Systems and platforms comprising engineered ribosome pool

Abstract

Disclosed herein are systems and platforms for recombinant protein expression. The systems and platforms include engineered cells or a lysate prepared from the engineered cell, in which the engineered cell includes (a) an exogenous nucleic acid expression vector including at least one RNA (rRNA) gene from at least one E. coli rRNA operon selected from A, B, C, D, E, G, and H; and optionally, and (b) optionally a mutation in one or more endogenous rRNA operons A, B, C, D, E, G, and H in which the mutation results in a lack of expression of at least one rRNA gene in the operon, in which at least one rRNA gene of (a) is different than at least one rRNA gene of (b).

Description

SYSTEMS AND PLATFORMS COMPRISING ENGINEERED RIBOSOME POOLCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. U.S. 63 / 611,685, filed December 18, 2023. The contents of which is incorporated by reference herein in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant numbers W91 INF-22-2-0246 and W91 INF-16-1-0372 awarded by the Department of Defense. The government has certain rights in the invention.SEQUENCING LISTING STATEMENT

[0003] 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.BACKGROUND

[0004] 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.SUMMARY

[0005] In one aspect, a system, kit, or platform for recombinant protein expression is provided. The system 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. coli rRNA operon selected from A, B, C, D, E, G,and H, optionally in which the at least one rRNA gene includes a modification; (b) optionally, a mutation in one or more endogenous rRNA operons A, B, C, D, E, G, and H, wherein the mutation results in a lack of expression of at least one rRNA gene in the operon, in which at least one rRNA gene of (a) is different than at least one rRNA gene of (b). In some embodiments, the rRNA genes include 5S, 16S, and / or 23 S rRNA.

[0006] In some embodiments, 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. In some embodiments, the rRNA of the engineered ribosome pool is homogenous among the plurality of ribosomes in the pool. In some embodiments, 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. In some embodiments, the genes of the rRNA operon are present on one or more exogenous vectors in the cell.

[0007] In some embodiments, the engineered ribosome pool includes rRNA encoded by genes from different rRNA operons. The rRNA genes from different rRNA operons may include: (i) a 16S rRNA gene from a first rRNA operon; and (ii) a 23S rRNA gene from a second operon. In some embodiments, 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. In some embodiments, 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.

[0008] In some embodiments, 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.

[0009] In another aspect, a cell-free lysate is provided. In some embodiments, a cell-free protein synthesis platform includes a cell-free lysate.

[0010] In another aspect, 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. In some embodiments, operon D and / or operon C include amino acid deletions in helices H91 and / or H92. In some embodiments, one or more rRNA genes from operon H have been removed.

[0011] In another aspect, a method for expressing a protein of interest is disclosed. The method 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. In some embodiments, 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. In some embodiments, the method of expressing a protein of interest further includes contacting the cell-free protein synthesis platform with a translation template.

[0012] In yet another aspect, an expression construct is provided. 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. In some embodiments, 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.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] 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. (FIG. 1C) Single operon rRNA encoding plasmids show a wide range of activities in iSAT. End point of sfGFP expression, normalized to iSAT activity of reference operon B, mean of n=3.

[0014] 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. 2B) Operons A, B, C, G and H were able to singularly support life. Single-operon strains were normalized to OD600=1 and serial dilutions were spotted onto plates. Plates were imaged when the most dilute sample showed cell growth. Images are representative of n=3 assays. (FIG. 2C) 70S ribosomes purified from single-operon strains can be tested for sfGFP production in a ribosome-free lysate. (FIG. 2D) Purified 70S show a wide range of activity, with many variants showing an advantage over the mixed pool (MG1655). End point of sfGFP expression, normalized to iSAT activity of reference MG1655 pool, mean of n=3.

[0015] FIGS. 3A-3C. Engineered ribosomal operons highlight that some natively occurring rRNA sequences are low performing or inactive. (FIG. 3A) iSAT activities of combination operons. End point of sfGFP expression, normalized to iSAT activity of reference BBB, mean of n=3. (FIG. 3B) Helix 91 (H91) and 92 (H92) polymorphism case study to reverse non-consensus sequences in the PTC. (FIG. 3C) Reversing H91 and H92 polymorphisms to match the consensus helps recover activity of operons C and D in iSAT. End point of sfGFP expression, normalized to iSAT activity of reference BBB, mean of n=3.

[0016] FIGS. 4A-4F. Homogeneous ribosome pools increase protein biosynthesis yields relative to heterogeneous ribosome pools. (FIG. 4A) Standard S12 lysates for CFPS contain a heterogeneous ribosome pool. (FIG. 4B) S12 lysates for CFPS made from single-operon strains yield lysate expressing a homogeneous ribosome pool. (FIG. 4C) Homogeneous ribosome pool lysates show a wide range of sfGFP production, and some outperform standard S12 lysate from a mixed ribosome pool (MG1655). Operon sequence notation shown as 16S:23S:5S. Error bars indicating standard deviations of n=4 replicates. (FIG. 4D) Protein yields as determined by radioactive quantification of 14C-Leucine for a panel of proteins. Standard deviation shown for n=3 replicates. Statistical significance in expression between AAA and MG1655 lysate denoted by asterisk (*) as calculated by a student’s paired t-test with p<0.05. (FIG. 4E) AlphaLISA binding pattern of TRI2-2 interfacing with S6P. (FIG. 4F) AdhE2 expressed in AAA lysate shows expected butyraldehyde conversion efficiency. Standard deviation shown for n=3 replicates.

[0017] FIG. 5. 70S sfGFP expression in iSAT reaction background for all single-operon constructs purified. Data presented are means of n = 3 experiments with standard deviation shown.

[0018] FIG. 6. Amount of butanol produced by equivalent concentration of AdhE2 expressed in AAA and MG1655 lysate. Data presented are means of n = 3 experiments with standard deviation shown.DETAILED DESCRIPTION

[0019] Systems and platforms that include an engineered ribosome pool are disclosed. The systems and platforms described herein can be used for recombinant protein synthesis. In various aspects, the engineered ribosome pools in the systems and platforms disclosed herein allow for increased protein biosynthesis yields as compared to native ribosome pools.

[0020] Definitions

[0021] The disclosed subject matter may be further described using definitions and terminology as follows. The definitions and terminology used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0022] As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise. For example, the term “a substituent” should be interpreted to mean “one or more substituents,” unless the context clearly dictates otherwise.

[0023] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.

[0024] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other thanthe components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0025] The phrase “such as” should be interpreted as “for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0026] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e. ., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0027] All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.

[0028] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”

[0029] Polynucleotides and synthesis methods

[0030] The terms “nucleic acid” and “oligonucleotide,” as used herein, refer to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and to any other type of polynucleotide that is an N glycoside of a purine or pyrimidine base. There is no intended distinction in length between the terms “nucleic acid”, “oligonucleotide” and “polynucleotide”, and these terms will be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA. For use in the present methods, an oligonucleotide also can comprise nucleotide analogs in which the base, sugar, or phosphate backbone is modified as well as non-purine or non-pyrimidine nucleotide analogs.

[0031] 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.

[0032] The term “amplification reaction” refers to any chemical reaction, including an enzymatic reaction, which results in increased copies of a template nucleic acid sequence or results in transcription of a template nucleic acid. Amplification reactions include reverse transcription, the polymerase chain reaction (PCR), including Real Time PCR (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)), and the ligase chain reaction (LCR) (see Barany et al., U.S. Pat. No. 5,494,810). Exemplary “amplification reactions conditions” or “amplification conditions” typically comprise either two or three step cycles. Two-step cycles have a high temperature denaturation step followed by a hybridization / elongation (or ligation) step. Three step cycles comprise a denaturation step followed by a hybridization step followed by a separate elongation step.

