WO2024036508A1

WO2024036508A1 - Fusion protease, fusion expression vector, engineered bacterium, and method for producing n-acetylneuraminic acid

Info

Publication number: WO2024036508A1
Application number: PCT/CN2022/113029
Authority: WO
Inventors: 陈�峰; 劳永民
Original assignee: 深圳大学
Priority date: 2022-08-17
Filing date: 2022-08-17
Publication date: 2024-02-22

Abstract

Provided are a fusion protease, a fusion expression vector, an engineered bacterium, and a method for producing N-acetylneuraminic acid. The fusion protease comprises N-acetyl-D-glucosamine 2-epimerase (AGE), N-acetylneuraminic acid aldolase (NAL), and a linker (GGGGS)₆. The flexible linker (GGGGS) links two key enzymes, i.e., the AGE and the NAL, in whole-cell biosynthesis of N-acetylneuraminic acid, so that the formation of a polymer from the fusion protease is facilitated, the catalytic capability of the fusion protease is equivalent to that in a native free state, and the fusion protease can be used for catalytic synthesis of N-acetylneuraminic acid.

Description

Fusion protease, fusion expression vector and engineering bacteria, and production method of N-acetylneuraminic acid

Technical field

This application belongs to the field of bioengineering technology, and in particular relates to a fusion protease, fusion expression vector and engineering bacteria, as well as a production method of N-acetylneuraminic acid.

Background technique

The whole-cell biocatalytic synthesis of N-acetylneuraminic acid (Neu5Ac) mainly relies on N-acetyl-D-glucosamine-2-epimerase (AGE) and N-acetylneuraminic acid aldolase (NAL). Expression function, the process is as follows:

It can be seen from the above process that the AGE enzyme isomerizes the substrate N-acetylglucosamine (GlcNAc) into the intermediate product N-acetylmannosamine (ManNAc), and then the NAL enzyme condenses the substrate pyruvate and the intermediate product ManNAc into the final product Neu5Ac. The whole-cell biocatalytic synthesis of Neu5Ac has the characteristics of relatively simple operation, low cost, guaranteed three-dimensional structure, and high yield.

In the currently reported AGE and NAL co-expression systems, AGE and NAL are expressed separately with different promoters. The AGE enzyme is mainly expressed in the form of inclusion bodies. The proteins in the inclusion bodies are often misfolded and have no catalytic activity. Although NAL can The proportion of dissolved proteins is high, but the total amount of expressed protein is small, resulting in low overall catalytic efficiency. In addition, the reactions catalyzed by AGE and NAL are both reversible reactions, and the activity of NAL enzyme is mainly in the opposite direction. For example, the activity ratio of E. coli NAL enzyme cleavage and synthesis of Neu5Ac is about 3:1. Therefore, optimizing the co-expression system to improve the soluble expression of AGE and discovering NAL enzymes with high Neu5Ac synthetic activity are one of the improvement directions to increase Neu5Ac production. However, due to the inherent characteristics of AGE enzyme (inclusion body expression), the current effect of optimizing the soluble expression of AGE is extremely limited. Co-expression based on AGE and NAL can only increase the Neu5Ac production to 127.6±7.61g/ L, and with the increase in NAL copy number, the burden of E. coli must increase, which in turn inhibits the production of Neu5Ac.

technical problem

The purpose of the embodiments of this application is to provide a fusion protease, fusion expression vector and engineering bacteria and a production method of N-acetylneuraminic acid.

Technical solutions

The technical solutions adopted in the embodiments of this application are:

In a first aspect, a fusion protease is provided. The fusion protease includes N-acetyl-D-glucosamine-2-epimerase, N-acetylneuraminic acid aldolase and the N-acetyl-D-glucose. Linker linking amine-2-epimerase to the N-acetylneuraminic acid aldolase (GGGGS) _1-6 .

