Engineering strain for efficiently expressing recombinant human pepsinogen rhPGA5 as well as construction method and application of engineering strain

Abstract

The invention belongs to the technical field of gene engineering and enzyme engineering, and particularly relates to an engineering strain for efficiently expressing recombinant human pepsinogen rhPGA5 as well as a construction method and application of the engineering strain. The engineering strain is constructed by replacing genes to be knocked out in aspergillus niger with an hPGA5 expression cassette, while the genes to be knocked out are knocked out, the copy number of the hPGA5 gene is increased, and the constructed engineering strain can produce recombinant human pepsinogen rhPGA5 at high yield, can be used for industrial production of human pepsin, and has a good application prospect. The problems that in the prior art, the pepsinogen extraction process is complex, the source is limited, and the heterologous production yield is low are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering and enzyme engineering, and specifically relates to an engineering strain for efficiently expressing recombinant human pepsinogen rhPGA5, and a construction method and application thereof. Background Art

[0002] Human pepsin (hunan pepsin) is one of the main digestive enzymes in the human body and belongs to aspartic acid protease. They are expressed in the human body as inactive pepsinogen (PGA), which is the precursor of pepsin. Among them, human pepsinogen 5 (hPGA5) is the most important pepsinogen in the human body. Pepsinogen is activated into pepsin with digestive activity under acidic conditions. Pepsin is widely used and plays an important role in many fields. In medicine, firstly, pepsin can treat indigestion caused by excessive protein intake. Secondly, serum pepsinogen level is of great value in the diagnosis of gastric diseases. On the other hand, the presence of pepsin in saliva can be used as a basis for the diagnosis of reflux esophagitis. In the food industry, pepsin can be used in alcohol brewing, and can also be used as a clarifier and additive for juice and beer. In leather making technology, the application of pepsin to fur before making can make the leather softer and more comfortable. At present, the pepsin on the market is mainly extracted from the stomach tissues of mammals such as pigs, cattle, and sheep. Not only is the extraction process relatively complicated and the extraction cost relatively high, but its source is also obviously limited. Therefore, the use of genetic engineering to recombinantly express human pepsin has become the main method to solve the limited source and complex extraction process, but the current yield is low. For example, in 2006, Malik et al. expressed recombinant human pepsinogen A in Escherichia coli, but the expression level was only 100 μg / L (A novel fusion protein system for the production of native human pepsinogen in the bacterial periplasm. Protein Expression and Purification, 47 (2)); in 2017, Chen Dan et al. expressed human pepsinogen I in Hansenula, with an expression level of more than 100 mg / L (Secretionary expression, purification and application of recombinant human pepsinogen I in Hansenula. Chinese Journal of Biological Products; 30 (2)); in the same year, Guo Hui et al. expressed four porcine pepsinogen mutants in Pichia pastoris, with the highest expression level being 22.6 U / mL (Construction of a novel eukaryotic expression system for porcine pepsinogen mutants. Biotechnology; 27 (3)). Summary of the invention

[0003] In view of the above problems, the present invention provides an engineering strain for efficiently expressing recombinant human pepsinogen rhPGA5, and a construction method and application thereof. The present invention increases the copy number of the hPGA5 gene by replacing a specific gene to be knocked out in Aspergillus niger with an hPGA5 expression cassette. The obtained engineering strain can efficiently express recombinant human pepsinogen rhPGA5, and can be used for industrial production of human pepsin and obtain high yield.

[0004] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical scheme: The first aspect of the present invention provides an engineered strain for efficiently expressing recombinant human pepsinogen rhPGA5: the engineered strain is constructed by replacing the gene to be knocked out in Aspergillus niger with an hPGA5 expression cassette; the gene to be knocked out includes an α-glucosidase gene; the hPGA5 expression cassette consists of a filamentous fungal promoter, a signal peptide gene, an hPGA5 gene and a terminator; the hPAG5 gene encodes the amino acid sequence shown in SEQ ID NO.1.