[0033] The terms “target,” “target sequence”, “target region”, and “target nucleic acid,” as used herein, are synonymous and refer to a region or sequence of a nucleic acid which is to be amplified, sequenced, or detected.

[0034] The term “primer,” as used herein, refers to an oligonucleotide capable of acting as a point of initiation of DNA synthesis under suitable conditions. Such conditions include those in which synthesis of a primer extension product complementary to a nucleic acid strand is induced in the presence of four different nucleoside triphosphates and an agent for extension (for example, a DNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature.

[0035] 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.

[0036] Primers can incorporate additional features which allow for the detection or immobilization of the primer but do not alter the basic property of the primer, that of acting as a point of initiation of DNA synthesis. For example, primers may contain an additional nucleic acid sequence at the 5' end which does not hybridize to the target nucleic acid, but which facilitates cloning or detection of the amplified product, or which enables transcription of RNA (for example, by inclusion of a promoter) or translation of protein (for example, by inclusion of a 5’-UTR, such as an Internal Ribosome Entry Site (IRES) or a 3’-UTR element, such as a poly(A)n sequence, where n is in the range from about 20 to about 200). The region of the primer that is sufficiently complementary to the template to hybridize is referred to herein as the hybridizing region.

[0037] As used 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. Typically, 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.One of skill in the art will recognize that various factors, 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. Thus, 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.

[0038] As used herein, 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. RNA-dependent DNA polymerases also fall within the scope of DNA polymerases. Reverse transcriptase, which includes viral polymerases encoded by retroviruses, is an example of an RNA-dependent DNA polymerase. Known examples of RNA polymerase (“RNAP”) include, for example, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase and E. coli RNA polymerase, among others. The foregoing examples of RNA polymerases are also known as DNA-dependent RNA polymerase. The polymerase activity of any of the above enzymes can be determined by means well known in the art.

[0039] The term “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.

[0040] As used herein, “expression template” refers to a nucleic acid that serves as substrate for transcribing at least one RNA that can be translated into a sequence defined biopolymer (e.g., a polypeptide or protein). Expression templates include nucleic acids composed of DNA or RNA. Suitable sources of DNA for use a nucleic acid for an expression template include genomic DNA, cDNA and RNA that can be converted into cDNA. Genomic DNA, cDNA and RNA can be from any biological source, such as a tissue sample, a biopsy, a swab, sputum, a blood sample, a fecal sample, a urine sample, a scraping, among others. The genomic DNA, cDNA and RNA can be from host cellor virus origins and from any species, including extant and extinct organisms. As used herein, “expression template” and “transcription template” have the same meaning and are used interchangeably.

[0041] In certain exemplary embodiments, vectors such as, for example, expression vectors, containing a nucleic acid encoding one or more rRNAs or reporter polypeptides and / or proteins described herein are provided. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Such vectors are referred to herein as “expression vectors.” In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably. However, the disclosed methods and compositions are intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0042] Oligonucleotides and polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and / or modified nucleotides. Examples of modified nucleotides include, but are not limited to diaminopurine, S2T, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5- iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta- D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2- dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6- adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46- isopentenyl adenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, 2,6-diaminopurine and the like. Nucleic acid molecules may also be modified at the base moiety (e g., at one or more atoms that typically are available to form a hydrogen bond with a complementary nucleotide and / or at one or more atoms that are not typically capable of forming a hydrogen bond with a complementary nucleotide), sugar moiety or phosphate backbone.

[0043] As utilized herein, a “deletion” means the removal of one or more nucleotides relative to the native polynucleotide sequence. The engineered strains that are disclosed herein may include a deletion in one or more genes (e.g., a deletion in gmd and / or a deletion in waaL). Preferably, a deletion results in a non-functional gene product. As utilized herein, an “insertion” means the addition of one or more nucleotides to the native polynucleotide sequence. The engineered strains that are disclosed herein may include an insertion in one or more genes (e.g., an insertion in gmd and / or an insertion in waaL). Preferably, a deletion results in a non-functional gene product. As utilized herein, a “substitution” means replacement of a nucleotide of a native polynucleotide sequence with a nucleotide that is not native to the polynucleotide sequence. The engineered strains that are disclosed herein may include a substitution in one or more genes (e.g., a substitution in gmd and / or a substitution in waaL). Preferably, a substitution results in a non-functional gene product, for example, where the substitution introduces a premature stop codon (e.g., TAA, TAG, or TGA) in the coding sequence of the gene product. In some embodiments, the engineered strains that are disclosed herein may include two or more substitutions where the substitutions introduce multiple premature stop codons (e.g., TAATAA, TAGTAG, or TGATGA).

[0044] In some embodiments, the engineered strains disclosed herein may be engineered to include and express one or more heterologous genes. As would be understood in the art, a heterologous gene is a gene that is not naturally present in the engineered strain as the strain occurs in nature. A gene that is heterologous to E. coli a gene that does not occur in E. coli and may be a gene that occurs naturally in another microorganism or a gene that does not occur naturally in any other known microorganism (i.e., an artificial gene).

[0045] Peptides, Polypeptides, Proteins, and Synthesis Methods

[0046] As used herein, the terms “peptide,” “polypeptide,” and “protein,” refer to molecules comprising a chain a polymer of amino acid residues joined by amide linkages. The term “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 or W), andtyrosine (Tyr or Y) residues. The term “amino acid residue” also may include nonstandard or unnatural amino acids. The term “amino acid residue” may include alpha-, beta-, gamma-, and delta-amino acids.

[0047] In some embodiments, the term “amino acid residue” may include nonstandard or unnatural amino acid residues contained in the group consisting of homocysteine, 2-Aminoadipic acid, N- Ethylasparagine, 3-Aminoadipic acid, Hydroxylysine, P-alanine, P-Amino-propionic acid, allo- Hydroxylysine acid, 2-Aminobutyric acid, 3-Hydroxyproline, 4-Aminobutyric acid, 4- Hydroxyproline, piperidinic acid, 6- Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo- Isoleucine, 2-Aminoisobutyric acid, N-Methylglycine, sarcosine, 3-Aminoisobutyric acid, N- Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine, 2,4-Diaminobutyric acid, N- Methylvaline, Desmosine, Norvaline, 2,2'-Diaminopimelic acid, Norleucine, 2,3-Diaminopropionic acid, Ornithine, and N-Ethylglycine. The term “amino acid residue” may include L isomers or D isomers of any of the aforementioned amino acids.

[0048] Other examples of 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 p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-bromophenylalanine, a p-amino-L-phenylalanine, an isopropyl-L- phenylalanine, an unnatural analogue of a tyrosine amino acid; an unnatural analogue of a glutamine amino acid; an unnatural analogue of a phenylalanine amino acid; an unnatural analogue of a serine amino acid; an unnatural analogue of a threonine amino acid; an unnatural analogue of a methionine amino acid; an unnatural analogue of a leucine amino acid; an unnatural analogue of a isoleucine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, ufa hor, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or a combination thereof; an amino acid with a photoactivatable cross-linker; a spin-labeled amino acid; a fluorescent amino acid; a metal binding amino acid; a metal-containing amino acid; a radioactive amino acid; a photocaged and / or photoisomerizable amino acid; a biotin or biotin-analogue containingamino acid; a keto containing amino acid; an amino acid comprising polyethylene glycol or polyether; a heavy atom substituted amino acid; a chemically cleavable or photocl eavable amino acid; an amino acid with an elongated side chain; an amino acid containing a toxic group; a sugar substituted amino acid; a carbon-linked sugar-containing amino acid; a redox-active amino acid; an a-hydroxy containing acid; an amino thio acid; an a, a disubstituted amino acid; a P-amino acid; a y-amino acid, a cyclic amino acid other than proline or histidine, and an aromatic amino acid other than phenylalanine, tyrosine or tryptophan.

