Length: 987 bp

Description: N-acetylneuraminic acid aldolase

Properties: NAL primarily exists as a homotetramer in its native state, which is assembled from four identical subunits via non-covalent bonds. This tetrameric structure is an essential prerequisite for the enzyme to maintain its catalytic activity. The secondary structure of each subunit is characterized by the alternating arrangement of α-helices and β-sheets, forming the typical TIM barrel structure—a conserved folding pattern within the aldolase family. Eight parallel β-sheets constitute the core of the barrel, surrounded by eight α-helices, which provide a stable spatial framework for the catalytic active site.

Figure 1 Gene map of NAL

Usage and Biology: The primary function of NAL is to catalyze the reversible aldol cleavage of N-acetylneuraminic acid (NeuAc) into N-acetylmannosamine (ManNAc) and pyruvate, and it can also catalyze the reverse condensation reaction under specific conditions. Therefore, it plays a crucial role in the biosynthesis of sialic acids like NeuAc.

Cultivation: A single colony containing the NAL plasmid was inoculated into liquid LB medium supplemented and incubated overnight at 37 °C with constant shaking. Plasmid DNA was subsequently extracted and used as the template for PCR amplification of the NAL coding sequence using gene-specific primers under optimized thermal cycling conditions. The PCR products were analyzed by agarose gel electrophoresis.

Figure 2 Gel electrophoresis of NAL

Length: 54 bp

Properties: The template sequence is used to fuse the enzyme AGE and NAL. After ligating the AGE-Linker-NAL fragment into a plasmid, we will use the plasmid to express the fused protein inside the E.coli. The linker will provide flexibility between protein domains, ensuring proper folding and catalytic activity.

Figure 3 Gene map of Linker

Cultivation: A single colony containing the AGE-Linker-NAL plasmid was inoculated into liquid LB medium supplemented and incubated overnight at 37 °C with constant shaking. Plasmid DNA was subsequently extracted and used as the template for PCR amplification of the AGE-Linker-NAL coding sequence using gene-specific primers under optimized thermal cycling conditions. The PCR products were analyzed by agarose gel electrophoresis.

Figure 4 Gel electrophoresis of AGE-Linker-NAL

Length: 755 bp

Properties: After inserting this scaffold into the plasmid, we will use the plasmid to express the scaffold structure in E.coli. The scaffold provides a stable framework for assembling and positioning functional domains, thereby improving catalytic efficiency and structural stability.

Figure 5 Gene map of Scaffold

Cultivation: A single colony containing the scaffold plasmid was inoculated into liquid LB medium supplemented and incubated overnight at 37 °C with constant shaking. Plasmid DNA was subsequently extracted and used as the template for PCR amplification of the scaffold coding sequence using gene-specific primers under optimized thermal cycling conditions. The PCR products were analyzed by agarose gel electrophoresis to confirm the presence and expected size of the amplified fragment.

Figure 6 Gel electrophoresis of Scaffold

Composition: pETDuet backbone, AGE gene fragment, NAL gene fragment

Apparatus used: pETDuet plasmid, AGE gene fragment, NAL gene fragment, restriction endonucleases, and homologous recombination enzyme

Figure 7 Plasmid map of pETDuet-AGE-NAL

Engineering Principle:

As a vector with dual promoters, the pRSFDuet plasmid enables the simultaneous expression of two proteins. It contains the RSF origin of replication (ori) to ensure stable replication and also includes promoter/regulatory elements such as lacIq and T7 for regulating gene expression.

The AGE gene fragment (1262 bp) was amplified using PCR. The NAL gene fragment (987 bp) was amplified using PCR. Figure 8 shows the agarose gel electrophoresis results of the PCR. We could confirm that the DNA fragments of both target genes were consistent with the predicted sizes. This indicated that the target genes were successfully amplified.