In one embodiment, the fusion protease includes N-acetyl-D-glucosamine-2-epimerase, N-acetylneuraminic acid aldolase, and N-acetyl-D-glucosamine-2-epimerase. Linker linking isomerase and N-acetylneuraminic acid aldolase (GGGGS) ₄ .

In one embodiment, the amino acid sequence of N-acetyl-D-glucosamine-2-epimerase is shown in SEQ ID NO.1.

In one embodiment, the amino acid sequence of N-acetylneuraminic acid aldolase is shown in SEQ ID NO. 2.

In the second aspect, a fusion expression vector is provided, which expresses the fusion protease of the present application.

In one embodiment, the nucleotide sequence for expressing N-acetyl-D-glucosamine-2-epimerase in the fusion expression vector is shown in SEQ ID NO. 4, expressing N-acetylneuraminic acid aldol. The nucleotide sequence of the enzyme is shown in SEQ ID NO.5, and the nucleotide sequence of the expression linker (GGGGS) ₄ is shown in SEQ ID NO.6.

In one embodiment, the fusion expression vector is constructed by combining a nucleotide fragment that expresses N-acetyl-D-glucosamine-2-epimerase, a nucleotide fragment that expresses linkers (GGGGS) _1-6 , and The DNA of the nucleotide fragment of N-acetylneuraminic acid aldolase was inserted downstream of the promoter of pET-32a(+) plasmid.

In the third aspect, an engineering bacterium for producing N-acetylneuraminic acid is provided, and the engineering bacterium contains the fusion expression vector of the present application.

In a fourth aspect, a production method of N-acetylneuraminic acid is provided, including the following steps:

The engineering bacteria of the present application are induced and cultured under conditions without isopropylthiogalactopyranoside inducer, and then whole-cell biocatalytic product synthesis is performed in a reaction solution containing N-acetylglucosamine and sodium pyruvate.

In one embodiment, the step of inducing culture under conditions without isopropyl thiogalactopyranoside inducer includes: placing the engineering bacteria in LB liquid culture medium without isopropyl thiogalactopyranoside inducer, First, culture at 36-38°C and 150-250 rpm until OD ₆₀₀ = 0.6-0.8, and then induce culture at 20-30°C and 60-100 rpm for 22-26 hours.

In one embodiment, the step of performing whole-cell biocatalytic product synthesis in a reaction solution containing N-acetylglucosamine and sodium pyruvate includes: placing the engineered bacteria after induction and culture into the reaction solution, and concentrating the bacterial cells The density reaches OD ₆₀₀ = 28~32, and the product is synthesized; the concentration of N-acetylglucosamine in the reaction solution is 0.5~1.5M, the concentration of sodium pyruvate is 1.0~1.8M, and the temperature of product synthesis is 25~37 ℃, the rotation speed is 150~250rpm, and the time is 24~48 hours.

beneficial effects

The beneficial effect of the fusion protease provided by the embodiments of the present application is that the fusion protease uses flexible linkers (GGGGS) _1-6 to connect the two key enzymes for whole-cell biosynthesis of N-acetylneuraminic acid, namely AGE and NAL, which can confer existence The AGE and NAL in the protein-protein interaction have a certain swing range, which is conducive to the formation of polymers to ensure that their catalytic ability is equivalent to that in the natural free state. Therefore, such a fusion protease can be used to catalyze the synthesis of N-acetylneuraminic acid, which has great Good application prospects.

The beneficial effect of the fusion expression vector provided by the embodiments of the present application is that the fusion expression vector can express the unique fusion expression protease of the present application. Therefore, when the fusion expression vector is introduced into a host cell, whole-cell biocatalysis can be performed to synthesize N-acetylneuramine. acid, so it has good application prospects in the field of whole-cell biosynthesis of N-acetylneuraminic acid.

The beneficial effect of the engineering bacteria for producing N-acetylneuraminic acid provided in the embodiments of the present application is that the engineering bacteria contain the fusion expression vector of the present application, so whole-cell biocatalytic synthesis of N-acetylneuraminic acid can be performed based on the engineering bacteria.