[0049] As used herein, a “peptide” is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110). In some embodiments, a peptide as contemplated herein may include no more than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. A polypeptide, also referred to as a protein, is typically of length > 100 amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110). A polypeptide, as contemplated herein, 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.

[0050] 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 isoprenoid group such as farnesol or geranylgeraniol), amidation at C-terminus, glycosylation (e.g., the addition of a glycosyl group to either asparagine, hydroxylysine,serine, or threonine, resulting in a glycoprotein). 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).

[0051] The terms "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.

[0052] As stated above, the term "antibody" includes "antibody fragments" or "antibody-derived fragments" and "antigen binding fragments" which comprise an antigen binding domain. The term "antibody fragment" as used herein is intended to include any appropriate antibody fragment that displays antigen binding function, for example, Fab, Fab', F(ab')2, scFv, Fv, dsFv, ds-scFv, Fd, dAbs, TandAbs dimers, mini bodies, monobodies, diabodies, and multimers thereof and bispecific antibody fragments.

[0053] Cell-Free Protein Synthesis (CFPS)

[0054] The strains and systems disclosed herein may be applied to cell-free protein synthesis methods as known in the art. See, for example, U.S. Patent Nos. 5,478,730; 5,556,769; 5,665,563; 6,168,931; 6,869,774; 6,994,986; 7,118,883; 7,189,528; 7,338,789; 7,387,884; 7,399,610; 8,703,471; and 8,999,668. See also U.S. Published Application Nos. 2015-0259757, 2014-0295492, 2014-0255987, 2014-0045267, 2012-0171720, 2008-0138857, 2007-0154983, 2005-0054044, and 2004-0209321. See also U.S Published Application Nos. 2005-0170452; 2006-0211085; 2006-0234345; 2006- 0252672; 2006-0257399; 2006-0286637; 2007-0026485; 2007-0178551; and 2018-0016612. See also Published PCT International Application Nos. 2003 / 056914; 2004 / 013151; 2004 / 035605; 2006 / 102652; 2006 / 119987; and 2007 / 120932. See also Jewett, M.C., Hong, S.H., Kwon, Y.C., Martin, R.W., and Des Soye, B.J. 2014, “Methods for improved in vitro protein synthesis with proteinscontaining non-standard amino acids,” U.S. Patent Application Serial No.: 62 / 044,221 ; Jewett, M.C., Hodgman, C.E., and Gan, R. 2013, “Methods for yeast cell-free protein synthesis,” U.S. Patent Application Serial No.: 61 / 792,290; Jewett, M.C., J. A. Schoborg, and C.E. Hodgman. 2014, “Substrate Replenishment and Byproduct Removal Improve Yeast Cell-Free Protein Synthesis,” U.S. Patent Application Serial No.: 61 / 953,275; and Jewett, M.C., Anderson, M.J., Stark, J.C., Hodgman, C.E. 2015, “Methods for activating natural energy metabolism for improved yeast cell-free protein synthesis,” U.S. Patent Application Serial No.: 62 / 098,578. See also Guarino, C., & DeLisa, M. P. (2012). A prokaryote-based cell-free translation system that efficiently synthesizes glycoproteins. Glycobiology, 22(5), 596-601. The contents of all of these references are incorporated in the present application by reference in their entireties.

[0055] In certain exemplary embodiments, one or more of the methods described herein are performed in 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. Examples of 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.

[0056] In certain exemplary embodiments, 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.

[0057] The disclosed cell-free protein synthesis systems may utilize components that are crude and / or that are at least partially isolated and / or purified. As used herein, 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. The term “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.

[0058] Systems and Platforms Comprising Engineered Cells

[0059] Tn one or more aspects, the systems and platforms described herein can include an engineered cell. 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). As a non-limiting example, the engineered cell can be an E. coli cell.

[0060] As used herein, an rRNA operon is a segment of DNA that encodes at least ribosomal rRNA. In the case of E. coli, 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. By way of example, a single operon may comprise a single nucleic acid which encodes all three subunits. Alternatively, a single operon may comprise multiple nucleic acids which encode all three subunits collectively.

[0061] In 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.

[0062] A “native operon” as used herein comprises all three rRNA genes and comprises no mutations or modifications in the rRNA genes (subunit sequences). In some embodiments, a native operon includes all three rRNA subunit sequences in a single nucleic acid. In some embodiments, 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). Note that the E. coli B 16S operon is identical to the E 16S operon, and the B 23S operon is identical to the G 23S operon. Additionally, not that the E. coli 5S operon is identical across the B, C, and E operons.

[0063] 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. There are several options, well known in the art, for how an operon nucleic acid sequence can be engineered. By way of example but not by way of limitation, an engineered operon can be a native operon with one or more point mutations. Point mutations can be a base insertion, deletion, or substitution. In some embodiments, a point mutation preferably alters active sites in the ribosomes (e.g., binding sites, or sites with catalytic importance for ribosome function). In some embodiments, 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). In the same or alternative aspects, an engineered rRNA operon can include one or more nucleic acid deletions in helices H91 and / or H92. In various aspects, 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. In some embodiments, the point mutations can provide and / or enhance various functions, such as increasing protein expression. In some embodiments, 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).

[0064] Additionally or alternatively, an operon may comprise subunits from different rRNA operons. For example, 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. By way of example but not by way of limitation, 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. By way of example, 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. In certain aspects, 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.

[0065] Tn some embodiments, engineered operons comprise the 23 S subunit from the B operon (SEQ ID NO: 40), and 16S subunits selected from the other operons. In some embodiments, the 16S subunit is selected from the B operon (SEQ ID NO: 39), and the 23 S subunits are selected from the other operons.

[0066] In various aspects, 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. In the same or alternative embodiments, the E. coli genome can be modified / engineered to modify or delete one or more endogenous (genomic) rRNA operons or portions thereof.

[0067] In some embodiments, 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. By way of example, 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. In one example embodiment, one or more of rRNA operons C, D, or H are deleted / rendered inactive / non-expressed from the genome of an E. coli cell. In some embodiments, one or more of operons A and B are deleted / rendered inactive. In some embodiments, 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.

[0068] As used herein “rendered inactive” or “non-expressed”, refers to no expression (functional expression) of a gene product (e.g., rRNA), or activity of that gene product. Methods of detecting gene products (e.g., the presence or absence of rRNAs) and activity of such gene products are well-known in the art.

[0069] In some embodiments, the engineered cell may be engineered to alter a ratio of rRNAs from the same or different operons. By way of example but not by way of limitation, an engineered cell may comprise more 23 S rRNA from operon A, as compared to 23 S RNA from operon B, and more 16s rRNA from operon C as compared to 16S rRNA from operon D. Such engineered cells comprise an altered ratio of ribosomes pools comprising the altered ratios of the operons and / or the rRNAs in the cell. In some embodiments, such ribosome pools provide higher performance with respect toprotein biosynthesis, as compared to a native, non-engineered cell. In various embodiments, 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. In the same or alternative aspects, in the case of cell-free extracts, 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.

[0070] As used herein the term “ribosome pool” refers to a defined plurality of ribosomes. In some embodiments, a ribosome pool comprises the totality of ribosomes expressed in a cell. In some embodiments, 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).

[0071] In some embodiments a ribosome pool may comprise a wildtype ribosome pool (i.e., the totality of ribosomes from a non-engineered E. coli cell). In some embodiments 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.

[0072] In some embodiments, an engineered cell may comprise a “heterogenous ribosome pool” or a “homogenous ribosome pool.”

[0073] In some embodiments, 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.

[0074] In some embodiments, a ribosome pool may be “a heterogeneous ribosome pool.” This refers to a ribosome pool comprising one or more differences in the 16S, 23 S and / or 5S rRNA sequences among the plurality of ribosomes in the ribosome pool.

