A unique Zn(II)2-Cys6-type protein, KpeA, is involved in secondary metabolism and conidiation in Aspergillus oryzae

https://doi.org/10.1016/j.fgb.2019.02.004Get rights and content

Highlights

  • Library-based screening identified a novel Zn(II)2-Cys6 type protein KpeA.

  • KpeA has unique structure but wildly conserved among filamentous fungi.

  • Functionality of Zn(II)2-Cys6 motif in KpeA was confirmed by alanine substitution.

  • kpeA disruption led to increased kojic acid production and decreased conidiation.

  • kpeA disruption led to decreased brlA, abaA, and wetA expression.

Abstract

Aspergillus oryzae is an important microorganism in the bio- and food industries; therefore, understanding the mechanism underlying its secondary metabolism regulation is important for ensuring its safe use. Here, we screened a novel Zn(II)2-Cys6-type protein-encoding gene, AO090003001186, designated as kpeA (kojic acid production enhancement A), from an A. oryzae disruption mutant library of transcriptional regulators. kpeA is highly conserved among filamentous fungi and encodes a protein with Zn(II)2-Cys6 motif located in the middle of the sequence. Phylogenetic analysis revealed that KpeA was classified into a distal group compared to other fungal Zn(II)2-Cys6-type transcriptional regulators. A Cys to Ala substitution mutant of KpeA showed identical phenotype to the kpeA disruption strain, confirming that KpeA is novel type Zn(II)2-Cys6 binding protein. Colonies of the kpeA disruption strain (ΔkpeA) had longer aerial hyphae and showed decreased conidia production. Microscopic analysis suggested that the reduced vesicle size and conidial head formation in ΔkpeA strain account for the decreased conidia production. Transcriptional levels of brlA and downstream abaA and wetA were decreased in ΔkpeA strain. Moreover, ΔkpeA strain produced 6-fold more kojic acid than the control strains, and the expression of kojR and kojA was increased in ΔkpeA strain. Therefore, KpeA is a novel Zn(II)2-Cys6-type protein likely involved in conidiation and kojic acid production at the transcriptional level.

Introduction

Aspergillus oryzae is a filamentous fungus used for the production of traditional Japanese foods, such as sake, miso, and soy source, and is also used as a producer of industrial enzymes. Its close relative A. flavus is notorious for its unfavourable ability to produce a carcinogenic mycotoxin, aflatoxin. Therefore, so far, researches on the secondary metabolism of A. oryzae have been focused primarily on the establishment of a scientific basis for non-toxigenecity (Kusumoto et al., 1998, Kato et al., 2011, Kiyota et al., 2011). It has been demonstrated that a high fraction of A. oryzae strains have lost the ability to produce aflatoxin, aflatrem, and cyclopiazonic acid (CPA) due to partial loss of the genetic region responsible for the biosynthesis of the corresponding secondary metabolites (Kusumoto et al., 1998, Tominaga et al., 2006, Nicholson et al., 2009, Rank et al., 2012, Tokuoka et al., 2008, Shinohara et al., 2011, Kato et al., 2011), and the genetic determinants for non-toxigenecity, which were identified by these studies, guarantee the safety of A. oryzae for industrial use. On the other hand, A. oryzae produces several industrially important secondary metabolites, such as kojic acid (KA), penicillin, and deferriferrichrysin, and the biosynthesis genes for the secondary metabolites have been identified in recent years (Terabayashi et al., 2010, Marui et al., 2010, Yamada et al., 2003). Moreover, genome analysis discovered that A. oryzae harbors a higher number of genes involved in secondary metabolism than those in A. nidulans and A. fumigatus (Machida et al., 2005, Keller et al., 2005). Therefore, elucidation of the regulatory mechanism for the production of secondary metabolites in A. oryzae is important in its safe use.

In filamentous fungi, the expression of genes involved in secondary metabolism is considered to be regulated by two different mechanisms: first, a cluster specific transcription factor that resides within a secondary metabolite biosynthesis gene cluster and specifically controls the genes within the cluster; second, a so-called global regulator that concertedly controls several secondary metabolite gene clusters. The former type of transcription factors often harbors a Zn(II)2-Cys6 DNA-binding domain, which is found only in fungi. Among the relatives of A. oryzae, one of the well-described proteins with Zn(II)2-Cys6 DNA-binding domain is AflR, which co-regulates the genes in the aflatoxin biosynthesis gene cluster (Woloshuk et al., 1994, Payne et al., 1993, Chang et al., 1993). The latter, a global regulator, affects the gene expression of several secondary metabolite biosynthesis gene clusters in response to environmental signals, such as temperature, pH, nutrition, and light (Lind et al., 2016, Keller et al., 1997, Then and Brakhage, 1998, Michielse et al., 2014, Kim and Woloshuk, 2008, Purschwitz et al., 2008). A velvet complex primarily composed of LaeA, VeA, and VelB was found as a key regulator for secondary metabolism and sexual/asexual development (Bayram et al., 2008). The complex regulates a number of genes involved in secondary metabolism and development in response to light (Perrin et al., 2007, Bayram et al., 2008). Another key regulator of asexual development is BrlA (Adams et al., 1988). A recent study on A. fumigatus demonstrated that brlA regulates expression of the genes responsible for the production of the secondary metabolites gliotoxin, fumigaclavine, and endocrocin (Lind et al., 2018).

