The production of 5-HTP requires the enzyme Tryptophan Hydroxylase 1 (TPH1), which facilitates the conversion of L-tryptophan to 5-HTP. However, TPH1 enzymes tend to aggregate, becoming insoluble and unable to be expressed in E. coli. To address this, we have improved the TPH1 enzyme by converting it into monomeric-human Tryptophan Hydroxylase 1 (m-hTPH1), making it soluble and expressible in E. coli. The activity of m-hTPH1 is supported by its cofactor Tetrahydrobiopterin (BH4), which is regenerated by two coenzymes: hPCBD1 and hQDPR. Therefore, we have decided to express the enzymes m-hTPH1, hPCBD1, and hQDPR in E. coli.
To further enhance 5-HTP production, we fused these three enzymes with different Zinc Finger proteins: Zif268-rigid linker-hTPH1, PBSII-rigid linker-hPCBD1, and ZFa-hQDPR, enabling them to bind to a DNA scaffold via their respective motifs and cluster together. Finally, we verified the expression of these fusion proteins in E. coli strain BL21 to ensure successful protein production.
▲ Figure 1: Induction of protein expression with IPTG and confirmation of fusion protein size by SDS-PAGE.
After preparing the probiotics, we analyzed 5-HTP production using an ELISA kit. The bar graph illustrates 5-HTP production in E. coli systems engineered for 5-HTP biosynthesis. The concentrations of 5-HTP were compared under different conditions, using varying E. coli dry weights (1 mg/ml and 2 mg/ml).
The conditions tested include:
▲ Figure 2: Production of 5-HTP in E. coli systems engineered for 5-HTP biosynthesis.
At a dry weight of 1 mg/ml, the hTPH1, hPCBD1, and hQDPR system without external BH4 outperformed the pST39 control, while supplementation with external BH4 significantly increased 5-HTP production. At 2 mg/ml dry weight, all systems showed enhanced 5-HTP production, with the highest concentration observed in the hTPH1, hPCBD1, and hQDPR system supplemented with external BH4.
These results suggest that the engineered systems successfully produce 5-HTP, even without BH4 supplementation. However, the addition of external BH4 further amplifies 5-HTP synthesis, emphasizing the importance of cofactor availability in optimizing biosynthetic pathways.