Overexpression of lysozyme in t7 RNA polymerase-expressing host cells

Abstract

Disclosed herein are host cells engineered for enhanced cell health and improved productivity in T7 RNAP overexpression. Host cells, polynucleotides, polypeptides, and related methods for co-expressing lysozyme with T7 RNA polymerase are provided. Also disclosed are methods for preparing a bacterial cell extract with T7 RNA polymerase for use in cell-free protein synthesis.

Description

PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO OVEREXPRESSION OF LYSOZYME IN T7 RNA POLYMERASE- EXPRESSING HOST CELLS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 546,075, filed on October 27, 2023, and U.S. Provisional Application No.63 / 565,189, filed on March 14, 2024, and the disclosure of each of these applications is hereby incorporated by reference in its entirety herein for all purposes. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The Sequence Listing was created on October 15, 2024, is named 091200-1456899- 007210PC_ST26.xml, and is 18,060 bytes in size. BACKGROUND

[0003] T7 RNA polymerase (T7 RNAP) is the polymerase used by the T7 phage to transcribe its own genes for phage propagation. It is a highly active polymerase that has been adapted by scientific researchers for high level transcription and expression of proteins in prokaryotic systems (Elroy-Stein, O, and B Moss. “Cytoplasmic expression system based on constitutive synthesis of bacteriophage T7 RNA polymerase in mammalian cells.” Proceedings of the National Academy of Sciences of the United States of America vol.87,17 (1990): 6743-7. doi:10.1073 / pnas.87.17.6743; Conrad, B et al. “A T7 promoter-specific, inducible protein expression system for Bacillus subtilis.” Molecular & general genetics: MGG vol.250,2 (1996): 230-6. doi:10.1007 / BF02174183). T7 RNAP, either purified or present in complex mixtures, such as in a bacterial cell lysate, can also be used to produce RNA through in vitro transcription or to produce proteins through coupled transcription and translation. Because of these potential uses, there is growing interest in producing T7 RNAP in larger quantities with a robust process.

[0004] It can be challenging to produce T7 RNAP at scale because when it is expressed in large amounts, T7 RNAP can cause severe growth defects in the host cell. Because T7 RNAP drives the overexpression of target proteins in large amounts, an abundance of T7 RNAP in the cell diverts KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO resources away from cellular growth, thereby causing toxicity in the cell (Davanloo, P et al. “Cloning and expression of the gene for bacteriophage T7 RNA polymerase.” Proceedings of the National Academy of Sciences of the United States of America vol.81,7 (1984): 2035-9. doi:10.1073 / pnas.81.7.2035; Li, Zhaopeng, and Ursula Rinas. “Recombinant protein production- associated metabolic burden reflects anabolic constraints and reveals similarities to a carbon overfeeding response.” Biotechnology and bioengineering vol.118,1 (2021): 94-105. doi:10.1002 / bit.27553).

[0005] Most processes used for reagent T7 RNAP production rely on an inducible system, where expression of the T7 RNAP is induced by a chemical signal, such as lactose, or a physical signal, such as heat (Tabor, S, and C C Richardson. “A bacteriophage T7 RNA polymerase / promoter system for controlled exclusive expression of specific genes.” Proceedings of the National Academy of Sciences of the United States of America vol. 82,4 (1985): 1074-8. doi:10.1073 / pnas.82.4.1074). However, even in tightly controlled inducible systems, background levels of “leaky” protein expression that negatively impacts the host cell can still occur (Pothoulakis, G and Ellis, T. “Chapter Nine - Using Spinach Aptamer to Correlate mRNA and Protein Levels in Escherichia coli.” Methods in Enzymology, Academic Press vol. 550 (2015): 173-185. https: / / doi.org / 10.1016 / bs.mie.2014.10.047). Background T7 RNAP expression causes cellular stress and low productivity of T7 RNAP expression strains in typical manufacturing processes. The present disclosure provides host cells that were engineered for enhanced cell health and improved productivity in T7 RNAP overexpression and methods related to the production and use of T7 RNAP. SUMMARY

[0006] In a first aspect, the present disclosure provides a host cell comprising (a) a polynucleotide sequence encoding a lysozyme, wherein the lysozyme may be a wild-type lysozyme or may be a modified version, e.g., one that comprises a lysine to tyrosine substitution at position 128 relative to a sequence of SEQ ID NO: 6; and (b) a polynucleotide sequence encoding a T7 RNA polymerase (T7 RNAP). In some embodiments, the polynucleotide sequence encoding the T7 RNAP is on a plasmid. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO

[0007] In some embodiments, the lysozyme of the host cell comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 6. In some embodiments, the lysozyme of the host cell comprises an amino acid sequence having at least 95% identity to the sequence of SEQ ID NO: 6. In some embodiments, the lysozyme comprises a sequence of SEQ ID NO: 4.

[0008] In some embodiments, the polynucleotide sequence encoding the lysozyme is in the host cell genomic sequence. In some embodiments, the polynucleotide sequence encoding the lysozyme is in gor locus of the host cell genomic sequence. In some embodiments, the polynucleotide sequence encoding the lysozyme is in gntT locus of the host cell genomic sequence

[0009] In some embodiments, the polynucleotide sequence encoding the lysozyme is driven by a constitutive promoter. In some embodiments, the constitutive promoter driving the polynucleotide sequence encoding the lysozyme is an MTL promoter (pMTL).

[0010] In some embodiments, the plasmid is a high copy pUC vector. In some embodiments, the polynucleotide sequence encoding the T7 RNAP is driven by an inducible promoter. In some embodiments, the polynucleotide sequence encoding the T7 RNAP is driven by arabinose- sensitive response regulator, araC.

[0011] In some embodiments, the host cell further comprises a polynucleotide sequence encoding a peptide deformylase (PDF). In some embodiments, the polynucleotide sequence encoding the PDF is in the host cell genomic sequence. In some embodiments, the polynucleotide sequence encoding the PDF is in gntT locus of the host cell genomic sequence. In some embodiments, the polynucleotide sequence encoding the PDF is in the gor locus of the host cell genomic sequence.

[0012] In some embodiments, the polynucleotide sequence encoding the PDF is driven by a constitutive promoter. In some embodiments, the constitutive promoter driving the polynucleotide sequence encoding the PDF is an MTL promoter (pMTL).

[0013] In some embodiments, expression of ompT gene is disrupted in the host cell.

[0014] In some embodiments, the host cell is a bacterial cell. In some embodiments, the bacterial cell is Escherichia coli. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO

[0015] In a related aspect, the present disclosure provides a method of producing T7 RNAP comprising: (a) culturing the host cells the present disclosure under conditions that permit transcription of the lysozyme polynucleotide sequence and expression of the lysozyme; and (b) applying an induction signal to the host cells to induce transcription of the T7 RNAP polynucleotide sequence and expression of the T7 RNAP.

[0016] In a related aspect, the present disclosure provides a method of producing a bacterial cell extract comprising: (a) culturing the host cells of the present disclosure under conditions that permit transcription of the lysozyme polynucleotide sequence and expression of the lysozyme; (b) applying an induction signal to the host cells to induce transcription of the T7 RNAP polynucleotide sequence and expression of the T7 RNAP; and (c) isolating and disrupting the host cells to produce the bacterial cell extract; wherein the bacterial cell extract comprises T7 RNAP.

[0017] In a related aspect, the present disclosure provides a method of producing T7 RNAP comprising: (a) culturing host cells comprising a polynucleotide sequence encoding the T7 RNAP; (b) applying an impeller tip speed; (c) controlling dissolved oxygen (DO) levels by increasing the impeller tip speed, cascading air flow, cascading back pressure, and / or cascading pure oxygen flow; and (d) applying an induction signal to the host cells to induce transcription of the T7 RNAP polynucleotide sequence and expression of the T7 RNAP. In some embodiments, the impeller tip speed does not exceed 8.0 m / s.

[0018] In a related aspect, the present disclosure provides a method of producing a bacterial cell extract comprising: (a) culturing host cells comprising a polynucleotide sequence encoding the T7 RNAP; (b) applying an impeller tip speed; (c) controlling dissolved oxygen (DO) levels by increasing the impeller tip speed, cascading air flow, cascading back pressure, and / or cascading pure oxygen flow; (d) applying an induction signal to the host cells to induce transcription of the T7 RNAP polynucleotide sequence and expression of the T7 RNAP; and (e) isolating and disrupting the host cells to produce the bacterial cell extract. In some embodiments, the impeller tip speed does not exceed a 8.0 m / s. In some embodiments, the bacterial cell extract comprises T7 RNAP.

[0019] In some embodiments, the impeller tip speed does not exceed 8.0 m / s. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO

[0020] In some embodiments, the host cells are any one of the host cells of the present disclosure.

[0021] In some embodiments, the method at step (a) further comprises (a) use of a shear protectant agent; (b) inducing T7 RNAP expression at lower cell density; (c) a reduced medium feed rate; (d) a reduced fed-batch temperature; or (e) any combination thereof.

[0022] In some embodiments, the bacterial cell extract in step c) of the method is able to synthesize a target protein with a titer of at least 80% relative to a control bacterial cell extract after storage at about -20°C, about 2°C to 8°C, or about 20°C to 24°C for at least 6, 12, or 18 months.

[0023] In some embodiments, the host cells in step a) of the method demonstrate reduced growth defects before T7 RNAP expression is induced. In some embodiments, in step b) of the method, the induction signal is heat or a chemical. In some embodiments, the induction signal is arabinose.

[0024] In some embodiments, the host cells of the method achieve a higher final OD595 when compared to host cells that do not express lysozyme with T7 RNAP expression. In some embodiments, the host cells of the method achieve a lower culture viscosity when compared to host cells that do not express lysozyme with T7 RNAP expression.

[0025] In another aspect, the present disclosure provides a composition for cell-free protein synthesis comprising a bacterial cell extract produced according to a method of the present disclosure.

[0026] In another aspect, the present disclosure provides a method of cell-free protein synthesis comprising a composition of the present disclosure.

[0027] In some embodiments, a method of the present disclosure produces a T7 RNAP with high activity. In some embodiments, a composition of the present disclosure comprises a T7 RNAP with high activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 shows an exemplary SDS-PAGE gel analysis of E. coli cell lysates produced from shake flask fermentations. T7 RNA polymerase (T7 RNAP) expression was induced in strains KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO SBDG818, SBDG853, and SBDG867. An uninduced strain was used as a control for T7 RNAP expression. A molecular weight ladder (in kDa) is shown in the first lane on the left. The bands corresponding to the size of T7 RNAP is shown with an arrow.

[0029] FIG. 2 shows an exemplary SDS-PAGE gel analysis of E. coli cell lysates produced from fed-batch fermentations. T7 RNAP and lysozyme Y (lysY), with or without peptide deformylase (PDF), were expressed in strains SBDG818, SBDG853, and SBDG867. A molecular weight ladder (in kDa) is shown in the first lane on the left. The bands corresponding to the size of T7 RNAP, PDF, and lysY are shown with arrows.

[0030] FIG. 3 shows an exemplary SDS-PAGE gel analysis of E. coli cell lysates produced from fed-batch fermentations. T7 RNAP and lysY, with or without PDF, were expressed in strains SBDG818 and SBDG853. A molecular weight ladder (in kDa) is shown in the first lane on the left. (A) indicates a fed-batch fermentation at normal feed rate. (B) indicates a fed-batch fermentation at a 25% reduced feed rate. The bands corresponding to the size of T7 RNAP, PDF, and lysY are shown with arrows.

[0031] FIG. 4 shows an exemplary plot of green fluorescence protein (GFP) fluorescence units against percent T7 RNAP reagent lysate in a cell-free protein synthesis (CFPS) reaction. Error bars represent the standard deviation of endpoint signals for 3 replicates of the same condition.

[0032] FIG. 5 shows an exemplary plot of GFP fluorescence units against percent T7 RNAP reagent lysate in a CFPS reaction. Error bars represent the standard deviation of endpoint signals for 3 replicates of the same condition.

[0033] FIG.6 (top and bottom panels) shows oxygen (O2) levels over time (hours post-induction of protein expression) during T7 RNAP fermentation runs. The strains shown are SBDG178, SBDG818, and SBDG867. Top panel shows dissolved oxygen % in the fermentation culture. Bottom panel shows % enrichment with pure oxygen (O2, gas) supplied during fermentation.

[0034] FIG.7 shows growth profile of SBDG178 during production fermentation at 10 L and 1,500 L-scale.

[0035] FIG.8 shows fermenter impeller tip speed and T7 RNAP activity. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO

[0036] FIG.9 shows growth profile of SBDG178 during production fermentation at 1 L and 30 L scale with variable peak impeller tip speed.

[0037] FIG.10 shows growth and glucose profile of SBDG178 during GMP run 3-5.

[0038] FIG.11 shows cell pellets after harvest centrifugation.

[0039] FIG.12 shows exemplary maximum stir rates (rpm) and exemplary impeller tip speeds (m / s) that are associated with fermentation volumes of 0.25 L (“7”), 0.5 L (“1”), 1 L (“2”), 10 L (“3”), 30 L (“5”), 200 L (“6”), and 1,000 L (“4”).

[0040] FIG.13 shows OD595for strains SBDG867 (“4,” “5,” and “6”) and SBDG178 (“1,” “2,” and “3”) at impeller tip speeds 2.67, 4.71, or 7.07 m / s. OD595measurements are shown for fermentation times leading up to induction of T7 RNAP expression.

[0041] FIG.14 shows the amount of T7 RNAP (g / L) that was present in the fermentation broth for strains SBDG867 (“5,” “6,” “7,” “8,” “9,” and “10”) and SBDG178 (“1,” “2,” “3,” and “4”) at impeller tip speed 2.30, 2.67, 4.71, or 7.07 m / s. T7 RNAP amounts are shown for fermentation times leading up to induction of T7 RNAP expression. DETAILED DESCRIPTION I. Introduction

[0042] This application relates to the overexpression of T7 RNA polymerase (T7 RNAP) in host cells. Disclosed herein are bacterial host cells, polynucleotides, and related methods for producing and evaluating the T7 RNAP, as well as uses for reagent T7 RNAP that is produced from these materials and methods. II. Definitions

[0043] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0044] As used herein, “T7 RNA polymerase” (“T7 RNAP”) refers to an RNA polymerase from the T7 bacteriophage that catalyzes the synthesis of RNA from DNA in the 5’ to 3’ direction. T7 KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO RNAP transcribes only DNA that is downstream of a T7 promoter. T7 RNAP requires a double- stranded DNA template and Mg2+ion as a cofactor for the synthesis of RNA. T7 RNAP can be used to transcribe DNA that has been cloned into host cell chromosomes or vectors that have a T7 promoter.

[0045] As used herein, a “reagent T7 RNAP” refers to a preparation of T7 RNAP according to the present disclosure that can be used as an essential reagent in a chemical reaction. For example, in a cell-free protein synthesis (CFPS) system, a polymerase reagent is needed to transcribe genetic information into RNA. Reagent T7 RNAP may be used in a CFPS reaction as a source of polymerase to perform this first step in protein expression from a gene of interest. A lysate of bacterial cells expressing T7 RNAP competent for use in recombinant protein production in a CFPS system is a “reagent T7 RNAP.” As used herein, “reagent T7 RNAP” includes T7 RNAP that is purified from a lysate, as well as a lysate containing T7 RNAP.

[0046] As used herein, the term “lysozyme” refers to an enzyme that can cleave amide bonds in bacterial cell walls and can bind to T7 RNAP, thereby inhibiting transcription. As used herein, the term “T7 lysozyme” is the lysozyme of T7 bacteriophage. The term “lysozyme” includes T7 lysozyme, lysozyme variants of the present disclosure, and homologs from other species. The term “lysozyme” also includes lysozymes that have altered functions such as a defect in amidase activity, T7 RNAP binding, or both. For example, a “lysozyme” may be lacking in amidase activity but maintain the ability to bind to T7 RNAP. For example, the term “lysozyme” encompasses the lysY lysozyme variant, which lacks amidase activity yet retains T7 RNAP inhibition (Cheng, X et al. “The structure of bacteriophage T7 lysozyme, a zinc amidase and an inhibitor of T7 RNA polymerase.” Proceedings of the National Academy of Sciences of the United States of America vol.91,9 (1994): 4034-8. doi:10.1073 / pnas.91.9.4034). The lysozymes also may be defined as having similar amino acid sequences if they have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type lysozyme protein from any species. The sequence identity of a protein is determined using the BLASTP program with the defaults word length of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992). Exemplary “T7 lysozymes” include lysY and T7 lysozymes that comprise the following mutations in the lysozyme gene: '3.5, sup,_AK6, and AFK136. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO

[0047] The term “promoter” refers to a nucleic acid region or sequence that is capable of directing or driving expression of a coding sequence in a host cell. Promoters are involved in recognition and binding of RNA polymerase and other proteins that initiate transcription. A promoter can be located upstream and / or downstream from the start of transcription that is involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. The promoter can be a eukaryotic or a prokaryotic promoter. A promoter may be an inducible promoter or a constitutive promoter.

[0048] A “constitutive promoter” is a promoter that is active under most environmental and developmental conditions. Examples of constitutive promoters include, but are not limited to, a MTL promoter (pMTL), a CMV promoter, a U6 promoter, a PGK promoter, an EF-1Į promoter, and a SV40 promoter.

[0049] An “inducible promoter” is a promoter that is active under environmental or developmental regulation, for example, regulated by the presence or absence of an induction signal. Examples of inducible promoters include, but are not limited to, the pL promoter (induced by an increase in temperature), the BAD promoter (AraBAD promoter; pBAD; induced by the addition of arabinose to the growth medium), the tetracycline-controlled transcriptional activation system (Tet-On / Tet-Off, Bujard and Gossen, PNAS, 89(12):5547-5551 (1992)), the Lac switch inducible system (Wyborski et al., Environ Mol Mutagen, 28(4):447-58 (1996)), the ecdysone- inducible gene expression system (No et al., PNAS, 93(8):3346-3351 (1996)), the cumate gene- switch system (Mullick et al., BMC Biotechnology, 6:43 (2006)), and the tamoxifen-inducible gene expression (Zhang et al., Nucleic Acids Research, 24:543-548 (1996)). Furthermore, a Cre- loxP inducible system can also be used, as well as a Flp recombinase inducible promoter system, both of which are known in the art.

