Recombinant escherichia coli capable of secreting extracellular bcsz and construction method therefor

Abstract

Provided are a recombinant Escherichia coli with improved ability to secrete extracellular endoglucanase (BcsZ) and a construction method therefor. The recombinant Escherichia coli is constructed by using Escherichia coli JBZ-DH5α as a starting strain and inserting a membrane protein coding gene. The membrane protein gene is one of the following coding genes: a coding gene of zinc transporter permease znuB, a coding gene of DUF1449 family inner membrane protein ylaC, and a coding gene of UPF0410 family inner membrane protein ymgE.

Description

Escherichia coli recombinant bacteria capable of secreting extracellular BcsZ and construction method thereof

[0001] The present application claims priority to the Chinese patent application No. CN202410854331.5, filed on June 28, 2024, and entitled "Escherichia coli recombinant bacteria capable of secreting extracellular BcsZ and construction method thereof", the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application relates to the field of bioengineering technology, in particular to Escherichia coli recombinant bacteria capable of secreting extracellular BcsZ and construction method thereof. BACKGROUND

[0003] Endoglucanase (EC 3.2.1.4, BcsZ) is a major component of cellulase, which can hydrolyze soluble cellulose into reducing oligosaccharides. Due to the wide existence of cellulose, lichenin and cereal beta-d-glucan, the endoglucanase has many applications in industrial production, and it also has a place in the global enzyme market. Therefore, the production of cellulase has been paid more and more attention. Escherichia coli is usually selected as an engineering strain in production due to its rapid growth, direct nutritional requirements, perfect fermentation process, easy amplification and simple genetic background, and is often used for large-scale fermentation, for example, Escherichia coli is often used as a cell factory to produce chemicals such as 1,3-propanediol. At present, Escherichia coli is also commonly used for fermentation to produce endoglucanase.

[0004] However, due to the double-membrane structure (inner membrane and outer membrane) of Escherichia coli, its protein secretion ability is poor. Therefore, under normal circumstances, Escherichia coli cannot secrete cellulase outside the cell, which greatly limits the application of Escherichia coli in cellulase production. SUMMARY

[0005] The purpose of the present application is to overcome the shortcomings of the prior art, and to provide Escherichia coli recombinant bacteria capable of secreting extracellular BcsZ and construction method thereof. The present application improves the ability of the obtained Escherichia coli recombinant bacteria to secrete BcsZ by inserting a target coding gene into the target Escherichia coli, which can expand the application of Escherichia coli as an engineering bacterium.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0007] An Escherichia coli capable of secreting extracellular BcsZ, named JBZ-DH5α, was deposited with the Guangdong Microbial Culture Collection Center on May 30, 2024, and the deposit number is GDMCC NO: 64706, and the deposit address is No. 59 Building, 5th Floor, 100, Martyrs' Road, Guangzhou.

[0008] This application also provides a recombinant strain of Escherichia coli that can secrete extracellular BcsZ, using the aforementioned Escherichia coli JBZ-DH5α as the starting strain, into which a membrane protein gene is inserted.

[0009] Furthermore, the membrane protein gene is one of the following encoding genes: the encoding gene of zinc transporter permease znuB, the encoding gene of DUF1449 family inner membrane protein ylaC, and the encoding gene of UPF0410 family inner membrane protein ymgE.

[0010] Furthermore, the gene encoding the zinc transporter permease znuB is shown in SEQ ID NO:1, and its amino acid sequence is shown in SEQ ID NO:2;

[0011] The gene encoding the DUF1449 family inner membrane protein ylaC is shown in SEQ ID NO:3, and its amino acid sequence is shown in SEQ ID NO:4;

[0012] The gene encoding the UPF0410 family intima protein ymgE is shown in SEQ ID NO:5, and its amino acid sequence is shown in SEQ ID NO:6.

[0013] The method for constructing recombinant Escherichia coli that can secrete extracellular BcsZ as described above includes: inserting the membrane protein gene between the BamHI and HindIII restriction sites of the pTrc99A plasmid to obtain a recombinant plasmid; after sequencing verification of the recombinant plasmid, introducing it into competent Escherichia coli JBZ-DH5α cells by electroporation to obtain the recombinant bacteria.