[0075] In various aspects, an engineered cell can comprise one or more modifications that can provide and / or enhance various functions, such as increasing rRNA expression from one or more rRNA operons or one or more rRNA subunits. In one aspect, the one or more mutations / modifications can be present in a promoter sequence driving the expression of an operon. Additionally or alternatively, a strong, intermediate, or weak promoter may be swapped to modulate the expression of an operon or an operon subunit.

[0076] Cell Free Protein Synthesis Platform

[0077] In one or more aspects, the systems and platforms described herein can comprises a cell-free protein synthesis (CFPS) platform. Example CFPS systems in general are described above. In various aspects, the CFPS platforms can include one or more cell lysates derived from one or more of the cells described above. For instance, in an aspect, 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. By way of example but not by way of limitation, the cell lysate can be derived from a cell that harbors a construct encoding one or more engineered rRNA operons. In another aspect, 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.

[0078] As discussed above, the systems and platforms disclosed herein can be used for recombinant protein expression. In such aspects, the systems and platforms described herein can exhibit increased protein expression yields as compared to a wildtype ribosome pool of a host cell. By way of example but not by way of limitation, 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.

[0079] In one or more aspects, methods for forming a protein expression system are provided. In aspects, 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). In the same or alternative aspects, one couldform engineered rRNA operons, e g., an rRNA operon that includes at least two rRNA genes from different genomic operons, and / or can perform mutagenesis on an rRNA gene and / or an rRNA operon to engineer the expression system as needed.

[0080] Example Applications of the Systems and Platforms

[0081] In one aspect, the systems and platforms described herein can be used to increase total recombinant protein production in cell-free systems and / or in cell-based systems.

[0082] In various aspects, the systems and platforms described herein can be used to optimize and / or tailor rRNA sequences for improved expression of specific proteins.

[0083] In various aspects, the systems and platforms described herein can be used to prototype rRNA sequences to survey which is best suited for a specific protein.

[0084] Additional Embodiments

[0085] Embodiment 1. An engineered ribosome pool, the engineered ribosome pool comprising ribosomal RNA (rRNA) optimized sequences that lead to increased protein synthesis yields.

[0086] Embodiment 2. An engineered ribosome pool, wherein the ribosome pool is capable of increase protein biopolymer yields relative to a heterogenous pool of ribosomes.

[0087] Embodiment 3. A cell comprising an engineered ribosome pool with sequence optimized ribosomes.

[0088] Embodiment 4. A cell comprising an engineered ribosome pool with altered ratios of naturally occurring wild type ribosomes - either by overexpressing highly active ribosomes or suppressing sub-optimally active ribosomes.

[0089] Embodiment 5. A cell-free protein synthesis system, derived from cell extracts of engineered cells comprising an engineered ribosome pool with sequence optimized ribosomes.

[0090] 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.

[0091] Embodiment 7: A cell-free protein synthesis system, derived from cell extracts of engineered cells overexpressing highly performing ribosomes within a heterogeneous ribosome pool.

[0092] 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.

[0093] Further Embodiments:

[0094] 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.

[0095] 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.

[0096] Embodiment 3: The system of embodiment 1 or 2, wherein the engineered ribosome pool is a homogeneous ribosome pool.

[0097] Embodiment 4: The system of any one of embodiments 1-3, wherein the engineered ribosome pool comprises rRNA encoded by a single rRNA operon.

[0098] Embodiment 5: The system of embodiment 4, wherein the single rRNA operon is present on one or more constructs in the cell.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] Embodiment 10: The system of any one of embodiments 1-9, wherein the cell comprises a deletion of at least one genomic rRNA operon.

[0104] Embodiment 11 : The system of embodiment 10, wherein the cell is modified to delete all but one genomic rRNA operon.

[0105] Embodiment 12: The system of any one of embodiments 1-11, wherein the cell is an E. coll cell.

[0106] Embodiment 13: The system of embodiment 12, wherein one or more rRNA genes from operon D and / or operon C have been removed.

[0107] 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.

[0108] Embodiment 15: The system of any one of embodiments 12-14, wherein one or more rRNA genes from operon H have been removed.

[0109] Embodiment 16: A cell lysate prepared from the cell of any one of embodiments 1-15.

[0110] Embodiment 17: A cell-free protein synthesis platform, comprising the cell lysate of embodiment 16.

[0111] Embodiment 18: An E. coli cell, wherein one or more rRNA genes from operon D, operon C, or operon H have been removed.

[0112] Embodiment 19: The cell of embodiment 18, wherein operon D and / or operon C comprise amino acid deletions in helices H91 and / or H92.

[0113] Embodiment 20: The cell of embodiment 18 or 19, wherein one or more rRNA genes from operon H have been removed.

[0114] 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.

[0115] 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.

[0116] Embodiment 23: A method for recombinant protein expression, comprising using the cell- free protein synthesis platform of embodiment 17.

[0117] 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.

[0118] Embodiment 25: A construct comprising a promoter operably coupled to a polynucleotide encoding for an rRNA operon, wherein the rRNA operon comprises: 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.

[0119] Embodiment 26: The construct of embodiment 25, wherein 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.

[0120] Advantages

[0121] The systems and platforms described herein can provide a powerful new method to optimize recombinant protein expression systems. Previous methods to optimize expression systems have not focused on the ribosomal RNA sequence itself, although ribosomes are the core machinery of protein synthesis. The systems and platforms described herein can increase overall protein production byimproving average protein synthesis ability of ribosome pool and substituting less active rRNA sequences with high performing variants.

[0122] The systems and platforms described herein increase yields of recombinant proteins by optimizing the pool of ribosomes being expressed. This can be used to generate more efficient biomanufacturing systems and consequently lower costs of protein production, which could improve access to recombinant protein technology. This has applications in therapeutics production, as well as industrial protein expression efforts such as for enzyme purification.EXAMPLES

[0123] The following Examples are illustrative and should not be interpreted to limit the claimed subject matter.

[0124] Summary

[0125] 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 (rrn) in their genomes1. In 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 replication2.

[0126] The seven unique rRNA operons in E. coli have been studied through the lens of promoter strength1 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 cells5. Previous work has shown that certain operons, such as rrnE, have stronger promoters and are more highly expressed3. 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 activesite6. However, while studying differential transcription of rRNA sequences provides insight into the regulation of ribosome heterogeneity and specialization, it does not directly show the impact of rRNA sequence diversity on performance of molecular translation. If rRNA sequences produce functionally different ribosomes, this suggests that the rRNA sequences in the genomes of biomanufacturing strains could be manipulated to express optimized ribosome pools.

[0127] Studying if and how native rRNA sequences affect protein translation is limited by our ability to isolate and test ribosomes from specific operons as well as difficulties in controlling for the effects of operon promoter architecture and position in the genome. Previous work has explored inactivating rRNA operons in the genome to assess how cells performed with fewer ribosomal operons and found that having fewer instances of rRNA in the genome results in slower doubling times, but these findings were not controlled for the differences in genome position and promoter architecture7. An alternative approach to assess the impact of rRNA sequence on ribosome function would be purifying distinct ribosomes from cells. However, adding purification tags with which to isolate specific ribosomes would require genome engineering that is complicated by significant homology between rRNA operons8. In addition, 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 studies9,10. Circumventing these limitations, 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.

[0128] Here, we use iSAT to demonstrate how the heterogeneity of native rRNA sequences affects the activity of resulting ribosomes. We use in vitro rRNA prototyping and strain engineering methods to test individual rRNA operons and combinations of operon components. We demonstrate that ribosomes resulting from different operons display a wide range of activities when expressed and assembled both in vitro and in vivo, and some strains carrying homogeneous rRNA populations yield significantly improved protein synthesis over those from the parent strain for a panel of proteins. Our results suggest that ribosome pool engineering has the potential to improve biomanufacturing systems for many applications in synthetic biology, as well as elucidate deeper understanding of ribosome heterogeneity and evolution. Overall, this is a promising tool to optimize protein biosynthesis.

