Escherichia coli genetically engineered bacterium and high-throughput screening method of enzyme

Abstract

The invention relates to the technical field of bioengineering and protein engineering, in particular to an escherichia coli genetically engineered bacterium and a high-throughput screening method of an enzyme. According to the high-throughput screening method of the enzyme, the enzyme is displayed on the surface of escherichia coli, and the surface display efficiency of the enzyme is remarkably improved by optimizing an escherichia coli curli fimbriae system, so that the screening efficiency of the enzyme is improved, the screening steps are reduced, and the screening cost is reduced. The method solves the problems of need of cell disruption, complex operation, easy reduction of protein activity and low screening efficiency of a traditional screening method in an enzyme directed evolution process, and has the advantages of no need of cell disruption, simple and easy operation, high flux, specific and accurate enzyme activity determination, wide enzyme activity detection range, high screening efficiency and the like; good application prospects are realized.

Description

Technical Field

[0001] This invention relates to the fields of bioengineering and protein engineering, and in particular to a high-throughput screening method for genetically engineered Escherichia coli and enzymes. Background Technology

[0002] Enzyme activity is a key factor affecting enzyme catalysis or biocatalytic reactions. Directed evolution and high-throughput screening are important techniques for obtaining highly active enzymes. After obtaining enzyme mutant libraries through methods such as directed evolution, efficient screening methods are needed to quickly obtain highly active enzymes. However, currently, many intracellular expression enzymes still require cell disruption to detect activity during mutant library screening. Cell disruption is not only time-consuming and inefficient, but it can also adversely affect enzyme activity. If enzyme activity could be detected without disrupting cells, the efficiency of enzyme screening would be greatly improved.

[0003] Escherichia coli (E. coli) surface display technology enables the display of target proteins on the cell surface, allowing enzymatic reactions to be carried out using the bacterial cells as biocatalysts without the need for cell disruption and enzyme extraction. Studies have reported on the use of the E. coli curli pili system to display target proteins on the surface of E. coli biofilms. The synthetic pathway of E. coli curli pili mainly includes CsgA, CsgB, CsgC, CsgD, CsgE, CsgF, and CsgG; among them, CsgA protein is a major component of the K-12 E. coli biofilm curli system. However, the amount of protein currently displayed on the surface of the E. coli curli pili system remains low. To improve enzyme screening efficiency, it is necessary to further increase the amount of protein displayed on the surface of the E. coli curli pili system.

[0004] 2,5-Furandicarboxylic acid (FDCA) is an important bio-based C6 platform compound that can replace terephthalic acid (TPA) in the production of polyethylene terephthalate (PET), and further be used to produce a biodegradable and higher-performance bio-based material—polyethylene furan-dicarboxylate (PEF). However, the key enzyme in the biocatalytic production of FDCA from 5-hydroxymethylfurfural (HMF), 5-hydroxymethylfurfural oxidase (HMFO), suffers from low catalytic efficiency and poor thermal stability. To address the shortcomings of HMFO, directed evolutionary techniques can be used to screen for HMFO mutants more suitable for industrial applications. Developing efficient, high-throughput screening methods is crucial in this process.

[0005] Currently, the main screening methods for directed evolution of HMFO include the ThermoFAD assay and the vanillin colorimetric method. The former mainly uses the fluorescence change caused by the release of flavin cofactors in HMFO due to protein denaturation at higher temperatures to characterize the thermal stability of HMFO, but it can only characterize the structural changes of the protein and cannot characterize enzyme activity. The latter uses vanillin as a substrate and uses its own absorbance for detection, but this method cannot accurately characterize the HMFO-catalyzed HMF activity due to the different substrates. In addition, both of the above methods require cell lysis to release HMFO first. Summary of the Invention

[0006] This invention provides a high-throughput screening method for genetically engineered Escherichia coli and enzymes.

[0007] In a first aspect, the present invention provides a high-throughput screening method for enzymes, the method comprising: displaying the enzyme to be screened on the surface of Escherichia coli, using the Escherichia coli displaying the enzyme to be screened on the surface as a biocatalyst to catalyze the enzymatic reaction of the enzyme, and screening the enzyme by detecting the enzymatic reaction products; The process of displaying the enzyme to be screened on the surface of E. coli includes: constructing a mutant library of the enzyme using the encoding gene of the fusion protein of E. coli CsgA protein and the enzyme as a template; introducing the mutant library into E. coli genetically engineered bacteria; inactivating the CsgA gene in the E. coli genetically engineered bacteria, and enhancing the expression of the CsgB and / or CsgG genes.