The beneficial effect of the production method of N-acetylneuraminic acid provided by the embodiments of the present application is that in this production method, the engineering bacteria of the present application are first induced and cultured without inducers, and then in the presence of N-acetylglucosamine and sodium pyruvate. The product is synthesized in the reaction solution, so that N-acetylneuraminic acid can be produced with high efficiency, and it has good application prospects in the field of N-acetylneuraminic acid production.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required to be used in the description of the embodiments or exemplary technologies will be briefly introduced below. Obviously, the drawings in the following description are only for the purpose of the present application. For some embodiments, for those of ordinary skill in the art, other drawings can be obtained based on these drawings without exerting creative efforts.

Figure 1 is a schematic diagram of the spatial structure of the fusion protease provided in the embodiment of the present application, where a is a top view and b is a bottom view;

Figure 2 is a schematic diagram of the fusion expression vector provided by the embodiment of the present application;

Figure 3 is a schematic diagram of the co-expression vector provided in the comparative example of the present application.

Embodiments of the invention

In order to make the purpose, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present application and are not used to limit the present application.

It should be understood that in various embodiments of the present application, the size of the sequence numbers of the above-mentioned processes does not mean the order of execution. Some or all steps can be executed in parallel or one after another. The execution order of each process should be based on its function and order. The internal logic is determined and should not constitute any limitation on the implementation process of the embodiments of the present application.

The terminology used in the embodiments of the present application is only for the purpose of describing specific embodiments and is not intended to limit the present application. As used in the embodiments and the appended claims, the singular forms "a," "the" and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

The terms "first", "second", etc. are used for descriptive purposes only and are used to distinguish objects such as substances from each other, and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be called the second XX, and similarly, the second XX may also be called the first XX. Therefore, features defined as "first" and "second" may explicitly or implicitly include one or more of these features.

The first aspect of the embodiments of the present application provides a fusion protease. The fusion protease includes N-acetyl-D-glucosamine-2-epimerase, N-acetylneuraminic acid aldolase and N-acetyl-D-glucosamine-2-epimerase. Glucosamine-2-epimerase and N-acetylneuraminic acid aldolase linker (GGGGS) _1-6 .

In the natural state, AGE and NAL can form homodimers and homotetramers respectively through protein-protein interactions. However, the fusion protease provided in the embodiments of the present application uses a flexible linker (GGGGS) _1-6 [1-6 A series of GGGGS amino acid sequences] connects the two key enzymes for whole-cell biosynthesis of N-acetylneuraminic acid, namely AGE and NAL. This can give AGE and NAL a certain swing range in the presence of protein-protein interactions, which is conducive to the formation of polymers. , to ensure that its catalytic ability is equivalent to that of the natural free state. Therefore, such a fusion protease can be used to catalyze the synthesis of N-acetylneuraminic acid and has good application prospects.

In one embodiment, the fusion protease includes N-acetyl-D-glucosamine-2-epimerase, N-acetylneuraminic acid aldolase, and N-acetyl-D-glucosamine-2-epimerase. Linker linking isomerase and N-acetylneuraminic acid aldolase (GGGGS) ₄ . Among them, the N-terminus of the fusion protease is AGE and the C-terminus is NAL; or the N-terminus of the fusion protease is NAL and the C-terminus is AGE.

Specifically, AGE can be Anabaena sp.CH1 AGE (bAGE), and the amino acid sequence is as shown in SEQ ID NO.1; NAL can be NanA of E. coli K12substr.MG1655, Staphylococcus hominis ShNAL and Corynebacterium glutamicum ATCC 13032 CgNAL, this application is selected from CgNAL, and the amino acid sequence is shown in SEQ ID NO. 2.

The amino acid sequence of the linker is SEQ ID NO.3: GGGGSGGGGSGGGGSGGGGS.

