Global regulator DksA as a key target for naringenin biosynthesis identified via whole-cell directed evolution

Whole-cell directed evolution, which enables optimization across multiple cellular levels, is powerful for enhancing microbial biosynthetic capabilities. Naringenin is a central precursor for producing diverse flavonoid compounds. In this study, an FdeR-based naringenin whole-cell biosensor was developed, to enable directed evolution of the genome of naringenin cell factory and 4-coumarate:CoA ligase (4CL), a rate-limiting enzyme of naringenin biosynthetic pathway. In directed genome evolution, overexpression of the global regulatory protein DksA was found to enhance naringenin biosynthesis by 1.95-fold. Further directed evolution of DksA protein again doubled the naringenin production. Transcriptomic analysis of the strain regulated by the mutant DksA was performed, suggesting improved cellular robustness. In addition, a 4CL variant, C1E1, was identified with a 3.02-fold improvement in naringenin production compared with the wild-type enzyme, aided by directed enzyme evolution. Finally, naringenin production reached 797 mg/L in shake-flask cultures at 30 °C for 72 h. This study highlights the power of whole-cell directed evolution in optimizing both enzyme function and host performance in natural products hyperproduction.