[0005] Aspergillus niger is an important cell factory in industrial fermentation and has many advantages. First, Aspergillus niger is a strain that has been defined as Generally Recognized As Safe (GRAS) by the Food and Drug Administration (FDA) of the United States and has been widely used in the food industry. Secondly, Aspergillus niger also has strong protein secretion ability, the presence of post-translational modifications (protein glycosylation, proteolytic cleavage and formation of multiple disulfide bonds), and the ability to grow on a relatively simple and inexpensive matrix. The present invention uses Aspergillus niger as a starting strain, integrates the human pepsinogen gene hPGA5 expression cassette constructed by a filamentous fungus promoter and a signal peptide into a specific site in the Aspergillus niger genome, and while knocking out these genes to be knocked out, increases the copy number of the hPGA5 gene, and constructs an engineered strain containing different hPGA copy numbers and capable of high-yield rhPGA5. The engineered strain solves the problems of complex pepsinogen extraction process, limited sources, and low heterologous production yield in the prior art.

[0006] Preferably, the nucleotide sequence of the hPAG5 gene is as shown in SEQ ID NO.2.

[0007] Preferably, the signal peptide is selected from the signal peptide of a saccharifying enzyme gene, the signal peptide of an amylase gene, the signal peptide of a cellulase gene or the signal peptide of a cellobiohydrolase CHB gene. All of the above signal peptides have strong secretion ability.

[0008] Further preferably, the signal peptide gene is a signal peptide glaASP gene encoding an amino acid sequence as shown in SEQ ID NO.3.

[0009] More preferably, the nucleotide sequence of the signal peptide glaASP gene is shown as SEQ ID NO.4.

[0010] Preferably, the α-glucosidase gene includes the gene agdA (GenBank: EHA20839.1)、Gene agdC (GenBank: EHA23512.1)、Gene agdD (NCBI Reference Sequence: XM_059743974.1), gene yes (GenBank: EHA28539.1)、Gene agdF (GenBank: EHA19157.1) and gene agdG (GenBank: EHA23468.1).

[0011] Preferably, the gene to be knocked out also includes an amylase gene.

[0012] Further preferably, the amylase gene is a gene Amy (NCBI_Locus_tag:An05g02100).

[0013] Preferably, the genes to be knocked out also include genes related to the secretory pathway.

[0014] Further preferably, the secretory pathway-related gene is an ERAD pathway-related gene.

[0015] More preferably, the ERAD pathway-related genes include genes skin (NCBI_Locus_tag: An15g00640) and genes mnsA (NCBI_Locus_tag: An18g06220).

[0016] Preferably, the nucleotide sequence of the filamentous fungus promoter is as shown in SEQ ID NO.5 or SEQ ID NO.6, both of which have high activity and have substantially the same effect in the present invention.

[0017] Preferably, the terminator is terminator TTglucan693.

[0018] Preferably, the Aspergillus niger is Aspergillus niger An41683. Aspergillus niger An41683 is an industrial production strain with high saccharifying enzyme production.

[0019] The second aspect of the present invention provides a method for constructing the above-mentioned engineered strain, which specifically comprises the following steps: S1. Constructing a donor DNA fragment containing the hPGA5 expression cassette and a sgRNA knockout plasmid containing the corresponding gene to be knocked out according to CRISPR-Cas9 technology; S2. Co-transform the donor DNA fragment and the sgRNA knockout plasmid into Aspergillus niger protoplasts, extract the transformant genomic DNA, and identify by PCR to obtain the engineered strain expressing rhPGA5.

[0020] The construction method can be used to construct an engineering strain in which the gene to be knocked out is knocked out and a corresponding amount of hPGA5 is integrated.

[0021] Preferably, the method of transforming the donor DNA fragment and the sgRNA knockout plasmid into Aspergillus niger protoplasts in S2 is a protoplast transformation method.

[0022] Preferably, the construction method of the sgRNA knockout plasmid in S1 is: Synthesize two single-target sgRNA primer pairs of the gene to be knocked out, anneal them separately and Bbs I restriction enzyme digestion plasmid pUC57-sgRNA was connected to construct sgRNA expression cassette plasmids pUC57-target1-sgRNA and pUC57-target2-sgRNA. Plasmid pUC57-target2-sgRNA was used as a template to PCR amplify the target2-sgRNA expression cassette fragment. Xho Ⅰ / BamH Ⅰ After double restriction digestion, cloned into plasmid pUC57-target1-sgRNA Xho Ⅰ / Bgl II sites, construct the sgRNA expression cassette plasmid pUC57-target1-sgRNA-target2-sgRNA containing two targets; The plasmid pUC57-target1-sgRNA-target2-sgRNA was used Bgl Ⅱ / Nhe Ⅰ double digestion to obtain the tandem target1-sgRNA-target2-sgRNA expression cassette fragment, and use Bgl Ⅱ / No Ⅰ Double-enzyme digestion of vector pFC332, ligation of the double-enzyme digested vector and fragment, and then transformation into competent E. coli, screening of transformants, and extraction of plasmids from the verified correct transformants to obtain the sgRNA knockout plasmid.

