Integration of pathway balance and protein fusion enables de novo biosynthesis of (+)-bicyclogermacrene in Escherichia coli.

(+)-Bicyclogermacrene and its derivatives, with promising antimicrobial, anticancer, and insecticidal properties, hold significant potential for applications in pharmaceuticals, agriculture, and industry. However, traditional extraction methods from plant essential oils are unsustainable. In this study, we achieved the de novo biosynthesis of (+)-bicyclogermacrene using a metabolically engineered Escherichia coli strain. The biosynthetic pathway of (+)-bicyclogermacrene was partitioned into upstream and downstream modules to enable precise regulation. This was accomplished through the genome-integrated overexpression of the endogenous methylerythritol phosphate pathway to ensure an adequate supply of terpenoid precursors, which pulled the titer from the initial 11.3 mg/L to 50.1 mg/L. Production was further enhanced to 96.9 mg/L by fusion of downstream key genes to facilitate precursor channeling, along with expression level optimization to improve pathway efficiency. Additionally, NADPH supply was fine-tuned through overexpressing dehydrogenases to improve the overall metabolic balance and this approach achieved a titer of 119 mg/L. Following site-directed of (+)-bicyclogermacrene synthase, the engineered E. coli strain M6-36 produced 565 mg/L of (+)-bicyclogermacrene in a 5-L bioreactor, an approximately 50-fold increase from the initial. To the best of our knowledge, the obtained titer in this study represents the highest level ever reported for the production of (+)-bicyclogermacrene. This study demonstrates an effective approach for the heterologous biosynthesis of sesquiterpenoids in E. coli and provides a scalable platform for the sustainable production of terpenoid-derived valuable chemicals.