Multisite Cre-lox recombination enables regulatory mechanism elucidation and systematic engineering of echinocandin B...

Echinocandin B (ECB), a cyclic lipohexapeptide for synthesizing antifungal drugs, is produced by the nonribosomal peptide synthetase gene cluster in Aspergillus nidulans. However, industrial production remains limited by the inefficiency of production capacity, primarily due to the complexity of the biosynthetic pathway and the absence of multi-gene regulatory tools in filamentous fungi. Here, we established an orthogonal Cre-lox-based platform enabling single-site insertion of up to 30 kb and simultaneous dual-site integration of 10 kb DNA fragments in A. nidulans. Through precursor supplementation and targeted gene overexpression, we identified key enzymatic bottlenecks in the precursor biosynthetic pathway, including the oxygenases AniF, AniK, AniG, and the acyl-AMP ligase AniI. Combinatorial overexpression of these genes acted synergistically to increase ECB titers. We further addressed bottlenecks in natural amino acid biosynthesis by overexpressing feedback-resistant mutants of Hom3 (L-Thr pathway) and LeuC (L-Leu pathway). Additionally, we uncovered a temperature-dependent regulation mechanism whereby low temperature (25 °C) concurrently upregulates both the ECB biosynthetic gene cluster and odeA gene, encoding Δ12-oleic acid desaturase, thereby increasing linoleic acid availability for ECB production. Leveraging our multisite DNA-integration platform to rewire expression of these key genes, we increased ECB production to 3.5 ± 0.2 g/L in a 5-L fed-batch bioreactor, a 2.3-fold improvement that represents the highest titer reported in the literature to date. Our orthogonal dual-site integration strategy and the systematic optimization approach provide a valuable framework for metabolic engineering of complex natural products in filamentous fungi.