High-throughput screening method for enzymes and construction method for enzyme library

Abstract

Provided are a high-throughput screening method for enzymes and a construction method for an enzyme library. The construction method for an enzyme library comprises: separately placing a plurality of different DNA molecules in a protein in-vitro expression system for expression, the plurality of different DNA molecules respectively encoding different mutants of a same enzyme to obtain an expression product of each mutant; and the expression products from primary screening of the plurality of mutants jointly forming an enzyme library of the enzyme, wherein the plurality of DNA molecules are present in the form of plasmids or PCR products. The method simplifies the process of preparing protein-form enzyme libraries, and saves fermentation and storage resources; and the expression products can be directly used for enzyme screening without separation, so that the operation is simple and convenient, and the parallelism is better. Additionally, the implementation is not affected by the difficulty in expressing certain proteins in intracellular expression or by the toxicity of such proteins to cells, which makes it difficult to obtain expression products.

Description

High-throughput screening method of enzyme and construction method of enzyme library

[0001] This application is based on Chinese application No. 202410454006.X, filed on April 16, 2024, and Chinese application No. 202410490345.3, filed on April 23, 2024, and claims priority to them, and the disclosure of the Chinese application is incorporated by reference in its entirety. TECHNICAL FIELD

[0002] The present application relates to the field of enzyme engineering, in particular, to a high-throughput screening method of enzyme and a construction method of enzyme library. BACKGROUND

[0003] The construction of enzyme library and high-throughput screening of enzyme are very important for biological catalysis and biological transformation. Generally, the construction of enzyme library is in the form of enzyme solution or enzyme lyophilized powder, which has certain defects. For example, the stability of enzyme during storage, enzyme may lose activity during storage, resulting in inaccurate screening results, or even failure. The enzyme lyophilized powder form is to freeze-dry the enzyme solution and some additives in a 96-well plate to enhance the storage stability of the enzyme. When used, the enzyme powder needs to be dissolved again. Because the freeze-dried enzyme powder is easy to absorb moisture, dry nitrogen is generally used for protection, and it cannot be used repeatedly.

[0004] Moreover, in the process of high-throughput screening of enzyme, a plurality of potential enzymes are obtained after the first round of screening, and the obtained enzymes need to be rescreened, which will waste a backup enzyme. Multiple backup enzymes occupy a large amount of fermentation resources, freezing resources and storage resources.

[0005] Therefore, the poor storage stability of enzyme library in the prior art needs to be improved.

[0006] SUMMARY

[0007] The main purpose of the present application is to provide a high-throughput screening method of enzyme and a construction method of enzyme library to solve the problem of poor storage stability of enzyme library in the prior art.

[0008] In order to achieve the above purpose, according to one aspect of the present application, an enzyme library is provided, which comprises a plurality of enzymes, wherein each enzyme is stored in the form of a corresponding DNA molecule, and the DNA molecule is a plasmid or a PCR product.

[0009] Further, the enzymes in the enzyme library are industrial enzymes, preferably, the industrial enzymes comprise any one or more of the following proteases: transaminase, ketoreductase, lipase or monooxygenase; preferably, the industrial enzymes comprise any one or more of the following proteases: transaminase with an amino acid sequence as shown in SEQ ID NO: 1, ketoreductase with an amino acid sequence as shown in any one of SEQ ID NO: 2 or SEQ ID Nos: 6-25, lipase with an amino acid sequence as shown in SEQ ID NO: 3 or monooxygenase with an amino acid sequence as shown in SEQ ID NO: 4.

[0010] According to a second aspect of the present application, there is provided a method for constructing an enzyme library in the form of a protein, comprising: expressing a plurality of different DNA molecules in a protein in vitro expression system, the plurality of different DNA molecules encoding different mutants of the same enzyme respectively, to obtain expression products of each mutant; and screening the expression products of the plurality of mutants together to form an enzyme library of the enzyme; wherein the plurality of DNA molecules are in the form of plasmids or PCR products.

[0011] Further, the protein in vitro expression system is an E. coli in vitro expression system or a yeast in vitro expression system; preferably, the E. coli in vitro expression system comprises: a basic component, an energy-related component, an additive component, a cell extract and an RNase inhibitor, wherein, in the E. coli in vitro expression system, the basic component comprises: 19 kinds of amino acids at a concentration of 2 mM for each amino acid, 2 mM tyrosine, 14 mM magnesium acetate, 60 mM potassium acetate and 7 mM DDT; in the E. coli in vitro expression system, the energy-related component comprises: 1.2 mM AMP, 0.85 mM CMP, 0.85 mM GMP, 0.85 mM UMP, 15-83 mM PEP, 0.4-0.6 mM NAD, 4 mM potassium oxalate, 90 mM potassium glutamate, 2.5-10 mM magnesium glutamate; in the E. coli in vitro expression system, the additive component comprises: 1.5 mM spermidine and 157.33 mM HEPES; in the E. coli in vitro expression system, the concentration of the RNase inhibitor is 150 U / 450 μL; and the volume content of the cell extract in the E. coli in vitro expression system is 20-60%.

