Penicillium Janthinellum NCIM-1366 Shows Improved Biomass Hydrolysis Performance Over Trichoderma Reesei RUT-C30, and Secretome Analysis Reveals a Larger Number of CAZymes with Higher Induction Levels.

Major cost of bioethanol is attributed to enzymes employed in biomass hydrolysis. Lignocellulolytic enzymes are predominantly produced from the hyper cellulolytic mutant lamentous fungus Trichoderma reesei RUT-C30. Several decades of research have failed to provide an industrial grade organism producing higher titers of an effective synergistic biomass hydrolyzing enzyme cocktail. Penicillium janthinellum NCIM1366 was reported as a cellulase hyper producer and a potential alternative to T. reesei, but a comparison of their hydrolytic performance was seldom attempted. Results Hydrolysis of acid or alkali pretreated rice straw using cellulase enzyme preparations from P. janthinellum and T. reesei indicated 37 and 43 % higher glucose release respectively with P. janthinellum enzymes. A comparison of these fungi with respect to their secreted enzymes indicated that the crude enzyme preparation from P. janthinellum showed 28 % higher overall cellulase activity. It also had an exceptional 10-fold higher beta-glucosidase activity compared to that of T. reesei, leading to a lower cellobiose accumulation and thus alleviating the feedback inhibition. P. janthinellum secreted more number of proteins to the extracellular medium whose total concentration was 1.8 fold higher than T. reesei. Secretome analyses of the two fungi revealed more number of CAZymes and a higher relative abundance of cellulases upon cellulose induction in the fungus. The results revealed the ability P. janthinellum for ecient biomass degradation through hyper cellulase production, and it outperformed the established industrial cellulase producer T. reesei in the hydrolysis experiments. A higher level of induction, larger number of secreted CAZymes and a high relative proportion of BGL to cellulases could be the possible reasons for its performance advantage in biomass hydrolysis.

not been performed. In this paper, the cellulase production and hydrolysis e ciency of P. janthinellum NCIM1366 was compared with T. reesei RUT-C30. Secretome analyses performed using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) revealed higher number of CAZymes in P. janthinellum compared to T. reesei, and a higher relative abundance of cellulases upon induction using cellulose, which may explain the higher activity and better biomass hydrolytic performance of enzyme preparation from the fungus. Results 1. Cellulases from janthinellum perform better than T. reesei cellulases in the hydrolysis of rice straw.
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 signi cantly higher glucose release (Fig. 1). 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.

janthinellum produces higher enzyme titers compared to T. reesei
To know how each of the major components of cellulolytic system contribute to the hydrolytic e ciency 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 10 th day, but the FPAse activity of P. janthinellum (0.83 FPU/ml) was 28 % higher than that of T. reesei (0.65 FPU/ml) (Fig 2A). Peak endoglucanase activity of 18.17 IU/ml was shown by P. janthinellum on the 10 th day, whereas T. reesei showed an almost identical activity (17.1 IU/ml), but at 6 th day ( Fig 2B). However, the endo glucanase activity levels 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 (12.8 IU/ml) which steadily increased to 18.17 IU/ml on 10 th 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 IU/ml. P. janthinellum also showed the highest BGL activity (95.42 IU/ml) on the 10 th day ( Fig 2C). Also, the fungus produced 24.88 IU/ml activity on the 2 nd day, where T. reesei could elaborate only 1.68 IU/ml. These results are remarkable as the beta-glucosidase activity at peak levels by the two fungi is different by an almost 10fold 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 con rmed by a Zymogram analysis which showed a prominent BGL activity band in P. janthinellum, whereas the T. reesei BGL band was barely visible (Fig 2D).
3. 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 [10,11]. 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 (Fig 3). 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 4 th 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 e cient 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.

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 e ciency of the fungus in utilizing the polymer, as e cient 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 6 th day of growth, whereas P. janthinellum secreted the maximum protein on 10 th day of growth which was ~1.8times higher than T. reesei (Fig 4A). 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 signi cant elevation in secreted proteins upon cellulose induction ( Fig 4B). It was also observed that visibly, a greater number of extracellular proteins were secreted by P. janthinellum.

