Expression vector and use thereof

Abstract

Provided are an Escherichia coli expression vector and a use thereof. The Escherichia coli expression vector comprises at least one Escherichia coli homologous pgl gene. Provided is an engineered Escherichia coli strain, the genome of which comprises a mutation of a zwf gene. Also provided is a method for increasing the purity of a recombinant protein by using the vector and / or strain.

Description

Expression vectors and uses thereof

[0001] Cross-reference to related applications

[0002] This disclosure claims the benefit of and priority to Chinese Patent Application No. 202410958593.2, filed June 5, 2024, entitled “A Method for Improving the Purity of Recombinant Proteins in Escherichia coli and Applications Thereof,” the entire contents of which, including any sequence listing and drawings, are incorporated herein by reference in their entirety.

[0003] Incorporation by reference of electronic sequence listing

[0004] This disclosure contains an electronic sequence listing (“CIE25S0078PCT.xml,” which was created on June 5, 2025, in accordance with WIPO Standard ST.26 using the “WIPO Sequence” software, and is 76,270 bytes in size), which is incorporated herein by reference in its entirety. In accordance with WIPO Standard ST.26, the symbol “t” is used to represent both T in DNA and U in RNA (see “Table 1: List of Nucleotide Symbols,” the definition of the symbol “t” is “Thymine in DNA / Uracyl in RNA (t / u)”). Thus, in an electronic sequence listing prepared in accordance with ST.26, in any instance where the sequence is RNA, all T’s in the sequence should be considered as U’s. TECHNICAL FIELD

[0005] The present disclosure is in the field of genetic engineering and protein engineering, and specifically relates to expression vectors and uses thereof. BACKGROUND

[0006] Escherichia coli (E. coli) is a host commonly used to express proteins in research, diagnostics, therapeutics, and industrial applications. Current expression systems are capable of producing high yields of a wide range of proteins. The most commonly used E. coli strain in industrial processes is BL21(DE3) because it has multiple important properties, including (i) fast growth, (ii) low acetate production even when grown at high glucose concentrations, and (iii) high turnover of the tricarboxylic acid cycle.

[0007] Nonenzymatic posttranslational modification can be a result of inevitable interference between the bacterial metabolic pathways and the abundant heterologously expressed proteins. This modification leads to heterogeneity of the expressed proteins. For therapeutic proteins, efforts must be made to correct this metabolic interference to help ensure the highest level of protein quality. Nonenzymatic glycation is a class of posttranslational modifications that have important implications. This class of reactions has been extensively evaluated in higher eukaryotes and in prokaryotes.

[0008] Gluconoylation is a common post-translational glycosylation that can adversely affect the quality of recombinant proteins. This modification relies on the formation of 6- phosphogluconolactone (6-PGLac or 6-PGL), an intermediate of the pentose phosphate (PP) pathway, produced by glucose-6-phosphate dehydrogenase. As an effective electrophile, 6-PGL is expected to participate in non-enzymatic glycosylation reactions in vivo. 6-PGL is converted to 6-phosphogluconate by hydrolysis of phosphogluconolactonase (PGL) (EC 3.1.1.31). But if the conversion of 6-PGL is lacking, leading to the start of accumulation of 6-PGL, it can become an important inducer of gluconoylation of proteins in eukaryotes and prokaryotes. The impact of post-translational modification can be very significant. For example, glycosylation of alanine aminotransferase has been shown to significantly reduce its activity. Other researchers have demonstrated interference with protein crystallization. Gluconoylation caused by 6-PGL is therefore considered undesirable when expressing heterologous recombinant proteins with E. coli, the resulting by-product is an impurity that one would like to reduce or even eliminate.

[0009] It has been observed that E. coli host strain BL21(DE3) exhibits low endogenous PGL activity in vitro. Juan C. Aon et al. (Aon J C, Caimi RJ, Taylor AH, Lu Q, Oluboyede F, Dally J, Kessler MD, Kerrigan JJ, Lewis TS, Wysocki LA, Patel PS. 2008. Suppressing Posttranslational Gluconoylation of Heterologous Proteins by Metabolic Engineering of Escherichia coli. Appl Environ Microbiol 74: https: / / doi.org / 10.1128 / AEM.01790-07) proposed the introduction of a heterologous phosphogluconolactonase PGL from Pseudomonas aeruginosa in BL21(DE3) in an attempt to reduce the undesirable post-translational gluconoylation modification on recombinant proteins caused by 6-PGL.

[0010] However, P. aeruginosa is an opportunistic pathogen (Gellatly, S. L., & Hancock, R. E. (2013). Pseudomonas aeruginosa: new insights into pathogenesis and host defenses. Pathogens and disease, 67(3), 159-173. https: / / doi.org / 10.1111 / 2049-632X.12033) that can cause extensive severe opportunistic infections in patients with severe underlying diseases. These infections are characterized by a strong neutrophilic response leading to significant damage to host tissues and often also exhibit resistance to antibiotics, leading to death. Treatment of persistent infections is additionally hampered by adaptive resistance due to the growth status of the bacteria in the patient (including the ability of the microorganisms to grow in biofilms).

[0011] Therefore, when using E. coli as an expression host cell to produce recombinant proteins such as antibodies, polypeptide drugs for disease prevention, diagnosis or treatment, the expression of exogenous proteins from pathogenic bacteria in E. coli host cells, in turn, leads to the possibility of the presence of these exogenous proteins from pathogenic bacteria in the therapeutic recombinant proteins, which constitutes an additional safety risk and regulatory challenge (e.g. new drug approval registration regulations). Moreover, the protein stability and functional stability of these exogenous proteins in E. coli host cells and their effects on the growth of E. coli host cells, etc. are questionable.

[0012] In addition, although attempts have been made to avoid the undesirable modification caused by 6-PGL by introducing PGL to consume 6-PGL in downstream reactions, the production of 6-PGL has not been addressed at the source.

[0013] Therefore, there is a need in the art for new technologies to overcome the current safety, effectiveness and other problems to express recombinant proteins in E. coli, particularly recombinant proteins for disease prevention, diagnosis or treatment.

[0014] The citation or identification of any document in this disclosure is not an admission that such document is available as prior art to the present disclosure. Each reference cited or identified in this disclosure is incorporated by reference in its entirety. SUMMARY

[0015] The present disclosure provides various E. coli engineering solutions for the upstream and downstream biochemical reactions of 6-PGL, to address the deficiencies of the prior art solutions and to solve the needs in the field. The recombinant proteins produced by the E. coli of the present disclosure have significantly reduced degree of undesired glucosylation modification initiated by 6-PGL, and the purity of the recombinant proteins is significantly increased. The engineering means used does not involve introduction of heterologous proteins, significantly reducing the safety risk, especially when used to produce recombinant proteins for disease prevention, diagnosis or treatment, significantly reducing the regulatory challenges (e.g. new drug approval registration regulations).

