Reagents and methods for high throughput plate screening of 3-sialyltransferase mutants

Abstract

The present application relates to the field of biotechnology, and more particularly to a reagent and method for high-throughput plate screening of 3-sialyltransferase mutants. The present application realizes efficient screening of 3SL high-yield mutants in large-scale mutant libraries by adopting solid plate color screening technology combined with pH indicator. The method quickly identifies the target mutant by directly observing the color change on the plate, thereby omitting the cumbersome steps of strain construction, sequencing, transfer and shake flask culture in the traditional screening process, significantly improving the screening speed and efficiency. Compared with the prior art, the present application not only greatly shortens the experimental time and simplifies the operation process, but also reduces the requirements for human resources and equipment, providing great convenience for screening of enzyme mutant libraries. The technical advantage of the present application is that it is fast, efficient and low-cost, and is expected to be widely applied in the fields of biomedicine and food science.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to reagents and methods for high-throughput plate screening of 3-sialyltransferase mutants. Background Technology

[0002] Human milk oligosaccharides (HMOs) are the third largest solid component of breast milk after lactose and fat. They are complex mixtures of numerous oligosaccharides with diverse structures and functions, thus playing important biological roles. Research and application of HMOs have also made progress in China. In July 2023, the Chinese Institute of Food Science and Technology published the "Scientific Consensus on Human Milk Oligosaccharides (HMOs)," aiming to accelerate the approval and application of HMOs in China and provide consumers with authoritative guidance on scientifically understanding HMOs. Furthermore, it emphasized the importance of HMOs in improving the health and nutritional needs of infants and young children, noting that the safety and functionality of HMOs have been globally recognized. It also highlighted the necessity of adding HMOs to food, especially to infant formula, to compensate for the insufficient HMO content in cow's milk.

[0003] Sialyllactose (SL) is an acidic human milk oligosaccharide, classified into 3-SL and 6-SL types based on the binding position of sialic acid to lactose. As a major component of human milk oligosaccharides, 3-SL has demonstrated several beneficial effects on infants: it can reach the distal small intestine and colon intact, stimulating the growth of beneficial bacteria in the gut and maintaining intestinal flora balance; it inhibits the growth of harmful bacteria, binds to pathogens, and reduces the incidence of infection; it supports intestinal barrier function and promotes immune development and potential neurodevelopment.

[0004] The commonly used enzymatic method for producing 3-SL uses sialic acid (NeuAc) as the original substrate. CMP-NeuAc synthase (CSS) is used to convert SA and CTP into CMP-NeuAc. Then, sialate transferase (ST) is used to catalyze the transfer reaction between CMP-NeuAc and lactose to produce 3-SL.

[0005] Glycosyltransferases are a class of enzymes that catalyze glycosylation reactions in vivo, transferring sugar groups from an activated glycosyl donor to an acceptor molecule to form a glycosidic bond. Although glycosyltransferases play a crucial role in biosynthesis and metabolic regulation, several limitations remain in their research and application. These include the potential for low expression levels of naturally derived glycosyltransferases, restricting their industrial-scale application potential; insufficient catalytic efficiency for some glycosyltransferases to meet the demands of large-scale production; solubility issues in expression systems for certain glycosyltransferases; and the influence of environmental conditions (such as temperature and pH) on enzyme stability and activity. To address these limitations, directed evolution and rational or semi-rational design are needed to improve catalytic efficiency. Therefore, establishing a novel, sensitive, and rapid 3-SL detection method has significant market application value. While some high-throughput screening methods have been developed for enzyme screening and optimization, these methods may require further improvement to enhance screening efficiency and accuracy.

[0006] Current high-throughput screening methods include: using flow cytometry to screen for substrates and glycosylated products based on differences in the permeability of galactosyltransferase on the cell membrane surface; and using CRISPR / Cas9 technology to insert the luciferase coding sequence into the 3' end of the target protein gene in neuroblastoma cell lines, and measuring the fluorescence value to determine the effect of small molecules on the expression level of the target protein. In addition, fluorescence-activated cell sorting (FACS) and droplet microfluidic sorting (DMFS) technologies are specifically mentioned; they can be combined with mutagenesis to identify and separate live cells with high 3-SL yields, further improving screening efficiency. Although these methods are widely used in the biomedical and food fields, they all have high requirements for detection instruments, are cumbersome to operate, and significantly increase costs.