[0014] Furthermore, when the membrane protein gene is the encoding gene for zinc transporter permease znuB, the recombinant bacterium is named Escherichia coli OE-znuB, with the accession number GDMCC NO: 64717.

[0015] When the membrane protein gene is the encoding gene of the DUF1449 family inner membrane protein ylaC, the recombinant bacterium is named Escherichia coli OE-ylaC, and the preservation number is GDMCC NO: 64718.

[0016] When the membrane protein gene is the encoding gene of the UPF0410 family inner membrane protein ymgE, the recombinant bacterium is named Escherichia coli OE-ymgE, with accession number GDMCC NO: 64716 and accession address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] The present application first provides an Escherichia coli JBZ-DH5a capable of secreting extracellular BcsZ, but the ability of the Escherichia coli to secrete extracellular BcsZ is limited, then the Escherichia coli is used as a starting strain for modification, and a recombinant Escherichia coli with improved ability to secrete extracellular BcsZ is obtained, which can expand the application of Escherichia coli as an engineering strain in production, and also provides a reference for studying the mechanism of Escherichia coli secreting cellulase.

[0019] Biological preservation information

[0020] The Escherichia coli JBZ-DH5, which is classified as Escherichia coli, was preserved in the Guangdong Microbial Culture Collection Center on May 30, 2024, and the address is: No. 59, Building 5, 100, Martyrs' Road, Guangzhou, and the preservation number is: GDMCC NO: 64706;

[0021] The Escherichia coli OE-znuB, which is classified as Escherichia coli, was preserved in the Guangdong Microbial Culture Collection Center on June 3, 2024, and the address is: No. 59, Building 5, 100, Martyrs' Road, Guangzhou, and the preservation number is: GDMCC NO: 64717;

[0022] The Escherichia coli OE-ylaC, which is classified as Escherichia coli, was preserved in the Guangdong Microbial Culture Collection Center on June 3, 2024, and the address is: No. 59, Building 5, 100, Martyrs' Road, Guangzhou, and the preservation number is: GDMCC NO: 64718;

[0023] The Escherichia coli OE-ymgE, which is classified as Escherichia coli, was preserved in the Guangdong Microbial Culture Collection Center on June 3, 2024, and the address is: No. 59, Building 5, 100, Martyrs' Road, Guangzhou, and the preservation number is: GDMCC NO: 64716. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below.

[0025] FIG. 1 is a growth curve diagram of each recombinant strain in the present application;

[0026] FIG. 2 is a BSA standard curve in the present application;

[0027] FIG. 3 is a BcsZ protein concentration bar chart of each recombinant strain in the present application;

[0028] Figure 4 is a bar chart of the relative expression of genes in the recombinant bacteria of the present application. DETAILED DESCRIPTION

[0029] The present application is further illustrated by the following description and examples, and the modes thereof include but are not limited to the following examples.

[0030] The present application provides an Escherichia coli JBZ-DH5a strain capable of secreting extracellular BcsZ, named Escherichia coli JBZ-DH5a, and deposited with the Guangdong Microbial Culture Collection Center on May 30, 2024, with the accession number GDMCC NO: 64706 and the address of the depositary being No. 59 Building, 5th Floor, 100 Middle Liangzhan Road, Guangzhou.

[0031] Compared with the Escherichia coli strains screened from the rumens of cattle and sheep in Inner Mongolia, the BcsZ secretion ability of the Escherichia coli JBZ-DH5a of the present application is not as good as that of the rumen screening strains. Therefore, the inventors analyzed the transcriptome data of the rumen Escherichia coli strains ZH-4, N15-1, N16-2, Y4-2 and Y15-3, found three membrane protein coding genes related to BcsZ secretion, and carried out metabolic engineering modification on these key genes to obtain a metabolic engineering modified strain capable of improving the secretion amount of BcsZ.

[0032] As an embodiment, the membrane protein of the present application includes zinc transporter permease znuB, DUF1449 family inner membrane protein ylaC or UPF0410 family inner membrane protein ymgE.

