Genetically engineered bacterium for efficient expression and secretion of green fluorescent protein mediated protein glutaminase, construction method and application

Abstract

The invention provides a genetically engineered bacterium and a construction method of the genetically engineered bacterium, and the genetically engineered bacterium is characterized in that a green fluorescent protein sfGFP is used for mediating protein glutaminase, PG (protein glutaminase), PGF (protein glutaminase), PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF, PGF and PGF; eC 3.5. 1.44) is efficiently secreted and expressed in engineering bacteria, and belongs to the technical field of biological engineering. The method comprises the following steps: an obtained sfGFP gene is derived from an NCBI database (GenBank numbering: CP035486.1), an obtained PG zymogen gene PP (Propeptide-Protein glutaminase) is derived from a Chryseobacterium prion prgA gene (GenBank numbering: AB046594.1) in the NCBI database, a recombinant plasmid pHT01 / sfGFP-PP is introduced into bacillus subtilis WB800N to obtain an engineering bacterium, extracellular secretion expression of PP is realized, the sfGFP-PP is activated by trypsin to obtain PG, and the PG is subjected to enzyme activation to obtain the recombinant bacillus subtilis. The expression quantity and the secretion efficiency of the PG in the bacillus subtilis are greatly improved. The recombinant PG produced by the method can generate 23.5 U / mL enzymatic activity under activation of trypsin, can be used for improving protein functional characteristics, and has relatively high industrial production and application values.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme gene engineering, and particularly relates to a genetically engineered bacterium for efficiently expressing and secreting protein glutaminase (PG; EC 3.5.1.44) mediated by superfolder green fluorescent protein (sfGFP), and a construction method and application thereof. Background Art

[0002] As an important resource for alternative proteins, plant protein foods have gradually received increasing attention globally. In addition to having low heat and low fat, slow absorption, reducing the risk of obesity, and meeting the needs of vegetarian consumers, plant proteins are also beneficial for reducing carbon emissions from agriculture, achieving sustainable renewable development, and promoting ecological environmental protection. However, during food processing, due to the complexity of plant protein components, residual anti-nutrients, unpleasant flavors, and high levels of amide groups, the functional properties of plant proteins such as water solubility, emulsification, foaming, and gelation are poor, resulting in limited application scope and low utilization rate of plant-based foods. Physical and chemical treatment methods such as heat treatment, ultra-high pressure, ultrasonic waves, and chemical group modification are commonly used to improve the functional properties of plant proteins. However, the poor effects of these methods and potential chemical residues limit their wide application in improving the functionality of plant proteins. Enzymatic modification of proteins has gradually received attention due to its mild reaction and the safety of modified proteins. Among various plant protein modification additives, protein glutaminase (PG) is very valuable and affects the development and utilization of proteins, especially plant-derived proteins.

[0003] PG is a deaminase that specifically targets glutamine residues on proteins. It is isolated from the non-pathogenic Chryseobacterium proteolyticum 9670T and only specifically hydrolyzes glutamine residues on the side chains of proteins or peptides, without protease or transglutaminase activity, and does not cause peptide chain hydrolysis or cross-linking. PG has shown excellent results in improving the functional properties and processing of plant proteins. The solubility and emulsifying properties of oat protein, coconut protein, and soy protein isolate (SPI) treated with PG deamination have been significantly improved. Based on the great application value of PG, many researchers are committed to isolating and optimizing Chryseobacterium proteolyticum that produces PG to increase the yield of PG. It is reported that the PG produced by Chryseobacterium proteolyticum WG15 has the highest activity, reaching 2.91 U / ml (Reference: Enhanced protein glutaminase production from Chryseobacterium proteolyticum combining physico-chemical mutagenesis and resistance screening and its application to soybean protein isolates). In addition, due to the low yield of PG in Chryseobacterium proteolyticum, many studies have made it possible to heterologously express PG or PG-like in Bacillus subtilis, Corynebacterium glutamicum, and Escherichia coli, with enzyme activities reaching 3.23 U / mL, 26 U / mg, and 0.175 U / mL respectively, increasing the possibility of industrial production of PG. However, so far, the yield of PG still cannot meet the requirements of industrial production of PG.

[0004] Bacillus subtilis is a powerful host for homologous and heterologous expression of target proteins, widely used in the industrial production of enzymes and secondary metabolites, and recognized as a generally recognized as safe (GRAS) strain. In 2021, the PG gene prgA from Chryseobacterium proteolyticum was first expressed in Bacillus subtilis, with a maximum yield of 0.6 U / ml (Reference: Optimizing Protein-Glutaminase Expression in Bacillus subtilis). Subsequently, the expression cassette for producing PG was further optimized by using different combinations of promoters and signal peptides. However, it was found that even with the optimal combination expression cassette of the SecA promoter and the YdeJ signal peptide, the improvement in PG expression was still limited, with a maximum enzyme activity of 7.07 U / ml in the fermenter (Reference: Combinatorial engineering for efficient production of protein-glutaminase in Bacillus subtilis). Therefore, to avoid the limitation of the classical secretion pathway on the PG expression yield, it is necessary to further develop other new strategies to mediate the efficient secretion and expression of PG.

[0005] sfGFP is a super-folder green fluorescent protein obtained by subjecting green fluorescent protein (GFP) to six rounds of mutations (S30R, Y39N, N105T, Y145F, I171V, A206V). Its folding rate is 3.5 times higher than that of GFP. Research has shown that sfGFP can act as a "chaperone protein" to mediate the secretion of heterologous proteins, such as the antimicrobial peptide PG4, endo-β-N-acetylglucosaminidase H (Endo H), human arginase-1 (ARG1), and glutamate decarboxylase (GAD) in Escherichia coli (Reference: Non-peptideguided auto-secretion of recombinant proteins by super-folder greenfluorescent protein in Escherichia coli), and to mediate the secretion of phospholipase D in Bacillus subtilis (Reference: Construction of aSuper-Folder Fluorescent Protein-Guided SecretoryExpression System for the Production of Phospholipase D inBacillus subtilis). Interestingly, different from the secretion of proteins mediated by traditional signal peptides, these secretion and expression results were obtained without the guidance of signal peptides. In addition, atypical secretion of target proteins mediated by type I L-asparaginase has also been found in Bacillus subtilis, indicating that the potential strategy of target protein secretion mediated by known proteins in Bacillus subtilis is feasible without a signal peptide in the traditional sense. In addition, some studies have pointed out that the use of a highly efficient translation initiation site on the N-terminal fusion tag can enhance the expression of fusion proteins. Based on the high translation and folding efficiency of sfGFP, the fusion expression of sfGFP and PP may have a positive promoting effect on the expression and secretion of PG. Summary of the Invention

[0006] The present invention overcomes the defects and deficiencies of the prior art methods and provides a genetically engineered bacterium mediated by sfGFP (super-folder green fluorescent protein) for the highly efficient expression and secretion of PG (protein glutaminase), and its construction method and application.

