Identification of an essential regulator controlling the production of raw-starch-digesting glucoamylase in Penicillium oxalicum

Background Raw-starch-digesting glucoamylases (RSDGs) from filamentous fungi have great commercial values in starch processing; however, the regulatory mechanisms associated with their production in filamentous fungi remain unknown. Penicillium oxalicum HP7-1 isolated by our laboratory secretes RSDG with suitable properties but at low production levels. Here, we screened and identified novel regulators of RSDG gene expression in P. oxalicum through transcriptional profiling and genetic analyses. Results Penicillium oxalicum HP7-1 transcriptomes in the presence of glucose and starch, respectively, used as the sole carbon source were comparatively analyzed, resulting in screening of 23 candidate genes regulating the expression of RSDG genes. Following deletion of 15 of the candidate genes in the parental P. oxalicum strain ∆PoxKu70, enzymatic assays revealed five mutants exhibiting significant reduction in the production of raw-starch-digesting enzymes (RSDEs). The deleted genes (POX01907, POX03446, POX06509, POX07078, and POX09752), were the first report to regulate RSDE production of P. oxalicum. Further analysis revealed that ∆POX01907 lost the most RSDE production (83.4%), and that POX01907 regulated the expression of major amylase genes, including the RSDG gene POX01356/PoxGA15A, a glucoamylase gene POX02412, and the α-amylase gene POX09352/Amy13A, during the late-stage growth of P. oxalicum. Conclusion Our results revealed a novel essential regulatory gene POX01907 encoding a transcription factor in controlling the production of RSDE, regulating the expression of an important RSDG gene POX01356/PoxGA15A, in P. oxalicum. These results provide insight into the regulatory mechanism of fungal amylolytic enzyme production. Electronic supplementary material The online version of this article (10.1186/s13068-018-1345-z) contains supplementary material, which is available to authorized users.

Native RSDEs are primarily produced by filamentous fungi, such as Aspergillus sp. [3,4], Penicillium sp. [5], and Aureobasidium pullulans [6]. Recently, a novel RSDG (PoxGA15A) exhibiting suitable properties was identified in Penicillium oxalicum and showed remarkably broad pH stability and substrate specificity. Simultaneous saccharification and fermentation of either raw cassava or corn flour using the recombinant protein rPoxGA15A from Pichia pastoris combined with the presence of commercial α-amylase resulted in high fermentation efficiency (> 90%) [5]. However, both native PoxGA15A and rPoxGA15A production, as well as that of other RSDGs from fungi, such as Rhizopus sp. A-11 [7], Aspergillus fumigatus CFU-01 [8] and Laceyella sacchari LP175 [9], are too low, which limit their industrial application. Notably, the expression of fungal RSDG genes is strictly controlled by transcription factors (TFs) at the transcriptional level. Genetic engineering of fungal strains based on constructed TF-specific regulatory networks and targets represents an efficient method to improve RSDG production.
In this study, we employed RNA-seq and molecular genetic technologies to screen and identify novel regulators of RSDE production and RSDG gene expression in P. oxalicum. Transcriptomes from P. oxalicum grown in the presence of glucose or soluble corn starch (SCS) were profiled to identify candidate regulators of RSDE production. Subsequent knockout of candidate genes, measurement of enzyme activity in the resulting mutants, and expression analysis of amylase genes, including the RSDG gene PoxGA15A, were performed to identify novel regulators of RSDG gene expression in P. oxalicum.

Transcriptome profiling and screening of candidate regulators of RSDE production in P. oxalicum
Genome-wide screening of candidate regulators of RSDE production was undertaken through RNA-seq analysis of the transcriptome profiles of P. oxalicum grown on media containing glucose or SCS as the sole carbon source following a transfer from glucose. In the presence of glucose, carbon catabolite repression is activated and inhibits RSDE production in P. oxalicum, whereas SCS stimulates the secretion of RSDEs. Total RNAs were extracted from the mycelia of P. oxalicum grown on glucose or SCS for 4 h and then sequenced. Approximately 24 million clean reads at 100 bp in length (Accession Number SRP116594 in Sequence Read Archive [SRA] database) were generated from each sample, with > 90% mapped into the genome of P. oxalicum wild-type strain HP7-1 [16] (Additional file 1: Table S1). High Pearson's correlation coefficients among three biological replicates of P. oxalicum under each culture condition (r ≥ 0.96) (Additional file 2: Figure S1) indicated the reliability of the transcriptome data. Gene-expression levels were quantitatively analyzed according to fragments per kilobase of exon per million mapped reads (FPKM), and the differences were evaluated using NOISeq [17].

