MCS), (4) a plant selectable marker, (5) a bacterial selectable marker, (6) a promoter capable of expressing a gene of interest, and (7) a poly(A) signal.
  • each first binary vector comprises a first fluorescent protein which fluorescences with a first color
  • each second binary vector comprises a second fluorescent protein which fluorescences with a second color, wherein the first color and the second color are different.
  • the present invention provides for a host cell comprising a first binary vector and a second binary vector of the present invention.
  • the host cell further comprises a vir helper plasmid.
  • the host cell is an Agrobacterium species.
  • the Agrobacterium species is Agrobacterium tumefaciens.
  • An Agrobacterium strain comprising the first binary vector and the second binary vector is also known as BiBi strain.
  • the host cell is a bacterium that is not A. tumefaciens/fabrum, for example, Escherichia coli or a Rhizobium cell, such as Rhizobium rhizogenes.
  • each vector of the binary vector pair has a different selectable marker, such as different antibiotic resistance markers or genes.
  • antibiotic resistance markers or genes include, but are not limited, by the following: kanamycin resistance, spectinomycin resistance, ampicillin resistance, chloramphenicol resistance, tetracycline resistance, rifampicin resistance, carbenicillin resistance, gentamicin resistance, hygromycin resistance, and the like.
  • each binary vector comprises a gene of interest. The present invention provides for a library of binary vectors.
  • the library of binary vectors comprises a first library of first binary vectors, and a second library of second binary Attorney Docket: 2024-046-02 Lawrence Berkeley National Laboratory vectors, wherein the first library of first binary vectors and the second library of second binary vectors in combination comprise genes of interests of enzymes (and optionally transporters) of a biosynthetic pathway or a catabolic pathway.
  • the first library of first binary vectors comprises about half of the genes of the biosynthetic pathway or catabolic pathway; and the second library of second binary vectors comprise about the other half of the genes of the biosynthetic pathway or catabolic pathway.
  • the present invention provides for a library of host cells wherein each host cell comprises a first binary vector and a second binary vector from a library of binary vectors of the present invention, such that the library of host cells comprises all of the genes of interest encoding all of the enzymes (and optionally transporters) of a biosynthetic pathway or a catabolic pathway.
  • the library of host cells is a library of BiBi strains.
  • the library comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 binary vectors, wherein each binary vector comprises a gene of interest which encodes an enzyme or transporter, wherein the enzymes are of a biosynthetic pathway for producing a compound of interest, or catabolic pathway to breaking down a compound of interest.
  • the gene encodes a tranporter, such as a cell transmembrane tranporter or an organelle transporter, such as a plastid or mitochondrial transporter.
  • the transporter is for transporting a compound or molecule through a membrane, such as importing a compound or molecule into a cell or organelle, or exporting a compound or molecule out of a cell or organelle.
  • the transporter is for importing a compound or molecule into a cell or organelle, wherein the compound or molecule is a precursor for the biosynthesis of the compound of interest.
  • the transporter is for exporting a compound or molecule out of a cell or organelle, wherein the compound or molecule is the compound of interest.
  • the compound of interest is a compound or molecule that naturally synthesized or catabolized by the target host cell.
  • the precursor(s) for synthesizing the compound of interest are naturally synthesized by the target host cell, or the target host cell has been genetically modified to synthesize the precursor(s).
  • the compound of interest is a biofuel.
  • the vir helper plasmid comprises a nucleic acid encoding refactored minimized set of Agrobacterium virulence genes.
  • the Agrobacterium virulence genes are operatively linked to one or more promoters.
  • the vir helper plasmid comprises features or components described by U.S. Provisional Patent Application Ser.
  • the present invention provides for a method for introducing the genes of interest encoding for the enzymes of a biosynthetic pathway or a catabolic pathway into a target host cell comprising: (a) providing a library of host cells of the present invention, (b) contacting the library of host cells to a target host cell resulting in introducing the genes of interest into the target host cell, and (c) identifying one or more the target host cell that are capable of synthesizing or catabolizing the compound of interest.
  • the contacting step comprises a plurality of target host cells.
  • all of the target host cells are of the same species, or on the same plant, or on a plurality of plants of the same species.
  • the target host cell is a eukaryotic cell, such as a plant cell or fungal cell.
  • the genes of interest are stably integrated into the genome of the target host cell.
  • Agrobacterium is used to transfer transgenes encoded in plasmids called ‘binary vectors’ into plant cells. This makes it possible to use plants infiltrated with agrobacterium as a chassis for the in planta biosynthesis of molecules of biotechnological interest such as high- value secondary metabolites and protein complexes like antigens and antibodies. [0017] These molecules often require the expression of multiple (10 or more) transgenes, making it necessary to use multiple agrobacterium strains, each one responsible for delivering a different transgene. We discovered that different agrobacterium strains compete with each other in terms of transgene delivery, limiting the number of transgenes that can be expressed with this approach.
  • benthamiana line carrying a ubiquitously expressed nuclear-localized BFP transgene is used for agroinfiltration experiments.3 strains are mixed and infiltrated into the BFP-NLS line: two reporter strains carrying T-DNAs coding for a constitutively expressed nuclear-localized fluorescent proteins (GFP or RFP) and the Hygromycin resistance hpt gene in the same T-DNA and an empty vector strain whose T- DNA codes only for hpt.
