Sequencing and characterization of an L-asparaginase gene from a new species of Penicillium section Citrina isolated from Cerrado

The enzyme L-asparaginase (L-ASNase) is used in the treatment of Acute Lymphoblastic Leukemia. The preparations of this enzyme for clinical use are derived from bacterial sources and its use is associated with serious adverse reactions. In this context, it is important to find new sources of L-ASNase. In this work, the Placket-Burman Experimental Design (PBD) was used to determine the influence of the variables on the L-ASNase production then it was followed by a 28–4 Factorial Fractional Design (FFD). The results obtained from PBD have shown a range of L-ASNase activity, from 0.47 to 1.77 U/gcell and the results obtained from FFD have showed a range of L-ASNase activity, from 1.10 to 2.36 U/gcell. L-proline and ammonium sulfate were identified as of significant positive variables on this production enzyme by Penicillium cerradense sp. nov. The precise identification of this new species was confirmed by morphological characteristics and sequence comparisons of the nuclear 18S-5.8S-28S partial nrDNA including the ITS1 and ITS2 regions, RNA polymerase II, β-tubulin and calmodulin genomic regions. The genetic sequence coding for the L-ASNase was obtained after carrying out a full genome sequencing. The L-ASNase expressed by P. cerradense sp. nov may have promising antineoplastic properties.


Results
Two isolates of Penicillium were obtained from the soil of the Cerrado for morphological and molecular characterization. The amplification and sequencing of the partial rDNA (including the ITS), RPB2, β-tubulin and calmodulin regions revealed sequences of ca. 1.200, 800, 720, and 570 bp, respectively. The ITS and RPB2 sequences were used in the multigenic species identification. The β-tubulin and calmodulin gene sequences were not included in the phylogenetic analysis because these regions were unavailable for most of the previously described Penicillium species. The new sequences were deposited in GenBank under accession numbers MT006126, MT006127, MT416532 to MT416537. No topological conflicts were found among the phylogenetic trees based on each of the two partial genomic regions, and therefore, the data sets were concatenated (single gene trees are available in TreeBASE). For the multilocus analysis, 86 taxa were used (Table Supplementary S1), with alignments of RPB2 and ITS having 915 and 586 bp in length, respectively. The concatenate alignment (1501 bp) showed 891 conserved characters, 589 variable, and 513 phylogenetically informative sites. The GTR + I + G model was selected for RPB2 and ITS.
Based on morphological and molecular comparisons, a new species of Penicillium belonging to the section Citrina is proposed in this work (Fig. 1). Etymology: -This species refers to Cerrado. Colonies of this fungus have grown slowly in MEA culture medium (15 mm), PDA (30 mm), and SDA (37 mm) in 7 days, without aerial and superficial mycelium formation. In MEA the colonies were light green or greyish-green, velvety, white or absent colony edges and light-brown in reverse ( Fig. 2A). Conidiophores were formed abundantly, solitary, erect, hyaline, emerging from hypha, consisting of a stipe followed by a penicillate conidiogenic apparatus; hyaline and smooth stipe with 25 In PDA, the colony was greyish-green with a well-defined white border and light-brown roughness in reverse. Finally, in SDA the colony was greyish-green, light gray or brown coloration with white border and light-brown with intense roughness in reverse (Fig. 3).    Notes: -The new species groups in a distinct clade with P. sumatrense. Penicillium cerradense sp. nov. is phylogenetically close but clearly distinct of the P. sumatrense. The new species has conidiophores predominantly monoverticillate or biverticillate with two branches, abundant sclerotia, smaller stipe (85 μm) and phialides (5.0-8.0 × 2.0-3.0 μm), while P. sumatrense has conidiophores with 3-6 branches, absent sclerotia, larger stipe (up to 200 μm) and phialides (8.0-10 × 2.0-3.5 μm) 16 .

