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Heteroexpression of Aspergillus nidulans laeA in Marine-Derived Fungi Triggers Upregulation of Secondary Metabolite Biosynthetic Genes

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.


Introduction
Marine fungi are a prolific source of discovering new drugs with tremendous bioactivities [1][2][3][4][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][9][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 have been studied in many aspects of fungal biology, such as in asexual and sexual differentiation, in phenotype changes including pigmentation and sporulation [12,13], reducing [14] or increasing secondary metabolite production [11,15,16], and activation of cryptic gene clusters [13,17]. Numerous studies based on model Aspergillus nidulans LaeA have been carried out during the past several years, which proved its mechanisms ( Figure 1) and regulatory role in secondary metabolism [11,[18][19][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 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 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 regulatory genes has similar effects on the phenotype and SM production of strain Z5. Additionally, comparative transcriptome analysis showed a substantial effect of AnLaeA overexpression on the whole genome gene expression of Aspergillus sp. Z5. Hence, this study suggests that AnLaeA can be directly used as a transcription regulation tool to elicit the secondary metabolism of fungi. Further studies are in progress to overexpress AnLaeA in fungi belonging to different genera.

Bioinformatics Analysis of Az5LaeA and AnLaeA
The open reading frame (ORF) of Az5LaeA encodes a 352 amino acid peptide. BLASTP analysis showed that Az5LaeA (LaeA of Aspergillus sp. Z5, BK011996) shares a high degree of similarity with its orthologs of different genera (Table S1). Multiple sequence alignment of Aspergillus sp. Z5 LaeA showed conserved domain ( Figure S1). Protein sequence analysis by InterProScan revealed that it is a Sadenosyl-L-methionine-dependent methyltransferase (SAM MTase). Phylogenetic analysis of wellcharacterized AnLaeA (LaeA of A. nidulans, XP_658411) supported that LaeA is conserved in a broad range of species belonging to the genus Aspergillus and other genera of the phylum Ascomycota ( Figure 2). The AnLaeA exhibited high degree similarity (86.07%) with the Az5LaeA. With an idea to use AnLaeA as a transcription regulation tool, we first attempted to prove the conserved function of this global regulator, therefore functional analysis of both laeA genes was done in the marine-derived fungus Aspergillus sp. Z5.

Bioinformatics Analysis of Az5LaeA and AnLaeA
The open reading frame (ORF) of Az5LaeA encodes a 352 amino acid peptide. BLASTP analysis showed that Az5LaeA (LaeA of Aspergillus sp. Z5, BK011996) shares a high degree of similarity with its orthologs of different genera (Table S1). Multiple sequence alignment of Aspergillus sp. Z5 LaeA showed conserved domain ( Figure S1). Protein sequence analysis by InterProScan revealed that it is a S-adenosyl-L-methionine-dependent methyltransferase (SAM MTase). Phylogenetic analysis of well-characterized AnLaeA (LaeA of A. nidulans, XP_658411) supported that LaeA is conserved in a broad range of species belonging to the genus Aspergillus and other genera of the phylum Ascomycota ( Figure 2). The AnLaeA exhibited high degree similarity (86.07%) with the Az5LaeA. With an idea to use AnLaeA as a transcription regulation tool, we first attempted to prove the conserved function of this global regulator, therefore functional analysis of both laeA genes was done in the marine-derived fungus Aspergillus sp. Z5.

Overexpression of laeA Genes
LaeA-overexpression plasmids were constructed by placing AnLaeA or Az5LaeA coding regions under the control of A. nidulans gpdA promoter and trpC terminator with G418-resistance marker. The plasmid replicator AMA1 sequence was inserted into laeA-overexpression plasmids for autonomous plasmid replication. AMA1 (MATEs or mobile Aspergillus transformation enhancers) is derived from A. nidulans. This sequence does not encode any extended polypeptide. AMA1 sequence improves the transformation frequency up to 2000 times in contrast to the traditional integrated plasmids [24,25]. Az5LaeA and AnLaeA were overexpressed in Aspergillus sp. Z5 for function analysis. To validate the proposed method, AnLaeA was further overexpressed in the fungus Penicillium sp. LC1-4.