[0012] Further, in the E. coli in vitro expression system, the concentration of PEP is 30 mM; preferably, the content of NAD is 0.4 mM; preferably, the content of magnesium glutamate is 7.5 mM; and preferably, the volume content of the cell extract in the E. coli in vitro expression system is 33.3%.

[0013] Further, the enzymes in the enzyme library are industrial enzymes.

[0014] Further, the industrial enzyme comprises any one or more of the following proteases: transaminase, ketoreductase, lipase or monooxygenase; preferably, the industrial enzyme comprises any one or more of the following proteases: transaminase with the amino acid sequence shown in SEQ ID NO: 1, ketoreductase with the amino acid sequence shown in any one of SEQ ID NO: 2 or SEQ ID Nos: 6-25, lipase with the amino acid sequence shown in SEQ ID NO: 3 or monooxygenase with the amino acid sequence shown in SEQ ID NO: 4.

[0015] According to a third aspect of the present application, a high-throughput screening method of an enzyme is provided, comprising: screening each mutant in an enzyme library for performance by using the same substrate; wherein the enzyme library is any one of the enzyme libraries described above, or is constructed by the method for constructing an enzyme library described above.

[0016] Further, the performance screening can be directly performed without isolating and purifying the expression product of each mutant; preferably, the screening method further comprises a secondary screening based on the performance screening, and the secondary screening comprises: selecting the plasmid or PCR product of the mutant of the corresponding enzyme screened, and placing it in a protein in vitro expression system for expression to obtain a secondary screening expression product; and directly incubating the secondary screening expression product with the substrate for secondary screening.

[0017] Further, the performance screening comprises screening for any one or more of the following performances: catalytic activity, substrate specificity or conversion rate.

[0018] According to another aspect of the present application, the enzyme library described above is applied in enzyme storage and / or high-throughput screening of enzymes.

[0019] Compared with the enzyme library stored in the form of protein, the enzyme library stored in the form of DNA has high stability and long storage time, and from the perspective of industrial application, the storage amount and storage space can be reduced. In addition, the construction method of the enzyme library of the present application can directly obtain enzymes by using the enzyme library stored in the form of DNA in cooperation with the protein in vitro expression system, thereby simplifying the preparation process of the enzyme library in the form of protein and saving fermentation and storage resources; and the expression product can be directly used for enzyme screening without isolation, which is simple to operate and has better parallelism. In addition, it is not affected by the difficulty in expressing some proteins or the difficulty in obtaining expression products due to the toxicity to cells. BRIEF DESCRIPTION OF DRAWINGS

[0020] The drawings accompanying the specification of the present application form a part thereof, serve to provide further understanding of the present application, and together with the description of the exemplary embodiments of the present application and the explanation thereof serve to explain the present application, and do not constitute improper limitations on the present application. In the drawings:

[0021] Figure 1 shows a standard curve diagram drawn by sfGFP as a model protein in different concentrations in the experiment of optimizing in vitro expression system according to the present application;

[0022] Figure 2 shows the result diagram of Mg 2+ concentration optimization in the experiment of optimizing in vitro expression system according to the present application;

[0023] Figure 3 shows the result diagram of PEP concentration optimization in the experiment of optimizing in vitro expression system according to the present application;

[0024] Figure 4 shows the result diagram of cell extract proportion optimization in the experiment of optimizing in vitro expression system according to the present application;

[0025] Figure 5 shows the result diagram of NAD concentration optimization in the experiment of optimizing in vitro expression system according to the present application;

[0026] Figure 6 shows the result diagram of glutamine concentration optimization in the experiment of optimizing in vitro expression system according to the present application;

[0027] Figure 7 shows the result diagram of in vitro protein expression of sfGFP gene with different amounts of PCR products in Example 1 of the present application;

[0028] Figure 8 shows the result diagram of in vitro protein expression of PCR products of sfGFP gene including different lengths upstream of the start codon in Example 1 of the present application;

[0029] Figure 9 shows the result diagram of in vitro expression of protein sfGFP with plasmid as enzyme library storage form in Example 3 according to the present application;

[0030] Figure 10 shows the influence of different plasmid concentrations on the PCR product of protein sfGFP in Example 4 according to the present application;

[0031] Figure 11 shows the influence of different PCR product concentrations on in vitro expression of protein sfGFP in Example 4 according to the present application;

[0032] Figure 12 shows the influence of different concentrations of 20 amino acids on the synthesis amount of protein sfGFP in Example 5 according to the present application. DETAILED DESCRIPTION

[0033] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments.

[0034] As mentioned in the background section, the enzyme library in the prior art is generally in the form of enzyme solution or enzyme lyophilized powder (i.e., protein in liquid or solid powder form), to improve the poor storage stability of the enzyme library in this form, the present application provides a solution, i.e., the enzyme library is preserved in the form of plasmid or PCR product, and then added to the protein in vitro expression system through the plasmid, or the target gene fragment is obtained through PCR, and then the protein in vitro expression is carried out using the PCR product as a template, and the obtained enzyme can be directly used for screening without separation. When re-screening is needed, only a part of the plasmid template or the PCR product can be used. Since the plasmid or the PCR product is stored in the form of DNA, its storage stability is much higher than that of protein. Moreover, the amount of plasmid required for producing protein is only about 1 / 1000 of the amount of protein, so it can be stored for a long time, and the storage amount can be reduced, and the process of preparing the enzyme library is simplified, the fermentation and storage resources are saved, and the operation is simple and the parallelism is better.

