CN113278567A - Method for improving strain to promote recombinant protein production - Google Patents

Abstract

The invention provides a strain capable of improving expression of recombinant protein, belonging to escherichia coli, wherein the strain is obtained by integrating a glf gene, a zwf gene, a pgl gene, an alaA gene and an alaC gene into an escherichia coli strain lacking a ptsG gene, an araABD gene manipulation group, an araFGH gene and a ppc gene manipulation group.

Description

Method for improving strain to promote recombinant protein production

Technical Field

The present invention relates to an improved escherichia coli strain and a method for enhancing the production of recombinant proteins using the same, and more particularly, to an escherichia coli strain improved by a central metabolic pathway (central metabolism) of a reconstituted strain and a method for producing recombinant proteins using the same.

Background

Science and technology are changing day by day, and today's biotechnology is different from day to day. Things involved in the biotechnology field are otosmells, including food, chemicals and medical treatments. Currently, the biotechnology industry utilizes various biological platforms to develop industrial enzymes or medical proteins with high economic value, such as insulin, erythropoietin, lactic acid and antibiotics.

Plants, animals, insects and microorganisms are all common biological reaction platforms for protein production. The complete and high quality bioreactor platform must have the following characteristics, low cost is necessary, the platform is easy to operate, can be used in various environmental conditions, and can efficiently produce the target.

Escherichia coli (Escherichia coli) is one of the most commonly used systems for expressing foreign proteins at present due to its characteristics of easy culture, low cost and large protein production amount, wherein the production of proteins is performed by obtaining amino acid chains through DNA transcription and translation, and then correctly folding and modifying the amino acid chains to obtain proteins with specific functions, but the process consumes a large amount of energy, so that how to obtain energy and the efficiency of obtaining energy will have a considerable influence on the Escherichia coli to produce specific proteins.

How and how efficiently escherichia coli gains energy varies because the components taken in, such as carbon or nitrogen sources, and the components taken in pass through and are metabolized.

Glycerol, also known as glycerol or glycol, has a molecular formula of C3H8O3, is a colorless, odorless, sweet viscous liquid with a boiling point of 290 ℃, has strong water absorption, and is used in food industry, medicine, personal care products, antifreeze and chemical intermediate products. The production of glycerol can be synthesized by means of the chemical industry and from petrochemical feedstocks, but is also a by-product of many industries, such as the soap industry or biodiesel. The use of E.coli for the metabolism of glycerol eliminates the consumption of these industrial by-products and also converts them into other products of higher economic value. Such as succinic acid, lactic acid, and 1, 2-propanediol, and the like.

The metabolic pathway of glycerol in E.coli has been fully published, and the entry of glycerol from outside to inside of cells utilizes passive transport through cell membranes, and glycerol is transported to inside of cell membranes by glycerol uptake promoting protein (glpF), which belongs to water transport membrane protein, and water and glycerol are highly selective channel proteins. After glycerol enters the cell membrane, it is mainly divided into two different metabolic pathways, the anaerobic (glpF-gldA-dhaK) and the aerobic (glpF-glpK-glpD) pathways, which convert glycerol to dihydroxyacetone phosphate (DHAP).

In the anaerobic pathway, the gldA gene (glycerol dehydrogenase) converts glycerol into DHA (dihydroxyacetone) and generates NADH to supply energy, and then converts it into DHAP through dhaK gene (dihydroxyacetone kinases).

In the Aerobic pathway, Glycerol is phosphorylated to G3P (Glycerol-3-phosphate) by the glpK gene (Glycerol kinase), but ATP is consumed in the process and converted to DHAP by the glpD gene (Aerobic Glycerol-3-phosphate dehydrogenase).

Glucose plays an important role in the field of biology, being an energy source for activating cells and an intermediate product of metabolism. In the past literature, the metabolic pathway of glucose in E.coli has been fully published. The metabolic pathway of Glucose (Glucose) in E.coli is mainly a PTS system, which transports Glucose into the cell membrane by means of phosphotransfer, converts PEP (phosphoenolpyruvate) into Pyr (Pyruvate) by means of the ptsG gene (PTS system Glucose-specific EIICB component; PTS system Glucose-specific EIICB component), and phosphorylates extracellular Glucose to G6P (Glucose-6-phosphate; Glucose-6-phosphate) into the cell.

