An inducible expression system for plasmid-free production of proteins of interest

Abstract

A genome-based expression system for producing a protein of interest (POI) in a prokaryotic host, comprising at least an RNA polymerase (RNAP) gene, a gene encoding a protein of interest, which comprises a coding sequence, a promoter operably linked to the coding sequence (wherein the promoter is recognized by an RNA polymerase expressed from the RNA polymerase gene), and at least one lac operator (lacO) within the sequence of the promoter; and a lacI gene encoding a lac repressor protein (Lacl), which comprises a coding sequence, a lacI promoter operably linked to the lacI coding sequence (wherein the lacI promoter is a wild-type lacI promoter or a lacI promoter that increases Lacl expression, wherein the expression rate of the protein of interest is regulated by an inducer that binds to Lacl).

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to the field of plasmid-free inducible systems for the expression of a protein of interest in a prokaryotic host. The present invention further relates to methods of using such systems for the production of a protein of interest in a prokaryotic host.

[0002] 2. Background of the Invention In industrial protein production processes, gene regulation is a key requirement. Transcription rate is controlled by the interaction of the promoter with RNA polymerase (RNAP). Understanding and external regulation of this interaction is necessary to control the process and optimize product yield and quality. Reduced promoter strength can be beneficial, especially for difficult proteins such as antibody fragments, membrane proteins, or toxic proteins (1-3). The yield of the final product, a soluble and properly folded protein, is often not directly determined by the strength of the promoter system but rather depends on further processing of the peptide chain, such as translocation to the periplasm and proper disulfide bond formation.

[0003] The most prominent and well-studied mechanism of gene regulation is the lactose (lac) operon (4). In wild-type E. coli, the lac inhibitor (LacI) forms a homotetramer that binds to the lac-operator sequence (lacO) and represses transcription of the lacZYA operon (5). In the presence of lactose or the non-metabolizable isopropyl β-D-1-thiogalactopyranoside (IPTG), LacI changes conformation and can no longer bind to the lac-operator, resulting in induction of transcription. The lac-operator site is a DNA sequence with an inverted, symmetric repeat sequence (6).

[0004] The higher the symmetry, the higher the binding affinity of LacI to the operator sequence. An artificial, completely symmetrical lacO (sym-lacO) was found to bind LacI with the highest affinity (7), whereas the three wild-type operators lacO1, lacO2, and lacO3, which show approximate symmetry, showed lower affinities, resulting in the following order: sym-lacO>lacO1>lacO2>lacO3 (8). LacI binds simultaneously to both the first operator, lacO1, and either lacO2 or lacO3 through a DNA looping mechanism (9). LacO2 is located 401 bp downstream of lacO1, whereas lacO3 is only 92 bp upstream of lacO1 (10). The role of lacO2 remains unclear because its main contribution to repression results from DNA looping between lacO1 and lacO3 due to their closer proximity (8). Furthermore, the binding of lacO1 and lacO3 to LacI prevents the production of LacI itself. The 3' end of the lacI gene overlaps with lacO3. In the repressed state, transcription of lacI produces a truncated mRNA that is rapidly degraded by the cell. Due to this autoregulation, the concentration of LacI tetramers is about 40 molecules in induced cells and about 15 molecules in uninduced cells (11).

[0005] Several mutants of the LacI repressor protein and the pLacI promoter exist. Penumetcha et al. tested various combinations of repressor and promoter mutants in an effort to find a system with reduced transcriptional readthrough. They found that pLacI Q1 We report that use of the wild-type LacI repressor protein in combination with the promoter results in high levels of induction and low levels of transcriptional readthrough ( 34 ).

[0006] Oehler et al. tested the effect of systematic disruption of all three lac operators in the chromosomal lac operon of Escherichia coli on repression by the Lac repressor and report that the three operators of the lac operon cooperate in repression (35).

[0007] The tetrameric Lac repressor can simultaneously bind two lac operators on the same DNA molecule, which induces the formation of a DNA loop. Muller et al. report that repression increases significantly as the DNA length between the operators decreases (36).

[0008] The effects of placing the lac operator at different positions relative to the promoter of bacteriophage T7 RNA polymerase have been examined: transcription can be strongly repressed by the lac repressor bound to the operator 15 base pairs downstream from the RNA start site (37).

[0009] WO 2003 / 050240A2 discloses an expression system for producing a target protein in a host cell comprising a homologously integrated gene encoding T7 RNA polymerase and a non-integrated gene encoding the target protein.

[0010] One of the first applications of the lac regulatory mechanism was the pET system, which is today the most widely used E. coli expression system for recombinant protein production (12, 13). This system is based on the special interaction of the T7 RNA polymerase derived from T7 phage with the strong T7 promoter. The recombinase function of bacteriophage λ was used for site-specific insertion of the T7 RNA polymerase gene into the E. coli genome. Expression of the T7 RNA polymerase is controlled by the lacUV5 promoter, a variant of the lactose promoter that is insensitive to catabolic repression. Addition of isopropylthiogalactoside (IPTG) induces high levels of expression of the T7 RNA polymerase, which in turn transcribes target genes under the control of the T7 promoter. This orthogonal expression system gives very high product titers of recombinant proteins, which can be efficiently produced in E. coli. However, the extraordinary strength of the T7 expression system, especially when combined with high copy number plasmids, imposes an extreme metabolic burden on the host cell. When the gene of interest encodes a difficult protein, stress and metabolic burden often lead to reduced yields, shortened production periods, and even cell death (14, 15).

[0011] Plasmid-mediated stress effects, such as high gene dosage and transcription of antibiotic resistance genes, can be overcome by integrating a gene of interest (GOI), i.e., a gene encoding a protein of interest, into the host genome (16, 17).

[0012] WO 2008 / 142028 A1 discloses a method for producing a protein of interest, in which DNA encoding the protein of interest is integrated into a preselected site in the genome of a bacterial cell.

[0013] Striedner et al. describe a plasmid-free, T7-based Escherichia coli expression system in which target genes are integrated site-specifically into the host genome.

[0014] Genome-integrated T7-based expression systems offer significant advantages: compared to plasmid-based expression systems, there is no plasmid-mediated metabolic burden and no gene dosage variation during the production process. However, T7 RNA polymerase (RNAP) is prone to mutations under long-term production conditions. This was demonstrated by Striedner et al. (17) in chemostat cultures, where mutations in T7 RNA polymerase caused non-producing cell populations to grow more rapidly and therefore greatly reduced product yields.

[0015] Thus, there is a clear need in the art for improved inducible expression systems that provide improved expression rates, low basal expression, even at low inducer concentrations, and truly tunable expression rates at the cellular level.

[0016] Summary of the Invention It is an object of the present invention to provide an improved inducible system for plasmid-free production of a protein of interest with improved expression rate control and very low basal expression of the protein of interest.

[0017] The problem is solved by the present invention.

[0018] According to the present invention, at least a) RNA polymerase (RNAP) gene, b) a gene for the expression of a protein of interest (this is, a coding sequence encoding a protein of interest, - a promoter operably linked to said coding sequence, wherein said promoter is recognized by the RNA polymerase expressed from a); and - containing at least one lac operator (lacO) within the sequence of said promoter; and c) the lacI gene for expression of the lac repressor protein (LacI) (this is, -lacI coding sequence, - a lacI promoter operably linked to a lacI coding sequence, wherein the lacI promoter is selected from the group consisting of a wild-type lacI promoter or a lacI promoter that increases lacI expression, wherein the rate of expression of the protein of interest is regulated by an inducer that binds to LacI. The present invention provides a genome-based expression system for the production of a protein of interest in a prokaryotic host, comprising:

[0019] According to a specific embodiment, at least a) RNA polymerase (RNAP) gene, b) a gene for the expression of a protein of interest (this is, a coding sequence encoding a protein of interest, - a promoter operably linked to said coding sequence, wherein said promoter is recognized by the RNA polymerase expressed from a); and - containing a lac operator (lacO) within the sequence of said promoter, preferably lacO1); and c) the lacI gene for expression of the lac repressor protein (LacI) (this is, -lacI coding sequence, - a lacI promoter operably linked to the lacI coding sequence (wherein the lacI promoter is a lacI promoter that increases lacI expression, preferably it is a lacI promoter that increases lacI expression, Q a promoter, wherein the rate of expression of the protein of interest is regulated by an inducer that binds to LacI) The present invention provides a genome-based expression system for the production of a protein of interest in a prokaryotic host, comprising:

[0020] Specifically, the gene for expression of the protein of interest contains one lacO within a promoter sequence operably linked to the coding sequence, and the lacI promoter is a promoter that increases LacI expression.

[0021] According to a further specific embodiment, at least a) RNA polymerase (RNAP) gene, b) a gene for the expression of a protein of interest (this is, a coding sequence encoding a protein of interest, - a promoter operably linked to said coding sequence, wherein said promoter is recognized by the RNA polymerase expressed from a); and - at least two lac operators (lacO) separated by at least 92 bp, in particular 94 bp, where one lacO is within the promoter sequence and the other lacO is upstream of the promoter; and c) the lacI gene for expression of the lac repressor protein (LacI) (this is, -lacI coding sequence, - a lacI promoter operably linked to a lacI coding sequence, wherein the lacI promoter is a wild-type lacI promoter, and wherein the rate of expression of a protein of interest is regulated by an inducer that binds to LacI. The present invention provides a genome-based expression system for the production of a protein of interest in a prokaryotic host, comprising:

[0022] According to an alternative embodiment, at least a) an RNA polymerase (RNAP) gene in the host chromosome; b) a gene for the expression of a protein of interest (this is, a coding sequence encoding a protein of interest, - a promoter operably linked to said coding sequence, wherein said promoter is recognized by the RNA polymerase expressed from a); and - containing at least one lac operator (lacO) within the sequence of said promoter; and c) the lacI gene for expression of the lac repressor protein (LacI) (this is, -lacI coding sequence, - a lacI promoter operably linked to a lacI coding sequence, wherein the lacI promoter is selected from the group consisting of a wild-type lacI promoter and a lacI promoter that increases expression of lacI. In one embodiment, the affinity of lacI for one or more lacO / lacOs of b) is lower than the affinity of lacI for the lac operators lacO1 and lacO3 of the endogenous lac operon of the host, and the rate of expression of the protein of interest is regulated by an inducer that binds to LacI.

[0023] According to a further embodiment, at least a) an RNA polymerase (RNAP) gene in the host chromosome; b) a gene for the expression of a protein of interest (this is, a coding sequence encoding a protein of interest, - a promoter operably linked to said coding sequence, wherein said promoter is recognized by the RNA polymerase expressed from a); and - containing a lac operator (lacO) within the sequence of said promoter, preferably lacO1); and c) the lacI gene for expression of the lac repressor protein (LacI) (this is, -lacI coding sequence, - a lacI promoter operably linked to the lacI coding sequence (wherein the lacI promoter is a lacI promoter that increases the expression of lacI, preferably it is a lacI Q promoter) b) an inducible system for the plasmid-free production of a protein of interest in a prokaryotic host, comprising: a) a lacO promoter; and b) a lacO promoter that is a lacO promoter that is a lacO promoter.

[0024] According to a further specific embodiment of the present invention, at least a) an RNA polymerase (RNAP) gene in the host chromosome; b) a gene for the expression of a protein of interest (this is, a coding sequence encoding a protein of interest, - a promoter operably linked to said coding sequence, wherein said promoter is recognized by the RNA polymerase expressed from a); and - containing at least two lac operators (lacOs) separated by at least 92 bp, where one lacO is within the promoter sequence and the other lacO is upstream of the promoter; and c) the lacI gene for expression of the lac repressor protein (LacI) (this is, -lacI coding sequence, - a lacI promoter operably linked to a lacI coding sequence, wherein the lacI promoter is a wild-type lacI promoter and b) an inducible system for the plasmid-free production of a protein of interest in a prokaryotic host, comprising: a) an affinity of lacI for at least two lacOs of b) lower than the affinity of lacI for lac operators lacO1 and lacO3 of the host's endogenous lac operon; and b) an inducible system for the plasmid-free production of a protein of interest in a prokaryotic host, comprising:

[0025] In particular, the prokaryotic host is Escherichia coli (E. coli). Specifically, the host is the BL21 or K-12 strain of E. coli.

[0026] In particular, the RNA polymerase is a host-homologous RNA polymerase, particularly σ 70 E. coli RNA polymerase.

[0027] In particular, the promoter operably linked to the coding sequence encoding the protein of interest is T5, T5 N25 , T7A1, T7A2, T7A3, lac, lacUV5, tac, or trc, or a functional variant thereof having at least 20, 30, 40, 50, 60, 70, 80, or 90% sequence identity to T5, T7A1, T7A2, T7A3, lac, lacUV5, tac, or trc.