[0050] A nucleic acid is “operably linked” to another nucleic acid when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. Linking is accomplished by KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0051] The terms “nucleic acid” and “polynucleotide” are used interchangeably and as used herein refer to both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. The terms also include, but is not limited to, single- stranded and double-stranded forms of DNA. A reference to a nucleic acid sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. The term also includes codon-optimized nucleic acids that encode the same polypeptide sequence.

[0052] A “nucleic acid sequence” or a “polynucleotide sequence” includes a DNA or RNA sequence which can function as a template from which a polypeptide will be translated. It will be understood by those of skill in the art that a DNA polynucleotide sequence must first be transcribed into RNA, and that the RNA is translated into a polypeptide. DNA can be transcribed into RNA either in vivo or in vitro. The methods of in vitro transcription of a DNA template are well known in the art. In some embodiments, the DNA template is subject to simultaneous in vitro transcription and translation, such as in a cell-free protein synthesis reaction.

[0053] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. In some cases, the vector is a plasmid or is derived from a plasmid. A “vector” as used here refers to a recombinant construct in which a nucleic acid sequence of interest is inserted into the vector. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0054] An “expression cassette” refers to a polynucleotide sequence that directs host cell machinery to produce gene products. An expression cassette may contain one or more genes of interest and 5’ and 3’ regulatory sequences. The cassette may additionally contain at least one additional gene or genetic element to be co-transformed into the organism (i.e., a cell, plurality of KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO cells, tissue, or animal). Where additional genes or elements are included, the components are operably linked. Alternatively, the additional gene(s) or element(s) can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and / or recombination sites for insertion of the polynucleotides to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain a selectable marker gene. The expression cassette will include in the 5’ to 3’ direction of transcription: a transcriptional and translational initiation region (i.e., a promoter), a polynucleotide of the disclosure, and a transcriptional and translational termination region (i.e., termination region) functional in the cell or organism of interest. The promoters are capable of directing or driving expression of a coding sequence in a host cell. The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) may be endogenous or heterologous to the host cell or to each other.

[0055] The terms “polypeptide,” “peptide” or “protein” are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds. The terms also encompass polymers comprising L-amino acids, polymers comprising D-amino acids, or polymers comprising both L- and D-amino acids.

[0056] A polypeptide “variant,” as the term is used herein, is a polypeptide that typically differs from one or more polypeptide sequences specifically disclosed herein in one or more substitutions, deletions, additions and / or insertions.

[0057] A “substitution,” as used herein, denotes the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively. A “conservative substitution” refers to a substitution of an amino acid such that charge, polarity, hydropathy (hydrophobic, neutral, or hydrophilic), and / or size of the side group chain is maintained. Illustrative sets of amino acids that may be substituted for one another include (i) positively-charged amino acids Lys and Arg; and His at pH of about 6; (ii) negatively charged amino acids Glu and Asp; (iii) aromatic amino acids Phe, Tyr and Trp; (iv) nitrogen ring amino acids His and Trp; (v) aliphatic hydrophobic amino acids Ala, Val, Leu and Ile; (vi) hydrophobic sulfur-containing amino acids KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO Met and Cys, which are not as hydrophobic as Val, Leu, and Ile; (vii) small polar uncharged amino acids Ser, Thr, Asp, and Asn (viii) small hydrophobic or neutral amino acids Gly, Ala, and Pro; (ix) amide-comprising amino acids Asn and Gln; and (xi) beta-branched amino acids Thr, Val, and Ile. Reference to the charge of an amino acid in this paragraph refers to the charge at pH 6-7.

[0058] The term “recombinant” or “recombinantly”, refers to a biomolecule, e.g., a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the gene is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature. The term “recombinant” can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems, as well as proteins and / or mRNAs encoded by such nucleic acids.

[0059] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Thus, a host cell is a recombinant host cell and includes the primary transformed cell and progeny derived therefrom without regard to the number of passages.

[0060] The term “fermentation” refers to any process or any culture in which T7 RNAP can be produced by a host cell of the present disclosure. The term “fermentation” may be used interchangeably with the terms “production” and “culture.” The term “fermentation” does not strictly mean an anaerobic chemical process performed by cells to convert a substrate to a product. As used herein, “fermentation” includes all culture processes and parameters that may be used for the production of T7 RNAP, including cultures where oxygen is present.

[0061] The term “bacterial cell extract” refers to a bacterial cell lysate or a fraction thereof (e.g., clarified and / or filtered) that can be used to synthesize protein from a nucleic acid template. In other words, the bacterial cell extract contains an energy source, such as ATP, GTP and the like. The majority of the biological components in a bacterial cell extract are present in concentrations resulting from the lysis of the cells rather than having been reconstituted. A bacterial cell extract can be a portion of a lysate from which other cellular components of the lysate have been separated by centrifugation, filtration, selective precipitation, selective immunoprecipitation, KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO chromatography, or other methods. It also includes lysates or fractions thereof that contain exogenous material such as preservatives, stabilizers and reagents that enhance CFPS. A bacterial cell extract may be further altered such that the extract is supplemented with additional cellular components, e.g., amino acids, nucleic acids, enzymes, etc. The bacterial cell extract may also be altered such that additional cellular components are removed or degraded following lysis. The term “bacterial cell extract” can refer to a preparation of an in vitro reaction mixture able to transcribe DNA into mRNA and / or translate mRNA into polypeptides. The mixture may include ribosomes, an energy source (such as ATP, GTP, glucose, glutamate, or pyruvate), amino acids, and tRNAs. The mixture may be derived directly from lysed bacteria, from purified components or combinations of both.

[0062] “Cell-free protein synthesis” or “CFPS” refers to the in vitro synthesis of nucleic acids, polypeptides, small molecules and / or viral particles in a reaction mix comprising biological extracts and / or defined reagents. The CFPS reaction mixture will comprise a template for production of the macromolecule, e.g., DNA, mRNA, etc.; monomers for the macromolecule to be synthesized, e.g., amino acids, nucleotides, etc.; and co-factors, enzymes and other reagents that are necessary for the synthesis, e.g., ribosomes, uncharged tRNAs, tRNAs charged with natural and / or unnatural amino acids, polymerases, transcriptional factors, tRNA synthetases, etc.

[0063] As used herein, an “increase” or a “decrease” refers to a detectable positive or negative change in quantity from a comparison control, e.g., an established standard control (such as an extract that does not contain an overexpressed protein). An increase is a positive change that is typically at least 10%, or at least 20%, or 50%, or 100%, and can be as high as at least 2-fold or at least 5-fold or even 10-fold of the control value. For example, the term “increased protein expression,” when used in relation to a bacterial culture described herein, may refer to a bacterial culture having greater or more protein expression relative to a control bacterial culture that does not contain detectable protein expression. Similarly, a decrease is a negative change that is typically at least 10%, or at least 20%, 30%, or 50%, or even as high as at least 80% or 90% of the control value. For example, the term “decreased protein expression,” when used in relation to a bacterial culture described herein, refers to bacterial culture having less protein expression. Other terms indicating quantitative changes or differences from a comparative basis, such as “more,” “less,” “higher,” and “lower,” as well as terms indicating an action to cause such changes or KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO differences, such as “increase,” “promote,” “enhance,” “decrease,” “inhibit,” and “suppress,” are used herein in the same fashion as described above.

[0064] The term “comprise,” including variations such as “comprises” or “comprising,” is used to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.

[0065] The term “consists of” or “consisting of” is used to mean that a method, process or composition of matter (e.g., amino acid sequence) has the recited steps and / or components and no additional steps or components.

[0066] The term “consists essentially of” or “consisting essentially of” in the context of a protein sequence as used herein is to be construed non-exhaustively and is understood to mean a protein sequence wherein additional amino acids may be present wherein said additional amino acids constitute no more than about 10% of the total protein sequence or, in the alternative, no more than additional 1, 2, 3, 4, or 5 amino acids, for example, at either N-terminus or C-terminus of the protein sequence. When a protein is described as “consisting essentially of” an amino acid sequence, the protein should not include any additional components that would materially change the fundamental function or utility of the protein: for example, a protein “consisting essentially of” an amino acid sequence should not include a moiety that confers another function such as a detection signal or a therapeutic activity. On the other hand, a protein “consisting essentially of” an amino acid sequence does not exclude chemical modifications that improve stability of the conjugate.

[0067] The terms “relative to,” “corresponding to,” “determined with reference to,” or “numbered with reference to” when used in the context of the identification of a given amino acid residue in a polypeptide sequence, refers to the position of the residue of a specified reference sequence when the given amino acid sequence is maximally aligned and compared to the reference sequence. The polypeptide that is aligned to the reference sequence need not be the same length as the reference sequence.

[0068] As used herein, the term “about,” when modifying any amount, refers to the variation in that amount typically encountered by one of skill in the art, e.g., in protein synthesis experiments. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO For example, the term “about” refers to the normal variation encountered in measurements for a given analytical technique, both within and between batches or samples. Thus, the term about can include variation of + / - 1-10% of the measured value, such as + / - 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% variation of the measured value. The amounts disclosed herein include equivalents to those amounts, including amounts modified or not modified by the term “about.”

[0069] As used herein the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the protein” includes reference to one or more proteins and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs unless clearly indicated otherwise. III. Host Cells for Protein Expression

[0070] The host cells of the present disclosure are capable of expressing T7 RNAP in large quantities in liquid culture. In some embodiments, the toxic effects related to having an abundance of T7 RNAP in a host cell are reduced. In some embodiments, the host cell is capable of expressing large quantities of T7 RNAP and appears healthy or lacking in indicators of T7 RNAP toxicity, such as changes in shape, structure, form, size, texture, color, motility, cell proliferation rate, T7 RNAP production rate, and / or liquid culture viscosity. In some embodiments, the host cell is able to grow and divide normally on seed media composed of diluted fermenter media without undergoing morphological changes. In some embodiments, the host cell is able to grow and divide normally on seed media composed of diluted fermenter media without any media supplementation.

[0071] In some embodiments, the host cells are capable of consistent growth during seed stage, batch stage, or both seed stage and batch stage in production culture. In some embodiments, the host cells have lower oxygen requirements during T7 RNAP production, thereby requiring less oxygen supplementation. In some embodiments, a culture of the host cells does not become anoxic during T7 RNAP production.

[0072] In some embodiments, the host cell is less prone to cell lysis in culture. In some embodiments, due to decreased cell lysis, the culture of the host cells achieves a higher OD595 at KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO the end of a production or fermentation run (i.e., final OD595or terminal OD595). In some embodiments, the culture of the host cells is less viscous.

[0073] To start a liquid culture for T7 RNAP production, the host cell is cultured in a liquid medium, e.g., seed medium, that is suitable for producing a large number of healthy cells for subsequent protein production. The seed medium may be inoculated, for example, with a single colony picked from an agar plate, or an inoculum from a cell bank. Generally, the seed medium is a nutrient-rich liquid medium or broth. Once the culture in seed stage has a sufficient number of cells (as can be determined at an optical density at 595 nm; OD595), the seed culture is used to inoculate a large and fixed volume of liquid media for batch protein overexpression. During batch stage, the host cell is cultured for a certain amount of time with the nutrients that were provided at the beginning of batch stage. When T7 RNAP is being expressed by the host cells, T7 RNAP and by-products accumulate in the culture while the nutrients in the media are depleted by the cells. Eventually, cell growth slows down and the culture enters a stationary growth phase. Typically, the culture is harvested when it arrives at stationary growth phase.

[0074] In some embodiments, the host cell demonstrates a specific growth rate, ^, that is consistent during seed stage and / or batch stage when cultured in liquid media. In some embodiments, the specific growth rate stays about the same after induction of T7 RNAP overexpression. In some embodiments, the host cell can grow in seed media composed of diluted fermenter media. In some embodiments, when the host cell is cultured in diluted fermenter media, the host cell can maintain a specific growth rate that is similar to the growth of a host cell that is cultured in undiluted fermenter media. In some embodiments, when the host cell is cultured in diluted fermenter media that is not supplemented with additional nutrients, the host cell can maintain a specific growth rate that is similar to the growth of a host cell that is cultured in undiluted and un-supplemented fermenter media. In some embodiments, the host cell is able to grow and divide normally on seed media composed of diluted fermenter media without undergoing morphological changes. In some embodiments, the host cell is able to grow and divide normally on seed media composed of diluted fermenter media without any media supplementation and without the use of specialized equipment. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO

[0075] In some embodiments, the host cells are cultured in conditions for T7 RNAP production with lysozyme co-expression. In some embodiments, the host cells of the present disclosure, when compared to host cells that do not co-express lysozyme, are capable of much more consistent growth during seed stage, batch stage, or both seed stage and batch stage in production culture. In some embodiments, the toxicity associated with T7 RNAP is mitigated by the co-expression of lysozyme in the host cells. In some embodiments, the lysozyme binds to T7 RNAP, thereby reducing toxicity that is associated with overexpression of T7 RNAP.

[0076] In some embodiments, the culture of the host cells that express lysozyme is less viscous than the culture of host cells that do not express lysozyme. In some embodiments, increased viscosity is due to increased cell lysis. In some embodiments, the host cell that expresses lysozyme is less prone to cell lysis when compared to a host cell that does not express lysozyme. In some embodiments, due to decreased cell lysis, the culture of the host cells that express lysozyme achieves a higher OD595at the end of a production or fermentation run (i.e., final OD595or terminal OD595) than the culture of host cells that do not express lysozyme.

[0077] In some embodiments, the host cells are cultured in conditions for T7 RNAP production with PDF co-expression. In some embodiments, the PDF rescues the host cells from acetate toxicity. In some embodiments, co-expression of PDF with T7 RNAP production causes the host cell to produce less acetate than a host cell that does not co-express PDF. In some embodiments, PDF co-expression enables the host cells to grow to higher OD595 as compared to the OD595 of host cells that do not co-express PDF. In some embodiments, the host cells have lower oxygen requirements during T7 RNAP production, thereby requiring less oxygen supplementation. In contrast, host cells that do not co-express PDF have little to no measurable oxygen in the culture because the host cells deplete the oxygen in the culture despite oxygen supplementation in large quantities. A. Bacterial cells

[0078] The present disclosure provides bacterial host cells for the expression of T7 RNA polymerase (T7 RNAP). Suitable bacteria include gram-negative bacteria and gram-positive bacteria, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, and Pseudomonas such as P. aeruginosa, and Steptomyces. In some embodiments, the host cell is from an Escherichia species, such as Escherichia coli or a derivative thereof. In some embodiments, the host cell is any E. coli strain that is known to one of skill in the art. In some embodiments, the E. coli strain is an A (K-12), B, C or D strain.

[0079] The host cell may be used to produce a bacterial cell extract for use in CFPS reactions. The host cell may have reduced nuclease and / or phosphatase activity which increases cell-free synthesis efficiency. For example, the host cell can have mutations in the genes encoding the nucleases RNase E and RNase A. The strain may also have mutations to stabilize components of the CFPS reaction such as deletions in genes such as tnaA, speA, sdaA or gshA, which prevent degradation of the amino acids tryptophan, arginine, serine and cysteine, respectively, in a CFPS reaction. Additionally, the host cell may have mutations to stabilize the protein products of cell- free synthesis such as knockouts in the proteases ompT or lonP.

[0080] In various embodiments, the host cell has one or more polynucleotides that encode for T7 RNAP. In some embodiments, the host cell also has one or more polynucleotides that encode for one or more proteins that improve the overall health of the host cell. In some embodiments, the host cell also has one or more polynucleotides that encode for one or more proteins that ameliorate toxic effects associated with overexpression of T7 RNAP. In some embodiments, the host cell also has one or more polynucleotides that encode for one or more proteins that improve expression of T7 RNAP by the host cell. In various embodiments, the polynucleotides may be in the host cell genomic sequence, on a plasmid, or both. Polynucleotides of host cells are discussed below in detail.

[0081] In various embodiments, the host cell has is capable of expressing T7 RNAP. In some embodiments, the host cell is capable of expressing one or more proteins that improve the overall health of the host cell. In some embodiments, the host cell is capable of expressing one or more proteins that ameliorate toxic effects associated with overexpression of T7 RNAP. In some embodiments, the host cell is capable of expressing one or more proteins that improve overexpression of T7 RNAP by the host cell. In some embodiments, the one or more proteins is lysozyme, and / or peptide deformylase (PDF). Proteins of host cells are discussed below in detail. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO

[0082] Standard methods for engineering host cells and for expressing proteins from host cells are known to one of ordinary skill in the art. Standard methods in molecular biology are described in Maniatis et al. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Wu (1993) Recombinant DNA, Vol.217, Academic Press, San Diego, CA.); Ausubel, F. M., et al., Current Protocols in Molecular Biology (Supplement 99), John Wiley & Sons, New York (2012); Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology (Volume 152 Academic Press, Inc., San Diego, Calif.1987); and PCR Protocols: A Guide to Methods and Applications (Academic Press, San Diego, Calif.1990). Standard methods also appear in Bindereif, SchĘn, & Westhof (2005) Handbook of RNA Biochemistry, Wiley-VCH, Weinheim, Germany, which describes detailed methods for RNA manipulation and analysis, and Walker, J.M., (2009), and The Protein Protocols Handbook, 3rd ed., Humana Press, New York, N.Y., which describes detailed methods for protein manipulation and analysis. These disclosures are incorporated by reference herein. B. Polynucleotides

[0083] In some embodiments, a polynucleotide of the present disclosure encodes for a T7 RNAP. In some embodiments, the polynucleotide comprises an additional sequence on the 5’-end or the 3’-end of the sequence encoding the T7 RNAP that encodes for a His tag. In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more identity to the sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 1 without the portion of the sequence that encodes for a His tag. In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 1.

[0084] In some embodiments, the polynucleotide encoding a T7 RNAP is present within the host cell genomic sequence. In some embodiments, the polynucleotide in the genomic sequence is driven by an inducible promoter. In some embodiments, the polynucleotide in the genomic sequence is driven by constitutive promoter. In some embodiments, the polynucleotide encoding a T7 RNAP is on a plasmid. In some embodiments, the plasmid is pUC plasmid, pBAD plasmid, KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO pMTL plasmid, pT7 plasmid, a p15a plasmid, a pUC-based T7 plasmid, or a plasmid with any of the BAD, MTL, and pT7 promoters, or any combination thereof. In some embodiments, the plasmid is a plasmid with an inducible promoter. In some embodiments, the plasmid is a plasmid with a constitutive promoter.