  • RB T-DNA right border
  • LB left border.
  • C Schematic of the reporter strain titration experiments. Along the vertical axis, the infiltration OD of the reporter strains is kept constant while the total culture OD is increased using the empty vector strain.
  • FIG. 1 Cartoon of the metabolite competition model. Top: the reporter and the competitor strain start off in a nonpathogenic state and become capable of transformation upon consumption of a metabolite. Bottom: when the EV strain is added it deprives the reporter strain of the limiting metabolite.
  • B Cartoon of the spatial competition model. Top: each plant cell has a limited number of slots on its surface where bacteria can make contact. Bottom: Increasing the OD of the EV strain leaves fewer Attorney Docket: 2024-046-02 Lawrence Berkeley National Laboratory transformation slots available for the reporter strain.
  • C Fit of the models in (A) and (B) to the data in Figure 2, Panel A.
  • (C) Top: Cartoon showing an alternative cellular basis for the results in (A) where adding a second strain increases the fluorescence of those cells transformed by the reporter strain. Bottom: box plots showing the average GFP fluorescence of nuclei that were detected in the GFP channel in (B) (for (B) and (C) N 9 plants, one image per plant).
  • ⁇ VirE12 is a knockout for the VirE1 and VirE2 genes.
  • the ⁇ VirE12 complement strain carries a previously described complementation construct coding for the VirE1 and VirE2 genes in a Attorney Docket: 2024-046-02 Lawrence Berkeley National Laboratory plasmid. Box plots show the tissue-level RFP fluorescence.
  • Leaves were infiltrated with the GFP and RFP reporter strains mixed at an OD of 0.002 each, with (bottom) or without (top) the addition of the EV strain at an OD of 0.046.
  • F Mean ⁇ standard deviation of tissue-level fluorescence. Across all measurements, the OD of a GFP reporter strain was kept constant at 0.025 and the OD of a second coinfiltrated strain (EV or C58C1) was titrated to achieve varying total ODs, from 0.025 to 2.
  • reporter bacteria correspond to two strains, GFP-NLS in the pVS1 plasmid and RFP-NLS in the BBR1 plasmid, each at an OD of 0.001.
  • Bottom: a single BiBi reporter strain carrying two plasmids, GFP-NLS in pVS1 and RFP-NLS in BBR1. Blue arrowheads indicate example nuclei expressing both fluorescent proteins. White and black arrowheads indicate nuclei expressing only RFP or GFP, respectively. Scale bar 500 ⁇ m.
  • B Cartoon of the BiBi codelivery model. T-DNA expression is modeled as a two step process. First, the bacterium has to contact the plant cell, an event that follows Poisson-like statistics.
  • this bacterium can transfer the pVS1 plasmid with a probability p and the BBR1 plasmid with a probability r. Delivery of both plasmids is independent of the other, making the co-delivery probability p x r.
  • C Fraction of leaf epidermis cells expressing GFP or RFP as a function of reporter bacteria infiltration OD. The total OD was kept constant at 0.5 using EV. Solid lines show the best fit of the model in (B) which assumes that in all cases strains share the same values of ⁇ , p, and r.
  • the plasmids in (C) are used to create 4 different agrobacterium mixes.
  • the pVS1 mix consists of 14 strains carrying one pVS1 plasmid per strain.
  • the BBR1 mix is composed of 14 strains each of which carries one of the 14 transgenes in a BBR1 plasmid.
  • BiBi mixes consist of 7 strains carrying two plasmids per cell. In BiBi mix 1 the odd-numbered transgenes are launched from pVS1 and even ones from BBR1.
  • Figure 11 Related to Figure 3: Comparison of transformation rates by reporter strains using EV or C58C1 as a competitor strain.
  • the reporter strains were titrated in equal ratios using a third strain to keep the total OD constant, as described in Figure 1, Panel C.
  • the third competitor strain used was either EV or C58C1. Shown is the mean ⁇ SEM of the fraction of transformable cells expressing each reporter when EV (empty markers) or C58C1 (filled markers) was used. Dashed and solid lines correspond to Poisson fits to the data for EV and C58C1, respectively.
  • promoter refers to a polynucleotide sequence capable of driving transcription of a DNA sequence in a cell.
  • promoters used in the polynucleotide constructs of the invention include cis- and trans-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene.
  • a promoter can be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3' untranslated regions, or an intronic sequence, which are involved in transcriptional regulation.
  • These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) gene transcription.
  • Promoters are located 5' to the transcribed gene, and as used herein, include the sequence 5' from the translation start codon.
  • a "constitutive promoter” is one that is capable of initiating transcription in nearly all cell types, whereas a “cell type-specific promoter” initiates transcription only in one or a few particular cell types or groups of cells forming a tissue.
  • the promoter is secondary cell wall-specific and/or fiber cell-specific.
  • a "fiber cell-specific promoter” refers to a promoter that initiates substantially higher levels of transcription in fiber cells as Attorney Docket: 2024-046-02 Lawrence Berkeley National Laboratory compared to other non-fiber cells of the plant.