Taxonomy. Penicillium cerradense
L-asparaginase gene. The L-asparaginase gene was obtained from the whole genome sequencing of Penicillium cerradense isolate DCFS6a. The L-asparaginase gene was 1251 bp in length, and its nucleotides sequence was analyzed using the Clustal Omega and NCBI's BLAST programs. The nucleotides and amino acids sequences of Penicillium cerradense showed homology to asparaginase genes derived from Penicillium sizovae, Aspergillus niger and Aspergillus ibericus ( Fig. Supplementary S1), among other species. Based on amino acid sequences alignment, it was markedly different from other previously reported Penicillium spp. derived-L-asparaginases. It is noteworthy that in the analysis using the Clustal Omega and NCBI's BLAST programs, homology to L-asparaginase genes was found only in this unique sequence presented in this work, thus an enzymatic activity reported in this manuscript is encoded by the only gene identified for L-asparaginase in the P. cerradense sp. nov. Identifying significant variables affecting L-asparaginase production by. Penicillium cerradense sp. nov. using statistical design. The PBD provides initial indications of how each variable tends to influence the L-asparaginase production 17 and it is convenient especially when facing large number of factors that can potentially influence optimal or near optimum responses 18 . This design is recommended when more than eight factors are under investigation 18 . This model describes interaction among factors, and it is used to screen and evaluate the important factors that influence asparaginase production. The experiment was conducted to study the effect of each selected variable on the production of L-asparaginase. The design matrix selected for the screening of significant variables for L-asparaginase production and the corresponding response (Y) under culture medium conditions are shown in Table 1. The results obtained from PBD have shown a wide range of L-asparaginase activity, from 0.47 to 1.77 U/g cell . The maximum L-asparaginase activity was achieved on run number 9 with culture medium containing L-asparagine 3.0%, L-proline 3.0%, urea 0.1%, sodium nitrate 2.5%, yeast extract 0.1%, ammonium sulfate 1.5%, peptone 2.0%, glucose 0.2%, sucrose 0.2%, malt extract 0.5% and potassium chloride 0.01%.
The relationship between a set of independent variables and the response (Y) is determined by a mathematical model called the multiple regression model. The determination of the main effects was performed, and the results are presented in Table 2. Eight out of the eleven variables tested (L-asparagine, L-proline, urea, yeast extract, ammonium sulfate, peptone, glucose and sucrose), showed positive effect and improved L-asparaginase production, whereas variables such as sodium nitrate and potassium chloride decreased L-asparaginase activity. Table 1. L-asparaginase activity as Plackett-Burman experimental design for Penicillium cerradense. L-asparagine (X 1 ), L-proline (X 2 ), urea (X 3 ), sodium nitrate (X 4 ), yeast extract (X 5 ), ammonium sulfate (X 6 ), peptone (X 7 ), glucose (X 8 ), sucrose (X 9 ), malt extract (X 10 ) and potassium chloride (X 11 ).

Levels (%)
L-asparaginase activity (U/g cell ) www.nature.com/scientificreports/ of your response than the current model. The "Lack of Fit t-value" of 2.58 implies the Lack of Fit is significant. After the analyses of the ANOVA results, the next steps of the study were carried out using one variable at a time. L-proline and ammonium sulfate were identified as of significant positive variables on the production of L-asparaginase by Penicillium cerradense. The effect of L-proline in different concentrations is represented in Fig. 4. The L-proline substrate concentration of 9% differs significantly from 3 and 7%. The enzymatic activity at 9% corresponds to approximately 1.6 times greater than the one found in lower concentration. In the medium with 7% concentration the morphology of the grown biomass is different from the others at other concentrations. The fungus grew slowly and the final biomass showed a lower yield. The observed decrease in enzyme activity from 5 to 7% of substrate concentration could be related to this morphological difference in the growth of P. cerradense sp. nov. El-Enshasy et al. 19 identified the correlation of protein production capacity with morphological differences in the growth of Aspergillus niger in submerged culture medium. In this study, while evaluating the effect of L-proline in concentrations of 15% and 20%, a reduction in L-asparaginase activity was observed.
The same strategy used to verify the effect of L-proline substrate on the L-asparaginase activity was used to identify the effect of substrate ammonium sulfate in enzyme production. The effect of ammonium sulfate in different concentrations is represented in Fig. 5. The analyses were performed in triplicate and the results are presented as mean of enzyme activities with standard deviation for each run. The higher enzymatic activity was observed at 7% ammonium sulfate concentration. The effect of ammonium sulfate in concentrations of 10% and 15% suggested stabilization or decrease in L-asparaginase activity. However, as per as statistical analysis, no significant difference in L-asparaginase activities was identified for the different ammonium sulfate concentrations used in this study.