[0023] Further preferably, the reaction system of the enzyme digestion is:

[0024] The reaction conditions of the enzyme digestion are: 37°C for 3h and 65°C for inactivation.

[0025] The reaction system of the connection is:

[0026] The ligation reaction conditions were: ligation at 37°C for 30 min.

[0027] Preferably, the preparation method of the donor DNA fragment in S1 is: The fragment containing the promoter and signal peptide was amplified by PCR using the vector pMD19-T-Pglucan703-glaA as a template; Bgl II / BsrG I double-digest the vector pUC57 and the fragment, connect the digested vector and the fragment to construct a plasmid containing a signal peptide; The terminator fragment was obtained by PCR amplification using the vector pMD19-T-TTglucan693 as a template. No I / BamH I double-digest the plasmid containing the signal peptide and the terminator fragment, connect the digested plasmid and the fragment to construct a plasmid containing the signal peptide and the terminator; use BsrG I / No I. Double-digest the plasmid containing the signal peptide and terminator and the hPGA5 gene, connect the vector and fragments after enzyme digestion, and construct a plasmid containing the signal peptide, terminator and hPGA5 gene; The upstream homology arm and the downstream homology arm of the knockout site corresponding to the gene to be knocked out were amplified by PCR using the Aspergillus niger genome as a template. Xho I / Bgl II double enzyme digestion of the upstream homology arm and the plasmid containing the signal peptide, terminator and hPGA5 gene, ligation, transformation, screening of transformants, and then use BamH I / Indian III. Continue to digest the obtained plasmid and the downstream homology arm, connect again, transform, screen transformants, and obtain the donor DNA fragment plasmid containing the hPGA5 expression cassette; use Indian III / Xho I double-digests the donor DNA fragment plasmid containing the hPGA5 expression cassette to obtain the donor DNA fragment.

[0028] Further preferably, the PCR reaction system is:

[0029] The PCR reaction conditions were as follows: denaturation at 98°C for 2 min; then denaturation at 98°C for 30 sec, annealing at 58°C for 30 sec, and extension at 72°C for 30 sec for 35 cycles; and then extension at 72°C for 2 min.

[0030] The third aspect of the present invention provides the use of the above-mentioned engineered strain in the fermentation production of pepsin.

[0031] The fourth aspect of the present invention provides a method for producing pepsin by fermentation, wherein the above-mentioned engineered strain is used to produce pepsin by fermentation.

[0032] Preferably, after the engineered strain is cultured in a liquid culture medium until uniform small balls are grown, the mycelium is broken up to make the mycelium evenly distributed, and then inoculated into a YPD culture medium and cultured until the bacteria grow into small balls; the small ball-shaped bacteria are inoculated into a fermentation medium to ferment and produce pepsin.

[0033] Further preferably, the fermentation medium is a malt wort fermentation medium. The malt wort fermentation medium can directly adopt a commercially available finished malt wort medium commonly used in the art.

[0034] The beneficial effect of the present invention is that the present invention constructs a high-yield rhPGA recombinant strain of Aspergillus niger containing different copy numbers of hPGA5 by integrating the hPGA5 expression cassette into the specific gene site of Aspergillus niger. The enzyme activity of the fermentation broth far exceeds the existing technology and the starting strain, laying the foundation for the industrial production of human pepsin and having important commercial application value. The present invention uses Aspergillus niger An41683 as the starting strain, uses a highly active promoter and a signal peptide with strong secretion ability to construct an expression cassette of the human pepsinogen gene hPGA5, uses the CRISPR / Cas9 editing technology without selection markers to integrate the hPGA5 expression cassette into different sites of the Aspergillus niger An41683 genome, and constructs a series of Aspergillus niger engineered strains containing different hPGA copy numbers and capable of high pepsin production. The above recombinant strains are subjected to shake flask fermentation and the rhPGA5 enzyme activity is measured. On the 7th day of fermentation, the strain with the highest enzyme activity is AnrhPGA5-8, with an enzyme activity of up to 487U / mL, followed by AnrhPGA5-3, AnrhPGA5-5, The enzyme activities of AnrhPGA5-1, AnrhPGA5-2, AnrhPGA5-11, AnrhPGA5-10, AnrhPGA5-7, AnrhPGA5-6, AnrhPGA5-9, and AnrhPGA5-4 were 416.667U / mL, 378.333U / mL, 369.667U / mL, 358.667U / mL, 358.670U / mL, 352U / mL, 315.340U / mL, 273U / mL, 273U / mL, 266U / mL, and 243U / mL, respectively, which solved the problem of low production yield of pepsin produced by genetic engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is an example of the corresponding sgRNA contained in Example 1 of the present invention: knockout plasmid pFC332-agdA-2sgRNA; Figure 2 This is the exemplary plasmid pUC57-agdAUp-hPGA5-agdA Down containing the donor DNA fragment of the hPGA5 expression cassette in Example 1 of the present invention; Figure 3 This is a diagram showing the PCR identification results of the engineering strain in Example 1 of the present invention; Figure 4 This is a diagram showing the enzyme activity detection of rhPGA5 in fermentation broths of different engineered strains in Example 2 of the present invention. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work fall within the scope of protection of the present invention.