The second aspect of the embodiments of the present application provides a fusion expression vector. The fusion expression vector expresses the fusion expression protease of the embodiments of the present application.

The fusion expression vector provided by the embodiments of the present application can express the unique fusion expression protease of the embodiments of the present application. Therefore, the fusion expression vector can be introduced into a host cell to perform whole-cell biocatalytic synthesis of N-acetylneuraminic acid. Specifically, the embodiments of this application use NAL as a "soluble tag" and use fusion expression linkers (GGGGS) _1-6 to fuse and express AGE and NAL, thereby simultaneously increasing the solubility of AGE and the expression level of NAL to ensure that NAL and AGE The total amount and soluble protein amount are uniformly equal; in addition, the fusion of NAL and AGE can shorten the spatial distance between the two enzyme catalytic clefts. In theory, ManNAc, the product of the AGE enzyme, can more easily enter the catalytic cleft of the downstream NAL enzyme. Increasing the effective concentration of ManNAc is beneficial to the catalytic reaction in the synthesis direction of Neu5Ac, thereby improving the catalytic efficiency of the reaction system and the yield of Neu5Ac. Therefore, such a fusion expression vector has good application prospects in the field of whole-cell biosynthesis of N-acetylneuraminic acid.

In one embodiment, the fusion expression vector is constructed by combining a nucleotide fragment that expresses N-acetyl-D-glucosamine-2-epimerase, a nucleotide fragment that expresses linkers (GGGGS) _1-6 , and The DNA of the nucleotide fragment of N-acetylneuraminic acid aldolase was inserted downstream of the promoter of pET-32a(+) plasmid. Specifically, AGE, (GGGGS) _1-6 linker and NAL fragment (the N-terminus of the fusion protein is AGE and the C-terminus is NAL) or NAL, (GGGGS) _1-6 linker and AGE fragment (the N-terminus of the fusion protein is NAL) were combined by overlapping PCR. (NAL, AGE at the C terminus) to form a large fusion expression fragment; use In-Fusion ligase to insert the large fusion expression fragment between the T7 promoter and T7 terminator of pET-32a(+) to construct a series of fusion expression vectors .

The fusion expression vector provided by the embodiments of the present application not only uses NAL as a "soluble tag" to significantly increase the soluble expression of AGE; at the same time, it utilizes the leakage expression of the T7 promoter without adding isopropylthiogalactopyranoside inducer. It is ready for expression, reducing product synthesis costs and subsequent product purification costs, and only requires 1 copy of AGE and 1 copy of NAL, which can further reduce the host cell burden of the fusion expression vector.

In one embodiment, the nucleotide sequence expressing AGE in the fusion expression vector is shown in SEQ ID NO.4, and the nucleotide sequence expressing NAL is shown in SEQ ID NO.5.

The nucleotide sequence of expression linker (GGGGS) ₄ is shown in SEQ ID NO.6:

The third aspect of the embodiments of the present application provides an engineering bacterium that produces N-acetylneuraminic acid, and the engineering bacterium contains the fusion expression vector of the embodiments of the present application.

The third aspect of the present application provides an engineering bacterium that produces N-acetylneuraminic acid. The engineered bacterium contains the fusion expression vector of the present application. Therefore, whole-cell biocatalytic synthesis of N-acetylneuraminic acid can be performed based on the engineered bacterium.

Specifically, in the embodiments of the present application, the unique fusion expression vector of the present application can be introduced into E. coli DH5 through chemical transformation, positive clones are screened through colony PCR, and confirmed by sequencing; then the plasmid is inoculated, extracted, and introduced into the production host strain E. coli BL21 (DE3), the engineering bacteria producing N-acetylneuraminic acid in the embodiments of the present application were obtained. At the same time, homology modeling of the fusion protease was performed to reveal the principle of fusion expression over co-expression from the catalytic mechanism, and based on this screening, the theoretically optimal strain was obtained.