[0033] As an embodiment, the coding gene of the zinc transporter permease znuB of the present application is shown in SEQ ID NO: 1, and the amino acid sequence thereof is shown in SEQ ID NO: 2. As an embodiment, the present application inserts the coding gene shown in SEQ ID NO: 1 between the BamHI and HindIII enzyme cutting sites of the pTrc99A plasmid (with ampicillin resistance) to obtain a recombinant plasmid pTrc99A-znuB. After sequencing verification of the recombinant plasmid pTrc99A-znuB, the recombinant plasmid pTrc99A-znuB is introduced into the competent cells of the Escherichia coli JBZ-DH5a by electroporation, and a recombinant strain is obtained, denoted as OE-znuB.

[0034] As an implementation form, the coding gene of the DUF1449 family inner membrane protein ylaC is shown as SEQ ID NO: 3, and the amino acid sequence is shown as SEQ ID NO: 4. As an implementation form, the coding gene shown as SEQ ID NO: 3 is inserted into the BamHI and HindIII enzyme cutting sites of the pTrc99A plasmid (with ampicillin resistance) to obtain a recombinant plasmid pTrc99A- ylaC. After the recombinant plasmid pTrc99A-ylaC is verified by sequencing, the recombinant plasmid pTrc99A-ylaC is introduced into the competent cells of Escherichia coli JBZ-DH5α by electroporation, and the recombinant bacteria are obtained, which are recorded as OE-ylaC.

[0035] As an implementation form, the coding gene of the UPF0410 family inner membrane protein ymgE is shown as SEQ ID NO: 5, and the amino acid sequence is shown as SEQ ID NO: 6. As an implementation form, the coding gene shown as SEQ ID NO: 5 is inserted into the BamHI and HindIII enzyme cutting sites of the pTrc99A plasmid (with ampicillin resistance) to obtain a recombinant plasmid pTrc99A-ymgE. After the recombinant plasmid pTrc99A-ymgE is verified by sequencing, the recombinant plasmid pTrc99A-ymgE is introduced into the competent cells of Escherichia coli JBZ-DH5α by electroporation, and the recombinant bacteria are obtained, which are recorded as OE-ymgE.

[0036] SEQ ID NO: 1 (znuB gene): 5'-ATGATTGAATTATTATTTCCCGGTTGGTTAGCCGGGATCATGCTCGCCTGTGCCGCGGGTCCGCTGGGTTCGTTTGTAGTCTGGCGTCGTATGTCTTATTTCGGTGATACGCTGGCTCATGCCTCATTACTTGGCGTCGCGTTTGGTTTGTTGCTGGACGTGAATCCATTCTATGCGGTGATTGCCGTTACGCTGCTGCTGGCGGGCGGTCTGGTATGGCTGGAGAAGCGTCCACAGCTGGCGATCGACACGTTATTAGGGATTATGGCGCACAGTGCCCTGTCGCTGGGCCTGGTGGTCGTGAGTCTGATGTCTAATATTCGTGTTGATTTGATGGCTTACCTGTTCGGTGATTTGCTGGCAGTGACGCCAGAAGATCTCATCTCTATTGCGATTGGCGTGGTCATCGTGGTGGCTATTTTGTTCTGGCAATGGCGCAATTTGCTGTCAATGACGATAAGCCCGGATCTGGCGTTTGTTGATGGTGTGAAATTACAGCGAGTGAAATTGTTGTTGATGCTGGTGACGGCATTGACGATTGGTGTAGCGATGAAATTTGTCGGTGCGTTGATTATTACTTCGTTGCTGATTATTCCTGCTGCTACTGCGCGTCGCTTTGCCCGCACGCCGGAACAGATGGCTGGTGTCGCTGTTTTGGTGGGGATGGTGGCAGTGACTGGCGGTTTAACCTTTTCCGCGGTTTACGATACGCCGGCGGGTCCGTCGGTGGTGCTATGTGCGGCACTGTTATTTATTCTCAGTATGATGAAAAAGCAGGCCAGCTAA-3';