[0037] Currently, the pepsin available on the market is mainly extracted from the stomach tissues of mammals such as pigs, cattle, and sheep. The extraction process is relatively complicated, the extraction cost is relatively high, and its source is obviously limited. The use of genetic engineering to recombinantly express human pepsin can solve the problem of limited sources and complex extraction process, but the current yield is low.

[0038] To address this problem, an embodiment of the present invention provides an engineered strain that efficiently expresses recombinant human pepsinogen rhPGA5: the engineered strain is constructed by replacing the gene to be knocked out in Aspergillus niger with an hPGA5 expression cassette; the gene to be knocked out includes an α-glucosidase gene; the hPGA5 expression cassette consists of a filamentous fungal promoter, a signal peptide gene, an hPGA5 gene and a terminator; the hPAG5 gene encodes an amino acid sequence as shown in SEQ ID NO.1.

[0039] The embodiment of the present invention also provides a method for constructing the engineered strain.

[0040] The embodiment of the present invention also provides the use of the engineered strain in the fermentation production of pepsin.

[0041] The embodiment of the present invention also provides a method for producing pepsin by fermentation.

[0042] The scheme of the present invention is described below through specific embodiments.

[0043] The components and preparation methods of the culture medium used in the following examples are as follows: CD liquid medium: 2% glucose, 0.3% NaNO3, 0.2% KCl, 0.1% KH2PO4, 0.05% MgSO4·7H2O and 0.001% FeSO4·7H2O, adjusted to pH 5.5 with 5 mol / L KOH. Then add 0.05% agar powder to suspend the bacteria, and sterilize with high pressure steam at 115℃ for 20 minutes; YPD medium (g / L): 2% peptone, 1% yeast powder and 2% glucose, sterilized at 115℃ for 20min; PDA solid culture medium: Peel 200g of potatoes, remove the buds, cut into small strips and put them into an aluminum pot. Add 1000mL of water and boil for about 20-30 minutes until the potatoes are soft but not mushy. Filter with 6-8 layers of gauze, take the filtered juice into a beaker, add 20g of glucose and stir evenly, add water to make up to 1000mL, divide and add 2% agar powder at the same time, sterilize at 115℃ for 20min; 100 mg / mL Hygromycin B solution: Dissolve 1 g of hygromycin in 8 mL of ultrapure water. After fully dissolved, add ultrapure water to make up to 10 mL. The final concentration of hygromycin B in each culture medium is 100 μg / mL.

[0044] Aspergillus niger An41683 is an industrial production strain with high production of saccharifying enzymes. It does not express pepsin and its fermentation broth has no pepsin activity.

[0045] Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the materials, reagents, etc. used in the following examples can be obtained from commercial channels.

[0046] Example 1 This embodiment provides an engineering strain for efficiently expressing recombinant human pepsinogen rhPGA5 and a method for constructing the same.

[0047] Construction of knockout plasmid The knockout sites involved in this example include agdA , agdC , agdD , yes , agdF , agdG and Amy , skin , mnsA The corresponding sgRNA was designed online in CRISPOR (http: / / crispor.tefor.net / ) according to the selected knockout site, and the sequence is shown in Table 1. In order to improve the transformation efficiency, two sgRNA expression cassettes were connected in series to construct a multi-target knockout plasmid, and then the series sgRNA expression cassettes were connected to the plasmid pFC332 to construct the corresponding knockout plasmid.