Phenotype and Scanning Electron Microscope Analysis
The OE::AnLaeA and OE::Az5LaeA transformants of Aspergillus sp. Z5 showed significant phenotype differences, i.e., reduced spore production, low dry weight, and decreased radial growth compare to the negative control transformant nc10.5 ( Figure 3). Scanning electron microscopy (SEM) analysis also displayed differences in the distribution of spores and in mycelial and spore morphology. LaeA-overexpression transformants exhibited the occurrence of less spores, and more sulcate colonies with more pronounced spore roughening ( Figure 4).

Overexpression of laeA Genes
LaeA-overexpression plasmids were constructed by placing AnLaeA or Az5LaeA coding regions under the control of A. nidulans gpdA promoter and trpC terminator with G418-resistance marker. The plasmid replicator AMA1 sequence was inserted into laeA-overexpression plasmids for autonomous plasmid replication. AMA1 (MATEs or mobile Aspergillus transformation enhancers) is derived from A. nidulans. This sequence does not encode any extended polypeptide. AMA1 sequence improves the transformation frequency up to 2000 times in contrast to the traditional integrated plasmids [24,25]. Az5LaeA and AnLaeA were overexpressed in Aspergillus sp. Z5 for function analysis. To validate the proposed method, AnLaeA was further overexpressed in the fungus Penicillium sp. LC1-4.

Phenotype and Scanning Electron Microscope Analysis
The OE::AnLaeA and OE::Az5LaeA transformants of Aspergillus sp. Z5 showed significant phenotype differences, i.e., reduced spore production, low dry weight, and decreased radial growth compare to the negative control transformant nc10.5 ( Figure 3). Scanning electron microscopy (SEM) analysis also displayed differences in the distribution of spores and in mycelial and spore morphology. LaeA-overexpression transformants exhibited the occurrence of less spores, and more sulcate colonies with more pronounced spore roughening ( Figure 4). , and (D) spore production on solid GMM + G418. All phenotype analyses were done at 30 °C in the dark. All results are represented as the average ± SE of triplicate samples. For radial growth and dry weight, p-value between control nc10.5 and transformants is < 0.05. For spores production, pvalue between control nc10.5 and transformant OE::Az5LaeA 9.10 /OE::AnLaeA 11.19 is < 0.05. , and (D) spore production on solid GMM + G418. All phenotype analyses were done at 30 • C in the dark. All results are represented as the average ± SE of triplicate samples. For radial growth and dry weight, p-value between control nc10.5 and transformants is < 0.05. For spores production, p-value between control nc10.5 and transformant OE::Az5LaeA 9.10 /OE::AnLaeA 11.19 is < 0.05.

Secondary Metabolites Analysis
High-performance liquid chromatography (HPLC) analysis revealed the high production of a compound by OE::AnLaeA and OE::Az5LaeA transformants compared to the negative control transformant nc10.5 and wild type Aspergillus sp. Z5 ( Figure 5). The increased production of compound 1 was obvious by the high peak area in laeA-overexpression transformants ( Table 1). The native Az5LaeA-overexpression delivered more compound production compared to the heterologous Mar. Drugs 2020, 18, 652 5 of 18 AnLaeA-overexpression, but both laeA genes caused the upregulation of a similar compound that established the conserved function of this regulator. The structure of upregulated compound 1 in Aspergillus sp. Z5 was determined by comparing 1 H and 13 C NMR data and mass spectroscopy results with the literature [26,27]. Compound 1 was identified as 3, 3 -dihydroxy-5, 5 -dimethyldiphenyl ether, also known as diorcinol ( Figures S2 and S3). The molecular weight of the compound was determined by [M + H] ion peak at m/z 229.0863 in HR-ESI-MS spectrum ( Figure S4). Compound 1 possesses cytotoxic activity against the HCT116 human colon cancer cell line with IC 50 10µg. This is probably the first reported cytotoxic activity for this compound against the HCT116 human colon cancer cell line. After functional analysis in Aspergillus sp. Z5, AnLaeA was heteroexpressed in a fungus LC1-4 belonging to the genus Penicillium. Heteroexpression of AnLaeA in Penicillium sp. LC1-4 resulted in up to 4-fold high production of a previously identified compound 2 in transformant OE::AnLaeA Plc11.11 ( Figure 6) ( Table 2). The secondary metabolite results of this study provide a basis for engineering Aspergillus and Penicillium species to get a high yield of pharmaceutically important compounds such as diorcinol and quinolactacin.