[0008] The high-throughput screening method provided by this invention solves the problem of needing to first lyse E. coli to release the enzyme before performing an enzymatic reaction to detect enzyme activity by fusing a mutant library of the enzyme with the CsgA protein and displaying it on the surface of E. coli. This significantly improves the screening efficiency and throughput of mutant libraries. To increase the amount of enzyme displayed on the surface of E. coli, this invention regulates the curli pili system of E. coli and found that enhancing the expression of CsgB and CsgG genes alone or simultaneously can significantly increase the content of CsgA protein on the surface of E. coli, thereby increasing the amount of enzyme displayed on the surface of E. coli and improving the enzyme screening efficiency.

[0009] Compared to enhancing the expression of the CsgG gene alone, enhancing the expression of the CsgB gene alone, or simultaneously enhancing the expression of both the CsgB and CsgG genes, has a more effective effect on increasing the display level of the enzyme on the surface of *E. coli*. Therefore, preferably, the CsgA gene of the genetically engineered *E. coli* is inactivated, and the expression of the CsgB and CsgG genes is enhanced, or the expression level of CsgB is enhanced.

[0010] In this invention, enhancing the expression of CsgB and CsgG genes can be achieved through the following methods (1) and / or (2): (1) Increase the copy number of the gene; (2) Enhance the activity of transcriptional and / or translational regulatory elements of the gene.

[0011] In (1) above, increasing the copy number of the gene can be achieved by inserting the gene into the genome of Escherichia coli, or by introducing an expression plasmid carrying the gene.

[0012] In (2) above, the transcriptional regulatory elements include promoters, enhancers, etc. The translational regulatory elements include ribosome binding sites, etc. Enhancing the activity of the transcriptional and / or translational regulatory elements of the gene includes replacing the promoter of the gene with a more active promoter.

[0013] In some embodiments of the present invention, the expression is enhanced by introducing an expression vector containing the CsgB and / or CsgG genes.

[0014] Preferably, in the expression vector, the promoters of the CsgB and / or CsgG genes are constitutive promoters. These constitutive promoters can be any strong constitutive promoter found in *Escherichia coli*.

[0015] Preferably, the constitutive promoter is a Trc promoter.

[0016] Preferably, the expression vector is constructed using the pET28 series vector as a backbone.

[0017] Preferably, the expression vector is constructed using the pET28 series vector as a backbone by linking an expression cassette consisting of a constitutive promoter and CsgB and / or CsgG genes.

[0018] In this invention, the inactivation of the CsgA gene can be achieved by inserting, deleting, or replacing one or more bases in the CsgA gene.

[0019] In some embodiments of the present invention, the CsgA gene is inactivated by knocking out the CsgA gene.

[0020] In some embodiments of the present invention, the genetically engineered Escherichia coli contains an expression vector in which the CsgA gene is knocked out and the CsgB and / or CsgG genes are present.

[0021] In some embodiments of the present invention, the genetically engineered Escherichia coli contains an expression vector in which the CsgA gene is knocked out and the CsgB and CsgG genes are present.

[0022] In other embodiments of the present invention, the genetically engineered Escherichia coli contains a vector in which the CsgA gene is knocked out and the CsgB gene is expressed.

[0023] In this invention, the amino acid sequences and encoding gene sequences of the CsgA, CsgB, and CsgG proteins of *Escherichia coli* can be obtained from publicly available databases. Specifically, the accession number for the CsgA gene is NC_415560.1; the accession number for the CsgB gene is NC_415559.1; and the accession number for the CsgG gene is NC_415555.1.

[0024] Specifically, the amino acid sequences of CsgA, CsgB, and CsgG proteins are shown in SEQ ID NO.1-3, respectively.

[0025] In the above method, the mutant library is introduced into the genetically engineered Escherichia coli via an expression vector.

[0026] Preferably, in the expression vector, the expression of the mutant library is initiated by an inducible promoter. The inducible promoter may be an IPTG inducible promoter.

[0027] Optional expression vectors include expression vectors suitable for Escherichia coli and compatible with expression vectors of the CsgB and / or CsgG genes, including but not limited to pET series expression vectors, such as pET22 series vectors (pET-22b, pET-22a), etc.

