[go: up one dir, main page]

WO2025207168A2 - Systèmes et plates-formes comprenant un groupe de ribosomes modifiés - Google Patents

Systèmes et plates-formes comprenant un groupe de ribosomes modifiés

Info

Publication number
WO2025207168A2
WO2025207168A2 PCT/US2024/060598 US2024060598W WO2025207168A2 WO 2025207168 A2 WO2025207168 A2 WO 2025207168A2 US 2024060598 W US2024060598 W US 2024060598W WO 2025207168 A2 WO2025207168 A2 WO 2025207168A2
Authority
WO
WIPO (PCT)
Prior art keywords
rrna
operon
cell
engineered
ribosome
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/060598
Other languages
English (en)
Other versions
WO2025207168A3 (fr
Inventor
Camila KOFMAN
Jessica Alexandria WILLI
Micheal Christopher JEWETT
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Northwestern University
Original Assignee
Northwestern University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Northwestern University filed Critical Northwestern University
Publication of WO2025207168A2 publication Critical patent/WO2025207168A2/fr
Publication of WO2025207168A3 publication Critical patent/WO2025207168A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins

Definitions

  • a sequence listing (file name: 702581 02601 SL ST26, size: 181,870 KB; date generated: December 16, 2024) is hereby incorporated by reference in its entirety.
  • Ribosomes are macromolecular machines that play a central role in the synthesis of proteins by catalyzing peptide bond formation between amino acids in a sequence defined manner. They are composed of a small and large subunit (SSU and LSU) which contain both ribosomal RNA (rRNA) and ribosomal proteins (r-proteins). In E. coli, the 16S rRNA and 21 r-proteins make up the SSU, while the LSU is composed of the 23 S rRNA, 5S rRNA, and 33 r-proteins. Ribosomes have conventionally been thought of as uniform molecular assemblies even though most organisms carry multiple copies of unique rRNA-encoding operons (rm) in their genomes. In E. coli K-12 strain MG1655, for example, there are seven unique genomically encoded rRNA operons containing several polymorphisms and are named with letters A-E, G, and H in increasing distance from the origin of replication.
  • a system, kit, or platform for recombinant protein expression can include an engineered cell or cell lysate prepared from the engineered cell, in which the engineered cell includes: (a) an exogenous nuclei acid expression vector including at least one ribosome RNA (rRNA) gene from at least one E.
  • rRNA ribosome RNA
  • the rRNA genes include 5S, 16S, and/or 23 S rRNA.
  • the engineered E. coli cell, or a lysate prepared from the cell includes an engineered ribosome pool, the engineered ribosome pool including a plurality of ribosomes.
  • the rRNA of the engineered ribosome pool is homogenous among the plurality of ribosomes in the pool.
  • the engineered ribosome pool includes rRNA encoded by the same rRNA operon, i.e., E. coli rRNA operon A, B, C, D, E, G, or H.
  • the genes of the rRNA operon are present on one or more exogenous vectors in the cell.
  • the rRNA genes are encoded by a polynucleotide sequence selected from SEQ ID NO: 2-11, or a polynucleotide sequence that is at least 85 %, at least 90 %, at least 95 %, at least 98 %, or at least 99 % identical to the polynucleotide sequences of SEQ ID NOs: 2-11.
  • the cell includes a deletion of at least one genomic rRNA operon. In some embodiments, the cell is modified to delete all but one endogenous rRNA operon. In some embodiments, one or more rRNA genes from operon D and/or operon C have been removed. In some embodiments, there may be nucleic acid deletions in helices H91 and/or H92 of operon D and/or operon C. In some embodiments, one or more rRNA genes from operon H have been removed.
  • a cell-free lysate is provided.
  • a cell-free protein synthesis platform includes a cell-free lysate.
  • an engineered E. coli cell in which one or more rRNA genes from operon D, operon C, or operon H have been removed is provided.
  • operon D and/or operon C include amino acid deletions in helices H91 and/or H92.
  • one or more rRNA genes from operon H have been removed.
  • a method for expressing a protein of interest may include: (a) with a system including an engineered cell, transforming the cell with a nucleic acid construct encoding the protein of interest; or (b) with a system including a lysate of the engineered cell, contacting the lysate with a transcription template or a translation template.
  • the method of expressing a protein of interest may express a greater level of protein than a level of protein expressed in a non-engineered control cell, or a lysate from a non-engineered control cell.
  • the method of expressing a protein of interest further includes contacting the cell-free protein synthesis platform with a translation template.