Secondary Metabolites Analysis
High-performance liquid chromatography (HPLC) analysis revealed the high production of a compound by OE::AnLaeA and OE::Az5LaeA transformants compared to the negative control transformant nc10.5 and wild type Aspergillus sp. Z5 ( Figure 5). The increased production of compound 1 was obvious by the high peak area in laeA-overexpression transformants ( Table 1). The native Az5LaeA-overexpression delivered more compound production compared to the heterologous AnLaeA-overexpression, but both laeA genes caused the upregulation of a similar compound that established the conserved function of this regulator. The structure of upregulated compound 1 in Aspergillus sp. Z5 was determined by comparing 1 H and 13 C NMR data and mass spectroscopy results with the literature [26,27]. Compound 1 was identified as 3, 3′-dihydroxy-5, 5′dimethyldiphenyl ether, also known as diorcinol ( Figures S2, S3). The molecular weight of the compound was determined by [M + H] ion peak at m/z 229.0863 in HR-ESI-MS spectrum ( Figure S4). Compound 1 possesses cytotoxic activity against the HCT116 human colon cancer cell line with IC50 10µg. This is probably the first reported cytotoxic activity for this compound against the HCT116 human colon cancer cell line. After functional analysis in Aspergillus sp. Z5, AnLaeA was heteroexpressed in a fungus LC1-4 belonging to the genus Penicillium. Heteroexpression of AnLaeA in Penicillium sp. LC1-4 resulted in up to 4-fold high production of a previously identified compound Plc11.11

Transcriptome Analysis
To find out the genome-wide effects of AnLaeA overexpression, transcriptome analysis of nc10.5 and OE::AnLaeA 11.19 was done at 60 h in potato dextrose broth (PDB) media. A total of 98,179,444 raw reads were generated from strain nc10.5 and OE::AnLaeA 11.19 that accounts for about~15 GB of paired-end sequencing data. The raw data is available on the NCBI Sequence Read Archive (SRA) under the accession number PRJNA649367. The pre-processing of raw reads was done for removal of adaptor sequences and low-quality reads. Ninety-eight million high-quality reads were retained after Cutadapt software (NBIS, Uppsala University, Uppsala, Sweden) [28] filtering (Table S2). The results show that the quality and data volumes of transcriptome sequencing were relatively high that meet the requirements for subsequent data assembly and processing. For the assembly process, reads from each strain were pooled together to reconstruct all the transcripts. Using trinity with default parameters [29], a total of 33,747 assembled transcripts were generated with a mean size of 2131 bp. 18,923 unigenes of mean length 1368 bp were obtained for a total of 25,898,836 bases (Table S3). The range in unigene length was from 201 bp to 26,466 bp, and the unigene length distribution is shown in Figure S5.