Figure 8 Gel electrophoresis of AGE and NAL

Prior to constructing the pETDuet-AGE-NAL plasmid, we first completed the construction of pETDuet-AGE. This construction employed a homologous recombination approach. The colonies obtained after transformation are shown in Figure 4, indicating successful recombination and the acquisition of positive clones. Subsequently, we selected a single colony for colony PCR verification. Electrophoresis revealed a specific band at 1262 bp, matching the expected size, preliminarily confirming that the AGE fragment had been correctly recombined into the pETDuet vector. To further validate the construction accuracy, we extracted the recombinant plasmid and submitted it for sequencing. Sequence alignment confirmed that the AGE gene was correctly inserted with a fully intact reading frame, showing no frameshifts or mutations. This indicates the successful construction of the pETDuet-AGE plasmid, laying the foundation for subsequent introduction of the NAL gene and further dual-enzyme co-expression studies.

Figure 9 pETDuet-AGE Plasmid Verification Diagram

Building upon the successful construction of the pETDuet-AGE plasmid, we further employed homologous recombination to insert the NAL gene into the vector, thereby constructing the pETDuet-AGE-NAL recombinant plasmid. Clear single colonies were visible on the transformation plates, indicating successful recombination. Randomly selected colonies were subjected to PCR validation. Electrophoresis analysis of the amplified products revealed a specific band at the expected size (approximately 2249 bp), preliminarily confirming the correct insertion of the NAL gene. Subsequently, positive clone plasmids were extracted for sequencing analysis. The sequencing results fully matched the expected sequence, confirming that both the AGE and NAL genes were correctly integrated into the pETDuet vector with intact reading frames and no frameshift mutations. These results indicate the successful construction of the pETDuet-AGE-NAL plasmid, providing a reliable vector for subsequent dual-enzyme co-expression and catalytic function studies.

Figure 10 pETDuet-AGE-NAL Plasmid Verification Diagram

Cultivation, Purification, and SDS-PAGE: Protein expression was induced with IPTG (1 mM) in E. coli BL21(DE3), followed by cell lysis using a sonicator under cold conditions to preserve enzyme activity. The recombinant protein was purified using nickel affinity chromatography via its 6×His-tag. The purification process involved washing with low (buffer A) and high (buffer B) concentrations of imidazole. The collected fractions were analyzed by SDS-PAGE. As shown in Figure 11, distinct protein bands were observed in all lysate samples. These results confirmed that the AGE and NAL proteins were successfully expressed using the plasmid, enabling the reactions: conversion of GlcNAc to ManNAc (catalyzed by AGE) and subsequent conversion to NeuAc (catalyzed by NAL).

Figure 11 SDS-PAGE of pETDuet-AGE-NAL

Composition: pET28a backbone, AGE gene fragment, Linker gene fragment, NAL gene fragment

Apparatus used: pET28a backbone, AGE gene fragment, Linker gene fragment, NAL gene fragment, restriction endonucleases, Homologous recombination enzyme

Figure 12 Plasmid map of pET28a-AGE-Linker-NAL

Engineering Principle:

The pET28a plasmid is a widely used prokaryotic expression vector, primarily designed for high-level recombinant protein expression in E. coli. It carries a T7 promoter, which is tightly regulated by T7 RNA polymerase, enabling induced protein expression via IPTG. It also includes antibiotic resistance genes, such as kanamycin resistance, for selecting E. coli transformants containing the plasmid. The AGE-Linker-NAL gene fragment (2117 bp) was amplified using PCR, as shown in Figure 12, indicating the target gene was successfully amplified.

Figure 13 Gel electrophoresis of AGE-Linker-NAL

We further employed homologous recombination to sequentially assemble the AGE gene, Linker sequence, and NAL gene into the pET28a vector, constructing the pET28a-AGE-Linker-NAL recombinant plasmid. Following the transformation, uniformly distributed single colonies were observed on the agar plate, indicating successful recombinant transformation. Several colonies were randomly selected for colony PCR verification. Electrophoresis results showed a specific band at the expected size (approximately 2303 bp for the AGE-Linker-NAL fragment), preliminarily confirming the correct insertion of the three-gene fusion into the vector. To further validate construction accuracy, positive clone plasmids were extracted for sequencing analysis. Sequence results fully matched the expected sequence, confirming that the AGE, Linker, and NAL elements were correctly integrated into the pET28a vector as designed, with precise codon transitions and no frameshift mutations. These results confirm the successful construction of the pET28a-AGE-Linker-NAL triple fusion expression vector, providing essential material for subsequent studies investigating the regulatory role of the Linker sequence on the spatial conformation and catalytic efficiency of the dual enzymes.