[0035] On the basis of the above research findings, the applicant proposes the technical solution of the present application. In a typical embodiment, an enzyme library is provided, which comprises a plurality of enzymes, each of which is stored in the form of a respective corresponding DNA molecule, which is a plasmid or a PCR product.

[0036] As mentioned above, compared with the enzyme library stored in the form of protein, the use of DNA form storage not only has high stability and long storage time, but from the perspective of industrial application, it can reduce the storage amount and save storage space. At the same time, it can be directly obtained by cooperating with the protein in vitro expression system, which simplifies the process of preparing the enzyme library, saves fermentation and storage resources; and the expression product can be directly used for enzyme screening without separation, which is simple to operate and has better parallelism. In addition, it is not affected by the influence of some proteins that are difficult to express or have toxicity to cells and are difficult to obtain expression products.

[0037] The enzyme in the above-mentioned enzyme library is preferably an industrial enzyme. At present, industrial enzymes are mostly stored in the form of enzyme solution or enzyme lyophilized powder. From the perspective of industrial application, the industrial enzymes used for high-throughput screening are stored in the form of DNA molecules, which has more industrial practical value. The specific industrial enzyme is not particularly limited in the present application, and any enzyme used for biocatalysis and / or biotransformation in industry is suitable for the present application. In a preferred embodiment, the industrial enzyme comprises any one or more of the following proteases: transaminase, ketoreductase, lipase or monooxygenase; more preferably, the industrial enzyme comprises any one or more of the following proteases: transaminase Asym-6890, ketoreductase Asym-2016 or ketoreductase as shown in any one of SEQ ID Nos: 6-25, lipase Asym-7231 and monooxygenase Monooxygeanse Asym-7047.

[0038] The amino acid sequence of the transaminase Asym-6890 is shown as SEQ ID NO: 1:

[0039] The amino acid sequence of the ketoreductase Asym-2016 is shown as SEQ ID NO: 2:

[0040] The amino acid sequence of the lipase Asym-7231 is shown as SEQ ID NO: 3:

[0041] The amino acid sequence of the monooxygenase Asym-7047 is shown as SEQ ID NO: 4:

[0042] It should be noted that the enzyme stored in the form of DNA molecule has low cost, occupies less resource or needs less freezing resource.

[0043] In a second typical embodiment, a method for constructing an enzyme library (in the form of protein) is provided, which comprises: expressing a plurality of different DNA molecules in a protein in vitro expression system respectively, the plurality of different DNA molecules respectively encoding different mutants of the same enzyme, to obtain an expression product corresponding to each mutant; and the expression products corresponding to the plurality of mutants collectively constituting the enzyme library of the enzyme; wherein the plurality of DNA molecules exist in the form of plasmids or PCR products.

[0044] The method for constructing the enzyme library uses the enzyme in the form of DNA to express the enzyme in the form of protein in the protein in vitro expression system to form the enzyme library. Such a method for constructing the enzyme library can be synthesized on demand, and on the basis of high and stable enzyme activity, it not only reduces the problem of poor long-term storage stability in the form of protein, but also reduces the storage space and cost.

[0045] The existing in vitro protein expression system can also be used in the present application. In order to further improve the in vitro expression effect, in a preferred embodiment, the in vitro protein expression system is an E. coli in vitro expression system or a yeast in vitro expression system; preferably, the E. coli in vitro expression system comprises: a basic component, an energy-related component, an additive component, a cell extract, and an RNase inhibitor, wherein, in the E. coli in vitro expression system, the basic component comprises: 19 kinds of amino acids, each at a concentration of 2 mM; 2 mM tyrosine; 14 mM magnesium acetate; 60 mM potassium acetate; and 7 mM DDT; in the E. coli in vitro expression system, the energy-related component comprises: 1.2 mM AMP, 0.85 mM CMP, 0.85 mM GMP, 0.85 mM UMP, 15-83 mM PEP, 0.4-0.6 mM NAD, 4 mM potassium oxalate, 90 mM potassium glutamate, and 2.5-10 mM magnesium glutamate; in the E. coli in vitro expression system, the additive component comprises: 1.5 mM spermidine and 157.33 mM HEPES; in the E. coli in vitro expression system, the concentration of the RNase inhibitor is 150 U / 450 μL; and the volume content of the cell extract in the E. coli in vitro expression system is 20-60%.

[0046] Compared with the in vitro protein expression system in the prior art, the above-mentioned preferred in vitro protein expression system of the present application can improve the expression amount of the protein. The above-mentioned cell extract refers to an E. coli cell extract, which mainly comprises ribosomes, RNA polymerase, transcription and translation proteins, and enzymes and cofactors for energy metabolism.