[0016] More specifically, the present disclosure enhances the downstream metabolic pathway of 6-PGL in E. coli by introducing an engineered recombinant protein expression vector comprising a specifically selected E. coli homologous pgl gene, reduces or eliminates the accumulation of 6-PGL and the resulting undesired modification, and improves the purity of the recombinant protein. Compared with the introduction of heterologous pgl gene and PGL protein from pathogenic bacteria, the safety is significantly improved, and in some embodiments, the purity of the recombinant protein is further improved. The present disclosure also reduces or eliminates the expression and function of glucose-6-phosphate dehydrogenase (G6PDH) by knocking out the zwf gene on the genome of E. coli, disrupting the upstream metabolic pathway of 6-PGL in E. coli, reducing or eliminating the production of 6-PGL from the upstream, thereby reducing or eliminating the accumulation of 6-PGL and the resulting undesired modification, and improving the purity of the recombinant protein. The engineered recombinant protein expression vector comprising a specifically selected E. coli homologous pgl gene of the present disclosure can be used in combination with conventional E. coli strains, or in combination with the zwf-knocked-out engineered E. coli strains of the present disclosure. Conversely, the zwf-knocked-out engineered E. coli strains of the present disclosure can be used in combination with conventional recombinant protein expression vectors that do not comprise pgl genes, or in combination with the engineered recombinant protein expression vector comprising a specifically selected E. coli homologous pgl gene of the present disclosure. In the case of combination of the engineered recombinant protein expression vector of the present disclosure with the engineered E. coli strain of the present disclosure, in some embodiments, the purity of the recombinant protein is further improved, and there is an additive or even synergistic effect.

[0017] In one aspect, the present disclosure provides an engineered recombinant protein expression vector of E. coli comprising at least one E. coli homologous pgl gene.

[0018] In some embodiments, the engineered recombinant protein expression vector is engineered based on pET28a plasmid.

[0019] In some embodiments, the pgl gene comprises a polynucleotide sequence encoding a protein having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14 and having 6-phosphogluconolactonase activity; or wherein the pgl gene comprises a polynucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 13 and encoding a protein having 6-phosphogluconolactonase activity.

[0020] In some embodiments, a promoter sequence is operably linked to the 5’ end of the pgl gene to form a promoter-pgl gene sequence; optionally the promoter sequence is a P Native rpl promoter sequence having a nucleotide sequence as set forth in SEQ ID NO: 6 or a P GAP rpl promoter sequence having a nucleotide sequence as set forth in SEQ ID NO: 7; optionally the promoter-pgl gene sequence is as set forth in SEQ ID NO: 4 or 5.

[0021] In some embodiments, a terminator sequence is operably linked to the 3’ end of the promoter-pgl gene to form a pgl expression cassette; optionally the terminator sequence has a nucleotide sequence as set forth in SEQ ID NO: 24; optionally the pgl expression cassette has a nucleotide sequence as set forth in SEQ ID NO: 25.

[0022] In some embodiments, the engineered recombinant protein expression vector further comprises a stability component; optionally wherein the stability component is a Cer element from a ColEl plasmid; optionally wherein the stability component comprises a polynucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16.

[0023] In some embodiments, the stability component is inserted into the engineered recombinant protein expression vector in a 3’-5’ or 5’-3’ orientation.

[0024] In some embodiments, the engineered recombinant protein expression vector does not comprise a F1 ori; optionally, wherein the F1 ori comprises a polynucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 17.

[0025] In some embodiments, the engineered recombinant protein expression vector comprises a multiple cloning site (MCS); optionally, wherein the MCS comprises a polynucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 15.

[0026] In some embodiments, the engineered recombinant protein expression vector comprises a gene of interest (GOI); optionally, wherein the GOI is inserted into the MCS or replaces part or all of the MCS.

[0027] In some embodiments, the GOI is a recombinant protein-encoding gene; optionally, the recombinant protein is a recombinant protein for disease prevention, diagnosis, or treatment, such as an antibody, a vaccine, an enzyme.

[0028] In some embodiments, the stability component is located between the MCS or the GOI and the pgl gene.

[0029] In some embodiments, the engineered recombinant protein expression vector comprises, from 5’-3’ direction, the MCS or the GOI, the stability component, and the pgl gene, and does not comprise a F1 ori.

[0030] In some embodiments, the engineered recombinant protein expression vector comprises a polynucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 21.

[0031] In some embodiments, the engineered recombinant protein expression vector comprises, from 5’-3’ direction:

[0032] (1) the MCS as set forth in SEQ ID NO: 15;

[0033] (2) the stability component set forth in SEQ ID NO: 16; and

[0034] (3) the promoter-pgl gene sequence set forth in SEQ ID NO: 4 or the pgl expression cassette set forth in SEQ ID NO: 25;

[0035] and does not include the F1 ori set forth in SEQ ID NO: 17;

[0036] wherein the engineered recombinant protein expression vector comprises a polynucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 21.

[0037] In some embodiments, the engineered recombinant protein expression vector comprises, in the 5’-3’ direction:

[0038] (1) the gene of interest;

[0039] (2) the stability component set forth in SEQ ID NO: 16; and

[0040] (3) the promoter-pgl gene sequence set forth in SEQ ID NO: 4 or the pgl expression cassette set forth in SEQ ID NO: 25;

[0041] and does not include the F1 ori set forth in SEQ ID NO: 17;

[0042] wherein the engineered recombinant protein expression vector comprises a polynucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 21.

[0043] In another aspect, the present disclosure provides an engineered E. coli strain comprising a mutation in a zwf gene on its genome, the mutation (e.g., a partial or complete knockout of the zwf gene) resulting in the zwf gene not expressing a protein having glucose-6-phosphate dehydrogenase (G6PDH) activity.

[0044] In some embodiments, the engineered E. coli strain is engineered on the basis of a B series (e.g., BL21(DE3) strain) or K-12 series (e.g., SHuffle T7 Express, W3110, or MG1655 strain) E. coli strain.

[0045] In some embodiments, the zwf gene comprises a polynucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2 and encodes a protein having glucose-6-phosphate dehydrogenase activity.

[0046] In some embodiments, the mutation of the zwf gene is introduced by homologous recombination using a zwf gene knockout fragment comprising two homology arms, each of which is identical to a portion of the zwf gene; optionally the zwf gene knockout fragment further comprises a sequence between the two homology arms; optionally the sequence comprises a resistance gene sequence, e.g., a resistance gene sequence encoding kanamycin, tetracycline, streptomycin, or chloramphenicol resistance.

[0047] In some embodiments, the zwf gene knockout fragment comprises a 5’ homology arm identical to the 5’ end of the zwf gene and a 3’ homology arm identical to the 3’ end of the zwf gene; optionally the zwf gene knockout fragment further comprises a sequence between the two homology arms; optionally the sequence comprises a resistance gene sequence, e.g., a resistance gene sequence encoding kanamycin, tetracycline, streptomycin, or chloramphenicol resistance.

[0048] In some embodiments, the zwf gene knockout fragment comprises a polynucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 10 or 11.