  • An expression construct may include a promoter operably coupled to a polynucleotide encoding for an rRNA operon, in which the rRNA operon include: i) a polynucleotide sequence that is at least 85 %, at least 90 %, at least 95 %, at least 98 % or at least 99 % identical to one or more of SEQ ID NOs: 1-11; or ii) one or more rRNA genes from different operons, where the rRNA genes are selected from the polynucleotide sequences of SEQ ID NOs: 36-56 or polynucleotide sequences that are at least 85 %, at least 90 %, at least 95 %, at least 98 %, or at least 99 % identical to the polynucleotide sequences of SEQ ID NOs: 36-56.
  • the promoter has a polynucleotide sequence that is at least 85%, at least 90 %, at least 95 %, at least 98 % or at least 99 % identical to SEQ ID NO: 57 or SEQ ID NO: 58.
  • FIGS. 1A-1C Genomic rRNA operons produce ribosomes that vary in protein biosynthesis activity.
  • FIG. 1A rm operons in the genome have different architectures and sequences. Residues that differ from the reference operon B sequence are highlighted in black; white boxes indicate tRNA genes.
  • FIG. lB iSAT reaction setup allows for expression, assembly, and testing of individual rRNA sequences in a ribosome-free lysate.
  • FIGS. 2A-2D Expression and assembly of single-operon ribosomes in vivo shows advantages over native heterogeneous ribosome pool.
  • FIG. 2A Single-operon strain selection process. Operon of interest is transformed into SQ171fg cells to replace the original rRNA copy, which is maintained on a SacB-containing plasmid.
  • FIG. 2A Single-operon strain selection process. Operon of interest is transformed into SQ171fg cells to replace the original rRNA copy, which is maintained on a SacB-containing plasmid.
  • FIG. 2B Operons A, B, C, G and H were able to singularly
  • Oligonucleotides can be prepared by any suitable method, including direct chemical synthesis by a method such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90-99; the phosphodiester method of Brown et al., 1979, Meth. Enzymol. 68:109-151; the diethylphosphoramidite method of Beaucage et al., 1981, Tetrahedron Letters 22:1859-1862; and the solid support method of U.S. Pat. No. 4,458,066, each incorporated herein by reference.
  • a review of synthesis methods of conjugates of oligonucleotides and modified nucleotides is provided in Goodchild, 1990, Bioconjugate Chemistry 1(3): 165-187, incorporated herein by reference.
  • a primer is preferably a single-stranded DNA.
  • the appropriate length of a primer depends on the intended use of the primer but typically ranges from about 6 to about 225 nucleotides, including intermediate ranges, such as from 15 to 35 nucleotides, from 18 to 75 nucleotides and from 25 to 150 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.
  • a primer need not reflect the exact sequence of the template nucleic acid, but must be sufficiently complementary to hybridize with the template. The design of suitable primers for the amplification of a given target sequence is well known in the art and described in the literature cited herein.
  • a primer is “specific,” for a target sequence if, when used in an amplification reaction under sufficiently stringent conditions, the primer hybridizes primarily to the target nucleic acid.
  • a primer is specific for a target sequence if the primer-target duplex stability is greater than the stability of a duplex formed between the primer and any other sequence found in the sample.
  • salt conditions such as salt conditions as well as base composition of the primer and the location of the mismatches, will affect the specificity of the primer, and that routine experimental confirmation of the primer specificity will be needed in many cases.
  • Hybridization conditions can be chosen under which the primer can form stable duplexes only with a target sequence.
  • the use of target-specific primers under suitably stringent amplification conditions enables the selective amplification of those target sequences that contain the target primer binding sites.
  • a “polymerase” refers to an enzyme that catalyzes the polymerization of nucleotides.
  • DNA polymerase catalyzes the polymerization of deoxyribonucleotides.
  • Known DNA polymerases include, for example, Pyrococcus furiosus (Pfu) DNA polymerase, E. coli DNA polymerase I, T7 DNA polymerase and Thermus aquaticus (Taq) DNA polymerase, among others.
  • RNA polymerase catalyzes the polymerization of ribonucleotides.
  • the foregoing examples of DNA polymerases are also known as DNA-dependent DNA polymerases.
  • promoter refers to a cis-acting DNA sequence that directs RNA polymerase and other trans-acting transcription factors to initiate RNA transcription from the DNA template that includes the cis-acting DNA sequence.
  • amino acid residue includes but is not limited to amino acid residues contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (He or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Vai or V), tryptophan (Trp