[0007] PG is a deamidase newly discovered in recent years, which is naturally fermented by the food safety strain Chryseobacterium proteolyticum. The full-length PG enzyme gene encodes a signal peptide (SP) containing 21 amino acids, a propeptide (Pro) containing 114 amino acids, and a mature peptide (mPG) containing 185 amino acids. Due to the presence of the propeptide in the Pro-mPG (PP) zymogen containing the propeptide and the mature peptide, Pro can shield the activity of mPG. Therefore, PP is the inactive form of the PG enzyme. The relative molecular mass of the mature peptide mPG is 19861 Da, which can specifically catalyze the conversion of glutamine residues in the side chain of proteins or polypeptide chains into glutamate residues and release NH4 + , thus showing deamidation activity towards substrate proteins and effectively enhancing functional properties such as the solubility of proteins. However, at present, whether it is Chryseobacterium proteolyticum that naturally produces PG or other strains that heterologously express PG, there is a bottleneck in the low yield of PG, which is not sufficient to meet the requirements of industrial production. Herein, the present invention proposes a new strategy for enhancing the expression and secretion of PG mediated by sfGFP. By constructing an expression cassette of the sfGFP and PP fusion protein without a signal peptide, and activating the activity of PG by trypsin cleavage in the extracellular supernatant of the engineered bacteria, the structure and activation process of sfGFP-PP were demonstrated. The efficient secretion and expression of PP mediated by sfGFP promoted the industrialization and application of PG in the food field.

[0008] In the present invention, 1. The PG enzyme gene itself carries a signal peptide. The usual idea is to use its own signal peptide for expression, but using its own signal peptide in Bacillus subtilis cannot achieve extracellular secretion expression. 2. Replacing the signal peptide of the PG enzyme with the signal peptide of Bacillus subtilis can express the PG enzyme in Bacillus subtilis, but its expression level is very low. 3. Replacing the signal peptide of Bacillus subtilis with green fluorescent protein as the "leader protein" or "chaperone protein" for extracellular secretion expression of PG can achieve high-efficiency expression on the basis of extracellular secretion expression, and the expression level is greatly improved. In summary, first, it is an innovation in the design of the expression idea that spans; second, a considerable part of proteins are actually not suitable for this expression mode, and the attempt to express PG in this mode and succeed is an innovation.

[0009] The present invention provides a genetically engineered bacterium for efficiently secreting and expressing PG mediated by sfGFP. The genetically engineered bacterium is a recombinant Bacillus subtilis WB800N capable of highly expressing and secreting the sfGFP-PP fusion protein. Specifically, the genetically engineered bacterium uses the pHT01 plasmid as the expression vector for the sfGFP-PP fusion gene. Its extracellular supernatant contains the sfGFP-PP fusion protein, and mature PG with protein glutaminase enzyme activity can be obtained after trypsin digestion, and its enzyme activity is 23.5 U / ml.

[0010] The present invention also provides a method for constructing a genetically engineered bacterium for efficiently secreting and expressing PG mediated by sfGFP, comprising the following steps:

[0011] Step 1: Obtain the coding gene of superfolder green fluorescent protein (sfGFP gene) and the protein glutaminase zymogen gene (PP gene);

[0012] Step 2: Use fusion PCR technology to synthesize the fusion expression gene of sfGFP gene and PP gene (sfGFP-PP), and ligate it with the double-digested linearized pHT01 plasmid vector DNA fragment to obtain the pHT01 / sfGFP-PP expression vector;

[0013] Step 3: Transfer the pHT01 / sfGFP-PP expression vector obtained in Step 2 into competent Bacillus subtilis WB800N by electroporation to obtain the genetically engineered bacterium.

[0014] In Step 3, it also includes the step of screening the genetically engineered bacterium through a chloramphenicol resistance plate.

[0015] In Step 1, the coding gene of the superfolder green fluorescent protein, abbreviated as the sfGFP gene, has a nucleotide sequence as shown in SEQ ID NO.1. The full length of this gene is 714 bp (excluding the stop codon TAA), with a G+C content of 42%, encoding 238 amino acids, and the protein sequence is as shown in SEQ ID NO.2. The sfGFP gene is derived from the NCBI database, GenBank accession number: CP035486.1, and the sfGFP gene DNA fragment is obtained by gene synthesis.

[0016] SEQ ID NO.1:

[0017] ATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGAT

[0018] GGTGATGTTAATGGGCACAAATTTTCTGTCCGTGGAGAGGGTGAAGGTGATGCTACAAAC

[0019] GGAAAACTCACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCGTGGCCAACA

[0020] CTTGTCACTACTCTGACCTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAAC

[0021] GGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTT

[0022] CAAAGATGACGGGACCTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTG

[0023] TTAATCGTATCGAGTTAAAGGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAA

[0024] ACTCGAGTACAACTTTAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAATGG

[0025] AATCAAAGCTAACTTCAAAATTCGCCACAACGTTGAAGATGGTTCCGTTCAACTAGCAGA

[0026] CCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTAC

[0027] CTGTCGACACAATCTGTCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTT

[0028] CTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAA

[0029] SEQ ID NO.2:

[0030] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPT

[0031] LVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLV

[0032] NRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHY

[0033] QQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK

[0034] The protein glutaminase zymogen gene, which is the propeptide coding gene of protein glutaminase, abbreviated as the PP gene, has a nucleotide sequence as shown in SEQ ID NO.3. The full length of this gene is 897 bp (excluding the stop codon TAA), with a G+C content of 50%, encoding 299 amino acids. The protein sequence encoded by the PP gene is as shown in SEQ ID NO.4.

[0035] SEQ ID NO.3:

[0036] GATTCCAACGGGAATCAGGAAATCAACGGAAAGGAAAAACTAAGTGTAAATGATTCT

[0037] AAGCTGAAAGATTTCGGAAAGACTGTACCGGTAGGGATAGACGAAGAAAACGGAATGAT

[0038] AAAGGTGTCATTTATGTTAACTGCGCAATTCTATGAAATTAAGCCGACCAAAGAAAATGA

[0039] GCAGTATATCGGAATGCTTAGACAGGCTGTTAAGAATGAATCTCCTGTACACATTTTCTTA

[0040] AAGCCTAATAGCAATGAAATAGGAAAAGTGGAGTCTGCAAGTCCGGAAGACGTAAGATA

[0041] TTTTAAAACGATCCTGACAAAAGAAGTAAAAGGGCAAACCAATAAATTGGCGAGTGTAA

[0042] TTCCTGATGTAGCTACATTAAATTCTTTATTCAATCAAATAAAGAATCAGTCTTGCGGTACC

[0043] TCTACGGCGTCCTCACCATGCATCACATTCAGATATCCTGTAGACGGATGTTATGCAAGAG

[0044] CCCATAAGATGAGACAAATCTTAATGAACAACGGCTATGACTGTGAAAAACAATTTGTAT

[0045] ACGGAAACCTAAAGGCATCAACAGGAACTTGCTGTGTGGCGTGGAGCTACCACGTTGCA

[0046] ATATTGGTAAGCTATAAAAATGCTTCCGGAGTAACGGAAAAAAGAATTATTGATCCTTCAC

[0047] TATTTTCAAGCGGTCCTGTAACAGATACAGCATGGAGAAACGCTTGCGTTAACACCTCTT

[0048] GCGGATCTGCATCCGTTTCCTCTTATGCTAATACTGCAGGAAATGTTTATTACAGAAGTCCT

[0049] AGTAATTCTTACCTGTATGACAACAATCTGATCAATACCAACTGTGTACTGACTAAATTTTC

[0050] ACTGCTTTCCGGATGTTCTCCTTCACCTGCACCGGATGTATCCAGCTGTGGATTT

[0051] SEQ ID NO.4:

[0052] DSNGNQEINGKEKLSVNDSKLKDFGKTVPVGIDEENGMIKVSFMLTAQFYEIKPTKENE

[0053] QYIGMLRQAVKNESPVHIFLKPNSNEIGKVESASPEDVRYFKTILTKEVKGQTNKLASVIPDV

[0054] ATLNSLFNQIKNQSCGTSTASSPCITFRYPVDGCYARAHKMRQILMNNGYDCEKQFVYGNLK

[0055] ASTGTCCVAWSYHVAILVSYKNASGVTEKRIIDPSLFSSGPVTDTAWRNACVNTSCGSASVSS

[0056] YANTAGNVYYRSPSNSYLYDNNLINTNCVLTKFSLLSGCSPSPAPDVSSCGF

[0057] The PP gene is derived from the prgA gene of Chryseobacterium proteolyticum in the NCBI database, GenBank accession number: AB046594.1. The DNA fragment of the PP gene was obtained by gene synthesis and amplified by PCR. Among them,

[0058] Primer sequence F1(PP): ATGGATGAGCTCTACAAAGATTCCAACGGGAATCAG (SEQ ID NO.7), R1(PP): TTAAAATCCACAGCTGGA (SEQ ID NO.8).

[0059] The DNA template used for PCR is the DNA fragment of the prgA gene of Chryseobacterium proteolyticum, and the DNA polymerase used is the high-fidelity PrimeSTAR HS DNA polymerase.

[0060] In step two, the fusion expression gene of the sfGFP gene and the PP gene, abbreviated as the sfGFP-PP gene, is a fusion gene of the sfGFP gene and the PP gene. Its nucleotide sequence is shown in SEQ ID NO.5. The full length of this gene is 1611 bp (excluding the stop codon TAA), the G+C content is 46%, encoding 537 amino acids. The protein sequence encoded by the sfGFP-PP gene is shown in SEQ ID NO.6.

[0061] SEQ ID NO.5:

[0062] ATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGAT

[0063] GGTGATGTTAATGGGCACAAATTTTCTGTCCGTGGAGAGGGTGAAGGTGATGCTACAAAC

[0064] GGAAAACTCACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCGTGGCCAACA

[0065] CTTGTCACTACTCTGACCTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAAC

[0066] GGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTT

[0067] CAAAGATGACGGGACCTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTG

[0068] TTAATCGTATCGAGTTAAAGGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAA

[0069] ACTCGAGTACAACTTTAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAATGG

[0070] AATCAAAGCTAACTTCAAAATTCGCCACAACGTTGAAGATGGTTCCGTTCAACTAGCAGA

[0071] CCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTAC

[0072] CTGTCGACACAATCTGTCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTT

[0073] CTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAAGATTCCA

[0074] ACGGGAATCAGGAAATCAACGGAAAGGAAAAACTAAGTGTAAATGATTCTAAGCTGAAA

[0075] GATTTCGGAAAGACTGTACCGGTAGGGATAGACGAAGAAAACGGAATGATAAAGGTGTC

[0076] ATTTATGTTAACTGCGCAATTCTATGAAATTAAGCCGACCAAAGAAAATGAGCAGTATATC

[0077] GGAATGCTTAGACAGGCTGTTAAGAATGAATCTCCTGTACACATTTTCTTAAAGCCTAATA

[0078] GCAATGAAATAGGAAAAGTGGAGTCTGCAAGTCCGGAAGACGTAAGATATTTTAAAACG

[0079] ATCCTGACAAAAGAAGTAAAAGGGCAAACCAATAAATTGGCGAGTGTAATTCCTGATGTA

[0080] GCTACATTAAATTCTTTATTCAATCAAATAAAGAATCAGTCTTGCGGTACCTCTACGGCGT

[0081] CCTCACCATGCATCACATTCAGATATCCTGTAGACGGATGTTATGCAAGAGCCCATAAGAT

[0082] GAGACAAATCTTAATGAACAACGGCTATGACTGTGAAAAACAATTTGTATACGGAAACCT

[0083] AAAGGCATCAACAGGAACTTGCTGTGTGGCGTGGAGCTACCACGTTGCAATATTGGTAAG

[0084] CTATAAAAATGCTTCCGGAGTAACGGAAAAAAGAATTATTGATCCTTCACTATTTTCAAGC

[0085] GGTCCTGTAACAGATACAGCATGGAGAAACGCTTGCGTTAACACCTCTTGCGGATCTGCA

[0086] TCCGTTTCCTCTTATGCTAATACTGCAGGAAATGTTTATTACAGAAGTCCTAGTAATTCTTA

[0087] CCTGTATGACAACAATCTGATCAATACCAACTGTGTACTGACTAAATTTTCACTGCTTTCC

[0088] GGATGTTCTCCTTCACCTGCACCGGATGTATCCAGCTGTGGATTT

[0089] SEQ ID NO.6:

[0090] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPT

[0091] LVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLV

[0092] NRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHY

[0093] QQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYKDSNGN

[0094] QEINGKEKLSVNDSKLKDFGKTVPVGIDEENGMIKVSFMLTAQFYEIKPTKENEQYIGMLRQ

[0095] AVKNESPVHIFLKPNSNEIGKVESASPEDVRYFKTILTKEVKGQTNKLASVIPDVATLNSLFNQ

[0096] IKNQSCGTSTASSPCITFRYPVDGCYARAHKMRQILMNNGYDCEKQFVYGNLKASTGTCCVA

[0097] WSYHVAILVSYKNASGVTEKRIIDPSLFSSGPVTDTAWRNACVNTSCGSASVSSYANTAGNV

[0098] YYRSPSNSYLYDNNLINTNCVLTKFSLLSGCSPSPAPDVSSCGF

[0099] The sfGFP-PP gene DNA fragment is derived from fusion PCR technology, wherein,

[0100] Primer sequence F1(sfGFP-PP): CCAATTAAAGGAGGAAGGATCCATGAGCAAAGGAGAAG AAC(SEQID NO.9);

[0101] R1(sfGFP-PP): GCCCCGGGGACGTCGACTCTAGATTAAAATCCACAGCTGGATAC (SEQ ID NO.10).

[0102] In step two, the DNA templates used for fusion PCR are the sfGFP gene and the PP gene, and the DNA polymerase used is the high-fidelity PrimeSTAR HS DNA polymerase.

[0103] In step two, an expression vector pHT01 / sfGFP-PP was also constructed. The construction method of the pHT01 / sfGFP-PP expression vector includes: double-digesting the vector pHT01 with DNA restriction enzymes BamH I and Xba I to obtain a linearized pHT01 DNA fragment. The linearized pHT01 DNA fragment and the sfGFP-PP DNA fragment are ligated and circularized under the catalysis of a DNA recombinase by using the DNA end homologous recombination technology to obtain the pHT01 / sfGFP-PP expression vector.