  • a "secondary cell wall-specific promoter” refers to a promoter that initiates substantially higher levels of transcription in cell types that have secondary cell walls, e.g., lignified tissues such as vessels and fibers, which may be found in wood and bark cells of a tree, as well as other parts of plants such as the leaf stalk.
  • a promoter is fiber cell-specific or secondary cell wall-specific if the transcription levels initiated by the promoter in fiber cells or secondary cell walls, respectively, are at least 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 500-fold, 000-fold higher or more as compared to the transcription levels initiated by the promoter in other tissues, resulting in the encoded protein substantially localized in plant cells that possess fiber cells or secondary cell wall, e.g., the stem of a plant.
  • Non- limiting examples of fiber cell and/or secondary cell wall specific promoters include the promoters directing expression of the genes IRX1, IRX3, IRX5, IRX7, IRX8, IRX9, IRX10, IRX14, NST1, NST2, NST3, MYB46, MYB58, MYB63, MYB83, MYB85, MYB103, PAL1, PAL2, C3H, CcOAMT, CCR1, F5H, LAC4, LAC17, CADc, and CADd.
  • a promoter is substantially identical to a promoter from the lignin biosynthesis pathway.
  • a promoter originated from one plant species may be used to direct gene expression in another plant species.
  • a polynucleotide or amino acid sequence is "heterologous" to an organism or a second polynucleotide or amino acid sequence if it originates from a foreign species, or, if from the same species, is modified from its original form.
  • a polynucleotide encoding a polypeptide sequence when said to be operably linked to a heterologous promoter, it means that the polynucleotide coding sequence encoding the polypeptide is derived from one species whereas the promoter sequence is derived from another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g., is a genetically engineered coding sequence, e.g., from a different gene in the same species, or an allele from a different ecotype or variety, or a gene that is not naturally expressed in the target tissue).
  • operably linked refers to a functional relationship between two or more Attorney Docket: 2024-046-02 Lawrence Berkeley National Laboratory polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence.
  • a promoter or enhancer sequence is operably linked to a DNA or RNA sequence if it stimulates or modulates the transcription of the DNA or RNA sequence in an appropriate host cell or other expression system.
  • promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting.
  • the terms “host cell” of “host organism” is used herein to refer to a living biological cell that can be transformed via insertion of an expression vector.
  • the terms "expression vector” or “vector” refer to a compound and/or composition that transduces, transforms, or infects a host cell, thereby causing the cell to express nucleic acids and/or proteins other than those native to the cell, or in a manner not native to the cell.
  • An "expression vector” contains a sequence of nucleic acids (ordinarily RNA or DNA) to be expressed by the host cell.
  • the expression vector also comprises materials to aid in achieving entry of the nucleic acid into the host cell, such as a virus, liposome, protein coating, or the like.
  • the expression vectors contemplated for use in the present invention include those into which a nucleic acid sequence can be inserted, along with any preferred or required operational elements. Further, the expression vector must be one that can be transferred into a host cell and replicated therein.
  • Particular expression vectors are plasmids, particularly those with restriction sites that have been well documented and that contain the operational elements preferred or required for transcription of the nucleic acid sequence. Such plasmids, as well as other expression vectors, are well known to those of ordinary skill in the art.
  • polynucleotide and “nucleic acid” are used interchangeably and refer to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
  • a nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, nucleic acid analogs may be used that may have alternate backbones, comprising, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press); positive backbones; non- Attorney Docket: 2024-046-02 Lawrence Berkeley National Laboratory ionic backbones, and non-ribose backbones.
  • nucleic acids or polynucleotides may also include modified nucleotides that permit correct read-through by a polymerase.
  • Polynucleotide sequence or “nucleic acid sequence” includes both the sense and antisense strands of a nucleic acid as either individual single strands or in a duplex. As will be appreciated by those in the art, the depiction of a single strand also defines the sequence of the complementary strand; thus the sequences described herein also provide the complement of the sequence. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
  • the nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc.
  • bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc.
  • the compound of interest is a compound naturally produced by the host cell. In some embodiments, the compound of interest is a compound not naturally produced by the host cell. In some embodiments, the compound of interest is a biofuel or bioproduct, or any other organic compound, and the corresponding biosynthetic enzyme(s) for producing the compound of interest thereof, are described and taught in U.S. Patent Nos.
  • PCT/US2014/48293 PCT/US2018/049609, PCT/US2017/036168, PCT/US2018/029668, PCT/US2008/068833, PCT/US2008/068756, PCT/US2008/068831, PCT/US2009/042132, PCT/US2010/033299, PCT/US2011/053787, PCT/US2011/058660, PCT/US2011/059784, PCT/US2011/061900, PCT/US2012/031025, Attorney Docket: 2024-046-02 Lawrence Berkeley National Laboratory and PCT/US2013/074214 (all of which are incorporated in their entireties by reference).