Five novel regulators are required for RSDE production in P. oxalicum
To investigate the regulatory roles of these 23 candidate TF-encoding DEGs in RSDE production in P. oxalicum, homologous recombinant technology was employed for their deletion from the parental strain ∆PoxKu70 [16]. Eleven deletion mutants (∆POX00852, ∆POX01907, ∆POX03446, ∆POX03789, ∆POX05041, ∆POX06509, ∆POX07078, ∆POX07522, ∆POX07938, ∆POX09088, and ∆POX09752) were successfully constructed in this present study, and four deletion mutants (∆POX02944, ∆POX04860, ∆POX05726, and ∆POX06425) had been constructed in the previously published work [21]. These 11 newly constructed deletion mutants in this study were verified by polymerase chain reaction (PCR) using specific primers (Additional file 5: Figure S2a and Additional file 6: Table S3). Both ∆POX03789 and ∆POX02944 were unable to produce spores (data not shown), which was consistent with previously described results [19,20]. Assays of RSDE activity undertaken on all of the deletion mutants, except for ∆POX03789 and ∆POX02944, grown on medium containing SCS as the sole carbon source for 4-6 days after direct inoculation revealed that five deletion mutants (∆POX01907, ∆POX03446, ∆POX06509, ∆POX07078, and ∆POX09752) showed significant reduction in RSDE production relative to the parental strain ∆PoxKu70; ranking from 30 to 83.4% (P ≤ 0.05, Student's t test) (Fig. 2a). This represents the first report showing that these five DEGs were involved in RSDE production in P. oxalicum (Table 1). Strikingly, the mutant ∆POX01907 showed higher losses of RSDE activity (83.4% at day 4 and 80.0% at day 6) relative to the other four mutants and was subsequently selected for further study. To exclude the possibility that multiple copies of POX01907-deletion cassette were integrated into the ∆PoxKu70 genome, the mutant ∆POX01907 was further confirmed by Southern hybridization analysis (Additional file 5: Figure S2b) using specific probes (Additional file 6: Table S3).

POX01907 regulates RSDE production in P. oxalicum following SCS induction
To elucidate POX01907-specific regulatory roles in RSDE production, P. oxalicum strains ∆POX01907 and ∆PoxKu70 were grown on medium containing SCS as the sole carbon source for 2-4 days after a transfer from glucose, followed by real-time investigation. The results revealed 43.5%-71.3% reduction in RSDE production by the mutant ∆POX01907 relative to that observed in the parental strain ∆PoxKu70 (Fig. 2b), which was consistent with our previous analyses.
To confirm the reduction in RSDE production in ∆POX01907 as being a result of POX01907 deletion, a complementary strain (CPOX01907) was constructed Screening and identification of novel regulatory genes required for the production of raw starch-degrading enzymes (RSDEs) in Penicillium oxalicum. a RSDE activities in strains with deletion of candidate regulatory genes and grown in medium containing soluble corn starch as the sole carbon source for 4-6 days after direct inoculation. The parental strain ∆PoxKu70 was used a control. Asterisks indicate significant differences (**P < 0.01; *P < 0.05) between the deletion mutants and the parental strain ∆PoxKu70 according to Student's t test. b, c RSDE activities in the deletion mutants ∆POX01907 and ∆PoxKu70 and the complementary strain CPOX01907. P. oxalicum strains were cultivated in medium containing soluble corn starch for 2-4 days after transfer from glucose. Asterisks indicate significant differences (**P < 0.01) between ∆POX01907 and ∆PoxKu70 or CPOX01907, as assessed by Student's t test and confirmed by PCR (Additional file 5: Figure S2c) using specific primer pairs (Additional file 6: Table S3). Enzyme assays indicated no significant difference in RSDE production between CPOX01907 and ∆PoxKu70 ( Fig. 2c).

Deletion of POX01907 promotes P. oxalicum mycelial growth during the late stage of SCS induction
Equal amounts of fresh spores collected from P. oxalicum ∆PoxKu70, the deletion mutant ∆POX01907, and the complementary strain CPOX01907 were inoculated on solid-medium plates in the presence of glucose, soluble corn starch as the sole carbon source, and potato dextrose agar (PDA), respectively, and cultured at 28 °C for 5 days. The results indicated that colony diameter of ∆POX01907 on all tested plates was significantly larger than that of ∆PoxKu70 and CPOX01907, which showed similar colony diameter (Fig. 3a). Moreover, the colony color of ∆POX01907 was lighter than that of ∆PoxKu70 and CPOX01907, which shared similar colony color (Fig. 3a).
Additionally, we measured mycelial biomass in the three P. oxalicum strains grown in liquid media containing glucose or SCS. The mycelial weight of ∆POX01907 grown in glucose medium was similar to that of ∆PoxKu70 at 60-h post-inoculation and increased slightly after 60 h (Fig. 3b). The mycelial biomass of ∆POX01907 in SCS medium was slightly lower relative to that of ∆PoxKu70 at 36-h post-inoculation but increased significantly after 36 h (Fig. 3c).