[0104] Specifically, the composition and content of the reagents for preparing the PCR reaction solution of the PrimeSTAR high-fidelity DNA polymerase are: 10 μl of 5x PrimeSTAR Buffer, 4 μl of dNTP, 1 μl of the DNA templates of the sfGFP gene and the PP gene, 1 μl of each of the upstream and downstream primers, 0.5 μl of PrimeSTAR DNA polymerase, and 32.5 μl of dH2O; the DNA end homologous recombination technology is to ligate and circularize the linear DNA of the linearized pHT01 vector and the sfGFP-PP DNA fragment obtained by fusion PCR through a DNA recombinase; the DNA recombinase is the 2×Uniclone Seamless Cloning Mix DNA recombinase produced by Beijing Jinsha Biotechnology Co., Ltd.

[0105] Among them, the mass ratio of the linearized pHT01 DNA fragment to the sfGFP-PP DNA fragment obtained by fusion PCR is 1:1 to 5:1. Preferably, the above fragment ratio is 2.5:1.

[0106] In step three, a genetically engineered bacterium expressing the sfGFP-PP gene was also constructed. The construction method includes: transferring the pHT01 / sfGFP-PP expression vector into the competent Bacillus subtilis WB800N by electroporation to obtain the genetically engineered bacterium. The specific steps are as follows:

[0107] Step 3.1: Preparation of electrocompetent Bacillus subtilis: Inoculate wild-type Bacillus subtilis WB800N into LB liquid medium, shake it in a 37°C incubator until the OD600 reaches 1, ice-bath for 10 min, centrifuge at 4000 rpm for 10 min, discard the supernatant, and repeatedly rinse the bacterial cells in ETM electrotransformation buffer pre-cooled at 4°C, repeating 2 - 3 times to obtain the electrocompetent Bacillus subtilis WB800N.

[0108] Step 3.2: Electroporation of electrocompetent cells: Take 60 μl of electrocompetent cells and add them to a pre-cooled electroporation cuvette containing the pHT01 / sfGFP-PP expression vector, mix well and ice-bath for 5 min, and finally perform one-time electroporation on the electroporator. The parameters of the electroporator are set as follows: 2.0 - 2.1 kV, 25 - 30 μF, 200 - 220 Ω, 2 - 2.2 mm; preferably, the parameters of the above electroporator are set as: 2.0 kV, 25 μF, 200 Ω, 2 mm.

[0109] Step 3.3: Resistance screening of genetically engineered bacteria: Add the bacterial solution after electroporation to RM recovery medium, recover it at 37°C for 3 - 3.5 h, preferably 3 h, and spread it on a chloramphenicol-resistant plate. The next day, pick monoclonal colonies on the chloramphenicol-resistant plate and inoculate them into LB medium, culture them at 37°C and 200 rpm for 12 h, extract the vector in the strain for DNA sequencing, and if the sequencing result is correct, it is the correct genetically engineered bacteria.

[0110] The present invention also provides a method for identifying the high-efficiency expression of sfGFP-PP in genetically engineered bacteria. Streak the genetically engineered bacteria expressing sfGFP-PP and the control bacteria not expressing sfGFP-PP on a chloramphenicol-resistant plate for activation, and culture them at 37°C for 12 h. The next day, pick the genetically engineered bacteria and the control bacteria and inoculate them into LB medium containing chloramphenicol resistance, culture them at 37°C and 200 rpm for 12 h; transfer the genetically engineered bacteria and the control bacteria in LB medium to TB medium at a transfer amount of 2% and continue to culture for 3 h. At this time, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 1 mM to the genetically engineered bacteria and the control bacteria respectively to induce the expression of sfGFP-PP, and continue to culture for 12 h - 30 h for sampling. The sampling time points are 12 h, 18 h, 20 h, 22 h, 24 h, 26 h, 28 h, and 30 h, and perform PG activity detection.

[0111] The present invention also provides a method for identifying the efficient secretion of sfGFP-PP expressed by a genetically engineered bacterium. When inducing the expression of sfGFP-PP in the engineered bacterium in TB medium, bacterial liquid samples at different time points are taken. After centrifuging the samples at 12,000 rpm for 1 - 5 min, the culture supernatant is collected for standby; the obtained bacterial cells are resuspended in PBS buffer with the same volume as the initial bacterial liquid, centrifuged at 12,000 rpm for 5 min, and washed 2 - 3 times repeatedly. Preferably, it is washed 3 times. Subsequently, it is resuspended again with the same volume and broken by an ultrasonic crusher. After centrifuging the broken sample at 12,000 rpm for 5 min, the soluble supernatant of the broken bacterial cells is collected for standby. The culture supernatant and the soluble supernatant of the bacterial cells are added with 1 mg / mL trypsin for the activation of PG and the detection of enzyme activity.

[0112] Specifically, the composition of the ETM electroporation buffer is: 0.5 M sorbitol, 0.5 M mannitol, 10% (V / V) glycerol; the composition of the RM recovery medium is: LB medium containing 0.5 M sorbitol and 0.38 M mannitol;

[0113] The composition of the LB medium is: 10 g / L tryptone; 5 g / L yeast extract; 10 g / L NaCl; the ETM electroporation buffer, the RM recovery medium, and the LB medium are all autoclaved at 121 °C for 20 min.

[0114] The final concentration of chloramphenicol in the chloramphenicol-resistant plate is 10 μg / ml.

[0115] The composition of the TB medium is: 2 g / L tryptone; 24 g / L yeast extract; 4 mL / L glycerol; 2.2 g / L KH2PO4: 9.4 g / L K2HPO4; autoclaved at 115 °C for 20 min.

[0116] And / or, the IPTG induction time is 28 h - 32 h, preferably 28 h.

[0117] And / or, the relative molecular mass of the sfGFP-PP fusion protein is 60 kDa, and the relative molecular mass of the mature PG is 19.8 kDa.

[0118] The present invention also provides a recombinant expression vector, which is the recombinant expression vector pHT01 / sfGFP-PP obtained according to the above-mentioned method for constructing a genetically engineered bacterium.

[0119] The present invention also provides a genetically engineered bacterium, which is a genetically engineered bacterium prepared according to the above-mentioned method for constructing a genetically engineered bacterium, and it is a recombinant Bacillus subtilis WB800N containing the pHT01 / sfGFP-PP recombinant expression vector.

[0120] The present invention also provides the expression of a fusion gene of sfGFP-PP, which is the expression of a fusion gene of sfGFP and the PG precursor PP. The steps are to use isopropyl-β-D-thiogalactoside to induce the recombinant Bacillus subtilis WB800N to express the sfGFP-PP fusion protein.

[0121] The present invention also provides a fusion protein sfGFP-PP, which is a fusion protein of sfGFP and the PG precursor PP.

[0122] The present invention also provides the application of the above-mentioned genetically engineered bacterium for highly expressing and secreting PG mediated by sfGFP, the above-mentioned method for constructing the genetically engineered bacterium for highly expressing and secreting PG mediated by sfGFP, the above-mentioned pHT01 / sfGFP-PP recombinant expression vector, the above-mentioned expression of the fusion gene of sfGFP-PP, or the above-mentioned fusion protein sfGFP-PP in the preparation of protein glutaminase.