[0129] Activity of ribosomes derived from single rRNA operons varies widely

[0130] The goal of our work was to characterize the sequence effect of natively occurring rRNA operons on recombinant protein production. As a model, we focused on the seven distinct genomically encoded rRNA operons from E. coli K-12 strain MG1655 (FIG. 1A). These operons are largely the same in sequence but differ by a total of 21 unique point mutants in the 16S rRNA, 34 in the 23S rRNA, and 3 in 5S rRNA. These mutations are present in all domains of the 23 S rRNA and exist on both the 5’ and 3’ ends of the 16S rRNA. Notably, the operons even have sequence differences in the rRNA that forms the catalytic active site of the ribosome, or peptidyl-transferase center (PTC).

[0131] We used an in vitro ribosome assembly and translation (iSAT) platform to study how rRNA operon sequence differences impacted protein production. iSAT enables one-pot co-activation of rRNA transcription, assembly of rRNA with native r-proteins into E. coli ribosomes, and the synthesis of functional proteins from these ribosomes in a crude SI 50 extract lacking native ribosomes12. iSAT allows researchers to prototype different rRNA sequences by simply changing the input DNA that codes for the rRNA of interest. Previously, iSAT has been used to carry out mutation mapping of the 70S ribosome14, enable assessment of computationally designed ribosomes13,16, and evolve the ribosome for new function17.

[0132] Typically, in the field of ribosome engineering and in past work using the iSAT system, the model operon rrnB is usedl x 20; the architecture of the pT7rmB plasmid (Table 1; SEQ ID NO: 1) was thus used as a template, and rRNA fragments from other operons were exchanged into this plasmid (Table 2, SEQ ID NOS: 2-11; Table 3, SEQ ID NOS 12-30). With each distinct operon on individual plasmids, we assembled separate iSAT reactions for each operon supplementing a ribosome-free SI 50 lysate with the operon plasmid, T7-superfolder green fluorescent protein (sfGFP) plasmid (reporter), ribosomal proteins, and energy mix (FIG. IB)15. The reactions are incubated at 37 °C where the transcribed rRNA is assembled into a ribosome and tested for the ability to synthesize sfGFP. We found that the seven naturally occurring rRNA operons in E. coll produce ribosomes exhibiting a wide range of protein synthesis activity. Operons B and E (16S:23S:5S) yielded the highest amount of sfGFP, and operons D and H yielded the lowest amount (FIG. 1C), with operon D being nonfunctional, surprisingly.

[0133] We next investigated whether functional variation of ribosomal operons is also observed in living E. coli by constructing strains that express only one rRNA operon sequence instead of the 7native sequences. This process involves transforming rRNA-carrying plasmids into strain SQ171fg, which was evolved from the “Squires” SQ171 strain19,21. The SQ171fg has all 7 genomic rRNA copies removed and lives off an rRNA sequence encoding a tethered ribosome, Ribo-T v219on a plasmid. This Ribo-T v2 plasmid also contains SacB and an antibiotic resistance gene, which can both be used as selection markers (FIG. 2A)22. If the rRNA variant of interest is able to support cell growth, the original plasmid can be cured, effectively replacing the original RT-v2-SacB plasmid with a plasmid carrying the rRNA variant of interest.13All 7 operons were transformed and selected for in this way, resulting in 5 single-operon strains (FIG. 2B). Single operon strain development for operons D and H were not successful, as the original SacB plasmid was not able to be cured, indicating that rmD and H sequences are unable to support life. The successful single-operon strains showed similar growth phenotypes, and the failure of D and H to support life correlated with their respective activity in iSAT (FIG. 1C).

[0134] We then compared translation activity of ribosomes derived from each operon to a wild-type heterogenous pool of ribosomes. 70S ribosomes were purified from the single-operon strains A, B, C, E, and G, as well as from the parent strain MG1655, which natively expresses all seven rRNA operons. Reactions were prepared in vitro with purified ribosomes, ribosome-free lysate, reporter plasmid, and reagent mix (FIG. 2C). The activity of all 70S variants was quantified by quantifying the purified sfGFP synthesis, measured in RFU (relative fluorescent units). While operon A, B, and E ribosomes performed better than the MG1655 70S ribosomes, C and G showed lower sfGFP production than the MG1655 pool. This, in combination with the failure of D and H to support life, suggests that the MG1655 70S translation capacity may be diluted by less active variants, specifically C, D, G, and H. Replacing weaker variants with the most active ribosomes could optimize the overall ribosome pool and enable higher efficiency protein production systems.

[0135] Removing rRNA polymorphisms recovers translation activity

[0136] To investigate why E. coli might maintain copies of rRNA operons that yield low-activity ribosomes such as those from D and H, we designed an experiment to ask whether pairing the SSU and LSU rRNAs from different operons could recover activity of operons that were less active. In living cells, it is possible that the SSU from one operon could associate with a LSU from a different operon to form a 70S ribosome because translation of a messenger-RNA (mRNA) is initiated first bythe association of a SSU with the 5’ end of the mRNA, followed by the LSU co-associating to form a translationally competent complex23.

[0137] To build operon combinations, we made constructs that combined 16S and 23 S rRNA genes individually from each operon with counterparts from operon B to test in iSAT. All constructs carry the 5S rRNA sequence from the model B operon. As some of the 16S and 23 S rRNA sequences are the same between different operons, this resulted in only 10 unique sequences including that of the B operon (e.g., the 23 S rRNA sequence of the B operon matches that of the G operon). When the unique combination constructs were tested for sfGFP production in iSAT, we found that 6 of 9 pairs yielded ribosomes that produced at least 20% as much sfGFP as the control from operon B (FIG. 3A). However, the 23 S rRNA of operons C and D yielded a significant decrease in activity when paired with the 16S rRNA from operon B. Similarly, the 16S rRNA sequence from operon H resulted in a severe drop in sfGFP production. These results indicate that polymorphisms in individual subunit rRNAs may be responsible for the negative impact on translation ability.

[0138] The 23 S rRNA sequences from operons C and D are particularly interesting because both carry polymorphisms in the PTC that differentiate them from consensus operon B (FIG. 3B). The PTC, located in domain V of the ribosomal RNA, is one of the most sequence conserved and catalytically important regions of rRNA24. Operon C contains a single nucleotide polymorphism in Helix 91 (H91) resulting in the loss of a Watson-Crick (WC) base pair, which is the strongest possible RNA base pair interaction25(FIG. 3B). Similarly, operon D contains a deletion and a single nucleotide polymorphism in Helix 92 (H92), effectively removing two of the three WC base pairs present in the consensus sequence. Both helices H91 and H92 compose part of the functionally important and highly sequence conserved region of the “accommodation corridor”26. Additionally, our previous work has shown these two helices to be highly sensitive to mutations, especially when mutations result in a loss of WC base pairing interactions15.

[0139] To test whether these specific polymorphisms are the source of decreased ribosome activity, we synthesized individual plasmids containing rrnC and rrnD with single motifs of interest mutated to match the consensus sequence of operon B and tested them in iSAT (FIG. 3C). We found that the resulting ribosome activity more than doubled when the single nucleotide polymorphism in H91 of operon C was reversed to the Operon B sequence. In a more extreme case, the activity of operon Dchanged from being undetectable to achieving nearly 80% of operon B’s activity when the polymorphisms in H92 were reversed to the sequence of rrnB. We then reverted other polymorphisms in operon D (in H62 and H78) but did not observe activity from these ribosomes. These data indicate that the mutations of helices H91 and H92 are largely responsible for the decrease in ribosome activity from operons C and D suggest that optimization of ribosome pools (i.e., removing these low-activity rRNA sequences and replacing them with high performing variants) could improve protein production capacity in E. coli.

