Integrating Membrane Permeability Engineering and Enzyme Design for Efficient Production of Ergothioneine in <i>E. coli</i>
Jiahuan Ling, Rui Chen, Minghai Wang, Chun-Xiao Yan, Ruomu Xia, Lihui Zhang
Abstract
Ergothioneine (EGT) is a powerful and natural antioxidant, which can protect cells in the human body from oxidative damage. Numerous studies have attempted to enhance the heterologous synthesis. However, research on EGT transport out of Escherichia coli cells and the wild-type enzyme from Trichoderma reesei is rare. Here, membrane permeability engineering and protein engineering are combined to improve EGT production in E. coli . After metabolic engineering to enhance precursor supply, we deleted the genes involved in lipopolysaccharide biosynthesis to increase the membrane permeability, which promoted EGT production. Further, a mutant of Tregt2 E155C with improved catalytic capacity was created, and the expression level of enzymes involved in EGT synthesis was optimized. Finally, fermentation parameters were systematically tuned to maximize the EGT production. The engineered strain MT9-PET-T1/RSF-T2 E155C produced 334.20 ± 6.33 mg/L EGT during 48 h of shake-flask fermentation, corresponding to an 8.4-fold increase compared with wild-type strain MT1. When scaled up to 5 L bioreactors, the strain achieved a final EGT titer of 4.06 g/L within 96 h (42.29 mg/L/h). Our work shows significant implications for EGT synthesis and is also a reference for manipulating E. coli to synthesize other biomolecules.