[0007] Directed evolution encompasses in vivo chemical mutagenesis, error-prone polymerase chain reactions (CPRTs), and in vitro saturated / unsaturated site-directed mutagenesis. These methods allow for the easy construction of a library containing a large number of mutants. Therefore, the ability to efficiently and rapidly screen for beneficial mutations from a vast library of protein mutants is crucial to the success rate of directed enzyme evolution. However, traditional screening methods require significant manpower and time, making them increasingly inadequate for the challenges facing enzyme engineering. High-throughput screening methods, however, are rapidly developing as an important technology in directed enzyme evolution.

[0008] The application of directed evolution as a highly efficient biocatalyst faces numerous challenges, including limited substrate range, thermal stability, solubility, and low substrate affinity. Besides rational protein engineering methods, directed evolution remains crucial for designing proteins to adapt to manufacturing processes, inevitably resulting in large libraries containing primarily inactive or low-activity variants. Therefore, to accelerate the directed evolution process, providing sensitive, efficient, and low-cost detection methods is essential. This would avoid the time-consuming and technically demanding analysis of each variant using HPLC or GC, while saving on the expensive equipment required for FACS and DMFS techniques. Summary of the Invention

[0009] In view of this, the technical problem to be solved by the present invention is to provide reagents and methods for high-throughput plate screening of 3-sialyltransferase mutants.

[0010] This invention provides a reagent for screening 3-sialyltransferase mutants, comprising: a buffer solution, cytidine monophosphate-sialic acid, lactose, and a pH indicator.

[0011] During the cellular catalytic reaction that produces 3-SL, the pH value in the reaction system gradually decreases as the production of cytidine monophosphate and glycosidic bonds increases. The 3-sialyltransferase mutant exhibits high activity and can more efficiently catalyze the transfer reaction between CMP-NeuAc and lactose to generate more 3-SL, thereby lowering the pH value. This invention utilizes this characteristic to make a preliminary assessment of the activity of the 3-sialyltransferase mutant by observing changes in pH value.

[0012] The reagent provided by this invention can identify bacterial colonies, eliminating the need to construct each mutant strain or construct a large number of mutant libraries for screening in the traditional enzyme mutant screening process, and then screen them after enzyme production. Moreover, it avoids the possibility of contamination caused by transfer and cumbersome steps such as cell disruption, greatly simplifying the screening process.

[0013] In this invention, the selection of the pH indicator is the most important screening reagent. Therefore, this invention screens feasible pH indicators from the prior art. In this invention, the pH indicator needs to change color within the range of 6.0 to 7.5. For example, the color change range of the pH indicator is 6 to 8, or 6.2 to 8.2, or 6.5 to 8.5, or 6 to 7.6, or 6.8 to 8.8, or 6.0 to 7.0 or 7.0 to 9.0. In some embodiments, the pH indicator is at least one of bromothymol blue, neutral red, or catechol purple. Preferably, the color change range of the pH indicator is 6 to 7.6. More preferably, the pH indicator is bromothymol blue.

[0014] In some embodiments, the concentration of bromothymol blue in the screening reagent is 0.1–1 mM, for example, 0.1–0.5 mM; or 0.3–0.5 mM; or 0.4–0.5 mM. Preferably, the concentration of bromothymol blue is 0.4 mM.

[0015] In this invention, the buffer solution needs to be used in conjunction with a pH indicator to avoid interfering with the intracellular reaction and to prevent affecting the color change. The buffer solution in the reagents of this invention is preferably a buffer solution with a pH of 7.5–8.0. For example, the buffer solution is Tris-HCl, phosphate buffer, or TM buffer. Experiments have shown that the most effective buffer solution is Tris-HCl buffer, wherein the concentration of Tris-HCl is 30–50 mM, preferably 45–55 mM, and more preferably 50 mM in the screening reagent.