[0140] Single operon derived ribosomes pools increase protein biosynthesis yields

[0141] We next sought to use sequence-optimized ribosomes to increase protein biosynthesis yields. As a model, we explored this strategy in the context of cell-free protein synthesis (CFPS). CFPS is an attractive approach to produce proteins in vitro without the need to maintain cell growth27’28. In recent years, CFPS has matured to impact a variety of applications in diagnostics, biomanufacturing, and educational kits, among others29 39. Typically, the ribosome-containing lysates (S12 lysates) for CFPS are made from bacterial strains harboring multiple rRNA operons, producing lysate with a heterogeneous ribosome pool (FIG. 4A). Here, we sought to assess if protein synthesis could be increased by creating CFPS-capable lysates that do not contain ribosomes derived from these deleterious operons (e g., operon D).

[0142] We made cell-free lysates derived from source strains with homogenous ribosome pools (i.e., each cell expressing the same three rRNA subunits) (FIG. 2A; FIG. 4B). We chose single operons that produced ribosomes with a relative activity over 50% in iSAT (FIG. 1C; FIG. 3 A) as well as two low- performing operons (BCB and HBB, notation 16S :23 S : 5 S rRNA) as negative controls. Of note, operon sequence BHB, which performed well in iSAT, was unable to support life and so could not be prepared as a single-operon lysate. We then set up CFPS reaction with these lysates and measured sfGFP production (FIG. 4C). We found that single operon lysates (same three rRNA subunits), produced more protein than lysates made from the parent strain (MG1655) with a pool of 7 operons. The lysate containing only operon A rRNA (AAA) showed nearly a 3 -fold increase in protein production. Ribosomes built in cells and tested in cell-free translation had similar profiles (FIG. 5).

[0143] We then took the highest performing lysate (AAA) and used it to express a panel of five proteins that differ in size, function, and structure (FIG. 4D). We chose proteins representing diversefields of interest for industrial and medical applications (e.g., genetic engineering (Cas9), vaccines (CRM 197), antibodies / protein binders (TRI2-2), bacteriophages (MS2), and sustainable chemical production (AdhE2)). (Table 4; SEQ ID NOs 31-35). Of the five proteins tested, four showed a statistically significant increase in yield and one had comparable expression when expressed in the AAA lysate compared to the MG1655 lysate.

[0144] We next assayed two of the proteins for activity. We used an amplified luminescent proximity homogeneous assay (AlphaLISA)40to detect the binding of TRI2-2, a multivalent minibinder protein, to the trimeric HexaPro SARS-Cov-2 S glycoprotein (S6P)34(FIG. 4E). AlphaLISA detected a characteristic binding interaction between CFPS-expressed minibinder TRI2-2 and target S6P. We also confirmed the functionality of the aldehyde-alcohol dehydrogenase (AdhE2) by measuring conversion of butyraldehyde to butanol (FIG. 4F)41. AdhE2 expressed in AAA showed a net conversion rate of -35%, matching previously reported values41. Notably, attaining this same butanol yield using MG1655 lysate to express AdhE2 required more than twice the CFPS reaction volume (FIG. 6). These findings highlight that using a lysate for CFPS containing only AAA rRNA sequences is beneficial for improving functional protein yields of a wide variety of proteins.

[0145] Methods

[0146] 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 described43. As some rRNA sequences between different operons match (for example, operons E and B have identical 23 S rRNA sequences), only 12 total rRNA constructs were purchased- ABB, BEB, CBB, DBB, GBB, HBB, BAB, HBB, BCB, BDB, BEB, and BHB (16S:23S:5S). Primers were designed to amplify the 16S and 23S rRNA sequences from the sequence-verified Twist plasmids, and combined into the AAB / BBB / CCB / DDB / EEB / GGB / HHB sequences as well as the mixed-operon 16S and 23 S rRNA combination constructs using Gibson assembly. 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.

[0147] 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 repressor44Plasmids were cloned into chemically competent POP2136 cells45, grown at 30°C, and purified using the Zymo Miniprep Kit.

[0148] DNA constructs for the expression of the proteins in CFPS were made using the pJLl backbone plasmid as previously described46and purified using the Zymo Midiprep Kit.

[0149] SI 50 lysate preparation: S150 lysate was prepared as previously reported43. One liter of 2X YTPG medium (containing 18 g / 1 of glucose) was inoculated with 10 mb of an overnight culture of MRE600. 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.

[0150] After thawing on ice for 20 minutes, S30 buffer was added at a ratio of 5 mb per 1g of cell mass, and then the cells were resuspended by vortexing until fully in solution. 100 pl of HALT Protease Inhibitor Cocktail was added per 10 mb of cell suspension, and 75 pl of Takara Recombinant RNAse Inhibitor was added per 4g of dry cell mass. Cell lysis was achieved using a C3 Avestin Homogenizer at a pressure of approximately 25,000 psig. Following lysis, a second aliquot of Takara Recombinant RNase Inhibitor was added. The resulting mixture was centrifuged at 12,000xg at 4°C for 15 minutes to remove cell debris. The supernatant was then layered on top of an equivalent volume of 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.

[0151] The samples were then spun in an ultracentrifuge at 90,000xg for 18 hours. After this first spin, the supernatant was carefully transferred to fresh Ti70 tubes and spun for 3 additional hours at 150,000xg. The pellets (ribosome pellets) remaining in the first tubes were used to purify the r-proteinsfor TP70. After the second spin, the top 2 / 3 of the supernatant was collected and transferred into MWCO = 3.5 K dialysis tubing (SnakeSkin) and dialyzed 2 x 1.5 hr x 3 L of S150 Extract Buffer at 4°C. For the third dialysis, 3 L of fresh SI 50 Extract Buffer was used to dialyze overnight (12-15 hrs). S 150 extract was concentrated using Centripreps (3 kDa MWCO) until A260 = 25 and A280 = 15. Extract was aliquoted and flash frozen in liquid nitrogen. TP70 was prepared from the ribosome pellets as previously described17.

[0152] SOI 71 transformations and plasmid selections: Electrocompetent E. coli SQ171fg cells, harboring RiboT-v2 rRNA on a pCSacB plasmid and kanamycin resistance (KanR)22,47, were prepared and stored in 50 pL aliquots. The SQ171fg strain is a modified E. coli strain with all seven rRNA operons deleted from its genome. The pCSacB / KanR plasmid contains the sequence for RiboT- v219, which functions as the sole rRNA operon in the cell. To remove the original pCSacB-RiboT-v2 plasmid and introduce pAM552 plasmids carrying the rRNA sequence of interest and an ampicillin resistance gene, selection was performed by plating on sucrose and carbenicillin (Cb). Successful selection was confirmed by checking the strain's resistance to Kan.

[0153] 50 ng of purified mutant pAM552 plasmid transformed into the SQ171fg electrocompetent cells. The cell / plasmid mixture was then incubated in 850 pL of SOC in a E5-mL microcentrifuge tube at 37°C with shaking at 250 rpm for 1 hour. After the incubation, 270 pL of the cell recovery was transferred to 2 mb of Super Optimal broth with Catabolite repression (SOC) supplemented with 50 pg / Ll Cb (Cbso) and 0.25% sucrose in a 14-mL plastic culture tube. The tubes were incubated overnight at 37°C for 16-18 hours. After incubation, the tubes were centrifuged at room temperature for 5 minutes at 4000xg. 2 mL of clear supernatant was removed, and the remaining cell pellet was concentrated into the remaining 270 pl. 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 ZymoPURE™ II Plasmid Midiprep Kit. The midiprepped plasmids were then subjected to Sanger sequencing to confirm that the operon sequence was as expected.

