- janthinellum secretes higher amount of proteins compared to T. reesei
Cellulose is a large polymer and utilization of it requires secretion of enzyme by microorganisms to process it outside the cell, so that the simple sugars derived from its breakdown can be taken inside. The total secreted protein concentration in presence of cellulose is indicative of the efficiency of the fungus in utilizing the polymer, as efficient cellulose digestion typically requires a milieu of different enzyme activities, in addition to cellulases. T. reesei showed a maximum protein secretion of 0.28 mg/ml on the 6th day of growth, whereas P. janthinellum secreted the maximum protein on 10th day of growth which was ~1.8times higher than T. reesei (Figure 1A). At all the time points tested, extracellular protein concentration was higher in P. janthinellum. SDS PAGE of the extracellular fractions from both fungi under un-induced (glucose grown) and induced (cellulose grown) conditions indicated significant elevation in secreted proteins upon cellulose induction (Figure 1B). It was also observed that visibly, a greater number of extracellular proteins were secreted by P. janthinellum.
- janthinellum shows lesser cellobiose accumulation in the hydrolysis medium which is indicative of its better beta-glucosidase activity
Cellobiose, the intermediate product of enzymatic cellulose hydrolysis, is produced through action of exoglucanases and is the substrate for cellobiase/beta-glucosidase. Cellobiose accumulation can lead to product inhibition of upstream enzymes (endoglucanases and cellobiohydrolases) thus slowing down the whole hydrolytic process [15,16]. T. reesei is known to have limited cellobiase/beta glucosidase (BGL) activity, and while it may be advantageous for the organism in tight regulation of cellulose metabolism while growing on natural substrates, it is a disadvantage for the biomass hydrolyzing enzyme cocktails produced using the fungus and often the T. reesei enzyme’s lack of BGL activity is compensated by addition of BGL enzyme from other organisms. In this study, it was observed that the cellobiose accumulation in the hydrolysis mixtures was higher for the T. reesei enzyme compared to P. janthinellum, indicating an incomplete digestion due to the inherent low BGL activity of the former (Figure 2). For acid pretreated rice straw, there was little or no cellobiose accumulation observed during the 24h of hydrolysis, in the case of P. janthinellum enzyme, while about 5 mg/ml cellobiose was observed consistently from 4th hour onwards in the case of T. reesei. In alkali pretreated rice straw hydrolysis, the cellobiose concentration increased from 2.42 mg/ml in 4h to 7.45 mg/ml in 24h for T. reesei while the maximum cellobiose accumulation in the case of P. janthinellum was only 0.76mg/ml at 12h after which it again decreased. This indicated the efficient removal of cellobiose, from the reaction medium by P. janthinellum enzyme, which could be accounted for by the almost 10-fold higher beta-glucosidase activity in the fungus. The results are an indicative of an optimum enzyme cocktail from a single fungus that outperforms the conventional cellulase producer.
- Cellulases from janthinellum perform better than T. reesei cellulases in the hydrolysis of pretreated biomass
Both the dilute acid and dilute alkali pretreated rice straw were hydrolyzed better by P. janthinellum cellulases compared to enzymes from T. reesei, indicated by a significantly higher glucose release (Figure 3A and Figure 3B). At 24h, glucose release by T. reesei and P. janthinellum cellulases from acid pretreated rice straw were 12.94 ± 0.8 mg/ml and 17.69 ± 0.47 mg/ml respectively, the latter showing a 37 % higher glucose release. Similar results were observed for alkali pretreated rice straw, where P. janthinellum enzyme released 27.24 ± 0.22 mg/ml of glucose which was 43 % higher than the T. reesei cellulase. Also, the glucose release for both the acid and alkali pretreated biomasses was higher with P. janthinellum cellulase at all the measured time points. Total sugar release from acid pretreated rice straw, calculated as the sum of the concentrations of glucose, xylose, arabinose and mannose in the hydrolysis mixture, was 17.88±0.5 mg/ml for T. reesei enzyme and 22.41 ±0.23 mg/ml for P. janthinellum enzyme. For alkali pretreated rice straw, the hydrolysate sugar contents were 29.49±0.57 mg/ml and 32.94±0.87 mg/ml respectively for T. reesei and P. janthinellum enzymes (Figure 3A and Figure 3B). P. janthinellum enzyme released 25% higher glucose and 11% higher total sugars from alkali pretreated rice straw sugars compared to T. reesei enzyme.