Because the production of the target protein is an extremely energy-consuming process and requires a large amount of energy in the process of producing the target protein, the scheme is expected to strengthen the metabolic pathways of glycerol and glucose by a gene recombination technology and match with a strict gene regulation system to obtain higher energy obtaining efficiency, so that a novel method for producing the protein is developed, and the activity and the production efficiency of the target protein can be improved.

Disclosure of Invention

In view of the above-mentioned needs, the present invention utilizes metabolic engineering to alter the glycerol metabolic pathway and glucose metabolic pathway of BAD-5 strain (Zei Wen Wang et al, A glucose-sensitive T7 expression system for full-induced expression of proteins at a sub-fermentation level of L-arabinosins. journal of Agricultural and Food chemistry.59:6534-6542) to construct a method for improving the production of recombinant proteins.

According to an object of the present invention, an improved strain and a method for enhancing the production of recombinant proteins using the strain are provided. The strain belongs to Escherichia coli. The strain is obtained by integrating the glf gene, zwf gene and pgl gene into an escherichia coli strain lacking the ptsG gene, the araABD gene manipulation group and the araFGH gene manipulation group.

Preferably, said strain further comprises an alaC gene.

Preferably, said strain further comprises an alaA gene.

Preferably, the strain capable of improving the expression of the recombinant protein has the characteristics of LacZ:PBAD-T7 gene 1Ptrc-araE Δ araBAD Δ ptsG Δ araFGH.

Preferably, said strain is free of the ppc gene.

According to another object of the present invention, there is provided a method for enhancing the production of a recombinant protein, wherein the recombinant protein is produced by culturing a strain capable of enhancing the expression of the recombinant protein as described above in a medium containing a carbon source.

Preferably, the strain further comprises one or a combination of the alaC gene and the alaA gene.

Preferably, the medium comprising a carbon source comprises one or a combination of glycerol and glucose.

Preferably, said strain is free of the ppc gene.

Preferably, the medium comprising a carbon source comprises glutamic acid.

Drawings

The present invention will become more readily apparent to those of ordinary skill in the art from the following detailed description when taken in conjunction with the drawings, wherein:

FIG. 1 is a schematic diagram of the structure of plasmid pET-TrChHDT; and

FIG. 2 is a schematic diagram of a central metabolic pathway of a strain capable of enhancing expression of a recombinant protein according to an embodiment of the present disclosure.

Detailed Description

The present application constructs a strain capable of improving recombinant protein expression by metabolic engineering by studying BAD-5 strain (Zei Wen Wang et al, A glucose-sensitive T7 expression system for full-induced expression of proteins at a sub-culturing level of L-arabinosins. journal of Agricultural and Food chemistry.59:6534-6542) grown on glycerol.

The present case uses the BAD-5 strain carrying the plasmid pET-TrChHDT (BAD-5/pET-TrChHDT) as a substrate. FIG. 1 is a schematic diagram of the structure of plasmid pET-TrChHDT. As can be seen from FIG. 1, the plasmid pET-TrChHDT carries a fusion gene consisting of trxA, D-hydantoinase gene (HDT) and chitin-binding domain (ChBD) under the control of the T7 promoter (PT7), which is obtained by amplifying DNA containing HDT-ChBD from plasmid pChHDT by PCR using promoters (cgaattccatatggatatcatcatcaagaacg and gtgactggtgagtactcaacc) (Jong-Tzer Chem and Yun-Pen Chano.2005. chitin-binding domain based immobilized of D-hydantoinase. J. Biotechnol.2005; 117: 267-.

The strain BAD-5 (Zei Wen Wang et al, A glucose-sensitive T7 expression system for full-induced expression of proteins at a sub-evaluation level of L-arabinosine. journal of Agricultural and Food chemistry.59:6534-6542) has the properties as shown in Table 1 below.