  • nonstandard or unnatural amino acids include, but are not limited, to a p- acetyl-L-phenylalanine, a p-iodo-L-phenylalanine, an O-methyl-L-tyrosine, a p- propargyloxyphenylalanine, a p-propargyl-phenylalanine, an L-3-(2-naphthyl)alanine, a 3 -methylphenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAcpP-serine, an L- Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, a p- acyl-L-phenylalanine,
  • a polypeptide may comprise, but is not limited to, 100, 101, 102, 103, 104, 105, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1 100, about 1200, about 1300, about 1400, about 1500, about 1750, about 2000, about 2250, about 2500 or more amino acid residues.
  • a peptide or polypeptide as contemplated herein may be further modified to include nonamino acid moieties.
  • Modifications may include but are not limited to acylation (e.g., O-acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., the addition of an iso
  • glycation Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine).
  • polysialylation e.g., the addition of polysialic acid
  • glypiation e.g., glycosylphosphatidylinositol (GPI) anchor formation
  • hydroxylation e.g., hydroxylation
  • iodination e.g., of thyroid hormones
  • phosphorylation e.g., the addition of a phosphat
  • antibody or “antibody molecule” are used herein interchangeably and refer to immunoglobulin molecules or other molecules which comprise an antigen binding domain.
  • the term “antibody” or “antibody molecule” as used herein is thus intended to include whole antibodies (e.g., IgG, IgA, IgE, IgM, or IgD), monoclonal antibodies, chimeric antibodies, humanized antibodies, and antibody fragments, including single chain variable fragments (ScFv), single domain antibody, and antigen-binding fragments, genetically engineered antibodies, among others, as long as the characteristic properties (e.g., ability to bind RNA-DNA hyrbids) are retained.
  • whole antibodies e.g., IgG, IgA, IgE, IgM, or IgD
  • monoclonal antibodies e.g., chimeric antibodies, humanized antibodies, and antibody fragments, including single chain variable fragments (ScFv), single domain antibody, and antigen-binding fragments, genetically
  • one or more of the methods described herein are performed in a vessel, e g., a single, vessel.
  • a vessel e g., a single, vessel.
  • the term “vessel,” as used herein, refers to any container suitable for holding on or more of the reactants (e.g., for use in one or more transcription, translation, and/or glycosylation steps) described herein.
  • vessels include, but are not limited to, a microtitre plate, a test tube, a microfuge tube, a beaker, a flask, a multi-well plate, a cuvette, a flow system, a microfiber, a microscope slide and the like.
  • physiologically compatible (but not necessarily natural) ions and buffers are utilized for transcription, translation, and/or glycosylation, e g., potassium glutamate, ammonium chloride and the like.
  • Physiological cytoplasmic salt conditions are well-known to those of skill in the art.
  • the disclosed cell-free protein synthesis systems may utilize components that are crude and/or that are at least partially isolated and/or purified.
  • the term “crude” may mean components obtained by disrupting and lysing cells and, at best, minimally purifying the crude components from the disrupted and lysed cells, for example by centrifuging the disrupted and lysed cells and collecting the crude components from the supernatant and/or pellet after centrifugation.
  • isolated or purified refers to components that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated.
  • an engineered cell refers to a cell comprising one or more genetic modification, such as one or more genetically modified ribosomal RNA (rRNA) operons.
  • the genetic modification may include an engineered modification of the native nucleic acid of the cell (e.g., engineered mutation to a gene to alter the expression, function, or both of the encoded polypeptide or encoded nucleic acid), or the engineered addition of nucleic acids encoding native, mutant, or heterologous polypeptides (e.g., via one or more expression vectors).
  • the engineered cell can be an E. coli cell.
  • an rRNA operon is a segment of DNA that encodes at least ribosomal rRNA.
  • the rRNA operons encode the subunits (rRNA genes) 16S, 23 S, and 5S.
  • the phrase “a single operon” can refer to a native (e.g., wildtype) operon or a genetically modified operon, so long as the operon includes all three subunits.
  • a single operon may comprise a single nucleic acid which encodes all three subunits.
  • a single operon may comprise multiple nucleic acids which encode all three subunits collectively.
  • E. coli there are seven ribosomal rRNA operons, named with the letters A-E, G, and H; each operon includes a 16S, 23 S and a 5S subunit. In native genomic operons, the three subunits are interspersed with tRNA subunits.
  • Operon B is considered the consensus operon (e.g., contains no mutations or polymorphisms; mutations in polymorphisms in operons A, C-E, G, and H are defined relative to operon B). There are several polymorphisms in the operon sequences encoding rRNA subunits that affect the activity of the resulting ribosome.
