Abstract illustration of an enzyme cascade producing CTP.

Revolutionizing CTP Production: A Novel Enzyme Cascade

Mira Elwood in Tech & Innovation December 2025 • 3 min read.

"Harnessing the power of a three-enzyme system to drive down costs and boost efficiency in industrial CTP manufacturing."

In various industries, the enzymatic synthesis of cytidine triphosphate (CTP) and other nucleotides is crucial. However, the reliance on ATP, and its high cost, in these processes necessitates more economical solutions. Researchers have been actively seeking ways to reduce expenses and enhance the efficiency of ATP-dependent biosynthetic reactions.

Traditional methods for ATP regeneration often fall short. Some systems utilize whole cells, which, although cost-effective, suffer from stability issues and generate unwanted byproducts. Other systems rely on enzymatic reactions, but these can be hampered by product inhibition, high reagent costs, and instability.

The study introduces a novel one-pot polyphosphate kinase (PPK) system, designed to minimize costs and maximize efficiency in CTP production. By leveraging a three-enzyme cascade—CMP kinase (CMK), nucleoside-diphosphate kinase (NDK), and PPK—along with an in vitro polyphosphate-based ATP regeneration system, this innovative approach paves the way for more sustainable and scalable CTP manufacturing.

Unlocking Efficiency: The Three-Enzyme Cascade

Abstract illustration of an enzyme cascade producing CTP.

The core of this innovation lies in its three-enzyme cascade. Each enzyme plays a vital role in the CTP production process:

To optimize the system, researchers carefully selected and engineered each enzyme:

PPK (Polyphosphate Kinase): Sourced from twenty different organisms, PPK was made soluble through fusion expression and co-expression with molecular chaperones. This ensured the enzyme was readily available for the reaction.
CMK (CMP Kinase) & NDK (Nucleoside-Diphosphate Kinase): These enzymes were optimized using fusion expression, the tac-pBAD system, a Rosetta host, and codon optimization. This multi-pronged approach maximized their activity and stability.

After 24 hours, the concentration of CDP and CTP reached 3.8 ± 0.2 and 6.9 ± 0.3 mM respectively, achieving a yield of approximately 79%. The molar conversion rate of CTP was 51%, a 100% increase compared to traditional methods. This demonstrates the potential of the new system in enhancing CTP production.

A Greener Future for Nucleotide Synthesis

The development of this one-pot ATP regeneration system marks a significant step forward in sustainable biomanufacturing. By utilizing polyphosphate kinase for CTP production, this innovative approach lowers costs and improves efficiency.

Beyond cost savings, this system offers several advantages, including simplified downstream processing and improved enzymatic reaction balance. The optimized enzyme ratios guarantee maximum yield and enhanced CTP production.

This PPK-based energy regeneration system has the potential to be applied to various synthetic processes involving high-energy compounds, which could revolutionize other areas of industrial biotechnology.

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This article is based on research published under:

DOI-LINK: 10.1007/s10529-017-2427-x, Alternate LINK

Title: Novel One-Pot Atp Regeneration System Based On Three-Enzyme Cascade For Industrial Ctp Production

Subject: General Medicine

Journal: Biotechnology Letters

Publisher: Springer Science and Business Media LLC

Authors: Junzhi Wang, Cheng Zheng, Tianyi Zhang, Yingmiao Liu, Zhuopei Cheng, Dong Liu, Hanjie Ying, Huanqing Niu

Published: 2017-08-31

Everything You Need To Know

What is the role of each enzyme in the three-enzyme cascade used for cytidine triphosphate (CTP) production?

The core innovation involves a three-enzyme cascade: CMP kinase (CMK), nucleoside-diphosphate kinase (NDK), and polyphosphate kinase (PPK). CMP kinase (CMK) and nucleoside-diphosphate kinase (NDK) work together to convert CMP into CTP, while polyphosphate kinase (PPK) regenerates ATP from polyphosphate, making the process more sustainable and cost-effective. These enzymes are optimized to work together efficiently in a single reaction vessel.

How does the one-pot ATP regeneration system enhance the sustainability of cytidine triphosphate (CTP) manufacturing?

The system uses a three-enzyme cascade—CMP kinase (CMK), nucleoside-diphosphate kinase (NDK), and polyphosphate kinase (PPK). By integrating these enzymes with an in vitro polyphosphate-based ATP regeneration system, the approach reduces the need for costly ATP, thereby paving the way for more sustainable and scalable CTP manufacturing. The use of polyphosphate as a phosphate donor, facilitated by polyphosphate kinase (PPK), is key to this cost reduction.

What specific methods were used to optimize the enzymes polyphosphate kinase (PPK), CMP kinase (CMK), and nucleoside-diphosphate kinase (NDK) in this system?

Researchers optimized polyphosphate kinase (PPK) by sourcing it from twenty different organisms and making it soluble through fusion expression and co-expression with molecular chaperones to ensure it was readily available for the reaction. CMP kinase (CMK) and nucleoside-diphosphate kinase (NDK) were optimized using fusion expression, the tac-pBAD system, a Rosetta host, and codon optimization to maximize their activity and stability.

How does using polyphosphate kinase (PPK) contribute to lowering costs in cytidine triphosphate (CTP) production?

The one-pot ATP regeneration system leverages the enzyme polyphosphate kinase (PPK) to use polyphosphate as a more economical alternative to ATP. This innovative approach reduces production costs and enhances efficiency, making the enzymatic synthesis of cytidine triphosphate (CTP) more viable for large-scale manufacturing. It addresses the limitations of traditional methods by avoiding product inhibition and reducing reagent costs.

What were the quantitative results demonstrating the performance of the new enzyme cascade system for cytidine triphosphate (CTP) production compared to traditional methods?

This system achieved a CTP molar conversion rate of 51%, which represents a 100% increase compared to traditional methods. After 24 hours, the concentration of CDP and CTP reached 3.8 ± 0.2 and 6.9 ± 0.3 mM respectively, achieving a yield of approximately 79%. This substantial improvement highlights the potential of this approach in enhancing cytidine triphosphate (CTP) production, offering a more efficient and cost-effective solution for nucleotide synthesis.