[0049] In some embodiments, the mutation of the zwf gene is a substitution of the zwf gene with a sequence as set forth in SEQ ID NO: 3, or the mutation of the zwf gene is a complete deletion of the zwf gene.

[0050] In yet another aspect, the present disclosure provides a system comprising:

[0051] (a) a recombinant protein expression vector that does not comprise at least one E. coli homologous pgl gene and an engineered E. coli strain according to the present disclosure;

[0052] (b) an E. coli strain in which a zwf gene expresses a protein having glucose-6-phosphate dehydrogenase activity and a recombinant protein expression vector engineered according to the present disclosure; or

[0053] (c) a recombinant protein expression vector engineered according to the present disclosure and an engineered E. coli strain according to the present disclosure.

[0054] In some embodiments, the E. coli strain in which a zwf gene expresses a protein having glucose-6-phosphate dehydrogenase activity is a B series (e.g., a BL21(DE3) strain) or a K-12 series (e.g., a SHuffle T7 Express, W3110, or MG1655 strain).

[0055] In some embodiments, the recombinant protein expression vector that does not comprise at least one E. coli homologous pgl gene is a T7 promoter-based expression vector (e.g., a pET-28a vector) or a lactose operon promoter-based expression vector (e.g., a pBR322 vector) comprising a gene of interest that encodes a recombinant protein.

[0056] In some embodiments, wherein the purity of the recombinant protein produced is higher than the purity of a recombinant protein produced by a control recombinant E. coli strain, wherein the control recombinant E. coli strain comprises (1) a zwf gene expressing a protein having glucose-6-phosphate dehydrogenase activity and (2) a recombinant protein expression vector comprising a gene of interest that encodes a recombinant protein and does not comprise at least one E. coli homologous pgl gene; optionally, wherein the purity of the recombinant protein produced by a system according to the present disclosure is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, or 99.8%; or optionally, wherein the purity of the recombinant protein produced by a system according to the present disclosure is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% higher than the purity of the recombinant protein produced by the control recombinant E. coli strain.

[0057] In yet another aspect, the present disclosure provides a method of increasing the purity of a recombinant protein, wherein the method comprises:

[0058] (i) cloning a gene of interest (GOI) encoding a recombinant protein into (a) an expression vector that does not comprise at least one E. coli homologous pgl gene to obtain a recombinant protein expression vector comprising the gene of interest (A), or cloning into (b) a recombinant protein expression vector engineered according to the present disclosure to obtain a recombinant protein expression vector comprising the gene of interest (B);

[0059] (ii) transforming the recombinant protein expression vector comprising the gene of interest (A) into an engineered E. coli strain according to the present disclosure to obtain a recombinant E. coli strain (C); or transforming the recombinant protein expression vector comprising the gene of interest (B) into an E. coli strain or an engineered E. coli strain according to the present disclosure to obtain a recombinant E. coli strain (D); and

[0060] (iii) culturing the recombinant strain (C) or (D) to produce the recombinant protein;

[0061] the purity of the recombinant protein produced by the recombinant E. coli strain is higher than the purity of a recombinant protein produced by a control recombinant E. coli strain;

[0062] wherein the control recombinant E. coli strain comprises (1) a zwf gene expressing a protein having glucose-6-phosphate dehydrogenase activity and (2) a recombinant protein expression vector comprising the gene of interest and not comprising at least one E. coli homologous pgl gene;

[0063] optionally, the purity of the recombinant protein produced by step (iii) is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, or 99.8%; or optionally, the purity of the recombinant protein produced by step (iii) is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% higher than the purity of a recombinant protein produced by the control recombinant E. coli strain.

[0064] In some embodiments, the control E. coli strain is a B-series (e.g., a BL21(DE3) strain) or a K-12 series (e.g., a SHuffle T7 Express, W3110, or MG1655 strain) E. coli strain.

[0065] In some embodiments, the recombinant protein expression vector that does not comprise at least one E. coli homologous pgl gene is a T7 promoter-based expression vector (e.g., a pET-28a vector) or a lactose operon promoter-based expression vector (e.g., a pBR322 vector) comprising the gene of interest.

[0066] In yet another aspect, the present disclosure provides an E. coli pgl gene expression cassette comprising an E. coli pgl coding sequence and a promoter operably linked to the E. coli pgl coding sequence, wherein the E. coli pgl coding sequence comprises a polynucleotide sequence encoding a protein having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14 and having 6-phosphogluconolactonase activity, or wherein the E. coli pgl coding sequence comprises a polynucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 13 and encoding a protein having 6-phosphogluconolactonase activity.

[0067] In some embodiments, the sequence of the promoter is set forth in SEQ ID NO: 4 or 5.

[0068] In some embodiments, the E. coli pgl gene expression cassette further comprises a terminator operably linked to the E. coli pgl coding sequence, the sequence of the terminator being set forth in SEQ ID NO: 24.

[0069] In some embodiments, the E. coli pgl gene expression cassette comprises a polynucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 25 and encoding a protein having 6-phosphogluconolactonase activity.

[0070] In yet another aspect, the present disclosure provides a recombinant protein expression vector comprising an E. coli pgl gene expression cassette of the present disclosure and a sequence encoding a recombinant protein; optionally the expression vector is a plasmid.

[0071] In another aspect, this disclosure provides a recombinant protein expression host comprising the recombinant protein expression vector of this disclosure; optionally, the expression host is *Escherichia coli*, such as B-series (e.g., BL21(DE3) strain) or K-12-series (e.g., SHuffle T7 Express, W3110, or MG1655 strain) *Escherichia coli* strains.

[0072] In another aspect, this disclosure provides a method for producing a recombinant protein, comprising (1) culturing a recombinant protein expression host of this disclosure under suitable conditions; and (2) harvesting the recombinant protein from the culture of the recombinant protein expression host.

[0073] This disclosure also includes the following embodiments:

[0074] In one aspect, this disclosure provides a modified Escherichia coli host cell containing at least one Escherichia coli homologous pgl gene.

[0075] In some embodiments, the homologous pgl gene is introduced into the E. coli host cell in plasmid form or integrated into the genome of the E. coli host cell.

[0076] In some implementations, homologous pgl genes (e.g., ECP) Native -pgl gene or ECP GAP The pgl gene was spliced ​​into the expression vector pACYC184 and introduced into E. coli cells.

[0077] In some implementations, homologous pgl genes (e.g., ECP) Native -pgl gene or ECP GAP The pgl gene is knocked into the genome of E. coli (B series or K-12 series) using gene editing technology. The gene editing technology can be homologous recombination or a CRISPR-Cas editing system.

[0078] In some embodiments, the E. coli host cell further includes an auxiliary sequence operatively linked to the 5' end of the homologous pgl gene.

[0079] In some implementations, the auxiliary sequence is a promoter sequence.

[0080] In some embodiments, the auxiliary sequence is a nucleotide sequence such as P shown in SEQ ID NO:6. Native The sequence or nucleotide sequence is as shown in SEQ ID NO:7. GAP sequence.