[0028] In some embodiments of the present invention, the expression vector is pET-22b(+).

[0029] In some embodiments of the present invention, the CsgA protein is fused to the N-terminus of the enzyme, and the CsgA protein and the enzyme are linked via a GS-rich short peptide linker. A preferred linker is GGGGS (SEQ ID NO. 6).

[0030] In some embodiments of the present invention, the method for constructing the expression vector of the mutant library includes: ligating the coding gene of the fusion protein of the CsgA protein and the enzyme to the expression vector, followed by constructing the mutant library. The method for constructing the mutant library includes error-prone PCR, site-directed saturation mutagenesis, etc. The above-mentioned mutant library construction is performed only on the enzyme, that is, only introducing mutation sites into the enzyme.

[0031] In this invention, the starting strain of the genetically engineered Escherichia coli is Escherichia coli K12 or a derivative thereof. Preferably, it is Escherichia coli BL21 or a derivative thereof. More preferably, it is Escherichia coli BL21(DE3).

[0032] Preferably, the enzyme is 5-hydroxymethylfurfural oxidase.

[0033] In this invention, the 5-hydroxymethylfurfural oxidase can be any species-derived 5-hydroxymethylfurfural oxidase. Preferably, it is derived from *Methylovorus sp.* MP688. Preferably, the amino acid sequence of the 5-hydroxymethylfurfural oxidase is shown in SEQ ID NO.4.

[0034] In this invention, 5-hydroxymethylfurfural oxidase can be the wild type of the enzyme or a mutant of the enzyme. The mutant can originate from directed evolution of the enzyme, including error-prone PCR or site-directed saturation mutagenesis.

[0035] Preferably, the enzymatic reaction uses 5-hydroxymethylfurfural as a substrate, and the activity of 5-hydroxymethylfurfural oxidase is characterized by detecting the production of H2O2.

[0036] This invention optimizes the enzyme activity detection method for 5-hydroxymethylfurfural oxidase, utilizing H2O2 generated during the catalytic process of 5-hydroxymethylfurfural oxidase to characterize enzyme activity. Simultaneously, it develops two high-throughput screening methods based on fluorescence and colorimetric methods, broadening the range of detectable enzyme activities.

[0037] In some embodiments of the present invention, the enzymatic reaction system includes: Escherichia coli cells displaying the enzyme to be screened, 5-hydroxymethylfurfural, L-tyrosine, and horseradish catalase on the surface; after the enzymatic reaction is completed, the fluorescence intensity is detected under the conditions of λex = 330 nm and λem = 415 nm.

[0038] Preferably, the enzymatic reaction system comprises: 80-120 μL of Escherichia coli bacterial culture displaying the enzyme to be screened, 15-25 μL of 45-55 mM HMF, 8-12 μL of 45-55 mM L-tyrosine, 15-25 μL of 0.8-1.2 mg / mL horseradish peroxidase, and 1×PBS to bring the system to 200 μL.

[0039] In other embodiments of the present invention, the enzymatic reaction system comprises: Escherichia coli cells displaying the enzyme to be screened, 5-hydroxymethylfurfural, ABTS, and horseradish catalase; and absorbance measured at 405 nm after the enzymatic reaction is completed.

[0040] Preferably, the enzymatic reaction system comprises: 80-120 μL of Escherichia coli bacterial culture displaying the enzyme to be screened, 15-25 μL of 45-55 mM HMF, 8-12 μL of 0.8-1.2 mM ABTS, 15-25 μL of 0.8-1.2 mg / mL horseradish peroxidase, and 1×PBS to bring the system to 200 μL.

[0041] Preferably, the temperature of the enzymatic reaction is 28-32°C.

[0042] Preferably, the preparation of the *E. coli* cells displaying the enzyme to be screened on their surface includes: culturing the *E. coli* cells displaying the enzyme to be screened on their surface at 36-37°C until the OD value reaches 100°C. 600 Add an inducer when the concentration is 0.6-0.8, induce culture at 16-18℃, and then collect the bacterial cells.

[0043] Secondly, the present invention provides a genetically engineered Escherichia coli strain in which the CsgA gene is inactivated and the expression of the CsgB and / or CsgG genes is enhanced.

[0044] Preferably, the expression is enhanced by introducing an expression vector containing the CsgB and / or CsgG genes.