[0085] In some embodiments, the polynucleotide encoding a T7 RNAP is on a high copy vector. In some embodiments, a host cell comprising a high copy vector produces higher amounts of T7 RNAP than a host cell comprising a low copy vector. In some embodiments, the high copy vector is a pUC vector. In some embodiments, the high copy vector is a pUC-based T7 plasmid.

[0086] In some embodiments, the polynucleotide encoding a T7 RNAP is on a low copy vector. In some embodiments, a host cell comprising a low copy vector produces lower amounts of T7 RNAP than a host cell comprising a high copy vector. In some embodiments, the host cell comprising a low copy vector demonstrates less stability in culture before an induction signal for T7 RNAP expression is applied. In some embodiments, the host cell comprising a low copy vector has elongated cell morphology when cultured in shake flask media. In some embodiments, the low copy vector is a p15a vector.

[0087] In some embodiments, the polynucleotide encoding a T7 RNAP is on a high copy vector. In some embodiments, a host cell comprising a high copy vector produces higher amounts of T7 RNAP than a host cell comprising a low copy vector or a host cell that expresses T7 RNAP from the chromosome. In some embodiments, the low copy vector is a pUC vector.

[0088] In some embodiments, the polynucleotide of the present disclosure encodes for any protein suitable for improving the health of the host cell, ameliorating toxic effects associated with T7 RNAP overexpression, and / or improving overexpression of T7 RNAP by the host cell.

[0089] In some embodiments, the polynucleotide encodes for a lysozyme. In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more identity to a wild-type lysozyme nucleic acid sequence from any species. In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more identity to the sequence of SEQ ID NO: 5. In some embodiments, the polynucleotide comprises a nucleic acid sequence of a wild-type KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO lysozyme from any species and a tyrosine at position 128 when the sequence of SEQ ID NO: 5 is used as a reference sequence. In some embodiments, the polynucleotide comprises a nucleic acid sequence of SEQ ID NO: 5 and a tyrosine at position 128 of SEQ ID NO: 5. In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the polynucleotide encodes for lysozyme S (lysS). In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the polynucleotide encodes for lysozyme Y (lysY).

[0090] In some embodiments, the polynucleotide encoding a lysozyme is present within the host cell genomic sequence. In some embodiments, the polynucleotide encoding a lysozyme is in the gntT locus of the host cell genomic sequence. In some embodiments, the polynucleotide encoding a lysozyme is in the gor locus of the host cell genomic sequence. In some embodiments, the polynucleotide in the genomic sequence is driven by an inducible promoter. In some embodiments, the polynucleotide in the genomic sequence is driven by constitutive promoter. In some embodiments, the polynucleotide encoding a lysozyme is on a plasmid. In some embodiments, the plasmid is pUC plasmid, pBAD plasmid, pMTL plasmid, pT7 plasmid, a p15a plasmid, a pUC-based T7 plasmid, or a plasmid with any of the BAD, MTL, and pT7 promoters, or any combination thereof. In some embodiments, the plasmid is a plasmid with an inducible promoter. In some embodiments, the plasmid is a plasmid with a constitutive promoter. In some embodiments, the lysozyme coding sequence is in the gor locus of the host cell genomic sequence and is driven by the constitutive MTL promoter (pMTL).

[0091] In some embodiments, the polynucleotide encodes for peptide deformylase (PDF). In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more identity to a wild-type PDF nucleic acid sequence from any species. In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more identity to the sequence of SEQ ID NO: 7. In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 7.

[0092] In some embodiments, the polynucleotide encoding a PDF is present within the host cell genomic sequence. In some embodiments, the polynucleotide encoding a PDF is in the gntT locus KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO of the host cell genomic sequence. In some embodiments, the polynucleotide encoding a PDF is in the gor locus of the host cell genomic sequence. In some embodiments, the polynucleotide in the genomic sequence is driven by an inducible promoter. In some embodiments, the polynucleotide in the genomic sequence is driven by constitutive promoter. In some embodiments, the polynucleotide encoding a PDF is on a plasmid. In some embodiments, the plasmid is pUC plasmid, pBAD plasmid, pMTL plasmid, pT7 plasmid, a p15a plasmid, a pUC-based T7 plasmid, or a plasmid with any of the BAD, MTL, and pT7 promoters, or any combination thereof. In some embodiments, the plasmid is a plasmid with an inducible promoter. In some embodiments, the plasmid is a plasmid with a constitutive promoter. In some embodiments, the PDF coding sequence is in the gntT locus of the host cell genomic sequence and is driven by the constitutive MTL promoter (pMTL).

[0093] In some embodiments, the host cell may have polynucleotides encoding a lysozyme and a PDF. In some embodiments, the polynucleotide encoding the lysozyme is present in the host cell genomic sequence and the polynucleotide encoding the PDF is on a plasmid. In some embodiments, the polynucleotide encoding the lysozyme is on a plasmid and the polynucleotide encoding the PDF is present in the host cell genomic sequence. In some embodiments, the polynucleotides encoding both the lysozyme and the PDF are present in the host cell genomic sequence. In some embodiments, the polynucleotides encoding both the lysozyme and the PDF on one or more plasmids. In some embodiments, the plasmid is pUC plasmid, pBAD plasmid, pMTL plasmid, pT7 plasmid, a p15a plasmid, a pUC-based T7 plasmid, or a plasmid with any of the BAD, MTL, and pT7 promoters, or any combination thereof. In some embodiments, the lysozyme coding sequence is in the gor locus of the host cell genomic sequence, the PDF coding sequence is in the gntT locus of the host cell genomic sequence, and each of the lysozyme coding sequence and the PDF coding sequence is driven by the constitutive MTL promoter (pMTL). i. Promoters

[0094] A variety of promoters may be used to drive the expression of a polynucleotide of the present disclosure. Promoters that can be used may be any appropriate promoter sequence suitable for a prokaryotic host cell, which shows transcriptional activity, including mutant, truncated, and hybrid promoters, and may be obtained from polynucleotides encoding extracellular or KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO intracellular polypeptides either endogenous (native) or heterologous (foreign) to the host cell. The promoter may be a constitutive promoter or an inducible promoter.

[0095] In embodiments where the promoter is an inducible promoter, expression of the encoded product occurs when an induction signal is applied to the host cells. In some embodiments, the protein of the polynucleotide is expressed at detectable levels after an induction signal is applied. In some embodiments, the protein of the polynucleotide is not detectable before application of an induction signal.

[0096] Suitable prokaryotic promoters for use in accordance with the present disclosure include, but are not limited to, the promoters of Pc0, PL59, MTL, AraBAD (BAD), lac, T3, T7, lambda PrƍP1ƍ, trp, the spc ribosomal protein operon promotor Pspc, the ȕ-lactamase gene promotor Pbla of plasmid pBR322, the PL promoter of phage ^, the replication control promoters PRNAI and PRNAII of plasmid pBR322, the P1 and P2 promoters of the rrnB ribosomal RNA operon, the tet promoter, and the pACYC promoter. Tetracycline-regulated transcriptional modulators and CMV promoters are described in WO 96 / 01313, U.S. Pat. Nos.5,168,062 and 5,385,839. A heat shock promoter, e.g., the E. coli sigma 32 subunit of RNA polymerase, may also be used.

[0097] In some embodiments, the promoter is an AraBAD promoter (pBAD) and the induction signal is arabinose. In these embodiments, pBAD is driven by arabinose-sensitive response regulator, araC, which is sensitive to the presence of arabinose and plays a dual role as an activator of gene expression in the presence of arabinose and a repressor of gene expression in the absence of arabinose.

[0098] In some embodiments, the promoter is a tetracycline promoter and the induction signal is tetracycline.

[0099] In some embodiments, the promoter is a heat shock promoter and the induction signal is heat. Examples of heat shock promoters include lac promoter, trc promoter, and rhamnose, and their respective induction signals are lactose or allolactose, isopropyl E-D-1-thiogalactopyranoside (IPTG), and rhamnose.

[0100] In some embodiments, the promoters may have different strength in terms of the amount of expression it can produce. Promoters can be a medium-strength promoter, a weak promoter, or KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO a strong promoter. The strength of a promoter can be measured as the amount of transcription of a gene product initiated at that promoter, relative to a suitable control. For constitutive promoters directing expression of a gene product in an expression construct, a suitable control could use the same expression construct, except that the ‘wild-type’ version of the promoter, or a promoter from a ‘housekeeping’ gene, is used in place of the promoter to be tested. In some embodiments, different proteins expressed in E. coli are under the control of promoters of different strengths to ensure the proteins, e.g., T7 RNAP, lysozyme, and / or PDF, are expressed at appropriate levels. In some embodiments, a strong promoter T7 is used to drive the expression of a protein to ensure maximal yield. In some embodiments, the E coli host cell expresses a T7 RNAP under the control of the AraBAD promoter (pBAD), which allows tight regulation and control of the protein of interest, e.g., through the addition or absence of arabinose. See, for example, Guzman et al., J. Bacteriol. July 1995177 (14): 4121-4130.

[0101] In some embodiments, the promoter driving the expression of the polynucleotide sequence encoding a T7 RNAP is a constitutive promoter. In some embodiments, the promoter driving the expression of the polynucleotide sequence encoding a T7 RNAP is an inducible promoter. In some embodiments, the inducible promoter is driven by the arabinose-sensitive response regulator (araC). In some embodiments, the inducible promoter responds to arabinose as the induction signal.

[0102] In some embodiments, the promoter driving the polynucleotide sequence encoding a lysozyme is a constitutive promoter. In some embodiments, the constitutive promoter is an MTL promoter (pMTL). In some embodiments, the promoter driving the polynucleotide sequence encoding a lysozyme is an inducible promoter.

[0103] In some embodiments, the strength of the promoter that drives the polynucleotide sequence encoding lysozyme has an impact on the amount of T7 RNAP that is produced. In different embodiments, the promoter is a strong promoter, a medium-strength promoter, or a weak promoter. In some embodiments, a promoter of any strength results in a host cell that produces T7 RNAP. In some embodiments, the promoter is a strong promoter. In some embodiments, a strong promoter results in a host cell that produces more T7 RNAP than a weak or medium-strength promoter. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO

[0104] In some embodiments, the promoter driving the polynucleotide sequence encoding a PDF is a constitutive promoter. In some embodiments, the constitutive promoter is an MTL promoter (pMTL). In some embodiments, the promoter driving the polynucleotide sequence encoding a PDF is an inducible promoter. ii. Methods of Introducing Mutations to a Host Cell Genome

[0105] In some embodiments, gene modifications to a host cell can be performed with homologous recombination. Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two different molecules of nucleic acids that have similar or identical sequences (i.e., the sequences are homologous). In some embodiments, the homologous recombination method is lambda Red homologous recombination or lambda Red mediated recombineering. See, e.g., Murphy, EcoSal Plus 2016, Vol. 7, 10.1128 / ecosalplus.ESP- 0011-2015(1). The bacteriophage lambda Red homologous recombination system can be useful because (1) it can recombine linear DNA containing regions of homology of greater than 50 base pairs, (2) it can be easily generated by co-infection of genetically-marked phages, and (3) the lambda Red proteins can manipulate bacterial chromosomes and artificial chromosomes with efficiencies that could not be achieved with restriction enzymes. Id.

[0106] In some embodiments, gene modifications to a host cell can be performed with site- specific recombination. Site-specific recombination uses enzymes possessing both endonuclease activity and ligase activity and the enzymes recognize a certain part of DNA sequences and replace it with any other corresponding DNA sequences. See, e.g., Yang W. and Mizuuchi K., Structure, 1997, Vol.5, 1401-1406(9). Site-specific recombination systems are well known in the art, e.g., Int / att system from bacterio ^ phage, Cre / LoxP system from PI bacteriophage and FLP- FRT system from yeast are well developed site-specific recombination systems.

[0107] Non-limiting examples of methods of introducing site specific recombination to various proteins disclosed herein include the Cre / Lox and Flp / Frt recombination systems. Both systems are well known in the art. For instance, site-specific integration into bacterial chromosomes has been reported (see, e.g., Sauer et al., Proc. Natl. Acad. Sci. USA, 85.5166-5170 (1988); Fukushige et al., Proc. Natl. Acad. Sci. USA, 89.7905-7907 (1992); Baubonis et al., Nucleic Acids Research. 21, 2025-2029 (1993); Hasan et al., Gene, 150.51-56 (1994); Golic et al., Cell.5_9, 499-509 KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO (1989); Sauer, Mol. Cell. Biolo.1, 2087-2096 (1987); Sauer et al., Methods: Companion to Methods in Enzymol. 4., 143-149 (1992); Sauer et al., The New Biologist.2., 441-449 (1990); Sauer et al., Nucleic Acids Res.17.147-161 (1989); Qin et al., Proc. Natl. Acad. Sci. USA, 91. 1706-1710 (1994); Orban et al., Proc. Natl. Acad. Sci. USA, 89, 6861-6865 (1992)). Specific deletions of chromosomal sequences and rearrangements have also been engineered, and excision of foreign DNA as a plasmid from ^ vectors is presently possible (see, e.g., Barinaga, Science. 265, 27-28 (1994); Sauer, Methods in Enzvmol.225.890-900 (1993); Sauer et al., Gene, 70.331- 341 (1988); Brunelli et al., Yeast, 1309-1318 (1993); Invitrogen (San Diego, Calif.) 1995 Catalog, 35; Clontech (Palo Alto, Calif.) 1995 / 1996 Catalog, 187-188). Cloning schemes have been generated so that recombination either reconstitutes or inactivates a functional transcription unit by either deletion or inversion of sequences between recombination sites (see, e.g., Odell et al., Plant Physiol.106.447-458 (1994); Gu et al., Cell.73.1155-1164 (1993); Lakso et al., Proc. Natl. Acad. Sci. USA, 89.6232-6236 (1992); Fiering et al., Proc. Natl. Acad. Sci. . USA, 90.8469-8473 (1973); O'Gorman et al., Science.251, 1351-55 (1991); Jung et al., Science, 259, 984-987 (1993)).

[0108] Genes encoding the Cre or Flp recombinases can be provided in trans under the control of either constitutive, inducible or developmentally-regulated promoters. Purified recombinase can also be introduced (see, e.g., Baubonis et al., supra; Dang et al., Develop. Genet.13, 367-375 (1992); Chou et al., Genetics.131. 643-653 (1992); Morris et al., Nucleic Acids Res.19.5895- 5900 (1991)).

[0109] In some embodiments, the genomic manipulations disclosed herein are performed with a modified site-specific recombination protocol from Kirill A. Datsenko and Barry L. Wanner Proc Natl Acad Sci USA.2000 Jun. 6; 97(12): 6640-6645. In one embodiment, knocking out a gene for example, ompT, treAF, or T7 RNAP, can be performed as follows. A PCR amplicon was generated comprising an antibiotic resistance gene flanked by two FRT sites and homology extensions, H1 and H2, which are homologous to the two ends of the gene to be knocked out. After transforming cells with this PCR product, the gene to be knocked out is then replaced by the antibiotic resistance gene through Red-mediated recombination in these flanking homology regions. After selection, the resistance gene can be eliminated using a helper plasmid expressing the FLP recombinase, which acts on the directly repeated FRT (FLP recognition target) sites KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO flanking the resistance gene. The Red and FLP helper plasmid can be simply cured by growth at 37°C because they are temperature-sensitive replicons.

[0110] In some embodiments, gene knockout is performed using a CRISPR / Cas system. The CRISPR / Cas system uses a Cas protein and at least one to two ribonucleic acids that are capable of directing the Cas protein to a sequence in a target gene, e.g., ompT, treAF, and T7 RNAP, to remove the gene. Methods of using CRISPR / Cas system to eliminate gene expression are well known and described in e.g., US. Pat. Pub. No.2014 / 0170753.

[0111] Additional methods of knocking out a target gene include, but are not limited to, transcription activation of the effector nuclease (Transcription Activator-Like Effector Nuclease, TALEN) technology and a zinc finger nuclease (Zinc-Finger Nuclease, ZFN). These methods are also well known in the art.

[0112] Knocking-in a gene, such as T7 RNAP, can be performed by standard molecular cloning techniques that are well-known for one skilled in the art.

[0113] Host cells carrying the desired gene modifications can be sequenced to confirm that the desired modifications are present. In some cases, whole genome sequencing can be performed to determine the location of the insertion, mutation, or deletion in the chromosomes. iii. Vectors

[0114] A nucleic acid encoding a protein of interest, e.g., T7 RNAP, lysozyme, or PDF, can be inserted into a replicable vector for expression in the bacteria, e.g., E. coli, under the control of a suitable prokaryotic promoter. Many vectors are available for this purpose and one of skilled in the art can readily determine the selection of appropriate vector. Besides the gene(s) of interest, a vector typically comprises one or more of the following: a signal sequence, an origin of replication, one or more marker genes, and a promoter. C. Polypeptides

[0115] The host cells of the present disclosure are useful for the overexpression of various polypeptides or proteins. In particular, the host cells are useful for the overexpression of T7 RNAP. In some embodiments, a protein expressed by the host cell is a T7 RNAP. In some embodiments, the T7 RNAP comprises a His tag, which is an amino acid sequence comprising six KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO histidines. The His tag may be on the N-terminus or the C-terminus of T7 RNAP, or both. In some embodiments, the T7 RNAP comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more identity to the sequence of SEQ ID NO: 2. In some embodiments, the T7 RNAP comprises the amino acid sequence of SEQ ID NO: 2 without a His tag portion. In some embodiments, the T7 RNAP comprises the amino acid sequence of SEQ ID NO: 2.

[0116] In some embodiments, the host cell also expresses one or more proteins that improve host cell productivity in overexpressing T7 RNAP. Methods for evaluating productivity are discussed below in detail.

[0117] In some embodiments, the host cell also expresses one or more proteins that improve the overall health of the host cell. In some embodiments, the one or more proteins ameliorate toxic effects associated with overexpression of T7 RNAP. In some embodiments, the overall health of a host cell and amelioration of T7 RNAP toxic effects can be determined by observing the shape, structure, form, size, texture, color, motility, T7 RNAP production rate, and / or proliferation rate of the cell, as well as the viscosity of the liquid culture of the host cell. In some embodiments, an indicator of toxicity is when one or more elements in the expression cassette or vector is eliminated or disrupted.

