Recombinant yeast expression system of trypsin free from enterokinase activation as well as construction method and application of recombinant yeast expression system

Abstract

The invention provides a recombinant yeast expression system of trypsin free from enterokinase activation, a construction method and application of the recombinant yeast expression system, modified trypsinogen coding nucleic acid, a recombinant yeast host cell, active trypsin and a production system. The propeptide sequence is connected with a mature enzyme sequence; and a mature enzyme sequence, wherein a basic amino acid residue is contained between the propeptide sequence and the mature enzyme sequence. According to the technical scheme, the problem that in the prior art, recombinant trypsin production must depend on exogenous enterokinase for activation can be effectively solved.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and enzyme engineering, specifically to a recombinant yeast expression system for trypsin that is immune to enterokinase activation, as well as a method for constructing the system and its application in the preparation of active trypsin. Background Technology

[0002] Trypsin (EC3.4.21.4) is a serine protease that specifically cleaves the peptide bonds at the carboxyl terminus of lysine and arginine residues. It is widely used in biopharmaceuticals, cell culture, proteomics research, food and leather industries.

[0003] Currently, commercial trypsin is mainly extracted from animal pancreas, which presents challenges such as pathogen contamination risk, large batch-to-batch variability, and purification difficulties. Recombinant expression in microorganisms using genetic engineering technology is an effective way to address these issues. However, recombinant expression of trypsin faces a key bottleneck: natural trypsin is synthesized in an inactive zymogen form and must be activated in vitro by enterokinase cleavage to obtain activity. Enterokinase is expensive, and adding an activation step not only increases production costs and process complexity but may also introduce impurities, affecting the purity and uniformity of the final product. Furthermore, wild-type trypsin is prone to autolysis and inactivation, exhibiting poor stability. Although we have previously obtained novel trypsin molecules with excellent anti-autolysis properties through mutation (see patent number ZL202010414080.0), the core process bottleneck of its production, which relies on exogenous enterokinase activation, remains unresolved. Therefore, developing a recombinant expression technology that can directly produce active trypsin without relying on exogenous enterokinase is of great significance for reducing production costs, simplifying the process, and promoting its industrial application.

[0004] Yeast expression systems, particularly Pichia pastoris, are commonly used platforms for producing eukaryotic recombinant proteins. They possess highly efficient endogenous protein processing systems. This invention rationally designs the secretion signal and propeptide sequence of trypsinogen to construct a novel expression system that enables trypsinogen activation to be completed by the yeast's endogenous processing system, thereby completely eliminating dependence on exogenous enterokinases. Summary of the Invention

[0005] The purpose of this invention is to overcome the deficiency in existing technologies where the production of recombinant trypsin relies on exogenous enterokinase activation. It provides a complete technical solution for the direct fermentation and secretion of active trypsin without enterokinase activation, including its core components, construction method, and applications. Specifically, by rationally modifying the propeptide sequence and / or signal peptide sequence of trypsinogen, it can be correctly recognized and efficiently cleaved by the yeast host's endogenous protease processing system, thereby directly producing active mature trypsin. No additional exogenous enterokinase is required during the entire production process, thus simplifying the process and reducing costs.

[0006] The technical solution for achieving the objective of this invention is as follows: This invention provides a method for producing active trypsin, comprising the following steps: (a) Expressing a modified trypsinogen in a yeast host, wherein the modification enables the trypsinogen to be processed by endogenous proteases of the yeast host to generate an active mature trypsin, and the production method does not depend on the addition of exogenous enterokinase for activation. (b) The active trypsin is recovered from the culture.

[0007] Furthermore, the modification includes altering the precursor peptide sequence of trypsinogen to introduce or optimize a protease cleavage site between the precursor peptide and the mature peptide sequence. Preferably, the protease cleavage site is a recognition site of the yeast Kex2 protease, more preferably containing an arginine (R) site, for example by introducing an arginine (R) codon between the precursor peptide and the mature peptide sequence.

[0008] Furthermore, the modification also includes modifying the signal peptide sequence of the trypsinogen to promote its proper cleavage by yeast signal peptidase, for example, by deleting lysine (K) at position 84 and / or arginine (R) at position 85.

[0009] The present invention provides a modified trypsinogen polypeptide sequence, which can be secreted and expressed in a yeast culture system without the need for exogenous enterokinase activation and has activity.