  • a “native operon” as used herein comprises all three rRNA genes and comprises no mutations or modifications in the rRNA genes (subunit sequences).
  • a native operon includes all three rRNA subunit sequences in a single nucleic acid.
  • a native operon includes all three subunits on more than one nucleic acid sequence.
  • a native operon thus encodes the wild-type subunits of operon A (SEQ ID NOS: 36-38), operon B (SEQ ID NOS: 39-41), operon C SEQ ID NOS: 42, 43, 41), operon D (SEQ ID NOS: 45-47), operon E (SEQ ID NOS: 39, 49, 41), operon G (SEQ ID NOS: 51,40,53), or operon H (SEQ ID NOS: 54-56).
  • the E. coli B 16S operon is identical to the E 16S operon
  • the B 23S operon is identical to the G 23S operon.
  • the E. coli 5S operon is identical across the B, C, and E operons.
  • an “engineered” or “genetically modified” rRNA refers to an rRNA that comprises a mutation/modification compared to the native rRNA sequence, and is encoded by a genetically modified (engineered) rRNA subunit in the corresponding operon.
  • an operon nucleic acid sequence can be engineered.
  • an engineered operon can be a native operon with one or more point mutations. Point mutations can be a base insertion, deletion, or substitution.
  • a point mutation preferably alters active sites in the ribosomes (e.g., binding sites, or sites with catalytic importance for ribosome function).
  • the point mutations can be present in a portion of the rRNA that forms the catalytic active site of the ribosome, or peptidyl-transferase center (PTC).
  • an engineered rRNA operon can include one or more nucleic acid deletions in helices H91 and/or H92.
  • the one or more nucleic acid deletions in helices H91 and/or H92 can be in operon C, operon D, and/or operon H from E. coli.
  • the point mutations can provide and/or enhance various functions, such as increasing protein expression.
  • a point mutation may reverse a polymorphism in an operon (e.g., reverse a polymorphism in the D operon to match the B operon sequence).
  • an operon may comprise subunits from different rRNA operons.
  • an engineered operon can include a 16S rRNA gene (subunit) from a first operon, and a 23 S rRNA gene from a second rRNA operon.
  • the sequence for 16S can be selected from SEQ ID NOS: 36, 39, 42, 45, 51 , or 54, or a sequence that is at least 85 %, at least 90 %, at least 95 %, at least 98 % or at least 99 % identical to those SEQ ID NOS.
  • the sequence for 23S can be selected from SEQ ID NOS: 37, 40, 43, 46, 49, or 55 or be a sequence that is at least 85 %, at least 90 %, at least 95 %, at least 98 % or at least 99 % identical to those SEQ ID NOS.
  • the sequence for 5S can be selected from SEQ ID NOS: 38, 41, 47, 53, or 56 or be a sequence that is at least 85 %, at least 90 %, at least 95 %, at least 98 % or at least 99 % identical to those SEQ ID NOS.
  • an engineered operon comprises an operon in which the 16S subunit is from operon A (SEQ ID NO: 36), the 23S subunit is from operon B (SEQ ID NO: 40), and the 5S subunit is from operon B (SEQ ID NO: 42).
  • Subunits of the seven operons can be genetically manipulated and combined to produce one or more engineered operons comprising a 16S, 23 S, and a 5S rRNA subunit.
  • the engineered operon can comprise a polynucleotide sequence that is at least 85 %, at least 90 %, at least 95 %, at least 98 % or at least 99 % identical to one or more of SEQ ID NOs: 1-11.
  • engineered operons comprise the 23 S subunit from the B operon (SEQ ID NO: 40), and 16S subunits selected from the other operons.
  • the 16S subunit is selected from the B operon (SEQ ID NO: 39), and the 23 S subunits are selected from the other operons.
  • the engineered rRNA operon can be engineered into one or more expression constructs and introduced into a host cell, such as an E. coli cell.
  • a host cell such as an E. coli cell.
  • the E. coli genome can be modified/engineered to modify or delete one or more endogenous (genomic) rRNA operons or portions thereof.
  • an “engineered cell” may further include a modification to remove or inactivate one or more native (e.g., endogenous) rRNA operons, or to inactivate one or more genes (eliminate the expression of one or more subunits) of a native (e.g., endogenous) operon.
  • one or more endogenous rRNA operons or one or more endogenous rRNA genes from one or more rRNA operons may be functionally deleted (i.e., mutated to inactivate), thereby providing an engineered cell.
  • one or more of rRNA operons C, D, or H are deleted/rendered inactive/non-expressed from the genome of an E. coli cell.
  • one or more of operons A and B are deleted/rendered inactive.
  • one or more subunits of one or more operons A, B, C, D, E, G, or H are deleted or rendered inactive or nonexpressed, and/or one or more complete operons A, B, C, D, E, G, or H are deleted or rendered inactive or non-expressed.
  • rendered inactive or “non-expressed”, refers to no expression (functional expression) of a gene product (e.g., rRNA), or activity of that gene product.