[0048] (1) Construction of plasmid pUC57-target1-sgRNA-target2-sgRNA: First, use BbsI digested the plasmid pUC57-sgRNA, and annealed with the designed corresponding primers to synthesize double-stranded DNA fragments. The fragments were connected to the digested vector, transformed, and verified to construct two single-target sgRNA expression cassette plasmids pUC57-target1-sgRNA and pUC57-target2-sgRNA. Then, the designed primers sgRNA LF and sgRNAL-R (as shown in Table 2) were used to PCR amplify the target2-sgRNA expression cassette with the plasmid pUC57-target2-sgRNA as a template. Xho Ⅰ / BamH Ⅰ Double enzyme digestion of target2-sgRNA expression cassette fragment, Xho Ⅰ / Bgl Ⅱ Double-digest the plasmid pUC57-target1-sgRNA, cut the gel to recover the vector and fragment, connect the digested vector and fragment to construct the sgRNA expression cassette plasmid pUC57-target1-sgRNA-target2-sgRNA containing two targets.

[0049] Table 1XXX-sgRNA sequences

[0050] Table 2 Primer sgRNA LF, sgRNA LR sequences

[0051] (2) Construction of plasmid pFC332-target-2sgRNA: The plasmid pUC57-target1-sgRNA-target2-sgRNA obtained above was used Bgl Ⅱ / No Ⅰ Double enzyme digestion to obtain the tandem target1-sgRNA-target2-sgRNA expression cassette fragment, and use Bgl Ⅱ / No Ⅰ Double-digest the vector pFC332, connect the double-digested vector and fragment. Then transform the competent E. coli and screen the transformants. First, perform colony PCR verification on the transformants. After the colony PCR verification is correct, pick the transformants and culture them overnight in a 5mL LB test tube for 14-16 hours to extract the plasmid pFC332-XXX-2sgRNA. Bgl II and No Ⅰ Double enzyme digestion verification was performed. After verification was correct, pFC332-XXX-2sgRNA plasmid was obtained, and the results are shown in Table 3. The enzyme digestion system and ligation system are shown in Tables 4 and 5.

[0052] Table 3 pFC332-XXX-2sgRNA plasmids

[0053] Table 4 Enzyme digestion system

[0054] Table 5 Connection system

[0055] Taking pFC332-agdA-2sgRNA as an example, its plasmid map is as follows Figure 1 shown.

[0056] 2. Construction of donor DNA fragment plasmid containing hPGA5 expression cassette First, a plasmid pUC57-Pglucan703-glaASP (27aa)-hPGA5-TTglucan693 containing an hPGA5 gene expression cassette was constructed, and then the upstream and downstream homologous arms of the gene to be knocked out were connected to the upstream and downstream of the expression cassette to construct a plasmid containing an hPGA5 donor DNA fragment.

[0057] (1) Construction of hPGA5 expression cassette pUC57-Pglucan703-glaASP(27aa)-hPGA5-TTglucan693: Primers PS-Bgl II-F and PS-BsrG IR were designed (as shown in Table 6), and PCR amplification was performed using the vector pMD19-T-Pglucan703-glaA (kept in this laboratory, for the construction process, see Chen Yongjia's 2019 master's thesis: Cloning and functional analysis of the glucoamylase promoter) as a template to obtain a fragment Pglucan703-glaASP (27aa) containing a promoter sequence as shown in SEQ ID NO.6 and a signal peptide sequence as shown in SEQ ID NO.4. Bgl II / BsrG I double-digested the vector pUC57 and the fragment Pglucan703-glaASP (27aa), cut the gel to recover the vector and fragment, connected the digested vector and fragment to construct the plasmid pUC57-Pglucan703-glaASP (27aa). Then designed primers T-Nhe IF and T-BamH IR, and used the vector pMD19-T-TTglucan693 (kept in this laboratory, for the construction process, see Chen Yongjia's 2019 master's thesis: Cloning and functional analysis of the glucoamylase promoter) as a template for PCR amplification to obtain the terminator fragment TTglucan693, and used No I / BamHI double-digested the vector pUC57-Pglucan703-glaASP (27aa) and the fragment TTglucan693, cut the gel to recover the vector and fragment, and ligated the vector and fragment after digestion to construct the plasmid pUC57-Pglucan703-glaASP (27aa)-TTglucan693. Then, the target gene hPGA5 was synthesized using BsrG I / No I double-digested the vector pUC57-Pglucan703-glaASP(27aa)-TTglucan693 and the target gene hPGA5, cut the gel to recover the vector and fragment, connected the digested vector and fragment, and successfully constructed the plasmid pUC57-Pglucan703-glaASP(27aa)-hPGA5-TTglucan693.