[0081] Among them, P Nativeis a sequence 96 bp upstream of gapA in the genome of E. coli (SEQ ID NO: 7). GAP is a sequence 96 bp upstream of gapA in the genome of E. coli (SEQ ID NO: 7).

[0082] In some embodiments, the E. coli host cell comprises an EC PglA gene or a nucleotide sequence as set forth in SEQ ID NO: 4. Native In some embodiments, the E. coli host cell comprises an EC PglA gene or a nucleotide sequence as set forth in SEQ ID NO: 4. GAP In some embodiments, the E. coli host cell comprises an EC PglA gene or a nucleotide sequence as set forth in SEQ ID NO: 4.

[0083] In some embodiments, the E. coli host cell comprises an EC PglA gene or a nucleotide sequence as set forth in SEQ ID NO: 4. Native In some embodiments, the E. coli host cell comprises an EC PglA gene or a nucleotide sequence as set forth in SEQ ID NO: 4. GAP In some embodiments, the E. coli host cell comprises an EC PglA gene or a nucleotide sequence as set forth in SEQ ID NO: 4.

[0084] In some embodiments, the E. coli host cell further comprises a mutated zwf gene, wherein the mutation renders the zwf gene non-functional. In the present disclosure, the mutation can be achieved by gene editing technology. The gene editing technology can be homologous recombination technology or CRISPR-Cas editing system.

[0085] In some embodiments, the mutation comprises an insertion mutation, a deletion mutation, or a substitution mutation of the zwf gene.

[0086] In some embodiments, the nucleotide sequence of the zwf gene is as set forth in SEQ ID NO: 2.

[0087] In some embodiments, the zwf gene is subjected to a substitution mutation, wherein the zwf gene is replaced by a substitution sequence as set forth in SEQ ID NO: 3.

[0088] In some embodiments, the zwf gene is subjected to a deletion mutation, wherein the zwf gene is deleted of a partial sequence as set forth in SEQ ID NO: 1.

[0089] In some embodiments, the E. coli host cell is an E. coli cell of B series or K-12 series.

[0090] In some embodiments, the B series comprises BL21(DE3) strain, and the K-12 series comprises W3110 and MG1655 strains.

[0091] In another aspect, the present disclosure provides a method of constructing an engineered Escherichia coli host cell, comprising introducing into the Escherichia coli host cell at least one Escherichia coli homologous pgl gene.

[0092] In some embodiments, the homologous pgl gene is introduced into the Escherichia coli host cell in the form of a plasmid or integrated into the genome of the Escherichia coli host cell.

[0093] In some embodiments, the method further comprises operably linking an auxiliary sequence to the 5' end of the homologous pgl gene. For example, the auxiliary sequence is a promoter sequence.

[0094] In some embodiments, the auxiliary sequence is a nucleotide sequence of a P Native glcD sequence as set forth in SEQ ID NO: 6 or a P GAP glcD sequence as set forth in SEQ ID NO: 7.

[0095] In some embodiments, the method comprises introducing into the Escherichia coli host cell an EC P Native glcD gene as set forth in SEQ ID NO: 4 or an EC P GAP glcD gene as set forth in SEQ ID NO: 5.

[0096] In some embodiments, the method comprises introducing into the Escherichia coli host cell an EC P Native glcD gene as set forth in SEQ ID NO: 4 and an EC P GAP glcD gene as set forth in SEQ ID NO: 5.

[0097] In some embodiments, the method further comprises mutating a zwf gene on the genome of the Escherichia coli host cell to render the zwf gene non-functional.

[0098] In some embodiments, the mutation comprises an insertion mutation, a deletion mutation, or a substitution mutation.

[0099] In some embodiments, the nucleotide sequence of the zwf gene is as set forth in SEQ ID NO: 2.

[0100] In some embodiments, the mutation comprises replacing the zwf gene with a replacement sequence as set forth in SEQ ID NO: 3.

[0101] In some embodiments, the mutation comprises deleting from the zwf gene a partial sequence as set forth in SEQ ID NO: 1.

[0102] In some embodiments, the E. coli host cell is a B series or K-12 series E. coli cell. For example, the B series includes BL21(DE3) strain, and the K-12 series includes W3110 and MG1655 strains.

[0103] In yet another aspect, the present disclosure provides an engineered E. coli host cell obtained according to the method of the present disclosure.

[0104] In yet another aspect, the present disclosure provides a method for improving protein purity, comprising expressing a recombinant protein using the engineered E. coli host cell as described in the present disclosure.

[0105] In some embodiments, the recombinant protein has improved purity compared to using a wild-type E. coli host cell.

[0106] In some embodiments, the purity of the recombinant protein is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 99.9%. For example, the purity of a recombinant protein expressed using a wild-type BL21(DE3) strain is only 82%, while the purity of a recombinant protein expressed using the engineered E. coli host cell of the present disclosure is at least 99%, or even as high as 99.8%.

[0107] In yet another aspect, the present disclosure provides use of the E. coli host cell according to the present disclosure in recombinant protein expression.

[0108] In some embodiments, the recombinant protein has improved purity compared to using a wild-type E. coli host cell. In some embodiments, the purity of the recombinant protein is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 99.9%.

[0109] In yet another aspect, the present disclosure provides a method for improving the purity of a recombinant protein, comprising expressing the recombinant protein using an engineered E. coli host cell; wherein the engineered E. coli host cell comprises a mutated zwf gene, the mutation rendering the zwf gene non-functional.

[0110] In some embodiments, the mutation comprises an insertion mutation, a deletion mutation, or a substitution mutation of the zwf gene; for example, the zwf gene is substituted with a substitution sequence as set forth in SEQ ID NO: 3, or the zwf gene is deleted of a partial sequence as set forth in SEQ ID NO: 1.

[0111] In some embodiments, the recombinant protein has an increased purity compared to using a wild-type E. coli host cell; for example, the recombinant protein has a purity of at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 99.9%.

[0112] Any embodiment of any aspect can combine with any embodiment of any other aspect without exceeding the scope of the present disclosure. BRIEF DESCRIPTION OF DRAWINGS

[0113] An understanding of certain features and advantages of the present disclosure will be obtained by reference to the following detailed description and drawings, which sets forth illustrative embodiments in which the principles of the present disclosure can be utilized, and in which:

[0114] Figure 1 shows the verification of the knockout of the zwf gene in E. coli using PCR. Among them, the wild type sample refers to the product obtained by PCR amplification using primers of BL21(DE3) wild type; the zwf gene completely knockout sample refers to the product obtained by PCR amplification using primers of BL21(DE3) zwf gene completely knockout strain; the zwf gene partial knockout sample refers to the product obtained by PCR amplification using primers KOseq-F (SEQ ID NO: 8) and KOseq-R (SEQ ID NO: 9) of BL21(DE3) zwf gene partial knockout strain.