[0016] This invention also optimizes and screens the ratio and concentration of lactose and cytidine monophosphate-sialic acid (CMAP). A lactose concentration of 20–30 mM is preferred to allow for a more complete and rapid reaction. For example, the lactose concentration in the screening reagent is 20–25 mM, 22–27 mM, or 25–30 mM. Preferably, the lactose concentration in the screening reagent is 23–25 mM. More preferably, the lactose concentration is 24 mM. A CMAP concentration of 15–25 mM is also preferred to allow for a more complete and rapid reaction. For example, the lactose concentration in the screening reagent is 17–23 mM, 18–24 mM, or 16–24 mM. Preferably, the lactose concentration in the screening reagent is 19–21 mM. More preferably, the lactose concentration is 20 mM.

[0017] In this invention, the screening reagents include: Tris-HCl buffer, cytidine monophosphate-sialic acid, lactose, and bromothymol blue.

[0018] In some embodiments, 30–50 mM Tris-HCl buffer, 15–25 mM cytidine monophosphate-sialic acid, 20–30 mM lactose, and 0.1–1 mM bromothymol blue are used.

[0019] The screening reagent described in this invention is a solid reagent, and this invention is not limited thereto. If it is a solid reagent, the reagent also includes agarose. The mass fraction of the agarose is 0.5% to 1.0%. In the embodiments, an agarose mass fraction of 0.8% is more conducive to the penetration of the screening reagent.

[0020] In a specific embodiment, the screening reagent includes: 50mM Tris-HCl buffer at pH=8.0, 24mM lactose, 20mM cytidine monophosphate-sialic acid, 0.4mM bromothymol blue, and 0.8% agarose.

[0021] The preparation of the screening reagent of the present invention includes boiling Tris-HCl buffer, cytidine monophosphate-sialic acid, lactose, bromothymol blue and agarose to 100°C and then cooling to about 50°C to pour into plates to obtain a solid screening reagent.

[0022] In this invention, the screening reagent also includes a nitrocellulose membrane.

[0023] The present invention does not limit the pore size of the nitrocellulose membrane, which can permeate the culture medium or screening reagent, thereby allowing the colonies to come into contact with the reagent or culture medium.

[0024] The reagents provided by this invention enable simultaneous culture and screening without affecting colony growth. Screened colonies can be directly picked, propagated, and detected. Therefore, this invention provides the application of the reagents described above in screening 3-sialyltransferase mutants.

[0025] The present invention also provides a method for screening 3-sialyltransferase mutants, comprising contacting colonies with the reagent and determining the activity of 3-sialyltransferase based on color changes.

[0026] The method described in this invention specifically includes:

[0027] Step 1) Spread the bacterial culture onto a solid culture medium for incubation;

[0028] Step 2) Imprint colonies onto the nitrocellulose membrane, then place the membrane with the colony side facing up onto a solid culture medium for incubation.

[0029] Step 3) Place the nitrocellulose membrane containing colonies from Step 2) onto the reagent as described above, and observe the color change after the reaction.

[0030] In this invention, the colonies do not come into direct contact with the screening reagent. Instead, they pass through a nitrocellulose membrane, allowing the colonies to come into contact with the permeated reagent, thereby causing a color change.

[0031] In this invention, the bacterial culture is *Escherichia coli* transformed with a mutant fragment. Its construction includes methods such as genetic engineering or mutagenesis; this invention does not limit the specific method used.

[0032] In the embodiments, it was constructed by genetic engineering methods. The main steps included transferring the obtained nucleic acid encoding the 3-sialyltransferase mutant into competent E. coli cells via plasmid, and then inducing the expression of the 3-sialyltransferase mutant with IPTG after culture.

[0033] In this invention, the solid culture medium used in step 1) is LB solid culture medium containing 50 μg / ml kanamycin.

[0034] In this invention, the solid culture medium used in step 2) is LB solid culture medium containing 50 μg / ml kanamycin and 0.5 mM IPTG, and the culture conditions include culturing at 30°C for 8 h.