  • the compound of interest is a terpene, isoprenoid, carboxylic acid, lactone, trimethylpentanoic acid, 1-deoxyxylulose 5-phosphate, 1-deoxy-D-xylulose 5- phosphate (DXP), fatty acid, or derivatives thereof, alkyl lactone, lactam, isoprenyl alkanoate, 3- me t h y l - 2 - b u t e n - 1 - o l , 3 - me t h y l - 3 - b u te n - 1 - o l , an d 3 - me t h y l - b u t a n - 1 -o l , f a t t y ac i d e s t e r , a l p h a - o o
  • the compound of interest is any product or intermediate in the mevalonate (MVA) pathway, including any compound from acetyl-CoA to mevalonate.
  • the biosynthetic enzyme(s) are phosphomevalonate decarboxylase (PMD), phosphatase, AtoB, hydroxymethylglutaryl-CoA synthase (HMGS), hydroxymethylglutaryl-CoA reductase (HMGR), and/or mevalonate kinase (MK).
  • the biosynthetic enzyme(s) is a polyketide synthase.
  • the promoters are each independently constitutive or inducible.
  • the promoters are promoters described herein.
  • the refactored minimized set of Agrobacterium virulence genes at least excludes: (1) the virA and virG genes, as these genes are regulatory genes; (2) the virB1 gene (Berger et al., J. Bacteriol.176(12): 3646-3660, 1994); and, (3) the virE12 gene (which can be replaced with the Agrobacterium rhizogenes GALLS gene (Hodges et al., J. Bacteriol. 191(1): 355-364, 2009).
  • the target host cell is a eukaryotic cell, such as a plant or fungal cell.
  • the plant is a tobacco plant.
  • the fungal cell is a Rhodosporidium cell, such as Rhodosporidium toruloides.
  • the fungal cell is torulosis’s a yeast.
  • the yeast is Saccharomyces species, such as a Saccharomyces cerevisiae.
  • the promoter linked to the gene of interest is a constitutive promoter or a tissue-specific promoter.
  • tissue-specific promoters under developmental control include promoters that initiate transcription only (or primarily only) in certain tissues, such as vegetative tissues, cell walls, including e.g., roots or leaves.
  • tissue-specific promoters include promoters that initiate transcription only (or primarily only) in certain tissues, such as vegetative tissues, cell walls, including e.g., roots or leaves.
  • a variety of promoters specifically active in vegetative tissues, such as leaves, stems, roots and tubers are known.
  • promoters controlling patatin the major storage protein of the potato tuber
  • the ORF13 promoter from Agrobacterium rhizogenes that exhibits high activity in roots can also be used (Hansen, Mol. Gen.
  • Other useful vegetative tissue-specific promoters include: the tarn promoter of the gene encoding a globulin from a major taro (Colocasia esculenta L. Schott) corm protein family, tarin (Bezerra, Plant Mol. Biol.28:137-144, 1995); the curculin promoter active during taro corm development (de Castro, Plant Cell 4:1549-1559, 1992) and the promoter for the tobacco root-specific gene TobRB7, whose expression is localized to root meristem and immature central cylinder regions (Yamamoto, Plant Cell 3:371-382, 1991).
  • Leaf-specific promoters such as the ribulose biphosphate carboxylase (RBCS) promoters can be used.
  • RBCS ribulose biphosphate carboxylase
  • the tomato RBCS1, RBCS2 and RBCS3A genes are expressed in leaves and light-grown seedlings, only RBCS1 and RBCS2 are expressed in developing tomato fruits (Meier, FEBS Lett.415:91-95, 1997).
  • a ribulose bisphosphate carboxylase promoters expressed almost exclusively in mesophyll cells in leaf blades and leaf sheaths at high levels (e.g., Matsuoka, Plant J.6:311-319, 1994), can be used.
  • Another leaf- specific promoter is the light harvesting chlorophyll a/b binding protein gene promoter (see, e.g., Shiina, Plant Physiol.115:477-483, 1997; Casal, Plant Physiol.116:1533-1538, 1998).
  • the Arabidopsis thaliana myb-related gene promoter (Atmyb5) (Li, et al., FEBS Lett. 379:117-1211996), is leaf-specific.
  • the Atmyb5 promoter is expressed in developing leaf trichomes, stipules, and epidermal cells on the margins of young rosette and cauline leaves, and in immature seeds.
  • Atmyb5 mRNA appears between fertilization and the 16 cell stage of embryo development and persists beyond the heart stage.
  • a leaf promoter identified in maize e.g., Busk et al., Plant J.11:1285-1295, 1997) can also be used.
  • Another class of useful vegetative tissue-specific promoters are meristematic (root tip and shoot apex) promoters.
  • the "SHOOTMERISTEMLESS” and “SCARECROW” promoters which are active in the developing shoot or root apical meristems, (e.g., Di Laurenzio, et al., Cell 86:423-433, 1996; and, Long, et al., Nature 379:66-69, 1996); can be used.
  • Another useful promoter is that which controls the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase HMG2 gene, whose expression is restricted to meristematic and floral (secretory zone of the stigma, mature pollen grains, gynoecium vascular tissue, and fertilized ovules) tissues (see, e.g., Enjuto, Plant Cell.7:517- 527, 1995). Also useful are kn1-related genes from maize and other species which show Attorney Docket: 2024-046-02 Lawrence Berkeley National Laboratory meristem-specific expression, (see, e.g., Granger, Plant Mol.