[0037] SEQ ID NO: 2 (znuB protein): MIELLFPGWLAGIMLACAAGPLGSFVVWRRMSYFGDTLAHASLLGVAFGLLLDVNPFYAVIAVTLLLAGGLVWLEKRPQLAIDTLLGIMAHSALSLGLVVVSLMSNIRVDLMAYLFGDLLAVTPEDLISIAIGVVIVVAILFWQWRNLLSMTISPDLAFVDGVKLQRVKLLLMLVTALTIGVAMKFVGALIITSLLIIPAATARRFARTPEQMAGVAVLVGMVAVTGGLTFSAVYDTPAGPSVVLCAALLFILSMMKKQAS*;

[0038] SEQ ID NO: 3 (ylaC gene): 5'-ATGACCGAAATACAACGCCTGCTGACCGAAACGATTGAGTCTCTGAATACCCGCGAAAAACGCGACAACAAACCCCGCTTTAGTATCAGTTTTATCCGTAAACATCCGGGGCTGTTTATCGGTATGTACGTTGCTTTTTTTGCCACCCTGGCGGTGATGTTGCAGTCCGAAACGCTGTCAGGCTCTGTCTGGCTACTGGTTGTATTATTTATCCTGCTTAATGGTTTCTTCTTTTTCGATGTCTACCCACGCTACCGCTATGAAGATATCGACGTGCTGGATTTCCGCGTTTGCTATAACGGCGAATGGTACAACACGCGCTTTGTACCTGCCGCGCTGGTTGAAGCCATCTTGAACTCTCCGCGTGTCGCGGATGTTCATAAGGAACAACTGCAAAAAATGATCGTCCGTAAAGGTGAACTGTCTTTTTACGATATTTTTACCCTCGCTCGCGCCGAATCAACATCTTAA-3';

[0039] SEQ ID NO: 4 (ylaC protein): MTEIQRLLTETIESLNTREKRDNKPRFSISFIRKHPGLFIGMYVAFFATLAVMLQSETLSGSVWLLVVLFILLNGFFFFDVYPRYRYEDIDVLDFRVCYNGEWYNTRFVPAALVEAILNSPRVADVHKEQLQKMIVRKGELSFYDIFTLARAESTS*;

[0040] SEQ ID NO: 5 (ymgE gene): 5'-ATGGGAATTATTGCCTGGATTATTTTTGACCTGATAGCCGGCATTATCGCCAAGCTAATCATGCCGGGGCGTGATGGTGGTGGATTTTTCCTGACCTGTATTCTCGGGATAGTCGGTGCGGTGGTCGGCGGCTGGCTGGCGACCATGTTTGGCATTGGCGGCTCCATCAGTGGTTTTAATCTGCACAGCTTCCTGGTGGCGGTGGTGGGAGCTATTCTCGTTCTGGGCATATTCCGCCTCCTGCGAAGAGAATAA-3';

[0041] SEQ ID NO: 6 (ymgE protein): MGIIAWIIFDLIAGIIAKLIMPGRDGGGFFLTCILGIVGAVVGGWLATMFGIGGSISGFNLHSFLVAVVGAILVLGIFRLLRRE*.

[0042] The ability of each of the obtained recombinant bacteria to secrete endoglucanase was also determined, including:

[0043] 1. Fermenting each of the recombinant bacteria

[0044] Each of the obtained recombinant bacteria was activated in a resistant medium plate, and then inoculated into a seed culture medium, and cultured at 37°C for 12-16 h under 180 rpm shaking to obtain a seed liquid. Each of the seed liquids was inoculated into an induction culture medium, and cultured at 37°C for 12-16 h under 180 rpm shaking to obtain an induced bacteria liquid.

[0045] The solvent of the seed culture medium was water, and the solute was: tryptone 10 g / L, sodium chloride 10 g / L, yeast extract powder 5 g / L; the pH value was 7.0.

[0046] The solvent of the induction medium is water, and the solute is: 10 g / L of trypsin peptone, 10 g / L of sodium chloride, 5 g / L of yeast extract powder, and 5 g / L of xylan; the pH value is 7.0.

