Nylon (Polyamide, PA), as one of the world’s most important polymer materials, is widely used in textiles, automobiles, electronics, aerospace, and other fields [1]. However, its traditional production mode relies heavily on fossil raw materials and is accompanied by problems of high energy consumption, high pollution, and high emissions. In particular, the large amount of waste salt, greenhouse gases, and other chemical pollutants generated during the chemical production process pose a continuous threat to the global environment and human health [2]. Against the global backdrop of pursuing "carbon neutrality" and green manufacturing, the development of bio-based PAs to replace petroleum-based PAs has become a core focus [3].
Through research, we found that the market demand for bio-based PAs is constantly expanding, with huge potential for future development. The global bio-based PA market was valued at 399.8 million US dollars in 2024 and is expected to reach 801.6 million US dollars by 2030, with a compound annual growth rate (CAGR) of 12.3% during the analysis period from 2024 to 2030 [4]. However, the existing fully bio-based PA products are mainly concentrated in long-chain PAs, while short-chain PAs are still in the active R&D stage and have not been applied on a large scale.
As a fully bio-based short-chain PA, PA54 can be polymerized from bio-based monomer materials—cadaverine and succinate—and exhibits excellent mechanical and processing properties [5]. Nevertheless, the traditional biosynthetic method requires two types of bacterial strains to produce the two monomer materials separately, and still faces problems such as complex processes, large consumption of acids and alkalis, and high output of waste salt [6-7]. We aim to overcome this limitation and develop a more efficient and environmentally friendly process for producing cadaverine and succinate.
This project adopts a green and low-carbon biosynthetic route, using a single engineered strain to simultaneously produce the two monomers of PA54—cadaverine and succinate. The acid-base properties of these two substances can achieve mutual neutralization, thereby significantly reducing the addition of acid-base adjustment reagents and the generation of waste salts. Furthermore, the CO2 produced by decarboxylation during cadaverine synthesis is fixed in situ for succinate synthesis, enabling green production.
In our project, E. coli NT1003 was selected as the chassis cell. This strain has been genetically modified to accumulate lysine. As lysine is the precursor of cadaverine, which can be converted into cadaverine by decarboxylase, it perfectly meets the needs of our project. To realize the co-synthesis of cadaverine and succinate in a single cell, four modules were designed:Switch---Controllable expression of CadA; Engineering CadA---Synthesis of cadaverine; Cofactor--- Improvement of succinate; An intelligent cell---Production of cadaverine-succinate.
In the first module, building upon the cadaverine–succinate co-production platform previously established in E. coli NT1003 by Gao et al. (2022), we sought to explore alternative regulatory strategies. To this end, we designed and constructed a dynamic genetic circuit using a temperature-responsive switch based on TlpA39 and encapsulin protein elements to regulate lysine decarboxylase CadA. In this system, cells grow and accumulate lysine under aerobic conditions; upon switching to anaerobic fermentation, the expression of CadA is activated to convert lysine into cadaverine, while enabling simultaneous succinate production.
In the second module, we aimed to engineer the key enzyme of CadA. Combined with in silico simulation analysis and design, the modification strategis were intended to improve the catalytic efficiency and stability to promote the synthesis of cadaverine from lysine.
In the third module, to meet the demand for reducing cofactors in succinate synthesis, we overexpressed exogenous GAP (glyceraldehyde-3-phosphate dehydrogenase) to increase intracellular reducing power NADPH. Additionally, we further overexpressed transhydrogenase to convert NADPH to NADH, thereby satisfying the requirement for succinate synthesis.
In the last module, we integrated the temperature-sensitive switch, CadA and the cofactor modules and successfully constructed an intelligent cell factory that can simultaneously synthesize cadaverine and succinate from glucose.
In summary, we constructed an intelligent single-cell factory for the simultaneous synthesis of cadaverine and succinate, which provides a reference for the green and low-carbon biosynthesis of polyamide monomers.
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