[0010] Furthermore, the polypeptide is as shown in SEQ ID NO. 5 or SEQ ID NO. 7, or is a variant obtained by replacing, deleting, or adding one or more conserved amino acids in regions other than the Kex2 cleavage site (arginine site) and the active site of mature trypsin, based on the sequence shown in SEQ ID NO. 5 or SEQ ID NO. 7, and the variant retains the function of being secreted and expressed and having activity in a yeast culture system without the addition of exogenous enterokinase.

[0011] The present invention provides a modified trypsinogen-encoded nucleic acid for use in the aforementioned method, wherein the modification enables a polypeptide expressed by the nucleic acid in a yeast host to be processed into active trypsin by the host's endogenous proteases, without relying on exogenous enterokinase.

[0012] Furthermore, the nucleotide sequence encoding the nucleic acid is as shown in SEQ ID NO. 6 or SEQ ID NO. 8, or is a variant that retains the same function, obtained by one or more synonymous codon substitutions and / or conserved modifications at non-Kex2 cleavage sites and in key regions of immature enzyme activity based on SEQ ID NO. 6 or SEQ ID NO. 8.

[0013] This invention provides a recombinant expression vector containing the aforementioned encoded nucleic acid, and a recombinant yeast host cell containing the vector or integrated with the encoded nucleic acid. Preferably, the host cell is Pichia pastoris (…). Pichia pastoris (, more preferably Pichia pastoris GS115 strain).

[0014] The present invention provides a production system for active trypsin, which comprises the above-mentioned recombinant yeast host cell.

[0015] The present invention provides an active trypsin produced by the above method or produced by the above host cells.

[0016] This invention provides the application of the active trypsin, or a production system containing the active trypsin, in fields requiring trypsin activity, including but not limited to biopharmaceuticals, food processing, feed additives, leather softening, and scientific research.

[0017] The beneficial effects of this invention are: This application involves the targeted modification of the trypsinogen polypeptide sequence, introducing a basic amino acid residue between the propeptide sequence and the mature enzyme sequence. This allows the polypeptide, after expression in yeast host cells, to be efficiently recognized and precisely cleaved by the host's own endogenous proteases, directly releasing naturally active mature trypsin without the need for in vitro activation by exogenous enterokinase. This design not only simplifies the production process of recombinant trypsin, reducing production costs and the complexity of purification steps, but also avoids the potential impact of exogenous enzyme residues on product purity and safety, significantly improving product yield and bioactivity consistency. Simultaneously, the introduction of this basic amino acid residue strictly preserves the integrity of the trypsin's natural active structure, ensuring that its enzymatic properties are not compromised. This enables the efficient and self-activated production of this modified trypsinogen in yeast expression systems, providing a practical and feasible technical path for industrial-scale green biomanufacturing. Specific effects are summarized below: 1. The fermentation process used in this invention is revolutionary and simplified, completely eliminating the expensive and cumbersome exogenous enterokinase activation step, and simplifying the traditional "expression-purification-activation-re-purification" process to "expression-purification", which significantly reduces production costs and time.

[0018] 2. The mature trypsin product prepared by this invention has high activity. The modified engineered bacteria can directly secrete highly active mature trypsin. Experiments show that the enzyme activity during shake-flask fermentation is 7.1-8.3 times higher than that of the original strain; the peak enzyme activity in a 7L fermenter can reach more than 2162.4 U / mL.

[0019] 3. The recombinant yeast expression system provided by this invention has strong versatility. This strategy has been successfully applied to the expression of porcine and human trypsin, proving the universality of its technical solution.

[0020] 4. The recombinant yeast of this invention has good prospects for industrialization. The expression system provided is stable, efficient and easy to scale up, providing a brand-new solution for the large-scale and low-cost production of active trypsin. Attached Figure Description

[0021] Figure 1 A schematic diagram of the strategy for constructing mutants.

[0022] Figure 2 Electrophoresis diagram of nucleic acid PCR products of porcine trypsin precursor mutant: Lane M, marker; Lane 1: porcine trypsin precursor mutant.

[0023] Figure 3 Electrophoresis image of PCR product of porcine trypsin signal peptide mutant. Lane M: marker; Lane 1: signal peptide mutant.

[0024] Figure 4 This study compares the trypsin activity of the original strain and the modified strain during shake-flask fermentation.

[0025] Figure 5 The graph shows the changes in trypsin activity during the fermentation of porcine trypsin mutant strains in a 7L tank.