[0058] Table 6 Sequences of primers PS-Bgl II-F, PS-BsrG IR, T-Nhe IF, T-BamH IR

[0059] (2) Construction of donor DNA fragment plasmid containing hPGA5 expression cassette: Corresponding primers were designed about 1000 bp upstream and downstream of both ends of the knockout site. The sequences are shown in Table 7. First, the upstream and downstream homology arms of the corresponding knockout site were obtained by PCR amplification using the Aspergillus niger genome as a template. The PCR products were observed by agarose gel electrophoresis to see if they were consistent with the theoretical band size. If the band size was correct, the upstream and downstream homology arms were cut and recovered. Then, the upstream and downstream homology arms were connected in sequence to the hPGA5 expression cassette plasmid pUC57-Pglucan703-glaASP(27aa)-hPGA5-TTglucan693. First, use Xho I / Bgl II double enzyme digestion of upstream homology arm and plasmid pUC57-Pglucan703-glaASP (27aa) -hPGA5-TTglucan693, gel cutting to recover linear plasmid and fragments, ligation, transformation, screening of transformants, and verification. After verification, plasmid pUC57-hPGA5-XXX-Up was obtained, and then used BamH I / Indian III continued to digest the plasmid and downstream homology arms, cut the gel to recover the linear plasmid and fragments, connect, transform, screen and verify the transformants. After verification, the donor DNA fragment plasmid pUC57-XXXUP-hPGA5-XXXDown containing the hPGA5 expression cassette was obtained. The results are shown in Table 8.

[0060] Table 7 Sequences of primers XXX-Up-XXX-F, XXX-Up-XXX-R, XXX-Down-XXX-F, XXX-Down-XXX-F

[0061] Table 8 Plasmids containing donor DNA fragments of hPGA5 expression cassette

[0062] Taking pUC57-agdAUp-hPGA5-agdA Down as an example, the plasmid map is as follows Figure 2 shown.

[0063] use Indian III / Xho The plasmid pUC57-XXXUP-hPGA5-XXXDown obtained by double enzyme digestion of the above strain was obtained, and the donor DNA fragment was recovered by gel excision.

[0064] The above PCR reaction system is shown in Table 9.

[0065] Table 9 PCR reaction system

[0066] Reaction conditions: denaturation at 98°C for 2 min; then denaturation at 98°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C for 30 sec, for 35 cycles; then extension at 72°C for 2 min; finally storage at 4°C and recovery.

[0067] 3. Construction and screening of recombinant strains Pick a single colony of Aspergillus niger An41683 from a fresh Aspergillus niger An41683 bacterial plate and inoculate it into CD liquid medium, culture it at 30℃ for 3-4 days, then inoculate it into YPD medium at a 4% inoculum, culture it at 30℃, 180r / min for 2-3 days until uniform bacterial pellets grow, and prepare protoplasts. The required knockout plasmid and donor DNA fragment are simultaneously transferred into protoplasts for transformation. After the transformants grow out, pick a single colony on the transformant plate and place it on a PDA hygromycin B plate, culture it at 30℃ for 3-4 days until the colonies grow up, pick a small amount of bacteria into 50μL 10×TE, stir the bacteria, and then use the freeze-thaw method to quickly extract the genome, and use the extracted genome as a template for PCR verification. The PCR verification primers are: downstream primer xxx-Down-Hind III-R, and the upstream primer is PGA5 verification-F, as shown in Table 10.

[0068] Table 10 Primer xxx-Down-Hind III-R, PGA5 Verification-F sequence

[0069] The PCR identification results of the engineered bacteria with the highest copy number (i.e., AnrhPGA5-11 in Table 11) are as follows: Figure 3 As shown (1-9 are genes agdA 、a gdC , agdD , yes , agdF , agdG , Amy , skin , mnsA The correct length of each primer pair is as follows: agdA-Down is 1794bp, agdC-Down is 1631bp, agdD-Down is 2101bp, agdE-Down is 2097bp, agdF-Down is 1646bp, agdG-Down is 2098bp, amyA-Down is 1578bp, derA-Down is 1601, and mnsA-Down is 1751bp, which is consistent with the theoretical expected results, indicating that the recombinant strain was successfully constructed. A total of 11 engineered strains with different copy numbers were successfully constructed. The names and genotypes of the engineered strains are shown in Table 11.