In this study, we aimed to determine the factor regulating secondary metabolism by using an A. oryzae disruption mutant library of transcriptional regulators. We focused on KA, which is one of the beneficial secondary metabolites produced by A. oryzae. KA and its derivatives are utilized as antibacterial, antifungal, and anti-melanosis agents in several fields such as the medical, food, agriculture, and cosmetic industries (Nohynek et al., 2004, Kotani et al., 1976, Baláž et al., 1993, Saruno et al., 1979, Chen et al., 1991, Noh et al., 2009, Lee et al., 2006). The regulatory factor KojR has been identified as a cluster-specific transcription factor for KA biosynthesis (Marui et al., 2010), and kojR is regulated by LaeA and HstD likely at the chromatin level in A. oryzae (Kawauchi et al., 2013, Oda et al., 2011). However, the factors regulating KA biosynthesis remain unknown.

Screening of a novel regulatory factor using a set of gene disruption strain has been reported for Neurospora crassa (Colot et al., 2006), and several novel genes relevant to carbon metabolism, development, and environmental signal response have been successfully identified (Coradetti et al., 2012, Gonçalves et al., 2011, Chinnici et al., 2014, Nargang et al., 2012, Watters et al., 2018). This approach is advantageous as a novel factor can be isolated without prior knowledge. In the case of A. oryzae, a mutant library composed of gene disruption strains of regulatory proteins were constructed and used in several studies. Through mining, a novel gene, ecdR, involved in conidiation has been identified (Jin et al., 2011). Furthermore, FlbC was rediscovered as a regulator for specific gene expression in solid state culture of A. oryzae (Tanaka et al., 2016). Here, we identified a regulatory protein involved in secondary metabolism in A. oryzae based on screening against the A. oryzae disruption mutant library. The protein that we found in this study has not been characterized in any other organism, indicating that this approach is rather effective for discovering a novel protein and its related unidentified mechanisms.

Section snippets

Strains

An A. oryzae disruption mutant library of transcriptional regulators including gene disruption strains of nsdDnsdD), creBcreB), lreAlreA), and AO090003001186 (ΔkpeA) was used. The pyrG-complemented RkuptrP2-1ΔAF strain (Δku70::ptr1+, pyrG+, ΔcypX-pksA), designated as E-F1, was used as the control strain (Ogawa et al., 2010). The gene complementation strain of kpeA (kpeA+ strain) and overexpression strain of kpeA (OE strain) were constructed. RkuptrP2-1ΔAF (Δku70::ptr1+, ΔpyrG, ΔcypX-

Screening of regulatory genes involved in KA production using the A. oryzae disruption mutant library

An A. oryzae disruption mutant library of transcriptional regulators was used in the screening experiments. The library includes over 500 A. oryzae transformants harboring a disrupted locus of the gene encoding (putative) a transcriptional regulator. All mutant strains including the kpeA disruption strain (ΔkpeA) were constructed from A. oryzae strain RkuptrP2-1ΔAF (Δku70::ptr1+, ΔpyrG, ΔcypX-pksA), a derivative of A. oryzae RIB40 (Takahashi et al., 2008). To minimize the effect of genetic

Discussion

In this study, we conducted a screening experiment for a regulatory factor of KA production in A. oryzae and found a novel Zn(II)2-Cys6-type protein, KpeA, which is widely conserved among filamentous fungi. KpeA is involved in KA production and conidiation via regulation of the expression of kojR and brlA, respectively.

KpeA is unique with respect to the position of the Zn(II)2-Cys6 DNA-binding domain. Generally, the Zn(II)2-Cys6 DNA-binding domain is located in the N-terminal region of a

References (67)

  • G.J. Nohynek et al.

    An assessment of the genotoxicity and human health risk of topical use of kojic acid [5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one]

    Food Chem. Toxicol.

    (2004)
  • M. Ogawa et al.

    Genetic analysis of conidiation regulatory pathways in koji-mold Aspergillus oryzae

    Fungal Genet. Biol.

    (2010)
  • J. Purschwitz et al.