[0026] Figure 6 SDS-PAGE images of extracellular proteins at different time points during fermentation in a 7L tank using engineered bacteria containing a porcine trypsin mutant. Lane 1: pPIC9K empty plasmid control bacteria; Lanes 2-6: extracellular protein samples at fermentation times of 32 h, 44 h, 56 h, 68 h, and 80 h, respectively.

[0027] Figure 7 This is an SDS-PAGE image of the fermentation supernatant of a human trypsin mutant engineered strain. Lane 1: pPIC9K empty plasmid control strain; Lane 2: human trypsin mutant engineered strain. Detailed Implementation

[0028] The present invention will now be described in detail with reference to the embodiments and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0029] The present invention provides a recombinant yeast expression system for trypsin that is not activated by enterokinase. The system modifies the trypsin precursor by introducing a Kex2 protease recognition site between the precursor sequence and the mature enzyme sequence. The signal peptide supporting its secretory expression is modified by deleting the Kex2 protease recognition site from the signal peptide sequence. This modification enables the trypsinogen to be processed by the yeast host's endogenous proteases, thereby directly generating active mature trypsin without relying on the addition of exogenous enterokinase for activation.

[0030] The present invention provides a recombinant yeast expression system for trypsin that is immune to enterokinase activation, wherein the precursor peptide is modified by introducing an arginine (R) site at its C-terminus. Preferably, the yeast α-factor signal peptide sequence is used, with the lysine (K) at position 84 and / or the arginine (R) at position 85 deleted.

[0031] The present invention provides a modified trypsinogen polypeptide sequence, wherein the polypeptide is a variant obtained by replacing, deleting or adding one or more conserved amino acids in regions other than the Kex2 cleavage site of the precursor peptide and the active site of mature trypsin, based on the sequence shown in SEQ ID NO: 5 or SEQ ID NO: 7, and the variant retains the function of being secreted and expressed in a yeast culture system without the addition of exogenous enterokinase and having activity.

[0032] The modified trypsinogen-encoded nucleic acid provided by the present invention is a variant obtained by one or more synonymous codon substitutions and / or conserved modifications to non-Kex2 cleavage sites and key regions of immature enzyme activity based on SEQ ID NO:6 or SEQ ID NO:8, and the variant retains the function of processing into active trypsin without relying on exogenous enterokinase.

[0033] The present invention also provides a recombinant yeast host strain comprising a recombinant expression system, or having integrated the coding nucleic acid of claim 5 into its genome. The yeast host cell is Pichia pastoris (…). Pichia pastoris Pichia pastoris strain GS115 is preferred.

[0034] A system for producing active trypsin, comprising fermentation of the above-mentioned recombinant yeast host strain.

[0035] The present invention also provides an active trypsin, which is expressed by a strain containing the above expression system.

[0036] The active trypsin produced by the above methods has applications in feed processing, food processing, leather processing, biopharmaceuticals, cell culture, or proteomics research.

[0037] The core and inventive point of this invention lies in the specific rational design of the propeptide and signal peptide to construct cleavage sites that can be efficiently recognized by yeast endogenous proteases (such as Kex2), thereby achieving endogenous processing instead of exogenous activation. To verify the universality of the strategy of this invention, the human trypsinogen sequence was modified according to the same principle. The core modification strategy of this invention is universal and independent, and is equally applicable to any other trypsinogen sequence, including but not limited to wild-type sequences or other variants that have undergone different modifications.

[0038] Reagents and materials: Pichia pastoris GS115, vector pPIC9K, and Escherichia coli JM109 are all commonly used or commercially available in this field. The preparation of culture media (YPD, BMGY, BMMY, etc.) and fermentation methods are standard techniques in this field.

[0039] Example 1 Methods for Assaying Trypsin Enzyme Activity N-benzoyl-L-arginine ethyl ester (BAEE) was used as a substrate to determine trypsin activity. At pH 7.6 and 25°C, absorbance changes were monitored at 253 nm. One BAEE enzyme activity unit was defined as an increase of 0.001 ΔA253 per minute. The specific procedure was as follows: 3 mL of 0.25 mM BAEE substrate was added, along with 125 μL of 1 mM HCl and 75 μL of appropriately diluted enzyme solution. The mixture was immediately stirred, and the rate of absorbance change over 210 seconds was recorded. Enzyme activity (U / mL) and specific activity (U / mg) were calculated.