[0118] In some embodiments, a protein expressed by the host cell is a lysozyme or a variant thereof. In some embodiments, the lysozyme binds to T7 RNAP. In some embodiments, the lysozyme inhibits T7 RNAP. In some embodiments, the lysozyme lacks amidase activity yet retains T7 RNAP binding. In some embodiments, the lysozyme lacks amidase activity yet retains T7 RNAP inhibition. In some embodiments, the lysozyme comprises a lysine to tyrosine substitution at position 128 (K128Y) of its amino acid sequence relative to the sequence of SEQ ID NO: 6. In some embodiments, the lysozyme comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more identity to the sequence of SEQ ID NO: 6. In some embodiments, the lysozyme comprises a lysine to tyrosine substitution at position 128 of its polypeptide sequence relative to the sequence of SEQ ID NO: 6, and an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more identity to the sequence of SEQ ID NO: 6. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO In some embodiments, the lysozyme comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the lysozyme is lysozyme Y (lysY).

[0119] In some embodiments, a protein expressed by the host cell is peptide deformylase (PDF). In some embodiments, the PDF removes the formyl group of N-terminus nascent polypeptides as they are synthesized from the ribosome. In some embodiments, when a host cell of the present disclosure is cultured under conditions that lack an induction signal for the expression of T7 RNAP, the burden of producing PDF prevents extra energy and carbon from entering overflow metabolism towards acetate production. In some embodiments, PDF expression reduces acetate production. In some embodiments, the PDF comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more identity to the sequence of SEQ ID NO: 8. In some embodiments, the PDF comprises the amino acid sequence of SEQ ID NO: 8. IV. Methods for T7 RNA Polymerase Overexpression A. Culturing Bacteria

[0120] Methods for culturing bacteria and use of bacteria for protein overexpression are well known to those skilled in the art. Bacteria suitable for culture and overexpression of T7 RNAP include gram-negative bacteria and gram-positive bacteria, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, and Pseudomonas such as P. aeruginosa, and Steptomyces. A bacteria derived from any strain of these bacteria can be used in the methods of the disclosure. In some embodiments, the bacteria used in cultures and in protein overexpression are from an Escherichia species, such as Escherichia coli or a derivative thereof. In some embodiments, the E. coli strain is an A (K-12), B, C or D strain. Suitable media for culturing bacteria and for protein overexpression are well-known to one of ordinary skill in the art. Examples of media recipes may be found, for example, in Allikian et al., (2019). Fundamentals of Fermentation Media. In: Berenjian, A. (eds) Essentials in Fermentation Technology. Learning Materials in Biosciences. Springer, Cham; doi.org / 10.1007 / 978-3-030-16230-6_2. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO B. Protein Overexpression

[0121] Methods for protein overexpression in bacteria are known to one of ordinary skill in the art. Any bacterial host cell of the present disclosure may be cultured for the overexpression of T7 RNAP. The host cell can be cultured for protein overexpression as follows. The host cell is grown in an overnight culture in any of a number of growth media and under growth conditions that are well known in the art and easily optimized by a practitioner for growth of the particular host cell. In general, host cells are grown in media until they reach balanced exponential growth phase or stationary phase. This can be between 106to 109cells per ml. In some embodiments, the culture is harvested when the pH of the culture rises above a set point indicating the depletion of glucose in the media. The bacterial culture can be grown to an OD595-600of 10 to 60, depending on the host cell that is used. In some embodiments, the host cell is cultured at a growth rate of about 0.06 to about 0.6 to about 0.8 doublings per hour. In some embodiments, the host cell is cultured in culture plates, test tubes, shake flasks, bioreactors, or fermenters.

[0122] Specific growth rate is a useful parameter for monitoring or evaluating the behavior of a host cell in a liquid culture. Specific growth rate, which may be expressed in units of ^ or h-1, is the rate of increase of biomass of a cell population per unit of initial biomass, and can be determined according to the following formula –Specific growth rate can also be modulated in a fed-batch operation to achieve maximal protein expression. In some embodiments, the growth rate is at least 0.05 ^, at least 0.1 ^, at least 0.15 ^, at least 0.2 ^, or at least 0.3 ^. In some embodiments, the growth rate is maintained at about 0.05 ^, about 0.1 ^, about 0.15 ^, about 0.2 ^, or about 0.3 ^.

[0123] The host cells can be grown in medium containing glucose and phosphate, where the glucose is present at a concentration of at least about 0.25% (weight / volume), more usually at least about 1%; and usually not more than about 4%, more usually not more than about 2%. An example of such media is 2YTPG medium, however, one of skill in the art will appreciate that many culture media can be adapted for this purpose, as there are many published media suitable for the growth of bacteria such as E. coli, using both defined and undefined sources of nutrients. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO Optimal media and growth conditions are known for specific species. For example, E. coli are commonly grown in YT broth (yeast extract and tryptone) or variants thereof. The media can be defined (synthetic) or complex (undefined).

[0124] Host cells can be transfected or transformed with expression or cloning vectors and cultured in conventional nutrient media modified as appropriate for expressing particular proteins and for preparing bacterial lysates, as described herein.

[0125] In some instances, the host cells are cultured in aerobic conditions to induce protein expression, and then the culture is switched to anaerobic conditions, for example by bubbling nitrogen, argon, etc. through the culture medium.

[0126] When large amounts of host cells are needed, continuous culturing means are employed instead of batch systems which are closed. These continuous systems involve a feed mechanism to maintain a flow of reagents and nutrients and may remove waste as well as isolate the end-product as part of the process. Optimally, this permits the cells to be grown at a constant biomass concentration for extended periods. Two well-known systems are chemostats and turbidostats. In the chemostat system, sterile media is fed in at a constant rate while media containing bacteria is removed at the same rate. The turbidostat system uses a photocell to measure absorbance or turbidity and regulates the inflow of sterile media and outflow of bacteria according to preset signals. Batch systems are also of interest, where additional reagents may be introduced to prolong the period of time for active synthesis. A reactor can be run in any mode such as batch, extended batch, semi-batch, semi-continuous, fed-batch and continuous, and which will be selected in accordance with the application purpose.

[0127] Methods of culturing bacterial host cells are described in, e.g., Zawada et al., Biotechnol. Bioeng., 108(7):1570-1578 (2011); Zawada, J. “Preparation and Testing of E.coli S30 In vitro Transcription Translation Extracts”, Douthwaite, J.A. and Jackson, R.H. (eds.), Ribosome Display and Related Technologies: Methods and Protocols, Methods in Molecular Biology, vol.805, pp. 31-41 (Humana Press, 2012); Jewett et al., Molecular Systems Biology: 4, 1-10 (2008); Shin J. and Norieaux V., J. Biol. Eng., 4:8 (2010).

[0128] In some instances, an engineered E. coli strain (e.g., engineered K-12 derived E. coli strain KGK10) is cultured to mid-log phase (OD595 of about 45 or about 140 g / L of cell wet KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO weight) using glucose and amino acid fed-batch fermentation at a maximal growth rate of about 0.7 h-1. Glucose can be increased during culturing such that there is excess glucose during harvest. See, e.g., Zawada et al., Biotechnol. Bioeng., 108(7): 1570-1578 (2011).

[0129] In some embodiments, the host cell is cultured under conditions that permit expression of T7 RNAP. In some embodiments, T7 RNAP expression is constitutive. In some embodiments, T7 RNAP expression is inducible. In some embodiments, T7 RNAP expression occurs when an induction signal is applied to induce transcription of the T7 RNAP polynucleotide sequence and expression of the T7 RNAP protein. Any induction signal may be used so long as it corresponds to the inducible promoter that drives T7 RNAP expression. In some embodiments, the induction signal is heat or a chemical. In some embodiments, the induction signal is arabinose. In some embodiments, T7 RNAP protein is not expressed in detectable amounts because the induction signal is absent.

[0130] In some embodiments, T7 RNAP expression is induced at an OD595of about 80, about 90, or about 100. In some embodiments, T7 RNAP expression is induced at an OD595of about 80- 100. In some embodiments, T7 RNAP expression is induced at a lower OD595to reduce shear stress. In some embodiments, T7 RNAP expression is induced at a lower OD595 of about 50, about 60, or about 70. In some embodiments, T7 RNAP expression is induced at a lower OD595 of about 50-80.

[0131] In some embodiments, the host cell appears healthy or lacking in growth defects before T7 RNAP expression is induced. In some embodiments, the host cell appears healthy or lacking in growth defects after T7 RNAP expression is induced. In some embodiments, a healthy host cell is indicated by the shape, structure, form, size, texture, color, motility, T7 RNAP production rate, liquid culture viscosity, and / or proliferation rate of the host cell. In some embodiments, a host cell appears healthy even when cultured in diluted fermenter media without nutrient supplementation. In some embodiments, a healthy host cell demonstrates normal cell division, e.g., cells that are lacking in elongated shapes or other morphology indicative of non-diving cells. In some embodiments, a healthy host cell is not concatenated. In some embodiments, a healthy host cell is indicated by retention of an expression cassette or vector. In some embodiments, a healthy host KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO cell is indicated by high levels of T7 RNAP expression. In some embodiments, high levels of T7 RNAP expression is indicated by the titer, productivity, and / or yield of a culture of the host cell.

[0132] In some embodiments, the host cell is cultured under conditions that permit expression of lysozyme. In some embodiments, lysozyme expression is constitutive. In some embodiments, lysozyme expression is inducible. In some embodiments, lysozyme expression occurs when an induction signal is applied to induce transcription of the lysozyme polynucleotide sequence and expression of the lysozyme protein. Any induction signal may be used so long as it corresponds to the inducible promoter that drives lysozyme expression. In some embodiments, the induction signal is heat or a chemical. In some embodiments, lysozyme protein is not expressed in detectable amounts because the induction signal is absent.

[0133] In some embodiments, the culture of the host cells that express lysozyme is less viscous than the culture of host cells that do not express lysozyme. In some embodiments, increased viscosity is due to increased cell lysis. In some embodiments, the host cell that expresses lysozyme is less prone to cell lysis when compared to a host cell that does not express lysozyme. In some embodiments, the culture of the host cells has a viscosity that is no greater than 1.1, no greater than 1.2, no greater than 1.3, no greater than 1.4, no greater than 1.5, no greater than 1.6, no greater than 1.7, no greater than 1.8, no greater than 1.9, no greater than 2.0, no greater than 2.1, no greater than 2.2, no greater than 2.3, no greater than 2.4, no greater than 2.5, no greater than 2.6, no greater than 2.7, no greater than 2.8, no greater than 2.9, no greater than 3.0, no greater than 3.1, no greater than 3.2, no greater than 3.3, no greater than 3.4, no greater than 3.5, no greater than 3.6, no greater than 3.7, no greater than 3.8, no greater than 3.9, or no greater than 4.0 mPA*S.

[0134] In some embodiments, the culture of the host cells that co-express lysozyme achieves a higher OD595at the end of a production or fermentation run (i.e., final OD595or terminal OD595) than the culture of host cells that do not co-express lysozyme. In some embodiments, the host cells that express lysozyme can achieve higher OD595 due to decreased cell lysis in culture. In some embodiments, the culture of the host cells that co-express lysozyme achieves a terminal OD595 of at least 90, at least 100, at least 110, at least 120, or at least 130. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO

[0135] In some embodiments, the host cell is cultured under conditions that permit expression of PDF. In some embodiments, PDF expression is constitutive. In some embodiments, PDF expression is inducible. In some embodiments, PDF expression occurs when an induction signal is applied to induce transcription of the PDF polynucleotide sequence and expression of the PDF protein. Any induction signal may be used so long as it corresponds to the inducible promoter that drives PDF expression. In some embodiments, the induction signal is heat or a chemical. In some embodiments, PDF protein is not expressed in detectable amounts because the induction signal is absent.

[0136] In some embodiments, co-expression of PDF with T7 RNAP production allows the host cell to mitigate toxicity associated with accumulating acetate in the culture. In some embodiments where host cells do not co-express PDF with T7 RNAP production, the accumulating amounts of acetate in liquid culture can inhibit cell growth. The accumulating acetate can prevent a host cell from growing to target high cell densities for protein overexpression. Therefore, the host cells are fed at a reduced medium feed rate, e.g., at a low targeted exponential feed rate, such as an exponential feed rate of about 0.215 h-1, to keep acetate production at a minimum. In some of these embodiments, the host cells produce significant and detrimental amounts of acetate, even when fed at low feed rates. Unfortunately, low feed rates ultimately lead to low yields of T7 RNAP. In contrast, host cells that co-express PDF with T7 RNAP production can grow to higher OD595despite accumulating acetate in the culture.

[0137] In some embodiments, the toxicity associated with acetate accumulation is mitigated by the co-expression of PDF in the host cell. In some embodiments, when the host cells are cultured for T7 RNAP production, the host cells co-express PDF and produce less acetate than host cells that do not co-express PDF. In some embodiments, the culture of the host cells that co-express PDF grow to a higher terminal OD595 than the culture of host cells that do not co-express PDF. In some embodiments, the culture of the host cells that co-express PDF achieves a terminal OD595 of at least 90, at least 100, at least 110, or at least 120.

[0138] In some embodiments, during fed-batch fermentation or protein production, the host cells are fed at a feed rate of at least 0.05 h-1, at least 0.06 h-1, at least 0.07 h-1, at least 0.08 h-1, at least 0.09 h-1, at least 0.10 h-1, at least 0.11 h-1, at least 0.12 h-1, at least 0.13 h-1, at least 0.14 h-1, at least KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO 0.15 h-1, at least 0.16 h-1, at least 0.17 h-1, at least 0.18 h-1, at least 0.19 h-1, at least 0.20 h-1, at least 0.21 h-1, at least 0.22 h-1, at least 0.23 h-1, at least 0.24 h-1, at least 0.25 h-1, at least 0.26 h-1, at least 0.27 h-1, at least 0.28 h-1, at least 0.29 h-1, or at least 0.30 h-1. In some embodiments, during fed- batch fermentation or protein production, the host cells are fed at a reduced feed rate to reduce shear stress. In some embodiments, during fed-batch fermentation or protein production, the host cells are fed at a reduced feed rate of about 0.05 h-1, about 0.06 h-1, about 0.07 h-1, about 0.08 h-1, about 0.09 h-1, about 0.10 h-1, about 0.11 h-1, or about 0.12 h-1. In some embodiments, during fed- batch fermentation or protein production, the host cells are fed at a reduced feed rate of about 0.05 h-1-0.12 h-1, 0.05 h-1-0.1 h-1, or 0.07 h-1-0.12 h-1. In some embodiments, during fed-batch fermentation or protein production, the host cells are fed at a feed rate that achieves a specific growth rate of at least 0.05 ^, at least 0.06 ^, at least 0.07 ^, at least 0.08 ^, at least 0.09 ^, at least 0.10 ^, at least 0.11 ^, at least 0.12 ^, at least 0.13 ^, at least 0.14 ^, at least 0.15 ^, at least 0.16 ^, at least 0.17 ^, at least 0.18 ^, at least 0.19 ^, at least 0.20 ^, at least 0.21 ^, at least 0.22 ^, at least 0.23 ^, at least 0.24 ^, at least 0.25 ^, at least 0.26 ^, at least 0.27 ^, at least 0.28 ^, at least 0.29 ^, or at least 0.30 ^.

[0139] The cultivation of microorganisms requires a sufficient supply of oxygen to the cells. Oxygen supply can be problematic in liquid cultures because the solubility of oxygen is significantly lower compared to glucose and other nutrient components in the liquid media. In a liquid fermentation, oxygen is supplied to the cells by air bubbles through spargers and headspace, and is then dispersed into the liquid by mixing. In some embodiments, an insufficient oxygen supply is a factor in a fermentation not being able to arrive at a target biomass concentration.

[0140] Dissolved oxygen (DO) is a key driver affecting culture growth in bioreactors. However, cell lines and strains can have different oxygen needs. For example, an aerobic culture of mammalian cell cultures and many microbial cultures consume oxygen and an effective bioreactor DO control system is therefore required to keep the dissolved oxygen concentration stable. In some examples, an aerobic culture may require a DO concentration of over 30%. DO concentrations may be controlled via adjusting the agitation via impellers, the influx of gases including air and oxygen using gas spargers, and / or gas composition. In contrast, an anaerobic fermentations of certain microorganisms are performed without oxygen. In anaerobic KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO fermentations, the removal of oxygen from bioreactors can be achieved through sparging nitrogen or other anaerobic gasses.

[0141] In some embodiments, a host cell that co-expresses PDF and / or lysozyme with T7 RNAP production has lower oxygen demand when compared to a host cell that does not co-express PDF and / or lysozyme. In some embodiments, during T7 RNAP production phase, the bioreactor can maintain a setpoint for dissolved oxygen in the liquid culture and the host cell can maintain productivity in overexpressing T7 RNAP. In some embodiments, the bioreactor can maintain at least 20%, at least 30%, or at least 40% of dissolved oxygen in the liquid culture while requiring no more than 5%, no more than 10%, no more than 20%, no more than 30%, no more than 40%, no more than 50%, or no more than 60% of supplemented oxygen or oxygen enrichment, i.e., O2gas. In some embodiments, a culture of the host cell does not become anoxic during T7 RNAP production.

[0142] In some embodiments, the DO setpoint is fixed, for example, at about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%. In some embodiments, the DO setpoint is fixed, for example, at about 10-50%, about 10%-30%, about 20- 40%, or about 30-50%. In some embodiments, DO is controlled by a DO cascade. As used herein, a DO cascade refers to a specific sequence of parameter changes that are employed by the bioreactor to increase the DO levels within the bioreactor. As the cascade progresses, different parameters, e.g., increased agitation, aeration, oxygen enrichment, and back pressure, of increasing or decreasing intensity are initiated to ensure DO control is sufficient to optimize yield, e.g., protein overexpression. In some embodiments, one parameter is increased until it reaches its maximum intensity before another parameter is increased. In some embodiments, when one parameter is increased, the remaining parameters are held constant or reduced. In some embodiments, the intensity of a parameter is increased incrementally or gradually (as in a gradient), in a stepwise fashion, or a combination thereof.