[0035] In this invention, the reaction conditions in step 3) include standing at 28°C for 2 hours.

[0036] In this invention, the method of judging the activity of 3-sialyltransferase based on color change includes: if the colony is blue or green, the 3-sialyltransferase activity is weak; if the colony turns yellow, the 3-sialyltransferase activity is strong.

[0037] This invention achieves highly efficient screening of high-yield 3SL mutants in large-scale mutant libraries by employing solid-plate colorimetric screening technology combined with pH indicators. This method rapidly identifies target mutants by directly observing color changes on the plate, thus eliminating the cumbersome steps of traditional screening processes such as strain construction, sequencing, transfer, and shake-flask culture, significantly improving screening speed and efficiency. Compared to existing technologies, this invention not only significantly shortens experimental time and simplifies the operation process but also reduces the requirements for human resources and equipment, providing great convenience for the screening of enzyme mutant libraries. The technical advantages of this invention lie in its speed, efficiency, and low cost, and it is expected to be widely applied in the fields of biomedicine and food science. Attached Figure Description

[0038] Figure 1 Screening using a colorimetric plate containing 10 mM Tris-HCl;

[0039] Figure 2 Screening of colorimetric plates containing 30 mM Tris-HCl;

[0040] Figure 3 Screening using a colorimetric plate containing 50 mM Tris-HCl;

[0041] Figure 4 The color development results of Example 1 are shown;

[0042] Figure 5 The color development results of Example 2 are shown;

[0043] Figure 6 The color development results of Example 3 are shown;

[0044] Figure 7 The liquid chromatography profile of the control group F314 mutant is shown.

[0045] Figure 8 The liquid phase spectrum of the Y314 mutant is shown. Detailed Implementation

[0046] This invention provides reagents and methods for high-throughput plate screening of 3-sialyltransferase mutants. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0047] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0048] In this invention, "and / or" describes the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone.

[0049] In this invention, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of a single item or a plurality of items.

[0050] In this invention, the terms "comprising," "including," and "having" are used interchangeably to indicate the inclusiveness of a solution, meaning that the solution may contain elements other than those listed. It should also be understood that the use of "comprising," "including," and "having" herein also provides for solutions that are "composed of" or "as shown."

[0051] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise explicitly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0052] In a specific embodiment of this invention, the pH range of bromothymol blue is 6-7.6. When the initial pH of the screening plate is around 7.5, preliminary experiments showed that if a superior mutant is present, the pH within the bacteria gradually decreases to below 7, resulting in a visible change in the bacteria from blue to green to yellow. This method eliminates the need for traditional enzyme mutant screening processes, which require constructing each mutant strain or building a large number of mutant libraries for culture, enzyme production, and subsequent screening. This method necessitates several culture and transfer steps, as well as cell disruption. This invention eliminates the need for large-scale bacterial selection and 96-well plate culture. The configured chromogenic plate has good biocompatibility, combining induced expression, growth, catalytic reaction, and enzyme activity colorimetric detection simultaneously on the plate. This enables rapid and efficient screening of effective mutant strains.

[0053] The amino acid sequence of wild-type ASL involved in this invention is as follows:

[0054] MGLKKACLTVLCLIVFCFGIFYTFDRVNQGEPVNLIFCYTILQMKVAERIMAQHPGERFYVVLMSENRNEKYDYYFNQIKDKAERAYFFHLPYGLNKSFNFIPTMAELKVKAMLLPKVKRIYLASLEKVSIAAFLSTYPDAEIKIFDDGTGNLIQSSSYLGDEFSVNGTIKRNFARMMI GDWSIAKTRNASDEHYTIFKGLKNIMDDGRRKMTYLPLFDASELKAGDETGGTVRILLGSPDKEMKEISEKAAKNFNIQYVAPHPRQTYGLSGVTTLNSPYVIEDYILREIKKNPHTRYEIYTFFSGAALTMKDFPNVHVYALKPASLPKDYWLKPVYALFTQSGIPILTFDDKD(SEQ ID NO:1)

[0055] The nucleic acid sequence of wild-type ASL involved in this invention is as follows:

[0056]

[0057] The test materials used in this invention are all commercially available products. It should be understood that in the various embodiments of this application, the sequence numbers of the above processes do not imply a specific order of execution. Some or all steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application. The invention is further illustrated below with reference to embodiments:

[0058] Example 1: Screening of colorimetric plate formulations:

[0059] I. Solution preparation:

[0060] 1) 500 mM tris(hydroxymethyl)aminomethane (Tris) was adjusted to pH 8 with hydrochloric acid (HCl) to obtain a 500 mM Tris-HCl buffer solution with pH = 8.