Discussion
Filamentous fungi are producers of a range of primary and secondary metabolites; therefore, they are commercially exploited as cell sources for the production of a wide variety of enzymes. These high productivity characteristics of filamentous fungi are related to their abilities to grow at high rates and to high biomass densities  The results are presented as mean of enzyme activities with standard deviation for each run (n = 9). *9% ≠ 3% and 7% of the proline concentration (p < 0.05). 7% vs 9% (p = 0.0004); 3% vs 9% (p < 0.0001).  15 reported that only two species have been deposited in a culture collection center, A. terreus MTCC 1782 strain and R. miehei CAU432. The molecular identification of a fungus was mentioned in one study, which species was identified based on 18S rRNA sequence analysis. The production of L-asparaginase by the fungus Penicillium cerradense, which was identified in this study as a new fungus species belonging to the genus Penicillium, represents an unprecedented work.
Besides that, the analysis of the results obtained by PBD and FFD made it possible to identify L-proline and ammonium sulfate substrates as significant independent variables with positive effect on L-asparaginase production by Penicillium cerradense. In similar studies investigating the culture medium components with filamentous fungi A. terreus, the variables L-proline and ammonium sulfate also had positive effects on L-asparaginase enzyme production after optimization of culture media 24 . Dias and Sato 25 , working with another species of the genus Aspergillus built a Plackett-Burman design for selection of significant variables and obtained a result where temperature, inoculum concentration, and pH of the culture medium presented a significant and positive effect for the proposed model.
In a strategy for optimizing L-asparaginase production by P. cyclopium applying experimental design, during the initial design phase it was identified that ammonium sulfate had a significant positive effect, in addition to the variables MgSO 4 .7H 2 O and KCl 26 . In a study with A. terreus 27 , in the analysis of the effects of nitrogen sources on the production of L-asparaginase, higher enzymatic activities were identified in the presence of ammonium sulfate substrate in solid culture medium, and the largest yield was obtained by supplementing sucrose (1%), ammonium sulfate (1%), NaCl (1%) and L-asparagine (1%). It was observed that L-proline and ammonium sulfate positively affect L-asparaginase production, however enzyme activity is inhibited at high concentrations of these substrates. In an optimization study of L-asparaginase production by Saccharomyces cerevisiae, Lang et al. (1997) verified the amount of nitrogen source in the medium and discussed the effect of undesirable increase of ammonium ions in cellular metabolism. The excess of ammonia molecules can lead to catabolic nitrogen repression in cells 28 .
Among the statistical designs found in the literature, L-asparagine and L-proline were the amino acids most used as nitrogen source as well as substrate inducer for asparaginase production, these results could be founded in the studies 12,25,26,[29][30][31][32][33] .
Phylogenetic analyses combined with morphological comparisons revealed a new species of Penicillium, P. cerradense sp. nov., isolated from the soil of the Cerrado and with potential for L-asparaginase production. The L-asparaginase gene was sequenced and this identification in eukaryotic sources is important in an effort to find new biopharmaceuticals with fewer side effects for leukemia treatment. The substrates L-proline and ammonium sulfate were found to have positive effects on the production of L-asparaginase by the fungus.

Methods
Fungal strain. Based on work previously developed by the research group at the Laboratory of Quality Control and Natural Products-Faculty of Health Sciences, University of Brasília, Brasília, Brazil-the production of L-Asparaginase by filamentous fungi obtained from soil and plant species of the Cerrado biome was addressed. A former screening of fungi revealed that the isolates addressed in this study have shown potential to produce L-asparaginase (personal communication).
The two isolates were obtained from soil samples of the Cerrado (14°58′10.21″S, 48°1′19.43″W) collected in Água Fria de Goiás city, Goiás state, Brazil. A hyphal-tip culture of each isolate was obtained on Potato Dextrose Agar (PDA) and were deposited within the scope of the SisBiota Brasil (National System of Research in DNA extraction, PCR amplification and sequencing. Genomic DNA was extracted from a pure culture originally grown on PDA at 25 ± 1ºC for 7 days. Fungal mycelium was scraped from colony margins and DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). Polymerase Chain Reaction (PCR) was performed with partial sequences of the nuclear 18S-5.8S-28S partial nrDNA, including the ITS1 and ITS2 regions (ITS), with the primers V9G 34 and LR5 35 ; RNA polymerase II subunit 2 (RPB2) with the primers 5F2 36 and 7Cr 37 ; β-tubulin (TUB) with the primers T1 38 and Bt2b 39 , and calmodulin (CAL) using primers Cal228F 40 and Cal2Rd 41 . Amplification was performed with an initial denaturation of 95ºC for 1 min and 30 s, followed by 35 cycles of 95ºC for 20 s, annealing at 53ºC (for ITS), 54ºC (RPB2), 55ºC (β-tubulin) or 59ºC (calmodulin) for 45 s, initial extension at 72ºC for 45 s and at 72ºC for 5 min on final extension. PCR products were analyzed in 1% agarose electrophoresis gels stained with GelRed (Biotium Inc., Hayward, CA, USA) in a TAE 1X buffer and visualized under UV light to check for amplification size and purity. The PCR products were purified and sequenced by Macrogen (South Korea). For whole genome sequencing, total genomic DNA was extracted from mycelia grown under submerged fermentation for 3 days at 30 °C. DNA concentration and quality were determined by Nanodrop ND-1000 spectrometer and agarose electrophoresis gel. The genome of isolate DCFS6a was sequenced with the Illumina HiSeq 2500 platform (StabVida, Lisbon, Portugal).