[0047] In order to further improve the expression amount of the protein, in a preferred embodiment, in the E. coli in vitro expression system, the concentration of PEP is 30 mM; preferably, the content of NAD is 0.4 mM; preferably, the content of magnesium glutamate is 7.5 mM; and preferably, the volume content of the cell extract in the E. coli in vitro expression system is 33.3%.

[0048] In the prior art, there are also individual documents reporting in vitro protein expression systems, and the protein expression amount is generally low, and in addition to model proteins such as green fluorescent protein GFP or its variants, other biocatalytic enzyme proteins are rarely reported. The protein yield of the commercially available in vitro expression kit is also very low, only at the level of tens of mg / mL, which can be used for proteomics research, but due to the too small amount of protein, it is difficult to meet the catalytic application requirements of industrial enzymes. The above-mentioned in vitro protein expression system optimized by the present application can achieve a yield of 1-2 mg / mL for many proteins without special optimization, and further optimization is expected to obtain higher protein expression levels, thereby meeting the needs of enzyme high-throughput screening.

[0049] The enzymes in the enzyme library can be any protease, and more preferably, an industrial enzyme. In some preferred embodiments, the industrial enzyme comprises any one or more of the following proteases: transaminase, ketoreductase, lipase, and monooxygenase, and more preferably, is selected from any one or more of the following: transaminase Asym-6890 (the amino acid sequence is shown in SEQ ID NO: 1), ketoreductase Asym-2016 (the amino acid sequence is shown in SEQ ID NO: 2), lipase Asym-7231 (the amino acid sequence is shown in SEQ ID NO: 3), and monooxygenase Monooxygenase Asym-7047 (the amino acid sequence is shown in SEQ ID NO: 4), or a ketoreductase shown in any one of SEQ ID Nos: 6-25.

[0050] In a third typical embodiment, a high-throughput screening method of enzymes is provided, which comprises: expressing a plurality of different DNA molecules in a protein in vitro expression system, respectively, the plurality of different DNA molecules encoding different mutants of the same enzyme, respectively, to obtain an expression product corresponding to each mutant; the expression products corresponding to the plurality of mutants collectively forming an enzyme library of the enzyme; and screening the performance of each enzyme in the enzyme library using the same substrate; wherein the plurality of DNA molecules exist in the form of plasmids or PCR products.

[0051] The high-throughput screening method of enzymes is not directly screening the enzymes having catalytic activity on the substrate from the enzyme library in the form of proteins, but is screening the enzyme activity by expressing the enzymes in the form of DNA in the screening process using the protein in vitro expression system. Although this method needs to express the enzyme library to be screened first, this method of screening enzymes not only does not need to save the enzymes in the form of proteins, but also has high activity of the enzymes expressed on site, and does not cause the decrease of enzyme activity due to long storage time.

[0052] In a preferred embodiment, the performance of each enzyme can be directly screened without separation and purification of the expression product corresponding to each enzyme (without separation and purification of the enzyme, the production efficiency can be greatly improved in industrial production), and preferably, the screening method comprises a further screening step in addition to the performance screening, wherein the further screening step comprises: taking the plasmid or PCR product of the corresponding enzyme, expressing it in the protein in vitro expression system to obtain a further screening expression product; and directly mixing and incubating the further screening expression product with the substrate to perform performance further screening (the corresponding enzyme is expressed again from the DNA to perform further screening, which can ensure the stability of the enzyme activity in the further screening process, so that the enzyme is synthesized and used on site, and not stored for a certain period of time before further screening, i.e., the difference between the two screenings is a certain period of time, which may have a certain impact on the activity of the enzyme saved in the form of protein before and after the two screenings.

[0053] In the present application, the specific screening criteria or index during the high-throughput screening of enzymes can be determined according to actual needs. In a preferred embodiment, the performance of the enzyme includes any one or more of the following: catalytic activity, substrate specificity, or conversion rate.

[0054] In a fourth typical embodiment, the application provides the use of the enzyme library described above in enzyme storage and / or high-throughput screening. The use of the preserved enzyme library in the form of the DNA molecule of the present application has the advantages of higher stability, simple and efficient screening process, and is more suitable for application in high-throughput screening of enzymes, in addition, it also has the advantages of small storage space and lower cost.

[0055] The beneficial effects of the present application will be further illustrated in conjunction with specific examples. It should be noted that in the following examples, the in vitro protein expression system used is the in vitro protein expression system of Escherichia coli (see Table 1, last column, system pH is 7-8, generally pH 7.5. Reaction time is 3-16h, generally 4h), and sfGFP (superfolder Green fluorescent protein) is used as an example for screening. Table 1 is obtained by optimizing the in vitro expression system (system 1 to system 3) in the prior art.

