Towards a reduced-background cell factory: disruption of a PKS-NRPS hybrid gene (AlepA) abolishing the production of 2-pyridones in Aspergillus oryzae.

Authors

Ruya Yin, Ziqi Zhai, Mengwei Zhang, Yifei Qin, Jianing Wang, Xingrui Liang, Yanxue Peng, Xuan Zhang, Dan Xu, Ligang Zhou, Daowan Lai

Published in

World journal of microbiology & biotechnology. Volume 42. Issue 8. Jul 10, 2026. Epub Jul 10, 2026.

Abstract

Aspergillus oryzae NSAR1 is a versatile and commonly used heterologous expression host. Typically, this host is used to express exogenous biosynthetic genes for secondary metabolites (SMs). However, the metabolite profile of A. oryzae itself is not clearly understood. In our course to enrich minor metabolites from heterologous expression using in situ macroporous resins, two metabolites were found to be significantly enhanced in A. oryzae itself. These metabolites were isolated, purified, and identified as 2-pyridones, including leporin B (1), and protoleporin A (2), as the major compounds, along with a minor of leporin C (3). The structural elucidation was performed by detailed NMR and HRMS analysis, and by comparison with the literature. This was the first observation of leporin-type 2-pyridones from A. oryzae, among which compound 2 was a new compound. Leporin B was found to display inhibition against Phytophthora sojae. A putative biosynthetic gene cluster (BGC, Aolep) harboring a backbone PKS-NRPS encoding gene (AolepA) was identified for these metabolites through genome mining. Subsequently, AolepA was knocked out using homologous recombination via conventional gene knockout (KO) and CRISPR-Cas9 mediated gene-editing system. Successful disruption of AolepA was achieved using both strategies leading to abolish the production of 2-pyridones completely. The KO mutants showed a similar phenotype as compared to the parent strain, regarding the growth and sporulation. The utility of these mutants as a new chassis was demonstrated by heterologous expression of a 1,3,6,8-tetrahydroxynaphthalene synthase. Hence, a genetic dereplication version of A. oryzae (NSAR1∆L) was generated, featuring a reduced background in endogenous SM production, which should facilitate the study of exogenous genes in SM biosynthesis by alleviating undesired metabolic burden, simplifying the detection and purification of heterologous compounds.

PMID:
42429871
Bibliographic data and abstract were imported from PubMed on 10 Jul 2026.

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