TABLE 1

As can be seen from Table 1 above, the BAD-5 strain used in the present case is an E.coli strain lacking ptsG gene, araABD gene manipulation group, and araFGH gene manipulation group.

The strain capable of improving the expression of the recombinant protein is obtained by metabolic engineering of a BAD-5 strain. Furthermore, the application obtains a strain capable of improving the expression of the recombinant protein by integrating the glf gene, the zwf gene and the pgl gene into the BAD-5 strain lacking the ptsG gene, the araABD gene manipulation group and the araFGH gene manipulation group.

Integration of the glf gene can enhance the glucose transport function of BAD-5 strain, thus increasing protein activity and yield through increased glucose consumption. The zwf gene is a gene encoding glucose-6-phosphate dehydrogenase, glucose-6-phosphate dehydrogenation (G6PD) can catalyze glucose-6-phosphate (glucose-6-phosphate) to be oxidized into 6-phosphoglucono-delta-lactone (6-phosphoglucono-delta-lactone) in the Pentose phosphate pathway (pentase phosphate pathway) and simultaneously reduce Nicotinamide Adenine Dinucleotide Phosphate (NADP) into NADPH, and gluconolactonase encoded by the pgl gene can promote hydrolysis of 6-phosphogluconate to 6-phosphogluconate, which can be oxidized and decarboxylated by 6-phosphogluconate dehydrogenase to generate NADPH.

Therefore, by integrating the glf gene, zwf gene and pgl gene, a strain which can enhance expression of recombinant protein and can effectively utilize glucose to increase the utilization rate of reducing equivalents can be provided.

The present case further integrates one or a combination of both of the alaC gene and the alaA gene into the above described BAD-5 strain comprising the glf gene, zwf gene and pgl gene and lacking the ptsG gene, the araABD gene panel, and the araFGH gene panel. Since the two glutamate pyruvate aminotransferases encoded by the alaC gene as well as the alaA gene are responsible for carrying out the transamination reaction from glutamate and pyruvate to form alanine associated with alpha-ketoglutarate (alpha-KG). Expression of both glutamate pyruvate aminotransferases can be enhanced by further including in the strain either or both of the alaC gene and the alaA gene in combination. By supplying α -KG to complement tricarboxylic acid (TCA) cycle intermediates for biosynthesis, recombinant proteins can be produced without the ppc gene. Strains lacking the ppc gene retain phosphoenolpyruvate (PEP). Under physiological conditions, PEP is usually converted to pyruvate through a pyruvate kinase mediated substrate level phosphorylation reaction, resulting in more pyruvate and ATP, which can further increase the activity and yield of recombinant proteins.

Fig. 2 is a schematic diagram of a central metabolic pathway of a strain capable of enhancing expression of a recombinant protein according to an embodiment of the present disclosure, wherein the strain capable of enhancing expression of a recombinant protein comprises an alaC gene and an alaA gene. Fig. 2 shows that the strains according to the embodiments of the present disclosure can improve the expression of recombinant proteins, and the metabolic pathways of glycerol and glucose in the presence of glucose and glycerol. In FIG. 2, glf denotes glucose transporter, G6P denotes glucose-6-phosphate, F6P denotes fructose-6-phosphate, PEP denotes phosphoenolpyruvate, PYR denotes pyruvate, Acetyl-CoA denotes Acetyl-CoA, OAA denotes oxaloacetate, CIT denotes citric acid, PGA denotes glyceraldehyde-3-phosphate, DHAP denotes dihydroxyacetone phosphate, Glu denotes glutamic acid, Ala denotes alanine, and α -KG denotes α -ketoglutaric acid.

The present invention further provides a method for producing a recombinant protein, comprising the step of culturing the strain capable of enhancing expression of the recombinant protein in a medium containing a carbon source and glutamic acid to produce the recombinant protein, wherein the recombinant protein is hydantoinase (hdt), and the medium containing the carbon source comprises glycerol and glucose.