The choice of substrate for the hydrolysis reaction often affects the efficiency of glucose release, and enzyme performance could be different on different biomass substrates. Two different biomasses with significantly different properties, viz. sugar cane bagasse and Eucalyptus leaves pretreated using acid or alkali, were used as substrates for testing hydrolytic efficiency of enzymes from both the fungi. Both the biomasses, regardless of the method of pretreatment were hydrolyzed better by P. janthinellum enzyme, indicated by higher glucose release. For acid pretreated sugarcane bagasse, the glucose release at 24 h was 24.19±0.34 for P. janthinellum enzyme and 15.54±0.76 for T. reesei enzyme, while in the case of alkali pretreated biomass, it was 22.16±0.35 and 17.87±0.91 respectively for P. janthinellum and T. reesei. Similar results were obtained for eucalyptus leaves, for which the glucose release across treatments were less compared to other biomass types (Figure 3C and Figure 3D).
- janthinellum produces higher enzyme titers compared to T. reesei
To know how each of the major components of cellulolytic system contribute to the hydrolytic efficiency of P. janthinellum cellulase cocktail, standard cellulase assays were performed, on the enzymes produced by the fungi. Extracellular enzyme production in this case was carried out using the same medium and under identical conditions of growth. Secreted enzymes from both fungi were analyzed for the total cellulase, endo-glucanase and beta-glucosidase activities. Both T. reesei and P. janthinellum showed maximum cellulase activity on the 10th day, but the FPAse activity of P. janthinellum (0.83 FPU/ml) was 28 % higher than that of T. reesei (0.65 FPU/ml) (Figure 4A). Peak endoglucanase activity of 21.72 IU/ml was shown by P. janthinellum on the 12th day, whereas T. reesei showed maximum activity (15.55 IU/ml) at 10th day (Figure 4B). T. reesei showed an endoglucanase activity of 12.93 IU/ml even at 6th day, but the levels were raised only upto 15.55 IU/ml on 10th day and were not sustained at further time points, probably indicating a feedback inhibition through glucose accumulation. P. janthinellum on the contrary, had a lower initial endo glucanase activity (9.55 IU/ml) which steadily increased to 21.72 IU/ml on 12th day and showed an ascending trend. The largest difference in enzyme activity between the two fungi was observed in the case of beta glucosidase (BGL) activity. Highest BGL activity in the case of T. reesei was 10.15 U/ml. P. janthinellum also showed the highest BGL activity (95.42 U/ml) on the 10th day (Figure 4C). Also, the fungus produced 24.88 U/ml activity on the 2nd day, where T. reesei could elaborate only 1.68 U/ml. These results are remarkable as the beta glucosidase activity at peak levels by the two fungi is different by an almost 10-fold margin. Also, it becomes evident that the expression of BGL sets in early in the P. janthinellum which would allow it to hydrolyze cellulose faster and prevent cellobiose accumulation, which in turn may help to overcome an early setting in of feedback inhibition. The results were also confirmed by a Zymogram analysis which showed a prominent BGL activity band in P. janthinellum, whereas the T. reesei BGL band was barely visible (Figure 4D).
- Comparative secretome analysis of cellulose induced cultures confirm secretion of a relatively larger number of CAZymes and lignocellulose active enzymes by janthinellum
The observed cellulase activity and hydrolysis activity are contributed by the extracellular enzymes in both organisms. As both cultures showed maximum filter paper activity on 10th day of inoculation in cellulose medium, it was speculated that the maximum repertoire of enzymes are secreted at that time point. For comparison glucose was selected as the non-inducing carbon source. The secreted proteins from both cultures, either grown with glucose as carbon source or upon induction with cellulose on the 10th day of growth, were identified and quantitatively analyzed by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) analysis. Supplementary Tables S1 and S2 lists all the proteins, their Uniprot accession number, molecular weights, number of unique peptides, normalized abundance in glucose and cellulose grown cultures and fold change of upon induction for both T. reesei RUT-C30 and P. janthinellum NCIM1366 cultures respectively. Our analysis detected a total of 53 proteins from T. reesei and 85 proteins from P. janthinellum in the 10th day secretome. The distribution of proteins according to their biological function is shown in Figure 5. Among them, 27 proteins from T. reesei (51 %) and 29 proteins from P. janthinellum (34 %) were predicted to have an N terminal signal peptide using SignalP5.0 server. The identification of proteins without a signal peptide in the secretome could be indicative of the presence of cell lysis, cell death, or secretion through unconventional mechanisms . Figure 6 shows the top 10 highly expressed proteins, as measured by the normalized abundance of their peptides in cellulose induced cultures, compared to the control (grown in glucose). Most of the highly expressed proteins from both organisms were directly involved in the lignocellulose degradation.