[0154] Constructs that did not yield colonies on LB-Suc5%-Cbioo plates underwent two subsequent transformation and selection attempts to confirm their inability to support life. Constructs that produced colonies on both antibiotics were investigated further by picking and spot plating additional colonies. If troubleshooting failed, transformations were repeated up to three times before concluding that the construct was unable to support life.

[0155] To confirm that the cells relied solely on the mutated ribosomes, overnight cultures of the successfully transformed SQ171fg strains were grown in 5 mL volumes, and total RNA was extracted using the QiagenTM RNeasy Mini kit. RT-PCRs were conducted using the Invitrogen™ SuperScript IV One-Step RT-PCR system to amplify regions of rRNA that contained mutations in the operons. The amplified products were then Sanger sequenced.

[0156] Ribosome purifications and testing: 500-mL of LB-Miller was induced with overnight culture of strain containing desired ribosomes, targeting an OD of 0.05. Cells were grown at 37 °C with 250 rpm until they reached an OD of 0.6-0.8. The cells were then pelleted via centrifugation for 10 minutes at 8,000xg at 4C. Supernatant was removed, and the pellet was resuspended by vortexing in 25-mL of Buffer A (20 mM Tris-chloride pH 7.2 at 4°C, 100 mM ammonium chloride, 10 mM magnesium chloride, 0.5 mM EDTA, 2 mM DTT). The pellet was washed in Buffer A for a total of three times, at which point the pellet was flash frozen and stored at -80°C.

[0157] The cell pellet was resuspended in 1-mL Buffer A per gram cell pellet and lysed by sonication (50% Amplitude, 45 seconds ON, 59 seconds OFF- 950 J per mL of suspension). The sonicated cell suspension was then diluted to a total volume of 13-mL in Buffer A and centrifuged for 10 minutes at 12,000xg. The supernatant (clarified lysate) was layered on top of 13-mL of Buffer B (20 mM Tris- HC1 pH 7.2, 100 mM NH4C1, 10 mM MgCh, 0.5 mM EDTA, 2 mM DTT, 37.7% sucrose) in a Ti70 ultracentrifuge tube. Samples were spun at 90,000xg for 18 hours, at which point the resulting pellet was resuspended in Buffer C (10 mM Tris-0 Ac pH 7.5, 60 mM NH4CI, 7.5 mM Mg(OAc)2, 0.5 mM EDTA, 2 mM DTT) and normalized to 25 pM. Ribosomes were added into blank iSAT reactions (no TP70 or pT7rrnB plasmid) to reach a final concentration of 4 pM.

[0158] S12 extract preparation: Cell growth for extract preparation was carried out as previously described46’54-55. Overnight cultures of strains used were to inoculate 100-mL of LB-Miller at an OD of 0.05. The cells were grown at 37 °C, 250 rpm and OD was monitored until they reached an OD of2.8. The culture was then spun down for 10 minutes at 12,000xg at 4C. The pellet was resuspended in 25-mL of S30 Buffer by vortexing and spun for 2 minutes at 12,000xg. This was repeated a total of three times, at which point the pellet was weighed and flash frozen in liquid nitrogen to be stored at - 80°C.

[0159] The pellet was thawed on ice and resuspended with 1-mL of S30 Buffer per gram of pellet in a 1.5-mL Eppendorf tube. The cells were then lysed via sonication (50% Amplitude, 45 seconds ON, 59 seconds OFF- 950 J per mL of suspension) and centrifuged for 10 minutes at 12,000xg. The supernatant was aliquoted and flash frozen for use as S12 extract.

[0160] AdhE2 activity quantification: CFPS reactions for the expression of pJL 1 -AdhE2 were set up with S12 lysates AAA and MG1655 as previously described50 51 52, and run overnight at 30 C. Negative control reactions were set up with pJLl-sfGFP, as the expression of sfGFP should not enable improved butyraldehyde conversion to butanol. Butyraldehyde conversion reactions then were assembled in 1.5-mL tubes to contain total AdhE2 concentrations of 0.075 pM by adding in corresponding volumes of overnight AAA and MG 1655 CFPS AdhE2 expression reactions (as quantified by14C -Leucine incorporation).

[0161] The overnight reactions were then mixed with 10 mM butyraldehyde, 10 mM Mg(Glu)2„10 mM NH4(Glu), 134 mM KGlu, and 500 mM BisTris buffer. NADH (20 mM) was added to initiate the reaction, and samples were quenched after one hour by adding an equivalent volume of 10% (w / v) trichloroacetic acid. Eppendorf tubes containing the quenched reactions were spun at maximum speed for 10 minutes, at which point the supernatant was transferred to HPLC vials. 5 pL of supernatant were injected into an Agilent 1290 HPLC with a Bio-Rad Fast Acid Analysis column held at 40 °C using 0.1% formic acid as the mobile phase flowing at 0.6 mL / min. Butanol concentrations were determined using refractive index values compared to a standard curve.

[0162] AlphaLISA assay: The AlphaLISA assay leverages the use of proprietary donor and acceptor beads that enable detection of protein-protein interactions based on bead proximity40. AlphaLISA assays were run based on a previously published protocol34in a 50 mM HEPES pH 7.4, 150 mM NaCl, 1 mg / mL BSA, and 0.015% v / v Triton X-100 buffer (“Alpha buffer”). Reaction components were dispensed into a ProxiPlate-384 Plus (PerkinElmer 6008280) destination plate from a 384-well Polypropylene 2.0 Plus source microplate (Labcyte, PPL-0200) using an Echo 525 liquid acousticliquid handler. The assays were run in a solution of 50 mM HEPES pH 7.4, 150 mM NaCl, 1 mg / mL BSA, and 0.015% v / v Triton X-100 buffer (“Alpha buffer”). Anti-FLAG donor beads (PerkinElmer) were used to immobilize TRI2-2 protein, which was expressed with a sFLAG tag on its C -terminus48. His-tagged stabilized trimeric S protein (S6P) (Aero SPN-C52H9) which has previously been shown to bind TRI2-234, was immobilized onto the acceptor bead. The final concentrations of the donor and acceptor beads were 0.08 and 0.02 mg / mL, respectively. S6P and CFPS reactions to produce TRI2-2 were cross titrated with final dilutions ranging from 25 nM to 0 nM for S6P and 20-fold to 6400-fold dilutions for TRI2-2 and incubated for one hour at room temperature. The donor and acceptor beads were then added to the wells and incubated for an additional hour at room temperature. Chemiluminescence was measured on a Tecan Infinite M1000 Pro using the AlphaLISA filter with an excitation time of 100 ms, an integration time of 300 ms, and a settle time of 20 ms after 10 min of incubation inside the instrument as previously reported49.

[0163] iSAT reactions: iSAT reactions were assembled with four 5-pL replicates per rRNA construct being tested based on previous work12. Reactions contained 8 mM magnesium glutamate, 10 mM ammonium glutamate, 130 mM potassium glutamate, 0.85 mM each of GTP, UTP, and CTP, 1.2 mM ATP, 34 pg / mL folinic acid, 0.171 mg / mL E. coli tRNA, 0.33 mM NAD, 0.27 mM CoA, 4 mM oxalic acid, 1 mM putrescine, 1.5 mM spermidine, 57 mM HEPES, 2 mM 20 amino acids, 37 mM PEP, -300 nM total protein of the 70S ribosome (TP70), 60 pg / mL T7 RNA polymerase, 0.50 pL of PEG- 8000 40% (SigmaAldrich), and 1.83 pL SI 50 extract (in a 5 pL reaction). The pJLl-sfGFP plasmid concentration was 6.27 ng / pL and the pT7rrn plasmid concentration was 20.78 ng / pL.