  • a gene product e.g., rRNA
  • Methods of detecting gene products e.g., the presence or absence of rRNAs
  • activity of such gene products are well-known in the art.
  • altering ratios of ribosomes in a ribosome pool and/or rRNAs in the ribosome pool can be achieved by genetic modification of the cell to alter operon availability (e.g., deleting, silencing, or replacing low performing ribosomes or rRNAs, and/or increasing copy numbers of high performing ribosomes and/or rRNAs), overexpression of high-performing ribosomes and/or rRNAs from a plasmid, and the like.
  • purified and/or isolated ribosomes from one or more strains having an engineered ribosome pool can be combined to achieve the desired ratio(s) of rRNAs and rRNA-containing ribosomes.
  • ribosome pool refers to a defined plurality of ribosomes.
  • a ribosome pool comprises the totality of ribosomes expressed in a cell.
  • a ribosome pool comprises a combination of ribosomes from more than one cell (e.g., ribosomes isolated from different modified cells and optionally unmodified cells, and/or ribosomes provided in the form of a lysate from different modified cells and optionally unmodified cells).
  • a ribosome pool may comprise a wildtype ribosome pool (i.e., the totality of ribosomes from a non-engineered E. coli cell).
  • a ribosome pool may comprise an “engineered ribosome pool”, i.e., the ribosomes are derived from or produced by or present in a cell comprising at least one engineered/genetically modified rRNA operon, such that the ribosome pool comprises a non-wildtype ratio of rRNA in the ribosomes.
  • an engineered cell may comprise a “heterogenous ribosome pool” or a “homogenous ribosome pool.”
  • a ribosome pool may be “a homogenous ribosome pool.” This refers to a ribosome pool wherein each ribosome in the pool comprises the same rRNA sequences, i.e., each 23 S, 16S, and 5S rRNA sequence in the ribosome pool is the same.
  • the rRNA subunits may be from the same or from different operons.
  • the systems and platforms described herein can comprises a cell-free protein synthesis (CFPS) platform.
  • CFPS cell-free protein synthesis
  • the CFPS platforms can include one or more cell lysates derived from one or more of the cells described above.
  • the CFPS platform can include a cell lysate derived from an engineered cell or cells, the engineered cell or cells comprising an engineered ribosome pool.
  • the cell lysate can be derived from a cell that harbors a construct encoding one or more engineered rRNA operons.
  • the CFPS system can include purified or isolated ribosomes that have been added to a CFPS system or cell lysate, where the ribosomes were purified or isolated from one or more cells having an engineered ribosome pool.
  • the systems and platforms disclosed herein can be used for recombinant protein expression.
  • the systems and platforms described herein can exhibit increased protein expression yields as compared to a wildtype ribosome pool of a host cell.
  • the protein yield of an engineered ribosome pool as disclosed herein can be 1 fold, 2 fold, 3 fold, 4 fold, or 5 fold greater than the protein yield from a wildtype (control) ribosome pool.
  • the method can include determining the protein expression activity of individual genomic rRNA operons in a cell of interest. For example, in an E. coli cell with seven different rRNA operons, one would assay each individual rRNA operon for protein expression activity, e.g., using one or more the methods disclosed in the Examples herein. In such an aspect, once the activity of each rRNA operon is determined, one could delete any rRNA operons that perform poorly (e.g., express lower levels of protein compared to another rRNA operon).
  • systems and platforms described herein can be used to increase total recombinant protein production in cell-free systems and/or in cell-based systems.
  • systems and platforms described herein can be used to prototype rRNA sequences to survey which is best suited for a specific protein.
  • Embodiment 2 An engineered ribosome pool, wherein the ribosome pool is capable of increase protein biopolymer yields relative to a heterogenous pool of ribosomes.
  • Embodiment 3 A cell comprising an engineered ribosome pool with sequence optimized ribosomes.
  • Embodiment 5 A cell-free protein synthesis system, derived from cell extracts of engineered cells comprising an engineered ribosome pool with sequence optimized ribosomes.
  • Embodiment 6 A cell-free protein synthesis system, derived from cell extracts of engineered cells with genomic modifications to remove sub-optimally performing ribosomes from the ribosome pool.
  • Embodiment 7 A cell-free protein synthesis system, derived from cell extracts of engineered cells overexpressing highly performing ribosomes within a heterogeneous ribosome pool.
  • Embodiment 8 A cell-free protein synthesis system, derived from cell extracts with heterogeneous ribosome pool, doped with additional highly performing ribosomes purified from a homogeneous strain.