[0056] Table 1:

[0057] The cell extract in the above table is obtained by the following method:

[0058] Activate the BL21 Star (DE3) strain and streak out single colonies. Inoculate the activated BL21 Star (DE3) monoclonal into 50 ml of LB liquid medium, incubate at 37°C, 200 rpm overnight. Incubate the overnight culture of BL21 Star (DE3) in 400 ml of 2xYT medium to an initial OD600 = 0.1. Add IPTG to a final concentration of 0.5 mM and incubate at 37°C to an OD600 = 3.8-4.0. Collect the bacterial slurry: 5000g, 10°C centrifuge for 10 min. Slowly pour the supernatant and transfer the bacterial slurry to a 50 ml pre-cooled centrifuge tube. Add 30 ml of S30 buffer to the 50 ml centrifuge tube to resuspend the cells. 5000g, 10°C centrifuge for 10 min, remove the supernatant, and dry the water in the centrifuge tube with a clean filter paper. Add 1 ml of pre-cooled S30 buffer to 0.6 grams of bacterial slurry. Resuspend the cells, sonicate, and add 65 μl of 1M DTT to 5 ml of cell lysate. 12000 rpm, 4°C centrifuge for 10 min. After aliquoting, store at -80°C for use.

[0059] The steps for establishing and optimizing the cell-free protein synthesis system (also referred to as the protein in vitro expression system in this application) (1 mL system) are as follows:

[0060] Table 2:

[0061] The final concentrations of Solution A in Table 2 are: 1.2 mM ATP, 0.85 mM GMP, 0.85 mM UMP, 0.85 mM CMP, 31.50 ug / mL folinic acid, 170.60 ug / mL tRNA, 0.40 mM NAD, 0.27 mM cofactor A (CoA), 4 mM oxalic acid, 1 mM diammonium phosphate, 1.50 mM spermidine, and 57.33 mM HEPES buffer.

[0062] The final concentration of Solution B is: 10mM Mg(Glu)2, 10mM NH4(Glu), 130mM K(Glu), 2mM 20 amino acids, 0.03M phosphoenolpyruvate (PEP).

[0063] The system was reacted at 30°C and 220 rpm for 16 h.

[0064] Taking sfGFP as the model protein, the in vitro expression system in Table 2 was optimized with reference to the existing in vitro expression systems (systems 1 to 3) in Table 1, specifically including the optimization of Mg 2+ Optimization of the concentration of , optimization of the concentration of PEP, optimization of the proportion of cell extract in the entire reaction system, optimization of the dosage of NAD and optimization of the concentration of glutamate.

[0065] Fluorescence intensity was measured using excitation at 485 nm and emission at 525 nm in a 96-well plate. A 50 μL volume was used to measure the fluorescence intensity. The standard curve is shown in Figure 1. The results obtained under optimized conditions for each parameter are shown in Figures 2 to 6.

[0066] 1) System Mg 2+ The optimization results of the concentration of Mg are shown in Figure 2. 2+ sfGFP can be produced in vitro by protein expression systems with concentrations ranging from 2.5mM to 19.5mM. 2+ The effect is better when the concentration is 2.5mM to 10mM, and the optimal concentration is around 7.5mM.

[0067] 2) The results of system PEP concentration optimization are shown in Figure 3. The protein in vitro expression system can produce sfGFP when the PEP concentration is between 5mM and 83mM. When the PEP concentration is between 15mM and 83mM, the protein synthesis amount is better, and the sfGFP protein synthesis amount is the highest when the PEP concentration is 30mM.

[0068] 3) The proportion of cell extract in the whole reaction system is optimized as shown in Figure 4. The proportion of cell extract in the whole system can be 20% to 60% to produce the target protein, and a better result can be obtained when the proportion is more than 33%.

[0069] 4) The dosage of NAD is optimized as shown in Figure 5. NAD plays an important role in energy cycle. The expression is best when the concentration of NAD is 0.6 mM, and there is no big difference with 0.4 mM.

[0070] 5) The concentration of glutamine is optimized as shown in Figure 6. From the experimental results, glutamine does not play any role in the whole experiment, and the effect is better without adding glutamine.

[0071] Therefore, the in vitro expression system of the present application in Table 1 is obtained after optimization of the above parameters. The pH of the system is 7-8, generally pH 7.5. The reaction time is 3-16 h, generally 4 h.

[0072] Example 1

[0073] In 450 μL of the in vitro protein expression system (the system of the present application in Table 1), the PCR product of the sfGFP gene is used as the DNA template, and different amounts of PCR products are added for in vitro protein expression. As shown in Figure 7, the amount of protein produced by the in vitro protein expression system is relatively equal when the amount of PCR product is more than 22.5 μL to 90 μL, which indicates that the amount of PCR product in this range can produce target protein in parallel.

[0074] Further, the PCR product of the sfGFP gene is used as the DNA template, and different PCR products include different lengths upstream of the start codon, including 0 bp, 50 bp, 100 bp, 115 bp, 130 bp and 140 bp. When these PCR products are used as the DNA template in the reaction system, the length upstream of the start codon has little effect on the expression of sfGFP protein as shown in Figure 8. When the length upstream of the start codon is 50-100 bp, the amount of protein expression is the highest.

