NeuAc has a variety of functions, including promoting infants’ neurodevelopment and anti-inflammatory ability. We use two enzymes, AGE and NAL, to catalyze GlcNAc to form NeuAc. We designed plasmids that can express the enzymes, including optimization of codons and addition of several parts necessary for the expression of our target gene. These two enzymes will then be expressed by the plasmids, induced by IPTG, and their activity levels will finally be examined through functional tests. The entire process will be completed inside the bacteria using the method of whole-cell biocatalysis. The specific process is shown below (Figure 1), covering the engineering of target plasmids, protein expression, and functional tests.

Figure 1 The detailed process of the whole experiment

Design:

To construct the recombinant plasmid pETDuet-AGE-NAL, the plasmid pETDuet-AGE was first constructed to serve as the vector. The plasmid pETDuet-AGE was constructed by ligating the AGE with pETDuet, as shown in Figure 2. The plasmid carries a ribosome-binding site, His-tag, S-tag, AmpR gene, lac operon, as well as T7 promoter and terminator. The AmpR gene is used for selection via antibiotics. Homologous recombination was employed to construct pETDuet-AGE. This plasmid functions as an IPTG-inducible expression vector and contains two core regulatory elements: the lac operon and T7 promoter. The lac operon binds the lac repressor to block transcription, whereas the T7 promoter drives high-level protein expression upon IPTG induction and provision of T7 RNA polymerase. These elements jointly regulate the expression of downstream proteins tagged with His and S, which are used for subsequent protein purification.

Figure 2 The plasmid map of pETDuet-AGE

After obtaining pETDuet-AGE, pETDuet-AGE-NAL was constructed by ligating the NAL fragment with pETDuet-AGE using the same method. The plasmid map is shown in Figure 3 below.

Figure 3 The plasmid map of pETDuet-AGE-NAL

Build:

After designing the plasmid types used for expressing the protein, we first amplified the DNA inside using PCR technology. The sequences of AGE (1262 bp) and NAL (987 bp) were amplified with high specificity, showing single, discrete bands upon electrophoresis, as shown in Figure 4.

Figure 4 Gel electrophoresis of PCR amplified AGE and NAL.

To construct the recombinant plasmid pETDuet-AGE, we first performed restriction enzyme digestion to linearize the pETDuet-1 vector. In the electrophoresis results (Figure 5), all pETDuet-1 samples digested with NcoI/BamHI showed at least one clear and intact band. This indicates the successful linearization of the vector.

Figure 5 Double enzyme digestion of plasmid pETDuet

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 6 pETDuet-AGE Plasmid Verification Diagram

The same method was used to construct the plasmid pETDuet-AGE-NAL. As shown in Figure 7, the pETDuet-AGE plasmids digested with NdeI/XhoI showed at least one clear and intact band. This indicates the successful linearization of pETDuet-AGE.

Figure 7 Double enzyme digestion of plasmid pETDuet-AGE

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 8 pETDuet-AGE-NAL Plasmid Verification Diagram

Test:

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 9, a distinct protein band was observed in the elution solution, indicating that our plasmid successfully expresses the target protein.

Figure 9 SDS-PAGE of pETDuet-AGE-NAL

Learn:

The plasmid pETDuet-AGE was constructed primarily as an intermediate vector for the subsequent assembly of pETDuet-AGE-NAL and was not subjected to independent functional assays. Although the co-expression of non-fused AGE and NAL enzymes from this plasmid facilitated the synthesis of sialic acid, the reaction efficiency remained limited. ELISA quantification revealed that this configuration yielded the lowest product amount among all tested constructs. These results suggest that while functionally active, the delocalized expression of AGE and NAL is suboptimal. In contrast, the integrated systems—particularly those incorporating a linker or a scaffold—demonstrate significantly higher production efficiency, making them more suitable for effective sialic acid synthesis.