[0070] Table 11 Names and genotypes of starting strains and constructed engineered strains

[0071] Example 2 In this example, the pepsin activity in the fermentation broth of the engineered strain constructed in Example 1 was measured.

[0072] Select the correctly identified engineering strain and inoculate it into CD liquid medium, culture at 30℃ until uniform small balls grow (2-3 days), use a 5mL gun to suck and blow the bacteria until the bacteria are broken up, so that the hyphae are evenly distributed, inoculate 2mL of bacterial liquid into 50mL of YPD medium, culture at 30℃, 180 r / min, until the bacteria grow into small balls (2-3 days). Then inoculate 2mL of bacteria into 50mL of malt extract fermentation medium, culture at 30℃, 180 r / min, and ferment for 7 days.

[0073] Pepsin activity assay: Dissolve 2g of hemoglobin in 80mL of pure water, adjust the pH to 2 with 300mM HCl, and adjust the volume to 100mL to obtain a 2% (w / v) hemoglobin solution with pH=2. The specific method of enzyme activity determination is as follows: set up an experimental group and a control group. First, add 300µL of 2% hemoglobin to a 1.5mL centrifuge tube in the experimental group and the control group, respectively, and preheat at 37℃ for 5min. Then, add 600µL of 5% TCA (trichloroacetic acid) to the control group, and then add 100µL of fermentation supernatant, and only add 100µL of fermentation supernatant to the experimental group. The control group and the experimental group react at 37℃ for 10min, and the experimental group adds 600µL of TCA to terminate the reaction. After the reaction, centrifuge at 12000r / min for 5 min, take the supernatant and measure the absorbance at OD280nm. The enzyme activity determination formula is shown below.

[0074]

[0075] Where N is the dilution factor and ∆t is the reaction time.

[0076] The enzyme activity detection data of rhPGA5 in the fermentation broth of each strain on the 7th day of fermentation are as follows Figure 4 As shown, the strain with the highest enzyme activity on the 7th day of fermentation was AnrhPGA5-8, with an enzyme activity of up to 487 U / mL, followed by AnrhPGA5-3, AnrhPGA5-5, AnrhPGA5-1, AnrhPGA5-2, AnrhPGA5-7, AnrhPGA5-6, and AnrhPGA5-4, with enzyme activities of 416.667 U / mL, 378.333 U / mL, 369.667 U / mL, 358.667 U / mL, 273 U / mL, 273 U / mL, and 243 U / mL, respectively. The enzyme activities of ERAD-deficient PGA recombinant strains AnrhPGA5-9, AnrhPGA5-11, and AnrhPGA5-10 were 266 U / mL, 352 U / mL, and 358.67 U / mL, respectively.

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An engineered strain for efficiently expressing recombinant human pepsinogen rhPGA5, characterized in that: The engineered strain is constructed by replacing the gene to be knocked out in Aspergillus niger with an hPGA5 expression cassette; the gene to be knocked out includes an α-glucosidase gene; the hPGA5 expression cassette consists of a filamentous fungus promoter, a signal peptide gene, an hPGA5 gene and a terminator; the hPAG5 gene encodes an amino acid sequence as shown in SEQ ID NO.

1.

2. The engineered strain according to claim 1, characterized in that The nucleotide sequence of the hPAG5 gene is shown in SEQ ID NO.2; and / or The signal peptide is selected from the group consisting of a signal peptide of a saccharifying enzyme gene, a signal peptide of an amylase gene, a signal peptide of a cellulase gene, and a signal peptide of a cellobiohydrolase CHB gene; and / or The gene to be knocked out further includes an amylase gene; and / or The genes to be knocked out also include genes related to the secretory pathway; and / or The nucleotide sequence of the filamentous fungus promoter is shown in SEQ ID NO.5 or SEQ ID NO.6; and / or The terminator is terminator TTglucan693; and / or The Aspergillus niger is Aspergillus niger An41683.

3. The engineered strain according to claim 1 or 2, characterized in that The signal peptide gene is a signal peptide glaASP gene encoding an amino acid sequence as shown in SEQ ID NO.3; and / or The α-glucosidase gene includes the gene aiZd ,Gene aiZd ,Gene aD ,Gene aiZd ,Gene f and genes aiD At least one of; and / or The amylase gene is a gene amyA ; and / or The secretion pathway related gene is an ERAD pathway related gene.