[0075] Example 2

[0076] The traditional enzyme library is constructed by expressing proteins in E. coli intracellularly, and then obtaining enzyme solution by breaking. Since the expression level of many proteins in E. coli is not ideal, the results obtained by enzyme library screening cannot truly reflect the advantages and disadvantages of the enzyme itself. In comparison, the E. coli in vitro protein expression system widens the range of enzyme library. It can not only express proteins that cannot be expressed in vivo, but also express proteins that are expressed as inclusion bodies in vivo.

[0077] Because the intracellular environment of E. coli is reductive, which is not conducive to the correct formation of disulfide bonds, E. coli expresses some proteins in the form of inclusion bodies. For other proteins, because the protein itself has a certain toxic effect on the host bacteria, it leads to little expression in vivo, or even no expression. The in vitro protein expression system is a non-reducing environment, which is more conducive to the correct formation of disulfide bonds and thus improves the synthesis level of disulfide bond-containing proteins. Moreover, without the obstruction of the cell membrane, the growth of living cells is not needed, so the expression effect of toxic proteins is better than that of intracellular expression.

[0078] In addition, in order to improve the expression level of proteins, the prior art also uses protein expression tags to express the target proteins. Among them, SUMO is a protein expression tag, which is essentially a small ubiquitin-like modifier protein. It is one of the important members of the ubiquitin (ubiquitin) polypeptide chain superfamily. Studies have found that SUMO can be used as a fusion tag and a molecular chaperone for recombinant protein expression. It not only can further improve the expression level of the fusion protein, but also has the functions of resisting protease hydrolysis, promoting the correct folding of the target protein, and improving the solubility of the recombinant protein. In addition, SUMO also has an important application, that is, it can be used to completely remove the tag protein to obtain a natural protein. Because the SUMO protease can recognize the complete SUMO tag protein sequence and can efficiently cut the SUMO from the fusion protein. After removing the SUMO, through affinity chromatography, the tag protein part is removed, and the recombinant protein which is the same as the natural protein can be obtained. Therefore, the SUMO tag is often used together with other tags as a specific enzyme hydrolysis site.

[0079] Enfuvirtide is a 36-amino-acid polypeptide active substance, and SUMO+Enfuvirtide is a fusion-expressed protein. SUMO-Enfuvirtide cannot be expressed in the intracellular environment of E. coli, and even after adjusting the expression conditions, it cannot be expressed. This may be due to the instability of its mRNA structure. However, when using the in vitro protein expression system in the prior art (i.e. system 3 in Table 2 described above) to express it, the protein expression level is 1 mg / mL.

[0080] Transaminase is a kind of biological enzyme with high efficiency and wide application. However, some transaminases are expressed in the form of inclusion bodies or not expressed in E. coli. Transaminase Asym-6890 (amino acid sequence as shown in SEQ ID NO: 1) is expressed in the form of inclusion bodies in E. coli. Reducing the expression temperature (for example, 16℃) can partially express soluble expression, and the soluble expression amount is only about 10wt%. Using the in vitro protein expression system in the prior art (i.e. system 3 in Table 1) to express it, the soluble expression is significantly improved, and the expression amount can reach 1.5mg / mL. See the following table for details.

[0081] Table 3:

[0082] Example 3

[0083] Green fluorescent protein (GFP for short) is a protein composed of about 238 amino acids, which can be excited from blue light to ultraviolet and emit green fluorescence. In cell biology and molecular biology, the green fluorescent protein (GFP) gene is often used as a reporter gene. sfGFP is the abbreviation of superfolder GFP, which is a well-designed folding version of GFP (compared with the 238 amino acid GFP, its amino acid sequence has the following 5 point mutations: F99S, M153T, V163A, F64L, S65T), which shows greater tolerance to chemical denaturants and extreme temperatures.

[0084] Using sfGFP as the target protein, using the pJL1-sfGFP plasmid (the specific nucleic acid sequence of the pJL1 plasmid is SEQ ID NO: 5) containing the gene as the enzyme storage form, using the in vitro expression system of the present application in Table 1 (pH = 7.5, reaction temperature is 16℃, reaction time is 4h) for in vitro expression, the results are shown in Figure 9.

[0085] The nucleic acid sequence of the pJL1 plasmid is shown in SEQ ID NO: 5:

[0086] Storage in the form of plasmid reduces the amount of protein that needs to be stored by 1 / 1000; on the other hand, the storage stability of plasmid is higher than that of protein, and the stability difference between different plasmids is not obvious. In addition, even if the concentration of the plasmid has decreased to a certain extent during storage, the concentration of the plasmid has a certain difference between the plasmids. As can be seen from Figure 9, the in vitro protein expression level does not differ much within a very wide range of plasmid concentrations (for example, 1ng / μL to 16ng / μL), and will remain uniform.

[0087] As can be seen from Fig. 9, the effect of plasmid concentration on protein synthesis shows that the in vitro protein expression system can express the target protein sfGFP with good parallelism when the plasmid concentration is kept in the range of 2-16 ng / μL.

[0088] Example 4

[0089] In addition to using the stored plasmid itself, the PCR product obtained by one-step PCR amplification using the plasmid can also be directly used for protein production in the in vitro protein expression system, which can further reduce the amount of plasmid used to less than 1 / 1000.