[0045] Preferably, in the expression vector, the promoters of the CsgB and / or CsgG genes are constitutive promoters.

[0046] Preferably, the constitutive promoter is a Trc promoter. The nucleotide sequence of the Trc promoter is shown in SEQ ID NO. 5.

[0047] Preferably, the expression vector is constructed using the pET28 series vector as a backbone.

[0048] Thirdly, the present invention provides an Escherichia coli that displays a target protein on its surface, wherein the Escherichia coli that displays the target protein on its surface is obtained by introducing a fusion protein encoding gene into the above-described Escherichia coli genetically engineered bacteria; the fusion protein comprises CsgA protein and the target protein.

[0049] Preferably, the introduction of the fusion protein encoding gene is achieved by introducing an expression vector containing the fusion protein encoding gene.

[0050] Preferably, the target protein is 5-hydroxymethylfurfural oxidase.

[0051] The Escherichia coli genetically engineered strain and the Escherichia coli with surface display of target protein provided by the present invention can achieve a higher CsgA protein content on its surface, thereby significantly improving the surface display efficiency of the target protein.

[0052] Fourthly, the present invention provides the application of the above-described Escherichia coli genetically engineered bacteria in the bacterial surface display of target proteins or in the high-throughput screening of enzymes.

[0053] In the above applications, when the genetically engineered Escherichia coli is used for bacterial surface display of the target protein or for high-throughput screening of enzymes, a fusion protein containing CsgA protein and the target protein is introduced into the genetically engineered Escherichia coli.

[0054] In some embodiments of the present invention, the CsgA protein is fused to the N-terminus of the target protein, and the CsgA protein and the target protein are linked by a GS-rich short peptide linker. A preferred linker is GGGGS.

[0055] Fifthly, the present invention provides Escherichia coli for displaying target proteins on bacterial surfaces, high-throughput screening of enzymes, or as a biocatalyst, as described above.

[0056] The beneficial effects of this invention include at least the following: This invention provides a high-throughput screening method for enzymes. This method displays the enzyme on the surface of Escherichia coli and significantly improves the surface display efficiency of the enzyme by optimizing the E. coli curli pili system, thereby improving the enzyme screening efficiency and reducing screening steps. This method solves the problems of traditional screening methods in the directed evolution of enzymes, which require cell disruption, are complex to operate, and are prone to causing reduced protein activity and low screening efficiency. It has the advantages of not requiring cell disruption, being simple and easy to operate, having high throughput, specific and accurate enzyme activity measurement, a wide enzyme activity detection range, and high screening efficiency. Attached Figure Description

[0057] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0058] Figure 1 This is a map of the overexpression vectors of the CsgB and CsgG genes in Example 1 of the present invention.

[0059] Figure 2 This is a schematic diagram of the HMFO high-throughput screening method in Embodiment 6 of the present invention.

[0060] Figure 3 This is a map of the CsgA-HMFO fusion expression vector in Example 6 of the present invention.

[0061] Figure 4 This is a schematic diagram of the reaction process in Example 6 of the present invention, in which HMFO catalyzes the production of FDCA and H2O2 from HMF. Detailed Implementation

[0062] A high-throughput screening method for 5-hydroxymethylfurfural oxidase is provided in a specific embodiment of the present invention, comprising the following steps: constructing a curli-fimbria-enhanced E. coli BL21(DE3)-curli(+); constructing a fusion expression vector containing CsgA protein and 5-hydroxymethylfurfural oxidase; constructing a mutant library of 5-hydroxymethylfurfural oxidase using the fusion expression vector; transferring the mutant library into E. coli BL21(DE3)-curli(+), and then performing induced expression culture, using the induced expression cultured bacteria as a biocatalyst to catalyze HMF, and detecting enzyme activity.

[0063] Preferably, the enzyme activity can be characterized by H2O2 generated during the HMFO-catalyzed HMF reaction.

[0064] In the above method, a mutant library is constructed for the HMFO portion of the CsgA-HMFO fusion protein.

[0065] The above method for constructing the curli-fibrillated Escherichia coli BL21(DE3)-curli(+) includes: knocking out the CsgA gene in E. coli BL21(DE3) to obtain E. coli BL21(DE3)-CsgA(-); and overexpressing the CsgB and / or CsgG genes in E. coli BL21(DE3)-CsgA(-) to obtain E. coli BL21(DE3)-curli(+).

