Search

Natural secondary metabolites are medically, agriculturally, and industrially beneficial to humans. For mass production, a heterologous production system is required, and various metabolic engineering trials have been reported in Escherichia coli and Saccharomyces cerevisiae to increase their production levels. Recently, filamentous fungi, especially Aspergillus oryzae, have been expected to be excellent hosts for the heterologous production of natural products; however, large-scale metabolic engineering has hardly been reported. Here, we elucidated candidate metabolic pathways to be modified for increased model terpene production by RNA-seq and metabolome analyses in A. oryzae and selected pathways such as ethanol fermentation, cytosolic acetyl-CoA production from citrate, and the mevalonate pathway. We performed metabolic modifications targeting these pathways using CRISPR/Cas9 genome editing and demonstrated their effectiveness in heterologous terpene production. Finally, a strain containing 13 metabolic modifications was generated, which showed enhanced heterologous production of pleuromutilin (8.5-fold), aphidicolin (65.6-fold), and ophiobolin C (28.5-fold) compared to the unmodified A. oryzae strain. Therefore, the strain generated by engineering multiple metabolic pathways can be employed as a versatile highly-producing host for a wide variety of terpenes., (© 2024. The Author(s).)

Mushroom terpenoids are biologically and chemically diverse fungal metabolites. Among them, melleolides are representative sesquiterpenoids with a characteristic protoilludane skeleton. In this study, we applied a recently established hot spot knock-in method to elucidate the biosynthetic pathway leading to 1α-hydroxymelleolide. The biosynthesis of the sesquiterpene core involves the cytochrome P450 catalyzing stepwise hydroxylation of the Δ 6 -protoilludene framework and a stereochemical inversion process at the C5 position catalyzed by short-chain dehydrogenase/reductase family proteins. The highlight of the biosynthesis is that the flavoprotein Mld7 catalyzes an oxidation-triggered double-bond shift accompanying dehydration and acyl-group-assisted substitution with two different nucleophiles at the C6 position to afford the Δ 7 -protoilludene derivatives, such as melleolide and armillarivin. The complex reaction mechanism was proposed by DFT calculations. Of particular importance is that product distribution is regulated by interaction with the cell membrane., (© 2023 Wiley-VCH GmbH.)

Previously, we succeeded to produce the core structure of the host-selective ACR toxin (1) on brown leaf spot on rough lemon when the polyketide synthase ACRTS2 gene was heterologously expressed in Aspergillus oryzae (AO). To confirm the production of 1 in AO, the detection limit and suppressing decarboxylation were improved, and these efforts led us to conclude the direct production of 1 instead of its decarboxylation product. During this examination, minor ACR-toxin-related metabolites were found. Their structure determination enabled us to propose a decarboxylation mechanism and a novel branching route forming byproducts from the coupling of the dihydropyrone moiety of 1 with the acetaldehyde and kojic acid abundant in AO. The involvement of putative cyclase ACRTS3 in the chain release of linear polyketide was excluded by the coexpression analysis of ACRTS2 and ACRTS3. Taken together, we concluded that the production of 1 in AO is solely responsible for ACRTS2., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

UstYa family proteins (DUF3328) are widely and specifically distributed in fungi. They are known to be involved in the biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs) and nonribosomal peptides, and possibly catalyze various reactions, including oxidative cyclization and chlorination. In this study, we focused on phomopsin A, a fungal RiPP consisting of unique nonproteinogenic amino acids. Gene knockout experiments demonstrated that three UstYa homologues, phomYc, phomYd, and phomYe, are essential for the desaturation of amino acid moieties, showing unprecedented function among UstYa family proteins. Sequence similarity network analysis indicated that their amino acid sequences are highly diverged and that most remain uncharacterized, paving the way for genome mining of fungal metabolites with unique modifications., (© 2021 Wiley-VCH GmbH.)