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

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).

Taxonomy

Penicillium cerradense sp. nov. Cruvinel, Magalhães, P. O., Pinho. (Fig. 2).

MycoBank: MB 835,241.

GenBank: ITS = MT006126, RPB2 = MT416532, TUB = MT416533, CAL = MT416534, L-ASNase = MT742156.

Systematic position: Ascomycota, Pezizomycotina, Eurotiomycetes, Eurotiomycetidae, Eurotiales, Aspergillaceae.

Type: —BRAZIL, Goiás, Água Fria de Goiás. 14°58′10.21′′S, 48°1′19.43″W, on soil of Cerrado, 30 January 2009, coll. F. G. Siqueira (holotype in dried culture UB23977, ex-type DCFS6a).

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 25.0‒240.0 × 2.0‒3.0 μm (average 85.0 × 2.5 μm; Fig. 2B‒D). The penicillated conidiogenic apparatus measured 7.0‒16.0 μm in length and 4.0‒14.0 μm in width. Conidiophore were predominantly monoverticillate, when biverticillate presented 2 branches with 10.0‒20.0 × 2.0‒3.0 μm (average 13 × 2.5 μm), each branch finished in the production of 2‒4 (− 8) phialides. Ampulliform phialides was predominantly in four, hyaline, aseptate, 5.0‒8.0 × 2.0‒3.0 μm (average 7.0 × 2.5 μm; Fig. 2E‒F). Conidia catenulate, subglobose or strongly ellipsoidal, hyaline, smooth with 1.5‒3.0 × 2.0‒3.0 μm (average 2.5 × 2.5 μm; Fig. 2G‒H). Globose sclerotia, solitary to abundant, superficial or immersed in the culture medium, pigmented, pale brownish to brownish with 150.0‒340.0 × 130.0‒320.0 μm (average 250 × 200 μm; Fig. 2I). Sexual form not observed.

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).

Additional specimen examined: BRAZIL, Goiás, Água Fria de Goiás. 14°58′10.21″S, 48°1′19.43″W, on soil of Cerrado, 30 January 2009, coll. F. G. Siqueira (culture DCFS6b).

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.

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 production¹⁷ and it is convenient especially when facing large number of factors that can potentially influence optimal or near optimum responses¹⁸. This design is recommended when more than eight factors are under investigation¹⁸. 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. The significant variables (p < 0.1) for L-asparaginase activity were identified as L-proline (p = 0.0395) and potassium chloride (p = 0.0768).

The experiment was conducted for the eight variables which presented positive effect. The results obtained from FFD showed a wide range of L-asparaginase activity, from 1.10 to 2.36 U/g_cell. The maximum L-asparaginase activity was achieved in run number 10 with culture medium containing L-proline 5.0%, peptone 2.0%, sucrose 0.2%, urea 2.0%, ammonium sulfate 2.0%, yeast extract 1.5%, glucose 1.0%, L-asparagine 3.0% (Table 3). Statistical analysis of the response was performed, and the results are presented in Table 4. The variables with positive effect such as L-proline, ammonium sulfate and L-asparagine induced the highest level of L-asparaginase production, whereas the variables with negative effect (peptone, sucrose, urea, yeast extract and glucose) decreased the activity. The significant variable (p < 0.05) identified was ammonium sulfate (p = 0.02). After 20 runs, L-asparaginase activity was calculated using the proposed model equation: Prediction L-asparaginase activity = 1.92 + 0.00375X1 − 0.045X2 − 0.31X3 − 0.042X4 + 0.29X5 − 0.15X6 − 0.17X7 + 0.04X8. Level of significance of 95%. R² = 0.5894 and predict R² = − 0.31. A negative predict R² implies that the overall mean is a better predictor 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.