[0047] The resistant medium plate: adding ampicillin (final concentration of 100 μg / mL) and 20 g / L of agar powder in the seed medium.

[0048] 2. Determining the growth curve of each recombinant strain

[0049] The recombinant strains OE-znuB, OE-ylaC and OE-ymgE were respectively cultured to the logarithmic phase (OD value was about 0.8), and the logarithmic phase bacterial liquid was respectively taken, inoculated into fresh 100 mL LB liquid medium containing ampicillin resistance (i.e. adding ampicillin in the seed medium, and making the final concentration of ampicillin 100 μg / mL) at an inoculation ratio of 2%, and samples were taken every six hours, three biological repeats were performed for each sample, and 0.5 mM IPTG was added to the bacterial liquid after the sixth hour sampling to induce the expression of the target gene, and the growth curve of each recombinant strain was drawn.

[0050] The growth curve results are shown in FIG. 1, in which OE-empty is the JBZ-DH5a E. coli recombinant strain without plasmid recombination. Due to three biological repeat experiments, the average value ± error bar of three data was used to draw the growth curve graph when the data was processed. As can be seen from FIG. 1, compared with OE-empty, the overexpression of znuB, ylaC and ymgE genes has no obvious effect on the growth of the recombinant strain.

[0051] 3. Determination of BcsZ concentration

[0052] S1: Preparation of protein sample

[0053] The strain was cultured to the logarithmic phase, inoculated into 100 mL fresh induction medium (water as solvent, solute: 10 g / L of trypsin peptone, 10 g / L of sodium chloride, 5 g / L of yeast extract powder, and 5 g / L of xylan; pH value is 7.0) at an inoculation amount of 2%, and then 100 μL of ampicillin solution was added, and fermentation culture was carried out at 37°C and 180 rpm. When the OD reached about 0.6, 0.5 mM IPTG solution was added to each medium, and then 16°C overnight induction was carried out for 12 h. The overnight induced bacterial liquid was collected, centrifuged at 4°C and 5000 rpm for 15 min to obtain the fermentation liquid supernatant, which was denatured at 95°C for 10 min. Then SDS-PAGE gel electrophoresis was carried out, and the target band was recovered using a protein gel recovery kit for standby use.

[0054] S2: Drawing bovine serum albumin (BSA) standard curve

[0055] Take appropriate amount of BSA mother liquor (purchased, concentration of 0.22 mg / mL), with phosphate buffer saline solution respectively prepared into concentration of 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL and 50 μg / mL BSA solution, total volume 50 μL, phosphate buffer saline solution as blank control. 200 μL of coomassie brilliant blue solution is added to the above five different concentrations of BSA system, fully mixed, room temperature for 5 min, then the absorbance value at 595 nm is measured, the standard curve is drawn, and the results are shown in Figure 2.

[0056] S3: Determination of BcsZ protein concentration

[0057] Take 50 μL of the sample in step S1, then add 200 μL of coomassie brilliant blue solution, fully mix, room temperature for 5 min, measure the absorbance value at 595 nm, calculate the protein concentration according to the BSA standard curve, detect the relative expression amount of protein coding gene, and the protein results are shown in Figures 3 and 4. As can be seen from Figures 3 and 4, compared with the control group OE-empty, the overexpression of OE-znuB, OE-ylaC and OE-ymgE genes can significantly increase the concentration of BcsZ of the strain, and the screened recombinant bacteria OE-znuB, OE-ylaC and OE-ymgE can significantly improve the secretion amount of BcsZ.

[0058] The above examples are only one of the preferred embodiments of the present application, and should not be used to limit the protection scope of the present application, but any modification or polishing without substantial meaning made in the main design idea and spirit of the present application, the technical problems solved are still consistent with the present application, and should be included in the protection scope of the present application.

Claims

1. An Escherichia coli JBZ-DH5α strain that secretes extracellular BcsZ, with accession number GDMCC NO: 64706.

2. A recombinant *Escherichia coli* capable of secreting extracellular BcsZ, characterized in that, It includes the Escherichia coli JBZ-DH5α as described in claim 1 and the membrane protein encoding gene inserted into the Escherichia coli JBZ-DH5α.