[0066] Preferably, overexpression of the CsgB and / or CsgG genes is achieved by introducing an expression vector containing the CsgB and / or CsgG genes. The vector used can be a pET-28 series vector, such as pET-28a(+).

[0067] In the above method, the construction of the mutant library can be carried out using error-prone PCR technology or site-directed saturation mutagenesis technology, etc., using the CsgA-HMFO fusion protein expression vector as a template, introducing the mutation site into the HMFO gene to construct the mutant library.

[0068] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0069] In the following examples, the accession number for the CsgA gene is NC_415560.1; the accession number for the CsgB gene is NC_415559.1; the accession number for the CsgG gene is NC_415555.1; the accession number for the CsgE gene is NC_415557.1; and the accession number for the CsgF gene is NC_415556.1.

[0070] Example 1 Construction of genetically engineered Escherichia coli (1)

[0071] This embodiment provides a genetically engineered Escherichia coli strain, the construction method of which is as follows: S1: The CsgA gene in the genome of E. coli BL21(DE3) was knocked out using CRISPR-Cas9 gene editing technology to obtain E.coli BL21(DE3)-CsgA(-); S2: Simultaneous overexpression of the CsgB and CsgG genes of *E. coli* BL21(DE3) using the Trc promoter, i.e., the Trc promoter and the CsgB and CsgG genes are ligated into the expression plasmid pET-28a to construct the overexpression plasmid (structural diagram shown in Figure 1). Figure 1 As shown in the figure, the overexpression plasmid was transformed into E. coli BL21(DE3)-CsgA(-) to obtain curli fimbriae-enhanced E. coli E. coli BL21(DE3)-CsgA(-)-curli(BG+).

[0072] Example 2 Construction of genetically engineered Escherichia coli (2)

[0073] This embodiment provides a genetically engineered Escherichia coli strain. The construction method differs from that in Example 1 only in that: in step S2, the CsgB gene is overexpressed using the Trc promoter to obtain the curli-fibrillated E. coli BL21(DE3)-CsgA(-)-curli(B+).

[0074] Example 3 Construction of genetically engineered Escherichia coli (3)

[0075] This embodiment provides a genetically engineered Escherichia coli strain. The construction method differs from that in Example 1 only in that: in step S2, the CsgG gene is overexpressed using the Trc promoter to obtain the curli-fibrillated E. coli BL21(DE3)-CsgA(-)-curli(G+).

[0076] Example 4 Construction of genetically engineered Escherichia coli (4)

[0077] This embodiment provides a genetically engineered Escherichia coli strain, the construction method of which differs from that of Example 1 only in that: the CsgE gene is overexpressed using the Trc promoter to obtain the curli-fibrillated E. coli BL21(DE3)-CsgA(-)-curli(E+).

[0078] Example 5: Construction of genetically engineered Escherichia coli (5)

[0079] This embodiment provides a genetically engineered Escherichia coli strain, the construction method of which differs from that of Example 1 only in that: the CsgF gene is overexpressed using the Trc promoter to obtain the curli-fibrillated E. coli BL21(DE3)-CsgA(-)-curli(F+).

[0080] Example 6

[0081] This embodiment provides a high-throughput screening method for HMFO, the steps of which are as follows (partial process is as follows). Figure 2 (As shown): S1: The CsgA gene and the HMFO gene were fused and inserted into the expression plasmid pET-22b to construct the CsgA-HMFO fusion expression vector (as shown). Figure 3 Primers were designed to perform error-prone PCR on the HMFO gene in the CsgA-HMFO fusion expression vector to obtain a mutant library plasmid. A non-mutated CsgA-HMFO fusion expression vector was also used as a wild-type control for HMFO.

[0082] S2: The mutant library plasmids were transformed into the curli fimbrial enhanced Escherichia coli and strain E. coli BL21(DE3)-CsgA(-) in Examples 1-5, respectively. Single colonies were picked and constructed into 96-well plates containing 200 μL of LB medium.

[0083] S3: Incubate the mutant library from step S2 at 37°C and 800 rpm until OD. 600 When the concentration of the induced protein is 0.6-0.8, IPTG is added, and the culture is induced at 17℃ and 800rpm for 24h.