  • Embodiment 1 A system for recombinant protein expression, comprising: a cell comprising an engineered ribosome pool, wherein the engineered ribosome pool is different than a native pool.
  • Embodiment 2 The system of embodiment 1, wherein the engineered ribosome pool comprises rRNA sequences that are different than rRNA sequences of the native ribosome pool.
  • Embodiment 3 The system of embodiment 1 or 2, wherein the engineered ribosome pool is a homogeneous ribosome pool.
  • Embodiment 4 The system of any one of embodiments 1-3, wherein the engineered ribosome pool comprises rRNA encoded by a single rRNA operon.
  • Embodiment 5 The system of embodiment 4, wherein the single rRNA operon is present on one or more constructs in the cell.
  • Embodiment 6 The system of embodiments 4 or 5, wherein the single rRNA operon is an engineered operon comprising at least two rRNA genes from different rRNA operons.
  • Embodiment 7 The system of embodiment 6, wherein the at least two rRNA genes from different rRNA operons comprise: i) a 16S rRNA gene from a first rRNA operon; and ii) a 23S rRNA gene from a second rRNA operon.
  • Embodiment 8 The system of any one of embodiments 4-7, wherein the single rRNA operon comprises a polynucleotide sequence that is at least 85 %, at least 90 %, at least 95 %, at least 98 % or at least 99 % identical to one or more of SEQ ID NOs: 1-11.
  • Embodiment 9 The system of any one of embodiments 4-7, wherein the single rRNA operon comprises rRNA genes from different operons, where the rRNA genes are selected from the polynucleotide sequences of SEQ ID NOs: 36-56 or polynucleotide sequences that are at least 85 %, at least 90 %, at least 95 %, at least 98 %, or at least 99 % identical to the polynucleotide sequences of SEQ ID NOs: 36-56.
  • Embodiment 10 The system of any one of embodiments 1-9, wherein the cell comprises a deletion of at least one genomic rRNA operon.
  • Embodiment 11 The system of embodiment 10, wherein the cell is modified to delete all but one genomic rRNA operon.
  • Embodiment 12 The system of any one of embodiments 1-11, wherein the cell is an E. coll cell.
  • Embodiment 13 The system of embodiment 12, wherein one or more rRNA genes from operon D and/or operon C have been removed.
  • Embodiment 14 The system of embodiments 12 or 13, wherein amino acid deletions in helices H91 and/or H92 of operon D and/or operon C have been performed.
  • Embodiment 15 The system of any one of embodiments 12-14, wherein one or more rRNA genes from operon H have been removed.
  • Embodiment 16 A cell lysate prepared from the cell of any one of embodiments 1-15.
  • Embodiment 17 A cell-free protein synthesis platform, comprising the cell lysate of embodiment 16.
  • Embodiment 18 An E. coli cell, wherein one or more rRNA genes from operon D, operon C, or operon H have been removed.
  • Embodiment 19 The cell of embodiment 18, wherein operon D and/or operon C comprise amino acid deletions in helices H91 and/or H92.
  • Embodiment 20 The cell of embodiment 18 or 19, wherein one or more rRNA genes from operon H have been removed.
  • Embodiment 21 A method for recombinant protein expression, comprising transforming a construct encoding a protein into the cell of any one of embodiments 1-15 and/or the cell of any one of embodiments 18-20.
  • Embodiment 22 The method of embodiment 21, wherein a level of the protein expressed is greater than a level of the protein expressed in a cell having a native ribosome pool.
  • Embodiment 23 A method for recombinant protein expression, comprising using the cell- free protein synthesis platform of embodiment 17.
  • Embodiment 24 The method of embodiment 23, wherein a level of the protein expressed is greater than a level of the protein expressed in a cell-free protein synthesis system having a native ribosome pool.
  • Ribosomes have conventionally been thought of as uniform molecular assemblies even though most organisms carry multiple copies of unique rRNA-encoding operons (rrn) in their genomes 1 .
  • rrn unique rRNA-encoding operons
  • E. coli K-12 strain MG 1655 for example, there seven genomically encoded rRNA operons containing several polymorphisms and are named with letters A-E, G, and H in increasing distance from the origin of replication 2 .
  • the seven unique rRNA operons in E. coli have been studied through the lens of promoter strength 1 3,4 ; it is known that the rRNA operon promoters are among the strongest in the genome, responsible for more than 70% of total RNA synthesis in fast-growing cells 5 .
  • Previous work has shown that certain operons, such as rrnE, have stronger promoters and are more highly expressed 3 .
  • Other studies have shown that specific rRNA genes, such as the 16S rRNA of rrnH, are more highly expressed in response to nutrient limitation and result in a ribosome population that is more resistant to tetracycline, a class of antibiotics that blocks tRNAs from interacting with the ribosome’s active site 6 .