4. The engineered strain according to claim 3, characterized in that The ERAD pathway-related genes include genes derA and genes mnS ; and / or The nucleotide sequence of the signal peptide gene is shown in SEQ ID NO.

4.

5. The method for constructing an engineered strain according to any one of claims 1 to 4, characterized in that: The specific steps include: S1. Constructing a donor DNA fragment containing the hPGA5 expression cassette and a sgRNA knockout plasmid containing the corresponding gene to be knocked out according to CRISPR-Cas9 technology; S2. Co-transform the donor DNA fragment and the sgRNA knockout plasmid into Aspergillus niger protoplasts, extract the transformant genomic DNA, and identify by PCR to obtain the engineered strain expressing rhPGA5.

6. The construction method according to claim 5, characterized in that: The method of co-transforming the donor DNA fragment and the sgRNA knockout plasmid into Aspergillus niger protoplasts in S2 is a protoplast transformation method.

7. The construction method according to claim 5, characterized in that: The construction method of the sgRNA knockout plasmid described in S1 is: Synthesize two single-target sgRNA primer pairs of the gene to be knocked out, anneal them separately and Bbs I restriction enzyme digestion plasmid pUC57-sgRNA was connected to construct sgRNA expression cassette plasmids pUC57-target1-sgRNA and pUC57-target2-sgRNA. The target2-sgRNA expression cassette fragment was amplified by PCR using plasmid pUC57-target2-sgRNA as a template. XOt Ⅰ / Bm Ⅰ After double restriction digestion, cloned into plasmid pUC57-target1-sgRNA XOt Ⅰ / Bgl II sites, construct the sgRNA expression cassette plasmid pUC57-target1-sgRNA-target2-sgRNA containing two targets; The plasmid pUC57-target1-sgRNA-target2-sgRNA was used Bgl Ⅱ / Nhe Ⅰ double digestion to obtain the tandem target1-sgRNA-target2-sgRNA expression cassette fragment, and use Bgl Ⅱ / Nhe Ⅰ Double-enzyme digestion of vector pFC332, connecting the double-enzyme digested vector and the fragment, and then transforming into competent E. coli, screening transformants, and extracting plasmids from the verified correct transformants to obtain the sgRNA knockout plasmid; and / or The preparation method of the donor DNA fragment described in S1 is: The fragment containing the promoter and signal peptide was obtained by PCR amplification using the vector pMD19-T-Pglucan703-glaA as a template; use Bgl II / BnB I double-digest the vector pUC57 and the fragment, connect the digested vector and the fragment to construct a plasmid containing a signal peptide; The terminator fragment was obtained by PCR amplification using the vector pMD19-T-TTglucan693 as a template. Nhe I / Bm I double-digest the plasmid containing the signal peptide and the terminator fragment, connect the digested plasmid and the fragment to construct a plasmid containing the signal peptide and the terminator; use BnB I / Nhe I. Double-digesting the plasmid containing the signal peptide and terminator and the hPGA5 gene, connecting the vector and fragments after enzyme digestion to construct a plasmid containing the signal peptide, terminator and hPGA5 gene; The upstream homology arm and the downstream homology arm of the knockout site corresponding to the gene to be knocked out were amplified by PCR using the Aspergillus niger genome as a template. XOt I / Bgl II double enzyme digestion of the upstream homology arm and the plasmid containing the signal peptide, terminator and hPGA5 gene, ligation, transformation, screening of transformants, and then use Bm I / Hind III. Continue to digest the obtained plasmid and the downstream homology arm, connect again, transform, screen transformants, and obtain the donor DNA fragment plasmid containing the hPGA5 expression cassette; use Hind III / XOt I double-digests the donor DNA fragment plasmid containing the hPGA5 expression cassette to obtain the donor DNA fragment.

8. Use of the engineered strain according to any one of claims 1 to 4 in the fermentation production of pepsin.

9. A method for producing pepsin by fermentation, characterized in that: The engineered strain according to any one of claims 1 to 4 is used to ferment and produce pepsin.

10. The method according to claim 9, characterized in that After the engineered strain is cultured in a liquid culture medium until uniform small balls are grown, the mycelium is broken up to make the mycelium evenly distributed, and then inoculated into a YPD culture medium and cultured until the bacteria grow into small balls; the small ball-shaped bacteria are inoculated into a fermentation culture medium to ferment and produce pepsin.

Images