Enzymatic CMP-Neu5Ac Synthesis via In Situ Cytidine Activation
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Solution Overview
Problem
The existing methods for producing cytidine 5′-monophospho-N-acetyl-neuraminic acid (CMP-Neu5Ac) are costly due to the high expense of starting materials like cytidine-5′-monophosphate (CMP) and cytidine-5′-triphosphate (CTP), and are inefficient due to inhibition by high concentrations of phosphorylated cytidine nucleotides.
Innovation Solution
A cost-effective and efficient method for producing CMP-Neu5Ac using low-cost substrates such as N-acetyl-D-glucosamine, pyruvate, cytidine, and polyphosphate in a single reaction mixture with a set of enzymes including N-acylglucosamine 2-epimerase, N-acetylneuraminate lyase, N-acylneuraminate cytidylyltransferase, uridine kinase, uridine monophosphate kinase, and polyphosphate kinase 3, where the uridine kinase in situ converts cytidine to CMP to control inhibitory concentrations of phosphorylated cytidine nucleotides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If high concentrations of phosphorylated cytidine nucleotides (CMP, CTP) are used as starting materials, then the production of CMP-Neu5Ac can proceed, but the reaction is inhibited and production efficiency decreases
Solution Approach 1:
The patent applies preliminary action by pre-converting cytidine to CMP using uridine kinase before the main CSS reaction occurs. This ensures that CMP is available at the right time and concentration, preventing the inhibition problem while maintaining productivity. The uridine kinase step is performed first to generate the necessary activated form of cytidine without creating excessive inhibitory concentrations.
Solution Approach 2:
The patent uses cytidine as an intermediary substance that is gradually converted to CMP by uridine kinase. This intermediary approach allows the system to avoid direct use of high concentrations of phosphorylated cytidine nucleotides, thereby reducing inhibition while still enabling the CSS reaction to proceed efficiently through controlled intermediate formation.
2Productivity
If expensive starting materials like CMP and CTP are used, then CMP-Neu5Ac can be produced, but production cost increases significantly
Solution Approach 1:
The patent employs cheap short-living objects by using cytidine (a low-cost substrate) instead of expensive CMP or CTP as the starting material. Cytidine is converted in situ to CMP through the action of uridine kinase, providing a cost-effective alternative that maintains production capability while significantly reducing material costs.
Solution Approach 2:
The patent applies parameter changes by altering the chemical form of the cytidine substrate from its activated phosphorylated forms (CMP, CTP) to the unphosphorylated form (cytidine). This parameter change reduces cost while the enzymatic conversion by uridine kinase ensures that the reactive phosphorylated forms are generated in situ at appropriate concentrations for the CSS reaction.
3Productivity
If existing enzymatic methods are used, then CMP-Neu5Ac production is achieved, but the process requires multiple separate reaction steps
Solution Approach 1:
The patent merges multiple separate enzymatic steps into a single integrated reaction system. The uridine kinase and CSS enzymes work together in one reaction mixture, allowing cytidine to be converted to CMP and then to CMP-Neu5Ac in a unified process. This reduces the number of separate reaction steps while maintaining or improving productivity through streamlined operations.
Solution Approach 2:
The patent applies universality by designing a multi-functional enzymatic system where uridine kinase and CSS work together in a single reaction mixture. This multi-functional approach allows the system to perform both the activation of cytidine to CMP and the subsequent synthesis of CMP-Neu5Ac in one integrated process, reducing complexity while maintaining synthesis capability.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method enables the production of CMP-Neu5Ac in a cost-effective and efficient manner by utilizing low-cost substrates and minimizing inhibitory effects, thereby enhancing the efficacy of the multi-enzymatic cascade reaction.
Implementation Method 1
wherein the uridine kinase (UDK) transfers in situ the cytidine to a cytidine 5'-monophosphate (CMP)
Implementation Method 2
an N-acylneuraminate cytidylyltransferase (CSS)
Implementation Method 3
an N-acetylneuraminate lyase (NAL)
Implementation Method 4
N-acylglucosamine 2-epimerase (AGE)
Data Source
AI summary
The present invention relates to a method for producing cytidine 5′-monophospho-N-acetyl-neuraminic acid (CMP-Neu5Ac, 1) from low-cost substrates N-acetyl-D-glucosamine (GlcNAc), pyruvate, cytidine and polyphosphate in a single reaction mixture with a set of optionally immobilized or optionally co-immobilized enzymes comprising N-acylglucosamine 2-epimerase (AGE), an N-acetylneuraminate lyase (NAL), an N-acylneuraminate cytidylyltransferase (CSS), a uridine kinase (UDK), a uridine monophosphate kinase and a polyphosphate kinase 3 (PPK3). Further, said process may be adapted to produce Neu5Acylated i.e. sialylated biomolecules and biomolecules including a saccharide, a peptide, a protein, a glycopeptide, a glycoprotein, a glycolipid, a glycan, an antibody, and a glycoconjugate, in particular, an antibody drug conjugate, and a carbohydrate conjugate vaccine, or a flavonoid.