[0123] In the specific embodiment, in the present invention, through genetic engineering and fusion PCR techniques, the method for heterologous expression of PG is innovated. Compared with the control bacterium, the genetically engineered bacterium in the present invention utilizes the high-efficiency folding and translation efficiency of sfGFP itself. After fusion expression with PG, the problems of difficult heterologous expression and low yield of PG are solved. The present invention achieves the highest PG enzyme activity reported in the current literature, up to 23.5 U / mL, and can secrete PG enzyme into the extracellular supernatant by itself without the need for a conventional signal peptide to guide the secretion of PG, simplifying the production process that requires breaking bacterial cells.

[0124] The beneficial effects of the present invention include: The present invention proposes a new strategy for improving the expression and secretion of PP mediated by sfGFP. sfGFP has dual functions of a signal peptide and a molecular chaperone protein in Bacillus subtilis.

[0125] After fusing sfGFP with the N-terminus of PP, compared with the control bacterium, the genetically engineered bacterium in the present invention shows a high-efficiency PP expression ability. The PG enzyme activity after activating PP by trypsin digestion reaches 23.5 U / mL, which is more than 8 times the highest level of PG expression reported in shake-flask culture at present, greatly promoting the process of PG in the industrial production field.

[0126] In the present invention, in addition to significantly increasing the yield of PG, in terms of extracellular secretion, sfGFP guides the transmembrane transport of PP, achieving a secretion efficiency of more than 90%. This high-efficiency secretion and expression mode promotes the yield of PG mediated by sfGFP, thus solving the problem of low efficiency of natural or heterologous expression.

[0127] In the present invention, the fusion expression pattern also simplifies the detection of PG production, providing a perfect solution for the expression and detection of PG or other target proteins.

[0128] The creativity and advancement of the present invention lie in: a genetically engineered bacterium for highly efficient secretion and expression of PG mediated by sfGFP and its construction method, including the following characteristics:

[0129] In the present invention, sfGFP is used as the leader peptide for the secretion of the PG precursor PP, replacing the signal peptide for the secretion and expression of the traditional PP, and guiding the efficient extracellular secretion of PP.

[0130] The present invention utilizes the high translation and folding efficiency of sfGFP to mediate the highly efficient expression of the PG precursor PP. Compared with the secretion and expression of PP mediated by the traditional signal peptide SamyQ in Bacillus subtilis, the expression efficiency is increased by more than 30 times.

[0131] The present invention clarifies the process by which the fusion protein sfGFP-PP is gradually secreted from the intracellular to the extracellular after being expressed in the engineered bacterium, and explains the process and mechanism of the secretion of sfGFP-PP.

[0132] The present invention explains the activation process by which the fusion protein sfGFP-PP is activated by exogenous trypsin to become the mature mPG (mature PG) and exerts the deamidation activity of the PG enzyme, and explains the feasibility of sfGFP-PP as a fusion protein to increase the yield of the PG enzyme from the mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0133] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those skilled in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0134] Figure 1 Agarose gel electrophoresis diagram of the DNA fragment of the sfGFP-PP gene obtained by fusion PCR.

[0135] Figure 2 SDS-PAGE diagram (left) of the culture supernatant and PG enzyme activity detection diagram (right) of the control bacterium and the genetically engineered bacterium after 28 h of IPTG induction.

[0136] Figure 3 Diagram of the PG enzyme activity detection results of the soluble supernatant (left) and the culture supernatant (right) after trypsin digestion of the sonicated cells of the genetically engineered bacterium at different times of IPTG induction. Samples of the control bacterium at 12 h and 30 h are used as the control group.

[0137] Figure 4 SDS-PAGE detection of sfGFP-PP expressed by genetically engineered bacteria and activated by trypsin into PG at different times. Specific implementation mode

[0138] Combined with the following specific embodiments and drawings, the invention will be further described in detail. The processes, conditions, experimental methods, etc. for implementing the present invention, except for the specifically mentioned content below, are all common knowledge and well-known common sense in the art, and the present invention has no particularly restricted content.

[0139] The present invention provides a genetically engineered bacterium and a method for constructing the same, which efficiently secretes and expresses protein glutaminase (PG; EC 3.5.1.44) in the engineered bacterium through a green fluorescent protein (superfolder green fluorescent protein, sfGFP), belonging to the field of bioengineering technology. The method is as follows: The obtained sfGFP gene is derived from the NCBI database (GenBank accession number: CP035486.1), and the obtained proenzyme gene PP (Propeptide-Protein glutaminase) of PG is derived from Chryseobacterium proteolyticum (GenBank accession number: AB046594.1). By introducing the recombinant plasmid pHT01 / sfGFP-PP into Bacillus subtilis WB800N, the above-mentioned genetically engineered bacterium is obtained, and the extracellular secretion expression of PP is realized. After activation of sfGFP-PP by trypsin, PG is obtained, greatly improving the expression level and secretion efficiency of PG in Bacillus subtilis. The recombinant PG produced by this method can generate an enzyme activity of up to 23.5 U / mL under the activation of trypsin, and can be used to improve the functional properties of proteins, having strong industrial production and application value.

[0140] The present invention provides a genetically engineered bacterium for efficiently secreting and expressing protein glutaminase through a green fluorescent protein. The genetically engineered bacterium is a recombinant Bacillus subtilis WB800N capable of highly expressing and secreting the sfGFP-PP fusion protein. The genetically engineered bacterium uses the pHT01 plasmid as the expression vector of the sfGFP-PP fusion gene. The extracellular supernatant expressed by it contains the sfGFP-PP fusion protein, and the mature PG with protein glutaminase enzyme activity can be obtained after digestion with trypsin, and its enzyme activity can reach 23.5 U / ml.

[0141] The present invention also provides a method for constructing the above-mentioned genetically engineered bacterium, and the method includes the following steps:

[0142] Step 1: Obtain a green fluorescent protein gene, abbreviated as the sfGFP gene, whose nucleotide sequence is shown in SEQ ID NO.1; obtain the protein glutaminase zymogen gene, abbreviated as the PP gene, whose nucleotide sequence is shown in SEQ ID NO.3;

[0143] Step 2: Fuse the sfGFP gene and the PP gene described in the first step into one DNA and clone it onto the expression vector pHT01 to obtain the pHT01 / sfGFP-PP recombinant expression vector;

[0144] Step 3: Transfer the pHT01 / sfGFP-PP recombinant expression vector described in the second step into the competent cells of Bacillus subtilis WB800N, and screen to obtain the recombinant Bacillus subtilis strain WB800N;

[0145] Subsequent to step 3, isopropyl-β-D-thiogalactoside is used for induction, and the recombinant Bacillus subtilis strain WB800N expresses the sfGFP-PP fusion protein, which is activated by trypsin to obtain the active mature protein glutaminase.