The fourth aspect of the embodiments of the present application provides a method for producing N-acetylneuraminic acid, which includes the following steps:

The above-mentioned unique engineering bacteria in the embodiments of the present application are induced and cultured under conditions without isopropylthiogalactopyranoside inducer, and then whole-cell biocatalysis is performed in a reaction solution containing N-acetylglucosamine and sodium pyruvate. Product synthesis.

The embodiments of the present application provide a production method of N-acetylneuraminic acid. The production method involves inducing and culturing the engineering bacteria of the present application without isopropylthiogalactopyranoside inducer, and then incubating it with N-acetylglucosamine. Whole-cell biocatalytic synthesis of Neu5Ac product is carried out in the reaction solution with sodium pyruvate, so that N-acetylneuraminic acid can be produced with high efficiency, and it has good application prospects in the field of N-acetylneuraminic acid production.

In one embodiment, the step of inducing culture under conditions without isopropyl thiogalactopyranoside inducer includes: placing the engineering bacteria in 4.5L LB liquid culture medium without isopropyl thiogalactopyranoside inducer. , first culture at 36-38°C, 150-250 rpm until OD ₆₀₀ = 0.6-0.8, and then induce culture at 20-30°C, 60-100 rpm for 22-26 hours. Further, by optimizing the induction culture conditions of the strain, the conditions for induction culture include: 0mM isopropylthiogalactopyranoside, 25°C, 80rpm, induction for 24 hours; under these conditions, soluble expression fusion of engineered bacteria can be better induced Protease.

In one embodiment, the step of performing whole-cell biocatalytic product synthesis in a reaction solution containing N-acetylglucosamine and sodium pyruvate includes: placing the engineered bacteria after induction and culture into the reaction solution, and concentrating the bacterial cells The density reaches OD ₆₀₀ = 28~32, and the product is synthesized; among them, the concentration of N-acetylglucosamine in 1L reaction solution is 0.5~1.5M, the concentration of sodium pyruvate is 1.0~1.8M, and the temperature of product synthesis is 25~ 37℃, rotation speed is 150~250rpm, time is 24~48 hours. Further, the fusion protein expression of the strain and the conditions for whole-cell catalytic synthesis of Neu5Ac can be optimized. The conditions for the synthesis of the Neu5Ac product include: the concentration of N-acetylglucosamine is 1.2M, the concentration of sodium pyruvate is 1.6M, and the reaction temperature of the product synthesis. The temperature is 30°C, the rotation speed is 200 rpm, and the reaction time for product synthesis is 24 to 48 hours. Furthermore, when the OD ₆₀₀ of the engineered bacteria is 0.6-0.8, the above-mentioned induction culture is performed; when the induced-cultured engineering bacteria are concentrated to OD ₆₀₀ = 28-32, the above-mentioned product synthesis reaction is performed. Under the above conditions, the maximum production efficiency of N-acetylneuraminic acid from the engineered bacteria can be increased to 7.73±0.56g/L/h, and the titer of N-acetylneuraminic acid can be as high as 149.72±8.52g/L, which is higher than the current The highest reported yield would be an increase of more than 17%.

In one embodiment, in order to reduce the metabolic consumption of the substrate GlcNAc in E. coli, site-specific gene editing can be performed on E. coli to knock out key genes in the metabolic bypass pathway related to the substrate GlcNAc; to knock out the genes in the Neu5Ac product transport and metabolic bypass pathways. Key genes; in addition, pyruvate chassis cells can be constructed, which can reduce the use of pyruvate substrate and reduce the production cost of Neu5Ac; the copy number of the fusion protease in the E. coli genome can also be optimized to further increase the production of Neu5Ac, and finally obtain Neu5Ac Super-accumulating engineering bacteria can use fermentation tanks to optimize the conditions for whole-cell biocatalytic production of Neu5Ac by Neu5Ac super-accumulating bacteria to achieve mass production of Neu5Ac.