[0040] Example 2 Design and synthesis of trypsinogen coding sequence for enterokinase activation Based on the existing porcine trypsinogen sequence (amino acid sequence as shown in SEQ ID NO.1, corresponding nucleic acid sequence as shown in SEQ ID NO.2) with excellent anti-autolysis properties and its α-factor signal peptide sequence expressed in yeast (amino acid sequence as shown in SEQ ID NO.3, corresponding nucleic acid sequence as shown in SEQ ID NO.4), the following rational modification design was carried out (see the schematic diagram of the construction strategy). Figure 1 ): Mutant 1: Trypsin-kex2, introduces an arginine (R) codon at the sequence boundary between the precursor and mature peptides, thus forming a complete "KR" site with the existing upstream lysine (K). This site is a highly efficient cleavage site for the yeast Kex2 protease. The amino acid sequence of this mutant is shown in SEQ ID NO.5, and the gene sequence is shown in SEQ ID NO.6.

[0041] Mutant 2: Trypsin-kex2-cut, based on the Trypsin-kex2 design, further deletes the codons for lysine (K) at position 84 and arginine (R) at position 85 in the α-factor signal peptide sequence to eliminate basic amino acids that may interfere with the proper processing of the signal peptidase. The amino acid sequence of this mutant is shown in SEQ ID NO.7, and the gene sequence is shown in SEQ ID NO.8. Based on the sequences shown in SEQ ID NO.2 and SEQ ID NO.4, the designed mutations can be introduced using conventional site-directed mutagenesis techniques such as overlap extension PCR to obtain the genes shown in SEQ ID NO.6 and SEQ ID NO.8. Alternatively, the genes can be directly synthesized by a company.

[0042] Example 3 Construction of recombinant expression vectors The gene sequences of the mutants Trypsin-kex2 and Trypsin-kex2-cut were amplified by PCR, and the nucleic acid electrophoresis images are shown below. Figure 2 and Figure 3 As shown in the figure, the amplified mutant Trypsin-kex2 gene fragment and the pPIC9K vector were digested and purified with EcoRI and NotI, respectively, and then ligated with T4 DNA ligase to construct the recombinant plasmid pPIC9K-Try-kex2. Similarly, the mutant Trypsin-kex2-cut gene fragment and the pPIC9K vector were digested and ligated with BamHI and EcoRI to construct the recombinant plasmid pPIC9K-Try-kex2-cut. The ligation product was transformed into E. coli JM109, and the correct recombinant plasmid was obtained after antibiotic selection, colony PCR, and sequencing verification.

[0043] Example 4 Construction of recombinant Pichia pastoris donor strain Linearized recombinant plasmids pPIC9K-Try-kex2 and pPIC9K-Try-kex2-cut were electrotransformed into Pichia pastoris GS115 competent cells. Transformants were plated on MD plates, screened, and then transferred to YPD plates containing gradient concentrations of G418 for high-copy transformant selection. Genomic DNA was extracted from positive clones for PCR verification, and the PCR products were sequenced to ensure correct integration of the target gene. Finally, recombinant engineered bacteria GS115 / pPIC9K-Try-kex2 and GS115 / pPIC9K-Try-kex2-cut were obtained.

[0044] Example 5 Shake-flask fermentation and activation-free verification The engineered bacteria and the empty vector control bacteria were inoculated and shake-flask fermentation was induced. Crucially, no exogenous enterokinase was added throughout the entire fermentation and subsequent treatment process. After fermentation, the supernatant was collected by centrifugation, and trypsin activity was directly determined using the method in Example 1. The results are as follows: Figure 4 As shown, the enzyme activity in the fermentation supernatant of the control strain was extremely low (157.09 U / mL), indicating that yeast could hardly secrete active trypsin without modification. In contrast, the supernatant enzyme activities of the engineered strains GS115 / pPIC9K-Try-kex2 and GS115 / pPIC9K-Try-kex2-cut reached 1114.6 U / mL and 1308.6 U / mL, respectively, which were 7.1 times and 8.3 times that of the control. This result directly proves that the modification described in this invention achieves efficient secretion of active trypsin under enterokinase activation.

[0045] Example 6 7L fermenter scale preparation The engineered strain GS115 / pPIC9K-Try-kex2-cut, which showed better performance, was used for fermentation in a 7L tank. A glucose batch-methanol fed-batch induction strategy was employed. Figure 5 As shown, after approximately 36 hours of methanol induction (56 hours of fermentation), the enzyme activity in the fermentation broth reached a peak of 2162.4 U / mL. SDS-PAGE analysis ( Figure 6 The results showed a distinct protein band at approximately 24.4 kDa, consistent with the theoretical molecular weight of trypsin. This embodiment demonstrates that the expression system provided by the present invention has good scalability and can achieve industrial-scale production of high-concentration active trypsin.