The advantages of the present invention will be described in detail through examples

In both the following comparative examples and examples, protein production was carried out using M9G medium consisting of M9 salt (6g/L Na2HPO4, 3g/L KH2PO4, 0.5g/L NaCl, 1g/L NH4Cl, 1mM MgSO4.7H2O and 0.1mM CaCl2) and 2g/L L-glutamic acid.

Comparative example 1

The strain BAD-5/pET-TrChHDT was inoculated into a flask (150mL) containing 15mL of M9G medium supplemented with 6g/L glycerol and 15. mu.g/mL ampicillin, and the cell density reached 0.08 at OD 550. The bacterial cultures were incubated at 37 ℃ in a rotary shaker set at 200 rpm. When the OD550 reached about 0.3, the strain was induced to produce protein by adding 30. mu.M of L-arabinose to the medium. After 6 hours of induction, the induced bacteria reached 0.8 at OD 550.

Protein production assay of strains

1mL of the bacterial culture was harvested by centrifugation and resuspended in 1mL of 0.1MTri-HCl buffer (pH 8.0). Cells were disrupted by sonication, then centrifuged. The supernatant (i.e., cell-free extract (CFX)) was recovered and assayed for CFX by Bio-Rad protein assay reagents for protein analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The yield of HDT was estimated using an image analyzer GAS9000 (UVItech). Meanwhile, HDT activity was measured by adding 10uL of CFX to a reaction solution (1mL) composed of 0.1M Tri-HCl buffer (pH 8.0), 6mM D, L-hydroxyphenylhydantoin (HPH), and 0.5mM MnCl 2. The reaction was carried out at 40 ℃ for 15 minutes and then terminated by transferring to 100 ℃ for 10 minutes. The concentration of HPH was measured by High Performance Liquid Chromatography (HPLC). Analysis showed that strain BAD-5/pET-TrChHDT produced soluble TrxA-HDT-ChBD with a yield of 28mg/L and an enzyme activity of 1.2U.

Comparative example 2

Strain N30, derived from strain BAD-5, was taken, which had enhanced expression of glpF (encoding glycerol transporter), gldA and dhaKLM (patent I537382; ZL 201410553469.8). The strain N30 was then transformed with the plasmid pET-TrHDTCh to give the strain N30/pET-TrChHDT.

The strain N30/pET-TrChHDT was cultured on M9G medium containing 6g/L glycerol. The cell density of the strain N30/pET-TrChHDT is 1.8 at OD550, and the yield and the enzyme activity of the soluble TrxA-HDT-ChBD reach 58mg/L and 3U.

Example 1

DNA (i.e., HDT gene) containing a fusion gene linked to PT7 (phage T7 promoter) was recovered from the plasmid pET-TrChHDT by NdeI-SacI digestion (NdeI-SacI digest). Subsequently, the recovered DNA was ligated into plasmid pPhi80-Km (Chung-Jen Chiang et al, reproduction-free and marker methods for genetic insertions of DNAs in phase attachment sites and controlled expression of chromogenes in Escherichia coli.Biotechnol Bioeng.2008; 101: 985. sup. the plasmid pPhi-TrHDTCh was obtained by chimeric DNA of PT 7-linked fusion gene, i.e., HDT gene, into the preprophyte mosaic site (expression site) of strain N30 by plasmid pPhi-TrHDTCh using recombinase Int of phage phi80 according to the homologous recombination of recombinase Int of the above reference, followed by insertion of plasmid pCP20(K.D.Datsenko B.L.Wal. and genetic insertion of plasmid DNA of Escherichia coli, expression cassette, expression site, expression cassette 11. the expression of plasmid DNA of plasmid 11. sup. 12. mu. origin, plasmid DNA of Escherichia coli, plasmid expression cassette, plasmid 11. sup. 12. plasmid, plasmid DNA and plasmid expression cassette, plasmid expression site of plasmid 11. 12. origin, plasmid DNA of plasmid, plasmid 11. sup. 12. plasmid, plasmid DNA, plasmid, producing strain N30-HDT.

The strain N30-HDT is cultured on an M9G culture medium containing 6g/L of glycerol, and the yield and the activity of the TrxA-HDT-ChBD can respectively reach 62mg/L and 3.4U.

