Sialic acids are a diverse family of nine-carbon α-keto sugars that occupy the terminal positions of glycoproteins and glycolipids in most vertebrate cells ^[1]. Among these, N-acetylneuraminic acid (Neu5Ac) is the predominant form in humans and plays indispensable roles in cellular recognition, immune modulation, signal transduction, and host–pathogen interactions ^[2-3]. The structural diversity and biological ubiquity of sialic acids make them crucial regulators of molecular and cellular processes, while their location on the cell surface highlights their importance in both normal physiology and disease ^[4-5].

One of the most intensively studied roles of Neu5Ac is its contribution to neurological development. Human breast milk is uniquely enriched in sialylated oligosaccharides, and Neu5Ac has been identified as a critical nutrient for infant brain growth and cognitive functions ^[6-8]. Animal studies demonstrate that dietary supplementation with Neu5Ac enhances learning and memory, likely through its incorporation into gangliosides and polysialylated neural cell adhesion molecules, which are essential for synaptic plasticity and neuronal connectivity ^[9-10]. Clinical studies further suggest that infants fed with formulas supplemented with sialylated glycans display improved cognitive performance compared to those fed non-supplemented formulas ^[11]. These findings position Neu5Ac not merely as a nutrient but as a conditional requirement for optimal neurodevelopment.

Beyond neurology, Neu5Ac has broad biomedical implications. Altered sialylation patterns are associated with cancer progression, immune evasion, and viral infection, making Neu5Ac and its derivatives valuable both as diagnostic biomarkers and as therapeutic targets ^[2,12]. For example, influenza viruses recognize sialylated glycans as cell entry receptors, and sialic acid analogs have been developed as antiviral drugs ^[3]. In oncology, over-sialylation correlates with metastasis and poor prognosis, suggesting Neu5Ac-related pathways as potential intervention points ^[5]. Collectively, these observations underscore the multifaceted significance of Neu5Ac in health and disease.

Despite its importance, the supply of Neu5Ac remains highly constrained. Extraction from natural sources, such as bird’s nest or mammalian tissues, is limited, expensive, and unsustainable ^[13]. Chemical synthesis of Neu5Ac, though theoretically feasible, requires complex multi-step protection–deprotection strategies, yielding low overall efficiency and making it economically impractical for large-scale production ^[14]. These challenges have motivated the exploration of biotechnological production routes.

Among biological methods, enzymatic and microbial synthesis of Neu5Ac have shown promise ^[10,15]. A widely investigated pathway involves the cascade of N-acetylglucosamine-2-epimerase (AGE) and N-acetylneuraminate lyase (NAL), which convert N-acetylglucosamine (GlcNAc) into Neu5Ac in the presence of pyruvate ^[16]. Whole-cell catalysis using engineered Escherichia coli has been demonstrated to achieve Neu5Ac production; however, this approach suffers from two fundamental bottlenecks: (1) both reactions are reversible, resulting in incomplete conversion and low overall yield, and (2) the process requires supplementation of high concentrations of pyruvate, which not only increases production costs but also imposes cytotoxic stress on host cells ^[17-18]. Furthermore, E. coli expression systems often encounter issues such as inclusion body formation, metabolic burden, and competition from byproduct pathways ^[19].

To address these limitations, researchers have increasingly turned to synthetic biology strategies that focus on the spatial and stoichiometric organization of enzymes. Fusion proteins linked by flexible or rigid peptides have been shown to enhance substrate channeling by bringing enzymes into close proximity, thereby reducing intermediate diffusion and reaction reversibility ^[14-15]. Protein scaffolds, such as the SpyTag/SpyCatcher system, provide modular platforms for precise control of enzyme ratios, enabling dynamic optimization of multi-enzyme cascades ^[18]. These approaches, inspired by natural metabolon organization, offer powerful tools to improve the efficiency of Neu5Ac biosynthesis.

Moreover, advances in metabolic engineering have suggested that redirecting intracellular fluxes can significantly reduce reliance on external substrates. Strengthening glycolysis and incorporating pyruvate-producing enzymes into the cascade may enable host strains to self-supply pyruvate, thereby alleviating the need for costly supplementation ^[13,19]. Adaptive laboratory evolution and strain selection also represent promising strategies for enhancing tolerance to Neu5Ac accumulation and substrate overload ^[12].

