Clarification of the biosynthetic gene cluster involved in the antifungal prodrug echinocandin B and its robust production in engineered Aspergillus pachycristatus

Abstract

Echinocandin antifungals exhibit high efficacy against drug-resistant strains due to their unique mechanism of action. The production of their semi-synthetic precursors relies solely on microbial metabolism, leading to elevated production costs. Anidulafungin, an excellent echinocandin drug, is derived from echinocandin B (ECB), which is industrially produced by Aspergillus pachycristatus. However, the genes involved in the actual ECB biosynthesis remain unclear, which hinders yield improvements through engineered strains. This study systematically investigated the putative ECB biosynthetic gene cluster using genomic and transcriptomic profiling combined with gene editing. Among the 18 putative genes previously reported, only a 13-gene cluster (ecdA, ecdG-J, htyA-F) was found to be actively involved in ECB biosynthesis, while the remaining 5 genes (ecdB-F) were non-essential and functioned independently. Notably, we identified that htyC and htyD were involved in L-homotyrosine biosynthesis, while HtyF catalyzed the C4 hydroxylation of 3S-hydroxyl-L-homotyrosine. Most importantly, EcdJ was identified as a crucial global transcriptional activator regulating the ECB gene cluster. Deletion of ecdJ silenced all related genes and abolished ECB production. Accordingly, overexpressing ecdJ alone or combining ecdA and htyF together significantly enhanced ECB yield. Under optimized liquid fermentation conditions, ECB production in the OEecdJ strain achieved 841 ± 23.11 mg/L. Solid-state fermentation further enhanced the ECB yield to 1.5 g/L, which is 7.7-fold higher than that of the wild-type strain under initial liquid fermentation conditions. This study has thoroughly elucidated the functions of key genes involved in the ECB biosynthesis and provided effective strategies for enhancing antifungal prodrug-ECB production, achieving the highest ECB production in an engineering A. pachycristatus strain.