Glucose is still the best fermentable sugar in E.coli. Thus, protein production in strain N30-HDT was investigated in the presence of glucose and glycerol. Strain N30-HDT was grown on M9G medium containing glucose and glycerol (3 g/L each). As a result, the strain N30-HDT consumed about 0.5g/L of glucose and 1g/L of glycerol and produced TrxA-HDT-ChBD with a yield and an enzyme activity of 80mg/L and 4.7U, respectively.

Example 2

The strain N30G-HDT was produced by chimerizing glf into the prephilic mosaic position of the chromosome of strain N30-HDT using the plasmid pHK-glf (Chung-Jen Chiang et al, Systematic approach to engineering Escherichia coli genes for co-solubilization of the glucose-xylose mix. J. Agric. food Chem.2013,61, 7583-K7590) in accordance with the procedure of example 1.

Strain N30G-HDT was used to produce proteins with M9G medium containing glucose and glycerol (3 g/L each). The strain N30G-HDT respectively consumes 1g/L of glucose and glycerol, and the yield and the activity of TrxA-HDT-ChBD respectively reach 92mg/L and 5.2U.

Example 3

A DNA fragment comprising in sequence a partial upstream region of the zwf gene, an anti-kanamycin gene, a P.lamda.PR (phage. lamda. PR promoter), and a 5' -end region of the zwf gene was amplified using a plasmid pPR-zwf (Mukesh Saini et al, Systematic engineering for expression of the n-butanol. 9:69) according to the above reference, transformed into the strain by the electroporation method, and then transformed into a plasmid pKD46(K.D.Datsenko and B.L.Wanner, One-P expression of chromosogenes in Escherichia coli K-12using products. proct. Natc.Act. Natl.S.97. USA) by homologous recombination of the amplified DNA fragment with the recombinase expressed by the homologous recombination of the rDNA fragment P.lamda.S.S.S.S.S.S.70, PCR, amplified by the homologous recombination of the rDNA fragment with the recombinase gene, the recombinase, the plasmid pPR, the plasmid pKD gene, the anti-kanamycin gene, the plasmid pKD gene, the plasmid, the, plasmid pTH19-creCs (Central et al,2012.Genomic engineering of Escherichia coli by the phase attachment site-based integration system with mutant loxP sites. Proc Biochem 47:2246-2254) was then used to express Cre recombinase to remove the kanamycin-resistant gene flanked by loxP sequences. Then, a partial upstream region of DNA comprising pgl gene in order, the kanamycin-resistant gene, P.lamda.PR (phage. lamda.PR promoter), the 5' -end region of the pgl gene were amplified in the same manner using plasmid pSPL-pgl (Mukesh Saini et al, Systematic engineering for expression of n-butanol. Biotechnology for Biotechnology.2016, 9:69), the strain was transformed by electroporation, the plasmid pKD46(K.D.Datsenko and B.L.Wanner, One-P expression of chromosomal genes in Escherichia coli K-12. PCR, the recombinase gene expression of the streptomycin Escherichia coli strain, U.S. S. Pat. No. 11, USA, the recombinase gene expression of homologous recombination plasmid DNA expressing the recombinase gene was amplified by means of the plasmid pSPL-pgl-P expression of the plasmid Escherichia coli strain, S.97, Natamycin, USA, U.97.S. Pat. S. Pat. 6697, the kanamycin-resistant gene flanked by loxP sequences was removed by fusion of the P.lamda.PR with the pgl gene, followed by expression of the Cre recombinase using plasmid pTH19-creCs (Chiang et al,2012.Genomic engineering of Escherichia coli by the phase attachment site-based integration system with mutant loxP sites. Proc Biochem 47:2246-2254) to produce strain N30G 4-HDT. Strain N30G4-HDT was grown on M9G medium containing glucose and glycerol (3G/L each). The strain N30G4-HDT effectively utilized two sugars, 1.8G/L glucose and 2.4G/L glycerol, respectively. The yield of soluble TrxA-HDT-ChBD reaches 170mg/L, the activity is 30U, and the pyruvate production reaches 4 mM.