[0061] 2) LB liquid medium: 1% peptone, 1% sodium chloride, 0.5% yeast extract.

[0062] 3) LB solid medium: 1% peptone, 1% sodium chloride, 0.5% yeast extract, 1.5% agar powder.

[0063] II. Three bacterial strains known in the laboratory to have different sialyl transferase activities were cultured separately in LB liquid medium containing 50 μg / ml kanamycin resistance. After bacterial growth, they were harvested according to OD... 600 The measured absorbance values ​​were mixed 1:1:1 and diluted to an appropriate concentration. The diluted solution was then spread onto LB solid medium containing 50 μg / ml kanamycin resistance and incubated overnight at 37°C to obtain single colonies of suitable density.

[0064] 3. Divide the 80cm nitrocellulose membrane (0.45μm) into three parts and cover each part onto the overnight culture plate (do not cover the same area repeatedly). Then, with the side with the colony print facing up, paste it onto an induction plate containing 50μg / ml kanamycin and 0.5mM IPTG. Induce expression at 30℃ for 8h.

[0065] IV. Add cytidine monophosphate-sialic acid (CMP-SA) to a final concentration of 20 mM, 24 mM lactose, and 0.4 mM bromothymol blue solution (BTB) to Tris-HCl systems at pH=8 (10 mM, 30 mM, and 50 mM, respectively). Finally, add 0.8% agarose powder. Mix the colorimetric solutions thoroughly and boil to dissolve the agarose. After cooling to approximately 50°C, quickly pour the mixture into plates. Once the plates have completely solidified, attach a nitrocellulose membrane containing induced bacterial cells to the colorimetric plate (colony side up). Incubate at 28°C for 2 hours and observe the colorimetric results.

[0066] The results are as follows Figures 1-3 When the Tris-HCl concentration was 10 mM and 30 mM, the sensitivity of the colorimetric reaction was low; single colonies of reacting mutants all turned yellow, making it difficult to screen out mutants with higher enzyme activity. It is worth noting that when the Tris-HCl concentration was 50 mM, single colonies could show different colors, and the sensitivity was greatly improved.

[0067] Example 2: Construction and Screening of Mutants

[0068] I. Construction of the ASL target gene. The target gene ASL was synthesized and then inserted into the pET28a expression vector. The pET28a-ASL original plasmid was formed using two sites of NdeI and XhoI restriction endonucleases.

[0069] Solution preparation:

[0070] 1) 500 mM tris(hydroxymethyl)aminomethane (Tris) was adjusted to pH 8 with hydrochloric acid (HCl) to obtain a 500 mM Tris-HCl buffer solution with pH = 8.

[0071] 2) LB liquid medium: 1% peptone, 1% sodium chloride, 0.5% yeast extract.

[0072] 3) LB solid medium: 1% peptone, 1% sodium chloride, 0.5% yeast extract, 1.5% agar powder.

[0073] II. Using Schrödinger's algorithm for residue mutation scanning, mutations at the L123 site enhance the affinity of ASL enzyme for lactose. This site is located at the active site entrance; mutation of this site can lengthen the side chain, increase steric hindrance, and narrow the active site entrance, thereby increasing the stability of lactose binding at the active site.

[0074] Design degenerate primers: 5'-3' direction:

[0075] PF1-AAACGCATTTATNNKGCGAGCCTGGAAA (SEQ ID NO: 3),

[0076] PR1-GCTCCGCMNNATAAATGCGTTTCACTTTC (SEQ ID NO: 4).