  • the Arabidopsis thaliana KNAT1 promoter (see, e.g., Lincoln, Plant Cell 6:1859-1876, 1994) can be used.
  • the promoter is substantially identical to the native promoter of a promoter that drives expression of a gene involved in secondary wall deposition.
  • promoters examples include promoters from IRX1, IRX3, IRX5, IRX8, IRX9, IRX14, IRX7, IRX10, GAUT13, or GAUT14 genes.
  • Specific expression in fiber cells can be accomplished by using a promoter such as the NST1 promoter and specific expression in vessels can be accomplished by using a promoter such as VND6 or VND7. (See, e.g., PCT/US2012/023182 for illustrative promoter sequences).
  • the promoter is a secondary cell wall-specific promoter or a fiber cell-specific promoter.
  • the promoter is from a gene that is co-expressed in the lignin biosynthesis pathway (phenylpropanoid pathway).
  • the promoter is a C4H, C3H, HCT, CCR1, CAD4, CAD5, F5H, PAL1, PAL2, 4CL1, or CCoAMT promoter.
  • the tissue-specific secondary wall promoter is an IRXl, IRX3, IRX5, IRX8, IRX9, IRX14, IRX7, IRX10, GAUT13, GAUT14, or CESA4 promoter.
  • tissue-specific secondary wall promoters and other transcription factors, promoters, regulatory systems, and the like, suitable for this present invention are taught in U.S. Patent Application Pub. Nos.2014/0298539, 2015/0051376, and 2016/0017355.
  • a tissue-specific promoter may drive expression of operably linked sequences in tissues other than the target tissue.
  • a tissue-specific promoter is one that drives expression preferentially in the target tissue, but may also lead to some expression in other tissues as well.
  • each GOI is operatively linked to a promoter that is activated by the transcription activator.
  • each GOI is a biosynthetic gene that expresses an enzyme that catalyzes the biosynthesis of a compound of interest, or an intermediate thereof.
  • Agrobacterium pathogenesis involves the transfer of a DNA segment into host plant cells. This feature has made Agrobacterium the cornerstone of both basic and applied plant genetic engineering from its very inception. As the sophistication of the scientific problems that require Agrobacterium-mediated transformation increases, it becomes critical to achieve a quantitative and predictive understanding of DNA transfer at the level of single plant cells. Such a framework would make it possible to better design and interpret experiments and could help identify engineering bottlenecks.
  • Equation 0 describes the frequency of the number of k occurrences of a random, recurring event within an interval of, for example, space or time. It assumes that the probability of occurrence per interval is a constant, implying that events are completely independent of each other.
  • Equation 0 The only free parameter in Equation 0 is ⁇ , the average number of events per interval, that is, the total number of events divided by the total number of intervals.
  • the original application of the Poisson distribution to pathogenesis is often ascribed to Ellis and Delbruck who used it to model the distribution of the number of bacteriophages infecting single E. coli.
  • the Poisson ‘interval’ corresponds to a single bacterium cell and an ‘event’ is defined as the infection by a single infectious agent. This allows calculating lambda as Attorney Docket: 2024-046-02 Lawrence Berkeley National Laboratory Equation 1) [0066] Obtaining ⁇ in this way can prove to be technically challenging because it requires counting the number of infectious agents and host cells in an experiment.
  • ⁇ may also encompass other, more subtle aspects such as the fraction of the infiltrated OD corresponding to bacterial cells that are viable and/or potentially capable of transformation.
  • this model had not been explicitly tested in the context of agroinfiltration, although researchers did use it implicitly at an intuitive level. For example, it was assumed that by increasing the density of bacteria a larger fraction of leaf cells would get transformed and that more cells would get transformed by multiple bacteria. To test if the predicted probability distribution from Equation 0 conforms to experiments it would be necessary to experimentally obtain k, namely, the number of different bacteria that infect each plant cell. This is technically very challenging because it would require labeling tens of different T-DNAs to then determine which ones are present in each plant cell.
  • the BFP channel was used to identify leaf epidermis nuclei to then determine what fraction of these nuclei were infected by the reporter strains based on whether GFP and/or RFP expression could be detected (Fig.1, Panel C).
  • Fig.1, Panel C the reporter strains based on whether GFP and/or RFP expression could be detected.
  • nuclei For example, at a GFP reporter strain OD of 0.025, about 80% of nuclei express GFP when the total OD is 0.5 (i.e. EV strain added at an OD is 0.475) but this fraction decreases to ⁇ 40% at a total OD of 2 (where EV is added at an OD of 1.975).
  • This decrease in the transformation rate with increasing total OD is reflected in the fact that the estimated values of the probability constant ⁇ obtained from these fits differ across total ODs.
  • the fitted ⁇ value decreases from ⁇ 100 for a total OD of 0.05 to ⁇ 6 for a total OD of 3 (Fig.1, Panel D).
  • bacteria still can be described as transforming plant cells independently of each other. This result is inconsistent with there being subpopulations of plant cells that have a higher susceptibility to transformation. It is also inconsistent with infection by one bacterium affecting the transformation susceptibility of plant cells. [0076] Our findings so far may appear contradictory: on the one hand the probability of transformation of a given strain decreases by increasing the total bacterial OD. Yet, bacteria Attorney Docket: 2024-046-02 Lawrence Berkeley National Laboratory still transform host cells independently of one another at any OD.