Example 4

PCR amplification of alaA was performed with primers RC15193(acatatgtcccccattgaaaaatcc) and RC15194 (actcgagattacagctgatgataaccag). The PCR DNA was integrated into plasmid pET20b (Novagene Co.) by NdeI-XhoI digestion (NdeI-XhoI digest) to obtain plasmid pET20 bI-AlaA. At the same time, alaC was amplified by PCR using primers RC15191(cgaattcaggagaggaaattatggctgacac) and RC15192 (tgggccctcagattattccgcgttttcgtgaatatg). The PCR DNA was integrated into the plasmid pND707 by ApaI-EcoRI digestion (ApaI-EcoRI digest) (Love, C.A. et al, Stable high-copy-number bacterial. lambda. promoter vectors for overproduction of proteins in Escherichia coli. Gene 1996,176, 49-53.) to obtain the plasmid pND-AlaC. A DNA containing alaA was recovered from plasmid pET20bI-AlaA by XbaI-XhoI cleavage (XbaI-XhoI cut), and integrated into the corresponding site of plasmid pND-AlaC to obtain plasmid pND-AlaAC containing a DNA fragment of P.lambda.PL (phage. lambda.PL promoter), alaC gene, and alaA gene (P.lambda.PL-alaC-alaA) in this order. In addition, P.lamda PL-alaC-alaA-containing DNA was amplified from plasmid pND-AlaAC by PCR using primers (agctaaggatccctcacctaccaaacaatgc and agcatcgccagtcactatgg). The resulting PCR DNA was incorporated into the plasmid pLam-Lox-Km (Chung-Jen Chiang et al, Genomic engineering of Escherichia coli by the phase attachment site-based integration system with mutant loxP sites. Process biochemistry.2012; 47: 2246-. The P lambda PL-alaC-alaA containing DNA was chimerized to the pre-phage mosaic position of strain N30G4-HDT using plasmid pLam-AlaCA to enhance the expression levels of both enzymes according to the procedure of example 1, followed by expression of the Cre recombinase to remove the kanamycin-resistant gene flanked by loxP sequences using plasmid pTH19-CreCs (Chiang et al,2012, Genomic engineering of Escherichia coli by the phase attachment site-based integration system with mutant loxP sites. Proc Biochem 47: 2246-2254). Next, the ppc gene was removed, DNA internally embedded with the kanamycin-resistant gene was amplified from strain JW3928-1 (. DELTA.ppc-742:: kan) (E.coli genetic stock center) using primers (agcgtcgtgaatttaatgacg and ccgaatgtaacgacaattcc), transformed into the strain by electroporation, plasmid pKD46(K.D.Datsenko and B.L.Wanner, One-step inactivation of chromosogenes in Escherichia coli K-12using PCR products, Proc.Natl.Acad.Sci.USA.2000,97: 6640. cake 6645.) expressing lambda Rad recombinase, and the amplified DNA internally embedded with the kanamycin-resistant gene was mutually replaced with the chromosomal ppc gene of the strain by homologous recombination of the Rad to disrupt the ppc gene of the transformed strain, resulting in HDN-5G-5.

Strain N30G5-HDT was unable to grow on glucose and glycerol without glutamate due to the lack of the ppc gene. Growth was restored on M9G medium (containing glutamic acid) containing glucose and glycerol (3 g/L each). As a result, the strain N30G5-HDT produced soluble TrxA-HDT-ChBD, and the yield and activity reached 253mg/L and 42.8U, respectively. Pyruvate was reduced to less than 1 mM.

The results of protein activity and yield of comparative examples 1 and 2 and examples 1 to 4 are collated in the following tables 2 and 3, wherein table 2 is the results of protein activity and yield of comparative examples 1 and 2 and example 1 in the glycerol-containing M9G medium, and table 3 is the results of protein activity and yield of examples 1 to 4 in the glucose-and glycerol-containing M9G medium. In tables 2 and 3 below, Glu represents glucose, Gly glycerol, Δ Glu represents consumed glucose, Δ Gly represents consumed glycerol, and # indicates that the strain carries the plasmid pET-TrChHDT.