[0077] Perform amplification in a PCR instrument according to the following procedure:

[0078] primer PF1 (10μM) 1μL primerPR1 (10μM) 1μL template(40ng) 0.5μL 2xphantamaxmastermix 10μL <![CDATA[ddH2O]]> Up to 20μL

[0079] PCR procedure:

[0080]

[0081] After PCR, the PCR products were subjected to gel electrophoresis. PCR-positive products were purified by gel extraction. 4 μL of the homologous recombination product was transferred into 30 μL of DH5α competent cells. The cells were incubated on ice for 30 min, followed by heat shock at 42°C for 45 s, and then on ice for 2-3 min. 500 μL of antibiotic-free LB medium was added, and the cells were incubated on a shaker for 45 min. The mixture was then centrifuged and spread onto LB agar plates containing 50 μg / ml kanamycin. The plates were incubated overnight at 37°C for 12-16 hours. The cells were washed off the plates with antibiotic-free LB medium, and the mixed plasmid was extracted using a kit. 5 μL of the mixed plasmid was transferred into 100 μL of BL21(D3) competent cells. The cells were incubated on ice for 30 min, followed by heat shock at 42°C for 45 s, and then on ice for 2-3 min. Next, add 500 μL of antibiotic-free LB medium, and after shaking culture for 1 hour, take 150 μL and spread it on an LB solid plate containing 50 μg / ml kanamycin. Incubate overnight at 37°C for 12-16 hours.

[0082] A 0.45 μm nitrocellulose membrane with a diameter of 80 cm was placed on a plate with a suitable colony density and labeled. Then, the membrane with the colony-printed side facing up was pasted onto an induction plate containing 50 μg / ml kanamycin and 0.5 mM IPTG, and expression was induced at 30°C for 8 h.

[0083] III. Preparation of colorimetric plates:

[0084] Add 20 mM cytidine monophosphate-sialic acid (CMP-SA), 24 mM lactose, and 0.4 mM bromothymol blue solution (BTB) to a 50 mM pH=8 Tris-HCl system, and finally add 0.8% agarose powder. After thoroughly mixing the chromogenic solution, boil to dissolve the agarose. Cool to approximately 50°C and quickly pour into plates. Once the plates have completely solidified, attach a nitrocellulose membrane containing induced bacterial cells to the chromogenic plate (colony side up). Incubate at 28°C for 2 hours and observe the chromogenic results. Figure 4 The results showed that the overall color was green, and no superior mutants were found.

[0085] Example 3

[0086] I. Same as Example 2

[0087] Second, by constructing a correlation network with catalytic residues, we selected the site T196, which has a high influence on it, and performed saturation mutations to screen for mutation types that positively affect catalytic residues and improve catalytic activity.

[0088] Design degenerate primers, 5'-3' direction:

[0089] PF2-GATGAACATTATNNKATTTTTAAAGGCCTGA (SEQ ID NO:5), PR2-CTTTAAAAAATMNNATAATGTTCATCGCTCG (SEQ ID NO:6).

[0090] Perform amplification in a PCR instrument according to the following procedure:

[0091] primer PF2 (10μM) 1μL primerPR2 (10μM) 1μL template(40ng) 0.5μL 2xphantamaxmastermix 10μL <![CDATA[ddH2O]]> Up to 20μL

[0092] PCR procedure:

[0093]

[0094] After PCR, the PCR products were subjected to gel electrophoresis. PCR-positive products were purified by gel extraction. 4 μL of the homologous recombination product was transferred into 30 μL of DH5α competent cells. The cells were incubated on ice for 30 min, followed by heat shock at 42°C for 45 s, and then on ice for 2-3 min. 500 μL of antibiotic-free LB medium was added, and the cells were incubated on a shaker for 45 min. The mixture was then centrifuged and spread onto LB agar plates containing 50 μg / ml kanamycin. The plates were incubated overnight at 37°C for 12-16 hours. The cells were washed off the plates with antibiotic-free LB medium, and the mixed plasmid was extracted using a kit. 5 μL of the mixed plasmid was transferred into 100 μL of BL21(D3) competent cells. The cells were incubated on ice for 30 min, followed by heat shock at 42°C for 45 s, and then on ice for 2-3 min. Next, add 500 μL of antibiotic-free LB medium, and after shaking culture for 1 hour, take 150 μL and spread it on an LB solid plate containing 50 μg / ml kanamycin. Incubate overnight at 37°C for 12-16 hours.

