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

Akira Yoshimi,Hideaki Koike,Keietsu Abe,Nozomi Nagano,Myco Umemura,Masayuki Machida

doi:10.1186/s13568-016-0181-4

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.

Highlights

IntroductionThe koji mold Aspergillus oryzae is an important filamentous fungus used in the traditional Japanese fermentation industry to produce sake (rice wine), shoyu (soy sauce), and miso (soybean paste) (Machida et al 2008)
The koji mold Aspergillus oryzae is an important filamentous fungus used in the traditional Japanese fermentation industry to produce sake, shoyu, and miso (Machida et al 2008)
Expression of ustR in A. oryzae In the wild-type strain, no transcript of the ustR gene was detected at the time points examined (Fig. 1), whereas in the ustR expression (ustREX) strains the ustR transcript was detectable in CDEm and V8 media (Fig. 1)

Summary

Introduction

The koji mold Aspergillus oryzae is an important filamentous fungus used in the traditional Japanese fermentation industry to produce sake (rice wine), shoyu (soy sauce), and miso (soybean paste) (Machida et al 2008). Aspergillus oryzae has a high potential for secretion of industrially important enzymes such as amylases and proteases (Kobayashi et al 2007; Machida et al 2008). Its long history of extensive use in the food industry has placed A. oryzae on the list of Generally Recognized as Safe (GRAS) organisms compiled by the Food and Drug Administration (FDA) in the USA (Abe et al 2006). The safety of this organism is supported by the World Health Organization (WHO) (FAO/WHO 1987). A. oryzae may be a suitable host for production of heterologous proteins and secondary metabolites with important medical activities (Kobayashi et al 2007; Machida et al 2008; Sakai et al 2008)

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