Figure 14 Colony growth, gel electrophoresis result, and DNA sequencing of pET28a-AGE-Linker-NAL

Cultivation, Purification, and SDS-PAGE: Protein expression was induced with IPTG (1 mM) in E. coli BL21(DE3), followed by cell lysis using a sonicator under cold conditions to preserve enzyme activity. The recombinant protein was purified using nickel affinity chromatography via its 6×His-tag. The purification process involved washing with low (buffer A) and high (buffer B) concentrations of imidazole. The collected fractions were analyzed by SDS-PAGE. As shown in Figure 15, a single distinct band was detected in the lane loaded with the elution solution. This result is consistent with the expected molecular weight, indicating that the AGE-Linker-NAL were successfully expressed and purified in the elution fractions under the current experimental conditions.

Figure 15 SDS-PAGE result of pET28a-AGE-Linker-NAL

Composition: pET28a backbone, Protein scaffold gene fragment

Apparatus used: pET28a backbone, Protein scaffold gene fragment, restriction endonucleases, and homologous recombination enzyme.

Figure 16 Plasmid map of pET28a-Scaffold

Engineering Principle:

The pET28a plasmid is a widely used prokaryotic expression vector, primarily designed for high-level recombinant protein expression in E. coli. It carries a T7 promoter, which is tightly regulated by T7 RNA polymerase, enabling induced protein expression via IPTG. It also includes antibiotic resistance genes, such as kanamycin resistance, for selecting E. coli transformants containing the plasmid. The Scaffold gene fragment (775 bp) was amplified using PCR, as shown in Figure 17, indicating the target gene was successfully amplified.

Figure 17 Gel electrophoresis of Scaffold

We proceeded to employ homologous recombination to clone the artificial scaffold protein gene into the pET28a vector, constructing the pET28a-Scaffold recombinant plasmid. Clear single colonies were observed on the plate post-transformation, indicating successful transformation. Subsequently, multiple colonies were randomly selected for colony PCR validation. Electrophoresis results showed a specific band at the expected size (approximately 755 bp), providing preliminary evidence that the Scaffold gene had been successfully inserted into the vector. To further confirm construction accuracy, we extracted positive clone plasmids for sequencing analysis. The sequencing results fully matched the expected sequence, confirming that the Scaffold gene was correctly integrated into the pET28a vector with an intact coding frame, free of frameshifts or mutations. These results indicate the successful construction of the pET28a-Scaffold plasmid, laying the foundation for subsequent studies investigating the regulatory role of scaffold proteins in multi-enzyme assembly and metabolic pathway efficiency.

Figure 18 Colony growth, gel electrophoresis result, and DNA sequencing of pET28a-Scaffold

Cultivation, Purification, and SDS-PAGE: Protein expression was induced with IPTG (1 mM) in E. coli BL21(DE3), followed by cell lysis using a sonicator under cold conditions to preserve enzyme activity. The recombinant protein was purified using nickel affinity chromatography via its 6×His-tag. The purification process involved washing with low (buffer A) and high (buffer B) concentrations of imidazole. The collected fractions were analyzed by SDS-PAGE.

Figure 19 SDS-PAGE result of pET28a-Scaffold+AGE-NAL

The activities of AGE and NAL were qualitatively analyzed using TLC at two time points: 30 and 60 minutes. As shown in Figure 20, in the AGE-catalyzed reactions, the band corresponding to the non-denatured enzyme at 30 minutes migrated similarly to the product reference, while the denatured enzyme at 60 minutes co-migrated with the substrate. This indicates that 30 minutes was sufficient for optimal AGE activity under these conditions. For NAL, the TLC results showed separation primarily at the substrate positions, suggesting that the current mobile phase may not be ideal for resolving NAL-derived products due to their high polarity.