Taken together, these findings highlight both the importance and the challenge of Neu5Ac production. On one hand, Neu5Ac represents a critical nutrient and biomedical molecule with far-reaching applications in nutrition, medicine, and biotechnology. On the other hand, current production methods remain inefficient and costly, limiting accessibility. Inspired by recent advances in enzyme assembly and host engineering, our project seeks to innovate Neu5Ac biosynthesis by redesigning the AGE–NAL cascade through synthetic biology.

During the biosynthetic reaction of sialic acid, AGE solely catalyzes the first step, which involves the phosphorylation of ManNAc to ManNAc-6P, while NAL exclusively mediates the second step, namely the aldolization of ManNAc-6P to Neu5Ac. Each single enzyme can only complete a partial step in sialic acid synthesis and cannot independently produce the final product. Using synthetic biology techniques, we developed different kinds of plasmids that can ensure the co-expression of AGE and NAL. Additionally, we use the fusion protein technology and protein self-assembly technology to assemble AGE and NAL into a multi-enzyme complex, aiming to resolve the issues of substrate-product balance and mass transfer between the two enzymes during the dual-enzyme catalytic process.

The first stage of the experiment consisted of molecular cloning and protein expression assays. We amplified the target genes AGE, NAL, Linker, and Scaffold using PCR technology, followed by recombinant expression technology to obtain the recombinant plasmids. Colony PCR and DNA sequencing are used to verify the successful cloning and sequence accuracy of the recombinant plasmids containing the target genes. Subsequent steps involved protein expression and purification, through which the target proteins were successfully isolated.

In the second stage of our research, we performed functional tests with TLC, ELISA, and HPLC. First, TLC experiments were conducted to screen out enzyme constructs capable of undergoing reactions and appropriate reaction conditions. Subsequently, ELISA and HPLC were used to detect the concentration of sialic acid produced by the reactions.

The overarching goal of our project is to develop an efficient, sustainable, and scalable platform for Neu5Ac biosynthesis by addressing the inherent bottlenecks of current enzymatic and microbial systems. Specifically, our work focuses on three complementary objectives as follows.

First, we aim to boost yield and catalytic efficiency by improving cooperation between AGE and NAL. Through co-expression in E. coli BL21(DE3), we seek to enable self-assembly of the two enzymes into functional complexes that mitigate substrate–product imbalances and reduce inter-enzyme mass transfer limitations. By optimizing the expression ratio of AGE to NAL, we anticipate achieving significantly higher conversion rates of GlcNAc to Neu5Ac compared to conventional systems.

Second, we intend to reduce production costs and simplify biosynthetic processes. This involves both molecular and process-level improvements: (i) streamlining synthesis steps through fusion protein constructs that enhance substrate channeling, and (ii) employing scaffold-based assemblies that provide modular and tunable control of enzyme stoichiometry. In parallel, we will explore metabolic engineering strategies to strengthen intracellular pyruvate supply, thereby lowering the need for external supplementation and reducing cytotoxic side effects. Together, these measures are expected to substantially improve the economic feasibility of Neu5Ac production.

Third, our goal extends beyond Neu5Ac itself. By leveraging protein scaffold systems and linker-based fusion designs, we aim to establish a versatile toolkit for multi-enzyme cascade optimization. This toolkit will be applicable not only to sialic acid biosynthesis but also to the production of other valuable biomolecules, including complex carbohydrates and glycoengineered precursors. In this way, our project aspires to create a generalizable framework for modular enzyme assembly in synthetic biology.

Ultimately, the successful implementation of these goals will have broad implications. For nutrition, our work could contribute to the development of improved infant formula enriched with Neu5Ac, providing neurodevelopmental benefits comparable to human milk ^[7,9]. For medicine, scalable Neu5Ac production will facilitate research and application in antiviral therapies, cancer diagnostics, and glycoengineering of therapeutic proteins ^[2-3]. More broadly, our strategies will demonstrate how principles of spatial organization and stoichiometric regulation can be harnessed to optimize multi-enzyme pathways, paving the way toward more sustainable and cost-effective biomanufacturing.

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