[0143] In some embodiments, a DO cascade comprises increased agitation (also termed “cascading agitation”). Typically, increased agitation is the first step of a DO cascade. A DO cascade increases agitation speeds in a bioreactor in order to maintain conditions for cell growth and product formation as a fermentation progresses. After agitation speeds reach a maximum KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO intensity, the agitation speed is held at that maximum setpoint while other fermentation parameters are increased to continue maintaining conditions, including DO levels, that allow for cell growth and product formation as a fermentation continues. In some embodiments, the maximum agitation speed is the intensity that will allow the cells in the culture to achieve a target titer, productivity, and / or yield. In some embodiments, the maximum agitation speed is the maximum intensity that the cells in the culture can tolerate without unfavorable effects on cell vitality, titer, productivity, and / or yield. In some embodiments, the maximum agitation speed is the maximum intensity that the bioreactor is capable of. Thus, in some embodiments, to maintain a DO setpoint, agitation is increased until it reaches a maximum agitation speed before the other parameters are changed, e.g., before aeration is increased, before oxygen enrichment is increased, and / or before back pressure is decreased.

[0144] In contrast, in some embodiments, a DO cascade comprises maintaining lower agitation speeds. In these embodiments, to maintain a DO setpoint, agitation speeds are not increased substantially. Instead, in these embodiments, agitation speeds are maintained at a minimum agitation speed limit. Lower agitation speeds can decrease the shear stress experienced by the cells in a liquid culture, but the lower agitation speeds must still allow for proper mixing of the liquid culture. Thus, in some embodiments, a DO cascade comprises agitation speeds at a minimum agitation speed (i.e., the speed is not too slow so that the culture is mixed sufficiently), and if the agitation speed is increased, the agitation speeds that do not exceed a minimum agitation speed limit (i.e., the speed is not too fast to avoid agitating the culture too much). In some embodiments, a DO cascade comprises maintaining agitation speeds at the minimum agitation speed limit while other parameters are changed (e.g., aeration is increased, oxygen enrichment is increased, and / or back pressure is decreased) to maintain a DO setpoint. In general, the minimum agitation speed limit is less than the maximum agitation speed limit. In some embodiments, a DO cascade comprising a minimum agitation speed limit produces greater cell vitality, titer, productivity, and / or yield than a DO cascade comprising a maximum agitation speed limit as discussed above.

[0145] Agitation is achieved using an impeller that is moved by a motor. Tip speed is the distance that a point on the edge of an impeller travels in a set amount of time. Tip speed is a function of the impeller’s diameter and rpm. Impeller tip speed serves as a measurement of the KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO shear rate produced by the impeller blades in a cell culture medium. In some embodiments, impeller tip speed can be calculated according to the following formula – Tip speed = ^(^)(^^^ / 60)

[0146] In some embodiments, the maximum agitation speed is limited to a certain value, for example, to avoid extensive shear stress to the cells and / or to provide sufficient mixing of the culture. In some embodiments, the maximum agitation speed is fixed at about 200 rpm, about 250 rpm, about 400 rpm, about 500 rpm, about 700 rpm, about 900 rpm, or about 1200 rpm. In some embodiments, the maximum agitation speed is fixed at about 200-1200 rpm, about 200- 500 rpm, about 400-700 rpm, about 600-900 rpm, about 800-1100 rpm, or about 900-1200 rpm. In some embodiments, the maximum agitation speed is measured as the maximum impeller tip speed. In some embodiments, the maximum impeller tip speed is fixed not to exceed about 2.2 m / s, about 2.4 m / s, about 2.6 m / s, about 2.8 m / s, about 3.0 m / s, about 3.2 m / s, about 3.4 m / s, about 3.6 m / s, about 3.8 m / s, about 4.0 m / s, about 4.2 m / s, about 4.4 m / s, about 4.7 m / s, about 5.0 m / s, about 5.2 m / s, about 5.4 m / s, about 5.6 m / s, about 5.8 m / s, about 6.0 m / s, about 6.2 m / s, about 6.4 m / s, about 6.6 m / s, about 6.8 m / s, about 6.9 m / s, about 7.0 m / s, about 7.1 m / s, about 7.2 m / s, about 7.3 m / s, about 7.4 m / s, about 7.6 m / s, about 7.8 m / s, about 8.0 m / s, about 8.2 m / s, about 8.4 m / s, about 8.6 m / s, about 8.8 m / s, or about 9.0 m / s. In some embodiments, the maximum impeller tip speed is fixed at about 2.2-4.7 m / s, about 2.2-3.8 m / s, about 3.0-4.0 m / s, about 3.5-4.5 m / s, about 4.0-4.7 m / s, about 5.0-6.0 m / s, about 6.0-7.0 m / s, about 6.5-7.5 m / s, about 7.0-8.0 m / s, about 6.9- 7.3 m / s, or about 7.0-7.2 m / s. In some embodiments, the maximum impeller tip speed limit is fixed at about 5.0-6.0 m / s, about 6.0-7.0 m / s, about 6.5-7.5 m / s, about 7.0-8.0 m / s, about 6.9-7.3 m / s, or about 7.0-7.2 m / s. In some embodiments, the maximum impeller tip speed limit does not exceed about 5.0 m / s, about 6.0 m / s, about 6.8 m / s, about 6.9 m / s, about 7.0 m / s, about 7.1 m / s. about 7.2 m / s, about 7.3 m / s, about 7.4 m / s, about 7.5 m / s, about 7.6 m / s, about 7.7 m / s, about 7.8 m / s, about 7.9 m / s, or about 8.0 m / s. In some embodiments, the impeller tip speed does not exceed about 5.0 m / s. In some embodiments, the impeller tip speed does not exceed about 6.0 m / s.

[0147] In some embodiments, a DO cascade comprises increased aeration or air sparging (also termed “cascading air flow,” “cascading aeration,” and “cascading air sparging”). When maximum agitation speed (discussed in the preceding paragraph) has been reached, aeration is increased. In KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO some embodiments, aeration is increased up to its maximum rate. In some embodiments, aeration is at least 500 sLPM, at least 550 sLPM, at least 600 sLPM, at least 650 sLPM, at least 700 sLPM, at least 750 sLPM, at least 800 sLPM, at least 850 sLPM, at least 900 sLPM, at least 950 sLPM, or at least 1,000 sLPM. In some embodiments, aeration is at least 500-1000 sLPM, at least 500- 750 sLPM, at least 650-900 sLPM, at least 800-1000 sLPM.

[0148] In some embodiments, a DO cascade comprises increased oxygen enrichment (also termed “cascading oxygen,” “cascading pure oxygen,” “cascading oxygen flow,” or “cascading pure oxygen flow”). The air that is sparged into the system (as discussed in the preceding paragraph) is gradually replaced by pure oxygen until only pure oxygen is sparged into the bioreactor. In some embodiments, pure oxygen is added to the system at about 0 sLPM, about 100 sLPM, about 200 sLPM, about 300 sLPM, about 400 sLPM, about 500 sLPM, about 600 sLPM, about 700 sLPM, about 800 sLPM, or about 900 sLPM. In some embodiments, pure oxygen is added to the system at about 0-900 sLPM, about 0-300 sLPM, about 200-500 sLPM, about 400-700 sLPM, or about 600-900 sLPM.

[0149] In some embodiments, a DO cascade comprises increased back pressure in addition to the increased agitation and aeration to maximize the dissolved oxygen in the cell culture.. In some embodiments, the back pressure is about 5 psi, about 10 psi, or about 15 psi. In some embodiments, the back pressure is about 5-15 psi, about 5-12 psi, about 7-15 psi, about about 5- 10 psi, or about 10-15 psi.

[0150] In some embodiments, the host cell is cultured in the presence of a compound that can function as a shear protectant agent. Shear stress is the mechanical force induced by the friction of liquid against the cell membrane. Shear stress can develop in a cell culture due to the presence of velocity gradients within the fluid flow. Thus, in some embodiments, the host cell is cultured in the presence of a shear protectant agent to decrease shear stress on the host cell. Many compounds can be used as a shear protectant agent, including, without limitations, Pluronic® L 61 (also known as Poloxamer 181) and / or Pluronic® F-68 (also known as Poloxamer 188). In some embodiments, the shear protectant agent is Pluronic® L 61 and / or Pluronic® F-68. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO V. Methods for Preparing, Using, and Evaluating T7 RNA Polymerase A. Preparation of Bacterial Cell Extracts with T7 RNA Polymerase

[0151] A bacterial cell extract derived from any host cell of the present disclosure can be isolated and disrupted for use in CFPS reactions. Methods of preparing a lysed bacterial cell extract are described in, e.g., Zawada, J. “Preparation and Testing of E.coli S30 In vitro Transcription Translation Extracts”, Douthwaite, J.A. and Jackson, R.H. (eds.), Ribosome Display and Related Technologies: Methods and Protocols, Methods in Molecular Biology, vol.805, pp. 31-41 (Humana Press, 2012); Jewett et al., Molecular Systems Biology, 4, 1-10 (2008); Shin J. and Norieaux V., J. Biol. Eng., 4:8 (2010).

[0152] In some embodiments, the bacterial cell extract contains T7 RNAP and the bacterial cell extract is applied as a reagent T7 RNAP in CFPS reactions. Once the bacterial culture is ready for harvest, it can be cooled to 2-8oC, usually on ice or through heat exchangers when the culture is of a large scale. The culture can be centrifuged to separate the spent media from the cell paste (cell slurry). Preferred centrifuges include disk stack centrifuges, tubular bowl centrifuges, and other centrifuges for large or small-scale bacterial cultures. In some embodiments, the bacterial culture is pelleted by centrifugation at greater than 14,000 x g for about 45 min at about 8-20oC twice in a tubular bowl centrifuge in continuous or batch mode or a disc stack continuous centrifuge with a maximum bowl speed of about 12,000 rpm and a feed flow rate of about 3.0-3.3 L / min.

[0153] The cell pellet or cell paste is typically resuspended in S30 buffer, any equivalent buffer solution, or water. S30 buffer comprises 10 mM Tris acetate, 14 mM magnesium acetate and 60 mM potassium acetate. In some embodiments, a 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 or more dilution (liquid: solid; ml of buffer: gram weight of cells) is made for washing. The cell paste can be washed again in S30 buffer or any equivalent buffer and centrifuged to remove any residual buffer. For small scale cultures, a second wash step is typically performed. At washing the cell pellet can be stored at -80º C for later use or further processed by lysis to produce a bacterial cell extract.

[0154] Harvested cells are lysed to produce a bacterial cell extract. If the harvested cells were frozen as cell pellets, the frozen cell pellets are resuspended and thawed in a suitable cell suspension buffer. The suspended cells are lysed by sonication, with a French press or with glass KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO beads, continuous flow high pressure homogenization, or any other method known in the art useful for efficient cell lysis.

[0155] In some embodiments, the cell suspension is homogenized or disrupted in a standard high-pressure homogenizer (e.g., an Avestin Emulsiflex C-55a Homogenizer) and / or microfluidizer (e.g., Microfluidics Microfluidizer) set at the appropriate pressure, such as 3,000 psi to produce a lysate. The homogenization step lyses the bacteria to release the necessary components required for protein synthesis, and in some aspects, formed inverted membrane vesicles provide energy for protein synthesis via respiration.

[0156] In some embodiments, the homogenizer pressure is at about 3,000-20,000 psi. In some embodiments, the homogenizer pressure is set at about 20,000 psi. In some embodiments, the speed (frequency setting) of the homogenizer is at about 20 Hz to about 60 Hz to produce flow rates of about 340 ml / min-1.0 L / min. Generally, flow rate is proportional to the frequency setting and can be varied independently from the homogenizing pressure. Preferably, the minimum speed setting for homogenizing steps is about 20 Hz with a flow rate of about 340 mL / min.

[0157] The cell lysate is then centrifuged or filtered to remove large cell debris, including DNA, and cells that have not been lysed. The lysate can be clarified by centrifugation such that from at least about 45% to about 85% or more, e.g., about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% of the cell solids are separated from the cell-free extract which is collected. In some embodiments, at least about 70%, 75%, 80%, 85%, 90%, or 95% of the cellular solids are separated by centrifugation. In some embodiments, the centrifugation is by a continuous centrifuge, e.g., disk stack centrifuge, tubular bowl centrifuge or appropriate centrifuge.

[0158] The clarified cell lysate can be filtered through one or more sterilizing grade filter membranes, e.g., a 0.45 μm filter membrane and / or a 0.22 μm filter membrane. A 0.45-μm filter membrane can be used first, and then a 0.22 μm filter membrane afterwards.

[0159] The filtered cell lysate can be aliquoted and frozen in liquid nitrogen before storing at - 80ºC. Optionally, a cell-free protein synthesis (CFPS) reaction mix, as described herein, can be added to the cell lysate prior to freezing. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO

[0160] Bacterial cell extracts of the present disclosure may also be used to supplement commercially-available bacterial cell extracts. Commercial bacterial cell extracts may be purchased from manufacturers such Promega Corp., Madison, WI; Agilent Technologies, Santa Clara, CA; GE Healthcare Biosciences, Pittsburgh, PA; Life Technologies, Carlsbad, CA; and Roche Diagnostics, Basel, Switzerland.

[0161] Bacterial cell extracts of the present disclosure may be stored in several formats for long periods of time before use. In some embodiments, the bacterial cell extract is stored at about - 20oC, about 2oC to 8oC, or about 20oC to 24oC (i.e., room temperature) for at least or equal to 3 to 18 months (e.g., at least or equal to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 months).

[0162] In some embodiments, the bacterial cell extract is dried prior to storage. In some embodiments, the bacterial cell extract is subjected to freeze drying, lyophilization, in situ vaporization, microwave radiation, or sublimation. In some embodiments, the bacterial cell extract is subject to a spray drying process where the spray dried bacterial cell extract essentially retains its physical and chemical stability and integrity upon storage. For example, a stable, spray dried bacterial cell extract refers to an extract that retains at least 75% of its initial capacity to synthesize a protein of interest when stored at about -20oC, about 2oC to 8oC, or about 20oC to 24oC (i.e., room temperature) for 6 months or more. Detailed descriptions of drying bacterial dried extracts are found in, for example, U.S. Application No. 18 / 231,204 and U.S. App. Publ. No. 2023 / 0167475.

[0163] In embodiments where the bacterial cell extract was dried as discussed in the preceding paragraph, the bacterial cell extract may require activation before being used in a protein synthesis reaction. The dried bacterial cell extract is reconstituted in a buffer or other liquid to form a liquid bacterial cell extract and exposed to heat. In some embodiments, the liquid bacterial cell extract is heated to about 20oC to 45oC for about 30 minutes to about 10 hours. In some embodiments, the liquid bacterial cell extract is heated to about 40oC for about 40 minutes. Activation improves protein expression in CFPS reactions. In some embodiments, the liquid bacterial cell extract is sterile-filtered before activation (i.e., heat-treatment). Activation of bacterial cell extracts is described in Groff, D., et al. (Development of an E. coli strain for cell-free ADC manufacturing. Biotechnology and Bioengineering, 119, 162– 175. doi.org / 10.1002 / bit.27961). KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO

[0164] In some embodiments, a thawed, activated, or rehydrated bacterial cell extract has high T7 RNAP activity. In some embodiments, a thawed, activated, or rehydrated bacterial cell extract in a CFPS reaction is able to synthesize a target protein of interest with a yield of at least 80%, at least 85%, at least 90%, at least 95% relative to a control extract. In some embodiments, the control extract is a fresh preparation of a bacterial cell lysate. In some embodiments, the control extract is a bacterial cell lysate that was not previously frozen. In some embodiments, the control extract is a bacterial cell lysate that was not previously dried. In some embodiments, the control extract is a bacterial cell lysate that was not previously thawed, activated, and / or rehydrated. B. Cell-Free Protein Synthesis

[0165] Bacterial cell extracts of the present disclosure may be used in cell-free protein synthesis (CFPS) reactions. CFPS systems have been used to generate various proteins including growth factors (Zawada et al., Biotechnol Bioeng, 108:1570-1578 (2011)), full-length antibodies and antibody fragments (Yin et al., mAbs, 4(2):217-225 (2012)) and antibody-drug conjugates (Zimmerman et al., Bioconjug Chem, 25(2):351-61 (2014)). In a generic CFPS reaction, a gene encoding a protein of interest is expressed in a transcription buffer, resulting in mRNA that is translated into the protein of interest in a CFPS extract and a translation buffer. The transcription buffer, cell-free extract and translation buffer can be added separately, or two or more of these solutions can be combined before their addition or added contemporaneously.

[0166] To synthesize a protein of interest in vitro, the bacterial cell extract at some point comprises an mRNA molecule that encodes the protein of interest. In some systems, mRNA is added exogenously after being purified from natural sources. In some systems, synthetic mRNA is prepared separately from cloned DNA using RNA polymerases (RNAPs) such as RNAP II, SP6 RNAP, T3 RNAP, T7 RNAP, RNAP III and / or phage derived RNAP. In some embodiments, the T7 RNAP used in the synthetic production of mRNA is produced according to methods of the present disclosure, as discussed above. In some embodiments, a bacterial cell extract comprising T7 RNAP according to the present disclosure is used to produce the synthetic mRNA, as discussed above and herein.

[0167] In other systems where both transcription and translation occur in the same reaction, the mRNA is produced in vitro from a template DNA. In some embodiments, the transcription and KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO translation systems are coupled or comprise complementary transcription and translation systems, which carry out the synthesis of both RNA and protein in the same reaction. In such in vitro transcription and translation systems, the bacterial cell extracts contain all the components (exogenous or endogenous) necessary both for transcription (to produce mRNA) and for translation (to synthesize protein) in a single system. In some embodiments, the bacterial cell extract is a bacterial cell extract with T7 RNAP as discussed in the present disclosure. In some embodiments, the bacterial cell extract is prepared according to methods as discussed in the preceding as well as below sections.

