We hypothesizedthat a KR encoded in the Fma gene cluster mightbe responsible for the stereospecific 5R reductionof the ketone during the biosynthesis of 1. Althoughno standalone KR is found in the Fma cluster shown in Figure A, Af490 encodes a partial PKSin which only the DH-KR domains are present. Upon initial discoveryof the fma cluster, the pseudo/partial PKS was assumedto be an inactive enzyme that had no likely role in the biosyntheticpathway. Although deletion of Af490 did not completelyabolish the production of 1, we did notice an 86% decreasein the titer of fumagillin (1) (Figure B). The incomplete abolishment of the biosynthesis of 1 may be due to the presence of endogenous KRs in A. fumigatus that have overlapping functions to that encodedby the pathway enzyme Af490 (Fma-KR). To investigate the role of Fma-KR, the 110kDa protein was therefore expressed as a 6xHis tag fusion and purifiedfrom S. cerevisiae (, Figure S6). Upon individual incubation of eachof the ketone derivatives 21, 22, or 23 with recombinant Fma-KR, we observed completeconversion to the corresponding 5R-hydroxy-containingcompounds, 9, 6, or 2, respectively(Figure A–C).
Reverse transcription quantitative real-time PCR (RT-qPCR) was used to determine the relative expression of the PKS genes of the biosynthesis gene clusters of cytochalasin E, patulin and pseurotin A ( for cytochalasin E, for patulin and for pseurotin A). Reverse transcription of RNA was performed using the iScript™ cDNA Synthesis Kit according to the manufacturer’s protocol (Biorad, Munich, Germany). Quickly, 1 µg of RNA was reverse-transcribed using a highly efficient RNase H+ MMLV reverse transcriptase and a mix of oligo dT and random hexamer primers.
steroid biosynthetic process (17)
Using FM2, VPA significantly increased the production of cytochalasin E after 48 and 72 h, TSA only after 48 h, whereas the presence of AZA resulted in a higher amount of cytochalasin E only after 72 h (A). Consistent with metabolite accumulation, the expression of the biosynthetic gene was highest in the presence of VPA and TSA after 48 h, and in the presence of AZA after 72 h (B and ). In addition, the combination of GlcNAc with TSA or butyrate slightly increased the production of cytochalasin E and the expression of after 48 h.
isoquinoline alkaloid biosynthetic process (4)
In addition, all SCCEs, HDAC inhibitors, as well as the DNMT inhibitor AZA and the chitin component GlcNAc, influenced SM biosynthesis in a media-, time- and target-dependent way. Furthermore, our data suggest that different regulatory components are involved in the transcription of mycotoxin biosynthesis genes, including pathway-specific activators and epigenetic regulators.
kynurenic acid biosynthetic process
We recently discoveredthe biosynthetic gene cluster for 1 in A. fumigatus. This fma clustercontains the first reported membranebound class I terpene cyclase (Fma-TC) that catalyzesthe formation of 3 from farnesyl pyrophosphate (FPP).The fma cluster also encodes a polyketide synthase(Fma-PKS) that synthesizes a dodecapentaenoate precursorthat is trans-esterified to 2 by the acyltransferaseFma-AT to yield the intermediate prefumagillin 4. It was proposed that the polyene portion of 4 would be oxidatively cleaved to yield 1. Followingour discovery, Wiemann et al. reported that the fma gene cluster is embedded within a supercluster on chromosome 8 of A. fumigatus that also encodes the biosynthetic pathwaysfor fumitremorgin and pseurotin. The fma gene cluster (Figure A) containsfour oxygenases that are most likely responsible for the oxidativetailoring steps that transform 3 to 1, including Af470 (Antibiotic Biosynthesis Monoxygenase superfamilymonooxygenase), Af480 (nonheme iron-dependent dioxygenase), Af510 (cytochrome P450 monooxygenase) and Af440 (flavin-binding monooxygenase/methyltransferase). An additionalredox enzyme in the fma gene cluster is the partialPKS (Af490), in which only the dehydratase (DH) andketoreductase (KR) domains are present. The mechanistic role of eachof these enzymes, especially that of the P450 (encoded by Af510), which is hypothesized to play a central role inthe conversion of 3 to 2, has remained unknown.