Highly reducing polyketide synthases (HR-PKSs) produce structurally diverse polyketides (PKs). The PK diversity is constructed by av ariety of factors,i ncluding the b-keto processing,c hain length, methylation pattern, and relative and absolute configurations of the substituents.W e examined the stereochemical course of the PK processing for the synthesis of polyhydroxyPKs such as phialotides,phomenoic acid, and ACR-toxin. Heterologous expression of aH R-PKS gene,atrans-acting enoylreductase gene,and atruncated non-ribosomal peptide synthetase gene resulted in the formation of al inear PK with multiple stereogenic centers.T he absolute configurations of the stereogenic centers were determined by chemical degradation followed by comparison of the degradation products with synthetic standards.As tereochemical rule was proposed to explain the absolute configurations of other reduced PKs and highlights an error in the absolute configurations of ar eported structure.T he present work demonstrates that focused functional analysis of functionally related HR-PKSs leads to ab etter understanding of the stereochemical course.

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 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 co-expression analysis of ACRTS2 and ACRTS3. Taken together, we concluded the production of 1 in AO is solely responsible for ACRTS2.

Highly reducing polyketide synthases (HR‐PKSs) produce structurally diverse polyketides (PKs). The PK diversity is constructed by a variety of factors, including the β‐keto processing, chain length, methylation pattern, and relative and absolute configurations of the substituents. We examined the stereochemical course of the PK processing for the synthesis of polyhydroxy PKs such as phialotides, phomenoic acid, and ACR‐toxin. Heterologous expression of a HR‐PKS gene, a trans‐acting enoylreductase gene, and a truncated non‐ribosomal peptide synthetase gene resulted in the formation of a linear PK with multiple stereogenic centers. The absolute configurations of the stereogenic centers were determined by chemical degradation followed by comparison of the degradation products with synthetic standards. A stereochemical rule was proposed to explain the absolute configurations of other reduced PKs and highlights an error in the absolute configurations of a reported structure. The present work demonstrates that focused functional analysis of functionally related HR‐PKSs leads to a better understanding of the stereochemical course.