In Vitro Validation of Specific Genes by qPCR
The relative expression level of velvet complex genes, diorcinol-synthesis related genes and different transcription factors (TFs) involved in the regulation of growth and secondary metabolism was checked at 60 h, 72 h, and 120 h. Both velvet complex genes veA and velB showed relatively high expression at 60 h and 120 h ( Figure 7A). According to the secondary metabolite results, OE::AnLaeA 11.19 exhibited high production of diorcinol. Feng and co-workers determined three enzymes from A. nidulans and defined their roles in the biosynthesis of diorcinol [27]. The biosynthetic mechanism of diorcinol includes a rare polyketide synthase (PKS), AN7909 (orsA) [30], which is responsible for production of diorcinolic acid, while an uncharacterized enzyme, AN7910 (orsF) [31], and a putative amidohydrolase, AN7911 (orsB) [30], are responsible for catalyzing regioselective decarboxylation of diorcinolic acid to produce diorcinol ( Figure 8). According to their findings, three genes, AN7912 (orsC), AN7913 (orsD), and AN7914 [32], of the Ors gene cluster are evidently not involved in the biosynthesis of diorcinol [27]. qPCR results of this study displayed differential expressions of diorcinol-synthesis related genes at different time points. Gene orsA (g2173.t1, ortholog of AN7909), orsB (g2171.t1, ortholog of AN7911), and orsF (g2172.t1, ortholog of AN7910) showed high expression in transformant OE::AnLaeA 11.19 at 60 h ( Figure 7A). These are the key genes involved in the biosynthesis of diorcinol. Gene expression analysis supported the high production of diorcinol in OE::AnLaeA 11.19 under heteroexpression of AnLaeA ( Figure 7A). LaeA has a broad impact on growth and secondary metabolism of fungi [11,33,34]. By comparing the Web Gene Ontology Annotation Plot (WEGO), enrichment of annotated unigenes, and AnLaeA upregulated genes, 26 GO categories were found enriched with AnLaeA upregulated genes ( Figure S10). The main categories related to secondary metabolism with AnLaeA upregulated genes include the "metabolic process" category (GO: 0008152, 33 genes, 1.41%), "translation regulator activity" category (GO: 0045182, 1 gene, 2.85%), and "transcription regulator activity" category (GO: 0030528, 3 genes, 2.45%). In the transcription regulation category, 2 genes involved in transcription regulation (g1727.t1, g742.t1) and CCAAT-binding transcription factor hapB [35] (g5897.t1) were found upregulated in transformant OE::AnLaeA 11.19 at 60 h. To further evaluate this category, the relative expression level of some of the key TFs was determined by qPCR ( Figure 7B). AflR (sterigmatocystin Zn 2 CyS 6 transcription factor) was upregulated in AnLaeA overexpression transformant OE::AnLaeA 11.19 at 72 h and 120 h. The other TFs, mtfA (C 2 H 2 transcription factor involved in the regulation of secondary metabolism) [36] and sclR (involved in sclerotial production) [37], showed significant upregulation at 60 h and 72 h, while sltA (C 2 H 2 transcription factor involved in morphogenesis and biosynthesis of sterigmatocystin) [38] was not significantly upregulated at these time points, but all three TFs were found downregulated at 120 h. McrA is a master regulator of secondary metabolism in the genus Aspergillus [39]. McrA was positively regulated by AnLaeA at 72 h and 120 h. AnLaeA overexpression also influenced the expression of llmf (negative regulator of development and sterigmatocystin) [40]. Llmf was downregulated at 60 h and 72 h, but showed upregulation at 120 h. GcnE (H3K9 acetyltransferase of SAGA complex) showed high expression at 120 h ( Figure 7B). time points, but all three TFs were found downregulated at 120 h. McrA is a master regulator of secondary metabolism in the genus Aspergillus [39]. McrA was positively regulated by AnLaeA at 72 h and 120 h. AnLaeA overexpression also influenced the expression of llmf (negative regulator of development and sterigmatocystin) [40]. Llmf was downregulated at 60 h and 72 h, but showed upregulation at 120 h. GcnE (H3K9 acetyltransferase of SAGA complex) showed high expression at 120 h ( Figure 7B).

Discussion
Filamentous fungi are a prospective resource of bioactive compounds. Many strategies have been introduced to explore the metabolic potential of these natural assets [8][9][10], but most of them are difficult to handle and require genome sequencing of the strain. Global regulators can improve the whole fungal natural product diversity [7]. Therefore, this study presents an easily attainable molecular method based on utilizing the global regulator of secondary metabolism LaeA from A. nidulans as a transcription regulation tool in filamentous fungi. This method involves heteroexpression of AnLaeA by an autonomously replicating plasmid in fungi. It is a very simple method and easy to perform. The significance of this method includes: (1) It does not require genome sequencing of host fungi; (2) this method is applicable to fungi belonging to the family Trichocomaceae; (3) if fungal strains are sensitive to the same drug marker, then the same plasmid can be transformed