[0146] In the first step, the sfGFP gene sequence is downloaded from the NCBI database and obtained by gene synthesis, and its GenBank accession number is CP035486.1; the PP gene is downloaded from the NCBI database and the prgA gene sequence is obtained by gene synthesis, and its GenBank accession number is AB046594.1, and is obtained by PCR amplification;

[0147] and / or;

[0148] In the second step, using the sfGFP gene and the PP gene as DNA templates, prepare a PCR reaction solution of PrimeSTAR HS DNA polymerase with high fidelity, and fuse the sfGFP gene and the PP gene by fusion PCR; clone the fused sfGFP-PP DNA fragment onto the expression vector pHT01 by DNA end homologous recombination technology to obtain the pHT01 / sfGFP-PP recombinant expression vector;

[0149] and / or;

[0150] In the third step, the pHT01 / sfGFP-PP recombinant expression vector is transferred into the competent Bacillus subtilis WB800N by electrotransformation method, and then positive colonies are screened by chloramphenicol-resistant agar plates to obtain the recombinant Bacillus subtilis WB800N.

[0151] The isopropyl-β-D-thiogalactoside-induced expression of the sfGFP-PP fusion protein is to induce the expression of the recombinant Bacillus subtilis strain in TB medium with IPTG and obtain the fermentation supernatant of the genetically engineered bacterium. Trypsin is added to the fermentation supernatant to activate sfGFP-PP, and the mature PG is obtained and its enzyme activity is detected.

[0152] The composition and content of the reagents for preparing the PCR reaction solution of the PrimeSTAR HS DNA polymerase are as follows: 10 μl of 5x PrimeSTAR Buffer, 4 μl of dNTP, 1 μl of the DNA templates of the sfGFP gene and the PP gene, 1 μl of each of the upstream and downstream primers, 0.5 μl of PrimeSTAR DNA polymerase, and 32.5 μl of dH2O; the DNA end homologous recombination technology is to ligate and circularize the linearized pHT01 vector and the sfGFP-PP DNA fragment obtained by fusion PCR through a DNA recombinase.

[0153] and / or;

[0154] The preparation method of the competent Bacillus subtilis WB800N is as follows: inoculate the wild-type Bacillus subtilis WB800N into LB liquid medium, incubate at a constant temperature, ice-bath, centrifuge, discard the supernatant, and repeatedly rinse the bacterial cells in pre-cooled ETM electroporation buffer for 2-3 times, which is the competent Bacillus subtilis strain WB800N.

[0155] and / or;

[0156] The specific process of electroporation of competent cells is as follows: add the recombinant expression vector pHT01 / sfGFP-PP to the competent Bacillus subtilis WB800N and transfer it to a pre-cooled electroporation cup at 4°C, electroshock once with an electroporator, immediately add RM recovery medium, recover at 37°C for 3 h to 3.5 h, and coat on a chloramphenicol-resistant plate.

[0157] and / or;

[0158] The final concentration of chloramphenicol in the chloramphenicol-resistant plate is 10 μg / ml; the final concentration of IPTG induction is 1 mM, and the induction time is 28 h to 32 h.

[0159] and / or;

[0160] The recombinant Bacillus subtilis strain is Bacillus subtilis WB800N containing the pHT01 / sfGFP-PP vector, which can express the sfGFP-PP fusion gene and produce the sfGFP-PP fusion protein with a relative molecular mass of 60 kDa.

[0161] and / or;

[0162] The fermentation supernatant of the genetically engineered bacterium is the supernatant obtained after removing the bacterial cells by centrifuging at 12,000 rpm for 1 min to 5 min after the genetically engineered bacterium is cultured in TB medium; trypsin is added to the fermentation supernatant to activate sfGFP-PP, promoting sfGFP-PP to be activated into active mature PG, with a relative molecular mass of 19.8 kDa, and the activity of the PG is detected.

[0163] The composition of the ETM electroporation buffer is: 0.5 M sorbitol, 0.5 M mannitol, 10% (V / V) glycerol.

[0164] The composition of the RM recovery medium is: LB medium containing 0.5 M sorbitol and 0.38 M mannitol.

[0165] Among them, the composition of the LB medium is: 10 g / L of tryptone; 5 g / L of yeast extract; 10 g / L of NaCl.

[0166] The ETM electroporation buffer, RM recovery medium and LB medium are all autoclaved at 121 °C for 20 min.

[0167] and / or;

[0168] The composition of the TB medium is: 2 g / L of tryptone; 24 g / L of yeast extract; 4 mL / L of glycerol; 2.2 g / L of KH2PO4: 9.4 g / L of K2HPO4; autoclaved at 115 °C for 20 min.

[0169] The present invention also provides a recombinant expression vector, and the recombinant vector is the recombinant expression vector pHT01 / sfGFP-PP constructed by the above method.

[0170] The present invention also provides a genetically engineered bacterium, and the genetically engineered bacterium is the genetically engineered bacterium prepared by the above method, which is the recombinant Bacillus subtilis strain WB800N containing the pHT01 / sfGFP-PP recombinant expression vector.

[0171] The present invention also provides an expression of a fusion gene of sfGFP-PP, and the expression of the fusion gene refers to the expression of the fusion gene of sfGFP and the PG precursor PP, and the steps are to induce the recombinant Bacillus subtilis WB800N to express the sfGFP-PP fusion protein using isopropyl-β-D-thiogalactoside.

[0172] The present invention also provides a fusion protein sfGFP-PP, and the fusion protein is a fusion protein of sfGFP and the PG precursor PP.

[0173] The present invention also provides the use of the above-mentioned genetically engineered bacterium, the above-mentioned method for constructing a genetically engineered bacterium, the above-mentioned recombinant expression vector, the above-mentioned genetically engineered bacterium, the above-mentioned method for expressing a fusion gene, or the above-mentioned fusion protein sfGFP-PP in the preparation of protein glutaminase.

[0174] In the present invention, the specific detection methods include:

[0175] (1) Detection of PG enzyme activity:

[0176] The determination of PG enzyme activity was referred to the method of Qu et al. with slight modifications (Qu, R., T. Dai, J. Wu, A. Tian, Y. Zhang, L. Kang, W. Ouyang, C. Jin, J. Niu, Z. Li, Z. Chang, D. Jiang, J. Huang and H. Gao, The characteristics of protein-glutaminase from an isolated Chryseobacterium cucumeris strain and its deamidation application. Front Microbiol, 2022.13: p. 969445). After adding 10 μl of the sample to be detected into a 96-well plate, 100 μl of Cbz-Gln-Gly (10.11 g / L) and 100 μl of trichloroacetic acid (TCA, 65.35 g / L) were added to each well of the experimental group and the control group, with 3 replicates in each group, and the reaction was carried out at 37 °C for 30 min. Then, 100 μl of TCA was added to the experimental group to terminate the reaction, and 100 μl of Cbz-Gln-Gly was added to the control group to make up the reaction system. Subsequently, 12 μl of the reaction solution of the above experimental group or control group was taken into a new 96-well plate, and 60 μl of chromogenic solution A (40.46 g / L phenol, 0.15 g / L sodium nitroprusside), 48 μl of distilled water, 30 μl of chromogenic solution B (49.94 g / L potassium hydroxide), and 60 μl of chromogenic solution C (200 g / L potassium carbonate, 8.37 ml / L sodium hypochlorite solution) were added respectively. At the same time, 12 μl of ammonium chloride solutions with different concentrations were added to equal volumes of each chromogenic solution and distilled water. An ammonia standard curve was plotted. The reaction was carried out at 37 °C for 20 minutes, and the absorbance value was measured at OD630. The enzyme activity was calculated according to the following formula:

[0177] PG enzyme activity (U / ml) = (A0 - A1) × 21 / 17.03 / 30 × k

[0178] In the formula, A0: absorbance value of the experimental group; A1: absorbance value of the control group; k: slope of the ammonia standard curve.