[0028] According to a preferred embodiment of the inducible system described herein, the lacI promoter is a promoter that increases the expression of LacI compared to a wild-type host, which is Q The lacI promoter is preferably a lacI promoter (SEQ ID NO: 1). In particular, the gene encoding the protein of interest contains only one lacO, preferably lacO1, and the lacI promoter is preferably a lacI promoter. Q (Sequence Number 1).

[0029] Preferably, the gene encoding the protein of interest comprises at least one lacO selected from the group consisting of lacO1, lacO2, or lacO3, and any combination thereof. In particular, the gene encoding the protein of interest comprises two lacOs, preferably lacO1 and lacO1, or lacO1 and lacO2, or lacO1 and lacO3.

[0030] In particular, at least one lac operator contained in the gene encoding the protein of interest is lacO1 (SEQ ID NO:3), lacO2 (SEQ ID NO:4), or lacO3 (SEQ ID NO:5).

[0031] In particular, at least one lac operator is a functional variant of lacO1, lacO2, or lacO3 having at least 65% sequence identity or a completely symmetrical lacO. In particular, the lac operator is a functional variant of lacO1, lacO2, or lacO3 having at least 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, or 95% sequence identity to wild-type lacO1, lacO2, or lacO3. In the alternative, the functional variant of lacO1, lacO2, or lacO3 comprises 1, 2, 3, 4, or 5 point mutations or 1, 2, 3, 4, or 5 base pair (bp) deletions.

[0032] In particular, the promoter operably linked to a coding sequence encoding a protein of interest comprises an early transcription sequence (ITS), preferably the native T7A1 early transcription sequence (SEQ ID NO:2).

[0033] According to the system provided herein, the expression rate of a protein of interest is regulated by an inducer that binds to LacI. In particular, LacI binds to at least one lacO, thereby suppressing the transcription of a gene encoding a protein of interest. In particular, the addition of an inducer that can bind to LacI disrupts the interaction of at least one lacO with LacI, thereby inducing the transcription of a gene encoding a protein of interest.

[0034] In particular, the inducer is selected from the group consisting of isopropylthiogalactoside (IPTG), lactose, methyl-β-D-thiogalactoside, phenyl-β-D-galactose, and ortho-nitrophenyl-β-galactoside (ONPG).

[0035] In particular, the promoter operably linked to the coding sequence expressing the protein of interest comprises an early transcription sequence, preferably a native T7A1 early transcription sequence. In particular, the early transcription sequence is not limited to the T7A1 early transcription sequence, but may be any early transcription sequence known to those skilled in the art.

[0036] According to a specific embodiment of the inducible system provided herein, the gene for expression of the protein of interest contains one lacO1 operator within the promoter sequence, operably linked to the native T7A1 early transcription sequence (SEQ ID NO:2) and the coding sequence, wherein the LacI promoter is lacI Q A promoter.

[0037] According to further specific embodiments of the inducible system provided herein, the gene of interest contains two lac operators separated by at least about 92 or 94 base pairs (bp), preferably at least about 103, 105, 114, 116, 125, 127, 136, 138, or 149 bp, where one lac operator is located within the sequence of a promoter operably linked to the coding sequence and a second lac operator is upstream of the promoter.

[0038] In particular, the gene encoding the protein of interest is a heterologous gene. In particular, the gene that is heterologous to the prokaryotic host is a recombinant gene that is introduced into the host.

[0039] According to a further specific embodiment, the gene encoding the protein of interest is a homologous gene. In particular, the gene homologous to the prokaryotic host comprises a coding sequence encoding the protein of interest, a promoter operably linked to the coding sequence, wherein the promoter is recognized by the RNA polymerase expressed from the gene in the host chromosome, and at least one lac operator (lacO) within the sequence of the promoter.

[0040] In particular, the gene homologous to the prokaryotic host is a recombinant gene, which is introduced into the host. According to yet a further specific embodiment, the gene homologous to the prokaryotic host is modified by replacing the promoter endogenous to the gene with the promoter described herein. Replacement also means the incorporation of the promoter described herein, such that it is operably linked to the endogenous homologous gene / polypeptide in the chromosome / genome of the host cell, where the native promoter of the endogenous homologous gene / polypeptide is inactivated by at least one point mutation in the native promoter. In particular, the promoter endogenous to the gene is replaced with the promoter described herein, which comprises at least one lacO in the promoter sequence, preferably at least two lacOs, where one lacO is in the promoter sequence and the second lacO is upstream of the promoter. In particular, the affinity of lacI for one or more lacO / lacOs of a promoter that replaces the endogenous promoter of a gene encoding a protein of interest is lower than the affinity of LacI for lacO1 and lacO3 of the endogenous lac operon.

[0041] In particular, the promoter operably linked to the coding sequence of the gene for expressing the protein of interest is a recombinant promoter.In particular, said promoter is not a wild-type lac promoter, but it can be a mutant of lac promoter.When the promoter described herein is a mutant of lac promoter, it comprises at least one lacO in its sequence, and in particular it comprises at least one lacO in the sequence of promoter element -10 to -35.

[0042] Further in this specification, a) culturing the host cells and inducing expression of the gene of interest by addition of an inducer; b) harvesting the protein of interest, and c) isolating and purifying the protein of interest, and optionally d) modifying the protein of interest, and e) Formulating the protein of interest The present invention provides a method for the plasmid-free production of a protein of interest in a prokaryotic host using the induction system described herein, comprising:

[0043] According to a specific embodiment of the system described herein, the gene for producing the protein of interest and / or the lacI gene for producing the lac repressor protein are contained in at least one expression cassette, which is preferably used to integrate the gene for producing the protein of interest and / or the lacI gene for producing the lac repressor protein into the chromosome of a prokaryotic host.

[0044] Also provided herein is an expression cassette comprising at least one heterologous gene designed to produce at least one heterologous protein of interest, the gene of interest being a) one or more coding sequences encoding one or more proteins of interest; b) a promoter operably linked to the coding sequence, and c) at least one lac operator (lacO) operably linked to the promoter; Includes.

[0045] In particular, the affinity of LacI for at least one lacO contained in the expression cassette is lower than the affinity of LacI for the lac operators lacO1 and lacO3 of the lac operon of the host cell, preferably the lac operon being endogenous to the host cell.

[0046] According to a specific embodiment of the expression cassette provided herein, the heterologous gene designed to produce at least one heterologous protein of interest comprises two lac operators at least 92 or 94 bp apart, where one lac operator is located within the promoter sequence and the second lac operator is upstream of the promoter. Preferably, the two lac operators are at least about 92 to 134 bp apart, preferably they are at least about 103, 105, 114, 116, 125, or 136, or 138, or 149 bp apart. In particular, the two lac operators are 92, 94, 103, 105, 114, 116, 125, 136, 138, or 149 bp apart.

[0047] According to a specific embodiment of the expression cassette provided herein, the heterologous gene designed to produce at least one heterologous protein of interest comprises a lacO1 operator within a promoter sequence operably linked to the coding sequence, and a native T7A1 early transcription sequence (SEQ ID NO:2). In particular, the expression cassette further comprises a heterologous lacI promoter, which is a lacI Q The promoter (SEQ ID NO:1).

[0048] Further in this specification, a) integrating the expression cassette into the chromosome of a prokaryotic host; b) culturing the host cells and inducing expression of the gene of interest by addition of an inducer; c) harvesting the protein of interest, and d) isolating and purifying the protein of interest, and optionally e) modifying the protein of interest, and f) Formulating the protein of interest The present invention provides a method for the plasmid-free production of a protein of interest in a prokaryotic host at a manufacturing scale using the expression cassette described herein, comprising:

[0049] According to specific embodiments of the methods and systems provided herein, the prokaryotic host contains an expression cassette integrated into an attachment site, preferably an attTn7, lacZ, recA, tufa, or attnB site. [Brief description of the drawings]

[0050] [Figure 1] Diagram of the integration cartridge. Expression of GFPmut3.1 is controlled by seven different promoter / operator combinations. The T7 expression system is used as a reference. Cartridges were cloned into the pET30a-cer vector (indicated with parentheses) or integrated into the attTN7 site (indicated with brackets) of the BL21 genome (B) rewritten as BL21Q (as described in Example 1) (BQ). In two promoter / operator combinations, the wild-type lacI promoter (lac wt) was replaced with the lacIQ promoter (lacIQ). LacO1* is a 2 bp truncated version of wild-type lacO1. Sym-lacO is a fully symmetric lac operator. +1T7A1+20 is the native early transcription sequence of the T7A1 promoter. Transcription is terminated by tZENIT (tZ). GFPmut3.1 is the coding sequence for expression of the GFPmut3.1 protein. lacO1 is wild-type lacO1. -35 and -10 are the -35 and -10 promoter regions of promoters A1 and T5, respectively. [Diagram 2] Promoter activity of different promoter / operator combinations under uninduced (0 mM IPTG) and induced (0.5 mM IPTG) conditions. Fluorescence of the reporter GFPmut3.1 (y-axis) was used to characterize the genomically integrated (A) and plasmid-based (B) expression systems. Integration cartridges cloned into the pET30a-cer vector are indicated by parentheses, and those integrated into the attTN7 site of the BL21 genome (B) recast as BL21Q (as described in Example 1) (BQ) are indicated by square brackets. [Diagram 3]Influence of lac operators on the expression of GFP and its regulatability, as depicted by the on-line fluorescence course of GFP (y-axis) in fed-batch-like microtiter cultures. The vertical dotted lines indicate the induction time. A-D: T5N25 promoter controlled by three lacOs (B<3lacO-T5>) (A), two lacOs (B<2lacO-T5>) (B), one lacO (B<1lacO-T5>) (C), and one lacO / lacIQ promoter (BQ<1lacO-T5>) (D). E-G: T7A1 promoter controlled by two lacOs (B<2lacO-A1>) (E), one lacO (B<1lacO-A1>) (F), and one lacO / lacIQ promoter (BQ<1lacO-A1>) (G). The T7 expression system is used as a reference (H). [Figure 4] Control of recombinant gene expression using various levels of inducer. B<2lacO-A1>, BQ<1lacO-A1>, and B3 <t7>Flow cytometric analysis of GFPmut3.1 expression in [Diagram 5] Diagram of lac-operator binding sites in the native lac operon (top) and the gene of interest (bottom). The promoter for the gene of interest is regulated by one lac-operator (A) or two lac-operators separated by 62 bp (B). [Figure 6-1] SEQ ID NOs referred to herein. [Figure 6-2] SEQ ID NOs referred to herein. [Figure 7] Effect of controlling recombinant expression rate on LacI concentration. (A) BL21 wild-type cells (lanes 1-3) and B<2lacO-A1> (lanes 4-6) were grown without IPTG (lanes 1 and 4), with 0.01 mM IPTG (lanes 2 and 5), and with 0.5 mM IPTG (lanes 3 and 6). Proteins from approximately 1.2 x 107 cells were separated by SDS-PAGE and analyzed by Western blot using anti-LacI antibody. (B) Fold change relative to BL21-wt at 0 mM IPTG is shown. Error bars indicate standard error of the mean (n=2). [Figure 8] B3 in carbon-limited exponential fed-batch cultures <t7-dftn2>Process characteristics and kinetics of product formation. Cultivation was carried out in a 1.5 L DASGIP® parallel bioreactor system with a final volume of 1.2 L. The vertical dotted line indicates the induction time. [Figure 9] BQ in carbon-limited exponential fed-batch cultures <a1-dftn2>Process characteristics and kinetics of product formation. Cultivation was carried out in a 1.5 L DASGIP® parallel bioreactor system with a final volume of 1.2 L. The vertical dotted line indicates the induction time.

[0051] Detailed Description Unless otherwise indicated or defined, all terms used herein have their usual meaning in the art, which will be apparent to those skilled in the art. For example, reference is made to standard handbooks, such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd Edition), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, "Genes IV", Oxford University Press, New York, (1990) and Janeway et al., "Immunobiology" (5th Edition or more recent edition), Garland Science, New York, 2001.

[0052] As used herein, the terms "comprise," "containing," "having," and "include" may be used synonymously and will be understood to be open-ended definitions that allow for additional members or moieties or elements. "Consisting of" is considered to be the narrowest definition that does not include further elements of the features of the definition that it comprises. Thus, "comprising" is broader in scope and includes the definition of "consisting of."

[0053] As used herein, the term "about" refers to the same numerical value or a numerical value that differs by + / - 5% of the given numerical value.