[0095] An 80 cm diameter nitrocellulose membrane was placed on a plate with a suitable colony density and labeled. Then, the membrane with the colony-printed side facing up was pasted onto an induction plate containing 50 μg / ml kanamycin and 0.5 mM IPTG, and expression was induced at 30°C for 8 h.

[0096] III. Preparation of colorimetric plates:

[0097] Add 20 mM cytidine monophosphate-sialic acid (CMP-SA), 24 mM lactose, and 0.4 mM bromothymol blue solution (BTB) to a 50 mM pH=8 Tris-HCl system, and finally add 0.8% agarose powder. After thoroughly mixing the chromogenic solution, boil to dissolve the agarose. Cool to approximately 50°C and quickly pour into plates. Once the plates have completely solidified, attach a nitrocellulose membrane containing induced bacterial cells to the chromogenic plate (colony side up). Incubate at 28°C for 2 hours and observe the chromogenic results. Figure 5 The results showed that the overall color was green, and no superior mutants were found.

[0098] Example 4

[0099] I. Same as Example 2

[0100] 2. The shape of the active site was redesigned based on the ligand using the enzyme design module in Rosetta. In the constructed mutant, the F314 mutation increased the cavity volume of the active site. After re-doping the mutant with lactose, the dosing results showed that the lactose binding site was deeper into the bottom of the active site, which is more conducive to the stable binding of lactose and SL09 enzyme.

[0101] Design degenerate primers: 5'-3' direction:

[0102] PF3-TGAAAGATNNKCCGAACGTGCATGTG (SEQ ID NO:7),

[0103] PR3-ACGTTCGGMNNATCTTTCATGGTCAGCG (SEQ ID NO:8).

[0104] Perform amplification in a PCR instrument according to the following procedure:

[0105]

[0106]

[0107] PCR procedure:

[0108]

[0109] After PCR, the PCR products were subjected to gel electrophoresis. PCR-positive products were purified by gel extraction. 4 μL of the homologous recombination product was transferred into 30 μL of DH5α competent cells. The cells were incubated on ice for 30 min, followed by heat shock at 42°C for 45 s, and then on ice for 2-3 min. 500 μL of antibiotic-free LB medium was added, and the cells were incubated on a shaker for 45 min. The mixture was then centrifuged and spread onto LB agar plates containing 50 μg / ml kanamycin. The plates were incubated overnight at 37°C for 12-16 hours. The cells were washed off the plates with antibiotic-free LB medium, and the mixed plasmid was extracted using a kit. 5 μL of the mixed plasmid was transferred into 100 μL of BL21(D3) competent cells. The cells were incubated on ice for 30 min, followed by heat shock at 42°C for 45 s, and then on ice for 2-3 min. Next, add 500 μL of antibiotic-free LB medium, and after shaking culture for 1 hour, take 150 μL and spread it on an LB solid plate containing 50 μg / ml kanamycin. Incubate overnight at 37°C for 12-16 hours.

[0110] An 80 cm diameter nitrocellulose membrane was placed on a plate with a suitable colony density and labeled. Then, the membrane with the colony-printed side facing up was pasted onto an induction plate containing 50 μg / ml kanamycin and 0.5 mM IPTG, and expression was induced at 30°C for 8 h.

[0111] III. Preparation of colorimetric plates:

[0112] In a 50 mM pH = 8 Tris-HCl system, add 20 mM cytidine monophosphate-sialic acid (CMP-SA), 24 mM lactose, 0.4 mM bromothymol blue solution (BTB), and finally add 0.8% agarose powder. After thoroughly mixing the chromogenic solution, boil to dissolve the agarose. Cool to approximately 50°C and quickly pour into plates. Once the plates have completely solidified, attach a nitrocellulose membrane containing induced bacterial cells to the chromogenic plate (colony side up). Incubate at 28°C for 2 hours and observe the chromogenic results. Figure 6 Among them, the single colonies with a yellower color are mutants with higher yields of 3-SL.