Figure 20 TLC for AGE and NAL under 30 and 60 minutes

To further validate the generation of ManNAc and NeuAc, we optimized the developing solvent system for thin-layer chromatography (TLC). After reacting at 42°C for 30 minutes, samples underwent two duplicate developments using freshly prepared developing solvent, with TLC plates thoroughly dried using a hair dryer after each development. Subsequently, TLC plates were immersed in a developing agent and heated at 130°C for 5 minutes for visualization.

Results showed that in the three distinct enzyme structures—AGE-NAL (A-N), AGE-Linker-NAL (A-L-N), and Scaffold + NAL-AGE (S+A-N)—no bands corresponding to ManNAc or NeuAc were detected in the A-N group. In contrast, both A-L-N and S+A-N groups exhibited distinct target product bands. This result indicates that introducing Linker or Scaffold modules into the enzyme structure effectively prevents reversible reactions during metabolism, thereby enhancing the synthetic efficiency of ManNAc and NeuAc.

Figure 21 TLC for AGE and NAL under 30 minutes

We conducted systematic TLC analysis of the enzymatic activities of three enzyme constructs—AGE-NAL (A-N), AGE-Linker-NAL (A-L-N), and Scaffold+NAL-AGE (S+A-N)—reacted for one hour at three temperature conditions: 25°C, 37°C, and 42°C. As shown in Figure 22A, the A-N construct yielded the highest amount of ManNAc in the 1-hour incubation group, with no detectable NeuAc product formation at this time point. This indicates that A-N tends to accumulate the intermediate ManNAc over shorter reaction times.

When incubation was extended to 2 hours, as shown in Figure 22B, both the A-L-N and S+A-N constructs exhibited distinct NeuAc bands whose intensity approached that of the target product reference band. This indicates that these constructs efficiently catalyze NeuAc synthesis upon extended reaction time. Compared to the 1-hour incubation group, the 2-hour incubation group exhibited more pronounced product bands at 37°C, indicating that appropriately extending the reaction time significantly enhances catalytic efficiency.

Collectively, these results indicate that the A-N construct favors ManNAc accumulation in short-duration reactions, while the A-L-N and S+A-N constructs promote final NeuAc production after extended reaction times, with 37°C and 2 hours representing optimal conditions. This phenomenon indicates significant differences in catalytic properties among different enzyme constructs. The introduction of linkers or scaffolds may promote the conversion of reaction intermediates into end products, thereby enhancing overall catalytic efficiency.

Figure 22 TLC for NAL-AGE (A-N), AGE-Linker-NAL (A-L-N), and Scaffold + NAL-AGE (S+A-N) under 25°C, 37°C, and 42°C for 1 hour,and 2 h

To evaluate the catalytic performance of the dual-enzyme system, an ELISA-based quantification assay was conducted.

Table 2. The standard table of Neu5Ac

A 4-parameter logistic regression (4PL) model was employed for standard curve fitting, with the equation shown in Figure 23, which exhibited a high correlation coefficient (R² = 0.9999).

Figure 23 The equation and the standard curve for the ELISA experiment

The absorbance measured in the experiment was substituted into the equation for calculation to obtain the initial detected concentration of sialic acid. The actual content of sialic acid in the samples was then derived by multiplying by the dilution factor (5 times), as shown in Table 3.

Table 3

The final results were plotted in Figure 24. For the enzyme construct A-N, the sialic acid synthesis efficiency was the highest at a concentration of 1 mg/mL, and decreased at medium to high concentrations (5-10 mg/mL). At a low concentration, the rate of the first-step reaction in sialic acid synthesis was slightly higher than that of the second-step reaction, resulting in the overall reaction proceeding toward sialic acid synthesis. Under high concentration conditions, the product sialic acid was decomposed into intermediate products by the NAL enzyme, which were further decomposed by the AGE enzyme.

For the enzyme construct A-L-N, the content of sialic acid showed an upward trend as the concentration increased. Compared with the maximum value of 1 mg/mL in the A-N group, the detection values at various concentrations in the A-L-N group were relatively stable. This indicated that the role of the Linker is to stabilize enzyme function rather than overactivate it.

