Enhancing erythromycin production in Saccharopolyspora erythraea through rational engineering and fermentation refinement: A Design‐Build‐Test‐Learn approach

Abstract Industrial production of bioactive compounds from actinobacteria, such as erythromycin and its derivatives, faces challenges in achieving optimal yields. To this end, the Design‐Build‐Test‐Learn (DBTL) framework, a systematic metabolic engineering approach, was employed to enhance erythromycin production in Saccharopolyspora erythraea ( S. erythraea ) E3 strain. A genetically modified strain, S. erythraea E3‐CymRP21‐dcas9‐sucC ( S. erythraea CS), was developed by suppressing the sucC gene using an inducible promoter and dcas9 protein. The strain exhibited improved erythromycin synthesis, attributed to enhanced precursor synthesis and increased NADPH availability. Transcriptomic and metabolomic analyses revealed altered central carbon metabolism, amino acid metabolism, energy metabolism, and co‐factor/vitamin metabolism in CS. Augmented amino acid metabolism led to nitrogen depletion, potentially causing cellular autolysis during later fermentation stages. By refining the fermentation process through ammonium sulfate supplementation, erythromycin yield reached 1125.66 mg L −1 , a 43.5% increase. The results demonstrate the power of the DBTL methodology in optimizing erythromycin production, shedding light on its potential for revolutionizing antibiotic manufacturing in response to the global challenge of antibiotic resistance.