We present a comprehensive modular genetic toolkit for the construction and optimization of E. coli cell factories dedicated to the biosynthesis of N-Acetylneuraminic Acid (NeuAc), a high-value sialic acid with significant applications in pharmaceuticals and nutraceuticals. Our collection features a series of standardized, fully characterized BioBrick-compatible parts. These include individual enzyme expression cassettes, a novel fusion enzyme construct, and a co-expression system, all designed for flexible and efficient pathway assembly. Beyond the parts themselves, we provide extensive characterization data (sequencing, SDS-PAGE, TLC activity assays) and a crucially improved protocol for thin-layer chromatography (TLC) analysis of NeuAc, solving a common problem in the field.
This toolkit is designed to be a plug-and-play solution for future iGEM teams and synthetic biologists aiming to enter the field of sialic acid production or to learn advanced metabolic engineering techniques. The relevant parts are listed in Table 1.
Table 1. Part contribution
|
Part Number |
Part Name |
Part Type |
Function Description |
Corresponding Toolkit Module |
Key Features |
|---|---|---|---|---|---|
|
BBa_K4846011 |
pETDuet-1 |
Basic Part |
Provides a standard pETDuet-1 vector backbone, containing multiple cloning sites and an ampicillin resistance gene, serving as the core skeleton for composite vector construction |
Core Enzyme Composite Vector, Expression System |
|
|
BBa_K5508004 |
AGE |
Basic Part |
Encodes N-acetylglucosamine-2-epimerase (AGE), which catalyzes the conversion of GlcNAc to ManNAc (the key enzyme for the first step of NeuAc synthesis) |
Core Enzyme Module, Fusion Protein, Co-expression System |
|
|
BBa_25FD9OTU |
NAL |
Basic Part |
Encodes N-acetylneuraminate lyase (NAL), which catalyzes the condensation of ManNAc and pyruvate to form NeuAc (the key enzyme for synthesis) |
Core Enzyme Module, Fusion Protein, Co-expression System |
|
|
BBa_25NRNS44 |
Linker-based enzyme fusion |
Basic Part |
Encodes a glycine-serine (G/S) flexible linker, used to connect AGE and NAL to form a fusion protein, realizing substrate channeling |
Fusion Protein System |
|
|
BBa_25PHTFUH |
Protein scaffold |
Basic Part |
Encodes a scaffold protein that can spatially localize free AGE and NAL enzymes, forming an artificial enzyme complex to improve catalytic efficiency |
Scaffold-Assisted Co-expression System |
|
|
BBa_25VDT58W |
pETDuet-AGE-NAL |
Composite Part |
A single vector carrying both AGE and NAL, co-expressed with the T7 promoter/operator of the vector |
Simplifies the vector system and avoids resistance conflicts of dual vectors |
|
|
BBa_25H3L7EW |
pET28a-AGE-Linker-NAL |
Composite Part |
The vector inserts the AGE-Linker-NAL to express a single fusion protein. |
Mimics natural metabolism, increases concentration, and reduces byproducts. |
|
|
BBa_25HYGDBT |
pET28a-Scaffold |
Composite Part |
The vector carries the coding gene of Protein scaffold, with T7 promoter, for separate expression of the scaffold protein. |
Used in conjunction with pETDuet-AGE-NAL to achieve spatial co-localization of enzymes |