Discussion
Filamentous fungi are a prospective resource of bioactive compounds. Many strategies have been introduced to explore the metabolic potential of these natural assets [8][9][10], but most of them are difficult to handle and require genome sequencing of the strain. Global regulators can improve the whole fungal natural product diversity [7]. Therefore, this study presents an easily attainable molecular method based on utilizing the global regulator of secondary metabolism LaeA from A. nidulans as a transcription regulation tool in filamentous fungi. This method involves heteroexpression of AnLaeA by an autonomously replicating plasmid in fungi. It is a very simple method and easy to perform. The significance of this method includes: (1) It does not require genome sequencing of host fungi; (2) this method is applicable to fungi belonging to the family Trichocomaceae; (3) if fungal strains are sensitive to the same drug marker, then the same plasmid can be transformed directly to elicit their secondary metabolism without the need of plasmid construction.
In this study, functional analysis of AnLaeA and Az5LaeA was conducted in the marine-derived fungus Aspergillus sp. Z5. AnLaeA-and Az5LaeA-overexpression caused characteristic changes in the phenotype and delivered increased production of a bioactive compound diorcinol in Aspergillus sp. Z5. Our phenotype and secondary metabolite results are consistent with other laeA-overexpression reports, such as overexpression of laeA ortholog caused reduced sporulation in A. fumisynnematus and altered the mycelial morphology of Monascus purpureus [15,16]. In the previous studies, native laeA-overexpression has also been reported with increased production of compounds including lavostatin, monacolin K, penicillin, and cyclopiazonic acid [11,15,16]. We found that heteroexpression of AnLaeA and native Az5LaeA exhibited similar phenotypic and metabolic effects, which proved that the function of this global regulator is conserved.
Emphasizing the regulatory effect of AnLaeA, overexpression of AnLaeA showed significant impact on the expression of velvet complex genes, diorcinol-synthesis related genes, and different TFs, which justified the results of phenotype analysis and secondary metabolite analysis of this study. This study also supports that LaeA positively regulates the Ors gene cluster. Moreover, the comparative transcriptome analysis revealed that heteroexpression of AnLaeA can influence the whole genome gene expression of host fungi due to the conserved function. The proposed method was further validated in fungus belonging to the genus Penicillium. Heteroexpression of AnLaeA caused increased production of quinolactacin A in Penicillium sp. LC1-4, which was the main compound of this strain ( Figure 6). Besides, we also tried to knockout Az5LaeA to activate some BGCs, but knockout was unsuccessful after several trials. According to our experience, knockout of global regulators may be challenging in some strains compared to the overexpression strategy.
In conclusion, heteroexpression of well-studied AnLaeA in a broad range of fungi serves as an efficient method to explore their metabolic potential. This study suggests that AnLaeA can be used as a transcription regulation tool in fungi.

Strains, Media, and Culture Conditions
Aspergillus sp. Z5 was isolated from the gut of marine isopod Ligia oceanica in our previous study [41], and Penicillium sp. LC1-4 was isolated from the intestine of a marine conch. A. nidulans RDIT 2.3 was obtained from Dr. Nancy Keller [11]. Strain Aspergillus sp. Z5 and Penicillium sp. LC1-4 were identified by an internal transcribed spacer (ITS) sequence, and the sequence data were deposited to GenBank under the accession number MN636770 and MT672583, respectively. Fungal strains were cultured and maintained on glucose minimal medium (GMM) agar plates [42] at 30 • C for 4 days. Spore suspensions were prepared in 0.1% Tween 80 and preserved as 25% v/v glycerol stocks at −80 • C. Liquid GMM supplemented with 0.5% yeast extract was used to harvest mycelia. GMM added with 1.2M D-sorbitol and supplemented with 100 µg/mL G418 (Solarbio, Shanghai, China) was used for screening of resistant transformants. For compound production, strains were cultured in PDB (200 g/L potatoes, 34 g/L ocean salts, and 20 g/L glucose) at 28 • C, 160 rpm for 10 days. Fungal strains used in this study are listed in Table 3.

Gene Cloning and Bioinformatics Analysis
The genomic sequence of AnLaeA was obtained from the Aspergillus genome database (http: //aspergillusgenome.org/). The 1255 bp open reading frame (ORF) of AnLaeA is comprised of two exons and one intron. Exons were amplified from genomic DNA of A. nidulans RDIT2.3 and joined together by modified double joint PCR method [43], followed by ligation into pMD TM 19-T Vector (TaKaRa, Beijing, China) to generate pISH1 plasmid. The Az5LaeA was found as AnLaeA orthologue. It is comprised of 1249 bp ORF and shares 86.07% identity with AnLaeA. Az5LaeA ORF was cloned from genomic DNA of Aspergillus sp. Z5 using specific primers (Table S5). The Az5LaeA was analyzed by an online BLAST search at the National Center for Biotechnology Information (NCBI) web site (https://blast.ncbi.nlm.nih.gov/Blast.cgi) [44]. Multiple sequence alignment of Az5LaeA coding sequence with other LaeA correlatives from diverse genera was done by ClustalW2 (EMBL-EBL, Hinxton, Cambridgeshire, UK) (https://www.ebi.ac.uk/Tools/msa/clustalw2/) [45]. The theoretical isoelectric point (pI) and molecular weight of Az5LaeA protein was determined by ExPASy (SIB, Switzerland) (https://web.expasy.org/compute_pi/) [46]. The conserved domain of Az5LaeA protein was scanned by the InterProScan (EMBL-EBL, Hinxton, Cambridgeshire, UK) (http://www.ebi.ac.uk/ interpro/search/sequence/) [47]. LaeA gene sequences of other correlatives from diverse genera were retrieved from NCBI to deduce the phylogenetic affiliation of well characterized AnLaeA. Phylogenetic analysis was carried out using MEGA7 software (https://megasoftware.net/) [48].