[0179] (2) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) experiment:

[0180] Mix 5× protein loading buffer with the sample protein solution to be detected for denaturation, and boil at 100 °C for 10 min. Then separate the protein bands of the sample by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The size of the protein bands of the sample to be detected is based on the protein marker (Protein Marker). The gel after electrophoresis is stained with Coomassie Brilliant Blue R250, and then decolorized multiple times with a decolorizing aqueous solution containing 10% glacial acetic acid and 10% ethanol, and the gel is imaged and analyzed under a gel imager.

[0181] Example 1: Construction of genetically engineered bacteria

[0182] Download the nucleic acid sequence of the sfGFP gene (GenBank accession number: CP035486.1) from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), and perform gene synthesis for later use. Download the nucleic acid sequence of the PP gene, the prgA gene (GenBank accession number: AB046594.1) from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), and perform gene synthesis. Use primers F1(PP), R1(PP) and high-fidelity Prime STAR HS DNA polymerase to perform a PCR reaction to amplify and recover the PP gene. Subsequently, using the sfGFP gene and the PP gene as DNA templates, use primers F1(sfGFP-PP), R1(sfGFP-PP) and high-fidelity Prime STAR HS DNA polymerase to perform a fusion PCR reaction to amplify and recover the sfGFP-PP gene. Further, use the restriction enzymes BamH I and Xba I to double-digest and linearize the circular vector pHT01 and recover it. Add 5 μl of the DNA fragment and 5 μl of the 2× DNA recombinase mixture to the reaction system at a dosage of linearized pHT01 DNA fragment (ng):sfGFP-PP DNA fragment (ng)=156:66, and react at 50 °C for 20 min to construct the pHT01 / sfGFP-PP expression vector and place it on ice for later use. Take the wild-type Bacillus subtilis WB800N cultured in LB medium at 37 °C and 200 rpm for 12 h, transfer it to a new LB medium at a transfer volume of 2% and culture it at 37 °C and 200 rpm until OD600=1; take all the bacterial liquid and ice-bath it for 10 min, then centrifuge at 4000 rpm for 10 min at 4 °C to collect the bacterial cells, wash the bacterial cells with an equal volume of pre-cooled ETM electroporation buffer, centrifuge at 4000 rpm for 10 min at 4 °C to remove the supernatant, and repeat 3 times to prepare competent cells. Add 60 μl of the ice-cold competent cells to a pre-cooled electroporation cup containing the pHT01 / sfGFP-PP recombinant expression vector, mix well and ice-bath for 5 min, and perform one electric shock on the electroporator. The parameters of the electroporator are set as: 2.0 kV, 25 μF, 200 Ω, 2 mm. Add the bacterial liquid after electroporation to the RM recovery medium, recover at 37 °C for 3 h, and spread it on a chloramphenicol-resistant plate. The next day, pick monoclonal colonies on the chloramphenicol-resistant plate (the final concentration of chloramphenicol is 10 μg / ml) and inoculate them into LB medium, culture at 37 °C and 200 rpm for 12 h, extract the vector in the strain for DNA sequencing, and if the sequencing result is correct, it is the correct genetically engineered bacterium.

[0183] The experimental results are as Figure 1 shown.

[0184] In this example, the experimental results show that: the electrophoretic band of the sfGFP-PP gene amplified by fusion PCR is between about 1611 bp, and the position and sequencing are correct.

[0185] Example 2: Inducing high-level expression of sfGFP-PP in engineered bacteria

[0186] The engineered bacteria expressing sfGFP-PP and the control bacteria not expressing sfGFP-PP were respectively streaked on a chloramphenicol-resistant plate for activation and cultured at 37 °C for 12 h. The next day, single colonies of the engineered bacteria and the control bacteria were picked from the plate and inoculated into LB medium containing chloramphenicol resistance, and cultured with shaking at 37 °C and 200 rpm for 12 h; the engineered bacteria and the control bacteria cultured in LB were transferred to TB medium at a transfer volume of 2%, and cultured at 37 °C and 200 rpm for 3 h. At this time, IPTG with a final concentration of 1 mM was added to the engineered bacteria and the control bacteria respectively to induce the expression of sfGFP-PP, and the culture was continued for 28 h. After the culture was completed, the bacterial solutions of the engineered bacteria and the control bacteria were taken, centrifuged at 12000 rpm for 5 min, and trypsin with a final concentration of 1 mg / mL (1:250) was added to the supernatant after centrifugation, and activated at 37 °C for 3 h, and the activated solution was taken for PG enzyme activity detection.

[0187] The experimental results are as Figure 2 shown.

[0188] In this example, the experiment shows that: the activity of the activated PG enzyme can reach 23.5 U / mL (right). The content and enzyme activity of sfGFP-PP expressed by the engineered bacteria are much higher than the highest levels reported in current research, indicating that sfGFP-PP can be highly expressed under the induction of IPTG.

[0189] Example 3: High-level secretion of sfGFP-PP by engineered bacteria

[0190] The engineered bacteria and the control bacteria were induced and cultured by adding ITPG in TB medium in the same method as in Example 1. The difference is that during the culture process, the bacterial solution of the control bacteria at 12 h and 30 h was used as the control group, and the bacterial solutions of the engineered bacteria at 12, 18, 20, 22, 24, 26, 28, and 30 h were taken, centrifuged at 12000 rpm for 5 min, and the supernatant after centrifugation was collected for standby; the obtained bacterial cells were resuspended with PBS buffer with the same volume as the initial bacterial solution, centrifuged at 12000 rpm for 5 min, washed 3 times repeatedly, and then resuspended with the same volume again and broken by an ultrasonic crusher. The broken sample was centrifuged at 12000 rpm for 5 min, and the soluble supernatant of the broken bacterial cells was collected for standby. Trypsin with a final concentration of 1 mg / mL (1:250) was added to the supernatant after centrifugation of the culture and the soluble supernatant of the broken bacterial cells, and activated at 37 °C for 3 h, and the activated solution was taken for PG enzyme activity detection.

[0191] The experimental results are asFigure 3 as shown

[0192] In this example, the experimental results show that: the activity of activated PG enzyme in the soluble supernatant of the bacterial cells gradually decreases from 12 h to 28 h and remains unchanged, and there is almost no enzyme activity in the cells after 28 h (left); on the contrary, the extracellular PG enzyme activity gradually increases from 12 h to 28 h and remains unchanged, and the extracellular enzyme activity reaches the highest after 28 h, about 26 U / mL (right), indicating that the vast majority of sfGFP-PP is gradually secreted from the cells to the extracellular with the increase of the culture time, that is, the ability of the engineered bacteria to efficiently secrete sfGFP-PP.

