Identification of the non-ribosomal peptide synthetase responsible for biosynthesis of the potential anti-cancer drug sansalvamide in Fusarium solani

Abstract

Sansalvamide is a cyclic pentadepsipeptide produced by Fusarium solani and has shown promising results as potential anti-cancer drug. The biosynthetic pathway has until now remained unidentified, but here we used an Agrobacterium tumefaciens-mediated transformation (ATMT) approach to generate knockout mutants of two candidate non-ribosomal peptide synthetases (NRPS29 and NRPS30). Comparative studies of secondary metabolites in the two deletion mutants and wild type confirmed the absence of sansalvamide in the NRPS30 deletion mutant, implicating this synthetase in the biosynthetic pathway for sansalvamide. Sansalvamide is structurally related to the cyclic hexadepsipeptide destruxin, which both contain an α-hydroxyisocaproic acid (HICA) unit. A gene cluster responsible for destruxin production has previously been identified in Metarhizium robertsii together with a hypothetical biosynthetic pathway. Using comparative bioinformatic analyses of the catalytic domains in the destruxin and sansalvamide NRPSs, we were able to propose a model for sansalvamide biosynthesis. Orthologues of the gene clusters were also identified in species from several other genera including Acremonium chrysogenum and Trichoderma virens, which suggests that the ability to produce compounds related to destruxin and sansalvamide is widespread.

Sponge-associated fungi from a mangrove habitat in Indonesia: species composition, antimicrobial activity, enzyme screening and bioactive profiling

“…4, it is noted that all isolates showed a clear band of NRPS genes. Although Fusarium is known to bring NRPS and PKS genes in its genome naturally (Hansen et al 2012;Romans-Fuertes et al 2016), this is the first report of the discovery of NRPS genes in Eutypella and Lasiodiplodia. It should also be noted that only a few peptides are reported from these genera (Wei et al 2014).…”

Biosynthesis of depsipeptides, or Depsi: The peptides with varied generations

“…129 To use a hydroxy acid as a building block substrate, both (1) availability and (2) selection of such substrates must be addressed by the BGC. (1) α-hydroxy acids are not common in primary metabolism, so in fungal systems that use α-hydroxy acids as NRPS substrates, the BGC often contains standalone ketoreductase (KR) proteins which convert α-keto acids to α-hydroxy acids in an NADPH-dependent reduction reaction [130][131][132][133][134] (Figure 4), with keto acids arising from branched-chain amino acid biosynthesis pathways 135 or from glycolysis. These KR proteins are not closely related to PKS KR domains, 128,136,137 but belong to the ApbA_C superfamily, which includes ketopantoate reductases.…”

“…Other depsipeptides for which ester bond formation occurs during elongation and is catalyzed by a C domain include the destruxins 131,132,142,143 and sansalvamide. 132 Destruxins are cyclic insecticidal molecules synthesized in fungal entomopathogens by a 6-module NRPS. 142 Module 2 is likely responsible for inserting the ester bond, starting with direct selection of the α-hydroxyisocaproic acid by the A domain.…”

“…142 Module 2 is likely responsible for inserting the ester bond, starting with direct selection of the α-hydroxyisocaproic acid by the A domain. 132,143,144 Timing of the ester bond formation step was controversial, with elongation 143 and terminal cyclization 131 both proposed. Cluster analysis of a BGC for a similar molecule, sansalvamide 132 lent support to the elongation proposal, which is now broadly accepted.…”

Combinatorial Synthesis and Biological Evaluation of Destruxins

“…Recently, the biosynthesis of destruxins has been reported, and revealed that the macrocyclic structure of destruxins could be biosynthetically provided by macrolactamization after the nonribosomal peptide synthesis of a cyclization precursor. 30,31) Thus, the results of the above biosynthesis suggest that macrolactamization is an effective method for the formation of a macrocyclic structure in destruxin synthesis. On the other hand, solid-phase synthesis is a powerful method for preparing a combinatorial library toward the elucidation of structure-activity relationships.…”