  • purification tags with which to isolate specific ribosomes would require genome engineering that is complicated by significant homology between rRNA operons 8 .
  • purification tags would only target the SSU or LSU individually rather than the formed 70S particle composed of both subunits, and the tag itself may have confounding effects on translation studies 9,10 .
  • the recently developed in vitro ribosome synthesis, assembly, and translation (iSAT) provides an approach to individually synthesize and assess activity of the unique, naturally occurring rRNA operons that exist in the E. coli genome" n .
  • iSAT in vitro ribosome assembly and translation
  • CFPS cell-free protein synthesis
  • AdhE2 expressed in AAA showed a net conversion rate of -35%, matching previously reported values 41 . Notably, attaining this same butanol yield using MG1655 lysate to express AdhE2 required more than twice the CFPS reaction volume (FIG. 6).
  • Plasmids Ribosomal operon sequences and annotations were acquired from the Escherichia coli K-12 substr. MG1655 reference genome (EcoCyc). rRNA-coding plasmids were constructed by mixing and matching fragments from synthetic plasmids ordered from Twist Biosciences within a pT7rrnB backbone as previously described 43 .
  • 5S polymorphisms were introduced via site-directed mutagenesis to result in pure-operon sequences AAA/BBB/CCC/DDD/EEE/GGG/HHH, and confirmed by Sanger sequencing. Plasmids were cloned into chemically competent DhlOB and purified using the Zymo Midiprep Kit and then further purified via ethanol precipitation using 0.5 M NELOAc for use in iSAT reactions.
  • Plasmids for expression of rRNA in vivo were assembled by cloning the rRNA sequence from the Twist plasmids and using Gibson assembly to insert it into a pAM-backbone plasmid, so that the rRNA expression is under the control of phage lambda promoter pL, regulated by the bacteriophage lambda cI857 repressor 44 Plasmids were cloned into chemically competent POP2136 cells 45 , grown at 30°C, and purified using the Zymo Miniprep Kit.
  • DNA constructs for the expression of the proteins in CFPS were made using the pJLl backbone plasmid as previously described 46 and purified using the Zymo Midiprep Kit.
  • SI 50 lysate preparation S150 lysate was prepared as previously reported 43 .
  • 2X YTPG medium containing 18 g/1 of glucose
  • the 1 L culture was incubated at 37°C with shaking at 250 rpm until the OD600 reached 2.8.
  • the culture was then immediately centrifuged at 5,000xg for 10 minutes at 4°C. Throughout the handling process, cells were kept on ice and as cold as possible. The supernatant was discarded and the resulting pellet was suspended in S30 buffer (10 mM TrisOAc pH 8.2, 14 mM Mg(OAc)2, 60 mM KOAc).
  • the cell resuspension was then subjected to two additional spins at 10,000xg for 3 minutes each. Between each spin, the supernatant was removed and the pellet was resuspended in 40 mb of fresh S30 buffer. Following the third spin, the pellets were weighed and immediately flash-frozen in liquid nitrogen before storing them at -80°C.
  • sucrose cushion buffer (20 mM Tris-HCl (pH 7.2 at 4°C), 100 mM NH4C1, 10 mM MgCk, 0.5 mM EDTA, 2 mM DTT, 37.7% sucrose) in Ti70 tubes.
  • the concentrated cell suspension was plated on lysogeny broth (LB) agar plates containing 5% sucrose and 100 pg/ml Cb. The plates were incubated at 37°C until colonies appeared. Eight colonies were selected from each plate and spotted onto two LB-agar plates, one containing Cbioo and the other containing Kamo. Colonies that grew successfully on Cbioo but not on Kanso were chosen and cultured overnight in LB with Cbioo for midiprep using the ZymoPURETM II Plasmid Midiprep Kit.
  • Table 2 16S and 23S rRNA sequence variants. Backbone sequence is maintained as that from pT7 BBB in Table 1 above.
  • Table 4 Plasmid sequences for protein expression panel.
  • Table 5 E. colt rRNA operons and sequences.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Sont divulgués des systèmes et des plates-formes pour l'expression de protéines recombinantes. Les systèmes et les plates-formes comprennent des cellules modifiées ou un lysat préparé à partir de la cellule modifiée, la cellule modifiée comprenant (a) un vecteur d'expression d'acide nucléique exogène comprenant au moins un gène d'ARN (ARNr) provenant d'au moins un opéron d'ARNr d'E. coli choisi parmi A, B, C, D, E, G et H; et (b) éventuellement une mutation dans un ou plusieurs opérons d'ARNr endogènes A, B, C, D, E, G et H dans lesquels la mutation entraîne une absence d'expression d'au moins un gène d'ARNr dans l'opéron, au moins un gène d'ARNr de (a) étant différent d'au moins un gène d'ARNr de (b).
PCT/US2024/060598 2023-12-18 2024-12-17 Systèmes et plates-formes comprenant un groupe de ribosomes modifiés Pending WO2025207168A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363611685P 2023-12-18 2023-12-18
US63/611,685 2023-12-18