Molecular Genetic Manipulations
The backbone of overexpression plasmids was amplified from pC-G418-YR plasmid (http: //www.addgene.org/61767/), that was comprised of resistant kanamycin (kanR) gene for Escherichia coli, URA3 gene for yeast, and resistant G418 (neoR/kanR) gene for fungi as selectable markers with Ori and 2µ origin of replication for E.coli and yeast, respectively. To construct autonomously replicating plasmids, AMA1 was obtained from ANEp2 plasmid (http://fgsc.net/) by restriction endonuclease (RE) Not I digestion. A constitutive gpdA promoter and trpC terminator were cloned from A. nidulans RDIT 2.3 using specific primers (Table S5). The ORF of Az5LaeA and the coding region of AnLaeA were placed between promoter and terminator to generate laeA-overexpression plasmids pISH9 and pISH11, respectively. A negative control plasmid pISH10 without laeA gene was also constructed to check the null effect plasmid elements. Plasmid maps are shown in Figure S11. All the PCR amplified and RE digested fragments were gel purified and transformed into Saccharomyces cerevisiae strain BJ5464 to generate circular plasmids by yeast homologous recombination [49]. Plasmids were transformed into competent E. coli strain DH5α for propagation and extracted to transform into fungi [13]. Plasmids details are shown in Table S6.

Fungal Transformation
Aspergillus sp. Z5 was transformed with negative, Az5LaeA-, and AnLaeA-overexpression plasmids. The strain was first grown on a GMM plate at 30 • C for 4 days. Aspergillus sp. Z5 germlings were prepared by culturing 5-6 mL 10 7 sp/mL in LMM at 30 • C, 200 rpm for about 12-14 h. Germlings were harvested by centrifugation at 5000 rpm, 4 • C, and washed twice with sterilized ddH 2 O. The harvested germlings were then digested to generate protoplasts by enzymatic preparation in 10 mL osmotic media (259.8 g/L MgSO 4 , 10 mM NaPhosphate buffer, pH 5.8 with 1M Na 2 HPO 4 ) as a buffer with following enzymes in respective concentrations: 0.01g/mL lysozyme (Sangon Biotech, Shanghai, China), 0.01 g/mL snailase (BBI Life Sciences, Shanghai, China), 0.01g/mL lysing enzyme (SIGMA, St. Louis, MO, USA), and 0.005 g/mL yatalase (TaKaRa, Beijing, China) at 30 • C, 80 rpm for 6 h. The solution after enzyme digestion was layered by slow release of 10 mL trapping buffer (109.3 g/L D-Sorbitol, 0.1 M Tris-HCI, pH 7.0) and centrifuge at 5000 rpm, 4 • C for 20 min. The clear white protoplast layer formed in the interface was collected and washed with STC buffer (218.6 g/L D-Sorbitol, 0.47 g/L CaC1 2 and 10 mM Tris-HCI, pH 7.5) at 5000 rpm, 4 • C for 10 min. For transformation, 100 µL protoplasts 10 7 p/mL was mixed with ≤ 75 µL (15-20 µg) transforming DNA (dissolved in STC buffer) and incubated on ice for 50 min. 1.25 mL polyethylene glycol (PEG) solution (60 g/L PEG, 50 mM CaC1 2 , 50 mM Tris-HCl, pH 7.5) was added into the transforming mixture, mixed well, and incubated for 20 min at room temperature. The final volume of the mixture was made up to 10 mL with STC buffer. Plating was done using SMM + G418 100 µg/mL agar media. 1 mL transforming mixture was poured per plate and overlaid with 3.5 mL 0.8% SMM agar top media. Plates were incubated at 30 • C in the dark for 2 days. pISH10 and pISH11 were transformed into Penicillium sp. LC1-4 by following the same transformation procedure, but with a different enzymic preparation, i.e., 0.0055g/mL lysing enzyme (SIGMA, St. Louis, MO, USA) and 0.0025g/mL yatalase (TaKaRa, Beijing, China) in 10 mL osmotic medium. Secondary screening of all transformants was done on GMM + G418 (100 µg/mL). Resistant transformants were cultured into LMM + G418 200 µg/mL for 2 days. Those transformants which were able to grow in the presence of the drug were subjected to further analysis. Extraction of genomic DNA was performed as defined by Green [50]. Confirmation of positive transformants was done by diagnostic PCR using gene specific primers (Table S5). Those transformants that contained the gpdA-laeA and neoR/kanR gene sequence were considered as positive laeA-overexpression transformants. While confirmation of negative control transformants was done by checking gpdA-trpC and neoR/kanR gene sequences.