[0164] The Echo 525 Acoustic Liquid Handler was used to assemble reaction components (separated into a master mix and individual rRNA plasmids to be tested) into 384-well nunc_267461 plates from a 384-well Polypropylene 2.0 Plus source microplate (Labcyte, PPL-0200). The nunc_267461 plate was then spun down and reactions were run in a plate reader at 37°C, measuring sfGFP fluorescence (excitation: 485 nm, emission: 528 nm) every 15 minutes and with constant shaking for 15 hours.

[0165] CFPS reactions: CFPS reactions were based on previous work50'33. 15-pL reactions were set up in triplicate on 384-well nunc_267461 plates. Reactions contained 8 mM magnesium glutamate, 10 mM ammonium glutamate, 130 mM potassium glutamate, 0.85 mM each of GTP, UTP, and CTP, 1.2 mM ATP, 34 pg / mL folinic acid, 0.171 mg / mL E. coli tRNA, 0.33 mM NAD, 0.27 mM CoA, 4mM oxalic acid, 1 mM putrescine, 1 .5 mM spermidine, 57 mM HEPES, 2 mM 20 amino acids, 0.03 M phosphoenolpyruvate, 36 pg / mL T7 RNA polymerase, 2.4 pL of S12 lysate, and 13.3 ng / pL of the pJLl backbone plasmid. Reactions were incubated at 30°C with continuous shaking for 15 hours and fluorescence (excitation: 485 nm, emission: 528 nm) was measured every 5 minutes for sfGFP expression. Measurements were recorded as relative fluorescent units (RFU).

[0166] Discussion

[0167] Past work had indicated that ribosomal operons in E. coli are differentially transcribed and that trends in transcription can change as a function of environmental stresses3,6. However, changes in translation activity arising from unique operon sequences have not been directly studied. By using an in vitro ribosome synthesis, assembly, and translation system we were able to determine for the first time that native rRNA sequence heterogeneity results in significant protein synthesis differences from the resulting ribosomes. In fact, some operons, like rrnD and rrnH, have no or little activity in iSAT and were not able to independently support life while others outperformed the natively expressed 7- operon mixture because of polymorphisms in rRNA sequence. By leveraging these findings, we were able to show proof-of-concept that homogeneous ribosome pools derived from high-performing, sequence-specific single operons improve protein expression in cell-free systems of a variety of proteins compared to heterogenous pool of wild-type ribosomes in CFPS.

[0168] Our finding that ribosomes derived from the rrnD and rrnH operons were non-functional was surprising, and suggests that the native E. coli ribosome pool maybe diluted with low-performing variants such as D and H. This begs the question of why the E. coli genome would retain ribosome variants of lesser fitness; heterologous ribosomes with suboptimal translation performance may contribute to cell survival in adverse conditions, such as being able to translate during nutrient starvation 6 or to fine tune translation while entering and exiting stationary phase hibernation42. It may be that laboratory cell growth conditions provide a non-natural environment where some ribosome variants perform better while others do not contribute to high protein yields, as was measured in this study. Further investigation of these specialized ribosome properties is needed.

[0169] In summary, this work demonstrates that functional activity variation exists across ribosomes derived from the seven rRNA operons in natively expressed ribosome pools in E. coli. We also illustrate the concept of ribosome pool engineering and show that some rRNA sequences haveincreased bulk protein biosynthesis yields. We suspect that ribosome pool engineering will enable engineering objectives in common workhorse organisms and strains by optimizing the ribosome pool to contain only the most productive sequences for specific applications both in vitro and in vivo. Looking forward, we anticipate that studying rRNA sequence-function relationships will build a deeper understanding of how ribosomes have evolved and how we might design specialized ribosomes for applications in biotechnology and synthetic biology.

[0170] References

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[0226] Table 1 : Plasmid sequence for pT7rrnBBB with backbone sequence bolded and underlined.Table 2: 16S and 23S rRNA sequence variants. Backbone sequence is maintained as that from pT7 BBB in Table 1 above.Table 3: Primers for cloning and sequence confirmation of rrn operon constructs.Table 4: Plasmid sequences for protein expression panel.Table 5: E. colt rRNA operons and sequences.Table 6: Sequences for in vitro plasmidTable 7: Sequences for in vivo plasmid

Claims

CLAIMS1. A system, kit, or platform for recombinant protein expression, comprising: an engineered Escherichia coli (E. coli) cell, or a lysate prepared from the engineered cell, the engineered cell comprising: a. an exogenous nucleic acid expression vector comprising at least one ribosomal RNA (rRNA) gene from at least one E. coli rRNA operon selected from A, B, C, D, E, G, and H, optionally wherein the at least one rRNA gene comprises a modification; b. optionally, a mutation in one or more endogenous rRNA operons A, B, C, D, E, G, and H, wherein the mutation results in a lack of expression of at least one rRNA gene in the operon; wherein at least one rRNA gene of (a) is different than at least one rRNA gene of (b).

2. The system of claim 1, wherein the rRNA genes comprise 5S, 16S, and / or 23 S rRNA.

3. The system of claim 1, wherein the engineered E. coli cell, or the lysate prepared from the cell, comprises an engineered ribosome pool, the engineered ribosome pool comprising a plurality of ribosomes.

4. The system of claim 3, wherein the rRNA of the engineered ribosome pool is homogeneous among the plurality of ribosomes in the pool.

5. The system of claim 4, wherein the engineered ribosome pool comprises rRNA encoded by the same rRNA operon, i.e., rRNA operon A, B, C, D, E, G, or H.

6. The system of claim 3, wherein genes of the rRNA operon are present on one or more exogenous expression vectors in the cell.

7. The system of claim 3, wherein the engineered ribosome pool comprises rRNA encoded by genes from different rRNA operons.

8. The system of claim 7, wherein the rRNA genes from different rRNA operons comprise: i) a 16S rRNA gene from a first rRNA operon; and ii) a 23 S rRNA gene from a second rRNA operon.

9. The system of claim 7, wherein 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.

10. The system of claim 7, wherein 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.

11. The system of any one of claims 1-10, wherein the cell comprises a deletion of at least one genomic rRNA operon.

12. The system of claim 10, wherein the cell is modified to delete all but one endogenous rRNA operon.

13. The system of claim 1, wherein one or more rRNA genes from operon D and / or operon C have been removed.

14. The system of claim 13, comprising nucleic acid deletions in helices H91 and / or H92 of operon D and / or operon C.

15. The system of any one of claims 11-14, wherein one or more rRNA genes from operon H have been removed.

16. The system of any one of claims 1-15 comprising a cell-free lysate.

17. A cell-free protein synthesis platform, comprising the cell-free lysate of claim 16.

18. An engineered E. coli cell, wherein one or more rRNA genes from operon D, operon C, or operon H have been removed.

19. The cell of claim 18, wherein operon D and / or operon C comprise amino acid deletions in helices H91 and / or H92.

20. The cell of claim 18 or 19, wherein one or more rRNA genes from operon H have been removed.

21. A method for expressing a protein of interest, the method comprising:(a) with the system of claim 1 comprising the engineered cell, transforming the cell with a nucleic acid construct encoding the protein of interest; or(b) with the system of claim 1 comprising the lysate of the engineered cell, contacting the lysate with a transcription template or a translation template.

22. The method of claim 21, wherein a level of the protein expressed is greater than a level of the protein expressed in a non-engineered control cell, or a lysate from a non-engineered control cell.

23. A method for expressing a protein of interest, comprising contacting the cell-free protein synthesis platform of claim 17 with a translation template.

24. The method of claim 23, wherein a level of the protein expressed is greater than a level of the protein expressed in a cell-free protein synthesis system comprising lysate from a nonengineered control cell.

25. An expression construct comprising a promoter operably coupled to a polynucleotide encoding for an rRNA operon, wherein the rRNA operon comprises: 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.

26. The construct of claim 25, wherein 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.

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