Phylogenetic analysis.
The new sequences were assembled and manually edited using Geneious v.8.1.9 (https:// www. genei ous. com/). To determine the Penicillium section which they shared the highest nucleotide identity with, the partial nucleotide sequences and the BLASTn algorithm were used to search the NCBI-Gen-Bank non-redundant nucleotide database. For species identification, a concatenate tree was reconstructed using the ITS and RPB2 sequences from deposited in the GenBank 16 L-asparaginase activity assay. In most of the microorganisms, L-asparaginase accumulates as an intracellular (periplasmic, cytoplasmic and membrane bound) product 49 . The L-asparaginase was determined by the formation of L-aspartic acid β-hydroxamate (AHA) from asparagine and hydroxylamine 50 with modifications. According to this method, a periplasmic activity of L-asparaginase can be quantified directly in the whole cell without previous extraction 51 . For analysis, the culture media were filtrated on Whattman #2 filter paper and the cells were washed twice with buffer Tris-HCl buffer (50 mM) pH 8.6 and used as samples. The reaction mixture was: 1.5 mL Tris-HCl buffer (50 mM) pH 8.6, 0.2 mL of L-asparagine solution (100 mM), 0.2 mL hydroxylamine solution (1 M), and 0.1 g biomass. After incubation at 37 °C for 30 min in a temperature-controlled bath, the reaction was ended by the addition of 0.5 mL of ferric chloride reagent 5.0% (w/w) FeCl 3 , 2.5% (w/v) TCA, and 0.33 M HCl. The reaction between the AHA and FeCl 3 led to a brown coloration that could be quantified by absorbance (500 nm). Sample blank was performed without the addition of L-asparagine and hydroxylamine hydrochloride solutions and all analyses were performed in triplicates. The results are presented as distribution of enzyme activities. The calibration curve was performed through multiple dilutions of a 5 mM AHA stock solution and the addition of appropriate amounts of FeCl 3 /TCA/HCl solution, ranging from 0.01 to 3.0 µmol of ferric AHA mL −1 . A unit of L-asparaginase activity (U/g cell ) corresponds to 1 µmol of AHA produced per minute per gram of sample cells.
Production culture for screening of variables. Submerse  www.nature.com/scientificreports/ disk of the mycelium of the fungus was deposited in the autoclaved media and kept in a temperature controlled orbital shaker at 30 °C and 120 rpm for 4 days. The culture media were filtrated on Whattman #2 filter paper and the cells were washed twice with buffer Tris-HCl buffer (50 mM) pH 8.6 and used as samples.

Evaluation of variables effect with statistical designs. Plackett-Burman experimental design
(PBD). The independent variables such as L-asparagine, L-proline, urea, sodium nitrate, yeast extract, ammonium sulfate, peptone, glucose, sucrose, malt extract and potassium chloride were considered to evaluate their effect on L-asparaginase production by Penicillium cerradense isolate DCFS6a. The culture media were prepared based on PBD 52 given in Table 5. The different factors were prepared in two levels: (− 1) for low and (+ 1) for high level. Eleven independent variables were screened in 16 combinations plus a triplicate of the central point, totaling 19 runs organized from the type matrix 2 K , K factors at 2 levels, considering the number of runs with n = 4t, where t is an integer. The matrix was built considering the minimum number of 4 trials more than the number of variables under study, allowing a degree of freedom to calculate the standard error 18 . To determine the significant effects, following the screening objective of the PBD, the fixed significance level was 10% (p < 0.1), minimizing the risk of excluding some important factor for the next step of the process 12 . However, the variables with positive effect in L-asparaginase production were fixed at a high level and those variables that had a negative effect were excluded to follow with FFD. This model (PBD) could provide us with initial indications of how each variable tends to influence L-asparaginase production, regardless the significancy of the variable 18 . The experimental design matrix and determination of the main effects were established by Protimiza Experimental Design software (https:// exper iment al-design. proti miza. com. br/).
Fractional factorial design (FFD). From the results of PBD, the independent variables with positive effect in L-asparaginase production (L-proline, peptone, sucrose, urea, ammonium sulfate, yeast extract, glucose and L-asparagine) were selected, regardless the significancy of the variable 18 , and the culture media were prepared to the FFD ( Table 6). The different factors were prepared in two levels: (− 1) for low and (+ 1) for high level. The FFD matrix was determined in a 2  factorial design, generating 16 combinations of the 8 variables and 4 replicates of the central point, totaling 20 runs. To determine the significant effects, following the objective of the FFD, the fixed significance level was 5% (p < 0.05). The experimental design matrix and analysis of variance were established by Protimiza Experimental Design software (https:// exper iment al-design. proti miza. com. br/). Table 5. Levels of the independent variables of the PBD experimental design to identify the influential factors on L-asparaginase production.  Table 6. Levels of the independent variables of the FFD to identify the influential factors on L-asparaginase production.