Since it appeared from the forgoing studies that the reason for better overall cellulolytic activity and hydrolytic efficiency of P. janthinellum could be its secretion of a larger number of proteins, most of which are known to be involved in lignocellulose hydrolysis, it was speculated that the organism could elaborate more cellulolytic enzymes and/or accessory proteins compared to the industrial workhorse - T. reesei. An analysis of the CAZymes in the total secretomes was performed to understand their distribution in the extracellular proteins of both fungi. Among the 53 secreted proteins detected in T. reesei, 20 were identified as CAZymes (Figure 5A) and the number of CAZymes identified in the 85 identified secreted proteins of P. Janthinellum were 27 (Figure 5B). The distribution of proteins among different CAZy families and the distribution of glycosyl hydrolase (GH) family proteins in both the fungi are shown in Figure 7. CAZymes from P. janthinellum were mostly GH family proteins except one in GT family. The CAZymes from T. reesei RUT-C30 were distributed to more CAZy families which included GH (Glycoside Hydrolases), CE (Carbohydrate Esterases), AA (Auxiliary Activities) and CBM (Carbohydrate Binding Module). In the case of GH family proteins, P. janthinellum secretome had almost double the number of different Glycoside Hydrolases compared to T. reesei. GH family proteins from P. janthinellum spanned over 12 GH subfamilies while for T. reesei it was 11 GH subfamilies. GH subfamilies 5, 6, 7 and 11 were detected in both secretomes while GH subfamilies 3, 4, 16, 17, 30 and 72 were detected only in T. reesei and GH subfamilies 2, 15, 27, 28, 36, 43, 55 and 75, were detected only in P. janthinellum.
Table 1 shows the list of CAZymes identified from the secretomes of P. janthinellum and T. reesei. Among the CAZymes detected, a total of 17 enzymes which are directly involved in cellulose hydrolysis were detected, of which 3 were common to both fungi, which were cellobiohydrolase1 (CBH1) (Uniprot accession: P62694, A0A088DLG0), cellobiohydrolase2 (CBH2) (P07987, F1CHI2) and endoglucanase 1 (EG-1) (A0A024SNB7, A0A0F7TSC9). The two cellobiohydrolases showed higher abundance in P. janthinellum and the endoglucanase showed higher abundance in T. reesei. Apart from the EG-1, two other endoglucanases, EG-II (P07982) and EG-V (A0A024S5P6), are identified from T. reesei. In P. janthinellum, 10 cellobiohydrolases and 2 endoglucanases were identified from the common cellulases. However, it may be noted that multiple peptide tags may be matching the same P. janthinellum NCIM 1366 gene sequence in reality, which may not be captured on analyzing against the genome (s) of other Penicillium species’ genomes as is the case here. While P. janthinellum exhibited 10 times the BGL activity of T. reesei, no beta glucosidases were identified in both the fungi. Therefore, there was no way to confirm if the higher BGL activity obtained experimentally for P. janthinellum correlates to a higher amount of the corresponding protein in the secretome. It was previously observed that the BGL proteins had high specific activities and minute quantities can give high hydrolytic efficiencies, even though their proteins were undetectable by conventional means.
There were 9 enzymes involved in hemicellulose degradation identified from the secretome of T. reesei, while 6 were identified in P. janthinellum, of which 2 were common with T. reesei. Chitin degrading enzymes were also identified from both secretomes but pectin degrading enzymes were identified only in P. janthinellum. Accessory activities known to aid cellulose hydrolysis in T. reesei, Swollenin (A0A024RZP7) and Lytic Polysaccharide Monooxygenase (A0A024SM10) were identified in the secretome of T. reesei, while these activities were not detected in P. janthinellum. In general, the relative abundance of most of the CAZymes were high in P. janthinellum compared to T. reesei and one of the cellobiohydrolases(A0A1Q5UFZ3) from GH7 family showed a very high relative abundance of 2.3 million. Though P. janthinellum did not show the accessory enzymes /activities in its secretome, it does not necessarily mean that the fungus lacks them, and further confirmations from the genome analysis is awaited.