Publications (2)

Publication Number Publication Date
WO2025207168A2 true WO2025207168A2 (fr) 2025-10-02
WO2025207168A3 WO2025207168A3 (fr) 2025-12-18

Family

ID=97219663

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/060598 Pending WO2025207168A2 (fr) 2023-12-18 2024-12-17 Systèmes et plates-formes comprenant un groupe de ribosomes modifiés

Country Status (1)

Country Link
WO (1) WO2025207168A2 (fr)

Similar Documents

Publication Publication Date Title
US11136574B2 (en) Methods for in vitro ribosome synthesis and evolution
US10118950B2 (en) Platforms for cell-free protein synthesis comprising extracts from genomically recoded E. coli strains having genetic knock-out mutations in release factor 1 (RF-1) and endA
US12157908B2 (en) Cell-free protein synthesis platforms derived from clostridia extracts
US11673921B2 (en) Cell-free protein synthesis platform derived from cellular extracts of Vibrio natriegens
US20170349928A1 (en) Methods for activating natural energy metabolism for improving yeast cell-free protein synthesis
US12305211B2 (en) Protein glycosylation sites by rapid expression and characterization of N-glycosyltransferases
EP3436471B1 (fr) Mutants de la recombinase
JP7048643B2 (ja) 無細胞タンパク質合成のためのプロモーター構築物
US20240027436A1 (en) Cell-free expression of antibodies, antigen-binding fragments thereof, and antibody derivatives
US20230015505A1 (en) Modular, cell-free protein expression vectors to accelerate biological design in cells
WO2025207168A2 (fr) Systèmes et plates-formes comprenant un groupe de ribosomes modifiés
JP7461652B2 (ja) 化合物ライブラリー及び化合物ライブラリーの製造方法
US20240384267A1 (en) Compositions and methods for multiplex decoding of quadruplet codons
Smalakyte et al. Filament formation activates protease and ring nuclease activities of CRISPR SAVED-Lon
US12421537B2 (en) Ribosome variants for sequence defined polymer synthesis
Sørensen et al. Structure-guided design of protein attachment points for functional augmentation of complex molecular machines
WO2025147710A1 (fr) Systèmes de traduction orthogonale et procédés d'identification de composants de associés
Chung Structural Insights Into Group II Intron Splicing and Retrotransposition
US20240294963A1 (en) Engineered n-glycosyltransferases with altered specificities
WO2020033416A1 (fr) Plateforme de transcription et de traduction combinée dérivées de chloroplastes végétaux
Xiang Expanding the genetic code in mammalian cells
HK1261197A1 (en) Recombinase mutants
HK1261197B (en) Recombinase mutants

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24932496

Country of ref document: EP

Kind code of ref document: A2