TABLE 2

TABLE 3

As can be seen from Table 2 above, the protein activity and the yield of strain N30 of comparative example 2 were superior to those of strain BAD-5 of comparative example 1, presumably because anaerobic catabolism consisted of reactions related to NADH production mediated by gldA and dhaKLM. Thus, strain N30 with glycerol catabolism showed better performance in terms of cell biomass and protein yield. The protein activity and yield of the strain N30-HDT of example 1 were similar to those of the strain N30 of comparative example 2, indicating that the use of the strain N30-HDT of example 1 can provide similar protein activity and yield to those of the strain N30 of comparative example 2 without the use of ampicillin.

As can be seen from Table 3 above, strain N30G-HDT with functional glf gene of example 2 improved protein activity and productivity compared to strain N30-HDT of example 1 due to the functional glf gene promoting the glucose metabolism ability of the strain. And as can be further seen from table 3, the strain N30G4-HDT with functional glf gene, zwf gene and pgl gene further improves protein activity and productivity, and it is evident that enhancing the phosphopentose pathway can increase NADPH production and increase the energetic state of the strain. The protein activity and the yield of the N30G5-HDT of the strain with the removed ppc gene and the functional glf gene, the zwf gene, the pgl gene, the alaA gene and the alaC gene are far superior to those of other strains, the strain lacking the ppc gene can retain phosphoenolpyruvate (PEP), and the PEP is generally converted into pyruvate through a substrate level phosphorylation reaction mediated by pyruvate kinase under physiological conditions, so that more pyruvate and ATP are caused; two glutamate pyruvate aminotransferases, encoded by the alaC gene as well as the alaA gene, are responsible for the transamination reaction from glutamate and pyruvate to form alanine associated with alpha-ketoglutarate (alpha-KG), supplementing tricarboxylic acid (TCA) cycle intermediates for biosynthesis by providing alpha-KG, and enhancing NADH and ATP production, enhancing the energy status of the strain.

From the above analysis results, it is clear that the recombinant protein having higher protein activity can be obtained in higher yield in an environment containing one or a combination of glycerol and glucose by using the strain capable of improving the expression of the recombinant protein.

The foregoing is by way of example only, and not limiting. It is intended that all equivalent modifications or variations not departing from the spirit and scope of the present invention be included in the claims.

Claims (10)

1. A strain capable of improving expression of recombinant protein belongs to Escherichia coli, and is characterized in that the strain capable of improving expression of recombinant protein is obtained by integrating a glf gene, a zwf gene and a pgl gene into an Escherichia coli strain lacking a ptsG gene, an araABD gene manipulation group and an araFGH gene manipulation group.

2. The strain capable of enhancing expression of a recombinant protein according to claim 1, wherein the strain capable of enhancing expression of a recombinant protein further comprises an alaC gene.

3. The strain capable of enhancing expression of a recombinant protein according to claim 1, wherein the strain capable of enhancing expression of a recombinant protein further comprises an alaA gene.

4. The strain capable of enhancing expression of a recombinant protein according to claim 1, wherein the strain capable of enhancing expression of a recombinant protein is free of ppc gene.

5. The strain capable of enhancing expression of a recombinant protein according to claim 1, wherein the strain capable of enhancing expression of a recombinant protein has the characteristics of LacZ:PBAD-T7 gene 1Ptrc-araE Δ araBAD Δ ptsG Δ araFGH.

6. A method for producing a recombinant protein, comprising the step of culturing the strain according to claim 1 in a medium containing a carbon source to produce the recombinant protein.

7. The method for producing a recombinant protein according to claim 6, wherein said strain further comprises one or a combination of the alaC gene and the alaA gene.

8. The method of producing a recombinant protein according to claim 7, wherein the medium comprising a carbon source comprises one or a combination of glycerol and glucose.

9. The method for producing a recombinant protein according to claim 7, wherein the strain capable of enhancing expression of the recombinant protein is free of the ppc gene.

10. The method of producing a recombinant protein according to claim 9, wherein the medium comprising a carbon source comprises glutamic acid.

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