[0054] Genome-integrated, i.e., plasmid-free, expression systems offer significant advantages. Compared with plasmid-based expression systems, there is no plasmid-mediated metabolic burden during the production process, and no gene dosage variation. However, the current state-of-the-art T7-based expression system, which utilizes a strong T7 promoter that relies on T7 RNA polymerase under the control of an inducible promoter, still suffers from significant shortcomings. The strength of the T7 expression system exerts extreme metabolic burden on the host cell. If the gene of interest encodes a difficult protein, the stress and metabolic burden often leads to reduced yield, shortened production period, and even cell death. Furthermore, the T7 expression system is prone to read-through because it exhibits significant basal expression and the T7 RNA polymerase is prone to mutation under prolonged production conditions.

[0055] The plasmid-free inducible expression system provided herein has the significant advantage that the expression rate can be adjusted at the single cell level, it shows very low basal expression, and it is very efficient in recombinant protein production. Moreover, it gives true control of expression rate, negligible basal expression, and high expression rate even at low inducer concentration, which is especially beneficial for the production of difficult proteins.

[0056] As used herein, the term "plasmid-free" or "genome-based" refers to an expression system of a protein of interest in a prokaryotic host, in which the gene for expression of the protein of interest is located in the genome of the host. In particular, the gene is an endogenous homologous gene located on the chromosome of the prokaryotic host, or a recombinant heterologous or homologous gene integrated into the chromosome of the prokaryotic host.

[0057] According to a particular embodiment, a gene for the expression of a protein of interest and optionally a lacI gene or a recombinant lacI promoter for the expression of a lac repressor protein are integrated into the genome of the host using one or more expression cassettes containing said genes.

[0058] In particular, further recombinant heterologous or homologous genes, such as genes encoding RNA polymerase or genes encoding accessory proteins, are introduced into the prokaryotic host, said further recombinant heterologous or homologous genes being either introduced into the host chromosome or present on a plasmid within the host cell.

[0059] The term "expression cassette" or simply "cassette", used synonymously with "expression cartridge" or simply "cartridge", refers to a linear or circular DNA construct that is to be integrated into a prokaryotic genome, e.g., a bacterial genome. As a result of integration, the expression host cell has an integrated expression cassette. Preferably, the cassette is a linear DNA construct that essentially contains a promoter, a gene of interest, a Shine-Dalgarno (SD) sequence (also called ribosome binding site (RBS)) immediately upstream of the gene of interest, and regions flanking the two ends that are homologous to genomic regions and allow homologous recombination. In addition, the cassette may contain other sequences, such as an antibiotic selection marker, a prototrophic selection marker or a fluorescent marker, a marker encoding a metabolic gene, a gene that improves the expression of a protein, or two flippase recognition target sites (FRTs) that allow the removal of certain sequences (e.g., antibiotic resistance genes) after integration.

[0060] The expression cassettes are synthesized and amplified by methods known in the art, in the case of linear cassettes, usually by standard polymerase chain reaction (PCR). Since linear cassettes are usually easy to construct, they are preferred for obtaining host expression cells used in the systems and methods provided herein. Furthermore, the use of linear expression cassettes offers the advantage that the genome integration site can be freely selected by the respective design of the homologous regions flanking the cassette. This allows the integration of linear expression cassettes to have a greater diversity in terms of genome regions.

[0061] An expression vector comprises an expression cassette as described herein, and optionally further comprises flanking regions homologous to the genomic integration site, a number of restriction enzyme cleavage sites, an early transcription sequence (ITS), and a transcription terminator, and optionally one or more selectable markers (e.g., amino acid synthesis genes, or genes conferring resistance to antibiotics such as ampicillin, kanamycin, chloramphenicol, or streptomycin), with these components operably linked to each other. A common type of vector is the "plasmid", which is generally a self-contained double-stranded DNA molecule that can readily accept additional (foreign) DNA, which can be easily introduced into a suitable host cell. In particular, the term "vector" or "plasmid" refers to a vehicle by which DNA or RNA sequences (e.g., foreign genes) can be introduced into a host cell, thereby transforming the host and promoting the expression (e.g., transcription and translation) of the introduced sequence.

[0062] The term "prokaryotic host" as used herein refers to a bacterial host, and in particular it refers to a bacterial host cell. In principle, there are no limitations regarding the choice of bacterial host cells, except for certain specific requirements detailed below. Bacterial host cells may be eubacteria (gram-positive or gram-negative) or archaea, as long as they allow genetic manipulation for the insertion of a gene of interest, advantageously for site-specific integration. Preferably, the bacterial host cells allow cultivation on a manufacturing scale. Preferably, the host cells have properties that allow cultivation up to high cell densities. Examples of bacterial host cells that have been shown to be suitable for recombinant industrial protein production are Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens and variants thereof, and Lactococcus lactis strains. Preferably, the host cell is E. coli.

[0063] A requirement for the host cell is that it contains an RNA polymerase capable of binding to the promoter controlling the gene encoding the protein of interest.

[0064] In certain embodiments, the host cell has a marker gene in its genome for selection purposes.

[0065] Another requirement for a host cell, in terms of site-specific gene insertion, is that it contains at least one genomic region (either a coding or any non-coding, functional or non-functional region, or a region with unknown function) whose sequence is known and which can be disrupted or otherwise manipulated to allow for the insertion of a heterologous sequence without harming the cell.

[0066] Regarding the integration locus, the expression system used in the present invention allows a wide variety: in principle, any locus with a known sequence can be selected, provided that the function of the sequence is unnecessary or can be supplemented if it is essential (e.g. in the case of auxotrophy).

[0067] Integration of the gene of interest into the bacterial genome can be accomplished by conventional methods, for example by using a concatenated cartridge containing flanking sequences homologous to a specific site on the chromosome, as described for example for the attTn7 site in (30). Furthermore, the use of a concatenated expression cartridge offers the advantage that the genomic integration site can be freely selected by the respective design of the homologous regions flanking the cartridge. Thereby, the integration of the concatenated expression cartridge allows a greater diversity in terms of genomic region. In a preferred embodiment, the integration of the concatenated cartridge is at an attachment site such as the attB site or the attTn7 site, which are well-documented integration sites. Examples of other integration methods useful in the present invention include, but are not limited to, those based on Red / ET recombination, as described for example in (31). Alternatively, the expression cassette can first be integrated into the genome of an intermediate donor host cell, which can then be subsequently introduced into the host cell by transduction with, for example, the P1 phage, as described in (32). The integration methods used herein are not limited to the above examples; rather, any integration method known in the art can be used.

[0068] The integration methods for obtaining host expression cells are not limited to integrating one gene of interest into one site in the genome; they allow for diversity in terms of both integration site and expression cassette. For example, more than one gene of interest can be inserted, i.e., two or more identical or different sequences under the control of the same or different promoters can be integrated into one or more different loci on the genome. For example, it allows the expression of two different proteins that form a heterodimeric complex. A heterodimeric protein is composed of two individually expressed subunit proteins, such as the heavy and light chains of a monoclonal antibody, or an antibody fragment.

[0069] Although the present invention allows for plasmid-free production of a protein of interest, it does not exclude the possible presence in the host expression cell of plasmids carrying sequences other than the gene of interest that are to be expressed, such as auxiliary proteins and / or recombinant proteins. Preferably, in such an embodiment, care should be taken to ensure that the advantages of the present invention are not overturned by the presence of plasmids, i.e. that the plasmids are present in low copy number and that the plasmids do not impose a metabolic burden on the cell.

[0070] The integration of one or more recombinant genes into genome results in a distinct and predetermined number of genes of interest per cell. In the embodiment of the present invention where one copy of a gene is inserted, this number is usually one (except when the cell contains more than one chromosome or genome, such as occurs transiently during cell division), whereas plasmid-based expression involves copy numbers up to several hundred. The expression system used in the method of the present invention utilizes an increased proportion of cellular synthetic capacity for recombinant protein production by freeing the host's metabolism from plasmid replication.

[0071] A particular advantage is that the inducible expression system described herein has no limitations in terms of induction levels. This means that the system cannot be "over-induced", as often happens with plasmid-based systems or systems that utilize strong promoters such as the T7 expression system. Genome-based expression systems allow for precise control of protein expression and are therefore particularly beneficial in combination with expression targeting pathways that rely or depend on well-controlled expression. In a preferred embodiment, the method of the invention comprises secretion (excretion) of the protein of interest from the bacterial cytoplasm into the periplasm and / or culture medium. An advantage of this embodiment is an optimized and sustained protein secretion rate, resulting in higher titers of secreted protein compared to prior art secretion systems. In particular, this can be achieved by fusing a signal peptide / a nucleotide sequence encoding a signal peptide N-terminal to the protein of interest, which leads to the protein of interest being transported to the host's transporter and causing translocation to the host's periplasm where it is cleaved by the host's signal peptidase. Any signal peptide known in the art can be used, such as, but not limited to, ompA-, pelB-, malE-, phoA-, dsbA-, lysC-, lolB-, pyrL-leader peptides.

[0072] As used herein, the term "RNA polymerase (RNAP) gene" refers to a gene expressing an RNAP, which is contained within the genome, e.g., within a plasmid, or within the chromosome of a prokaryotic host. Preferably, the gene expresses an RNAP that is endogenous to the prokaryotic host.

[0073] In bacteria, the same enzyme catalyzes the synthesis of mRNA and non-coding RNA (ncRNA). RNAP is a large molecule; the core enzyme has five subunits (about 400 kDa). To bind to a promoter, the RNAP core forms an RNA polymerase holoenzyme by associating with the transcription initiation factor sigma (σ). Sigma decreases the affinity of RNAP for non-specific DNA while increasing the specificity for the promoter, thus allowing initiation of transcription at the correct site. Thus, the complete holoenzyme has six subunits (about 450 kDa). The core enzyme is responsible for binding to template DNA and synthesizing RNA, which is complemented by the σ factor to form the holoenzyme, which recognizes the promoter sequence and initiates promoter-specific transcription.

[0074] According to a preferred embodiment, the prokaryotic host cell of the system described herein is an E. coli cell, and the RNAP is an RNAP endogenous to E. coli, most preferably it is σ 70 E. coli RNA polymerase. The σ subunit of bacterial RNA polymerase (RNAP) is required for promoter-specific transcription initiation. In E. coli and other gram-negative rods, the "housekeeping" or "first" sigma factor is σ 70 Every cell has a set of "housekeeping" sigma factors that keep essential genes and pathways running. When complexed with the RNAP core enzyme (subunit structure α2ββ'ω), different σ factors specialize in the recognition of different classes of promoters. σ 70 All genes recognized by σ contain a similar two-part promoter consensus sequence. The first σ factor in Escherichia coli, σ 70 typically direct transcription initiation from a promoter defined by two conserved hexameric DNA sequence elements, called the -10 and -35 elements because of their relationship to the transcription start site (position +1). With respect to the DNA bases that correspond to the start of the RNA transcript, consensus promoter sequences are characteristically centered 10 and 35 nucleotides prior to the transcription start (-10 and -35).

[0075] The term "expression" is understood as follows: Nucleic acid molecules containing a desired coding sequence of an expression product, such as a recombinant protein as described herein, and control sequences, such as a promoter, operably linked to the nucleic acid, may be used for the purpose of expression. A host transformed or transfected with these sequences is capable of producing the encoded protein. To effect transformation, the expression system may be included in a vector, however, most preferably, the relevant DNA is integrated into the host's chromosome.

[0076] The term "gene" as used herein refers to a DNA sequence that includes at least promoter DNA (optionally including operator DNA) and coding DNA that codes for a specific amino acid sequence for a particular polypeptide or protein. Promoter DNA is a DNA sequence that initiates, regulates, or otherwise mediates or controls the expression of coding DNA. The promoter DNA and coding DNA may be from the same gene or from different genes, and may be from the same organism or from different organisms.

[0077] As used herein, the term "recombinant" means "prepared by or resulting from genetic engineering." Recombinant hosts specifically include recombinant expression or cloning vectors, or they have been genetically engineered to contain recombinant nucleic acid sequences, particularly using nucleotide sequences that are foreign to the host. Recombinant proteins are produced by expressing the respective recombinant nucleic acids in the host.

[0078] There is no limitation regarding the protein of interest (POI). More specifically, the protein may be a polypeptide that does not naturally occur in the host cell, i.e. a heterologous protein, or may be a protein that is native to the host cell, i.e. a homologous protein to the host cell, but is produced, for example, by the integration of one or more copies of the nucleic acid sequence that codes for the homologous protein of interest into the genome or chromosome of the host cell by recombinant techniques, or by recombinant modification of the promoter sequence that controls the expression of the gene that codes for the protein of interest. The protein of interest may be a monomer, dimer, or polymer, and may be homomeric or heteromeric.

