Transcriptomic and secretomic integration

High throughput analysis such as transcriptomics and proteomics are important methodologies used to reveal a snap-shot view of alterations triggered by a specific cultivation condition. However, large-scale transcriptome surveys generally show only modest correlation between transcript expression and protein production as presented in Figure 10 (and additional file 9).

Figure 10. Transcriptome and secretome correlation. It was evaluated the overall secretome (A) and the CAZymes identified in the secretome (B). Numbers out of ovals indicate transcripts and its respective proteins that were not statistically significant considering the parameters described in Figure1 and Figure 7 captions.

Only small correlations among transcriptome and mycelial proteome were observed when N. crassa was grown on Avicel or in the absence of carbon source using a sucrose growth as control, the correlation values were 7 and 43% respectively [49]. In our analysis the correlation was 23% (Figure 10A) for the overall secretome, and although the methodologies utilized in this work were different from the cited results, it is evident that post-transcriptional, translational and protein degradation regulation contribute significativelly to protein abundance [49]. However these factors are less significant when CAZymes are the subjet, since the correlation for this group of proteins were 39% (Figure 10B).

We manually annotated the N. crassa genome CAZyme set taking into account the enzymes, characterized or not, described in CAZy database and the domain prediction performed by dbCAN (csbl.bmb.uga.edu/dbcan) [60]. The result was a genome with 401 predicted CAZymes of which almost all were visualized in our transcriptomic analysis, but only 188 were present in a statistical significant form. Zhao and collaborators showed that more than a half of the 103 fungi analyzed in they work posses more than 300 predicted CAZymes in their genomes [61], and although some families are described in all fungi their expression is highly variable, dependent of specie and growth conditions [61,62].

The data are presented dividing the enzymes in the GHs, GTs, CEs, AAs and the CBMs with no other assign domain predicted in the sequence. There are a great number of GHs and GTs that were not expressed in the conditions analyzed here (Figure 11A), however if the diversity of families were taken into account it is possible to notice that N. crassa reaches a considerable level of diverse enzyme production (Figure 11B). Interestingly, although present in the transcriptomic data, the only class that is not represented in the secretome analysis is the GT, probably because these enzymes are essentially intracellular and often membrane associated [63].

Figure 11. General vision of N.crassa CAZymes in ômics studies.

So far, it has been identified 131 proteins in N. crassa secretomes studies using Avicel, miscanthus, xylan from beechwood, pectin and orange peel powder [13,15,16]. Of them 23 are not among the 292 proteins described in this work, and most of them correspond to proteins induced exclusively by pectic substrates. Of the remaining 108 common proteins, the majority are CAZymes, although twenty were described as hypothetical. However, as we mannualy annotated the identified proteins this number decreased to five. Therefore, there are less hypothetical proteins in N. crassa secretome than generally described in literature, whereas among the transcripts this value still is high, reaching 25% considering Hide Markov Model prediction [49]. Even though proteomic surveys are considered under-representative in fungal research, they can provide direct information on proteins specie identity, location, posttranslational modifications [64], as exemplified by the hypothetical proteins.

Although the number of hypothetical proteins were lower than initially supposed, it is important to investigate their roles, as well as the case of the predicted proteins. Enzymes with auxiliary activities, important in carbohydrates oxidative degradation such as NCU08760 (AA9) and NCU08746 (AA13) were previously described as hypothetical proteins [13,20]. In this work it was highly produced a predicted AA3 (NCU09024) as shown in Table 5, so far only two AA3 were caracterized and only NCU00206 had been found in N. crassa lignocellulose secretomes [16,65] .

Table 5. Overview of identified AAs in the secretome.

