Abstract
Filamentous fungi are an abundant source of bioactive secondary metabolites (SMs). In many cases, the biosynthetic processes of SMs are not well understood. This work focuses on a group of SMs, the alkylcitric acids, each of which contains a saturated alkyl “tail,” and a citrate-derived “head.” We initially identified their biosynthetic gene cluster and the transcriptional regulator (akcR) involved in the biosynthesis of alkylcitrates in the filamentous fungus Aspergillus niger by examining the functional annotation of SM gene clusters predicted from genomic data. We overexpressed the transcription regulator gene akcR and obtained from one liter of culture filtrate 8.5 grams of extract, which are represented by seven alkylcitric acids as determined by NMR. Hexylaconitic acid A comprised 94.1% of the total production, and four of the seven identified alkylcitrates have not been reported previously. Analysis of orthologous alkylcitrate gene clusters in the Aspergilli revealed that in A. oryzae and A. flavus an in-cluster gene displays sequence similarity to cis-aconitate decarboxylase, the orthologue of which in A. niger, NRRL3_00504, is located on a different chromosome. Overexpression of the A. niger NRRL3_00504 and akcR genes together shifted the profile of alkylcitrates production from primarily hexylaconitic acids to mainly hexylitaconic acids, suggesting that NRRL3_00504 encodes an enzyme with hexyl aconitate decarboxylase activity. We also detected two additional, previously unreported, alkylcitric acids in the double overexpression strain. This study shows that phylogenomic analysis together with experimental manipulations can be used to reconstruct a more complete biosynthetic pathway in generating a broader spectrum of alkylcitric compounds. The approach adopted here has the potential of elucidating the complexity of other SM biosynthetic pathways in fungi.
Highlights
Filamentous fungi are a rich source of secondary metabolites (SMs)
To locate the gene cluster involved in alkylcitric acid production, we began by examining the structures of the alkylcitric acids that have previously been reported (Figure 1)
Using the secondary metabolite gene clusters annotated in the genome of A. niger NRRL3, Overexpression of Transcription Factor Gene NRRL3_11765 Leads to the Overproduction of Secondary Metabolites
Summary
Filamentous fungi are a rich source of secondary metabolites (SMs). Many fungal SMs have useful bioactive properties; for example, the antibiotic penicillin, the cholesterol-lowering drug lovastatin, and the anti-cancer compound griseofulvin (Fleming, 1929; Rubinstein et al, 1991; Panda et al, 2005). Genes involved in the biosynthesis of SMs are typically co-localized in the genome and they are referred to as SM gene clusters (Gacek and Strauss, 2012). A SM gene cluster can contain gene(s) encoding transcriptional regulator(s) which may facilitate the transcription of the entire cluster and start the process of SM biosynthesis (Fernandes et al, 1998). Overexpression of these in-cluster transcription factor genes can be a useful strategy to activate a SM gene cluster (Zabala et al, 2012)
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