[0079] Examples of proteins that can be produced by the method of the present invention include, but are not limited to, enzymes, regulatory proteins, receptors, peptides, such as peptide hormones, cytokines, membrane proteins, or transport proteins. The protein of interest can also be an antigen, an antigen-binding protein, an immunostimulatory protein, an allergen, a full-length antibody, or an antibody fragment or derivative, such as those used for vaccination, vaccines. The antibody derivative can be, for example, a single chain variable fragment (scFv), a Fab fragment, or a single domain antibody.

[0080] A DNA molecule encoding a protein of interest is also referred to as a "gene of interest." In particular, a gene of interest includes a DNA sequence encoding the protein of interest, a promoter operably linked to the coding sequence, and at least one lac operator within the promoter sequence.

[0081] Furthermore, the gene of interest encoding the protein of interest may be a naturally occurring or non-natural DNA sequence. One or more genes of interest may be under the control of a promoter as described herein. Alternatively, each gene of interest is under a promoter. The genes of interest may all be on the same expression cassette or on multiple expression cassettes. The protein of interest may be modified in any way. Non-limiting examples of modifications may be the insertion or deletion of a post-translational modification site, the insertion or deletion of a targeting signal (e.g., a leader peptide), fusion to a tag, a protein or protein fragment that facilitates purification or detection, a mutation that affects a change in stability or a change in solubility, or any other modification known in the art. In certain embodiments of the present invention, the recombinant protein is a biopharmaceutical, which may be any protein suitable for therapeutic or prophylactic purposes in a mammal.

[0082] As used herein, the term "promoter" refers to an expression control element that allows binding of RNA polymerase and initiation of transcription. In particular, a promoter operably linked to a gene of interest as described herein comprises at least one lac operator within its sequence. In particular, said at least one lac operator is located at elements -10 to -35, which are preferably located 10 and 35 nucleotides before the start of transcription (-10 and -35), as illustrated in Figure 1.

[0083] The lac promoter is the promoter of the lac operon, which controls the transcription of the three lac genes, namely lacZ, lacY and lacA. The wild-type lac promoter does not contain a lac operator in its sequence, since it does not contain a lac operator in the promoter elements from -10 to -35. Preferably, in the inducible expression system described herein, the lac promoter is an endogenous lac promoter, which contains an endogenous lac operator. According to a particular embodiment, one or more lac operators of the endogenous lac promoter are genetically modified to increase their binding affinity for the lac repressor molecule LacI. In particular, they are genetically modified such that their affinity for the lac repressor LacI is higher than that of the lac operator of the promoter operably linked to the gene of interest.

[0084] As used herein, a lacI promoter is a promoter operably linked to the coding sequence of the lacI gene. In particular, the inducible system described herein can be used with a wild-type lacI promoter or a genetically modified lacI promoter that increases expression of LacI, such as the exemplary lacI promoters described herein. Q The lacI promoter includes a lacI promoter. In particular, the lacI promoter is a constitutive promoter. In particular, any constitutive promoter stronger than the native lacI promoter may be used as the lacI promoter according to the present invention. In particular, any promoter stronger than the native lacI promoter may be used as the lacI promoter according to the system provided herein, such as, but not limited to, T5, T7A1, T7A2, T7A3, T7, dnaK / J, spac, bla, nptII, cat promoter.

[0085] The promoter operably linked to a gene encoding a protein of interest as described herein can be any inducible promoter that is recognized by RNAP encoded by an RNAP gene contained in the host's chromosome.

[0086] According to a particular embodiment of the invention, the gene of interest is expressed by a lac, lacUV5, tac, or trc promoter, a lac or lacUV5 promoter, a T5 promoter (Gentz ​​and Bujard, 1985), e.g., a T5 promoter. N25 , or the T7 promoter (Hawley and McClure, 1983), such as the T7C or T7D or T7A promoter, such as the T7A1, T7A2, or T7A3 promoters (all inducible by lactose or its analogues IPTG), or other promoters suitable for the expression of recombinant proteins, all of which use E. coli RNA polymerase. The sequences of such promoters are well known in the art, for example those described in Gentz ​​and Bujard, 1985 (33) or Hawley and McClure, 1983 (38). In particular, the sequences of the promoters have been modified to include within their sequence at least one lacO as described herein.

[0087] According to a particular embodiment, the promoters described herein are operably linked to a sequence encoding a protein of interest, which includes lacO within its sequence. In bacteria, promoter sequences typically contain two short sequence elements, which in wild-type promoters are typically located about 10 and 35 nucleotides upstream of the transcription start site. These sequences are conserved among many bacterial strains. For example, the sequence at -10 nucleotide (also called the -10 element) typically contains the consensus sequence TATAAT (SEQ ID NO: 34), and the sequence at -35 (also called the -35 element) has the consensus sequence TTGACA (SEQ ID NO: 35). Although the above consensus sequences are conserved on average, they are not found intact in all promoters. On average, only 3-4 base pairs out of 6 in each consensus sequence are found in any given promoter. Few natural promoters have been identified to date that have intact consensus sequences in both the -10 and -35 elements. In particular, artificial promoters in which the -10 and -35 elements are perfectly conserved transcribe less frequently than those that have several mismatches in the consensus sequence.

[0088] In particular, the promoters described herein contain at least one lacO at elements -10 to -35.

[0089] The term "inducer", used synonymously with "inducing agent", refers to a factor capable of resulting in the induction of transcription through direct or indirect regulation of promoter activity. In particular, as used herein, an inducer is any factor capable of binding to the lac repressor molecule and inhibiting its interaction with a gene operably linked to a gene of interest. Preferably, the inducer used herein is isopropylthiogalactoside (IPTG), lactose, methyl-β-D-thiogalactoside, phenyl-β-D-galactose, or ortho-nitrophenyl-β-galactoside (ONPG).

[0090] There are no restrictions on the mechanism by which the induction of protein expression is performed. For example, the inducer may be added as a single or multiple bolus or by continuous feeding, the latter also known as "inducer feeding". There are no restrictions on the time point at which the induction is performed. The inducer may be added at the start of the culture, or at the start of continuous nutrient feeding, or after the start of feeding. Inducer feeding can be accomplished either by including the inducer in the culture medium or by feeding it separately. The advantage of inducer feeding is that it allows the dosage of the inducer to be controlled, i.e., it allows the maintenance of a defined or constant amount of inducer dosage per constant number of genes of interest in the production system. For example, inducer feeding allows the dosage of the inducer to be proportional to the biomass, resulting in a constant ratio of inducer to biomass. The unit of biomass (on which the dose of inducer can be based) can be, for example, cell dry weight (CDW), wet cell weight (WCW), absorbance, total cell number (TCN; cells per volume), or colony forming units (CFU per volume), or an online monitored signal proportional to biomass (e.g., fluorescence, turbidity, light scattering, dielectric capacity, concentration of carbon dioxide in exhaust gas, etc.). In essence, the method of the present invention allows for accurate dose of inducer per any parameter or signal proportional to biomass, regardless of whether the signal is measured offline or online. Since the number of genes of interest per unit of biomass is defined and constant (one or more genes per cell), the result of this induction mode is a constant dose of inducer per gene of interest. As an additional advantage, the exact and optimal dose of the amount of inducer compared to the amount of biomass can be determined and optimized by experiment.

[0091] It may not be necessary to determine the actual biomass level by analytical methods. For example, it may be sufficient to add an amount of inducer based on previous cultures (historical biomass data). In another embodiment, it may be preferable to add an amount of inducer per unit of biomass, as theoretically calculated or predicted. For example, in a feeding-based culture (such as a fed-batch or continuous culture), it is well known that one unit of the growth-limiting component, usually the carbon source, in the feed medium will result in a certain amount of biomass.

[0092] Preferably, the inducer is used at a concentration ranging from 0.005 mM to 1 mM, even more preferably from 0.01 mM to 0.5 mM. In particular, the concentration of IPTG ranges from 1 to 100 μmol / g (cell dry weight).

[0093] As provided herein, hosts for use in the inducible expression systems described herein contain a lac operon, preferably a wild-type lac operon, and a lacI gene.

[0094] As referred to herein, the endogenous lac operon contains three genes, namely lacZ, lacY, and lacA. These genes are transcribed as a single mRNA under the control of a promoter. In addition to the three genes, the lac operon includes the lac promoter and the lac operators lacO1, lacO2, and lacO3. The lac promoter is a binding site for RNA polymerase. The lac operators are negative regulatory sites for the binding of the lac repressor protein. The operator overlaps with the promoter, and upon binding to the lac repressor protein, the RNA polymerase cannot bind to the promoter and initiate transcription. According to certain embodiments, the endogenous lac operon is modified to increase the binding affinity of LacI to at least one of the lac operators lacO1, lacO2, or lacO3. In particular, at least one of the lac operators lacO1, lacO2, or lacO3 is modified, ie, the endogenous lac operon contains a functional mutant of lacO1, lacO2, and / or lacO3 that has increased affinity for LacI.

[0095] The term "lacI gene" as used herein refers to a gene for the expression of lac repressor protein (also called lac inhibitor (LacI)) or any functional variant thereof having at least 30% sequence identity to lacI (SEQ ID NO: 26). In particular, the gene comprises a lacI coding sequence, a lacI promoter operably linked to the lacI coding sequence, wherein the lacI promoter is selected from the group consisting of a wild-type lacI promoter and a lacI promoter that increases the expression of lacI. In particular, the lacI gene expresses LacI or a functionally active variant thereof comprising at least 40, 50, 60, 70, 80, or 90% sequence identity to LacI (SEQ ID NO: 27). In particular, the lacI promoter that increases the expression of LacI is a strong promoter that increases the expression of LacI by at least 1.5-fold, 2-fold, 2.5-fold, or 5-fold, preferably 10-fold or more. In particular, it increases expression of LacI by at least 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or even 100-fold. Exemplary embodiments of the inducible system provided herein include the lacI promoter as the lacI promoter that increases expression of lacI. Q Contains the promoter. lacI Q The promoter contains a point mutation, a single C to T change, upstream of the native lacI gene within the promoter region, resulting in a 10-fold increase in mRNA transcription. The promoter for the lacI coding sequence may contain the native lacI start codon or any variant thereof. The lacI gene is preferably integrated into the host chromosomal DNA or contained in a single copy vector.

[0096] In wild-type E. coli, the lac repressor protein forms a homotetramer that binds to the lac operator sequence (lacO) and represses transcription of the lacZYA operon. In the presence of lactose or the non-metabolizable isopropyl β-D-1-thiogalactopyranoside (IPTG), LacI changes conformation and can no longer bind to the lac-operator, resulting in induction of transcription. The lac-operator site is a DNA sequence with an inverted, symmetric repeat sequence.

[0097] The higher the degree of symmetry, the higher the binding affinity of LacI to the operator sequence. An artificial, completely symmetrical lacO (sym-lacO) was found to bind LacI with the highest affinity, whereas the three wild-type operators lacO1, lacO2 and lacO3, which show approximate symmetry, showed lower affinities, resulting in the following order of affinity for LacI: sym-lacO>lacO1>lacO2>lacO3. LacI binds simultaneously to both the first operator lacO1 and either lacO2 or lacO3 through a DNA looping mechanism. LacO2 is located 401 bp downstream of lacO1, while lacO3 is only 92 bp upstream of lacO1. The main contribution to repression results from DNA looping of lacO1 and lacO3, due to the closer proximity of lacO1 and lacO3. Furthermore, the binding of lacO1 and lacO3 to LacI prevents the production of LacI itself. The 3' end of the lacI gene overlaps with lacO3. In the repressed state, transcription of lacI produces a truncated mRNA that is rapidly degraded by the cell. Due to this autoregulation, the concentration of LacI tetramers is about 40 molecules in induced cells and about 15 molecules in uninduced cells.

[0098] Lac operator sequences are well known in the art. Exemplary lac operator sequences are provided by SEQ ID NOs: 3-5.

[0099] Suitable variants of the nucleic acid or polypeptide sequences disclosed herein, particularly lacO1, lacO2, and lacO3, are functional variants that have the same type of activity (not with respect to the degree of activity) as the nucleic acid or polypeptide to which the sequence corresponds. Such activity can be tested according to the assays described in the Examples below, and according to methods known in the art.

[0100] The term "functional variant" or functionally active variant also includes naturally occurring allelic variants, as well as mutants or any other non-naturally occurring variants. As is known in the art, allelic variants are alternative forms of a nucleic acid or peptide characterized as having one or more nucleotide or amino acid substitutions, deletions, or additions that do not essentially alter the biological function of the nucleic acid or polypeptide.

