marine drugs Article Heteroexpression of Aspergillus nidulans laeA in Marine-Derived Fungi Triggers Upregulation of Secondary Metabolite Biosynthetic Genes Ishrat Khan, Wan-Lin Xie, Yu-Chao Yu, Huan Sheng, Yan Xu, Jia-Qi Wang, Sanjit Chandra Debnath , Jin-Zhong Xu , Dao-Qiong Zheng , Wan-Jing Ding and Pin-Mei Wang * Ocean College, Zhejiang University, Zhoushan 316021, China; [email protected] (I.K.); [email protected] (W.-L.X.); [email protected] (Y.-C.Y.); [email protected] (H.S.); [email protected] (Y.X.); [email protected] (J.-Q.W.); [email protected] (S.C.D.); [email protected] (J.-Z.X.); [email protected] (D.-Q.Z.); [email protected] (W.-J.D.) * Correspondence: [email protected] Received: 30 October 2020; Accepted: 15 December 2020; Published: 18 December 2020 Abstract: Fungi are a prospective resource of bioactive compounds, but conventional methods of drug discovery are not effective enough to fully explore their metabolic potential. This study aimed to develop an easily attainable method to elicit the metabolic potential of fungi using Aspergillus nidulans laeA as a transcription regulation tool. In this study, functional analysis of Aspergillus nidulans laeA (AnLaeA) and Aspergillus sp. Z5 laeA (Az5LaeA) was done in the fungus Aspergillus sp. Z5. Heterologous AnLaeA-and native Az5LaeA-overexpression exhibited similar phenotypic effects and caused an increase in production of a bioactive compound diorcinol in Aspergillus sp. Z5, which proved the conserved function of this global regulator. In particular, heteroexpression of AnLaeA showed a significant impact on the expression of velvet complex genes, diorcinol synthesis-related genes, and different transcription factors (TFs). Moreover, heteroexpression of AnLaeA influenced the whole genome gene expression of Aspergillus sp. Z5 and triggered the upregulation of many genes. Overall, these findings suggest that heteroexpression of AnLaeA in fungi serves as a simple and easy method to explore their metabolic potential. In relation to this, AnLaeA was overexpressed in the fungus Penicillium sp. LC1-4, which resulted in increased production of quinolactacin A. Keywords: Aspergillus; heteroexpression; LaeA; fungi; secondary metabolites 1. Introduction Marine fungi are a prolific source of discovering new drugs with tremendous bioactivities [1–5]. Many studies revealed the noteworthiness of these microorganisms as a prospective resource of pharmaceutically important antifungal, antibacterial, antiviral, anti-inflammatory, anticancer, enzyme inhibitor, and antitumor compounds [6]. Industrial investments in natural product research have decreased because conventional methods for the discovery of natural products cannot fulfil the increasing need of bioactive compounds in the healthcare system [7]. Many genetic manipulation methodologies, for instance, transcriptional regulation, epigenetic regulation, ribosome engineering, and heterologous expression, have been tested to analyze biosynthetic processes at different gene regulation levels in different fungi [8–10], but there is still a need to choose an easily attainable approach that could also work in those unstudied fungi whose genomes are not explored. Global regulators regulate the expression of secondary metabolite (SM) biosynthetic gene clusters [7]. The discovery of global regulator LaeA in Aspergillus spp. by Bok and Keller has revolutionized the understanding of fungal secondary metabolism [11]. The dynamic roles of LaeA Mar. Drugs 2020, 18, 652; doi:10.3390/md18120652 www.mdpi.com/journal/marinedrugs Mar.Mar. Drugs Drugs 20202020, 18, 18, ,x 652 2 of2 18 of 18 have been studied in many aspects of fungal biology, such as in asexual and sexual differentiation, inhave phenotype been studied changes in manyincluding aspects pigmentation of fungal biology, and sp suchorulation as in asexual[12,13], andreducing sexual [14] diff erentiation,or increasing secondaryin phenotype metabolite changes production including [11,15,16], pigmentation and and activa sporulationtion of cryptic [12,13 gene], reducing clusters [14 [13,17].] or increasing Numerous studiessecondary based metabolite on model production Aspergillus [11 nidulans,15,16], andLaeA activation have been of carried cryptic geneout during clusters the [13 past,17]. several Numerous years, whichstudies proved based its on mechanisms model Aspergillus (Figure nidulans 1) andLaeA regulatory have been role carried in secondary out during metabolism the past several [11,18–20]. years, In thewhich present proved study, its mechanismswe hypothesized (Figure that1) and if the regulatory function role of LaeA in secondary is conserved metabolism among [ 11filamentous,18–20]. In the present study, we hypothesized that if the function of LaeA is conserved