POX01907 dynamically regulates the expression of major amylase genes in P. oxalicum
To investigate the regulatory roles of POX01907 in the expression of amylase genes in P. oxalicum, real-time quantitative reverse transcription PCR (RT-qPCR) was performed using total RNA from ∆PoxKu70 and ∆POX01907 grown on SCS medium for 4 h-48 h after a shift from glucose. Three major amylase genes were selected for evaluation, including an α-amylase gene POX09352/Amy13A, the RSDG gene POX01356/Pox-GA15A, and a glucoamylase gene POX02412. The results revealed that transcript levels of POX01356/PoxGA15A and POX02412 in ∆POX01907 increased by 2752.1% and 506.0%, respectively, relative to those in the parental strain ∆PoxKu70 at 4 h post-SCS induction, whereas we did not observe changes in POX09352/Amy13A transcript levels. At 12 h, only POX01356/PoxGA15A continued to show elevation in transcript level in ∆POX01907 (by 212.3%), whereas POX09352/Amy13A transcript levels began to decrease (by 77.3%). The expression of all three genes was reduced by 71.3%-98.8% in ∆POX01907 after 12 h of SCS induction, although POX01356/Pox-GA15A transcript level showed no significant difference from that in the ∆PoxKu70 at 24 h (Fig. 4).
In the regulon of POX01907, 96 DEGs were identified as encoding CAZymes, including 35 from the glycoside hydrolase family, 11 from the glycosyl transferase family, seven from the carbohydrate esterase family, eight from families exhibiting auxiliary activity, one polysaccharide lyase, and 11 from carbohydrate-binding-module families. Among these, 35 were downregulated (− 4.2 < log 2 fold change < − 1.0) and 61 were upregulated (1.0 < log 2 fold change < 4.5) (Fig. 5c). The regulon mainly contained five genes encoding starch-degrading enzymes previously described in this study, 20 genes encoding CWDEs, and seven genes predicted to encode enzymes degrading chitin. Notably, ∆POX01907 showed significant upregulation of genes encoding most of the CWDEs and amylase, and downregulation of chitin-degrading genes under SCS induction, suggesting multiple-regulation of genes involved in degrading different carbohydrates, including starch, cellulose, hemicellulose, and chitin.

Discussion
In this study, we identified five novel regulatory genes (POX01907, POX03446, POX06509, POX07078, and POX09752) involved in mediating RSDE production in P. oxalicum through transcriptome profiling and genetic analysis. Further analyses confirmed that POX01907 regulated the expression of major amylase genes, including the RSDG gene PoxGA15A, as well as P. oxalicum mycelium growth in the presence of SCS. This represents the first report of POX01907 involvement in regulation of RSDG gene expression.
The SANT domain comprises an approximately 50-amino acid motif located in the subunits of many members of chromatin-remodeling complexes, such as Swi3, N-CoR, Ada2, and chromodomain-helicase-DNA binding protein 1 (Chd1), and consists of three α-helices arranged in a helix-turn-elix motif, with each α-helix containing a bulky aromatic residue and is similar to the Myb DNA-binding domain (DBD) [24]. However, the functions of SANT domains might be divergent from those of canonical Myb DBDs.
The SANT domain functions as a unique histone-interaction module that couples histone binding to enzyme catalysis and plays a central role in chromatin remodeling by regulating the activities of histone acetyltransferases and deacetylases to synergistically promote and maintain histone deacetylation [24]. SANT domains are capable of interacting with DNA (i.e., Saccharomyces cerevisiae Chd1 and Arabidopsis sp. Swc4 bind specific AT-rich DNA sequences in a non-canonical manner) [25,26], histones [24], and other proteins (i.e., Chd1 interacts with the transcription-elongation factors Rtf1, Spt4-Spt5, and Spt6-Pob3) [27]. Additionally, the SANT protein Ada2 from Trichoderma reesei is required for mycelial growth, sporulation, and the expression of cellulase genes [28]. In filamentous fungi, including P. oxalicum, the transcription of cellulase and amylase genes is often co-regulated by several TFs, such as PoxNsdD [29], PoxAmyR [30], and PoxHmbB [15]. In the present study, comparative analysis of transcriptomes indicated that POX01907 also regulated the transcription of several cellulase genes in P. oxalicum under the induction of SCS. Therefore, we speculated that POX01907 might play an essential role in the expression of amylase genes by interacting with chromatin-remodeling complexes, although this requires further confirmation. POX01907 dynamically regulated the expression of major RSDG and α-amylase genes similar to other known TFs identified previously in P. oxalicum [15,21,29,30] and was dependent upon the nutrient and energy needs of fungal cells. Transcriptome profiling indicated that POX01907 had minimal influence on the expression of genes involved in the glycolysis pathway. During the early period of P. oxalicum cultivation, fungal cells require trace amounts of glucose for development and growth, and POX01907 inhibited the expression of glucoamylase genes POX01356/PoxGA15A and POX02402, thereby avoiding carbon catabolite repression. Along with glucose consumption and cell proliferation, POX01907 initiated the transcription of genes encoding glucoamylases and α-amylases, resulting in sufficient enzyme secretion to promote the degradation of starch into glucose.