[0101] Functional variants can be obtained by sequence modifications in a polypeptide or nucleotide sequence, for example by one or more point mutations, which sequence modifications retain or improve the function of the unmodified polypeptide or nucleotide sequence when used in the combinations of the invention. Such sequence modifications can include, but are not limited to, (conservative) substitutions, additions, deletions, mutations, and insertions.

[0102] A point mutation is specifically understood as the engineering of a polynucleotide that results in the expression of an amino acid sequence that differs from the non-engineered amino acid sequence by substituting or exchanging, deleting, or inserting one or more singlet (non-conservative) or doublet amino acids with different amino acids.

[0103] An exemplary functional mutant of the lacO1 operator is a two base pair truncated form of wild-type lacO1, which has a 2 bp deletion at its 5' end, i.e., lacO * (SEQ ID NO: 6).

[0104] Transcription rate control, also referred to as fine tuning or "tunability" of protein production, is highly relevant in bioprocessing. Bioprocesses are designed to maximize the synthetic capacity of cells during the longest period to obtain properly folded and processed proteins. However, powerful expression systems, such as the T7 expression system, are known to exhibit "all-or-none" behavior, where reduced expression levels in partially induced cultures are a result of the formation of subpopulations of fully induced and uninduced cells. Such problems are solved by the inducible expression system described herein, which allows for tunability, particularly single-cell tunability. In the inducible expression system described herein, the affinity of LacI for at least one lacO of a promoter operably linked to a gene of interest is lower than the affinity of LacI for the lac operators lacO1 and lacO3 of the endogenous lac operon of the host. The binding constant (K a ) is higher than the binding constant for lacO in the lac operon, the first LacI molecule that is not inactivated by IPTG will preferentially bind to the lacO binding site of the gene of interest, rather than to lacO3 / lacO1 on the lac operon. Thus, the autoregulation of LacI will not intervene and more LacI molecules will be produced, which will lead to overregulation of the system, resulting in a complete cessation of transcription of the gene of interest in this cell. Especially at low inducer concentrations, such a system will result in at least two distinct subpopulations of cells that produce the gene of interest and those that do not, because such expression systems will stop their productivity but still continue to grow.

[0105] However, in the inducible expression system described herein, the binding constant (K a ) is lower than the binding constant for lacO in the lac operon. Therefore, LacI preferentially binds to the operator of the endogenous lac operon, preventing transcription of the three genes lacZ, lacY, and lacA, and through autoregulation of LacI, also preventing further production of LacI, resulting in a homogenous population at any given inducer concentration.

[0106] As used herein, the term "affinity" or "binding affinity" refers to the strength of association between a ligand and a receptor as defined by the dissociation constant and / or the binding constant. d ) is k off / k on is the equilibrium dissociation rate constant, defined as the ratio of off is the rate constant for dissociation of the ligand from the receptor, and k on is the rate constant for the association of the ligand to the receptor. The association constant (K a ) is K d It is the opposite of K. a If is high, K d is low and the ligand has high affinity for the receptors (fewer molecules are required to bind to 50% of the receptors).

[0107] Typically, the binder has at least d <10 -7 M, and in some cases, e.g., K d <10 -8 M, preferably K d <10 -9 Higher affinities are required, such as M, and even more preferred are K d <10 -10 It's M.

[0108] In the inducible expression system described herein, the binding affinity of LacI to one or more lacO / lacOs of a gene of interest is lower than the affinity of LacI to the lac operators lacO1 and lacO3 of the endogenous lac operon. In particular, lacI binds to the lac operators lacO1 and lacO3 with at least a K d <10 -7 M, preferably K d <10 -8 M, preferably K d <10 -9 K of M d and even more preferably, K d <10 -10 In particular, LacI is a 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100% or more increase in K in one or more lacO / lacOs of the gene of interest. d As a result, LacI binds to one or more lacO / lacOs of the gene of interest via the K of LacI for lacO1 and lacO3 of the endogenous lac operon. a K that is about 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, or 90% lower than a Combine with.

[0109] In particular, the binding affinity is determined by affinity ELISA assay. In certain embodiments, the binding affinity is determined by Biacore, Forte Bio, or MSD assay. In certain embodiments, the binding affinity is determined by kinetic methods. In certain embodiments, the binding affinity is determined by equilibrium / solution methods. Those skilled in the art can determine the appropriate parameters to determine the binding affinity of a ligand to a particular molecule. Binding affinity can be routinely determined by those skilled in the art.

[0110] "Percent sequence identity" or "percent amino acid sequence identity" as used herein is defined as the percentage of nucleotides or amino acid residues in a candidate sequence that are identical with nucleotides or amino acid residues in a particular nucleotide or polypeptide sequence to be compared (the "parent sequence"), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the percent sequence identity. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0111] The term "operably linked" as used herein refers to the association of nucleotide sequences on a single nucleic acid molecule, i.e., a vector, such that the function of one or more nucleotide sequences is affected by at least one other nucleotide sequence present on the nucleic acid molecule. For example, a promoter is operably linked to a coding sequence encoding a protein of interest if it can cause expression of the coding sequence. In particular, such nucleic acids that are operably linked to each other can be directly linked, i.e., without any additional elements or nucleic acid sequences in between, or can be indirectly linked by including spacer sequences or other sequences in between. In particular, in the context of the lac operator, being operably linked to a promoter refers to the ability of the lac operator to regulate the ability of the promoter to control the expression of a coding sequence under certain conditions, e.g., the lac operator can inhibit the promoter-dependent expression of the gene of interest when the lac repressor protein binds to the gene of interest.

[0112] The term "heterologous" as used herein with respect to a nucleotide or amino acid sequence or protein refers to a compound that is either foreign, i.e., "exogenous," e.g., not found in nature, to a given host cell; or that is found naturally, e.g., "endogenous," in a given host cell, however, in the context of, e.g., heterologous constructs utilizing heterologous nucleic acids, and is thereby referred to as "non-naturally occurring." Heterologous nucleotide sequences, as found endogenously, may also be produced in unnatural amounts within a cell, e.g., in amounts greater than expected or greater than found in nature. A heterologous nucleotide sequence, or a nucleic acid that includes a heterologous nucleotide sequence, may differ in sequence from an endogenous nucleotide sequence, but encode the same protein as found endogenously. In particular, a heterologous nucleotide sequence is one that is not found in the same relationship to a host cell in nature (i.e., "not naturally associated"). Any recombinant or artificial nucleotide sequence is understood to be heterologous. An example of a heterologous polynucleotide or nucleic acid molecule includes a nucleotide sequence that is not naturally associated with a promoter or that is operably linked to a coding sequence as described herein, e.g., to obtain a hybrid promoter. As a result, a hybrid or chimeric polynucleotide can be obtained. A further example of a heterologous compound is a polynucleotide or gene encoding a protein of interest that is operably linked to a transcriptional control element, e.g., a promoter, to which the coding sequence of the endogenous, native protein of interest is not normally operably linked.

[0113] The present invention further includes the following:

[0114] 1. At least a) RNA polymerase (RNAP) gene, b) a gene encoding a protein of interest (this is, -code sequence, - a promoter operably linked to said coding sequence, wherein said promoter is recognized by the RNA polymerase expressed from a); and - containing at least one lac operator (lacO) within the sequence of said promoter c) the lacI gene for expression of the lac repressor protein (LacI) (this is, -lacI coding sequence, - a lacI promoter operably linked to a lacI coding sequence, wherein the lacI promoter is a wild-type lacI promoter or a lacI promoter that increases LacI expression, wherein the rate of expression of a protein of interest is regulated by an inducer that binds to LacI. A genome-based expression system for the production of a protein of interest (POI) in a prokaryotic host, comprising:

[0115] 2. The genome-based expression system of item 1, wherein the gene encoding the protein of interest contains (i) one lacO within the promoter sequence, or (ii) one lacO within the promoter sequence and one lacO upstream of the first lacO.

[0116] 3. The genome-based expression system of item 1 or 2, wherein the gene encoding the protein of interest contains one lacO within the promoter sequence, and the lacI promoter is a promoter that increases LacI expression.

[0117] 4. The genome-based expression system of any one of items 1 to 3, wherein the gene encoding the protein of interest contains one lacO within the promoter sequence and one lacO upstream of the first lacO, and the lacI promoter is a promoter that increases LacI expression.

[0118] 5. The genome-based expression system of any one of items 1 to 4, wherein the prokaryotic host is Escherichia coli (E. coli).

[0119] 6. The genome-based expression system according to any one of items 1 to 5, wherein the host is E. coli of the BL21 or K-12 strain.

[0120] 7. The RNA polymerase is a heterologous or homologous RNA polymerase, preferably the RNA polymerase is a homologous RNA polymerase to the host, in particular it is an E. coli RNA polymerase, preferably σ 70 7. The genome-based expression system of any one of items 1 to 6, wherein the E. coli RNA polymerase is

[0121] 8. The promoter of item 1 b) is T5, T5 N25 8. The genome-based expression system of any one of items 1 to 7, wherein the expression system is selected from the group consisting of T7A1, T7A2, T7A3, lac, lacUV5, tac, or trc.

[0122] 9. The lacI promoter is a lacI promoter that increases LacI expression, which is Q 9. The genome-based expression system according to any one of items 1 to 8, wherein the promoter is (SEQ ID NO: 1).

[0123] 10. The genome-based expression system of any one of items 1 to 9, wherein the lac operator is lacO1 (SEQ ID NO: 3), lacO2 (SEQ ID NO: 4), or lacO3 (SEQ ID NO: 5).

[0124] 11. The genome-based expression system of item 10, wherein the lac operator is a functional mutant of lacO1, lacO2 or lacO3 having at least 65% sequence identity or a completely symmetrical lacO.

[0125] 12. The genome-based expression system of any one of items 1 to 11, wherein the promoter operably linked to a coding sequence encoding a protein of interest comprises an early transcription sequence (ITS), preferably the native T7A1 early transcription sequence (SEQ ID NO:2).

[0126] 13. The genome-based expression system of any one of items 1 to 12, wherein the inducer is selected from the group consisting of isopropylthiogalactoside (IPTG), lactose, methyl-β-D-thiogalactoside, phenyl-β-D-galactose, and ortho-nitrophenyl-β-galactoside (ONPG).

[0127] 14. A gene for expression of a protein of interest contains one lacO1 operator and a native T7A1 early transcription sequence (SEQ ID NO:2) within a promoter sequence operably linked to the coding sequence, wherein the lacI promoter is lacI Q 14. The genome-based expression system of any one of items 1 to 13, wherein the promoter is a promoter.

[0128] 15. The genome-based expression system of any one of items 1 to 14, wherein the gene of interest contains two lac operators separated by at least 92 or 94 base pairs (bp), preferably 103, 105, 114, 116, 125, 127, 134, 136, 138, or 149 base pairs, wherein one lac operator is located within a promoter sequence operably linked to the coding sequence and the second lac operator is upstream of the promoter.

[0129] 16. The genome-based expression system of any one of items 1 to 15, wherein the gene encoding the protein of interest is a heterologous gene.

[0130] 17. The system of any one of items 1 to 16, wherein at least one lac operator of the lac operon of the prokaryotic host has been genetically modified to increase its binding affinity for the lac repressor molecule LacI.

[0131] 18. a) inducing expression of the gene encoding the protein of interest by addition of an inducer; b) harvesting the protein of interest; c) isolating and purifying the protein of interest, and optionally d) modifying, and e) Formulating the protein of interest 18. A method for plasmid-free production of a protein of interest in a prokaryotic host using the genome-based expression system of any one of items 1 to 17, comprising:

[0132] 19. a) one or more coding sequences encoding one or more proteins of interest; b) a promoter operably linked to one or more coding sequences; and c) at least one lac operator (lacO) within the promoter sequence; (wherein the affinity of LacI for lacO in c) is lower than the affinity of LacI for the lac operators lacO1 and lacO3 of the endogenous lac operon of the host cell) An expression cassette comprising at least one heterologous gene designed to produce at least one heterologous protein of interest, comprising:

[0133] 20. The expression cassette of item 19, wherein the heterologous gene designed to produce at least one heterologous protein of interest comprises two lac operators, separated by 92 or 94 bp, wherein one lac operator is located within the promoter sequence and the second lac operator is upstream of the promoter.

[0134] 21.lacI Q 21. The expression cassette of item 19 or 20, further comprising a heterologous lacI promoter, which is the promoter (SEQ ID NO:1).