[0115] Figure 2 shows the expression of recombinant protein after knockout of zwf gene. Among them, lane 1: protein ladder; lane 2, 3: BL21(DE3) in which the zwf gene is completely knocked out, before the addition of inducer; lane 7, 8: BL21(DE3) in which the zwf gene is completely knocked out, after the addition of inducer; lane 4, 5: BL21(DE3) in which the zwf gene is partially knocked out, before the addition of inducer; lane 9, 10: BL21(DE3) in which the zwf gene is partially knocked out, after the addition of inducer; lane 6: BL21(DE3) wild type strain, before the addition of inducer; lane 11: BL21(DE3) wild type strain, after the addition of inducer.

[0116] Figure 3 shows the expression of recombinant protein after introduction of homologous pgl gene into E. coli. Lane 1: protein ladder; lane 2, 3: BL21(DE3) introduced with EC P Native -pgl gene, before the addition of inducer; lane 7, 8: BL21(DE3) introduced with EC P Native -pgl gene, after the addition of inducer; lane 4, 5: BL21(DE3) introduced with EC P GAP- pgl gene, before inducer addition; lanes 9, 10: BL21(DE3) introduced with EC P GAP - pgl gene, after inducer addition; lane 6: BL21(DE3) wild type strain, before inducer addition; lane 11: BL21(DE3) wild type strain, after inducer addition.

[0117] Figure 4 shows the comparison of the purity of recombinant protein expressed by E. coli before and after modification. The zwf gene is partially knocked out.

[0118] Figure 5 shows the comparison of the purity of recombinant protein TNF expressed by E. coli with or without pgl gene.

[0119] The drawings herein are for purposes of illustration only and are not necessarily drawn to scale. DETAILED DESCRIPTION

[0120] Further embodiments are illustrated in the following examples, which are for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0121] Unless otherwise specified, experimental methods used in the examples are routine.

[0122] Unless otherwise specified, the materials, reagents, and the like used in the examples are commercially available.

[0123] The following materials and experimental methods were used in the examples unless otherwise specified.

[0124] The sequences involved in the present disclosure are described as follows.

[0125] Example 1: Partially knock out zwf gene on the genome of E. coli

[0126] The design and use of recombinase tool plasmids and foldase tool plasmids are known in the art. The recombinase tool plasmid and foldase tool plasmid of the present embodiment are designed according to the lambda Red knockout system (Doublet et al., Antibiotic marker modifications of lambda Red and FLP helper plasmids, pKD46 and pCP20, for inactivation of chromosomal genes using PCR products in multidrug-resistant strains. J Microbiol Methods. 2008 Oct;75(2):359-61. doi: 10.1016 / j.mimet.2008.06.010. Epub 2008 Jun 21. PMID: 18619499).

[0127] (1) Transform the recombinase tool plasmid into the host cell BL21(DE3), and use LB solid medium containing 50 mg / L spectinomycin to screen positive transformants, and prepare electrocompetent cells.

[0128] (2) Mix the DNA fragment containing the zwf gene homologous arm, FRT site, and kanamycin resistance gene (SEQ ID NO: 10) with the competent cells in the previous step, and then add it to the electrotransformation cup (catalog number: 1652086, Bio-rad). Use the electrotransformation instrument (model: Gene Pulser Xcell, Bio-Rad) to perform the transformation. Use LB solid medium containing 50 mg / L kanamycin to screen positive transformants.

[0129] (3) Further verify the positive transformants using colony PCR and Sanger sequencing. The single clone in which the homologous fragment in the previous step has been integrated into the genome is passaged to culture, and the recombinase tool plasmid is discarded, and electrocompetent cells are prepared.

[0130] (4) Transform the foldase tool plasmid into the competent cells of the previous step, and use LB solid medium containing 50 mg / L spectinomycin to screen positive transformants. Further streak on LB solid medium (without antibiotics), and culture at 42°C to discard the foldase tool plasmid, and use colony PCR to confirm.

[0131] (5) The obtained positive monoclonal was inoculated on LB solid medium (without antibiotics), LB solid medium (with spectinomycin), and LB solid medium (with kanamycin) in parallel. Clones that only grew on LB solid medium (without antibiotics) were selected. Colony PCR and Sanger sequencing were used to confirm the partial knockout of the zwf gene. The PCR primers were KOseq-F (SEQ ID NO: 8) and KOseq-R (SEQ ID NO: 9).

[0132] As shown in FIG. 1, the PCR amplification product size of the BL21(DE3) zwf gene partial knockout strain was as expected, indicating that the strain genome was successfully modified.

[0133] Example 2: Complete knockout of the zwf gene on the genome of E. coli

[0134] The design and use of recombinase tool plasmids and foldase tool plasmids are known in the art. The recombinase tool plasmid and foldase tool plasmid of this example were designed according to the lambda Red knockout system (Doublet et al., Antibiotic marker modifications of lambda Red and FLP helper plasmids, pKD46 and pCP20, for inactivation of chromosomal genes using PCR products in multidrug-resistant strains. J Microbiol Methods. 2008 Oct;75(2):359-61. doi: 10.1016 / j.mimet.2008.06.010. Epub 2008 Jun 21. PMID: 18619499).

[0135] (1) The recombinase tool plasmid was transformed into the host cell BL21(DE3), and positive transformants were selected using LB solid medium containing 50 mg / L spectinomycin to prepare electrocompetent cells.

[0136] (2) The DNA fragment containing the zwf gene homologous arm, FRT site, and kanamycin resistance gene (SEQ ID NO: 11) was electrotransformed into the competent cells in the previous step according to Example 1, and positive transformants were selected using LB solid medium containing 50 mg / L kanamycin.

[0137] (3) Colony PCR and Sanger sequencing were used to further verify the positive transformants. The single clones in the previous step that were confirmed to have integrated the homologous fragments into the genome were passaged, and the recombinase tool plasmids were discarded to prepare electrotransformation competent cells.

[0138] (4) Transform the folding enzyme tool plasmid into the competent cells obtained in the previous step, and screen for positive transformants using LB solid medium containing 50 mg / L spectinomycin. Further, after streaking on LB solid medium (without antibiotics), the cells were cultured at 42°C to remove the folding enzyme tool plasmid, and confirmed by colony PCR.

[0139] (5) The obtained positive monoclonal clones were inoculated in parallel on LB solid medium (antibiotic-free), LB solid medium (with spectinomycin), and LB solid medium (with kanamycin). Clones that grew only on LB solid medium (antibiotic-free) were selected. Colony PCR and Sanger sequencing were used to confirm complete knockout of the zwf gene. The PCR primers were KOseq-F (SEQ ID NO:8) and KOseq-R (SEQ ID NO:9).

[0140] As shown in Figure 1, the PCR amplification product size of the BL21(DE3)zwf gene knockout strain is as expected, indicating that the strain's genome modification was successful.

[0141] Example 3: Introduction of ECP into Escherichia coli Native -pgl gene or ECP GAP -pgl gene plasmid

[0142] (1) Thaw BL21(DE3) competent Escherichia coli cells on ice.