  • This scaling parameter which can completely determine the transformation probability, is simply When all the reporter strain infiltration ODs are multiplied by this scaling parameter the curves of the fraction of transformed host cells collapse into a single curve. As expected, this master curve fits well to a Poisson distribution with an ⁇ value corresponding to the y-intercept of the fitted ⁇ 's as a function of the total OD, c ⁇ 100.
  • c One way to think of c is as the value of the transformation probability constant ⁇ in the absence of any competition, when the number of bacteria in the leaf is arbitrarily small. We next wished to test if this scaling factor is generalizable to new experimental settings different from the ones used to find this scaling property.
  • this limiting resource may correspond to a plant metabolite that bacteria must consume to become infectious.
  • the 'metabolite competition model' As an alternative mechanism we envisioned a 'spatial competition model' where the limiting host resource corresponds to the area on the surface of the plant cell that is accessible for bacteria to make contact and transfer the T-DNA. We note that these two models are not mutually exclusive. [0079] To explore if these mechanisms are consistent with our data, we first followed a mathematical modeling approach. To model the metabolite competition scenario, we assume that reporter and EV bacteria can exist in two states, infectious and noninfectious.
  • Infiltrated cells start in a noninfectious state but can become infectious inside the leaf upon consumption of a metabolite M. Those bacteria that can make the transition from noninfectious to infectious then go on to transform plant cells following a Poisson process with a single probability constant ⁇ .
  • This ⁇ should be a true constant, regardless of the data, OD of the competing EV strain and should also be equal to c, the rate in the absence of any competition shown in Figure 2.
  • ⁇ should be a true constant, regardless of the data, OD of the competing EV strain and should also be equal to c, the rate in the absence of any competition shown in Figure 2.
  • tissue-level results are quantitatively consistent with Attorney Docket: 2024-046-02 Lawrence Berkeley National Laboratory either of our resource competition models, we used each one of them to calculate the value of OD_sat that best fits the microscopy data.
  • tissue-level GFP fluorescence of a reporter strain can be increased by adding EV cells at a low OD (Fig.3, Panel F and Fig.4, Panel A). This was intriguing because all our data so far showed that the EV strain out-competes the reporter strains. To better understand the mechanistic basis of this phenomenon we asked whether cooperativity depends on the presence of the Ti plasmid. Adding C58C1 at any OD reduces the tissue-level GFP fluorescence driven by the reporter strain compared to a buffer only control, unlike EV which is able to increase it at low OD (Fig.3, Panel F and Fig.4, Panel A).
  • tissue-level leaf fluorescence represents an aggregate measurement of a number of distinct molecular processes such as the fraction of transformed host cells, the number of T-DNAs per host cell and the expression level of each of these T-DNAs.
  • C58C1 was able to compete but not cooperate.
  • competition by C58C1 is somewhat less effective than for the fraction of transformed host cells since at low OD of C58C1 the fluorescence of GFP nuclei is close to that of the buffer control (Fig.4, Panel C).
  • Fig.4, Panel C the buffer control
  • these single cell measurements confirm that cooperation requires pTi and show that it arises mainly from an increase in GFP output in cells that have already been transformed by the GFP reporter strain.
  • the GFP reporter strain whose activity is enhanced and the helper strain responsible for this enhancement are genetically identical, have the full set of effector vir genes and are thus fully capable of pathogenesis on their own.
  • a GV3101 strain lacking VirE1 and VirE2 could enhance the tissue-level expression of an RFP reporter strain compared to a buffer control.
  • Panel D the VirE12 mutant strain can still increase the fluorescence driven by the reporter RFP strain but to a lesser extent than GV3101. This decrease in cooperation can be partially restored by a VirE12 complementation transgene.
  • VirE1 and/or VirE2 both of which are secreted and act inside of the plant cell, partially contribute to cooperation.
  • Attorney Docket: 2024-046-02 Lawrence Berkeley National Laboratory [0087] These data are consistent with a scenario where transformation of a plant cell by a single agrobacterium can in some cases be limited by the vir gene dosage of this bacterium, perhaps due to non-genetic heterogeneity in vir gene expression. This implies that a single bacterium may also happen to have excess dosage of vir genes. If both kinds of bacteria contact the same plant cell, then the vir-poor bacterium can be aided by the vir-rich bacterium.
  • VirE12 and other genes encoded in pTi modify the extracellular environment at a larger scale and make the mesophyll in general more conducive to agrobacterium pathogenesis. If cooperation occurs at the level of single plant cells, we expect that the level of expression of GFP and RFP T-DNAs delivered by two different strains should correlate in individual nuclei. Further, this correlation should decrease when a third EV strain is added to the culture since vir-poor cells from the GFP and RFP strains can then take advantage of vir genes derived from the abundant EV strain. To test this prediction, we coinfiltrated the GFP and RFP reporter strains at a low OD of 0.002 with or without the EV strain at an OD of 0.046.
  • T-DNA delivery is a two-step process (Fig.5, Panel B).