Conclusions
Collectively, our results identified POX01907, a novel transcription factor gene responsible for regulating the production of RSDE through controlling the expression of the major RSDG gene POX01356/PoxGA15A, a glucoamylase gene (POX02412), and the α-amylase gene POX09352/Amy13A. These findings provide novel insights into the regulatory mechanism associated with fungal amylolytic enzymes production and their genes expression.

P. oxalicum strains and cultivation conditions
All P. oxalicum strains ( Table 2) were cultured on PDA plates at 28 °C for 6 days to obtain fungal spores, resuspended using 0.1% Tween 80, and the concentration of fungal spores adjusted to 1.0 × 10 8 /mL. P. oxalicum strains HP7-1 and ΔPoxKu70 were deposited in the China General Microbiological Culture Collection (Beijing, China) with Accession Numbers 10781 and 3.15650, respectively.
For measurement of RSDE production, P. oxalicum strains were cultivated according to previously described methods [5], with some modifications. Under nontransferring conditions, fresh spores (1.0 × 10 8 /mL) of P. oxalicum strains were directly inoculated into minimal medium containing SCS as the sole carbon source at 28 °C for 4-6 days. Under transferring conditions, P. oxalicum spores (1.0 × 10 8 /mL) were first inoculated into minimal medium containing glucose as the carbon source for 24 h, followed by transfer of an equal amount of mycelia from each P. oxalicum strain into minimal medium containing SCS as the carbon source for incubation at 28 °C for 2-4 days. For RNA-seq and RT-qPCR analyses, P. oxalicum strains were cultured for 4 h-48 h according to the methods described for transferring conditions.

Extraction of total DNA and RNA from P. oxalicum
Extraction of total DNA and RNA from P. oxalicum was performed as described previously [16]. Briefly, collected P. oxalicum mycelia were ground with liquid nitrogen, and lysate reagent [20 mM sodium acetate trihydrate, 10 mM ethylenediaminetetraacetic acid, 40 mM Tris-HCl, and 1% sodium dodecyl sulfate (pH 8.0)] was added and mixed. Phenol-chloroform was used to remove proteins. Total DNA was separated and collected by centrifugation at 11,300g for 10 min a Trizol RNA kit (Life Technologies, Carlsbad, CA, USA) was used to extract total RNA according to manufacturer instructions.

Construction of P. oxalicum gene-deletion mutants
Deletion of candidate genes from P. oxalicum was performed according to methods reported by Zhao et al. [16]. The knockout cassette for each candidate gene was constructed by fusion PCR and comprised a 1.9kb G418-resistance gene and approximately 2 kb of the upstream and downstream DNA fragments of the target gene, which were amplified by PCR using the corresponding primer pairs (Additional file 6: Table S4). The generated knockout cassette was introduced into parental strain ∆PoxKu70 protoplasts, and selected transformants were further confirmed by PCR and/or Southern blot using specific primer pairs and/or probes (Additional file 6: Table S4).

Mutant complementation
A complementary strain of the deletion mutant ∆POX01907 was generated as described previously [21]. The complementary cassette comprised approximately 2 kb of the upstream-and downstream-flanking sequences of an aspartic protease gene (POX05007) used as the integrative locus in the genome, 1.2 kb of a DNA fragment encoding the bleomycin-resistance gene, and 7.8 kb of the complementary gene containing the promoter, coding region, and terminator. These four DNA fragments were amplified by PCR using specific primer pairs (Additional file 6: Table S4) and subsequently ligated together using a pEASY-ui seamless cloning and assembly kit (TransGen Biotech, Beijing, China). The generated complementary cassette was introduced into fresh ∆POX01907 protoplasts, and the resulting complementary strains were further confirmed by PCR.