Design:

pET28a-AGE-Linker-NAL was constructed by ligating the AGE-Linker-NAL fragment with the pET28a plasmid, where pET28a served as the vector. As a widely used prokaryotic expression vector, pET28a is primarily designed for high-level recombinant protein expression in E. coli. It carries a T7 promoter—tightly regulated by T7 RNA polymerase—that enables IPTG-induced protein expression. Additionally, it contains antibiotic resistance genes (e.g., kanamycin resistance) for selecting E. coli transformants harboring the plasmid. Homologous recombination was employed for the construction of pET28a-AGE-Linker-NAL. This recombinant plasmid functions as an IPTG-inducible expression vector and contains two fundamental regulatory elements, the lac operon and the T7 promoter, with a mechanism identical to that of pETDuet-AGE. These elements control the expression of downstream His-tagged proteins, which are used for subsequent protein purification.

Figure 10 The plasmid map of pET28a-AGE-Linker-NAL

Build:

After designing the plasmid constructs for protein expression, we first amplified the target DNA using PCR technology. The AGE-Linker-NAL sequence was amplified with high specificity, showing a single, discrete band in electrophoresis, as shown in Figure 11. We can find out that the DNA fragment was approximately 2303 bp in length, which was consistent with the predicted size. This indicated that the target gene was successfully amplified.

Figure 11 Gel electrophoresis of PCR amplified AGE-Linker-NAL.

To construct the plasmid pET28a-AGE-Linker-NAL, restriction enzyme digestion was performed to linearize the pET28a vector. In the electrophoresis analysis results (Figure 12), the pET28a plasmids digested with NcoI/XhoI showed at least one clear and intact band. This indicates that the linearization of pET28a was successful.

Figure 12 Double enzyme digestion of plasmid pET28a

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 13 pET28a-AGE-Linker-NAL Plasmid Verification Diagram

Test:

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 14, a distinct protein band was observed in the elution solution, indicating that our plasmid successfully expresses the target protein.

Figure 14 SDS-PAGE of pET28a-AGE-Linker-NAL

Learn:

By constructing a fusion protein (AGE-Linker-NAL), the two enzymes are co-expressed in a spatially coordinated manner. This configuration enhances the channeling of the reaction intermediate, leading to a more efficient synthesis of sialic acid than the system employing separate AGE and NAL enzymes.

Design:

The plasmid pET28a-Scaffold was constructed by ligating the Scaffold gene into the pET28a vector, shown in Figure 15. The plasmid contains a ribosome-binding site, His-tag, KanR, lac operon, as well as T7 promoter and terminator. The KanR enables selection via antibiotics. The pET28a-Scaffold was constructed using homologous recombination. As an IPTG-inducible expression plasmid, it harbors two key regulatory elements, the lac operon and the T7 promoter, that function through the same mechanism as in pETDuet-AGE. These elements regulate the expression of downstream His-tagged proteins, which facilitate subsequent protein purification.

Figure 15 The plasmid map of pET28a-Scaffold

Build:

The Scaffold gene (775 bp) was PCR-amplified. We can find out that the DNA fragment of Scaffold is approximately 775 bp in length. The sequence of Scaffold was amplified with high specificity, showing a single, discrete band upon electrophoresis, as shown in Figure 16.

Figure 16 Agarose gel electrophoresis results of PCR for Scaffold

To construct the plasmid pET28a-Scaffold, the pET28a vector was linearized by restriction enzyme digestion with NcoI and HindIII. As shown in the electrophoresis analysis (Figure 17), the enzyme-digested pET28a plasmids displayed at least one distinct and intact band, confirming successful linearization of the vector.

Figure 17 Double enzyme digestion of plasmid pET28a

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 pET28a-Scaffold Plasmid Verification Diagram

Test:

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 19, a distinct protein band was observed in the elution solution, indicating that our plasmid successfully expresses the target protein.

Figure 19 SDS-PAGE of pET28a-Scaffold

Learn:

The scaffold protein mediates a structured interaction between AGE and NAL, forming a multi-enzyme complex. This complex demonstrated the highest catalytic efficiency in sialic acid production among all tested constructs, as quantified by ELISA.

1. Thin-Layer Chromatography (TLC)

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

3.2 Enzyme-linked immunosorbent assay (ELISA)

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

Table 1 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 2.

Table 2

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

3.3 High-Performance Liquid Chromatography (HPLC)

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