  • the bacterium has to make contact with the plant cell, which based on our previous results, we assume to be Poisson-like (i.e., a random process whose probability is proportional to the OD of the strain).
  • this bacterium can deliver a T-DNA that gets expressed but this step is not always successful, as revealed by the fact that BiBi strains do not always result in coexpression of GFP and RFP in individual nucleus (Fig.5, Panel A).
  • T-DNA delivery is a probabilistic step with a probability of p for the pVS1 T-DNA and r for the BBR1 T-DNA.
  • T-DNA delivery is a probabilistic step with a probability of p for the pVS1 T-DNA and r for the BBR1 T-DNA.
  • different T-DNAs harbored in the same BiBi strain are delivered completely independently of each other. This is, the probability that a pVS1 T-DNA is delivered is the same in a regular one-plasmid strain and a BiBi strain and the same is true for a BBR1 T-DNA. This implies that the probability of coexpressing the pVS1 and BBR1 T-DNAs given contact is p x r (Fig.5, Panel B).
  • BiBi strains can be deployed to increase the coexpression of multiple transgenes in metabolic engineering setups where 10-20 strains are coinfiltrated. Testing the impact of competition on an engineered metabolic pathway. [0097] Having shown that the BiBi system can be used to bypass bacterial competition and increase the fraction of plant cells that express multiple plasmids, we sought to explore its implications for metabolic engineering. Recently, tobacco has emerged as the primary platform for the discovery and commercial synthesis of complex, high-value plant metabolites. These molecules tend to require multiple enzymatic steps for their biosynthesis, all of which presumably need to co-occur in the same cell for the complete pathway to be reconstituted.
  • Glucoraphanin is a glucosinolate found in cruciferous vegetables such as Broccoli that is thought to have a number of beneficial effects for human health including anti-cancer properties. Efficient glucoraphanin synthesis in tobacco requires the expression of 14 genes: 13 enzymes and one transporter (Fig.6, Panel B). Importantly, focusing on a complex molecule that has received considerable interest in plant metabolic engineering would also allow us to determine to what extent BiBi strains may improve existing bioengineering efforts.
  • BiBi mix 1 consists of 7 BiBi strains carrying all the odd-numbered genes in the glucoraphanin pathway in the pVS1 vector and the even-numbered ones in the BBR1 vector.
  • BiBi mix 2 carries odd-numbered genes in the pVS1 vector and even-numbered genes in BBR1 (Fig.6, Panel D).
  • Agrobacterium is often used in plant science to deliver reporter constructs, usually in combination with other strains such as upstream effectors. Our finding that the expression of a transgene delivered by a strain can be increased or decreased depending on the OD of a second strain means that interactions between strains need to be taken into account when designing and interpreting this kind of experiment. As a general rule, it is a good idea to keep the total OD of bacteria constant using an empty vector strain when necessary.
  • Plasmid DNA transfection into protoplasts is routinely used to quantify the level of expression of reporter constructs in transactivation assays.
  • Protoplasts are also being used to express massive gene libraries. Interpreting and optimizing these experiments may benefit from the kind of quantitative dissection we engaged in here, particularly if the goal is to extract single cell data.
  • metabolic engineering has almost exclusively relied on agroinfiltration rather than protoplast transfection. Perhaps the fact that protoplasts lack plasmodesmata connections makes it much harder to reconstitute long pathways via intercellular sharing of intermediates.
  • MATERIALS AND METHODS Plasmids and Agrobacterium strains. [00113] All plasmids used in this study can be accessed from the JBEI public repository.
  • the BFP-NLS plasmid was created in a previous study.
  • the binary vectors used for the GFP-NLS, RFP-NLS, and the EV strain were based on the pCambia1300 backbone.
  • the BBR1 plasmids were custom made based on the XX backbone. All plasmids were created using standard Gibson assembly or Golden Gate assembly protocols. All plasmids were transformed into the GV3101::pMP90 Agrobacterium strain via electroporation. A list of all the plasmids used in this study can be found in the JBEI registry. Plant growth conditions.
  • Nicotiana benthamiana (tobacco) plants were grown in an indoor growth room kept at 60% humidity and 25 oC temperature under a 16/8 light/dark daily cycle and 120 ⁇ mol/m 2 s of light intensity. Plants were grown in Sunshine Mix #4 soil (Sungro) Attorney Docket: 2024-046-02 Lawrence Berkeley National Laboratory supplemented with 499 Osmocote 14-14-14 fertilizer (ICL) at 5 mL/L and infiltrated with agrobacterium cultures 29 days after sowing. Agrobacterium growth conditions. [00115] Agrobacterium glycerol stocks were streaked on LB plates containing antibiotics. The day before infiltration, single colonies were grown overnight in liquid LB containing antibiotics, shaking at 30°C.
  • cultures were diluted 1:10 in LB with the same antibiotic concentrations and grown for a few more hours under the same conditions until reaching an OD600 of 0.5-1.0.
  • the antibiotic concentrations used for all strains except C58C1 and strains carrying BBR1 plasmids were: 50 ⁇ g/ml Rifampicin, 50 ⁇ g/ml Kanamycin, and 30 ⁇ g/ml Gentamycin.