In one embodiment, this application adopts a two-step method to synthesize Neu5Ac:

Step 1, induction culture:

The engineering bacteria preserved in glycerol were streaked on a solid LB plate containing 100 μg/mL ampicillin, cultured overnight at 37°C for activation, single clones were picked, and inoculated into 150 mL of liquid LB containing 100 μg/mL ampicillin. Cultivate overnight at 37°C, 200 rpm until OD ₆₀₀ = 1.3~1.5. Pour into 4.5L liquid LB containing 100 μg/mL ampicillin at a ratio of 10%, and culture at 37°C and 200 rpm until OD ₆₀₀ = 0.6 to 0.8. Adjust the induction temperature to 25°C, 80rpm rotation speed, and induce for 24 hours.

Step 2, product synthesis reaction:

Centrifuge at 10000g for 3 minutes at 25°C to collect the cultured cells, add 1L reaction solution (formula: 100mM Tris-HCl, pH7.5; 10mMgCl ₂ , 1.2M GlcNAc, 1.6M sodium pyruvate), and adjust OD ₆₀₀ to 30 ; 30°C, 200rpm, react for 48 hours, and detect the Neu5Ac product titer by HPLC. HPLC detection conditions: Chromatographic column: aminex HPX-87H column (BioRad Laboratories, Beverly, Mass); column temperature 65°C; mobile phase: 0.056% H ₂ SO ₄ ; flow rate: 0.6mL·min-1; PDA detection wavelength: 210nm ;Injection volume: 10μL. Use HPLC to quantitatively analyze the Neu5Ac product and calculate the yield.

Description will be made below with reference to specific embodiments.

Example 1

A fusion protease (NAL_AGE fusion protease), including N-acetyl-D-glucosamine-2-epimerase (amino acid sequence is SEQ ID NO.1), N-acetylneuraminic acid aldolase (amino acid sequence It is SEQ ID NO.2) and the linker connecting N-acetyl-D-glucosamine-2-epimerase and N-acetylneuraminic acid aldolase (the amino acid sequence is SEQ ID NO.3). Among them, the N-terminal of the fusion protease is NAL and the C-terminal is AGE. The spatial structure is shown in Figure 1.

Example 2

A fusion expression vector, the sequence structure of which is shown in Figure 2. The fusion expression vector expresses the fusion protease of Example 1, and is specifically obtained by the following preparation method:

By overlapping PCR, 1 copy of NAL, (GGGGS) ₄ linker and 1 copy of AGE fragment were connected into a large fusion expression fragment (the nucleotide sequence is: SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.4) ; Use In-Fusion ligase to insert the large fusion expression fragment downstream of the T7 promoter of pET-32a(+) to construct a fusion expression vector.

Example 3

An engineering bacterium that produces N-acetylneuraminic acid is obtained by the following preparation method:

The fusion expression vector of Example 2 was introduced into E.coli DH5 using a chemical transformation method, positive clones were screened through colony PCR, and confirmed by sequencing; the plasmid was inoculated, extracted, and introduced into the production host strain E.coliBL21 (DE3).

Example 4

A production method of N-acetylneuraminic acid, including the following steps:

Step 1: Set up different gradients, optimize the culture conditions of the engineering bacteria in Example 3, including isopropylthiogalactopyranoside inducer concentration, induction duration, induction temperature and shaking rate, etc., to obtain AGE and NAL fusion proteins Under the soluble expression conditions, the induction temperature was 25°C, the concentration of the inducer isopropylthiogalactopyranoside was 0mM, the shaking speed was 80rpm, and the induction time was 24 hours. Specifically, the engineering bacteria were inserted into 4.5L liquid LB containing 100 μg/mL ampicillin at a ratio of 10%, and cultured at 37°C and 200rpm until OD ₆₀₀ = 0.6-0.8; the induction temperature was adjusted to 25°C and the rotation speed was 80rpm for induction culture. 24 hours.