[0193] Example 4: Activation of sfGFP-PP expressed by engineered bacteria

[0194] The sfGFP-PP in the 28-h culture supernatant of the engineered bacteria was activated with trypsin at a final concentration of 1 mg / mL for different times of 0, 15, 30, 60, 90, and 120 min to verify the effectiveness of activating sfGFP-PP into active PG.

[0195] The experimental results are as Figure 4 shown

[0196] In this example, the experimental results show that: by activating the sfGFP-PP in the culture supernatant of the engineered bacteria with 1 mg / mL trypsin for different times, it can be found that sfGFP-PP is gradually cleaved to produce independent sfGFP and PP, indicating that trypsin cleaves sfGFP-PP, and sfGFP can be effectively activated to produce PG activity.

[0197] The protection scope of the present invention is not limited to the above examples. Without departing from the spirit and scope of the inventive concept, the changes and advantages that can be conceived by those skilled in the art are included in the present invention, and the appended claims are used as the protection scope.

Claims

1. A genetically engineered bacterium that mediates the efficient secretion and expression of PG through sfGFP, characterized in that, The genetically engineered bacterium is recombinant Bacillus subtilis WB800N capable of highly expressing and secreting the sfGFP-PP fusion protein; the genetically engineered bacterium uses the pHT01 plasmid as the expression vector for the sfGFP-PP fusion gene, and the extracellular supernatant expressed by the genetically engineered bacterium contains the sfGFP-PP fusion protein, and mature PG with protein glutaminase enzyme activity can be obtained after digestion with trypsin, and its enzyme activity is 23.5 U / ml.

2. A method for constructing a genetically engineered bacterium for efficiently secreting and expressing protein glutaminase mediated by green fluorescent protein, characterized in that, The method includes the following steps: Step 1: Obtain the coding gene sfGFP gene of superfolder green fluorescent protein and the zymogen gene PP gene of protein glutaminase. Step 2: Use fusion PCR technology to synthesize the fusion expression gene sfGFP-PP gene of the sfGFP gene and the PP gene, and ligate it with the pHT01 plasmid vector DNA fragment after double digestion and linearization to obtain the pHT01 / sfGFP-PP expression vector. Step 3: Transfer the pHT01 / sfGFP-PP expression vector obtained in Step 2 into competent Bacillus subtilis WB800N by electroporation, and screen to obtain the genetically engineered bacterium, that is, recombinant Bacillus subtilis strain WB800N.

3. The construction method according to claim 2, characterized in that, In Step 1, the sfGFP gene is obtained by gene synthesis, and the nucleotide sequence of the sfGFP gene is shown in SEQ ID NO.1; the nucleotide sequence of the PP gene is shown in SEQ ID NO.2, and the PP gene is synthesized by gene synthesis and the required fragment is obtained by PCR amplification; the DNA polymerase used for the PCR amplification is the high-fidelity PrimeSTAR HS DNA polymerase; and / or, In Step 2, the sfGFP-PP gene is a fusion gene of the sfGFP gene and the PP gene, and the nucleotide sequence of the sfGFP-PP gene is shown in SEQ ID NO.5; the DNA polymerase used for the fusion PCR technology is the high-fidelity PrimeSTAR HS DNA polymerase; the pHT01 vector is double-digested with DNA restriction enzymes BamH I and Xba I, and the linearized pHT01 DNA fragment and the sfGFP-PP DNA fragment are ligated and circularized under the catalysis of DNA recombinase by DNA end homologous recombination technology to obtain the pHT01 / sfGFP-PP expression vector; and / or, Step 3 further includes the following sub-steps: Step 3.1: Preparation of electrocompetent Bacillus subtilis: Inoculate wild-type Bacillus subtilis WB800N into LB liquid medium, incubate at a constant temperature, ice-bath, centrifuge, discard the supernatant, and repeatedly wash the bacterial cells in pre-cooled ETM electroporation buffer for 2-3 times to obtain the electrocompetent Bacillus subtilis strain WB800N; Step 3.2: Electroporation of competent cells: Take electrocompetent cells and add them to a pre-cooled electroporation cuvette containing the pHT01 / sfGFP-PP expression vector, mix well and ice-bath, and finally perform one-time electroporation on the electroporator to obtain electrotransformed competent cells; Step 3.3, Resistance screening of genetically engineered bacteria: The bacterial solution containing competent cells after electrotransformation obtained in Step 3.2 is added to RM recovery medium, cultured for recovery, and spread on a chloramphenicol-resistant plate for screening to finally obtain the genetically engineered bacteria.

4. The construction method according to claim 3, characterized in that The composition and content of the PCR reaction solution preparation reagents of the PrimeSTAR HS DNA polymerase are as follows: 10 μl of 5x PrimeSTAR Buffer, 4 μl of dNTP, 1 μl of DNA templates of sfGFP gene and PP gene, 1 μl of each of the upstream and downstream primers, 0.5 μl of PrimeSTAR DNA polymerase, and 32.5 μl of dH2O.

5. The construction method according to claim 3, characterized in that The composition of the ETM electrotransformation buffer is: 0.5 M sorbitol, 0.5 M mannitol, 10% glycerol; The composition of the RM recovery medium is: LB medium containing 0.5 M sorbitol and 0.38 M mannitol; Among them, the composition of the LB liquid medium is: 10 g / L of tryptone; 5 g / L of yeast extract; 10 g / L of NaCl; The ETM electrotransformation buffer, RM recovery medium, and LB medium are all autoclaved at 121 °C for 20 min; and / or The final concentration of chloramphenicol on the chloramphenicol-resistant plate is 10 μg / ml; and / or The recombinant Bacillus subtilis strain is Bacillus subtilis WB800N containing the pHT01 / sfGFP-PP vector, which can express the sfGFP-PP fusion gene and produce the sfGFP-PP fusion protein, and the relative molecular mass of the sfGFP-PP fusion protein is 60 kDa.

6. A recombinant expression vector, characterized in that, The recombinant expression vector is the pHT01 / sfGFP-PP expression vector obtained by ligating and circularizing the linearized pHT01 DNA fragment and sfGFP-PP DNA fragment using the DNA end homologous recombination technique under the catalysis of a DNA recombinase as described in any one of claims 2-5.

7. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria prepared by the method according to any one of claims 2-5 are the recombinant Bacillus subtilis strain WB800N containing the pHT01 / sfGFP-PP recombinant expression vector.

8. A method for expressing a fusion gene of sfGFP-PP, characterized in that, The fusion gene expression refers to the expression of the fusion gene of sfGFP and the PG precursor PP. The fusion expression method uses isopropyl-β-D-thiogalactoside to induce the recombinant Bacillus subtilis WB800N to express the sfGFP-PP fusion protein, which is activated by trypsin to obtain the active mature protein glutaminase.

9. A fusion protein sfGFP-PP, characterized in that, The fusion protein is the fusion protein of sfGFP and the PG precursor PP.

10. Use of the genetically engineered bacteria as described in claim 1, the method for constructing the genetically engineered bacteria as described in any one of claims 2-5, the recombinant expression vector as described in claim 6, the genetically engineered bacteria as described in claim 7, the fusion gene expression as described in claim 8, or the fusion protein sfGFP-PP as described in claim 9 in the preparation of protein glutaminase.

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