[0168] A CFPS reaction can contain the following components: a template nucleic acid, such as DNA, that comprises a gene of interest operably linked to at least one promoter and, optionally, one or more other regulatory sequences (e.g., a cloning or expression vector containing the gene of interest) or a PCR fragment; an RNA polymerase that recognizes the promoter(s) to which the gene of interest is operably linked (e.g. T7 RNA polymerase) and, optionally, one or more transcription factors directed to an optional regulatory sequence to which the template nucleic acid is operably linked; ribonucleotide triphosphates (rNTPs); optionally, other transcription factors and co-factors therefor; ribosomes; transfer RNA (tRNA); other or optional translation factors (e.g., translation initiation, elongation and termination factors) and co-factors therefore; one or more energy sources, (e.g., ATP, GTP); optionally, one or more energy regenerating components (e.g., PEP / pyruvate kinase, AP / acetate kinase or creatine phosphate / creatine kinase); optionally factors that enhance yield and / or efficiency (e.g., nucleases, nuclease inhibitors, protein stabilizers, chaperones) and co-factors therefore; and; optionally, solubilizing agents. The reaction mix can also include amino acids and other materials specifically required for protein synthesis, including salts (e.g., potassium, magnesium, ammonium, and manganese salts of acetic acid, glutamic acid, or sulfuric acids), polymeric compounds (e.g., polyethylene glycol, dextran, diethyl aminoethyl dextran, quaternary aminoethyl and aminoethyl dextran, etc.), cyclic AMP, inhibitors of protein or nucleic acid degrading enzymes, inhibitors or regulators of protein synthesis, oxidation / reduction adjuster (e.g., DTT, ascorbic acid, glutathione, and / or their oxides), non-denaturing surfactants (e.g., Triton X-100), buffer components, spermine, spermidine, putrescine, etc. Components of such reactions are discussed in more detail in U.S. Patent Nos. 7,338,789; 7,351,563; 8,715,958; and 8,778,631. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO

[0169] Depending on the specific enzymes present in the extract, for example, one or more of the many known nuclease, polymerase, or phosphatase inhibitors can be selected for inclusion to advantageously improve synthesis efficiency.

[0170] Protein and nucleic acid synthesis typically requires an energy source. Energy is required for initiation of transcription to produce mRNA (e.g., when a DNA template is used and for initiation of translation high energy phosphate for example in the form of GTP is used). Each subsequent step of one codon by the ribosome (three nucleotides; one amino acid) requires hydrolysis of an additional GTP to GDP. ATP is also typically required. For an amino acid to be polymerized during protein synthesis, it must first be activated. Significant quantities of energy from high energy phosphate bonds are thus required for protein and / or nucleic acid synthesis to proceed.

[0171] An energy source is a chemical substrate that can be enzymatically processed to provide energy to achieve desired chemical reactions. Energy sources that allow release of energy for synthesis by cleavage of high-energy phosphate bonds such as those found in nucleoside triphosphates, e.g., ATP, are commonly used. When sufficient energy is not initially present in the synthesis system, an additional source of energy is preferably supplemented. Energy sources can also be added or supplemented during the in vitro synthesis reaction. Any source convertible to high energy phosphate bonds is especially suitable. ATP, GTP, and other triphosphates can normally be considered as equivalent energy sources for supporting protein synthesis. Other energy sources that may be considered include glucose, pyruvate, phosphoenolpyruvate (PEP), carbamoyl phosphate, acetyl phosphate, creatine phosphate, phosphopyruvate, glyceraldehyde-3- phosphate, 3-Phosphoglycerate and glucose-6-phosphate, that can generate or regenerate high- energy triphosphate compounds such as ATP, GTP, other NTPs, etc.

[0172] In some embodiments, the CFPS reaction is performed using the PANOx-SP system comprising NTPs, E.coli tRNA, amino acids, Mg2+acetate, Mg2+glutamate, K+acetate, K+glutamate, folinic acid, Tris pH 8.2, DTT, pyruvate kinase, T7 RNA polymerase, disulfide isomerase, phosphoenol pyruvate (PEP), NAD, CoA, Na+oxalate, putrescine, spermidine, and S30 extract. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO

[0173] In some embodiments, the CFPS reaction is performed using the Cytomim system comprising NTPs, E.coli tRNA, amino acids, Mg2+acetate, Mg2+glutamate, K+acetate, K+glutamate, folinic acid, Tris pH 8.2, DTT, pyruvate kinase, T7 RNA polymerase, disulfide isomerase, sodium pyruvate, NAD, CoA, Na+oxalate, putrescine, spermidine, and S30 extract. The Cytomim system is defined as a reaction condition performed in the absence of polyethylene glycol with optimized magnesium concentration. This system does not accumulate phosphate, which is known to inhibit protein synthesis. Detailed descriptions of the Cytomim system are found in, for example, U.S. Patent No.7,338,789; Jewett et al., Mol Syst Biol, (2008), 4:220; Spirin, A.S. and Swartz, J.R. (2008) Cell-free Protein Synthesis; Methods and Protocols, New Jersey:John Wiley & Sons. In some embodiments, the energy substrate for the Cytomim system is pyruvate, glutamic acid, and / or glucose. In some embodiments of the system, the nucleoside triphosphates (NTPs) are replaced with nucleoside monophosphates (NMPs).

[0174] In some embodiments, proteins containing a non-natural amino acid (nnAA) may be synthesized. In such embodiments, the reaction mix may comprise the non-natural amino acid, a tRNA orthogonal to the 20 naturally occurring amino acids, and a tRNA synthetase that can link the nnAA with the orthogonal tRNA. See, e.g., U.S. Patent No.8,715,958. Alternatively, the reaction mix may contain a nnAA conjugated to a tRNA for which the naturally occurring tRNA synthetase has been depleted. See, e.g., U.S. Patent No.8,778,631 and U.S. App. Publ. No. 2010 / 0184134. Various kinds of unnatural amino acids, including without limitation detectably labeled amino acids, can be added to cell-free protein synthesis reactions and efficiently incorporated into proteins for specific purposes. See, for example, Albayrak, C. and Swartz, JR., Biochem. Biophys Res. Commun., 431(2):291-5; Yang WC et al., Biotechnol. Prog., (2012), 28(2):413-20; Kuechenreuther et al., PLoS One, (2012), 7(9):e45850; and Swartz JR., AIChE Journal, 58(1):5-13.

[0175] In some embodiments, the CFPS reaction includes inverted membrane vesicles to perform oxidative phosphorylation. These vesicles can be formed during the high-pressure homogenization step of the preparation of cell extract process, as described herein, and remain in the extract used in the reaction mix. The presence of an active oxidative phosphorylation pathway can be tested using inhibitors that specifically inhibit the steps in the pathway, such as electron transport chain inhibitors. Examples of inhibitors of the oxidative phosphorylation pathway KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO include toxins such as cyanide, carbon monoxide, azide, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), and 2,4-dinitrophenol, antibiotics such as oligomycin, pesticides such as rotenone, and competitive inhibitors of succinate dehydrogenase such as malonate and oxaloacetate.

[0176] The CFPS reaction conditions may be performed as batch, continuous flow, or semi- continuous flow, as known in the art. The reaction conditions are linearly scalable, for example, the 0.3 L scale in a 0.5 L stirred tank reactor, to the 4 L scale in a 10 L fermenter, and to the 100 L scale in a 200 L fermenter. C. Methods for Evaluating Protein Expression

[0177] Various methods can be used to evaluate the expression level of the various proteins in the host cells of the present disclosure or in a CFPS reaction. Methods for measuring protein levels include, but are not limited to, Coomassie-stained polyacrylamide gel electrophoresis (PAGE), silver-stained polyacrylamide gel, capillary electrophoresis (e.g., Caliper LabChip), ELISA, immunoblotting, Western blotting, size exclusion chromatography, affinity chromatography, and mass spectrometry.

[0178] In some embodiments, the protein of interest is detected by an assay that measures the activity of that particular protein. For example, if the protein of interest is a kinase, the kinase may be detected by performing an enzymatic assay specific to detecting the conversion of substrate to product by that kinase. Illustrative examples of assays include luciferase assays and the chloramphenicol acetyl transferase assay. These assays measure the amount of functionally active protein that is expressed.

[0179] In some embodiments, the protein of interest is covalently linked with a detectable label. The amount of fluorescence signal detected in a cell, culture, or reaction may indicate the amount of protein that is present. Exemplary fluorophores that may be used as a detectable label include but are not limited to, cyanine dyes (e.g., Cy2, Cy3, Cy3B, Cy5, Cy5.5, Cy7, etc.), Alexa Fluor (AF) dyes (e.g., AF 647, AF 555, or AF 488), rhodamine dyes (e.g., fluorescein, FITC, Texas Red, ROX), ATTO dye (e.g., ATTO 532 or 655). Exemplary proteins that may be used as a detectable label include green fluorescent protein (e.g., GFP and enhanced GFP (eGFP)), yellow fluorescent proteins (e.g., YFP, Citrine, Venus, and Ypet), cyan fluorescent protein (e.g., ECFP, Cerulean, KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO CyPet, mTurquoise2) or photoactivabale fluorescent proteins, such as PAGFP, PSCFP, PSCFP2, Dendra, Dendra2, EosFP, tdEos, mEos2, mEos3, PamCherry, PAtagRFP, mMaple, mMaple2, and mMaple3. Other suitable fluorophores are known to those of ordinary skill in the art. Methods of detecting these labels, including their excitation and emission at specific wavelengths, are known to one of ordinary skill in the art.

[0180] In some embodiments, the protein of interest in a fluorescent protein, such as GFP, eGFP, YFP, Citrine, Venus, Ypet, ECFP, Cerulean, CyPet, mTurquoise2, PAGFP, PSCFP, PSCFP2, Dendra, Dendra2, EosFP, tdEos, mEos2, mEos3, PamCherry, PAtagRFP, mMaple, mMaple2, and mMaple3. In some embodiments, the protein of interest is GFP.

[0181] In some embodiments, radiolabeled amino acids are incorporated into the protein of interest. In some embodiments, the protein of interest may be monitored by the incorporation of radiolabeled amino acids, typically,35S-labeled methionine or14C-labeled leucine. Radiolabeled proteins can be visualized for molecular size and quantitated by autoradiography after electrophoresis or isolated by immunoprecipitation.

[0182] In some embodiments, a host cell of the present disclosure is cultured under conditions that permit transcription of a target polynucleotide sequence and expression of a corresponding protein. The titer of the liquid culture refers to the amount or mass (g) of the expressed protein in the total liquid volume (L) of the culture. Titer can be expressed in various units of mass per volume, e.g., mg / mL, mg / L, and g / L. Titer can also be used as a parameter for evaluating a CFPS reaction. Here, titer refers to the amount or mass (g) of the expressed protein in the total liquid volume (L) of the CFPS reaction.

[0183] Volumetric productivity and specific productivity are two useful productivity parameters for evaluating protein expression in a liquid culture. Volumetric productivity refers to how much protein is being produced and is calculated as the concentration of that product (e.g., g / L) over time (e.g., hour). For example, volumetric productivity can be expressed in units of g / L / hour. Specific productivity (Qp) is calculated based on the growth rate and volumetric productivity. Growth rate (^) is calculated as follows: KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO; where N is the viable cell density and t ix the cultivation time. Qp can then be calculated as follows:; where P is volumetric productivity.

[0184] When expressing a protein using a CFPS reaction, the activity of a bacterial lysate refers to the amount of protein produced in the reaction when that particular bacterial lysate is used as a reagent of the reaction. In some embodiments, the activity of a bacterial lysate is the yield of an expressed protein in a CFPS reaction. The activity of the bacterial lysate can be determined using assays such as performing CFPS to produce a model protein (a test protein) which can be measured. Methods for CFPS are described in detail in, e.g., Kim, D.M. and Swartz, J.R. Biotechnol. Bioeng. 66:180-8 (1999); Kim, D.M. and Swartz, J.R. Biotechnol. Prog. 16:385-90 (2000); Kim, D.M. and Swartz, J.R. Biotechnol. Bioeng.74:309-16 (2001); Swartz et al., Methods Mol. Biol. 267:169-82 (2004); Kim, D.M. and Swartz, J.R. Biotechnol. Bioeng.85:122-29 (2004); Jewett, M.C. and Swartz, J.R., Biotechnol. Bioeng. 86:19-26 (2004); Yin, G. and Swartz, J.R., Biotechnol. Bioeng. 86:188-95 (2004); Jewett, M.C. and Swartz, J.R., Biotechnol. Bioeng.87:465- 72 (2004); Voloshin, A.M. and Swartz, J.R., Biotechnol. Bioeng. 91:516-21 (2005).

[0185] In some embodiments, the yield of a protein of interest is determined by dual flow chromatography (DFC) using a Protein A resin. The Protein A resin can be packed between two thin frit screens in the tip of a single-use pipette tip. In some embodiments, the yield of a protein of interest is determined by passing the products of a CFPS reaction over a Protein A column, such as a PhyTip® column (Biotage®). The yield of a protein of interest can be expressed as weight / volume (e.g., mg / L or g / L) of a CFPS reaction or as a percentage of the yield of a control extract. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO

[0186] In some embodiments, the yield of a protein of interest is determined by performing High-performance liquid chromatography (HPLC) of the protein products from a CFPS reaction along with protein standards.

[0187] Another method of measuring the amount of protein produced in coupled in vitro transcription and translation reactions is to perform the reactions using a known quantity of radiolabeled amino acid such as35S-methionine,3H-leucine or14C-leucine and subsequently measuring the amount of radiolabeled amino acid incorporated into the newly translated protein. Incorporation assays will measure the amount of radiolabeled amino acids in all proteins produced in an in vitro translation reaction including truncated protein products. The radiolabeled protein may be further separated on a protein gel, and by autoradiography confirmed that the product is the proper size and that secondary protein products have not been produced.

[0188] Methods of measuring the capacity of an expression system to express a protein includes the14C Leu incorporation assay. In some embodiments, a method for measuring the protein synthesis activity of a spray-dried extract is the14C Leu incorporation assay.

[0189] Alternately, the yield of protein can be determined by running the protein labeled with14C Leu on a polyacrylamide gel using conventional techniques. The gel can be denaturing or non- denaturing, according to the polypeptide to be detected. Where a protein containing multiple subunits is to be detected, a non-denaturing gel is preferred. The yield of protein can be determined through specific binding assays such as enzyme linked immunosorbant assay (ELISA) or surface binding resonance (e.g., Biacore).

[0190] Alternatively, the yield of protein can be determined through whole or partial purification, such as using chromatography, coupled with protein quantitation, such as UV absorbance or BCA analysis. VI. Compositions and Kits

[0191] This disclosure also provides kits that comprise viable host cells of the present disclosure and optionally a growth media and / or instructions for use. In some embodiments, the kit may further comprise one or more reagents for producing T7 RNAP from the host cells and / or a KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO reagent T7 RNAP bacterial cell extract. In some embodiments, the kit may comprise a nucleic acid and / or a vector of the present disclosure.

[0192] In some embodiments, the kit may comprise one or more reagents for analysis of T7 RNAP and / or a reagent T7 RNAP bacterial cell extract that is produced according to the present disclosure.

[0193] In some embodiments, the kit may comprise reagents and / or instructions for CFPS. In some embodiments, the kit may comprise reagents and / or instructions for producing GFP – a control protein of interest – using a CFPS reaction. EXAMPLES Overview

[0194] The following Examples discuss modifications to E. coli host cells developed for reagent T7 RNAP manufacturing. The modifications enhance cellular health and productivity of T7 RNAP overexpression. Among the host cell modifications is the overexpression of Lysozyme Y (lysY), an inhibitor of T7 RNAP. The following examples demonstrate that overexpressing lysY in host cells engineered for T7 RNAP expression increases cell heath and reduces the negative impacts of toxic, background T7 RNAP production. The following Examples are offered to illustrate, but not to limit, the claimed invention. Example 1: Materials and Methods

[0195] The following reagents were used in the Examples that follow.KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WOKILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO “Qs” = add as much as needed to achieve desired result / outcome

[0196] The following E. coli strains are discussed in the Examples that follow.KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO

[0197] Gene knock-ins. Knockins were generated using lambda Red homologous recombination as described in Figueroa-Bossi et al., Cold Spring Harbor protocols vol.2023,9 601-606.1 Sep. 2023, doi:10.1101 / pdb.top107855c.

[0198] Cloning methods. In some E. coli strains in the Examples that follow, cells were transformed with a pUC_pBAD_T7 plasmid that encodes for a T7 RNAP with an N-terminal His tag. The cells were transformed using a heat shock protocol. After recovery, cells were plated on selective Luria Broth + Kanamycin plates and grown at 37°C for 16-18 hours. Single colonies were picked from plates and used for subsequent analysis.

[0199] Cell bank strains. E. coli strains for T7 RNAP expression were developed and tested for storage in the cell bank. Cells were picked from a single colony and used to inoculate a 3-mL culture tube with I17-SF shake flask media containing 50 ^g / mL of Kanamycin. After overnight growth, the culture tube was used to inoculate a 25-mL shake flask containing the same media. The cells were grown to an optical density at 595 nm (OD595) of 3-4.80% glycerol was added to shake flask at a ratio of 1:5. The cells were then aliquoted into 2 mL culture vials and flash frozen in liquid nitrogen and stored at -80°C. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO

[0200] T7 RNAP expression in shake flasks. A culture for each E. coli strain (e.g., SBDG818, SBDG853, and SBDG867 in Example 4 below) was grown from a single colony. Each culture tube contained 2 mL of Terrific Broth + Kanamycin (50 ^g / mL) and was incubated in an incubator-shaker overnight at 37°C with shaking at 250 revolutions per minute (rpm) for 16-18 hours. Each overnight culture was used to inoculate a larger test expression culture of 50 mL Terrific Broth + Kanamycin (50 ^g / mL) in a 250 mL flask to a starting OD595 of 0.1. The cells were grown until they reached an OD595 of 1.5-2.0, at which point arabinose was added to 0.2% to induce protein expression. Flasks were returned to an incubator-shaker set to 37°C with shaking at 250 rpm.

[0201] After a 2 to 3-hour expression period, cells were harvested by centrifugation in 50 mL Falcon tubes at 4°C and 7,000 x g for 5 minutes. Each cell pellet was weighed then resuspended in S30-5 buffer at a ratio of 5 mL per gram of wet cell pellet. Cells were centrifuged again at 4°C and 7,000 x g for 5 minutes then frozen prior to lysis.

[0202] The frozen cell pellets were thawed on ice then resuspended in S30-5 buffer at a ratio of 5 mL per gram of wet cell pellet. Cells were sonicated on ice for 1 minute total with pulses of 10 seconds-on and 10 seconds-off using an amplitude of 100%. After sonication, the whole cell lysates were centrifuged at 4°C and >20,000 x g for 10 minutes to pellet insoluble components. The soluble lysates were removed and stored in new tubes.

