Systematic small regulatory RNA guided pathway attenuation enables high-level riboflavin production in Escherichia coli.

Riboflavin is an essential vitamin widely used in pharmaceutical and feed applications, and its industrial production relies primarily on microbial fermentation. However, riboflavin biosynthesis in Escherichia coli is constrained by precursor availability and broader physiological regulation. In this study, a small regulatory RNA (sRNA)-mediated attenuation strategy was employed to systematically screen 91 genes across five functional modules. Twenty-three targets increased riboflavin production by more than 20%, and five key targets were further validated through genome-level modification. Combinatorial attenuation of rpe and yrfG, optimization of the nrfA-targeting sRNA expression cassette, and chromosomal reinforcement of the riboflavin operon were subsequently integrated to construct strain SN49M. Following fermentation medium optimization, SN49M produced 3153.3 mg/L riboflavin at 46 h in shake-flask cultivation with a yield of 193.4 mg/g glucose. In fed-batch fermentation, SN49M reached 20.5 g/L riboflavin within 72 h, with an overall yield of 178.9 mg/g glucose, representing the highest reported glucose-based riboflavin yield achieved by microbial fermentation. This work establishes a systematic and tunable framework for discovering and integrating metabolic and physiological perturbations, providing an effective strategy for improving carbon efficiency in microbial riboflavin production.