among filamentous fungi, then A. nidulans laeA can be used as a transcription regulation tool to upregulate biosynthetic fungi, then A. nidulans laeA can be used as a transcription regulation tool to upregulate biosynthetic gene clusters (BGCs) in fungi. The proposed method may exclude the need for genome sequencing gene clusters (BGCs) in fungi. The proposed method may exclude the need for genome sequencing techniques, and it could be a quick way to explore the metabolic potential of fungi. To demonstrate techniques, and it could be a quick way to explore the metabolic potential of fungi. To demonstrate the conserved role of LaeA, functional analysis of A. nidulans laeA (AnLaeA) and Aspergillus sp. Z5 the conserved role of LaeA, functional analysis of A. nidulans laeA (AnLaeA) and Aspergillus sp. Z5 laeA (Az5LaeA) was done in the host Aspergillus sp. Z5. It was observed that overexpression of both laeA (Az5LaeA) was done in the host Aspergillus sp. Z5. It was observed that overexpression of both regulatoryregulatory genes genes has has similar similar effects effects on thethe phenotypephenotype andand SM SM production production of of strain strain Z5. Z5. Additionally, Additionally, comparativecomparative transcriptome transcriptome analysis analysis showed showed a substant a substantialial effect eff ofect AnLaeA of AnLaeA overexpression overexpression on the on whole the genomewhole genomegene expression gene expression of Aspergillus of Aspergillus sp. Z5.sp. Hence, Z5. Hence, this study this suggests study suggests that AnLaeA that AnLaeA can be can directly be useddirectly as a usedtranscription as a transcription regulation regulation tool to elicit tool the to secondary elicit the secondary metabolism metabolism of fungi. Further of fungi. studies Further are in progressstudies to are overexpress in progress AnLaeA to overexpress in fungi AnLaeA belonging in fungi to different belonging genera. to different genera. Mechanisms of LaeA Member of Methyltransferase Epigenetics velvet complex activity VelB VeA LaeA Coordinate development Automethylation Turn heterochromatin into and secondary metabolism euchromatin in SM gene clusters Figure 1. Figure 1. SchematicSchematic representation of of LaeA LaeA mechanisms mechanisms [21 [21–23].–23]. 2. Results 2. Results 2.1. Bioinformatics Analysis of Az5LaeA and AnLaeA 2.1. Bioinformatics Analysis of Az5LaeA and AnLaeA The open reading frame (ORF) of Az5LaeA encodes a 352 amino acid peptide. BLASTP analysis showedThe open that Az5LaeAreading frame (LaeA (ORF) of Aspergillus of Az5LaeAsp. Z5, encodes BK011996) a 352 shares amino a high acid degree peptide. of similarityBLASTP analysis with showedits orthologs that Az5LaeA of different (LaeA genera of Aspergillus (Table S1). sp. Multiple Z5, BK011996) sequence shares alignment a high of degreeAspergillus of similaritysp. Z5 LaeA with its orthologsshowed of conserved different genera domain (Table (Figure S1). S1). Multiple Protein sequence sequence alignment analysis by of InterProScanAspergillus sp. revealed Z5 LaeA that showed it conservedis a S-adenosyl-L-methionine-dependent domain (Figure S1). Protein sequence methyltransferase analysis (SAMby InterProScan MTase). Phylogenetic revealed that analysis it is ofa S- adenosyl-L-methionine-dependentwell-characterized AnLaeA (LaeA ofmethyltransferaseA. nidulans, XP_658411) (SAM supportedMTase). Phylogenet that LaeA isic conservedanalysis of in well- a characterizedbroad range ofAnLaeA species (LaeA belonging of A. to nidulans the genus, XP_658411)Aspergillus andsupported other genera that LaeA of the is phylum conservedAscomycota in a broad range(Figure of2 ).species The AnLaeA belonging exhibited to the high genus degree Aspergillus similarity and (86.07%) other with genera the Az5LaeA.of the phylum With an Ascomycota idea to use AnLaeA as a transcription regulation tool, we first attempted to prove the conserved function of (Figure 2). The AnLaeA exhibited high degree similarity (86.07%) with the Az5LaeA. With an idea to this global regulator, therefore functional analysis of both laeA genes was done in the marine-derived use AnLaeA as a transcription regulation tool, we first attempted to prove the conserved function of fungus Aspergillus sp. Z5. this global regulator, therefore functional analysis of both laeA genes was done in the marine-derived fungus Aspergillus sp. Z5. Mar. Drugs 2020, 18, 652 3 of 18 Mar. Drugs 2020, 18, x 3 of 18 B8N406.2|Aspergillus flavus Q2ULA2.2|Aspergillus oryzae 99 AAX68414.1| Aspergillus parasiticus KAB8222746.1| Aspergillus novoparasiticus 100 XP_015402148.1| Aspergillus nomius KAE8138122.1| Aspergillus pseudotamarii KAB8230037.1| Aspergillus alliaceus XP_024703593.1| Aspergillus steynii 84 KAA8651618.1| Aspergillus