5.
Comparative secretome analysis of cellulose induced cultures con rm 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 lter paper activity on 10 th 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 10 th day of growth, were identi ed 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 10 th 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 identi cation 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 [12]. 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 e ciency 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 identi ed as CAZymes ( Figure 5A) and the number of CAZymes identi ed in the 85 identi ed 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 identi ed 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 identi ed from T. reesei. In P. janthinellum, 10 cellobiohydrolases and 2 endoglucanases were identi ed 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 identi ed in both the fungi. There was therefore no way to con rm if the higher BGL activity obtained experimentally for P. janthinellum corresponded to a higher amount of the corresponding protein in the secretome. It was previously observed that the BGL proteins had high speci c activities and minute quantities can give high hydrolytic e ciencies, even though their proteins were undetectable by conventional means.
There were 9 enzymes involved hemicellulose degradation identi ed from the secretome of T. reesei, while 6 were identi ed in P. janthinellum of which, 2 were common with T. reesei. Chitin degrading enzymes were also identi ed from both secretomes but pectin degrading enzymes were identi ed only in P. janthinellum. Accessory activities known to aid cellulose hydrolysis in T. reesei, Swollenin (A0A024RZP7) and Lytic Polysaccharide Monooxygenase (A0A024SM10) were identi ed 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 con rmations from the genome analysis is awaited.

Discussion
Lignocellulose degrading enzymes are critical in biomass conversion to biofuels and lamentous fungi are typically used for the production of these enzymes because of their ability to synthesize and secrete a wide array of plant cell wall degrading enzymes [13]. T. reesei RUT-C30 is the most widely used fungus for cellulase production, in the past several decades, and efforts for improving its enzyme production are still at large [14,15]. P. janthinellum NCIM 1366 is a mutant strain developed at the National Chemical Laboratory, Pune, India through classical mutagenesis and which exhibited enhanced cellulase production compared to the parent strain NCIM 1171 [9]. The extracellular enzyme preparation from the strain was found in this study to be more e cient than the T. reesei enzyme in the hydrolysis of pretreated rice straw. The cellulase system of the fungus is relatively unexplored and the present study aimed to study the cellulases from P. janthinellum and compare it with established cellulase hyper producer Trichoderma reesei RUT-C30.
Experiments were designed to assess the e ciencies of both cellulase preparations for the hydrolysis of rice straw, pretreated using the most common methods of dilute acid or dilute alkali treatment at high temperature. The enzyme from P. janthinellum hydrolyzed the pretreated biomass better, indicated by the higher glucose release. Interestingly, the glucose release from acid and alkali pretreated biomasses was respectively 37 % and 43 % higher compared to T. reesei. The results were surprising for a "new" cellulase producer to outperform the established industrial producer. Hence the extracellular enzyme preparations from both fungi were analyzed for their major component activities. These included endoglucanases (EGs), cellobiohydrolases (CBHs), and β-glucosidases (BGLs), which act in synergism to hydrolyze cellulose [16]. Since the parameters like media composition, pH, carbon source used etc., can in uence the quantity and variety of the cellulase components produced by fungi [17], the basic mineral salts medium of Mandels and Weber [18] with cellulose as the sole carbon source was used for both the organisms to obtain un-biased data. The results showed higher activity for all the three major components with peak activity on 10 th day of incubation. Among the enzymes, the largest difference in activity was observed for beta glucosidase (BGL), which was 10 fold higher in P. janthinellum. It is already recognized that the extracellular enzymes of T. reesei strains are limited in BGL activity for effective biomass hydrolysis [19]. Thus it may be speculated that the higher BGL activity may be one of the signi cant factors which can contribute to the higher hydrolytic e ciency shown by P. janthinellum cellulase. This was also supported by the fact that the hydrolysis using T. reesei cellulase accumulated higher concentration of cellobiose in the medium, indicative of an incomplete hydrolysis. Thus, unlike T.reesei enzyme which has to be supplemented with external BGL for hydrolysis reaction [20], P. janthinellum enzyme preparations may be used without the need for any blending or with minimal addition of synergistic BGL preparation (s). In addition to the major cellulases, the total extracellular proteins were also 1.8 times higher for P. janthinellum and the qualitative analysis by SDS-PAGE showed more number of proteins in the gel complementing this nding.
Proteomic approaches have been widely used in lamentous fungi for the identi cation of both intracellular and extracellular proteins [21]. The genome of T. reesei QM6a, which is the parent strain of RUT-C30 was rst sequenced in 2008 giving insight into its CAZyme system [22]. T. reesei is known to encode at least 10 cellulases, 16 hemicellulases and a total of around 400 CAZymes in its genome. But the composition of secretome varies depending on the carbon source used, culture conditions or experimental parameters. The rst proteome analysis of T. reesei RUT-C30 identi ed a total of 22 proteins using lactose as carbon source [23]. Another study, using different carbon sources identi ed 230 extracellular proteins and 90 CAZymes [24]. In the present study using a minimal mineral salt medium under identical conditions, a total of 53 proteins were identi ed from T. reesei secretome, while P. janthinellum secreted 85 different proteins. As expected, most of the proteins identi ed from both the fungi were related to biomass degradation. More number of CAZymes was identi ed from P. janthinellum secretome. CAZymes from T. reesei included 2 cellobiohydrolases, 3 endoglucanases, 9 hemicellulases and the accessory activities -Swollenin and Lytic Polysaccharide monooxygenase (LPMO). CAZymes from P. janthinellum were grouped into 12 cellobiohydrolases, 3 endoglucanases, and 6 hemicellulases. No beta glucosidases were identi ed from both secretomes to support the extremely higher beta glucosidase activity shown by P. janthinellum. However, the number of cellobiohydrolases and their relative abundance was very high in P. janthinellum. The proteins identi ed from the secretome may not be a complete representation of all the CAZymes secreted by the organism, as the study used only a single time point and pure cellulose as sole carbon source. The highest differentially expressed protein from T. reesei was the GH7 family endoglucanase EG-1, which showed 183567-fold increase in expression upon cellulose induction. However, CBH1 is known to be the major secreted protein of T. reesei on cellulose induction [25]. The difference in this study might be a result of the culture conditions and/or the time point of analysis. It could also result from the processing of samples where the insoluble cellulose fraction, which could bind the enzyme, was removed to obtain the supernatant used for analyses. The normalized fold difference shown by the most highly expressed protein from P. janthinellum was almost 2 million, and this was a cellobiohydrolase from GH7 family. While T. reesei secreted a wider variety of enzymes involved in lignocellulose hydrolysis, it was the P. janthinellum that secreted more glycosyl hydrolases and especially very high level of exoglucanases. The study provides preliminary information on the presence of all major cellulolytic and hemicelluloytic activities in the fungus and a very high induction in presence of cellulose, which could account for its enhanced hydrolytic performance.

