Functional characterization and manipulation of the apicidin biosynthetic pathway inFusarium semitectum

Abstract

SummaryApicidin is a cyclic tetrapeptide produced by certain isolates of Fusarium semitectum and has been shown to inhibit Apicomplexan histone deacetylase. An apicidin-producing strain (KCTC16676) of the filamentous fungus was mutated using an Agrobacterium tumefaciens-mediated transformation, resulting in 24 apicidin-deficient mutants. Three of the mutants had a T-DNA insertion in a gene that encodes a nonribosomal peptide synthetase (NRPS). Results of sequence, expression, and gene deletion analyses defined an apicidin biosynthetic gene cluster, and the NRPS gene was named as apicidin synthetase gene 1 (APS1). A 63 kb region surrounding APS1 was sequenced and analysis revealed the presence of 19 genes. All of the genes including APS1 were individually deleted to determine their roles in apicidin biosynthesis. Chemical analyses of the mutant strains showed that eight genes are required for apicidin production and were used to propose an apicidin biosynthetic pathway. The apicidin analogues apicidin E, apicidin D 2 and apicidin B were identified from chemical analysis of the mutants. The cluster gene APS2, a putative transcription factor, was shown to regulate expression of the genes in the cluster and overexpression of APS2 increased apicidin production. This study establishes the apicidin biosynthetic pathway and provides new opportunities to improve the production of apicidin and produce new analogues.

Genetic basis of destruxin production in the entomopathogen Metarhizium robertsii

“…The bialaphos-resistant strains 1.1 and 5.1, in which DXS appeared to be intact, were retained as phenotypic controls. Southern analysis confirmed the PCR analysis demonstrating that 2.1 and 4.1 carried a single (Hendrickson et al 1999) and LDKS (Kennedy et al 1999), fusarin C, FUSS (Song et al 2004), cytochalasan, CheA (Schumann and Hertweck 2007); and the O-methyltransferases involved in the biosynthesis of gliotoxin, GliM (Gardiner and Howlett 2005), aflatoxin, stcP (Kelkar et al 1996), and apicidin, APS6 (Jin et al 2010). Note that the four NMet domains in AbA are 100% identical to each other at the amino acid level, so only one was used for this analysis.…”

Enhancing Nonribosomal Peptide Biosynthesis in Filamentous Fungi

“…With genetic manipulation of fungal genomes becoming available, genetic and chemical pathways leading to NRPS-derived metabolites could be identified through relatively straightforward gene deletions of NRPS-encoding genes coupled with analytical screenings for loss of compound production. Examples of compounds and pathways that have been identified in this traditional fashion include those of cyclosporine [27], HC-toxin [29], AM-toxin [30], peptaibols [31], ergotpeptine [32], fusarin C [33], equisetin [34], peramine [35], sirodesmin [36], gliotoxin [37], fumitremorgin [38], tenellin [39], pseurotin A [40], cytochalasin [41], cyclopiazonic acid [42], aureobasidin A [43], fumiquinazolines [44], apicidin [45], tryptopquialanine [46], ochratoxin A [47], fumigaclavines [48], ardeemin [49], nidulanin A [50], and pneumocandin [51]. Although many of the aforementioned NRPS-derived fungal secondary metabolites display biological activity, the need for discovery of new antibiotics is becoming more urgent [52] and calls for innovative avenues of uncovering cryptic metabolites.…”

Reconstitution of the FK228 Biosynthetic Pathway Reveals Cross Talk between Modular Polyketide Synthases and Fatty Acid Synthase

“…Furthermore, cross talk between a type I FAS (FAS I) for primary metabolism and a secondary FAS I for HC-toxin biosynthesis had also been postulated in the fungal species Cochliobolus carbonum, where only one gene encoding the ␤-subunit of FAS I was found in the secondary metabolic gene cluster, while the ␣-subunit of FAS I necessary for HC-toxin biosynthesis might be recruited from the primary FAS I (1). Recently, an opposite example was reported in the apicidin biosynthetic pathway in Fusarium semitectum, where a gene encoding the ␣-subunit of FAS I is present in the apicidin biosynthetic gene cluster, while the ␤-subunit of FAS I necessary for apicidin biosynthesis is speculated to be recruited from the primary FAS I (16). Lastly, the ability by which the engineered SW07 strain produced 0.4 mg/liter of actual yield or a ca.…”