A unique Zn(II)2-Cys6-type protein, KpeA, is involved in secondary metabolism and conidiation in Aspergillus oryzae
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 nsdD (ΔnsdD), creB (ΔcreB), lreA (ΔlreA), 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
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Production of kojic acid by Aspergillus species: Trends and applications
2023, Fungal Biology ReviewsNew role of a histone chaperone, HirA: Involvement in kojic acid production associated with culture conditions in Aspergillus oryzae
2022, Journal of Bioscience and BioengineeringCitation 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).