Efficient Synthesis of Ergothioneine in <i>Escherichia coli</i> Through Identification of Bacterial‐Type Egt1 and Self‐Assemble of Pathway Enzymes

Ergothioneine (EGT) offers diverse physiological benefits and potential applications. Production of EGT by engineered bacterial strain has emerged as an eco-friendly and cost-effective alternative to traditional extraction and chemical synthesis methods. The fungal pathway requires only two steps and is simpler than the bacterial pathway, which typically requires three or five steps depending on the catalytic characteristics of EgtB. However, heterologous overexpression of fungal enzymes in Escherichia coli still represents a great challenge, which would limit the practical application of fungal pathway for EGT biosynthesis. In this study, a bacterial-type Egt1 with catalytic function similar to that of the fungal Egt1 was identified by genome data mining, enabling the construction of the simplest bacterial EGT biosynthetic pathway. The pathway was introduced into E. coli resulting in an EGT titer of 426 mg/L. Cip-based self-assemble of enzymes requires only one scaffold protein and occurs spontaneously, which has been employed to aggregate the rate-limiting enzymes of biosynthetic pathway to speed up the desired reaction via substrate channeling effects. The pathway enzymes were self-assembled using the CipB scaffold protein. The optimal engineered strain E48 produced 925 mg/L of EGT in shake flask after 72 h cultivation, with a productivity of 13 mg/(L·h). Finally, the production of EGT was scaled up in a 5 L fermenter, with an EGT titer reaching 1934 mg/L and a productivity of 36.5 mg/(L·h) after 55 h of fermentation. This study provides new approach for the efficient synthesis of EGT and other value-added bioactive molecules.