[0084] S4: Add 100 μL of the bacterial culture induced in step S3, 20 μL of 50 mM HMF, 10 μL of 50 mM L-tyrosine, and 20 μL of 1 mg / mL horseradish catalase to a 96-well fluorescent plate. Make up the volume to 200 μL with 1×PBS. Set the microplate reader to λex = 330 nm and λem = 415 nm for kinetic analysis. The enzyme activity assay involves the following enzymatic reactions: Figure 4 As shown.

[0085] The enzyme activity in the reaction system was calculated based on the H2O2 fluorescence standard curve.

[0086] The method for constructing the H2O2 fluorescence standard curve involves adding 10 μL of 50 mM L-tyrosine, 20 μL of 1 mg / mL horseradish peroxidase, and H2O2 solutions of different concentrations to a 96-well fluorescence plate, then bringing the volume to 200 μL with 1×PBS. The detection limit for H2O2 in this method is 0.1-25 μM. HMFO enzyme activity is defined as the amount of enzyme required to produce 1 μM H2O2 per minute. Based on the H2O2 fluorescence standard curve and the kinetics of the microplate reader, the shortest interval between two detections of the same sample is 2 seconds.

[0087] The HMFO enzyme activity detection range of the above method was determined to be 0.01-25U.

[0088] The wild-type HMFO enzyme activity in *E. coli* BL21(DE3)-CsgA(-)-curli(BG+)* of Example 1 was 19.3 U; in *E. coli* BL21(DE3)-CsgA(-)-curli(B+)* of Example 2, it was 18.4 U; in *E. coli* BL21(DE3)-CsgA(-)-curli(G+)* of Example 3, it was 13.6 U; in *E. coli* BL21(DE3)-CsgA(-)-curli(E+)* of Example 4, it was 9.5 U; and in *E. coli* BL21(DE3)-CsgA(-)-curli(E+)* of Example 5, it was... The wild-type HMFO enzyme activity in BL21(DE3)-CsgA(-)-curli(F+) was 11.7 U; while the wild-type HMFO enzyme activity in E. coli BL21(DE3)-CsgA(-) before curli system optimization was 7.6 U.

[0089] The detected wild-type enzyme activity reflects the surface display efficiency of HMFO in this curli-fimated *E. coli* strain. In high-throughput screening, 96-well plates are typically used as the culture system for the fermentation of target proteins by genetically engineered bacteria. This method has the advantage of enabling small-scale parallel experiments, significantly improving experimental efficiency. However, this also leads to lower protein yields in the initial screening stage. The optimized surface display system provided by this invention eliminates the need for cell lysis and protein release before enzyme activity detection, making the experimental operation more convenient; furthermore, it improves the surface display efficiency of the target protein, enhancing screening sensitivity.

[0090] Example 7

[0091] This embodiment provides a high-throughput screening method for HMFO, the steps of which are as follows (partial process is as follows). Figure 2 (As shown): S1: The CsgA gene and the HMFO gene are fused and ligated into the expression plasmid pET-22b to construct the CsgA-HMFO fusion expression vector; primers are designed to perform site-directed saturation mutagenesis on the HMFO gene in the CsgA-HMFO fusion expression vector to obtain the mutant library plasmid.

[0092] S2: The mutant library plasmids were transformed into the curli fimbrial enhanced Escherichia coli and strain E. coli BL21(DE3)-CsgA(-) in Example 1, and single colonies were picked and constructed into 96-well plates containing 200 μL of LB medium.

[0093] S3: Incubate the mutant library from step S2 at 37°C and 800 rpm until OD. 600When the concentration of the induced protein is 0.6-0.8, IPTG is added, and the culture is induced at 17℃ and 800rpm for 24h.

[0094] S4: Add 100 μL of the bacterial culture induced by S3, 20 μL of 50 mM HMF, 10 μL of 1 mM ABTS, and 20 μL of 1 mg / mL horseradish catalase to a 96-well plate. Make up the volume to 200 μL with 1×PBS. Set the microplate reader to OD=405nm for kinetic detection. Calculate the enzyme activity in the reaction system based on the H2O2 colorimetric standard curve.