Step 2: Use the above induction conditions to induce the fusion protein, collect the engineered bacterial cells that have been induced and expressed, then add the collected engineering bacteria to the production system of the reaction solution, adjust to OD ₆₀₀ = 30, and synthesize the Neu5Ac product. The production system of the reaction solution has a volume of 1L, which contains: 100mM Tris-HCl (pH7.5), 10mMgCl ₂ , 1.2M GlcNAc, and 1.6M sodium pyruvate. Reaction temperature: 30°C, shake the reaction system at a shaking speed of 200 rpm, reaction time: 48 hours.

The fusion protease was induced under the above conditions, and the final maximum production efficiency of Neu5Ac was 7.73±0.56g/L/h, and the titer of Neu5Ac reached 149.72±8.52g/L.

Comparative ratio

Step 1: Refer to Molecular Biotechnology, 2018, 60:427-434, and insert 2 copies of NAL downstream of the two T7 promoters of pETDuet-1; use overlapping PCR to connect the T7 promoter, 1 copy of AGE and the T7 terminator into The large fragment was inserted into the SphI restriction site of pETDuet-1 to construct the co-expression vector shown in Figure 3 (the sequences of NAL, AGE, promoter and terminator are all the same as those in Example 2).

Step 2: Set up different gradients and optimize the culture conditions of the engineering bacteria containing the co-expression vector of the above comparative example, including the concentration of isopropylthiogalactopyranoside inducer, induction time, induction temperature and shaking rate, etc., to obtain a co-expression vector. The soluble expression conditions for expressing AGE and NAL were as follows: the induction temperature was 25°C, the concentration of the inducer isopropylthiogalactopyranoside was 0.5mM, the shaking speed was 80rpm, and the induction time was 24 hours. Specifically, the proportional engineering bacteria were inserted into 4.5L of liquid LB containing 100 μg/mL ampicillin at a ratio of 10%, and cultured at 37°C and 200rpm until OD ₆₀₀ = 0.6-0.8; the induction temperature was adjusted to 25°C, and the inducer The concentration of isopropylthiogalactopyranoside was 0.5mM, the rotation speed was 80rpm, and the culture was induced for 24 hours.

Step 3: Use the above induction conditions to induce the protein of the comparative engineering bacteria, collect the induced expression bacteria, adjust to OD ₆₀₀ = 30, and synthesize the Neu5Ac product. The engineering bacteria are added to the reaction system for producing Neu5Ac for catalytic production. The production system: 100mM Tris-HCl (pH7.5), 10mMgCl ₂ , 1.2M GlcNAc, 1.6M sodium pyruvate, the volume is 1L. Reaction temperature: 30°C, shake the reaction system at a shaking speed of 200 rpm, reaction time: 48 hours.

The protease of the comparative engineering strain was induced under the above conditions, and the final maximum Neu5Ac production (i.e. titer) was 127.6±7.61g/L.

The above are only optional embodiments of the present application and are not used to limit the present application. Various modifications and variations may be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of this application shall be included in the scope of the claims of this application.