[0143] (2) 50 ng containing EC P Native -pgl gene expression sequence (SEQ ID NO:4) or ECP GAP The E. coli pgl expression plasmid (full-length sequences SEQ ID NO:27 and SEQ ID NO:28) containing the pgl gene expression sequence (SEQ ID NO:5) was added to 40 μL of thawed BL21(DE3) competent E. coli cells. After gentle mixing, the mixture was incubated on ice for 30 min. Native -pgl gene expression sequence (SEQ ID NO:4) or ECP GAP The -pgl gene expression sequences (SEQ ID NO:5) all contain the same E. coli pgl coding sequence (SEQ ID NO:13) but contain different promoter sequences, namely P Native Promoter (SEQ ID NO:6) and PGAP Promoter (SEQ ID NO: 7). There is also the same rrnB Tl terminator (SEQ ID NO: 24) downstream of the E. coli pgl coding sequence.

[0144] (3) The mixture from the previous step was heat shocked at 42°C for 90 s and placed on ice for 5 min.

[0145] (4) 900 μL of LB medium was added and incubated at 37°C, 220 RPM for 1 h.

[0146] (5) 200 μL of the bacterial solution was taken and evenly spread on LB solid medium containing chloramphenicol. After the plate was incubated at 37°C for 16 h, the colonies grown on the plate were E. coli into which one of the pgl expression plasmids was introduced.

[0147] Example 4: Expression of recombinant proteins in engineered E. coli

[0148] (1) Two BL21(DE3) strains into which the E. coli pgl expression plasmid was introduced (without knocking out the zwf gene), a BL21(DE3) strain in which the zwf gene was partially knocked out (without the pgl expression plasmid), a BL21(DE3) strain in which the zwf gene was completely knocked out (without the pgl expression plasmid), and a BL21(DE3) wild type strain were prepared into competent cells. The plasmid (SEQ ID NO: 12) for inducing expression of the recombinant protein urate oxidase (UOX) was transformed into each strain.

[0149] (2) The transformed strains were incubated in LB liquid medium at 37°C until the OD 600 1.0-2.0.

[0150] (3) The inducer IPTG (final concentration 0.2 mM) was added to induce expression of the recombinant protein, and incubated at 20°C for 17 hours.

[0151] (4) The OD 600 of the culture after induction was measured. 1 μL of the enzyme solution (catalog number 71110, Millipore) was added to every 15 OD of the bacterial solution, and lysed at 27°C, 800 RPM for 25 min. The lysed solution was centrifuged at 4°C, 13,000 RPM for 20 min. 20 μL of the supernatant was taken, 20 μL of 2x loading buffer was added, and heated in a 95°C metal bath for 5 min to prepare the SDS-PAGE sample.

[0152] (5) SDS-PAGE electrophoresis was performed and the expression of the recombinant protein was observed by taking photographs (see Figures 2 and 3).

[0153] For E. coli in which the partial sequence of the zwf gene (SEQ ID NO: 1) or the entire sequence of the zwf gene (SEQ ID NO: 2) was knocked out, a clear SGS-PAGE band appeared at the theoretical position of the recombinant protein to be expressed, proving the expression of the recombinant protein.

[0154] For E. coli into which the E. coli pgl expression plasmid driven by the P

[0155] Example 5: Purity detection of recombinant protein expressed by E. coli after high-density fermentation

[0156] (1) After culturing all the transformed strains in Example 4 at 37°C to OD 600 7-9, they were inoculated into a fermenter and cultured at 37°C for 7 hours.

[0157] (2) IPTG (final concentration 0.2 mM) was added for induction of expression of the recombinant protein, and the culture was incubated at 30°C for 16 hours.

[0158] (3) The bacterial cells were harvested and subjected to affinity purification to obtain the recombinant protein.

[0159] (4) The purity of the recombinant protein was analyzed by CE-SDS.

[0160] As shown in Figure 4, the recombinant protein expressed by the E. coli strain into which the E. coli pgl expression plasmid driven by the P Native promoter for expression of E. coli pgl had a purity of 99.8%, the recombinant protein expressed by the E. coli strain into which the E. coli pgl expression plasmid driven by the P GAP promoter for expression of E. coli pgl had a purity of 99.6%, and the recombinant protein expressed by the E. coli strain in which the zwf gene was partially knocked out had a purity of 99.7%, all of which were significantly higher than the purity of the recombinant protein expressed by the wild-type E. coli BL21 (DE3) (82.3%).

[0161] Example 6: Introduction of an expression plasmid containing a TNF-encoding gene and containing or not containing an E. coli pgl gene into E. coli

[0162] (1) The SHuffle T7 Express competent E. coli cells were thawed on ice.

[0163] (2) 50 ng of expression plasmid pWXEB2.0-WT-TNF (SEQ ID NO: 18; 5499 bp) containing the coding sequence (SEQ ID NO: 22) of the target protein TNF (SEQ ID NO: 23) and not comprising the E. coli pgl gene expression cassette (SEQ ID NO: 25; 1356 bp) and 50 ng of expression plasmid pWXEB4.0-WT-TNF (SEQ ID NO: 19; 6855 bp) containing the coding sequence (SEQ ID NO: 22) of the target protein TNF (SEQ ID NO: 23) and comprising the E. coli pgl gene expression cassette (SEQ ID NO: 25) were added into 40 μL of thawed SHuffle T7 Express competent E. coli cells, respectively, and mixed gently and placed on ice for 30 min.

[0164] The E. coli pgl gene expression cassette (SEQ ID NO: 25) consists of, from 5’ to 3’, a P Native a promoter (SEQ ID NO: 4), an E. coli pgl coding sequence (SEQ ID NO: 13) (encoding an E. coli pgl protein (SEQ ID NO: 14)), and a rrnB T1 terminator (SEQ ID NO: 24).

[0165] Both pWXEB2.0-WT-TNF and pWXEB4.0-WT-TNF contain a multiple cloning site (MCS; SEQ ID NO: 15) and a Cer element (SEQ ID NO: 16) and do not contain a F1 ori (SEQ ID NO: 17).

[0166] The difference between pWXEB2.0-WT-TNF and pWXEB4.0-WT-TNF is that the latter has the E. coli pgl gene expression cassette (SEQ ID NO: 25; 1356 bp).

[0167] (3) The mixture from the previous step was heat shocked at 42 °C for 90 s and placed on ice for 5 min.

[0168] (4) 900 μL of LB medium was added and incubated at 37 °C, 220 RPM for 1 h.

[0169] (5) 200 μL of the bacterial solution was taken and evenly spread on LB solid medium containing kanamycin. After incubating the plate at 37 °C for 16 h, the colonies grown on the plate were E. coli strains into which one of the two expression plasmids was introduced.

[0170] Example 7: Expression and purity analysis of TNF recombinant protein in E. coli

[0171] (1) The E. coli strain in Example 6 was cultured at 30°C to OD 600 4-7, then inoculated into a fermenter and cultured at 30°C for 20 hours, after which the culture temperature was lowered to 20°C.

[0172] (2) IPTG (final concentration 0.5 mM) was added to induce expression of the recombinant protein, and the culture was incubated at 20°C for 16 hours.

[0173] (4) A small sample of the bacterial cells was harvested, and the TNF recombinant protein was obtained after MMC purification.