[0113] Example 5

[0114] The positions of the yellowish single colonies were compared with those in the original plate (after membrane covering, incubation at 37°C for 4 hours, and then storage at 4°C). Single colonies from the original plate and pre-streaked controls (non-mutated strains) were cultured separately in 96-well plates. Single colonies were inoculated into 600 μL of LB medium containing 50 μg / ml kanamycin using a toothpick and incubated at 37°C and 220 rpm. When OD600 = 2, 20 μL of the bacterial culture was added to LB medium containing 50 μg / ml kanamycin and 0.3 mM IPTG inducer and incubated overnight at 37°C and 220 rpm. After incubation, the culture was centrifuged, and the 96-well plates containing the bacterial cells were frozen at -20°C.

[0115] Remove the cultured 96-well plate containing only bacterial cells from the -20℃ freezer, add 100μL of lysis buffer to each well, thoroughly resuspend the bacterial cells by pipetting, and then break them with the lysis buffer under the following conditions: 25℃, 800rpm, 2h.

[0116] lysis buffer formulation concentration Dosage Lysozyme / 20mg Tris-HCl 10mM, pH 8.0 19.8mL DnaseI 2000U / mL 2.5μL <![CDATA[MgCl2]]> 1M 0.2mL

[0117] A mixture of 30 mM CMP-SA, 36 mM lactose, and 50 mM Tris was thoroughly mixed, and 600 μL was added to a new 96-well plate. Then, 50 μL of each mutant containing lysis buffer was added to each well. The reaction was carried out at 28 °C and 220 rpm for 1 h. After the reaction, 100 μL of the sample was added to 60% acetonitrile for quenching and then validated by liquid chromatography. Subsequently, bacteria with high 3-SL yields detected by liquid chromatography were picked from the original plate, cultured, and sent for sequencing.

[0118] The strain that mutated F314 to Y314 produced the highest yield of 3-SL. Figures 7-8 The yield increased by 1.46 times compared to the control group. This demonstrates that the high-throughput screening method for flat plates of the present invention is efficient, rapid, and the results are reliable.

[0119] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Reagents for screening 3-sialyltransferase mutants, including: 30–50 mM Tris-HCl buffer, 15–25 mM cytidine monophosphate-sialic acid, 20–30 mM lactose, 0.1–1 mM bromothymol blue, and 0.5%–1% agarose; also includes nitrocellulose membrane.

2. The reagent according to claim 1, characterized in that, It includes: 50 mM Tris-HCl buffer, 24 mM lactose, 20 mM cytidine monophosphate-sialic acid, 0.4 mM bromothymol blue and 0.8% agarose.

3. A method for screening 3-sialyltransferase mutants, comprising contacting colonies with the reagent described in claim 1 or 2 and determining the activity of 3-sialyltransferase based on color changes.

4. The method according to claim 3, characterized in that: Specifically, it includes: Step 1) Spread the bacterial culture onto a solid culture medium for incubation; Step 2) Imprint colonies onto the nitrocellulose membrane, then place the membrane with the colony side facing up onto a solid culture medium to induce culture. Step 3) Place the nitrocellulose membrane cultured in Step 2) with the colony-containing side facing up onto the reagent described in claim 1 or 2, and observe the color change after the reaction.

5. The method according to claim 4, characterized in that, Step 1) The solid culture medium used for the culture is LB solid medium containing 50 μg / ml kanamycin. Step 2) The solid culture medium used for the culture is LB solid medium containing 50 μg / ml kanamycin and 0.5mM IPTG, and the culture conditions include induction culture at 30℃ for 8h; Step 3) The reaction conditions include standing at 28°C for 2 hours.

6. The method according to claim 4, characterized in that, The method of judging the activity of 3-sialyltransferase based on color change includes: if the colony is blue or green, the 3-sialyltransferase activity is weak; if the colony turns yellow, the 3-sialyltransferase activity is strong.

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