[0203] Cell growth and T7 RNAP expression in high density fermentation. The fermentation process began by taking a 2 mL cell bank vial and inoculating a shake flask with I17-SF shake flask media containing 50 ^g / mL of Kanamycin at about 8% (volume of solute / volume of solution; v / v.) seeding density. A second stage shake flask was started for overnight growth targeting an OD595of 2-4. This flask culture was used to inoculate a 250 mL bioreactor at a seeding density of 9% (v / v) in batch media, which consists of 50 ^g / mL of Kanamycin, 0.1% (v / v) A204 antifoam, and 3.2% (v / v) 5x I17 Media in DI H2O. The bioreactor temperature, dissolved oxygen, and pH setpoints were 30°C, 30%, and 7, respectively. Once the cells grew to an OD595 of 3-5 in the batch phase, the fed-batch phase began by feeding 5x I17 media at an exponential rate of 0.215 h-1. After 12 hours in fed-batch phase, the temperature of the bioreactor was increased to 37°C. An hour later, the induction phase began by adding L-Arabinose to a target concentration of KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO 4 g / L based on the current volume of the bioreactor and reducing the feed to a constant rate of 0.2 h-1. The induction phase was 3 hours before the cells were harvested. At the end of the fermentation, the culture was collected and centrifuged at 18,592 x g and 2-8°C for 20 minutes in a floor centrifuge. The supernatant was discarded and the cell pellets were frozen at -80°C.

[0204] Strains were also evaluated with a fermentation process utilizing a 25% reduced feed rate. With this process, the fed-batch phase began with feeding 5x I17 media at an exponential rate of 0.16125 h-1. After 17 hours in fed-batch phase, the temperature was increased to 37°C. An hour later, the induction phase began by addition of L-arabinose to a target concentration of 4 g / L based on the current volume of the bioreactor and reducing the feed to a constant rate of 0.15 h-1.

[0205] Production of T7 RNAP reagent lysate. Frozen cell pellets were thawed and washed by resuspension in S30 buffer at a ratio of 1:5 (weight of solute / weight of solution; w / w). The resuspended cells were centrifuged at 18,592 x g and 2-8° C for 20 minutes in a floor centrifuge. The supernatant was discarded, and the cell pellets were resuspended in S30-5 buffer at a ratio of 1:5 (w / w). The cell resuspension was passed through an Avestin Homogenizer (Emulsiflex-C5) twice at 14,000 psi to disrupt the cells and to generate the crude T7 RNAP reagent lysate. The crude lysate was clarified by centrifuging at 18,592-30,000 x g and 2-8° C for 30 minutes in a floor centrifuge. The supernatant was collected and centrifuged again at 18,592-30,000 x g and 2- 8° C for 30 minutes in a floor centrifuge. The clarified T7 RNAP reagent lysate was aliquoted, and then flash frozen in liquid nitrogen and stored at -80°C.

[0206] Cell-free protein synthesis. T7 RNAP activity in a T7 RNAP reagent lysate was assessed using a CFPS reaction to produce GFP. In this reaction, the T7 RNAP reagent lysate was the sole source of T7 RNAP. Each cell free reaction contained: (1) T7 RNAP lysate (37.5%), (2) GFP plasmid DNA containing the GFP coding sequence behind a T7 promoter (2 ^g / mL), (3) pyruvate (35 mM), (4) Mg glutamate (7.95 mM), and (5) a mixture of nucleotide monophosphates, amino acids, and other small molecules as previously described (Zawada et al., 2011). A master mix containing all components except the T7 RNAP lysate was generated and held on ice until the start of the assay.

[0207] Separately, a serial dilution of each T7 RNAP lysate was generated using S30-5 buffer so that each CFPS reaction would have a final concentration of 0.03125-2.5% T7 RNAP lysate while KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO keeping the volume of S30-5 constant at 5%. The master mix (95 ^L) was then aliquoted into individual wells of a UV-transparent clear 96-well plate held on ice. Finally, 5 ^L of diluted T7 RNAP reagent lysate was added to each well. Each lysate was tested in 3 separate replicates (one well per replicate) in the same experiment. The plate was covered with a breathable plate cover and transferred to a Tecan F200 Infinite Pro plate reader for kinetic analysis of the reaction. The plate reader was set to read at two sets of wavelengths (488 / 510 nm and 470 / 530 nm) for 17 hours with orbital shaking at an amplitude of 1 mm. The temperature was set to 25°C + / - 2°C throughout the reaction. At the end of the 17-hour incubation, data was analyzed by plotting GFP signal vs. time. GFP signal at the final timepoint was representative of T7 RNAP activity, so endpoint data was used as a readout of T7 RNAP lysate activity.

[0208] T7 RNAP expression in a midscale bioreactor. E. coli strains capable of expressing T7 RNAP were grown on an LB plate containing Kanamycin at 15-30°C. Once single colonies were formed, a single colony was picked and transferred in 7 mL of I17-SF Shake Flask Media in a 50 mL conical tube and then grown overnight at 30°C and 250 RPM in a shaker with an orbital diameter of 25 mm. Once the OD595 of the cells reached between 2-4, the cells were inoculated at a seeding density of 1.5-3.5% v / v in I17-SF Shake Flask Media. Components of I17-SF Shake Flask Media are disclosed in the tables above. All solutions in the tables were dissolved in DI H2O or water of better quality. Then, the cells in the shake flasks were grown at 30°C and 250 RPM in a shaker with an orbital diameter of 25 mm. Once reaching an OD595of 2-3, the cells were diluted down to 20% v / v in an 80% glycerol stock solution, mixed well for at least 30 seconds, aliquoted into 1 mL vials, flash frozen and stored in a -80°C freezer.

[0209] The fermentation process began by inoculating a shake flask with I17-SF Shake Flask Media with a cell bank inoculum at a 1:35 ratio of inoculum to a total starting volume (seeding density should be between 0.057-0.086 OD595). Once the cells reached an OD595 of 2-5, the cells were inoculated again into a second seed stage shake flask with I17-SF Shake Flask Media at an inoculation OD595 of around 0.007-0.07. Once the cells of the second stage seed flask reached an OD595 of 2-5, the batch phase began by transferring the cells from the shake flask and inoculating a 500 mL Biostat Qplus or 1L Biostat B bioreactor at a seeding density of 2.2% v / v in batched media, which consists of 0.1% v / v of P2000 antifoam and 3.2% v / v of 5x I17 Media in DI H2O. The target starting volume of the bioreactor was 220 mL for the Biostat Qplus and 500 mL for the KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO Biostat B. Components of 5x I17 Media Solution are disclosed in the tables above. All solutions in the tables were dissolved in DI H2O or water of better quality. The bioreactor setpoints for temperature, dissolved oxygen, and pH were 30°C, 50%, and 7 (deadband = 0.05), respectively. Once the cells grew to an OD595 between 2-5 after 6-9 hours in the batch phase, the fed-batch phase began by feeding the cells with 5x I17 Media using an exponential rate parameter of 0.13 h-1. The temperature setpoint was downshifted from 28°C to 30°C as well.

[0210] After 22 hours of the fed-batch phase, the temperature setpoint of the bioreactor was increased to 34°C, the exponential feed rate parameter was decreased to 0.07 h-1, and the feed rate was set to constant rate. An hour later, the induction phase began - a stock solution of 400 g / L L- Arabinose was added to a target concentration of 14.45 %w / w based on the starting volume of the bioreactor. For example, for starting volume of 500 mL, 7.23 mL of 400 g / L Arabinose was added to the bioreactor.

[0211] After 3 hours in the induction phase, the fermentations were harvested.1 ml from each culture was used for measuring viscosity of the culture. The viscosity was measured using a microVISC viscometer (RheoSense, San Ramon CA). The rest was collected in centrifuge bottles, mixed with S30-2 wash buffer at a 1:2 buffer to cell weight ratio, and spun down in a floor centrifuge at 18,592 x g and 2-8° C for 15 min. After the wash, the supernatant was discarded, and the cells were resuspended with S30-5 buffer at a 5:1 buffer to cell weight ratio. The cell resuspension was then passed through an Avestin Homogenizer (EmulsiFlex-C5) at 15000-17,000 Psi, twice, to disrupt the cells and generate crude lysate. The crude lysate was further clarified by floor centrifugation at 13,000-15,000 x g and 2-8° C for 30 minutes. The supernatant (clarified lysate) was collected and aliquoted in 50-mL falcon tubes, flash frozen and stored in -80°C. Example 2: Plasmid pUC_pBAD_T7 for Expression of T7 RNA Polymerase (T7 RNAP)

[0212] This Example discusses creating the pBAD-T7-His plasmid for T7 RNAP expression. High level expression of T7 RNAP requires expression from a tight promoter system and the use of a high copy origin of replication. The T7 RNAP gene with a N-terminal his tag (His Tag) (T7- His) was cloned into a low background pBAD vector with an AraBAD promoter (pBAD). This plasmid also had the gene for the arabinose sensitive response regulator, araC, a pUC high copy origin of replication, and a kanamycin resistance marker. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO Example 3: E. coli Strains for T7 RNA Polymerase Production i. SBDG347 Base Strain

[0213] The E. coli strain SBDG347 was used as the base strain for E. coli reagent strains for T7 RNAP production, as well as the control strains and other strains discussed in the Examples that follow. The strain names, their genotypes, and their descriptions are shown in Table 6 above.

[0214] In SBDG347, T7 RNAP was driven by the BAD (araB, araBAD) promoter (pBAD). The ompT was gene deleted so that the T7 RNAP that was expressed by the cell did not get cleaved by the OmpT protein. ii. SBDG804 – For Expression of T7 RNAP with PDF

[0215] The gene for peptide deformylase, PDF, with a constitutive pMTL promoter was knocked into the gntT genomic locus of SBDG347. This step produced strain SBDG804. PDF was introduced to the strain because it may mitigate acetate toxicity during T7 RNAP production by providing a sink for overflow metabolism products. iii. SBDG804, SBDG818 and SBDG867 – For Expression of T7 RNAP with PDF and LysY

[0216] In SBDG804, the lysY gene was knocked into the gor genomic locus with a pMTL promoter, producing strain SBDG805. SBDG805 was capable of co-expressing PDF and lysY. The effect of lysY on T7 RNAP overexpression is discussed in these Examples.

[0217] To produce strain SBDG818, strain SBDG805 was transformed with the pUC_pBAD_T7 plasmid of Example 2 (above). SBDG818 was capable of expressing T7 RNAP, PDF, and lysY. The T7 RNAP was expressed from the plasmid has an N-terminal His tag.

[0218] To produce strain SBDG867, strain SBDG805 was modified. First, the genomic copy of the T7 RNAP gene in SBDG805 was deleted to produce strain SBDG866. Then, SBDG866 was transformed with the pUC_pBAD_T7 plasmid of Example 2 (above) to produce strain SBDG867. SBDG867 is capable of expressing T7 RNAP, PDF, and lysY. The T7 RNAP in the cell was expressed from the pUC_pBAD_T7 plasmid and not from the genome, and the T7 RNAP had an N-terminal His tag. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO iv. SBDG825 – For Expression of T7 RNAP with LysY

[0219] Strain SBDG825 was also produced from the base strain SBDG347. Here, a lysY overexpression cassette was inserted into the gor locus of the SBDG347 genome. SBDG825 did not have the pUC_pBAD_T7 expression plasmid. Thus, in SBDG825, T7 RNAP was expressed only from its genome. v. SBDG853 – For Expression of of T7 RNAP with LysY and PDF

[0220] Strain SBDG853 was produced by transforming strain SBDG847 with the pUC_pBAD_T7 expression plasmid. SBDG853 is similar to SBDG867 except that SBDG853 does not express PDF. SBDG853 (with PDF) has higher acetate production than SBDG867 (without PDF). vi. SBDG847 – A Control Strain that Does Not Express T7 RNAP

[0221] Strain SBDG847 was produced from the base strain SBDG825. Here, the chromosomal copy of the T7 RNAP gene has been deleted. SBDG847 did not have the pUC_pBAD_T7 expression plasmid. Thus, SBDG847, has no T7 RNAP genes anywhere in the cell. Example 4: T7 RNA Polymerase (T7 RNAP) is Produced in Strains SBDG818, SBDG853, and SBDG867 in Shake Flasks

[0222] This Example discusses testing E. coli strains SBDG818, SBDG853, and SBDG867 for expression of T7 RNAP with lysY. As discussed in Example 3 and in Table 6 above, SBDG818 expressed T7 RNAP from the chromosome and the pUC_pBAD-T7 plasmid, while SBDG853 and SBDG867 expressed T7 RNAP from pUC_pBAD_T7 plasmid only. In all three strains, T7 RNAP that was expressed from the pUC_pBAD_T7 plasmid had an N-terminal His tag.

[0223] T7 RNAP expression of SBDG818, SBDG853, and SBDG867 were tested in shake flasks as disclosed in Example 1 above. The clarified, soluble lysates of SBDG818, SBDG853, and SBDG867 were analyzed for the presence of T7 RNA polymerase using reducing SDS-PAGE (FIG.1).

[0224] T7 RNAP was expressed in the SBDG818 and SBDG867 strains, which are both strains that co-expressed T7 RNAP with PDF and lysY. T7 RNAP was also expressed in SBDG853 KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO strain, which co-expressed T7 RNAP with lysY. T7 RNAP expression was greater in SBDG818 and SBDG867 (the strains with PDF) than in SBDG853 (the strain without PDF). As shown by SDS-PAGE (FIG.1), the presence of PDF increased the amount of T7 RNAP that was expressed. SDS-PAGE analysis provided relative levels of T7 RNAP expression that can be expected from fed-batch fermentations. Example 5: Long-term Stability of the T7 RNAP Strains SBDG818 and SBDG867

[0225] Stability testing of the T7 RNAP strains SBDG818 and SBDG867 was conducted by propagation of the strains through consecutive rounds of growth in shake flasks. Light microscopy was used to observe cell morphology. SBDG818 did not show any detectable elongated cell morphology after 30 generations of propagation from the cell bank. SBDG867 exhibited a low proportion of elongated cells after 30 generations of propagation from the cell bank. Example 6: Expression of T7 RNAP with LysY and PDF Produced Reagent Lysates with High Amounts of T7 RNAP

[0226] T7 RNAP reagent lysates were produced according to methods discussed in Example 1 above and analyzed for T7 RNAP expression by SDS-PAGE. T7 RNAP reagent lysates were produced from strains SBDG818, SBDG853, and SBDG867. Briefly, the strains were cultured at high density using a fed-batch fermentation protocol. Cells were harvested from the fermentations and T7 RNAP reagent lysates were produced from those cells.

[0227] As shown by SDS-PAGE (FIG.2), strain SBDG818 (which expressed T7 RNAP with PDF and lysY) produced higher levels of T7 RNAP than the control strain SBDG350 (without PDF and lysY).

[0228] SDS-PAGE also revealed that strain SBDG853, which had expression of lysY from its genome and T7 RNAP from the pBAD-T7-His plasmid only, had low levels of T7 RNAP when fed-batch growth rate of the fermentation was targeted at 0.2 ^ (SBDG853 (A) FIG. 3). This indicated that removal of the genomically-expressed T7 RNAP was not enough to overcome T7 RNAP toxicity associated with T7 RNAP expression from the plasmid at high cell density. However, when the fermentation fed-batch growth rate was reduced by 25%, SBDG853 was able to produce high levels of T7 RNAP (SBDG853 (B) in FIG.3). KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO

[0229] Strain SBDG867 (which expressed T7 RNAP with PDF and lysY) produced high levels of T7 RNAP in fed-batch conditions with the standard growth rate of 0.2 ^ (FIG.2).

[0230] The results demonstrate that expression of T7 RNAP with PDF and lysY produced larger amounts of T7 RNAP than when PDF was absent. Example 7: Reagent T7 RNAP Bacterial Lysates Produced Green Fluorescent Protein (GFP) in Cell-Free Protein Synthesis Reactions

[0231] The activities of reagent T7 RNAP bacterial lysates generated from strains SBDG818, SBDG853, and SBDG867 were confirmed. As discussed in Example 1 above, GFP was produced using CFPS reactions that contained reagent T7 RNAP bacterial lysate from each of these strains. The amount of fluorescence signal produced by the GFP that was synthesized using lysates from each strain was determined (FIGS.4 and 5). Fluorescence signal at the final timepoint corresponded to T7 RNAP activity. Thus, endpoint data was used as a readout of T7 RNAP lysate activity. An SBDG178 cell lysate, which has high T7 RNAP activity, was used as control lysate.

[0232] Strain SBDG818 produced high levels of GFP fluorescence, which indicated that it produced T7 RNAP bacterial lysate with high activity. This result was observed when SBDG818 was grown using a normal fed-batch feed rate (0.2 ^) and a 25% reduced fed-batch feed rate (FIG. 5). Strain SBDG853 showed equivalent activity when grown using a 25% reduced feed rate (FIG. 5).

[0233] Strain SBDG867 also produced high levels of GFP fluorescence, indicating that its T7 RNAP lysate had high activity (FIG. 4). In contrast, the base strain SBDG350 showed very low levels of GFP fluorescence, which indicated that it produced T7 RNAP lysate with low activity (FIG. 4).

[0234] These results demonstrate that E. coli strains that express T7 RNAP with lysY and PDF can produce reagent T7 RNAP bacterial lysate with high T7 RNAP activity. Example 8: T7 RNAP Expression in a Midscale Bioreactor

[0235] Expression of T7 RNAP was performed in a midscale bioreactor as disclosed in Example 1 above. The starting volume for a midscale bioreactor was from 200 mL to 500 mL. The strains were SBDG178, SBDG818, and SBDG867 (Table 6). SBDG818 expresses T7 RNAP from KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO the chromosome and from the pUC_pBAD_T7 plasmid; PDF and lysY are co-expressed. SBDG867 expresses T7 RNAP from the pUC_pBAD_T7 plasmid only; PDF and lysY are co- expressed.