Conclusions
Here, we provide the rst ever secretome analysis of Penicillium janthinellumNCIM1366 and its comparison with the established cellulase hyper producing industrial strain -Trichoderma reesei RUT-C30. The analyses have highlighted the better hydrolytic e ciency, enzyme activity, protein production and secretion e ciency of P. janthinellum, which indicates its potential as future industrial cellulase producer. Further exploration and a deeper understanding on the reasons of its better cellulase production warrants genome and transcriptome level studies on the fungus which is progressing and we aim to reveal soon. Further targeted genetic improvements is expected to improve its performance even more, providing a worthy alterative for T. reesei, or complement it in the cellulase applications for biomass conversion.  The carbon sources used were 0.1 % Lactose, 2% cellulose and 1.5 % Wheat bran and the medium was inoculated at 1% (v/v) level with a 1×10 6 spores/ml spore suspension. Cultivation was carried out at 30 ± 2 C and 200 rpm agitation. The extracellular crude enzyme from both cultures was collected after 10 days of incubation, and was assayed for total cellulase activity. Enzyme activity was expressed in Filter Paper Units (FPU).
Rice straw, pretreated using either dilute acid or alkali, was used for hydrolysis studies. For acid pretreatment, rice straw (20% w/v) was mixed with 10% w/w of H 2 SO 4 and was pretreated for 1h at 120 ± 2 °C. The biomass was cooled to room temperature and a slurry was made by adding 2× volume of water. The pH of the slurry was adjusted to 6.0 by adding 10N NaOH. Solid-liquid separation was performed using a nylon sieve and the biomass was washed twice with tap water. The biomass was used directly after correction of moisture or air dried at room temperature and stored until used. For alkali pretreatment, 20% w/v rice straw and 10% w/w NaOH was mixed and pretreated at 120 ± 2 °C for 1h. Water (2× volume) was added and the pH of the pretreated slurry was adjusted to 6 by adding 10N H 2 SO 4 .The biomass was then processed as described above.
The hydrolysis reaction was carried out in a volume of 20 ml in screw capped glass conical asks with the following conditions: 10 % w/w of dry biomass, 10 FPUs /g enzyme loading and 0.05 % w/w surfactant (Tween 80) loading. 0.5 % v/v of a commercial Penicillin /Streptomycin mixture (Himedia, India) was added to prevent any bacterial contamination. The hydrolysis reaction was carried out at 50 C for 24 h and samples were collected at 0, 4, 8, 12 and 24 hours of hydrolysis. The amount of glucose and cellobiose in the samples was determined using the HPLC as described previously [26].
c. Comparison of total cellulase activity, endoglucanase activity, and beta-glucosidase activity Both organisms were cultivated in the basic Mandels and Weber [18]  Cellulose (1% w/v) was used as the sole carbon source. The inoculum size used was 1% v/v of a 1×10 5 spores/ml suspension for both the cultures. Cultivation was carried out at 30 ± 2 C and 200 rpm agitation. Samples were collected from 2 nd day onwards, every 48h. Total cellulase activity (Filter Paper Activity) and endoglucanase (CMCase) activity was determined by the IUPAC method [27] and beta-glucosidase activity was determined as described by Rajasree et al [28].d. Zymogram analysis of extracellular proteins Native Poly-Acrylamide Gel Electrophoresis (PAGE) of the extracellular enzyme and Methyl umbelliferyl β-D-glucopyranoside (MUG) staining was performed as described by Rajasree et al. [28].
e. Comparison of extracellular protein production and secretome analysis Both organisms were grown in basic Mandels and Weber [18]medium with either Glucose or cellulose at 1% (w/v) level as sole carbon sources, as described above. Samples were collected at 48h intervals starting from 2 nd day. Total secreted proteins present in the samples were estimated by the Bradford method [29] with BSA as standard. SDS-PAGE was carried out as described by Laemmli [30], using 12% and 5% acrylamide for the resolving and the stacking gels, respectively. Proteins were visualized by staining with Coomassie Brilliant Blue R-250.
For secretome analyses, extracellular proteins were collected by centrifugation for 10 min at 4°C and 13400g after 10 days of incubation. The supernatants were further clari ed by ltration through 1 Glass micro ber lter. The collected proteins were dialyzed against 50 mM ammonium bicarbonate buffer and concentrations were normalized. Samples were digested using trypsin following the standard protocol [31]. The proteomic pro ling was performed in duplicates by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) at the Mass Spectrometry & Proteomics Core facility of Rajiv Gandhi Centre for Biotechnology, Trivandrum, India. The protein samples were subjected to in-solution trypsin digestion using sequence grade Trypsin (Sigma Aldrich, India). The LC/MS/MS analyses of the tryptic peptides were performed in a SYNAPT G2 High De nition Mass Spectrometer (Waters, UK), connected to a NanoACQUITY UPLC® chromatographic system (Waters, UK) for the separation of the peptides. The LC-MS/MS acquired raw data was analyzed by Progenesis QI for Proteomics V3.0 (NonLinear Dynamics, Waters, UK) for protein identi cation using the protein database of Trichoderma reesei and Penicillium downloaded from UniProt repository. Prediction of the presence of secretion signal motifs was achieved using SignalP 5.0 [32].
f. Identi cation of CAZymes from secretome data Annotation of CAZy (Carbohydrate Active Enzymes) family of proteins and as per CAZy database [33] was done through dbCAN2 meta server [34].