Phenotypic investigation of P. oxalicum strains
Equal amounts of fresh spores from P. oxalicum strains, including the deletion mutant ∆POX01907, the complementary strain CPOX01907, and the parental strain ∆PoxKu70, were inoculated on solid plates containing glucose or SCS as the sole carbon source or PDA and incubated at 28 °C for 5 days. Colonies were photographed using a Canon EOS 6D digital camera (Canon, Beijing, China).

Biomass determination for P. oxalicum strains
Fresh 1.0 × 10 8 spores from P. oxalicum strains, including the parental strain ∆PoxKu70 and the deletion mutant ∆POX01907, were inoculated into 100 mL of glucose or starch liquid medium, respectively, and cultured at 28 °C for 12 h-72 h. The hypha was harvested using a vacuum filter every 12 h and dried to a constant weight at 50 °C.

RNA-seq analysis
RNA-seq analysis was performed according to methods described by Zhao et al. [16]. Total RNA extracted from P. oxalicum strains was used to construct cDNA libraries, with each cDNA having an average length of 100 bp. The constructed cDNA libraries were subjected to evaluation using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and an ABI StepOnePlus realtime PCR system (Applied Biosystems, Forster City, CA, USA), and subsequently sequenced using an IIIumina HiSeq 4000 system (Illumina, San Diego, CA, USA). After quality control, the generated clean reads were mapped onto the P. oxalicum HP7-1 genome and functionally annotated using BWA v0.7.10-r789 (http://sourc eforg e.net/proje cts/bio-bwa/files /) and Bowtie2 v2.1.0 [31]. Gene-expression levels (FPKM) were calculated using RSEM v1.2.12 [22], and DEGs were screened and identified using NOISeq or DESeq2 [23]. Pearson's correlation coefficient was used to evaluate transcriptome reliability among three biological replicates of each sample.

Southern hybridization analysis
Southern hybridization analysis of the deletion mutant ∆POX01907 and the parental strain ∆PoxKu70 was performed as previously described [16]. Briefly, total DNA of each strain was extracted and digested with PstI (TaKaRa, Dalian, China). After separation on an 0.8% agarose gel, the generated DNA fragments were transferred to a Hybond-N + nylon membrane (GE Healthcare, Little Chalfont, UK). The probe used for Southern hybridization was amplified with the primers POX01907-probe-F and POX01907-probe-R (Additional file 4: Table S3). A DIG-High prime DNA labeling and detection starter kit (Life Technologies, Carlsbad, CA, USA) was used to investigate the hybridized bands.

RT-qPCR analysis
RT-qPCR was used to analyze differences in the expression levels of amylase genes between the deletion mutant ∆POX01907 and the parental strain ∆PoxKu70 according to a previously described method [16]. Total RNA from both ∆POX01907 and ∆PoxKu70 was extracted and used as a template to generate first-strand cDNA for RT-PCR using the PrimeScript RT regent kit with gDNA Eraser (TaKaRa). Each qPCR comprised a 20-μL volume, including 2.0 μL of the template for first-stand cDNA, 10 μL of SYBR Premix ExTaq II, 0.8 μL of 10 μM primer (either forward or reverse), and 6.4 μL of sterile water, subjected to initial denaturation for 3 min at 98 °C, followed by 40 cycles of 10 s at 98 °C and 30 s at 58 °C. Fluorescent signals were investigated at the end of each extension step at 80 °C according to the method described by Zhang et al. [10].

Enzyme activity and concentration
RSDE activity and concentration were measured as described previously [5]. Briefly, crude extract from P. oxalicum strains, including the deletion mutant ∆POX01907, the complementary strain CPOX01907, and the parental strain ∆PoxKu70, was added to 0.1 M citric acid/disodium hydrogen phosphate buffer (pH 4.5) containing 1.0% (w/v) raw cassava flour as the substrate, and the mixture was incubated at 65 °C for 30 min. Inactivated crude enzyme extract was used as the blank control. The generated reducing sugars were measured using the 3,5-dinitrosalicylic acid method [32] at 540 nm. One unit of enzymatic activity (U) was defined as the amount of enzyme required to produce 1 μmol of reducing sugars per min from the reaction substrate.
Intracellular enzyme concentration in P. oxalicum strains was measured using a Bradford assay kit (Pierce Biotechnology, Rockford, IL, USA) according to manufacturer instructions.

Statistical analysis
Microsoft Excel (Office 2016; Microsoft, Redmond, WA, USA) was used for the statistical analysis of all experimental data associated with enzyme production and gene transcription. Significance (P < 0.05 or P < 0.01) among samples was calculated using Student's t test.