[0236] SBDG178 was used as the control strain in this Example because it was a strain that did not express lysY. Previously, it was determined that during T7 RNAP production, SBDG178 formed long concatenated cells due to incomplete cell division and required specialized shake flask media to suppress these morphological changes. In this Example, mid-scale bioreactor production of T7 RNAP of SBDG867 and SBDG818 were compared to that of SBDG178 to determine whether the presence of lysY had an impact on fermentation oxygen requirements, final OD595, final broth viscosity, and final yield.

[0237] During the fermentation, SBDG178 required more oxygen than the lysY-expressing strains SBDG818 and SBDG867. FIG.6 shows that SBDG178 required aeration with pure oxygen late in the fermentation (bottom panel) to maintain sufficient dissolved oxygen levels (top panel). Even when pure oxygen was supplied (bottom panel), levels of dissolved oxygen levels in the SBDG178 tank dropped to 0% for an hour-long period (top panel). In contrast, strains SDG818 and SDG867, which expressed lysY, were able to maintain the dissolved oxygen setpoint of 30% using a maximum enrichment with pure oxygen of 60% or less. In addition, neither of these vessels went anoxic during the fermentation.

[0238] After 3 hours of induction, the fermentations were harvested as disclosed in Example 1 above. SBDG818 and SBDG867 each had a final OD595 that was almost 20% higher than the OD595 for SBDG178 (Table 7). All three strains were tested for T7 RNAP activity as disclosed in Example 1 above; all three strains had acceptable levels of T7 RNAP activity. The threshold of acceptable T7 RNAP activity in a lysate was defined as > 0.2 Sutro T7 units (STGU) / mL lysate. The most striking difference between SBDG178 and SBDG818 or SBDG867 was the final viscosity in the harvested broth. The broth for SBDG178 was 4 times more viscous, possible due to cell lysis. This elevated viscosity could potentially complicate downstream process, including centrifugation and filtration.KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO

[0239] The results demonstrate that the co-expression of lysY and PDF during the T7 RNAP production (i.e., strains SBDG818 or SBDG867) produced a higher final OD595and substantially reduced the final viscosity of the production culture when compared to T7 RNAP production in the absence of lysY (SBDG178). All three strains produced acceptable amounts of T7 RNAP, but the lysY expressing strains (SBDG818 or SBDG867) were much healthier as evidenced by the higher OD595 and lower final viscosity in Table 7 and lower oxygen demand seen in FIG. 6. This greatly simplifies the upstream and downstream process for T7 RNAP production. Example 9: Large Scale Manufacturing of T7 RNAP with E. coli Strain SBDG178

[0240] In this Example, E.coli strain SBDG178 was used for the production of T7 RNAP at 1,500 L scale. As described in Table 6 above and in the preceding Examples, SBDG178 expresses T7 RNAP from the chromosome and does not express lysY. The production fermentation started with a batch phase followed by a fed-batch phase and an induction phase where T7 RNAP was over-expressed. Previously, the T7 RNAP was expressed at 10 L-scale (Run 10 L-5 and Run 10 L- 6). However, during the first 1,500 L-run, the OD595plummeted shortly after the induction (“RNAP1 GMP” in FIG.7) with no detectable T7 RNAP expression as determined by T7 RNAP activity in the lysate (Table 8).

[0241] Failure mode effect analysis (FMEA) revealed that the strain was sensitive to shear forces in the fermentation. As shown in FIG.8, the maximum fermenter impeller tip speed is negatively correlated with T7 RNAP activity. Peak tip speed in the 1,500 L fermenter (“1500L” in FIG.8) was higher than peak tip speed at bench-scale (“0.5L” and “10L” in FIG.8), which resulted in growth stall and no product expression. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO

[0242] The failed run was reproduced by running SBDG178 in a 30 L fermenter with the peak tip speed set at 6.6 m / s to mimic the 1,500 L conditions. The other two 30 L runs with 2.2 m / s and 3.8 m / s peak tip speeds had similar and expected growth profiles – the cultures reached peak OD595 upon induction and peak OD595 was maintained until towards the end of the fermentation (“D302.2 m / s” and “D303.8 m / s” in FIG.9). However, the run with 6.6 m / s peak tip speed (“D30 6.6 m / s” in FIG.9) had a growth curve that deviated from the other two 30 L runs during early fed-batch phase. The growth for the run with 6.6 m / s peak tip speed never caught up with the pre- determined feed rate. The fermentation was pre-maturely terminated due to accumulation of glucose and acetate.

[0243] Additional 1 L satellite runs were set up in parallel with the 30 L runs. Due to the limitation of the impeller size, the maximum tip speed in the 1 L fermenter was 4.7 m / s. Even at this lower tip speed, deviated growth was observed, similar to the GMP1 run, although the run was completed after a full induction phase. Both end OD595and T7 RNAP activity were significantly lower than expected (Table 9). Therefore, it was determined that a lower peak tip speed should be used for SBDG178 during production fermentation to maximize the T7 RNAP production.*Runs with 30% DO setpoint. All other runs with 50% DO setpoint.

[0244] During GMP1 fermentation, which failed to produce T7 RNAP, dissolved oxygen (DO) was controlled by a cascade of agitation and a cascade of pure oxygen while constant air flow was KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO maintained (Table 10). Peak agitation speed reached 450 rpm which is equivalent to 8.6 m / s tip speed. In order to produce T7 RNAP at the 1,500 L scale in GMP2, agitation speed had to be maintained at a low level because SBDG178 was sensitive to shearing. It was determined that 200 rpm (3.8 m / s tip speed) was the minimum agitation speed that can be used for production fermentation while providing sufficient mixing of the culture.

[0245] In general, process methods for controlling DO in E. coli fermentations include increasing agitation, followed by increasing air flow and / or enrichment with pure oxygen. The new DO control strategy proposed and tested here was fixed agitation at 200 rpm, and cascading air flow, cascading back pressure, and cascading pure oxygen flow to maintain cell growth and ensure stable T7 RNAP expression. Since implementation of the new DO control strategy, three large-scale / commercial batches have been successfully manufactured with reproducible cell growth (FIG.10) and T7 RNAP activity (average 0.5 STGU / mL).

[0246] There are alternative ways to grow a shear-sensitive strain in addition to changing the DO control strategy mentioned above. First, use of a shear protectant agent in the E.coli culture, e.g., Pluronic® L 61 or Pluronic® F-68. Pluronic® L 61 was tested in the 1 L fermentation at 0.1% and 1% concentrations. The peak tip speed was kept at 4.7 m / s. As shown in Table 11 below, both concentrations were effective in maintaining higher final OD595 and achieved higher T7 RNAP expression compared to the control. The sliminess of the cell pellets was noticeably reduced (FIG. 11). Second, T7 RNAP expression can be induced at a lower cell density. Other possible ways include reducing the medium feed rate and / or reducing fed-batch temperature to slow down cell growth. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO

[0247] From a strain engineering perspective, control of leaky T7 RNAP expression of the production strain during fed-batch growth phase is important so that toxicity of T7 RNAP has minimal impact on cell health. As discussed in the preceding Examples, production strains capable of expressing lysY (see, e.g., strains SBDG818 and SBDG867 in Table 6) were engineered – overexpression of lysY in host cells engineered for T7 RNAP expression increased cell health and reduced the negative impacts of toxic, background T7 RNAP production. The large-scale manufacturing parameters discussed in this Example may apply to large-scale T7 RNAP production using lysY-expressing strains to reduce viscosity of the strain in liquid culture. Midscale manufacturing of T7 RNAP using SBDG867 is discussed in Example 10 below. Example 10: Midscale Manufacturing of T7 RNAP with E. coli Strain SBDG867 at High Tip Speed

[0248] This Example relates to the evaluation of the E. coli strain SBDG867 for T7 RNAP expression at higher impeller tip speeds that are typically observed in large-scale manufacturing. FIG.12 shows impeller tip speeds (m / s) that are often associated with bioreactor stir rates (rpm) for fermentation volumes of 0.25 L (“7” in FIG.12), 0.5 L (“1” in FIG.12), 1 L (“2” in FIG.12), 10 L (“3” in FIG. 12), 30 L (“5” in FIG. 12), 200 L (“6” in FIG.12), and 1,000 L (“4” in FIG.12). In large-scale manufacturing, e.g., in a 1,000 L fermentation, impeller tip speeds of up to 8.5 m / s are typically observed (“4” in FIG. 12). In this Example, fermentations were carried out at the 250 mL-scale with the Sartorius Ambr® 250 system (“7” in FIG.12), which allows for tip speeds KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO of up to 7 m / s and higher, such that strain tolerance for high tip speeds approaching 8.5 m / s can be analyzed.

[0249] Two strains were used in this Example – SBDG867 and SBDG178. As discussed above, SBDG867 expresses T7 RNAP from the pUC_pBAD_T7 plasmid. SBDG867 also expresses PDF and lysY. SBDG867 was evaluated and compared to SBDG178 as the control (parental) strain. SBDG178 expresses T7 RNAP from the chromosome and does not express lysY. See Table 6 above for detailed strain descriptions. As discussed in Example 9 above, SBDG178 did not produce T7 RNAP at the 1,500 L scale.

[0250] Fermentations for both strains were carried out in identical conditions except for impeller tip speeds. Maximum impeller tip speeds were as shown in Table 12 below. Each fermentation started with a batch phase followed by a fed-batch phase and an induction phase when T7 RNAP was over-expressed.

[0251] The results show that strain SBDG867 (i.e., the strain that overexpresses LysY) was more tolerant of higher impeller tip speeds than the control strain SBDG178. As shown in FIG. 13, high OD595was maintained by SBDG867 at all tips speeds (“4,” “5,” and “6” in FIG.13 for 2.67 m / s, 4.71 m / s, and 7.07 m / s, respectively). In contrast, OD595for SBDG178 decreased with increasing tip speed (“1,” “2,” and “3” in FIG.13 for 2.67 m / s, 4.71 m / s, and 7.07 m / s, respectively). At a maximum impeller tip speed of 7.07 m / s, SBDG867 had a higher final OD595 of 102 compared to a final OD595 of 66 for SBDG178 (see Table 12 below), which indicates that the SBDG867 cells did not lyse at the tip speed of 7.07 m / s.KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO

[0252] The amounts of T7 RNAP that were present in the broth for each fermentation were analyzed. As shown in FIG. 14, SBDG178 (the control strain) fermentations had high levels of T7 RNAP in the broth, which indicates that the cells had lysed and released the overexpressed T7 RNAP into the broth (“1,” “2,” “3,” and “4” in FIG. 14). In contrast, SBDG867 had lower levels of T7 RNAP in the broth, which indicates that the cells were tolerant of high tip speeds and did not lyse, thereby keeping the overexpressed T7 RNAP within the cells (“5,” “6,” “7,” “8,” “9,” and “10” in FIG. 14). Further, at 7.07 m / s tip speed, the lysate from SBDG867 had a higher T7 RNAP activity of 0.61 STGU / mL compared to 0.08 STGU / mL for the lysate from SBDG178. (In Table 12, compare Run IDs 240320-A3 and 240320-A6.)

[0253] In conclusion, the results indicate the SBDG867 was able to withstand higher impeller tip speeds (e.g., up to 7.07 m / s) while the SBDG178 parental strain could not. (In Table 12, compare Run IDs 240320-A3 and 240320-A6.) Because SBDG867 expressed lysY in addition to T7 RNAP, whereas SBDG178 did not express lysY, the results indicate that lysY overexpression provided resistance to higher impeller tip speeds and improved the health of the T7 expression strain SBDG867. Therefore, the SBDG867 strain provides an advantage over other strains for large-scale manufacturing of the reagent T7 RNAP. INFORMAL SEQUENCE LISTING

[0254] Unless noted otherwise, amino acid sequences are shown in the N-terminus to C- terminus direction and nucleic acid sequences are in the 5’ to 3’ direction. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WOKILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WOKILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WOKILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WOKILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO

[0255] All publications, issued patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0256] It is to be understood that this disclosure is not limited to the particular methodology, protocols, cell lines, animal species or genera, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present disclosure, which will be limited only by the appended claims. KILPATRICK TOWNSEND 778804912

Claims

PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO WHAT IS CLAIMED IS:

1. A host cell comprising: a) a polynucleotide sequence encoding a lysozyme and b) a polynucleotide sequence encoding a T7 RNA polymerase (T7 RNAP), wherein the polynucleotide sequence encoding the T7 RNAP is on a plasmid.

2. The host cell of claim 1, wherein the lysozyme comprises a lysine to tyrosine substitution at position 128 relative to a sequence of SEQ ID NO:

6.

3. The host cell of claim 1 or 2, wherein the lysozyme comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:

6.

4. The host cell of any one of claims 1-3, wherein the lysozyme comprises a sequence of SEQ ID NO:

4.

5. The host cell of any one of claims 1-4, wherein the polynucleotide sequence encoding the lysozyme is in the host cell genomic sequence.

6. The host cell of claim 5, wherein the polynucleotide sequence encoding the lysozyme is in gor locus of the host cell genomic sequence.

7. The host cell of claim 5, wherein the polynucleotide sequence encoding the lysozyme is in gntT locus of the host cell genomic sequence 8. The host cell of any one of claims 1-7, wherein the polynucleotide sequence encoding the lysozyme is driven by a constitutive promoter.

9. The host cell of claim 8, wherein the constitutive promoter driving the polynucleotide sequence encoding the lysozyme is an MTL promoter (pMTL).

10. The host cell of any one of claims 1-9Error! Reference source not found., wherein the plasmid is a high copy pUC vector. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO 11. The host cell of any one of claims 1-10Error! Reference source not found., wherein the polynucleotide sequence encoding the T7 RNAP is driven by an inducible promoter.

12. The host cell of any one of claims 1-11, wherein the polynucleotide sequence encoding the T7 RNAP is driven by arabinose-sensitive response regulator, araC.

13. The host cell of any one of claims 1-12, further comprising a polynucleotide sequence encoding a peptide deformylase (PDF).

14. The host cell of claim 13, wherein the polynucleotide sequence encoding the PDF is in the host cell genomic sequence.

15. The host cell of claim 14, wherein the polynucleotide sequence encoding the PDF is in gntT locus of the host cell genomic sequence.

16. The host cell of claim 14, wherein the polynucleotide sequence encoding the PDF is in the gor locus of the host cell genomic sequence.

17. The host cell of any one of claims 13-16, wherein the polynucleotide sequence encoding the PDF is driven by a constitutive promoter.

18. The host cell of claim 17, wherein the constitutive promoter driving the polynucleotide sequence encoding the PDF is an MTL promoter (pMTL).

19. The host cell of any one of claims 1-18, wherein expression of ompT gene is disrupted.

20. The host cell of any one of claims 1-19, wherein the host cell is a bacterial cell.

21. The host cell of claim 20, wherein the bacterial cell is Escherichia coli.

22. A method of producing T7 RNAP comprising: KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO a) culturing the host cells of any one of claims 1-21 under conditions that permit transcription of the lysozyme polynucleotide sequence and expression of the lysozyme; and b) applying an induction signal to the host cells to induce transcription of the T7 RNAP polynucleotide sequence and expression of the T7 RNAP.

23. A method of producing a bacterial cell extract comprising: a) culturing the host cells of any one of claims 1-21 under conditions that permit transcription of the lysozyme polynucleotide sequence and expression of the lysozyme; b) applying an induction signal to the host cells to induce transcription of the T7 RNAP polynucleotide sequence and expression of the T7 RNAP; and c) isolating and disrupting the host cells to produce the bacterial cell extract; wherein the bacterial cell extract comprises T7 RNAP.

24. A method of producing T7 RNAP comprising: a) culturing host cells comprising a polynucleotide sequence encoding the T7 RNAP; b) applying an impeller tip speed; c) controlling dissolved oxygen (DO) levels by increasing the impeller tip speed, cascading air flow, cascading back pressure, and / or cascading pure oxygen flow; and d) applying an induction signal to the host cells to induce transcription of the T7 RNAP polynucleotide sequence and expression of the T7 RNAP; wherein the impeller tip speed does not exceed 8.0 m / s.

25. A method of producing a bacterial cell extract comprising: a) culturing host cells comprising a polynucleotide sequence encoding the T7 RNAP; b) applying an impeller tip speed; c) controlling dissolved oxygen (DO) levels by increasing the impeller tip speed, cascading air flow, cascading back pressure, and / or cascading pure oxygen flow; d) applying an induction signal to the host cells to induce transcription of the T7 RNAP polynucleotide sequence and expression of the T7 RNAP; and e) isolating and disrupting the host cells to produce the bacterial cell extract; KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO wherein the impeller tip speed does not exceed a 8.0 m / s, and the bacterial cell extract comprises T7 RNAP.

26. The method of claim 24 or 25, wherein the impeller tip speed does not exceed 5.0 m / s.

27. The method of any one of claims 24-26, wherein the host cells are the host cells of any one of claims 1-23.

28. The method of any one of claims 22-27, wherein step a) further comprises: (a) use of a shear protectant agent; (b) inducing T7 RNAP expression at lower cell density; (c) a reduced medium feed rate; (d) a reduced fed-batch temperature; or (e) any combination thereof.

29. The method of claim 23 or 25, wherein the bacterial cell extract is able to synthesize a target protein with a titer of at least 80% relative to a control bacterial cell extract after storage at about -20°C, about 2°C to 8°C, or about 20°C to 24°C; and for at least 6, 12, or 18 months.

30. The method of any one of claims 22, 23, or 27, wherein at step a), the host cells demonstrate reduced growth defects before T7 RNAP expression is induced.

31. The method of any one of claims 22-30, wherein the induction signal is heat or a chemical.

32. The method of any one of claims 22, 23, or 27-31, wherein the host cells achieve a higher final OD595when compared to host cells that do not express lysozyme with T7 RNAP expression.

33. The method of any one of claims 22, 23, or 27-32, wherein the host cells achieve a lower culture viscosity when compared to host cells that do not express lysozyme with T7 RNAP expression. KILPATRICK TOWNSEND 778804912PATENT Attorney Docket No.091200-1456899-007210PC Client Ref. No.0219WO 34. The method of any one of claims 22-33, wherein the induction signal is arabinose.

35. A composition for cell-free protein synthesis comprising a bacterial cell extract produced according to the method of any one of claims 22-34.

36. A method of cell-free protein synthesis comprising the composition of claim 35.

37. The method of any one of claims 22-36, or the composition of claim 35, wherein the T7 RNAP has high activity. KILPATRICK TOWNSEND 778804912

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