[0095] The method for constructing the H2O2 colorimetric standard curve involves adding 10 μL of 1 mM ABTS, 20 μL of 1 mg / mL horseradish peroxidase, and different concentrations of H2O2 solution to a 96-well plate, then bringing the volume to 200 μL with 1×PBS. The detection limit for H2O2 in this method is 1-200 μM. HMFO enzyme activity is defined as the amount of enzyme required to produce 1 μM H2O2 per minute. Based on the H2O2 fluorescence standard curve and the kinetics of the microplate reader, the shortest interval between two detections of the same sample is 2 seconds.

[0096] The HMFO enzyme activity detection range of the above method was determined to be 10-200 U. When the HMFO enzyme activity is high, the colorimetric method described in this example can be used for enzyme activity determination and screening.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A high-throughput screening method for enzymes, characterized in that, The method includes: displaying the enzyme to be screened on the surface of Escherichia coli, using the Escherichia coli displaying the enzyme to be screened on the surface as a biocatalyst to catalyze the enzymatic reaction of the enzyme, and screening the enzyme by detecting the enzymatic reaction products; The process of displaying the enzyme to be screened on the surface of E. coli includes: constructing a mutant library of the enzyme using the encoding gene of the fusion protein of E. coli CsgA protein and the enzyme as a template; introducing the mutant library into E. coli genetically engineered bacteria; inactivating the CsgA gene in the E. coli genetically engineered bacteria, and enhancing the expression of the CsgB and / or CsgG genes.

2. The method according to claim 1, characterized in that, The expression is enhanced by introducing an expression vector containing the CsgB and / or CsgG genes.

3. The method according to claim 2, characterized in that, In the expression vector, the promoters of the CsgB and / or CsgG genes are constitutive promoters; Preferably, the constitutive promoter is a Trc promoter; More preferably, the expression vector is constructed using the pET28 series vector as a backbone.

4. The method according to any one of claims 1 to 3, characterized in that, The mutant library was introduced into the genetically engineered Escherichia coli via an expression vector; Preferably, in the expression vector, the expression of the mutant library is initiated by an inducible promoter.

5. The method according to any one of claims 1 to 4, characterized in that, The enzyme is 5-hydroxymethylfurfural oxidase; preferably, the enzymatic reaction is performed using 5-hydroxymethylfurfural as a substrate, and the activity of 5-hydroxymethylfurfural oxidase is characterized by detecting the production of H2O2. More preferably, the enzymatic reaction system includes: Escherichia coli cells displaying the enzyme to be screened, 5-hydroxymethylfurfural, L-tyrosine, and horseradish catalase on the surface; after the enzymatic reaction is completed, the fluorescence intensity is detected under the conditions of λex = 330 nm and λem = 415 nm. Alternatively, the enzymatic reaction system comprises: Escherichia coli cells displaying the enzyme to be screened, 5-hydroxymethylfurfural, ABTS, and horseradish catalase on the surface; and absorbance measured at 405 nm after the enzymatic reaction is completed.

6. The method according to claim 5, characterized in that, The preparation of the bacterial cells includes: culturing *E. coli* with the enzyme to be screened on its surface at 36-37°C until OD200. 600 Add an inducer when the concentration is 0.6-0.8, induce culture at 16-18℃, and then collect the bacterial cells.

7. A genetically engineered Escherichia coli bacterium, characterized in that, The CsgA gene of the genetically engineered Escherichia coli is inactivated, and the expression of the CsgB and / or CsgG genes is enhanced.

8. The genetically engineered Escherichia coli according to claim 7, characterized in that, The expression was enhanced by introducing an expression vector containing the CsgB and / or CsgG genes; Preferably, in the expression vector, the promoter of the CsgB and / or CsgG gene is a constitutive promoter; More preferably, the constitutive promoter is a Trc promoter; More preferably, the expression vector is constructed using the pET28 series vector as a backbone.

9. An Escherichia coli strain displaying a target protein on its surface, characterized in that, The E. coli displaying the target protein on the surface is obtained by introducing a fusion protein encoding gene into the E. coli genetically engineered bacteria as described in claim 7 or 8. The fusion protein comprises CsgA protein and the target protein; Preferably, the introduction of the fusion protein encoding gene is achieved by introducing an expression vector containing the fusion protein encoding gene; more preferably, the target protein is 5-hydroxymethylfurfural oxidase.

10. The application of the genetically engineered Escherichia coli of claim 7 or 8 or the Escherichia coli of claim 9 that displays the target protein on its surface for bacterial surface display of the target protein, high-throughput screening of enzymes, or as a biocatalyst.

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