[0135] 22. The expression cassette of any one of items 19 to 21, wherein the heterologous gene designed to produce at least one heterologous protein of interest comprises a lacO1 operator and a native T7A1 early transcription sequence (SEQ ID NO: 2) within a promoter sequence operably linked to the coding sequence.

[0136] 23.a. Integrating the expression cassette into the chromosome of a prokaryotic host; b. inducing expression of the gene encoding the protein of interest by the addition of an inducer; c. harvesting the protein of interest; d. isolating and purifying the protein of interest, and optionally e. modifying; and f. Formulating the Protein of Interest 23. A method for the plasmid-free production of a protein of interest in a prokaryotic host on a manufacturing scale using the expression cassette of any one of items 19 to 22, comprising:

[0137] 24. At least a) an RNA polymerase (RNAP) gene in the host chromosome; b) a gene encoding a protein of interest (this is, -code sequence, - a promoter operably linked to said coding sequence, wherein said promoter is recognized by the RNA polymerase expressed from a); and - containing at least one lac operator (lacO) within the sequence of said promoter c) the lacI gene, which encodes the lac repressor protein (LacI) (this is, -lacI coding sequence, - a lacI promoter operably linked to a lacI coding sequence (wherein the lacI promoter is a wild-type lacI promoter or a lacI promoter that increases LacI expression) a) an inducible system for plasmid-free production of a protein of interest (POI) in a prokaryotic host, comprising: b) a gene encoding LacI; c) a gene encoding LacI; d) a gene encoding LacI; e) a gene encoding LacI; d) a gene encoding LacI;

[0138] 25. The system of item 24, wherein at least one lac operator of the lac operon of the prokaryotic host has been genetically modified to increase its binding affinity for the lac repressor molecule LacI.

[0139] The examples described herein are illustrative of the present invention and are not intended to limit it. Various embodiments of the present invention have been described in accordance with the present invention. Many modifications and variations can be made to the techniques described and illustrated herein without departing from the spirit and scope of the present invention. Therefore, it should be understood that the examples are merely illustrative and do not limit the scope of the present invention.

[0140] Working Example Example 1: Overview and Materials and Methods Used in the Examples herein The aim of this study was to characterize σ 70 Two constitutive phage-derived promoters recognized by E. coli RNAP: T5 N25 and T7 A1 The aim of this study was to investigate the ability of the promoter sequences to express GFPmut3.1 in a BL21 expression system. The promoter sequences were modified to contain either one, two, or three lacO binding sites (SEQ ID NOs: 28-33). The seven promoter / operator combinations tested with the model protein GFPmut3.1 are shown in Figure 1. Expression strength, tunability, basal expression, and cell growth were investigated in plasmid-based and plasmid-free BL21 expression systems. The resulting sets of producing clones were cultivated under fed-batch-like conditions in microtiter fermenters and compared.

[0141] Strains and culture conditions. E. coli K-12 NEB5-α [fhuA2Δ(argF-lacZ)U169 phoA gln V44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17] was obtained from New England Biolabs (MA, USA) and used for all cloning procedures. Stranded DNA cartridges were integrated into the attTN7 site of the bacterial chromosome of E. coli BL21 [fhuA2 [lon] ompT gal [dcm] ΔhsdS] (New England Biolabs, MA, USA). For reference experiments, the same strains were transformed with the respective plasmids. The soluble protein GFPmut3.1 was used as a recombinant model protein (19).

[0142] Basic cloning procedures, such as restriction endonuclease (REN) digestion, agarose gel electrophoresis (AGE), ligation, and transformation of E. coli plasmids, were performed according to Sambrook et al. (24).

[0143] For cloning purposes, cells were routinely grown in M9ZB medium, recovered in SOC medium and plated on M9ZB agar. The following antibiotic concentrations were used: ampicillin (Amp) 100 μg / ml or 30 μg / ml, kanamycin (Kan) 50 μg / ml or 30 μg / ml, and chloramphenicol (Cm) 20 μg / ml or 10 μg / ml for plasmid-based and plasmid-free expression systems, respectively.

[0144] Culture conditions Strains were cultivated in 48-well Flower Plates® (m2p-labs, Bössweiler, Germany) in a BioLector microfermentation system as described by Torok et al. (23). Synthetic Feed-in-Time (FIT) Fed-batch medium (m2p-labs, Bössweiler, Germany) with glucose and dextran as carbon sources was used. 0.6% (v / v) glucose-releasing enzyme mix (EnzMiz) was added immediately before inoculation. GFPmut3.1 expression levels were monitored at 488 nm excitation and 520 nm emission. Signals are given in relative fluorescence units [rfu]. Cycle times for all parameters were 20 min. Initial cell densities were determined by OD 600 For inoculation, a flash frozen (-80°C) working cell bank (WCB) (OD 600 =2) were thawed and the biomass was harvested by centrifugation (7500 rpm, 5 min). Cells were washed with 500 μL of the corresponding medium to remove residual glycerol and centrifuged; the pellet was then resuspended in the whole culture medium. All cultures were prepared in triplicate at 30° C. for 22 h. Recombinant gene expression was induced 10 h after the start of the culture with 0.005 mM, 0.01 mM, or 0.5 mM IPTG, respectively.

[0145] For fed-batch fermentation, cells were grown in a 1.5 L (1.2 L working volume, 0.4 L minimum volume) DASGIP® Parallel Bioreactor System (Eppendorf, Germany) equipped with a standard control unit. pH was maintained at 7.0 ± 0.05 by addition of 12.5% ​​ammonia solution (Thermo Fisher Scientific, MA / USA); temperature was maintained at 37 ± 0.5 °C during the batch period and reduced to 30 ± 0.5 °C during the feed period. Dissolved oxygen (O2) levels were stabilized at > 30% saturation by controlling the agitation and aeration rates. Foaming was suppressed by addition of an antifoam suspending agent (Glanapon 2000, Bussetti, Austria). For inoculation, a deep-frozen (-80 °C) working cell bank vial was thawed and 1 ml (absorbance at 600 nm = 1) was aseptically transferred to the bioreactor.

[0146] Feeding was initiated when the culture, grown to 6 g cell dry mass (CDM) in 0.6 L batch medium, entered stationary phase. A fed-batch strategy with exponential carbon-limited substrate feeding was used to obtain a constant growth rate of 0.1 h-1 over 2.5 doubling times. The substrate feed was controlled by an exponential growth algorithm x = x0e with layered feedback control of the weight loss in the substrate bottle. μt The yield coefficient of cell dry weight with respect to glucose was 0.3 g / g, and the feed medium provided sufficient glucose and components to yield an additional 32 g cell dry weight. Induction of the expression system was performed by adding isopropyl-bD-thiogalactopyranoside (IPTG) to the reactor to obtain a concentration of 10 μmol / g cell dry weight. The preparation and composition of the minimal medium used in this experiment was previously described (17).

[0147] KK BL21 Q , or BQ lacI into E. coli BL21 (New England Biolabs, MA, USA) Q For promoter integration, the pETAmp-lacIq plasmid was constructed, which contains the ampicillin resistance gene flanked by FRT sites and the lacI Q The pET11a vector contains the lacI gene controlled by the promoter. The ampicillin resistance gene was amplified from pET11a using overhang PCR technique to add an FRT site and the restriction enzyme sites BamHI (5') and KpnI (3'). The following primers were used: BamHI-FRT-Amp-for and KpnI-FRT-Amp-rev.

[0148] The pBR322 origin of replication and lacI gene were amplified from pET30a using overhang PCR technique to add a C to T mutation in the lacI promoter and the restriction enzyme sites KpnI (5') and BamHI (3'). The following primers were used: KpnI-pBR322-for and BamHI-laciq-rev.

[0149] Stranded DNA cartridges for genomic integration were amplified using Q5® High Fidelity DNA Polymerase (New England Biolabs, MA / USA) according to the manufacturer's manual. The following primers were used: GI-lacIq-for and GI-lacIq-rev.

[0150] Integration into the bacterial chromosome occurred at the lac operon site of E. coli BL21 (New England Biolabs, MA / USA) harboring the pSIM5 plasmid as described by Sharan et al. (26).

[0151] Screening of positive clones and amplification of the integrated DNA cartridge were performed by basic colony PCR technique using OneTaq® DNA polymerase (New England Biolabs, MA / USA) according to the manufacturer's manual. The following primers were used: lacI / 1_ext and laci / 2_ext.

[0152] Primer AmpStop was used to sequence the amplified DNA integration cartridge.

[0153] BL21Q::TN7<1lacOA1-GFPmut3.1-tZ>, abbreviated to BQ<1lacO-A1> T7 A1 The promoter sequence was taken from (18) (P A1 / 04 The promoter was ordered as a gBlocks® gene fragment (Integrated DNA Technologies, IA / USA) containing a 2 bp truncated lacO1 sequence in the promoter region from -10 to -35. The promoter was then cloned into the pET30a-cer-tZENIT-GFPmut3.1 backbone. The new plasmid was named pETk1lacOA1tZ.c-GFPmut3.1.

[0154] Stranded DNA cartridges for genomic integration were amplified using Q5® High Fidelity DNA Polymerase (New England Biolabs, MA / USA) according to the manufacturer's manual. The following primers were used: TN7_1_pET30aw / oKanR_for and TN7_2_pET30a_for.

[0155] Integration into the bacterial chromosome was performed using E. coli BL21 carrying the pSIM5 plasmid as described by Sharan et al. (26). Q This occurred at the attTN7 site.

[0156] The following primers were used for screening positive clones: TN7 / 1_ext and TN7 / 2_ext.

[0157] The primers seq_MCS-for and seq_MCS-rev were used for sequencing the amplified DNA integration cartridge.

[0158] BL21Q::TN7<1lacOT5-GFPmut3.1-tZ>, abbreviated to BQ<1lacO-T5> T5 N25 The promoter sequence was taken from (18) and contains a 2-bp truncated lacO1 sequence in the promoter region from -10 to -35. N25 The initial transcriptional sequences (ITS) from +1 to +20 of T7 A1 (21). This promoter was ordered as a gBlocks® gene fragment (Integrated DNA Technologies, IA / USA) containing the 5' spacer sequence from pET30a and the restriction enzyme sites SphI (5') and XbaI (3') and subsequently cloned into the pET30a-cer-tZENIT-GFPmut3.1 backbone. The new plasmid was named pETk1lacOT5tZ.c-GFPmut3.1.

[0159] BL21::TN7<2lacOA1-GFPmut3.1-tZ> and BL21::TN7<2lacOT5-GFPmut3.1-tZ>, abbreviated to B<2lacO-A1> and B<2lacO-T5> lacI Q Besides the elevated level of lacI by the promoter, the second lacO can reduce basal expression by allowing DNA loop formation. For the addition of the second lacO1 sequence 62 bp upstream of the first lacO1, overhang PCR was performed using the templates pETk1lacOA1tZ.c-GFPmut3.1 or pETk1lacOT5tZ.c-GFPmut3.1, respectively. The forward primer (2lacO-for) contains the lac operator and the restriction enzyme site SphI (5'), and the reverse primer (2lacO-rev) contains the restriction enzyme site NdeI (3'). The new plasmids were named pETk2lacOA1tZ.c-GFPmut3.1 and pETk2lacOT5tZ.c-GFPmut3.1.

[0160] Integration into the bacterial chromosome occurred at the attTN7 site of E. coli BL21 (New England Biolabs, MA / USA).

[0161] Amplification and screening of concatenated DNA cartridges was performed as previously described.

[0162] Construction and characterization of promoter / operator combinations Basic cloning procedures, such as restriction endonuclease (REN) digestion, agarose gel electrophoresis (AGE), ligation, and transformation of E. coli plasmids, were performed according to Sambrook et al. (24). The lacI fragment was inserted into E. coli BL21 (New England Biolabs, MA / USA). Q For promoter integration, the plasmid pETAmp-lacIq was constructed, which contains the ampicillin resistance gene flanked by FRT sites and the lacI Q The plasmid pBR322 contains the lacI gene controlled by the pBR322 promoter (25). The pBR322 origin of replication and the lacI gene were amplified from pET30a using overhang PCR techniques to add a C to T mutation in the lacI promoter. Concatenated lacI for genomic integration Q The DNA cartridge was amplified using Q5® High Fidelity DNA Polymerase (New England Biolabs®, MA / USA) according to the manufacturer's manual. Integration into the bacterial chromosome occurred at the lac operon site of E. coli BL21 carrying the pSIM5 plasmid as described by Sharan et al. (26). This strain was BL21. Q It was named T7. A1 and T5 N25 The promoter sequence was adopted from Lanzer and Bujard (18) (P A1 / 04 and P N25 / 04 The promoters were ordered as gBlocks® gene fragments (Integrated DNA Technologies, IA / USA) containing a 2-bp truncated lacO1 sequence in the promoter region from -10 to -35. These promoters were then cloned into the pET30a-cer-tZENIT-GFPmut3.1 backbone, containing a 5' spacer sequence from pET30a and the restriction enzyme sites SphI (5') and XbaI (3'). The tZENIT terminator was described elsewhere (27). A second lacO1 sequence was added 62 bp upstream of the first lacO1 via overhang PCR. The 3lacO-T5 promoter / operator combination was taken from the pJexpress 401-406(T5) vector from ATUM, CA / USA. The linear DNA cartridges were transformed into E. coli BL21 or BL21 Q The plasmid was integrated into the attTN7 site of the bacterial chromosome.