TPM NORMALIZED SPECTRUM COUNTS

Gene Class AVICEL PASB RAW LIGNIN SUCROSE AVICEL PASB RAW LIGNIN SUCROSE

NCU03646 AA2 11 8 5 49 5 7 7 2 3 15

NCU05770a,b _{AA2 275 45 19 479 19 0 1 2 6 1}

NCU09024 AA3 124 111 106 47 18 22 29 29 5 0

NCU01395 a _{AA5 27 72 65 40 55 0 0 1 0 0}

NCU09267 a _{AA5 8 4 3 19 1 44 39 45 15 15}

NCU02948 AA6 328 334 333 1915 511 0 0 0 2 0

NCU01123b _{AA7 15 16 27 68 6 0 3 2 0 0}

NCU04108a,b _{AA7 8 163 197 84 11 0 14 13 12 0}

NCU09518 AA7 44 22 39 11 2 5 3 3 0 0

NCU00206a_{* AA8 1064 933 2080 8 2 86 125 66 0 0}

NCU00836a,b _{AA9 687 997 2630 13 1 1 13 20 0 0}

NCU01050 AA9 5198 2356 4274 2 1 8 13 9 0 0

NCU02240b _{AA9 2276 1930 3402 14 3 3 11 11 0 0}

NCU02916 a _{AA9 1465 1814 2805 46 14 2 4 3 0 0}

NCU07760a,b _{AA9 44 367 871 60 19 0 2 2 0 0}

NCU07898a,b _{AA9 3327 1053 3240 27 2 21 44 28 0 0}

NCU08760b _{AA9 3044 3209 4699 37 4 12 57 52 3 0}

NCU08746b _{AA13 61 58 41 43 8 4 1 1 0 0}

a _{Statistical significant difference among complex carbohydrates substrates in transcriptome analysis} b _{Statistical significant difference among complex carbohydrates substrates in secretome analysis} *NCU00206 is classified as both AA3 and AA8

Altough five of the identified AA enzymes in this secretome are among the twenty most produced proteins in complex carbohydrates substrates (Tables 3 and 5), possessing higher probability to be key enzymes in biomass degradation (as showed in section 3.3, where the goal was to compare the secretomes), it is not discarded the possibility of less abundant proteins to be key enzymes either. Phillips and collaborators showed that although only four proteins correspond to 65% of N. crassa secretome weight in microcrystalline cellulose growth, they are responsible for only 43% of the total activity, considering hydrolytic activity only [66].

Five AAs, corresponding to 28% of these enzymes found in secretome, are correlated considering transcriptome and secretome data, as showed in Table 5. Although this number is considered small for CAZymes as showed in Figure 10, the LMPOs (AA9) have a correspondence of 43%. Induction of these enzymes have been shown to be source-specific and time dependent [53,67], however as described in this work no significative differences were observed in overall CAZymes found among AVI, PASB and RAW.

The results presented in this research indicate that phosphoric acid pretreated sugarcane bagasse (PASB) do not affect significativelly the enzyme production in N. crassa, considering both transcriptome and secretome results in relation to RAW bagasse. The compositional effect of wheat straw preatreatment were evaluated in Aspergillus niger transcriptome, and it was observed beneficial effects by using untreated and ionic liquid pretreated straw, but not on hydrothermically pretreated straw as feedstock for CAZyme production [68], revealing that the use of a pretreatment procedure not always is indicated.

Surprisingly, Avicel transcriptome and secretome results were also close related to both sugarcane substrates, which is related to the complexit of mechanisms involved in lignocellulose degradation, but also to the early response evaluated in a 24h growth. Secretome time course analyses of Aspergillus niger growth on sorghum straw and Ceriporiopsis subvermispora growth on aspen wood revealed that larger number of enzymes are produced in initial times [69,70].

In summary, the three carbon sources composed by complex carbohydrates evaluated in this work were excelent inducers of CAZymes, and although more studies are needed using these sources in order to better understand the microorganism response, RAW is the best option for enzyme production, since the total amount of generated proteins is higher (crude extract quantified proteins). Similar results were observed for A. niger and T. reesei growth in culm and steam exploded pretreated sugarcane bagasse, in which the more