Claims

A fusion protease, wherein the fusion protease includes N-acetyl-D-glucosamine-2-epimerase, N-acetylneuraminic acid aldolase and the N-acetyl-D-glucosamine -Linker linking 2-epimerase to said N-acetylneuraminic acid aldolase (GGGGS) 1-6 .
The fusion protease of claim 1, wherein the fusion protease includes the N-acetyl-D-glucosamine-2-epimerase, the N-acetylneuraminic acid aldolase and the A linker (GGGGS) 4 connecting the N-acetyl-D-glucosamine-2-epimerase and the N-acetylneuraminic acid aldolase 4 .
The fusion protease according to claim 1, wherein the amino acid sequence of the N-acetyl-D-glucosamine-2-epimerase is as shown in SEQ ID NO.1.
The fusion protease according to claim 1, wherein the amino acid sequence of the N-acetylneuraminic acid aldolase is as shown in SEQ ID NO. 2.
A fusion expression vector, wherein the fusion expression vector expresses the fusion protease according to any one of claims 1-4.
The fusion expression vector according to claim 5, wherein the nucleotide sequence for expressing the N-acetyl-D-glucosamine-2-epimerase in the fusion expression vector is as shown in SEQ ID NO.4 shows that the nucleotide sequence expressing the N-acetylneuraminic acid aldolase is shown in SEQ ID NO.5, and the nucleotide sequence expressing the linker (GGGGS) 4 is shown in SEQ ID NO.6.
The fusion expression vector according to claim 4, wherein the fusion expression vector is composed of a nucleotide fragment containing the N-acetyl-D-glucosamine-2-epimerase and the linker. The nucleotide fragments of (GGGGS) 1 to 6 and the DNA expressing the nucleotide fragment of N-acetylneuraminic acid aldolase were inserted downstream of the promoter of pET-32a(+) plasmid.
An engineering bacterium that produces N-acetylneuraminic acid, wherein the engineering bacterium contains the fusion expression vector of claim 5.
The engineering bacterium according to claim 8, wherein the nucleotide sequence for expressing the N-acetyl-D-glucosamine-2-epimerase in the fusion expression vector is shown in SEQ ID NO.4 , the nucleotide sequence expressing the N-acetylneuraminic acid aldolase is shown in SEQ ID NO.5, and the nucleotide sequence expressing the linker (GGGGS) 4 is shown in SEQ ID NO.6.
The engineering bacterium according to claim 8, wherein the fusion expression vector contains a nucleotide fragment expressing the N-acetyl-D-glucosamine-2-epimerase and expresses the linker ( The nucleotide fragment of GGGGS) 1-6 and the DNA expressing the nucleotide fragment of N-acetylneuraminic acid aldolase were inserted downstream of the promoter of pET-32a(+) plasmid.
A production method of N-acetylneuraminic acid, characterized in that it includes the following steps:

The engineered bacterium according to claim 5 is induced and cultured under conditions without isopropylthiogalactopyranoside inducer, and then whole-cell biocatalysis is performed in a reaction solution containing N-acetylglucosamine and sodium pyruvate. synthesis.
The production method according to claim 11, wherein in the fusion expression vector in the engineering bacterium, the nucleotide sequence for expressing the N-acetyl-D-glucosamine-2-epimerase is as follows SEQ ID NO.4 is shown, the nucleotide sequence expressing the N-acetylneuraminic acid aldolase is shown in SEQ ID NO.5, and the nucleotide sequence expressing the linker (GGGGS) 4 is shown in SEQ ID Shown in NO.6.
The production method of claim 11, wherein the fusion expression vector in the engineered bacterium is produced by converting a nucleotide fragment containing the N-acetyl-D-glucosamine-2-epimerase into , the DNA fragment expressing the linker (GGGGS) 1-6 and the DNA fragment expressing the N-acetylneuraminic acid aldolase were inserted downstream of the promoter of the pET-32a(+) plasmid.
The production method according to claim 11, wherein the step of inducing culture under conditions without isopropyl thiogalactopyranoside inducer includes: placing the engineered bacteria in an environment without isopropyl thiogalactopyranoside inducer. In the LB liquid medium of glycoside inducer, first culture at 36-38°C and 150-250rpm to OD 600 = 0.6-0.8, and then induce and culture at 20-30°C and 60-100rpm for 22-26 hours.
The production method according to claim 11, wherein the step of performing whole-cell biocatalytic product synthesis in a reaction solution containing N-acetylglucosamine and sodium pyruvate includes: placing the engineered bacteria after induction and culture. In the reaction solution, concentrate the bacterial cell density to OD 600 =28-32, and perform product synthesis; wherein the concentration of N-acetylglucosamine in the reaction solution is 0.5-1.5M, and the concentration of sodium pyruvate is 1.0 ~1.8M, the product is synthesized at a temperature of 25-37°C, a rotation speed of 150-250rpm, and a time of 24-48 hours.