[0090] In this example, the PCR product of pJLl-sfGFP plasmid is used as a template for in vitro protein expression, and the in vitro expression system of the present application in Table 1 is used for expression, and the results are shown in Figs. 10 and 11.

[0091] As shown in Fig. 10, the effect of plasmid concentration on the PCR product shows that the amount of plasmid is kept in the range of 0.5 ng to 10 ng, and uniform bands can be obtained by PCR. Thus, when the storage time of the plasmid is longer, if there is some degradation of the plasmid, which causes uneven use of the plasmid, it will not affect the parallelism of the final results.

[0092] As shown in Fig. 11, the effect of PCR product concentration on protein synthesis shows that the in vitro protein synthesis system can express the target protein sfGFP with good parallelism when the amount of PCR product added is kept in the range of 22.5 μL to 90 μL.

[0093] Example 5

[0094] In this example, the effect of different concentrations of 20 amino acids on the synthesis of protein sfGFP in the in vitro protein expression system of the present application shown in Table 1 is further detected, and the results are shown in Fig. 12. As can be seen from Fig. 12, after the concentration of amino acids exceeds 1 mM, there is a higher synthesis capacity, and it is relatively parallel.

[0095] Example 6

[0096] In order to further detect the applicability of this method to other industrial enzymes, five different types of proteins are selected in this example for comparison of the storage stability of their plasmids and proteins.

[0097] The stability of the plasmid is represented by the comparison of the plasmid concentration after 1 year of storage with the initial concentration, and the stability of the protein is represented by the comparison of the protein activity after 1 year of storage with the initial activity. As shown in the following table, the storage stability of the plasmid is good, and there is no obvious decrease after 1 year of storage, while the stability of the protein is obviously lower, and the storage stability of different types of proteins is obviously different.

[0098] Table 4:

[0099] The sequence of SUMO-Enfuvirtide is as follows:

[0100] The gene sequence (SEQ ID NO: 47) is as follows:

[0101] The protein sequence (SEQ ID NO: 46) is as follows:

[0102] Example 7

[0103] Twenty kinds of ketoreductase (fermentation enzyme liquid freeze-dried powder, amino acid sequences as shown in SEQ ID NOs: 6-25, nucleotide sequences as shown in SEQ ID NOs: 26-45) prepared by a conventional method stored for 3 months and the in vitro protein expression system of the present application shown in Table 1 were used to screen the target compound ethyl acetoacetate, respectively.

[0104] The reaction conditions were as follows: reaction system 1 mL, 0.1M phosphate buffer pH 8.0, substrate ethyl acetoacetate 10 mg, coenzyme NAD / NADP each 1 mg, isopropyl alcohol 10 μL, freeze-dried enzyme powder dosage 1 mg, in vitro protein expression system 300 μL.

[0105] The screening results are shown in Table 5. As shown in the results of Table 5, the in vitro protein expression system (enzyme freeze-dried powder is replaced by pJL-ketoreductase plasmid, wherein the reaction system is 1 mL, 0.1M phosphate buffer pH 8.0, substrate ethyl acetoacetate 10 mg, coenzyme NAD / NADP each 1 mg, isopropyl alcohol 10 μL, in vitro protein expression system 300 μL) and the conventional method under the optimal conditions (reaction system 1 mL, 0.1M phosphate buffer pH 8.0, substrate ethyl acetoacetate 10 mg, coenzyme NAD / NADP each 1 mg, isopropyl alcohol 10 μL, freeze-dried enzyme powder dosage 1 mg) obtained the screening results are consistent, and the enzyme screening results obtained by the in vitro protein expression system are reliable.

[0106] Table 5:

[0107] The amino acid sequences of the 20 kinds of ketoreductases are as follows:

[0108] KRED1---SEQ ID NO: 6:

[0109] KRED2---SEQ ID NO: 7:

[0110] KRED3---SEQ ID NO: 8:

[0111] KRED4 - SEQ ID NO: 9:

[0112] KRED5 - SEQ ID NO: 10:

[0113] KRED6 - SEQ ID NO: 11:

[0114] KRED7 - SEQ ID NO: 12:

[0115] KRED8 - SEQ ID NO: 13:

[0116] KRED9 - SEQ ID NO: 14:

[0117] KRED10 - SEQ ID NO: 15:

[0118] KRED11 - SEQ ID NO: 16:

[0119] KRED12 - SEQ ID NO: 17:

[0120] KRED13 - SEQ ID NO: 18:

[0121] KRED14 - SEQ ID NO: 19:

[0122] KRED15 - SEQ ID NO: 20:

[0123] KRED16 - SEQ ID NO: 21:

[0124] KRED17 - SEQ ID NO: 22:

[0125] KRED18 - SEQ ID NO: 23:

[0126] KRED19 - SEQ ID NO: 24:

[0127] KRED20 - SEQ ID NO: 25:

[0128] The nucleotide sequences of the above 20 kinds of ketoreductases are as follows, respectively:

[0129] KRED1 - SEQ ID NO: 26:

[0130] KRED2 - SEQ ID NO: 27:

[0131] KRED3 - SEQ ID NO: 28:

[0132] KRED4 - SEQ ID NO: 29:

[0133] KRED5 - SEQ ID NO: 30:

[0134] KRED6 - SEQ ID NO: 31:

[0135] KRED7 - SEQ ID NO: 32:

[0136] KRED8 - SEQ ID NO: 33:

[0137] KRED9 - SEQ ID NO: 34:

[0138] KRED10 - SEQ ID NO: 35:

[0139] KRED11 - SEQ ID NO: 36:

[0140] KRED12 - SEQ ID NO: 37:

[0141] KRED13 - SEQ ID NO: 38:

[0142] KRED14 - SEQ ID NO: 39

[0143] KRED15 - SEQ ID NO: 40

[0144] KRED16 - SEQ ID NO: 41

[0145] KRED17 - SEQ ID NO: 42

[0146] KRED18 - SEQ ID NO: 43

[0147] KRED19 - SEQ ID NO: 44

[0148] KRED20 - SEQ ID NO: 45

[0149] From the above description, it can be seen that the above-mentioned embodiments of the present application achieve the following technical effects:

[0150] 1) The enzyme solution or enzyme powder stored in the enzyme library is replaced by a plasmid which is more stable and has less demand.

[0151] 2) Parallel enzyme data can be obtained by using plasmids and PCR products.

[0152] 3) The screening data of the enzyme library of the present application are consistent with the screening data obtained by traditional methods.

[0153] 4) The present application can be used for enzyme library construction and preservation of enzymes with poor storage stability.

[0154] 5) The evolution method provided by the present application saves more than 90% of the enzyme library construction cost and more than 90% of the enzyme library storage cost compared with traditional experiments.

[0155] 6) The evolution method provided by the present application is simple in steps and has better parallel data.

[0156] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for constructing an enzyme library, characterized by, The construction method comprises: putting a plurality of different DNA molecules into a protein in vitro expression system for expression, the plurality of different DNA molecules encoding different mutants of the same enzyme respectively, and obtaining expression products of each mutant; a collection of expression products of the plurality of mutants constitutes an enzyme library of the enzyme; wherein the plurality of DNA molecules exist in the form of plasmids or PCR products.

2. The construction method of claim 1, wherein, The protein in vitro expression system is an E. coli in vitro expression system or a yeast in vitro expression system; The E. coli in vitro expression system comprises: basic components, energy-related components, additive components, cell extracts and RNase inhibitors, wherein, In the E. coli in vitro expression system, the basic components comprise: 19 kinds of amino acids, each at a concentration of 2 mM; 2 mM tyrosine; 14 mM magnesium acetate; 60 mM potassium acetate; and 7 mM DDT; In the E. coli in vitro expression system, the energy-related components comprise: 1.2 mM AMP, 0.85 mM CMP, 0.85 mM GMP, 0.85 mM UMP, 15-83 mM PEP, 0.4-0.6 mM NAD, 4 mM potassium oxalate, 90 mM potassium glutamate, and 2.5-10 mM magnesium glutamate; In the E. coli in vitro expression system, the additive components comprise: 1.5 mM spermidine and 157.33 mM HEPES; In the E. coli in vitro expression system, the concentration of the RNase inhibitor is 150 U / 450 μL; The volume content of the cell extract in the E. coli in vitro expression system is 20-60%.

3. The construction method of claim 2, wherein, In the E. coli in vitro expression system, the concentration of PEP is 30 mM; The content of NAD is 0.4 mM; The content of magnesium glutamate is 7.5 mM; The volume content of the cell extract in the E. coli in vitro expression system is 33.3%.

4. The construction method according to any one of claims 1 to 3, characterized in that, The enzyme in the enzyme library is an industrial enzyme.

5. The construction method according to claim 4, characterized in that, The industrial enzyme comprises any one or more of the following proteases: transaminase, ketoreductase, lipase, or monooxygenase.

6. The construction method of claim 4, wherein, The industrial enzyme comprises a transaminase with an amino acid sequence as shown in SEQ ID NO: 1, a ketoreductase with an amino acid sequence as shown in SEQ ID NO: 2 or any one of SEQ ID Nos: 6-25, a lipase with an amino acid sequence as shown in SEQ ID NO: 3, or a monooxygenase with an amino acid sequence as shown in SEQ ID NO:

4.

7. A method of high-throughput screening of enzymes, characterized in that, The screening method comprises: performing performance screening on each mutant in the enzyme library using the same substrate; wherein the enzyme library is constructed by the construction method of any one of claims 1-6.

8. The screening method according to claim 7, characterized in that, Without separating and purifying the expression product of each mutant, the performance screening is directly performed, and the performance screening comprises screening any one or more of the following performances: catalytic activity, substrate specificity, or conversion rate.

9. The screening method according to claim 7, characterized by, On the basis of the performance screening, the screening method further comprises re-screening, which comprises: The plasmid or PCR product of the mutant of the corresponding enzyme selected is selected and placed in the protein in vitro expression system for expression, and the re-screening expression product is obtained; The re-screening expression product is directly mixed with the substrate for re-screening incubation.

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