Phenotype Analysis and Scanning Electron Microscopy
Phenotype analyses of Aspergillus sp. Z5 transformants were performed. In phenotype analysis, transformants were examined for their radial growth, dry weight, and spore production. To examine the radial growth, 5 µL (10 4 spores in total) of each transformant was point-inoculated onto GMM + G418 (100 µg/mL) agar plates. Colony diameter was measured from day 4 to day 7 of growth at 30 • C in the dark. Spore production was determined on GMM + G418 (100 µg/mL) agar plates. Briefly, 10 mL 0.8% molten agar top layer that contained 10 7 spores in total for each transformant was overlaid on GMM + G418 (100 µg/mL) plates. Plates were incubated at 30 • C for 7 days in the dark. An agar plug of 1.5 cm in diameter was removed with the help of a sterilized cork borer and homogenized in 2 mL 0.1% tween 80 to make a uniform spore suspension. Spores were counted using a Neubauer chamber. Dry weight was determined by inoculating 10 7 spores of each transformant in 4 mL LMM + G418 (100 µg/mL) and cultured at 30 • C for 4 days in the dark. Mycelia were collected and dried in a freeze dryer at −55 • C, 5 Pa for 3 h. Dried mycelia were kept at −80 • C over night, and the weight was determined. All experiments were performed in triplicate. Mycelial and spore morphology of transformant nc10.5, OE::Az5LaeA 9.8 and OE::AnLaeA 11.19 were observed by using scanning electron microscopy (SEM) (ZEISS Sigma, Jena, Germany). Transformants were point inoculated onto GMM + G418 (100 µg/mL) agar plates and cultured at 30 • C for 5 days in the dark. Samples were prepared as follows; the conductive double-sided adhesive carbon tape was stuck on small metal grids and then the grids were gently punched onto the growth of transformants to take the impression. Samples were observed at 500 ×, 5K×, and 20K× magnification to analyze the arrangement of spore chain, mycelial, and spore morphology.

Secondary Metabolite Analysis, Purification, and Identification of Compound
Secondary metabolite analysis was initially done on a small scale. The wild type of Aspergillus sp. Z5 was activated on GMM agar plates, while the negative control, OE::Az5LaeA, and OE::AnLaeA

Conclusions
In conclusion, the function of global regulator LaeA is conserved in filamentous fungi, as heterologous AnLaeA and native Az5LaeA overexpression showed similar effects on phenotype and secondary metabolism of the host strain. Comparative transcriptome analysis revealed that heteroexpression of AnLaeA can influence the whole genome gene expression and induce the secondary metabolism of host fungi. Therefore, AnLaeA can be used as a transcription regulation tool in other fungi.
Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/18/12/652/s1, Figure S1: Multiple sequence alignment of Az5LaeA with its orthologs by ClustalX2.1, Figure S2: 1 H NMR Spectrum of compound 1 in CD3OD, Figure S3: 13 C NMR Spectrum of compound 1 in CD 3 OD, Figure S4: LC-MS analysis of compound 1, Figure S5: (A) The range of unigene length; (B) Unigene length distribution, Figure S6: Top-hit species similarity of assembled unigenes, Figure S7: The Gene Ontology (GO) pathway classification of assembled unigenes, Figure S8: The KEGG pathway classification of assembled unigenes, Figure S9: The visualization of differentially expressed genes (DEGs), Figure S10: WEGO enrichment comparison between annotated unigenes and AnLaeA upregulated genes in transformant OE::AnLaeA 11.19 , Figure S11: Plasmid maps, Table S1: Comparison between Az5LaeA and its orthologs, Table S2: Summary of RNA-seq statistics, Table S3: Statistics of the de novo assembly of RNA-seq, Table S4: Annotation of unigenes against different databases,  Table S5: Primers used in this study, Table S6: List of plasmids used in this study, Table S7