  • Strains carrying BBR1 plasmids were grown using 50 ⁇ g/ml Rifampicin, 100 ⁇ g/ml Spectinomycin, and 30 ⁇ g/ml Gentamycin.
  • C58C1 was grown without antibiotics. Agroinfiltration.
  • Agrobacterium liquid cultures at an OD600 of 0.5-1.0 were spun down at 4K rcf for 10-20 minutes and then resuspended in a similar volume of infiltration buffer (10 mM MES pH5.6, 10 ⁇ mM MgCl 2 , 150 ⁇ M Acetosyringone). Cultures were incubated in infiltration buffer shaking for 1 hour at room temperature prior to the next OD 600 measurements. Next, a 1:5 dilution of each culture in infiltration buffer was prepared in 1 mL final volume and the OD 600 of this dilution was measured using a spectrophotometer. The final OD dilutions for infiltration were then prepared using infiltration buffer.
  • the filter sets used were: the DAPI filter for BFP (excitation 350 pm 50, emission 460 pm 50), the L5 filter for GFP (excitation 480 pm 40, emission 527 Attorney Docket: 2024-046-02 Lawrence Berkeley National Laboratory pm 30), and the TXR filter for RFP (excitation 560 pm 20, emission 630 pm38).
  • a 5x dry objective was used to acquire 2.641 mm 2 images of 2048 x 2048 pixels, resulting in a pixel size of 0.78 ⁇ m. In each sample, 5 z-sections were acquired every 20 ⁇ m.
  • the laser power was set to 17 % for all channels.
  • the camera exposure time was 800 ms. Imaging: Laser-scanning confocal microscopy.
  • All confocal images were acquired using a Zeiss LSM710 microscope. In each Z-slice, two sequential scans were used, one for BFP and RFP and another one for GFP. In the first scan, excitation wavelengths were 405 nm using the Diode and 568 nm using the InTune laser. The two emission windows in this first scan were 410-530 nm (for BFP) and 585-630 nm (for RFP). The second scan used the Argon 488 nm laser for excitation and an emission window of 494-581nm for GFP.
  • the frame size was 2048 x 2048 pixels with 1.5 Zoom using a 5x dry objective, resulting in a 1.133 mm 2 image with a pixel size of 0.55 ⁇ m.
  • Scans were performed bidirectionally at a speed set to 9, corresponding to a pixel dwell time of 0.39 ⁇ s and a scan time of 7.75 s/slice. Averaging during acquisition was done using 2 lines.
  • the laser intensities and gain settings were chosen to avoid detector saturation and were as follows. Laser power of 1.0 for 405 nm, 4.0 for 568 nm and 1.0 for 488 nm. Gain of 711 for BFP, 589 for RFP and 480 for GFP.
  • the pinhole was set to 1 AU in both scans.
  • guard cell nuclei the smallest nuclei in the epidermis

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Abstract

La présente invention concerne une bibliothèque de vecteurs binaires comprenant une première bibliothèque de premiers vecteurs binaires, et une seconde bibliothèque de seconds vecteurs binaires, qui, en combinaison, comprennent des gènes d'intérêt d'une voie de biosynthèse ou d'une voie catabolique. La présente invention concerne un procédé d'introduction des gènes d'intérêt codant les enzymes d'une voie de biosynthèse ou d'une voie catabolique dans une cellule hôte cible consistant à : (a) fournir une bibliothèque de cellules hôtes, (b) mettre en contact la bibliothèque de cellules hôtes avec une cellule hôte cible, ce qui conduit à l'introduction des gènes d'intérêt dans la cellule hôte cible, et (c) identifier au moins une cellule hôte cible permettant de synthétiser ou de cataboliser le composé d'intérêt.
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Citations (4)

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Publication number Priority date Publication date Assignee Title
US20030159184A1 (en) * 2000-02-08 2003-08-21 Fabijanski Steven F Novel methods and constructs for plant transformation
US20090265814A1 (en) * 2003-01-31 2009-10-22 Icon Genetics Ag Plant transformation with in vivo assembly of a sequence of interest
US20170191074A1 (en) * 2004-02-13 2017-07-06 Monsanto Technology Llc In vivo Assembly of Transcription Units
US20200255845A1 (en) * 2017-06-12 2020-08-13 Juan Antonio GARCIA ÁLVAREZ Binary vectors and uses of same

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030159184A1 (en) * 2000-02-08 2003-08-21 Fabijanski Steven F Novel methods and constructs for plant transformation
US20090265814A1 (en) * 2003-01-31 2009-10-22 Icon Genetics Ag Plant transformation with in vivo assembly of a sequence of interest
US20170191074A1 (en) * 2004-02-13 2017-07-06 Monsanto Technology Llc In vivo Assembly of Transcription Units
US20200255845A1 (en) * 2017-06-12 2020-08-13 Juan Antonio GARCIA ÁLVAREZ Binary vectors and uses of same

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Title
SZARZANOVICH ET AL.: "Binary vector copy number engineering improves Agrobacterium-mediated transformation", NATURE BIOTECHNOLOGY, vol. 1-15, 4 November 2024 (2024-11-04), XP038453383, DOI: 10.1038/s41587-024-02462-2 *

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