[0163] Offline expression analysis and quantification of GFPmut3.1 Recombinant GFPmut3.1 was quantified by ELISA according to Reischer et al. (28). SDS-PAGE analysis was performed as previously described (29).

[0164] Flow cytometry The percentage of GFPmut3.1 producing cells was determined using a Galius flow cytometer (Beckman Coulter, CA / USA). Cells were harvested 12 hours after inoculation and then diluted 1 / 2025 in PBS. Excitation of GFPmut3.1 fluorescence was performed using a sapphire laser of an optically excited semiconductor laser at 488 nm, and the subsequent emission was measured through the use of the FL1 channel (505-545). Data were recorded at approximately 300 events / s for 15,000 cells per sample and analyzed using the Calusa analysis software (Beckman Coulter).

[0165] Western blot and quantification of LacI Approximately 1.2×10 7 Cell extracts obtained from BL21-wt and B<2lacO-A1> cells were separated by SDS-PAGE as previously described (29). After separation, proteins were blotted onto the given membranes using the iBlot® Dry Blotting System according to the manufacturer's manual (Invitrogen™ / Thermo Fisher Scientific, CA / USA). Proteins were subsequently blocked with 3% nonfat dry milk in PBST (1× PBS Dulbecco's and 0.05% Tween 20) for 4 h at room temperature. Blots were then incubated with primary antibody (anti-LacIl antibody, clone 9A5 (Sigma-Aldrich / Merck, MO / USA) at 1:1000) for 1 h at room temperature. They were then incubated with secondary antibody conjugated to alkaline phosphatase (anti-mouse IgG (whole molecule)-Sigma A5153 (Sigma-Aldrich / Merck, MO / USA) at 1:2000 at room temperature for 1 h and developed with Sigma FAST™ BCIP® / NPT tablets (Sigma-Aldrich / Merck, MO / USA) according to the manufacturer's manual. Band intensity was quantified using ImageQuant TL software (GE Healthcare, IL / USA).

[0166] [Table 1]

[0167] [Table 2]

[0168] [Table 3]

[0169] Example 2: Productivity of host RNA polymerase-dependent promoter / operator combinations The T7 expression system is known to give high expression rates even from a single copy of the target gene integrated into the E. coli genome. First, the same productivity was achieved in the same experimental setting. 70 We tested whether the promoters could be reached by E. coli RNA polymerase-dependent promoters. Therefore, we used plasmid-free and plasmid-based T5 N25 and T7 A1 The promoter / operator combination was compared to the T7 expression system. Cells were grown in fed-batch-like conditions in microtiter fermentations over a period of 22 hours. GFP expression was induced after 10 hours with a single pulse of 0.5 mmol / L IPTG.

[0170] In all promoter / operator combinations, cells were able to sustain growth during a 12-h production period in microtiter fermentations. The average growth rate of μ=0.05 / h allowed for a direct comparison of T7 and host RNA polymerase-dependent promoters.

[0171] In the plasmid-based expression systems, the results of online fluorescence measurements of GFPmut3.1 were in a similar range to the T7 expression system for all promoter / operator combinations, except B(3lacO-T5) (Figure 2B). These results were confirmed by SDS-PAGE analysis. However, in the genome-integrated expression systems, very distinct differences of each promoter / operator combination could be observed (Figure 2A). Compared to the T7 expression system, the yield of GFPmut3.1 was 1.5-fold higher in the A1 expression system. In the genome-integrated T7 expression system, induction of GFP gene expression led to a specific product concentration (Y ) of 145 relative fluorescence units and approximately 135 mg / g of soluble GFPmut3.1. P / X ), and negligible amounts of inclusion bodies (IB). The same experiment with the A1 expression system yielded approximately 50 relative fluorescence units and 37 mg / g of inclusion body-free soluble GFPmut3.1.

[0172] The observed reduced productivity of B(3lacO-T5) and B<3lacO-T5> may result from a completely symmetric lac-operator (sym-lacO) (7) in the early transcription sequence (ITS), which has an effect on promoter avoidance and therefore productivity (21). This effect is less visible in the plasmid-based 3lacO-T5 expression system, where the high plasmid copy number compensates for the reduced promoter activity. However, in the plasmid-free expression system, the promoter activity is very low, so the three lacO forms for the A1 promoter were rejected. For the one and two lacO promoter / operator combinations, sym-lacO was replaced with the native early transcription sequence (+1 to +20) of the A1 promoter. This resulted in a 2.4-fold increase in productivity in the case of the T5 promoter. However, the reduction in lacO binding sites inevitably increased basal expression.

[0173] Example 3: Basal expression in a host RNA polymerase-dependent expression system For difficult proteins, even low basal expression can have deleterious effects on the host's metabolism. Sometimes, the transformation or integration cartridge of the plasmid leads to toxicity and transformants are difficult to obtain. Therefore, the tightness of gene regulation is an important quality criterion of an expression system.

[0174] In the plasmid-based system, the promoter controlled by one lac-operator (1lacO) showed the highest basal expression, especially under carbon-limited conditions, at a level of about 10 relative fluorescence units. Addition of a second lacO (2lacO), or lacI Q Increasing the inhibitor LacI by introducing the promoter reduced the basal expression of the A1 promoter by 50%. In the case of the T5 promoter, only the combination of three lac-operators (3lacO) reduced the basal expression to almost zero relative fluorescence units. In contrast to the plasmid-based expression systems, in all genomic integrated systems, a significant effect of the promoter / operator combination on the readthrough of the system could be observed. Either increasing the number of LacI molecules or adding a second lacO reduced the basal expression of the A1 expression system from 14 relative fluorescence units to almost no significant background expression, without any loss of productivity (Figure 2A). Although both promoters contain lac-operators at the same position, only increased levels of LacI molecules or three lac-operators sufficiently reduced the basal expression of the T5 expression system. A1 The promoter is T5 N25 Because the lac-repressor is recognized by RNA polymerase only half as efficiently as the lac-operator (20), and one lac-operator is located within the promoter sequence from the -10 to -35 promoter elements, the host RNA polymerase and the lac-repressor compete with each other for their respective binding sites, which determines how efficiently promoter activity is controlled by the repressor.

[0175] Example 4: Control of recombinant gene expression rate Transcription rate control, also called the fine tuning or "tunability" of protein production, is highly relevant in bioprocessing. Optimal bioprocesses are designed to maximize the synthetic capacity of the cell for the longest period of time to obtain properly folded and processed proteins. Depending on the physical properties and metabolic requirements of the desired product, the transcription rate must be adapted depending on the RNA stability, translation efficiency, folding, transport, and all other interactions in the system.

[0176] To evaluate the tunability of the promoter / operator combinations described herein, a series of fed-batch-like microtiter cultures at various IPTG levels were tested and compared to a plasmid-free T7 expression system. Induction was performed using a single pulse of 0.005, 0.01, and 0.5 mM IPTG. Online fluorescence measurements and end-point flow cytometry analysis were used to characterize the various promoter / operator combinations.

[0177] The expression system controlled by one lacO for gene regulation showed the highest basal expression but also the least pronounced scale of GFPmut3.1 expression at a given inducer concentration (Fig. 3C, F). The promoters with two lacOs showed sufficiently low basal expression, but they produced significantly less at lower inducer concentrations (Fig. 3B, E). The promoter / operator combinations 3lacO-T5 and 2lacO-A1 completely stopped the production of recombinant GFP after a certain time, independent of the inducer concentration (Fig. 3A, E). This behavior was not observed in the promoter / operator combinations with only one lacO. The promoter controlled by one lacO, lacI Q (Fig. 3D, G), and the T7 expression system (Fig. 3H), combine the desirable properties of low readthrough and tunability of the system.

[0178] However, T7 expression systems are known to exhibit an “all-or-none” behavior, and the reduced expression levels in partially induced cultures here are the result of the formation of subpopulations of fully induced and uninduced cells, as reviewed in (22). To answer the question, where single-cell regulativity in the host RNA polymerase-dependent expression system is possible, flow cytometric analysis of all promoter / operator combinations was performed. As shown in Figure 4, the genomically integrated T7 expression systems do not show any homogenous populations in partially induced cultures. In fact, a mixture of fully induced, partially induced, and uninduced cells was found especially at very low inducer concentrations. In the B<2lacO-A1> expression system, flow cytometric analysis revealed two clearly distinct subpopulations of producing and non-producing cells (Figure 4), since these expression systems stopped their productivity but still continued to grow. This behavior was also observed in B<3lacO-T5>. This was not the case for BQ<1lacO-A1>, where induction of GFP resulted in a homogenous population at any given IPTG concentration (FIG. 4).

[0179] Based on these findings, the complete cessation of productivity of all other expression systems when partially induced appears to be related to the autoregulation of the lac inhibitor. The lac-operon is regulated by three lacO binding sites (Figure 5A). LacI molecules bind either to lacO1 and lacO3 or to lacO1 and lacO2. LacO3 overlaps with the 3' end of the lacI gene. Binding of LacI to lacO1 and lacO3 leads to DNA looping, resulting in a truncated lacI mRNA molecule, which is digested by the cell. This results in a constant level of approximately 40 molecules in fully induced cells and approximately 15 molecules in uninduced cells.

[0180] The binding constant (K a ) is higher than the binding constant for lacO in the lac-operon, the first LacI molecule that is not inactivated by IPTG will preferentially bind to the lacO binding site of the gene of interest, instead of lacO3 / lacO1 on the lac-operon. Thus, the autoregulation of LacI does not intervene, and more LacI molecules are produced (Figure 5B). The whole system becomes overregulated, and as a result, production stops completely.

[0181] To support this hypothesis, we compared the effect of autoregulation on LacI levels in B<2lacO-A1> and BL21 wild-type (BL21-wt) cells. The LacI content in uninduced, partially induced, and fully induced cells was estimated using Western blot analysis. Band intensities were quantified and normalized by cell number (Figure 7).

[0182] In fully induced BL21 wild-type cells, the amount of LacI molecules was 3.5-fold higher than that in uninduced BL21 wild-type cells. Partial induction with 0.01 mM IPTG increased it by only 0.3-fold. The fold change of 3.5 in fully induced BL21-wt cells is in accordance with the results of Semsey et al. (11), who measured an average of 15 LacI molecules per cell in the absence of inducer and about 40 molecules in fully induced cells. In B<2lacO-A1>, the amount of LacI in uninduced and partially induced cells was clearly higher than that in BL21 wild-type. The yield of LacI was 2.3-fold higher in the absence of inducer and 2.7-fold higher in partially induced cells compared to BL21-wt. In fully induced cells, the yield of LacI was 4.0-fold, which is comparable to that of fully induced wild-type BL21.

[0183] Addition of 0.01 mM IPTG results in nearly half-maximal expression of GFPmut3.1 (Figure 3), but it has almost no effect on LacI levels. Apparently, LacI is still able to bind to lacO1 / lacO3 of the lac operon and thus maintain its autoregulation under these conditions. In addition, the lacI gene is transcribed from a weak promoter, resulting in the strong T7 A1 Unlike promoters, which give rise to approximately one new mRNA per cell generation (38), the high LacI levels in uninduced and partially induced B<2lacO-A1> cells clearly support our hypothesis of an autoregulatory influence of LacI on expression rate control in genomically integrated E. coli producer strains, as shown in (Figure).

[0184] The effect of LacI autoregulation was only observed in genome-integrated host RNA polymerase-dependent expression systems controlled by two or three lac operators. However, this effect was not observed in plasmid-based host RNA polymerase-dependent expression systems or in conventional T7 expression systems. The reason for this can be attributed to the equilibrium of the lac operators with respect to the LacI concentration. The T7 expression system has an additional lacI gene sequence in its DE3 lysogen, thus theoretically doubling the concentration of LacI per cell. The plasmid-based expression system used in this study is based on the pET expression system, which encodes an additional lacI gene sequence. This in turn results in an additional 15-20 lacI gene sequences depending on the plasmid copy number. However, the effect of LacI autoregulation on partially induced cells can also be observed in plasmid-based expression systems, such as in the case of the E. coli pAVEway™ expression system from Fujifilm Diosynth Biotechnologies (NC / USA). In this plasmid-based expression system, transcriptional control is provided by two perfectly symmetric lac operators, one of which is T7 A3 One is located upstream and one downstream of the promoter. The high affinity of LacI for the symmetric lac operator combined with its ability to form DNA loops results in very low basal expression but also a complete cessation of productivity in partially induced cultures.