3. The recombinant Escherichia coli according to claim 2, characterized in that, The membrane protein encoding gene is one of the following encoding genes: the encoding gene of zinc transporter permease znuB, the encoding gene of DUF1449 family inner membrane protein ylaC, and the encoding gene of UPF0410 family inner membrane protein ymgE.

4. The recombinant Escherichia coli according to claim 3, characterized in that, The amino acid sequence of the zinc transporter permease znuB is shown in SEQ ID NO:2; The amino acid sequence of the DUF1449 family inner membrane protein ylaC is shown in SEQ ID NO:4; The amino acid sequence of the UPF0410 family inner membrane protein ymgE is shown in SEQ ID NO:

6.

5. The recombinant Escherichia coli according to claim 4, characterized in that, The nucleotide sequence of the gene encoding the zinc transporter permease znuB is shown in SEQ ID NO:1; The nucleotide sequence of the gene encoding the DUF1449 family intima protein ylaC is shown in SEQ ID NO:3; The nucleotide sequence of the gene encoding the UPF0410 family intima protein ymgE is shown in SEQ ID NO:

5.

6. The recombinant Escherichia coli according to any one of claims 2 to 5, characterized in that, When the membrane protein is zinc transporter permease znuB, the recombinant Escherichia coli is Escherichia coli OE-znuB, with accession number GDMCC NO: 64717; When the membrane protein is ylaC, an inner membrane protein of the DUF1449 family, the recombinant Escherichia coli is Escherichia coli OE-ylaC, with accession number GDMCC NO: 64718. When the membrane protein is UPF0410 family inner membrane protein ymgE, the recombinant Escherichia coli is Escherichia coli OE-ymgE, with accession number GDMCC NO: 64716.

7. The method for constructing recombinant Escherichia coli according to any one of claims 2 to 6, characterized in that, The procedure includes the following steps: inserting the gene encoding the membrane protein between the BamHI and HindIII restriction sites of the pTrc99A plasmid to obtain a recombinant plasmid; after verifying the recombinant plasmid by sequencing, introducing it into competent Escherichia coli JBZ-DH5α cells by electroporation to obtain the recombinant Escherichia coli.

8. The use of membrane proteins in promoting the secretion of extracellular BcsZ by *Escherichia coli* JBZ-DH5α according to claim 1, characterized in that, The membrane protein is one of the following membrane proteins: zinc transporter permease znuB, DUF1449 family inner membrane protein ylaC, and UPF0410 family inner membrane protein ymgE. The amino acid sequence of the zinc transporter permease znuB is shown in SEQ ID NO:2; The amino acid sequence of the DUF1449 family inner membrane protein ylaC is shown in SEQ ID NO:4; The amino acid sequence of the UPF0410 family inner membrane protein ymgE is shown in SEQ ID NO:

6.

9. The application according to claim 8, wherein the nucleotide sequence of the gene encoding the zinc transporter permease znuB is shown in SEQ ID NO:1; The nucleotide sequence of the gene encoding the DUF1449 family intima protein ylaC is shown in SEQ ID NO:3; The nucleotide sequence of the gene encoding the UPF0410 family intima protein ymgE is shown in SEQ ID NO:

5.

10. A method for preparing BcsZ, characterized in that, Includes the following steps: The *Escherichia coli* JBZ-DH5α of claim 1 or the recombinant *Escherichia coli* of any one of claims 2 to 6 was cultured to the logarithmic growth phase, and then inoculated into an induction medium containing ampicillin resistance at a 2% inoculum size, and fermented at 37°C and 180 rpm; when the OD of the fermentation broth... 600nm Once the pH value reaches 0.6, 0.5 mM IPTG is added to the fermentation broth, and the mixture is induced at 16°C for 12 h to obtain the induced bacterial culture. The induced bacterial culture is centrifuged at 4°C and 5000 rpm for 15 min to obtain the fermentation broth supernatant. The fermentation broth supernatant contains BcsZ. The induction medium comprises 10 g / L tryptone, 10 g / L sodium chloride, 5 g / L yeast extract and 5 g / L xylan; pH is 7.0.

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