Expression of ustR and the Golgi protease KexB are required for ustiloxin B biosynthesis in Aspergillus oryzae

Abstract

Ustiloxin B, originally isolated from the fungus Ustilaginoidea virens, is a known inhibitor of microtubule assembly. Ustiloxin B is also produced by Aspergillus flavus and is synthesized through the ribosomal peptide synthesis pathway. In A. flavus, the gene cluster associated with ustiloxin B production contains 15 genes including those encoding a fungal C6-type transcription factor and ustiloxin B precursor. Although the koji mold Aspergillus oryzae, which is genetically close to A. flavus, has the corresponding gene cluster, it does not produce ustiloxin B, which may be explained by the fact that the gene encoding the transcription factor UstR is not expressed. Here, to investigate whether ustiloxin B can be produced by expressing ustR in A. oryzae, we constructed ustR expression (ustREX) strains and analyzed ustiloxin B production. In the ustREX strains, all genes in the cluster were up-regulated, in line with expression of ustR, and ustiloxin B produced. To elucidate whether the KexB protease is involved in the processing of the ustiloxin B precursor protein UstA, which has repeats of basic amino acid doublets resembling KexB target sites, we also constructed a ustREX strain with the ∆kexB genotype. Although ustR was expressed in this strain, ustiloxin B was barely detectable. This finding strongly suggests that KexB is required for ustiloxin B production.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0181-4) contains supplementary material, which is available to authorized users.

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“…It is very interesting to note that, e.g., ustiloxin B and ustilaginoidin C, have both been isolated from the rice false smut pathogen Villosiclava virens (= Ustilaginoidea virens ) even though they are not biosynthetically related. However, these two types of secondary metabolites have also been found in Aspergillus flavus (Umemura et al 2014 ; Tsukui et al 2015 ; Yoshimi et al 2016 ). This is remarkable as both unrelated fungi occur on rice.…”

Heterologous production of asperipin-2a: proposal for sequential oxidative macrocyclization by a fungi-specific DUF3328 oxidase

“…Each repeat is flanked by KR, which is a target site of Golgi protease, KexB, suggesting that AprA undergoes a similar proteolytic process to that known in the biosynthesis of ustiloxin B. 18 AprA is also modified by AprY and AprR to furnish 1. However, there is no information concerning the order of these posttranslational modifications.…”

<i>Aspergillus flavus</i> Secondary Metabolites: More than Just Aflatoxins

“…Upon further examination, the cluster gene ustA, was predicted to encode a protein containing an N-terminal signal sequence and 16-fold repeated peptide region containing YAIG repeats which represent the exact tetrapeptide sequence of ustiloxin B. Biosynthesis was proposed to proceed via processing of the UstA precursor protein by a Kex2 protease not present in the cluster that cleaves at nearby Lys-Arg residues on the C-terminal side of the YAIG tetrapeptide 17) . Subsequent work in A. oryzae confirmed that inactivation of the kexB endopeptidase gene resulted in almost complete loss of ustiloxin B 156) . The partially processed pre-protein is then believed to be processed in a stepwise manner by the products of ustP and ustH, both encoding peptidases, resulting in generation of the mature YAIG tetrapeptide.…”