[0174] (5) The purity of the TNF recombinant protein was detected by CE-SDS.

[0175] As shown in Figure 5, the purity of the TNF recombinant protein expressed using the pWXEB4.0-WT-TNF plasmid (92.5%) was significantly higher than the purity of the TNF recombinant protein expressed using the pWXEB2.0-WT-TNF plasmid (85.2%).

[0176] Example 8: Introduction of an expression plasmid containing a recombinant protein coding sequence with or without an E. coli pgl gene into a zwf knockout E. coli

[0177] (1) The partially or completely zwf knockout E. coli strain prepared in Example 1 or 2 was thawed on ice.

[0178] (2) 50 ng of an expression plasmid containing a recombinant protein coding sequence without an E. coli pgl expression cassette (SEQ ID NO: 25; 1356 bp) and 50 ng of an expression plasmid containing a recombinant protein coding sequence with an E. coli pgl expression cassette (SEQ ID NO: 25) were added to 40 μL of the thawed partially or completely zwf knockout E. coli cells, gently mixed, and then placed on ice for 30 min.

[0179] (3) The mixture from the previous step was heat shocked at 42°C for 90 s and then placed on ice for 5 min.

[0180] (4) 900 μL of LB medium was added, and the culture was incubated at 37°C at 220 RPM for 1 h.

[0181] (5) 200 μL of the bacterial solution was taken and evenly spread on LB solid medium containing kanamycin. After the plate was incubated at 37°C for 16 h, the colonies grown on the plate were the partially or completely zwf knockout E. coli strains into which one of the two expression plasmids had been introduced.

[0182] Example 9: Expression and purity analysis of recombinant proteins in E. coli

[0183] (1) The E. coli strain in Example 8 was cultured at 30°C to OD 600 4-7, then inoculated into a fermenter, and cultured at 30°C for 20 hours, after which the culture temperature was lowered to 20°C.

[0184] (2) IPTG (final concentration 0.5 mM) was added to induce expression of the recombinant protein, and the culture was incubated at 20°C for 16 hours.

[0185] (4) The bacterial cells were harvested, and the recombinant protein was obtained after MMC purification.

[0186] (5) The purity of the recombinant protein was determined by CE-SDS.

[0187] In the case of the combination of zwf knockout and introduction of E. coli pgl, the purity of the expressed recombinant protein can be further improved, and there is an additive or even synergistic effect.

[0188] Various modifications and variations to the described products, methods, and uses of the present disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the present disclosure. Although the present disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and that this is intended to be encompassed within the scope of the present disclosure. It is intended that various modifications and variations of the described products, methods, and uses of the present disclosure be included within the scope of the present disclosure and be protected by the following claims.

Claims

1. An engineered recombinant protein expression vector of Escherichia coli comprising at least one Escherichia coli homologous pgl gene.

2. The engineered recombinant protein expression vector of claim 1, wherein the engineered recombinant protein expression vector is engineered on the basis of a pET28a plasmid.

3. The engineered recombinant protein expression vector of any one of the preceding claims, wherein the pgl gene comprises a polynucleotide sequence encoding a protein having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14 and having 6-phosphogluconolactonase activity; or wherein the pgl gene comprises a polynucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 13 and encoding a protein having 6-phosphogluconolactonase activity.

4. The engineered recombinant protein expression vector of any one of the preceding claims, wherein a promoter sequence is operably linked to the 5' end of the pgl gene to form a promoter-pgl gene sequence; optionally the promoter sequence is a P Native GAP sequence of nucleotides set forth in SEQ ID NO: 7; optionally the promoter-pgl gene sequence is set forth in SEQ ID NO: 4 or 5.​ 5. The engineered recombinant protein expression vector of any one of the preceding claims, wherein a terminator sequence is operably linked to the 3’ end of the promoter-pgl gene to form a pgl expression cassette; optionally the nucleotide sequence of the terminator sequence is set forth in SEQ ID NO: 24; optionally the nucleotide sequence of the pgl expression cassette is set forth in SEQ ID NO:

25.

6. The engineered recombinant protein expression vector of claim 1, further comprising a stability component; optionally wherein the stability component is a Cer element from a ColEl plasmid; optionally wherein the stability component comprises a polynucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

16.

7. The engineered recombinant protein expression vector of any one of the preceding claims, wherein the stability component is inserted into the engineered recombinant protein expression vector in a 3’-5’ or 5’-3’ orientation.

8. The engineered recombinant protein expression vector of claim 1, which does not comprise a F1 ori; optionally, wherein the F1 ori comprises a polynucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

17.

9. The engineered recombinant protein expression vector of any one of the preceding claims, wherein the engineered recombinant protein expression vector comprises a multiple cloning site (MCS); optionally, wherein the MCS comprises a polynucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

15.

10. The engineered recombinant protein expression vector of any one of the preceding claims, wherein the engineered recombinant protein expression vector comprises a gene of interest (GOI); optionally, wherein the GOI is inserted into the MCS or replaces part or all of the MCS.

11. The engineered recombinant protein expression vector of any one of the preceding claims, wherein the GOI is a recombinant protein-encoding gene; optionally the recombinant protein is a recombinant protein for disease prevention, diagnosis, or treatment, such as an antibody, a vaccine, an enzyme.

12. The engineered recombinant protein expression vector of any one of the preceding claims, wherein the stability component is located between the MCS or the GOI and the pgl gene.

13. The engineered recombinant protein expression vector of any one of the preceding claims, which comprises, from 5’-3’ direction, the MCS or the GOI, the stability component, and the pgl gene, and does not comprise a F1 ori.

14. The engineered recombinant protein expression vector of any one of the preceding claims, wherein the engineered recombinant protein expression vector comprises a polynucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

21.

15. The engineered recombinant protein expression vector of any one of the preceding claims, which comprises, from 5’-3’ direction: (1) the MCS set forth in SEQ ID NO: 15; (2) the stability component set forth in SEQ ID NO: 16; and (3) the promoter-pgl gene sequence set forth in SEQ ID NO: 4 or the pgl expression cassette set forth in SEQ ID NO:

25. and does not comprise the F1 ori set forth in SEQ ID NO: 17; wherein the engineered recombinant protein expression vector comprises a polynucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

21.

16. The engineered recombinant protein expression vector of any one of the preceding claims, comprising, in the 5’-3’ direction: (1) the gene of interest; (2) the stability component set forth in SEQ ID NO: 16; and (3) the promoter-pgl gene sequence set forth in SEQ ID NO: 4 or the pgl expression cassette set forth in SEQ ID NO: 25; and does not comprise the F1 ori set forth in SEQ ID NO: 17; wherein the engineered recombinant protein expression vector comprises a polynucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

21.

17. An engineered E. coli strain comprising a mutation in a zwf gene on its genome, the mutation (e.g., a partial or complete knockout of the zwf gene) resulting in the zwf gene not expressing a protein having glucose-6-phosphate dehydrogenase (G6PDH) activity.