[0185] Given the autoregulation of the lac inhibitors, promoter / operator combinations could be successfully identified that fulfilled the desired properties, such as high expression rate, negligible basal expression, and true control of expression rate without complete cessation of productivity even at low inducer concentrations.

[0186] conclusion Transcriptional regulation in E. coli has received considerable attention because it is the first step in the process of recombinant protein production. Transcriptional control allows the cell to allocate its resources towards recombinant protein production, and tight and tunable control is essential for a successful bioprocess. Herein, it is demonstrated that in a plasmid-free expression system, the regulatory elements of the lac-operon must be well balanced to control the host RNA polymerase-dependent promoter. Three lac-operators reduce the basal expression to negligible amounts, but also reduce the recombinant production rate. The perfectly symmetric lacO in the early transcription sequence (ITS) prevents the escape of the RNA polymerase by the promoter. As shown by Hsu et al., the wild-type early transcription sequence of T7A1 exhibits purine reinforcement and one of the best promoter escape properties (21).

[0187] Promoters containing only one lacO show significantly higher promoter strength, but also higher systemic read-through. In promoter / operator combinations containing two lacOs, the two lacO1s, which are 62 bp apart at the site of the gene of interest, show very strong binding affinity to the repressor molecule, thus preventing the autoregulation of lacI, resulting in complete cessation of productivity in partially induced cells. However, the binding affinity can be reduced by using less symmetric lacOs, such as lacO3 or lacO2, or by changing the distance between them (see Example 5).

[0188] As demonstrated herein, lacI Q The combination of the promoter-driven elevated levels of intracellular LacI and one lacO results in high expression rates, low basal expression, and true tunability at the cellular level. This novel expression system is therefore particularly suitable for the production of difficult proteins, since there is no plasmid-mediated metabolic burden and gene stability is increased by the use of the host RNA polymerase.

[0189] Importantly, the inducible system described herein exhibits significantly improved expression rates, reduced basal expression, and true tunability compared to the T7 expression system (see, e.g., Figures 3 and 4). The inducible expression system described herein meets all the desired characteristics required for an efficient expression system, such as high expression rates, negligible basal expression, and true control over expression rates that are infinitely adjustable, even at low inducer concentrations.

[0190] Example 5: Control of recombinant gene expression rate in an inducible expression system containing two lacOs Strains: BL21::TN7<2lacO.xxA1-GFPmut3.1-tZ> and BL21::TN7<2lacO.xxT5-GFPmut3.1-tZ>, abbreviated to B<2lacO.xx-A1> and B<2lacO.xx-T5>.

[0191] For the addition of a second lacO2 sequence that is more than 62 bp away from the first lacO1, overhang PCR is performed using templates pETk1lacOA1tZ.c-GFPmut3.1 or pETk1lacOT5tZ.c-GFPmut3.1, respectively. The two lacO1 operators are 92, 103, 114 or 125 bp apart. The following primers 2lacO.92-for, 2lacO.103-for, 2lacO.114-for, and 2lacO.125-for contain the lac operators and the restriction enzyme site SphI (5'), and the reverse primer (2lacO-rev) contains the restriction enzyme site NdeI (3'). The new plasmids are named pETk2lacO.92A1tZ.c-GFPmut3.1, pETk2lacO.103A1tZ.c-GFPmut3.1, pETk2lacO.114A1tZ.c-GFPmut3.1, pETk2lacO.125A1tZ.c-GFPmut3.1 and pETk2lacO.92T5tZ.c-GFPmut3.1, pETk2lacO.103T5tZ.c-GFPmut3.1, pETk2lacO.114T5tZ.c-GFPmut3.1, pETk2lacO.125T5tZ.c-GFPmut3.1.

[0192] Integration into the bacterial chromosome occurs at the attTN7 site of E. coli BL21 (New England Biolabs, MA / USA).

[0193] Amplification and screening of the concatenated DNA cartridges is carried out as described above.

[0194] Example 6: Fab production using BQ<1lacO-A1> in fed-batch culture T7-based expression systems show sufficient inherent strength for high expression rates, even from a single copy. Significantly reduced expression rates are to be expected in systems with a single copy of the gene of interest under the control of a promoter specific for the host RNA polymerase. As a consequence, such systems will not be competitive when recombinant proteins have to be produced at high levels. The situation is different for antibody fragments and other difficult proteins, where the yield of the final product is not clearly determined by the strength of the promoter system, but by reasons that are currently not identified. To investigate these aspects, the BQ<1lacO-A1> expression system was selected for the production of the leader / Fab combination dsbA / FTN2 (dFTN2) and the B3 expression system, which produces the same leader / Fab combination. <t7>The cells were grown in fed-batch mode with a constant growth rate of 0.1 / h and a feeding of defined medium. The amount of cell dry weight to be produced in the experiment was predefined as 40 g cell dry weight. Recombinant gene expression was induced with one pulse of 10 μmol / g cell dry weight IPTG at 0.5 doubling time from the start of feeding.

[0195] The results in Figures 8 and 9 are shown as the specific content of total recombinant Fab per cell dry weight (mg / g), which is the sum of extracellular and intracellular Fab measured in the fermentation supernatant. In the T7-based system (Figure 8), induction of dFTN2 expression resulted in a maximum intracellular Fab concentration of 1.8 mg / g 11 hours after induction, which dropped to 0.7 mg / g at the end of fermentation (Figure 8, open diamonds). During this period, extracellular Fab increased from almost 0 mg / g to 2.2 mg / g (Figure 8, open triangles). This resulted in a maximum total Fab concentration of 3.5 mg / g 15 hours after induction, which dropped to 2.1 mg / g at the end of fermentation (Figure 8, black dots). The increase in extracellular Fab in the fermentation supernatant could be attributed to cell lysis, which could be confirmed by measuring the DNA content in the fermentation supernatant.

[0196] The same experiment with the BQ<1lacO-A1> expression system gave significantly improved results (Figure 9). The content of intracellular Fab could be maintained at 2.5 mg / g during the entire fermentation (Figure 9, open diamonds). The extracellular Fab content increased to 2.4 mg / g at the end of the fermentation (Figure 9, open triangles). This resulted in a maximum total Fab concentration of 4.7 mg / g at the end of the fermentation (Figure 9, black dots). Although the relative promoter strength of 1lacO-A1 is about 30% compared to T7, this expression system produced the same amount of total Fab as the strong T7 expression system up to 15 hours after the start of the feed, and exceeded the T7 system by two orders of magnitude at the end of the fermentation. These results clearly indicate that a reduced promoter strength can be beneficial for the production of difficult proteins, since it reduces the metabolic burden of the cells and stress-induced protein degradation.

[0197] References

Table 4

Claims

1. at least a) the RNA polymerase (RNAP) gene; b) a gene encoding a protein of interest (this is, - coding sequence, - a promoter operably linked to said coding sequence, said promoter being recognized by the RNA polymerase expressed from a); and - in the sequence of said promoter, one lac operator (lacO) is included within the sequence of the promoter element from -10 to -35 c) the lacI gene, which encodes the lac repressor protein (LacI) (this is, - coding sequence, - a lacI promoter operably linked to a lacI coding sequence, wherein the lacI promoter is a wild-type lacI promoter or a lacI promoter that increases LacI expression, wherein the rate of expression of a protein of interest is regulated by an inducer that binds to LacI. A genome-based expression system for the production of a protein of interest (POI) in a prokaryotic host, comprising:

2. 2. The genome-based expression system of claim 1, wherein the gene encoding the protein of interest contains one lacO within a promoter sequence and one lacO upstream of the first lacO.

3. A genome-based expression system as described in claim 1 or 2, wherein the lacI promoter is a promoter that increases LacI expression.

4. 4. The genome-based expression system of any one of claims 1 to 3, wherein the gene encoding the protein of interest contains one lacO within the promoter sequence and one lacO upstream of the first lacO, and the lacI promoter is a promoter that increases LacI expression.

5. The genome-based expression system according to any one of claims 1 to 4, wherein the prokaryotic host is Escherichia coli (E. coli).

6. The genome-based expression system according to any one of claims 1 to 5, wherein the RNA polymerase is a heterologous or homologous RNA polymerase.

7. The genome-based expression system of claim 6, wherein the RNA polymerase is σ 70 E. coli RNA polymerase.

8. The promoter of claim 1 b) is T5, T5 Ｎ２５ 8. The genome-based expression system of claim 1 , wherein the gene is selected from the group consisting of T7A1, T7A2, T7A3, lac, lacUV5, tac, and trc.

9. The lacI promoter that increases LacI expression comprises the lacI gene of SEQ ID NO:

1. Ｑ A genome-based expression system according to any one of claims 1 to 8, which is a promoter.

10. 10. The genome-based expression system of any one of claims 1 to 9, wherein the lac operator is lacO1 comprising SEQ ID NO:3, lacO2 comprising SEQ ID NO:4, or lacO3 comprising SEQ ID NO:5, or a functional variant thereof having at least 90% sequence identity or a completely symmetrical lacO.

11. The genome-based expression system according to any one of claims 1 to 10, wherein the promoter operably linked to a coding sequence encoding a protein of interest comprises an early transcription sequence (ITS).

12. The genome-based expression system of claim 11, wherein the early transcription sequence (ITS) is a native T7A1 early transcription sequence comprising SEQ ID NO:

2.

13. 13. The genome-based expression system of any one of claims 1 to 12, wherein the inducer is selected from the group consisting of isopropylthiogalactoside (IPTG), lactose, methyl-β-D-thiogalactoside, phenyl-β-D-galactose, and ortho-nitrophenyl-β-galactoside (ONPG).

14. The gene encoding the protein of interest contains one lacO1 operator in the promoter sequence operably linked to the coding sequence and a native T7A1 early transcription sequence comprising SEQ ID NO:2, and the lacI promoter is lacI Ｑ A genome-based expression system according to any one of claims 1 to 13, which is a promoter.

15. 14. The genome-based expression system of any one of claims 1 to 13, wherein the gene encoding the protein of interest contains two lac operators, separated by at least 92 base pairs (bp), where one lac operator is located within a promoter sequence operably linked to the coding sequence and a second lac operator is upstream of the promoter.

16. a) culturing the host cells and inducing expression of the gene encoding the protein of interest by addition of an inducer; b) harvesting the protein of interest, and c) isolating and purifying the protein of interest A method for the plasmid-free production of a protein of interest in a prokaryotic host using the genome-based expression system according to any one of claims 1 to 15, comprising:

17. a) one or more coding sequences encoding one or more proteins of interest; b) a promoter operably linked to one or more coding sequences; and c) Within the promoter sequence, one lac operator (lacO) within the sequence of the promoter element from -10 to -35 (wherein the affinity of lacI for lacO in c) is lower than the affinity of lacI for the lac operators lacO1 and lacO3 of the endogenous lac operon of the host cell) 1. An expression cassette for a genome-based expression system comprising at least one heterologous gene designed to produce at least one heterologous protein of interest, comprising:

18. The expression cassette of claim 17, wherein the heterologous gene designed to produce at least one heterologous protein of interest contains one lacO within the promoter sequence and one lacO upstream of the first lacO.

19. 19. The expression cassette of claim 17 or 18, wherein the heterologous gene designed to produce at least one heterologous protein of interest comprises two lac operators separated by at least 92 bp, wherein one lac operator is located within the promoter sequence and a second lac operator is upstream of the promoter.

20. lacI containing SEQ ID NO:1 Ｑ 20. The expression cassette of any one of claims 17 to 19, further comprising a promoter, a heterologous lacI promoter, wherein the heterologous gene designed to produce at least one heterologous protein of interest comprises a lacO1 operator within the promoter sequence operably linked to a coding sequence and a native T7A1 early transcription sequence comprising SEQ ID NO:

2.

21. a) integrating the expression cassette into the chromosome of a prokaryotic host; b) culturing the host cells and inducing expression of the gene encoding the protein of interest by addition of an inducer; c) harvesting the protein of interest; and d) isolating and purifying the protein of interest A method for producing a protein of interest in a prokaryotic host on a manufacturing scale using an expression cassette according to any one of claims 17 to 20, comprising:

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