Biosynthetic platform for orsellinic acid-derived meroterpenoids in Escherichia coli

Orsellinic acid (OSA)-derived meroterpenoids, that have an OSA backbone, are plant-derived natural products that have attracted considerable attention as pharmaceutical precursors because of their diverse pharmacological activities. Therefore, developing efficient microbial production methods is highly desirable. However, to date, only a few reports on the microbial production of OSA-derived meroterpenoids are available, and even for the precursor OSA, only minimal production levels (approximately 5 mg/L) have been achieved using engineered microbes. In this study, Escherichia coli was engineered to enable the de novo biosynthesis of OSA to establish an alternative production platform for OSA-derived meroterpenoids. The introduction of type III polyketide synthase and cyclase resulted in 1.4 mg/L production. CRISPR interference aimed at enhancing OSA production revealed that the knockdown of fadR, which is involved in malonyl-CoA consumption, was effective. Metabolome analysis was performed to evaluate the metabolic impact of the engineering strategies revealed malonyl-CoA depletion, indicating that its supply constituted a major bottleneck. Based on this insight, the overexpression of acetyl-CoA carboxylase, pantothenate kinase, and ATP citrate lyase was implemented, which increased OSA production to 202 mg/L under optimized cultivation conditions, representing a 145-fold improvement. Finally, introducing a plant-derived prenyltransferase enabled grifolic acid biosynthesis (2.5 μg/g-DCW), representing the first de novo production of OSA-derived meroterpenoids in E. coli. This study establishes E. coli as a versatile and scalable host for the biosynthesis of pharmacologically valuable meroterpenoids.