18. The engineered E. coli strain of any one of the preceding claims, engineered on the basis of a B series (e.g., a BL21(DE3) strain) or K-12 series (e.g., a SHuffle T7 Express, W3110, or MG1655 strain) E. coli strain.

19. The engineered E. coli strain of any one of the preceding claims, wherein the zwf gene comprises a polynucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2 and encodes a protein having glucose-6-phosphate dehydrogenase activity.

20. The engineered E. coli strain of any preceding claim, wherein the mutation of the zwf gene is introduced by homologous recombination using a zwf gene knockout fragment comprising two homology arms, wherein each homology arm is identical to a portion of the zwf gene; optionally the zwf gene knockout fragment further comprises a stretch of sequence between the two homology arms; optionally the stretch of sequence comprises a resistance gene sequence, for example a resistance gene sequence encoding kanamycin, tetracycline, streptomycin or chloramphenicol.

21. The engineered E. coli strain of any preceding claim, wherein the zwf gene knockout fragment comprises one 5’ homology arm identical to the 5’ end of the zwf gene and one 3’ homology arm identical to the 3’ end of the zwf gene; optionally the zwf gene knockout fragment further comprises a stretch of sequence between the two homology arms; optionally the stretch of sequence comprises a resistance gene sequence, for example a resistance gene sequence encoding kanamycin, tetracycline, streptomycin or chloramphenicol.

22. The engineered E. coli strain of any preceding claim, wherein the zwf gene knockout fragment comprises a polynucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 10 or 11.

23. The engineered E. coli strain of any preceding claim, wherein the mutation of the zwf gene is a substitution of the zwf gene with a sequence as set forth in SEQ ID NO: 3, or the mutation of the zwf gene is a complete deletion of the zwf gene.

24. A system comprising: (a) a recombinant protein expression vector that does not comprise at least one E. coli homologous pgl gene and an engineered E. coli strain according to any preceding claim; (b) an E. coli strain in which the zwf gene expresses a protein having glucose-6-phosphate dehydrogenase activity and a modified recombinant protein expression vector according to any preceding claim; or (c) a modified recombinant protein expression vector according to any preceding claim and an engineered E. coli strain according to any preceding claim.

25. The system of any preceding claim, wherein the E. coli strain in which the zwf gene expresses a protein having glucose-6-phosphate dehydrogenase activity is a B series (e.g. a BL21(DE3) strain) or a K-12 series (e.g. a SHuffle T7 Express, W3110 or MG1655 strain).

26. The system of any one of the preceding claims, wherein the recombinant protein expression vector that does not comprise at least one E. coli homologous pgl gene is a T7 promoter-based expression vector (e.g., a pET-28a vector) or a lactose operon promoter-based expression vector (e.g., a pBR322 vector) comprising a gene of interest encoding a recombinant protein.

27. Use of the system of any one of the preceding claims for producing a recombinant protein, wherein the purity of the recombinant protein produced is higher than the purity of a recombinant protein produced by a control recombinant E. coli strain, wherein the control recombinant E. coli strain comprises (1) a zwf gene expressing a protein having glucose-6-phosphate dehydrogenase activity and (2) a recombinant protein expression vector comprising a gene of interest encoding a recombinant protein and does not comprise at least one E. coli homologous pgl gene; optionally, wherein the purity of the recombinant protein produced by the system of any one of the preceding claims is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, or 99.8%; or optionally, wherein the purity of the recombinant protein produced by the system of any one of the preceding claims is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% higher than the purity of the recombinant protein produced by the control recombinant E. coli strain.

28. A method of increasing the purity of a recombinant protein, wherein the method comprises: (i) cloning a gene of interest (GOI) encoding a recombinant protein into (a) an expression vector that does not comprise at least one E. coli homologous pgl gene to obtain a recombinant protein expression vector comprising the gene of interest (A), or into (b) the engineered recombinant protein expression vector of any one of the preceding claims to obtain a recombinant protein expression vector comprising the gene of interest (B); (ii) transforming the recombinant protein expression vector comprising the gene of interest (A) into the engineered E. coli strain of any one of the preceding claims to obtain a recombinant E. coli strain (C); or transforming the recombinant protein expression vector comprising the gene of interest (B) into an E. coli strain or the engineered E. coli strain of any one of the preceding claims to obtain a recombinant E. coli strain (D); and (iii) culturing the recombinant strain (C) or (D) to produce the recombinant protein; wherein the purity of the recombinant protein produced is higher than the purity of a recombinant protein produced by a control recombinant E. coli strain; wherein the control recombinant E. coli strain comprises (1) a zwf gene expressing a protein having glucose-6-phosphate dehydrogenase activity and (2) a recombinant protein expression vector comprising the gene of interest and not comprising at least one E. coli homologous pgl gene; optionally, wherein the recombinant protein produced in step (iii) has a purity of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, or 99.8%; or optionally, wherein the recombinant protein produced in step (iii) has a purity that is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% greater than the purity of the recombinant protein produced by the control recombinant E. coli strain.

29. The method of any one of the preceding claims, wherein the control E. coli strain is a B series (e.g., a BL21(DE3) strain) or a K-12 series (e.g., a SHuffle T7 Express, W3110, or MG1655 strain) E. coli strain.

30. The method of any one of the preceding claims, wherein the recombinant protein expression vector not comprising at least one E. coli homologous pgl gene is a T7 promoter-based expression vector (e.g., a pET-28a vector) or a lactose operon promoter-based expression vector (e.g., a pBR322 vector) comprising the gene of interest.

31. An E. coli pgl gene expression cassette comprising an E. coli pgl coding sequence operably linked to a promoter, wherein the E. coli pgl coding sequence comprises a polynucleotide sequence encoding a protein having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14 and having 6-phosphogluconolactonase activity, or wherein the E. coli pgl coding sequence comprises a polynucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 13 and encoding a protein having 6-phosphogluconolactonase activity.

32. The E. coli pgl gene expression cassette of any one of the preceding claims, wherein the sequence of the promoter is set forth in SEQ ID NO: 4 or 5.

33. The E. coli pgl gene expression cassette of any one of the preceding claims, further comprising a terminator operably linked to the E. coli pgl coding sequence, the sequence of the terminator being set forth in SEQ ID NO:

24.

34. The E. coli pgl gene expression cassette of any one of the preceding claims, comprising a polynucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 25 and encoding a protein having 6-phosphogluconolactonase activity.

35. A recombinant protein expression vector comprising the E. coli pgl gene expression cassette of any one of the preceding claims and a sequence encoding a recombinant protein; optionally the expression vector is a plasmid.

36. A recombinant protein expression host comprising the recombinant protein expression vector of any one of the preceding claims; optionally the expression host is an E. coli, for example an E. coli strain of the B series (e.g., BL21(DE3) strain) or K-12 series (e.g., SHuffle T7 Express, W3110, or MG1655 strain).

37. A method of producing a recombinant protein, comprising (1) culturing the recombinant protein expression host of any one of the preceding claims under suitable conditions; (2) harvesting the recombinant protein from the culture of the recombinant protein expression host.

Images