    Functional and physical interaction of blue- and red-light sensors in Aspergillus nidulans

    Curr. Biol.

    (2008)
  • Y. Terabayashi et al.

    Identification and characterization of genes responsible for biosynthesisof kojic acid, an industrially important compound from Aspergillus oryzae

    Fungal Genet. Biol.

    (2010)
  • M. Tokuoka et al.

    Identification of a novel polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) gene required for the biosynthesis of cyclopiazonic acid in Aspergillus oryzae

    Fungal Genet. Biol.

    (2008)
  • T.H. Adams et al.

    Asexual sporulation in Aspergillus nidulans

    Microbiol. Mol. Biol. Rev.

    (1998)
  • M.A. Alam et al.

    Proteins interacting with CreA and CreB in the carbon catabolite repression network in Aspergillus nidulans

    Curr. Genet.

    (2017)
  • Š. Baláž et al.

    Relationship between antifungal activity and hydrophobicity of kojic acid derivatives

    Folia Microbiol.

    (1993)
  • Ö. Bayram et al.

    VelB/VeA/LaeA complex coordinates light signal with fungal development and secondary metabolism

    Science

    (2008)
  • M.T. Boylan et al.

    Isolation and physical characterization of three essential conidiation genes from Aspergillus nidulans

    Mol. Cell. Biol.

    (1987)
  • P.K. Chang et al.

    Cloning of the Aspergillus parasiticus apa-2 gene associated with the regulation of aflatoxin biosynthesis

    Appl. Environ. Microbiol.

    (1993)
  • P.K. Chang et al.

    Genome-wide analysis of the Zn(II)2Cys6 zinc cluster-encoding gene family in Aspergillus flavus

    Appl. Microbiol. Biotechnol.

    (2013)
  • J.S. Chen et al.

    Inhibitory effect of kojie acid on some plant and crustacean polyphenol oxidases

    J. Agric. Food Chem.

    (1991)
  • J.L. Chinnici et al.

    Neurospora crassa female development requires the PACC and other signal transduction pathways, transcription factors, chromatin remodeling, cell-to-cell fusion, and autophagy

    ProS One

    (2014)
  • H.V. Colot et al.

    A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors

    Proc. Natl. Acad. Sci. USA

    (2006)
  • S.T. Coradetti et al.

    Conserved and essential transcription factors for cellulase gene expression in ascomycete fungi

    Proc. Natl. Acad. Sci. USA

    (2012)
  • A. Garzia et al.

    Aspergillus nidulans FlbE is an upstream developmental activator of conidiation functionally associated with the putative transcription factor FlbB

    Mol. Microbiol.

    (2009)
  • K. Gomi et al.

    Integrative transformation of Aspergillus oryzae with a plasmid containing the Aspergillus nidulans argB gene

    Agric. Biol. Chem.

    (1987)
  • R.D. Gonçalves et al.

    A genome-wide screen for Neurospora crassa transcription factors regulating glycogen metabolism

    Mol. Cell. Proteomics

    (2011)
  • K.H. Han et al.

    The nsdD gene encodes a putative GATA-type transcription factor necessary for sexual development of Aspergillus nidulans

    Mol. Microbiol.

    (2001)
  • S. Ichinose et al.

    Improved α-amylase production by Aspergillus oryzae after a double deletion of genes involved in carbon catabolite repression

    Appl. Microbiol. Biotechnol.

    (2014)
  • N. Kato et al.

    Genetic safeguard against mycotoxin cyclopiazonic acid production in Aspergillus oryzae

    Chembiochem

    (2011)
  • M. Kawauchi et al.

    Fungus-specific Sirtuin HstD coordinates secondary metabolism and development through control of LaeA

    Eukaryot. Cell

    (2013)
  • Cited by (24)

    • New role of a histone chaperone, HirA: Involvement in kojic acid production associated with culture conditions in Aspergillus oryzae

      2022, Journal of Bioscience and Bioengineering
      Citation Excerpt :

      An A. oryzae disruption mutant library of transcriptional regulators (11,20,21) was used including gene disruption strains of nsdD (ΔnsdD), creB (ΔcreB), lreA (ΔlreA), sreA (ΔsreA), kpeA (ΔkpeA), and AO090012000864 (ΔhirA). Gene disruption of ΔnsdD, ΔcreB, ΔlreA, and ΔkpeA was confirmed in a previous study (11), and that of ΔsreA and ΔhirA was confirmed in this study (Fig. S1). The pyrG-complemented RkuptrP2-1ΔAF (Δku70:ptrA+, pyrG+, ΔcypX-pksA), designated as E-F1+, was used as the control strain (20).

    View all citing articles on Scopus
    View full text