[0046] Example 7 Universality verification of the technical solution—human trypsin To verify the universality of the strategy of this invention, the human trypsinogen sequence was modified according to the same principle (introducing the Kex2 site and optimizing the signal peptide; the modified amino acid sequence is shown in SEQ ID NO. 9, and the corresponding nucleic acid sequence is shown in SEQ ID NO. 10), constructing the engineered strain GS115 / pPIC9K-H-Try-kex2-cut with modified human trypsinogen. Fermentation was carried out in a 7L tank, and without the addition of enterokinase, the expression of the target protein in the fermentation supernatant was significant (…). Figure 7 The enzyme activity peak reached 2273.5 U / mL. This result successfully demonstrates that the "enterokinase-free activation" expression strategy of the present invention is applicable to trypsin from different sources and has broad application value.

[0047] Application example: The method for preparing recombinant trypsin without enterokinase activation provided by this invention only modifies the signal peptide and propeptide, without affecting the activity and properties of the trypsin. Therefore, it can be widely used in all fields that require trypsin to perform proteolytic activities, including but not limited to: In the fields of biopharmaceuticals and R&D: specific site cleavage and purification of recombinant proteins, preparation of Fab / F(ab')2 fragments by antibody enzymatic digestion, peptide drug production, and digestion of adherent cells in cell culture, etc.

[0048] In the food industry: it is used for protein hydrolysis modification, preparation of bioactive peptides, and clarification of wines and beverages.

[0049] In the feed industry: as a feed additive, it supplements endogenous digestive enzymes in animals and promotes protein absorption.

[0050] In the leather industry: used to soften leather, improving its elasticity and softness.

[0051] In the field of scientific research: as a key tool enzyme in proteomics and biochemistry research, it is used for sample digestion before protein sequencing. Those skilled in the art can directly use the trypsin provided by this invention based on the known application requirements described above, and can expect its excellent results.

Claims

1. A recombinant yeast expression system for trypsin that is immune to enterokinase activation, characterized in that: The trypsin precursor peptide was modified to introduce a Kex2 protease recognition site between the precursor peptide sequence and the mature enzyme sequence.

2. The recombinant yeast expression system for enterokinase-free trypsin according to claim 1, characterized in that: The modification of the trypsin precursor peptide involves introducing an arginine site at its C-terminus.

3. The recombinant yeast expression system for enterokinase-activated trypsin according to claim 1, characterized in that: The C-terminal recognition site of the Kex2 protease contains at least one set of lysine-K and arginine-R double amino acid sites.

4. The recombinant yeast expression system for enterokinase-activated trypsin according to claim 3, characterized in that: A variant is obtained by making one or more conserved amino acid substitutions, deletions, or additions at the arginine site of the Kex2 cleavage site of the precursor peptide sequence, and the variant retains its function of being secreted and expressed in a yeast culture system without the addition of exogenous enterokinase.

5. The recombinant yeast expression system for enterokinase-free trypsin according to claim 1, characterized in that: It also includes the α-factor signal peptide sequence of the modified yeast expression system, with the lysine at position 84 and / or the arginine at position 85 deleted from the α-factor signal peptide sequence.

6. The recombinant yeast expression system for enterokinase-free trypsin according to claim 5, characterized in that: The modification of the signal peptide includes the deletion of lysine K at position 84 and / or arginine R at position 85, followed by one or more synonymous codon substitutions at other positions, and / or conserved modifications at non-Kex2 cleavage sites and in key regions of immature enzyme activity, resulting in variants that retain the function of the peptide in processing into active trypsin without relying on exogenous enterokinase.

7. A modified trypsinogen polypeptide sequence, characterized in that: The polypeptide sequence is SEQ ID NO: 5 or SEQ ID NO:

7.

8. A modified trypsinogen-encoded nucleic acid, characterized in that: The nucleotide sequence is SEQ ID NO:6 or SEQ ID NO:

8.

9. A recombinant yeast host strain, characterized in that: It comprises the recombinant expression system according to any one of claims 1-4, or has the coding nucleic acid of SEQ ID NO.2 or SEQ ID NO.4 integrated into its genome.

10. The recombinant yeast host strain according to claim 9, characterized in that: The yeast host cell is Pichia pastoris (Pichia pastoris). Pichia pastoris Pichia pastoris strain GS115 is preferred.

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