For the enzyme construct S+A-N, all detection indicators were generally higher than those in the A-L-N group. At the concentration of 10 mg/mL, the detected value was second only to that of the A-N group at 1 mg/mL. Through the clustering effect, the Scaffold formed high-concentration A-N enzyme clusters locally, significantly improving the reaction efficiency. Meanwhile, by binding to the AGE and NAL enzymes, the Scaffold induced their conversion from an inactive conformation to an active conformation, which could directly enhance the catalytic efficiency of individual enzymes. This was the core reason why the detected values of the S+A-N group were higher than those of the A-L-N group.

In conclusion, the enzyme construction Scaffold+AGE-NAL can maximize the efficiency of sialic acid synthesis at medium to high concentrations, making it the optimal solution for efficiently increasing sialic acid production.

Figure 24 Concentration of Neu5Ac tested by the ELISA Kit calculated by the standard curve

The catalytic experiment of the A-N enzyme construct was illustrated in Figure 25A. The sialic acid content reached its highest level at a reaction time of 60 minutes across all three reaction temperatures. As the reaction time prolonged, the sialic acid yield exhibited a decreasing trend. We hypothesize that this phenomenon was associated with the reversibility of the catalytic reaction. When the content of the intermediate product ManNAc-6P accumulated to a certain extent, the reaction proceeded in the reverse direction. Consequently, the substrate for the second step of the reaction was reduced, leading to a decrease in the final yield of sialic acid. Under the condition of a reaction time of 60 minutes, the reaction temperature of 42 °C yielded the highest accumulation of sialic acid. Overall, the optimal reaction system for enzyme construct A-N is as follows: temperature 42 °C + reaction time 60 minutes.

The similar results were also observed when enzyme constructs A-L-N and S+A-N were used, as shown in Figure 25B and 25C. Notably, when the S+A-N enzyme construct was used, the accumulation of sialic acid rebounded under the conditions of 42 °C and a reaction time of 240 minutes. This may be attributed to the fact that the protein scaffold effectively reduced the occurrence of reversible reactions under such reaction conditions. Overall, the optimal reaction systems for both enzyme constructs A-L-N and S+A-N were as follows: temperature 42 °C + reaction time 60 minutes.

Figure 25 Sialic acid production by each enzyme construct under different reaction times and temperatures

The sialic acid production of the three enzyme constructs under the condition of 42 °C, 240 minutes was illustrated in Figure 26. Although the sialic acid content decreased as the reaction time prolonged, the enzyme construct S+A-N exhibited the smallest degree of decrease and achieved the highest final accumulated product concentration. This indicated that construct S+A-N can effectively reduce the occurrence of reversible reactions and was thus the optimal enzyme construct for producing sialic acid.

Figure 26 Sialic acid production by three enzyme constructs under 42 ℃, 240 minutes

Through detailed analysis of the HPLC peak chart in Figure 27, the successful generation of sialic acid and the significant catalytic advantage of the S+A-N construct can be further confirmed.

In the chromatogram of the S+A-N construct, a distinct and sharp peak appears at approximately 11 minutes retention time, which aligns perfectly with the characteristic peak of sialic acid in the standard sample. This peak exhibits symmetry and a large area, indicating high product concentration and excellent separation with no significant tailing.

In contrast, although product peaks are detectable at the same retention time for other constructs (e.g., A-N and A-L-N), their response intensity is significantly lower than that of S+A-N. These peaks are smaller and accompanied by minor impurity peaks. Particularly in the A-N sample, a chromatographic peak belonging to the intermediate ManNAc (retention time approximately 3 minutes) appeared before the target product peak, further indicating incomplete reaction and lower catalytic efficiency.

In summary, the HPLC quantitative results align with previous activity assessment conclusions: the S+A-N construct most effectively catalyzes sialic acid synthesis, yielding the highest product yield and optimal purity, fully demonstrating its superiority as a highly efficient catalytic module.

Figure 27 Chromatograms of sialic acid content under 42 ℃, 240 minutes