_Dr._Da-Hee_Ahn,_Distinguished_Professor_Sang_Yup_Lee.jpg)
<(From Left) Dr. Da-Hee Ahn, Distinguished Professor Sang Yup Lee>
Nylon is a representative plastic material used throughout our daily lives, from clothing to automobiles. However, most of its raw materials have been produced through petrochemical processes, resulting in large carbon emissions. KAIST researchers have developed a technology that can produce key nylon precursors in an eco-friendly way using microbes.
KAIST (President Kwang Hyung Lee) announced on the 31st of May that a research team led by Distinguished Professor Sang Yup Lee of the Department of Chemical and Biomolecular Engineering has developed an Escherichia coli-based modular platform capable of producing three key monomers (basic molecular units that make up polymers) of "nylon 6,6" and "nylon 6" — adipic acid, hexamethylenediamine, and epsilon-caprolactam — from "glycerol (an eco-friendly bio-based byproduct generated during biodiesel production)," a renewable carbon source, using systems metabolic engineering (a technology that designs and optimizes microbial metabolic pathways to maximize the production of desired substances).
"Nylon 6" is highly flexible and is used in clothing and films, while "nylon 6,6" has excellent strength and heat resistance and is used in automobiles and machinery parts. The numbers after the nylon name indicate the number of carbon atoms contained in the raw material molecules.
The core of this study is that the biosynthetic pathway was divided into upstream and downstream modules, with E. coli strains assigned different roles. The upstream strain was designed to produce adipic acid from glycerol, while the downstream strain was designed to convert it into hexamethylenediamine or epsilon-caprolactam, respectively. Through this, the research team succeeded in producing adipic acid and hexamethylenediamine, the key raw materials of nylon 6,6, and epsilon-caprolactam, the key raw material of nylon 6, within a single integrated platform.
To improve production efficiency, the researchers compared and validated various enzymes (proteins that promote chemical reactions in living organisms), including carboxylic acid reductases and transaminases, and applied the optimal combination, thereby improving hexamethylenediamine titer. In addition, in the epsilon-caprolactam production process, they designed a flexible-linker fusion enzyme that enhances reaction efficiency through efficient cofactor regeneration.In the upstream module, the team reconstructed the biosynthetic pathway (a series of reaction processes through which compounds are produced in living organisms) and improved the performance of key enzymes using artificial intelligence (AI), increasing production titer. As a result, they succeeded in producing adipic acid at a level of 6 grams per liter (g/L) in a fed-batch fermentation process.
The research team also applied a "delayed inoculation" strategy (time-staggered co-culture), in which the second strain is introduced later after sufficient adipic acid has first been produced, rather than adding the two types of E. coli simultaneously. This is a method of sequentially introducing microbes with different roles at different times.
When this strategy was applied to a fed-batch fermentation process (a fermentation method that increases productivity by supplying nutrients step by step), the team produced 230 milligrams per liter (mg/L) of hexamethylenediamine and 808 micrograms per liter (μg/L) of epsilon-caprolactam using only glycerol. Although the production amounts are not yet high, the research team explained that these results represent world-class performance among cases of direct production from glycerol.
This technology is significant in that it presents the possibility of producing nylon raw materials, which have relied on petrochemical processes, through bio-based methods.
The research team plans to further improve titer by combining AI-based enzyme design with additional systems metabolic engineering, and to expand the platform to produce various polymer raw materials (substances formed by the repeated bonding of multiple monomers).
Distinguished Professor Sang Yup Lee stated, "This study is meaningful in that it presents a modular microbial platform capable of producing key monomers required for nylon 6 and nylon 6,6 production from renewable carbon sources," adding, "We will continue to advance enzyme and metabolic flux engineering to improve titer and develop this into a core platform for sustainably producing various bio-based polymer raw materials."
The results of this study were published on May 4 in the Proceedings of the National Academy of Sciences (PNAS), with Dr. Da-Hee Ahn of the Department of Chemical and Biomolecular Engineering as the first author.
※ Paper title: "Metabolic engineering of Escherichia coli for the biosynthesis of nylon 6 and nylon 6,6 monomers"
Authors: Sang Yup Lee (KAIST, corresponding author), Da-Hee Ahn (KAIST, first author), Tong Un Chae (KAIST, second author), total of 3 authors
DOI: https://doi.org/10.1073/pnas.2535786123
This research was supported by the "Development of Platform Technologies of Microbial Cell Factories for the Next-Generation Biorefineries" project under the Petroleum Replacement Eco-Friendly Chemical Technology Development Program supported by the Ministry of Science and ICT, and by the "Development of Advanced Synthetic Biology Source Technologies for Leading the Biomanufacturing Industry" project under the Core Synthetic Biology Technology Development Program.
