Polyamide 54 (PA54), as a bio-based short-chain polyamide, can be polymerized from bio-based monomers cadaverine and succinate. Currently, traditional biosynthesis methods require two separate strains to synthesize cadaverine and succinate, respectively, which are plagued by problems such as complex processes, high consumption of acids and alkalis, and large output of waste salts.
The ultimate goal of our project is to construct an intelligent single-cell factory for the simultaneous synthesis of cadaverine and succinate, providing a reference for the green and low-carbon biosynthesis of PA monomers. The core concept of our project is an intelligent single E. coli cell factory, which can accumulate the precursor lysine during the aerobic growth phase, and efficiently converted into the target product cadaverine during the anaerobic phase through a dynamic regulatory switch, with simultaneous synthesis of succinate.
We successfully designed and constructed a themperature switch, whose core components consist of the repressor protein TlpA39 and its corresponding promoter PtlpA. At 33-37°C, this system successfully repressed the expression of lysine decarboxylase CadA, allowing lysine to accumulate. When the temperature was switched to above 39°C, the system was activated, and CadA began to be expressed, converting the accumulated lysine into cadaverine. This confirmed the effectiveness of the basic temperature-controlled switch, but there was a problem of leaky expression at lower temperatures.
To improve the precision and controllability of the themperature switch system, we first optimized the promoter controlling the expression of the repressor protein TlpA39 at the transcriptional level. Among them, PJ23106 showed the most significant improvement,increasing lysine accumulation by 25% from 4.07 g/L to 5.1 g/L during the aerobic phase. Subsequently, by introducing RNA thermometers (U7/U8), we added control at the translational level, further increasing lysine accumulation to 10.4 g/L (U7) and 11.9 g/L (U8), achieving an overall 2 to 2.3-fold increase. Specifically, lysine was hardly consumed in advance in the early stage, and then began to be gradually consumed after increasing the temperature. While increasing lysine accumulation, this also confirmed the feasibility of the synergistic synthesis of cadaverine and succinate in a single strain.
To ensure the efficient synthesis of cadaverine, we performed engineering modification on the key lysine decarboxylase CadA. Through rational design guided by in silico simulation calculations, we obtained the mutant Y13C/P36C with significantly improved catalytic efficiency, whose enzymatic activity was 113% higher than that of the wild type. This mutant exhibited better catalytic performance and stability than the wild type over a wider pH range and at higher substrate concentrations, providing a reliable guarantee for high cadaverine production.
Regarding succinate, our other co-produced product, it is a reductive product that requires the consumption of excess reducing power during synthesis. Based on the metabolic simulation results from the in silico team, we screened four NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenases (GAP) from different sources, and finally selected to express the NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase (CggapC) from Corynebacterium glutamicum ATCC 13032, thereby enhancing the intracellular regeneration of reducing power NADPH.
To balance the synthesis pathways of the two-stage products and avoid excessive metabolic burden, we further regulated the expression intensity through low-copy plasmids and introduced soluble pyridine nucleotide transhydrogenase (sthA) to promote the rational distribution and flow of cofactors. The results showed that the succinate titer increased by 28% relative to control; meanwhile, the lysine titer reached 14.9 g/L.This proves that cofactor engineering can successfully coordinate the metabolic flux distribution in the co-production process.
By integrating the temperature-sensitive switch, CadA and the cofactor modules, we successfully constructed an intelligent cell factory that can simultaneously synthesize 5.7 g/L cadaverine and 5.4 g/L succinate from glucose. It laid the foundation for the subsequent large-scale green production of PA54 salt.
The intelligent cell factory incorporates sensing, processing, and response capabilities that enable autonomous adaptation to environmental changes. The system can automatically adjust metabolic flux distribution based on substrate availability and product accumulation.
The integrated system achieved simultaneous production of both monomers with high yields and maintained metabolic balance throughout the fermentation process. The intelligent cell factory demonstrated the feasibility of our approach for industrial-scale bioproduction applications.
The figure1 below shows our improvements across engineering cycles.
In the future, we will continue our journey of single-cell PA54 synthesis. We continuously scale up the fermentation system and plan to optimize fermentation medium and strategies in 5 L fermentor to obtain a higher production(Figure2). In addition, we have designed a separation process to purify PA54 salts from the fermentation broth(Figure3). These efforts aim to provide theoretical support for the project's implementation.