Declarations
Ethics approval: This article does not contain any studies with human participants or animals performed by any of the authors Consent to publish: RKS, the corresponding author, declare on behalf of all authors that we do not have data pertaining to individuals Availability of data and materials: All essential data generated or analyzed during this study are included in this published article and its supplementary information les. More elaborate datasets generated during and/or analysed during the current study available from the corresponding author on reasonable request.
Competing interests: The authors declare that they have no competing interests Author's Contributions: ASR -Performed most of the experimental work and protein studies, MC, PKV and AA did data analyses and categorization, RKP did initiated the original studies on cellulase production from Pj and Tr and established protocols, DVG originally isolated the Pj culture, did classical mutations and also corrected manuscript, MS and AA did biomass pretreatment and hydrolysis of biomass, AP was involved in experiment planning and manuscript corrections, RKS conceptualized the work, planned experiments and work distribution, did data interpretation and graphing, drafted manuscript and performed corrections and coordinated the entire work.  Cellobiose accumulation during hydrolysis of biomass using T. reesei or P. janthinellum enzymes A) Acid pretreated rice straw B) Alkali pretreated rice straw Extracellular protein production by T. reesei and P. janthinellum in response to induction by cellulose A) Extracellular protein concentrations in T. reesei (Tr) and P. janthinellum (Pj) at different time points when cultivated in glucose and in cellulose B) SDS-PAGE showing differences in protein production by the two fungi. Lane Information -1) Page Ruler ® pre-stained protein ladder, 2) Tr grown in glucose 3) Tr. grown in cellulose as sole C source 4) Pj grown in glucose 5) Pj. grown in cellulose as sole C source.

Figure 5
Functional categories of proteins secreted on cellulose induction by P. janthinellum and T. reesei Legend: A) P. janthinellum B) T. reesei Figure 6 Most highly expressed proteins in the secretome of P. janthinellum and T. reesei, on cellulose induction Legend A) P. janthinellum B) T. reesei