Arabinose and cellobiose induce CAZymes in T. aurantiacus
Previously, fed-batch induction experiments led to the identification of xylose as a inducer of CAZymes in T. aurantiacus (12). During this previous fed-batch study, beechwood xylan and a variety of celluloses (Avicel, Sigmacell, bacterial cellulose) also induced CAZyme production in T. aurantiacus (12). Since both beechwood xylan and the plant-derived celluloses (Avicel, Sigmacell) contained xylose, the inductive effects of other sugars besides xylose in these substrates could not be deconvoluted (15). Therefore, we chose two additional lignocellulose-derived sugars to test as inducers: L-arabinose, which constitutes ~ 10% of beechwood xylan, and cellobiose, which is a common cellulase inducer and the product of cellobiohydrolase, (12). T. aurantiacus was grown first in a seed culture in D-glucose medium supplemented with soy meal peptone for 48 h and shifted to two sets of shake flasks; one set containing each individual sugar (0.5% w/v D-xylose, cellobiose and L-arabinose, referred to as batch culture set) and another set of flasks where the same amount of those sugars were continuously fed (fed-batch set) over a 3 day period (Fig. 1). The batch culture set also included triplicate beechwood xylan (0.5% w/v) cultures to compare the purified sugars with a complex substrate that previously found to be a good CAZyme inducer.
SDS-PAGE of culture filtrates demonstrated that the characteristic cellulase bandspreviously observed for T. aurantiacus (cellobiohydrolase, endoglucanase, and lytic polysaccharide monooxygenase) (3) were clearly visible for the fed-batch cultures, which had higher total protein titers than batch cultures (Fig. 1A-B). The three sugar feeds induced different enzymatic activities. High xylanase activities were found during C5 sugar feeding, while cellobiose fed-batch led only to a small increase in xylanase activity in the culture broth (Fig. 1C). Interestingly, cellobiosidase activity followed the same trend of high activity during C5 sugar feeding (Fig. 1D) while endoglucanase activities, measured through the CMCase assay, were in a comparable range for both the cellobiose and C5 fed-batch cultures (Fig. 1E). In contrast, the cellobiose fed-batch and batch cultured displayed higher beta-glucosidase activity than the C5 sugar cultures (Fig. 1F). The ability of the supernatants from the fed-batch cultures to release glucose from Avicel was also tested. In these experiments, the supernatant from the cellobiose-grown cultures had 72% higher glucose release than the xylose-grown cultures and 114% higher glucose release than the arabinose-grown cultures (Fig. 1G).
Development of minimal medium for T. aurantiacus protein production
The fed-batch experiments identified D-xylose, L-arabinose and cellobiose as inducers of CAZyme production. RNA-Seq experiments were designed to study gene expression patterns under these growth conditions to describe the cellular responses to these sugars. However, the T. aurantiacus induction experiments described above were performed with soy meal peptone, a complex nitrogen source containing sugars. Using peptones can pose challenges when using systems biology tools, since considerable differences among manufacturers and also batch effects for the same product can be found (26). While peptones were found to enable high protein production of T. aurantiacus, using a defined medium appeared more favorable for RNA-Seq experiments. Therefore, a minimal medium with a defined nitrogen source was designed in this study. Previously, T. aurantiacus cultivations have been performed on media containing complex nitrogen sources such as yeast extract or peptones (4, 9, 10, 20, 27–32). In only one study, T. aurantiacus was cultivated in Vogel’s minimal glucose medium (11). This medium supported poor growth in our hands, so the McClendon’s medium, which has been used for the above described CAZyme production was adapted to employ (NH4)2SO4 as the sole nitrogen source for a defined minimal medium to study T. aurantiacus induction.
T. aurantiacus growth on D-glucose with McClendon’s medium with soy meal peptone as the nitrogen source was compared with growth with (NH4)2SO4 (Fig. 2A-B). Growth of T. aurantiacus with (NH4)2SO4 as the nitrogen source in liquid medium was < 30% of the growth with soy meal peptone as measured by mycelial biomass. Growth with (NH4)2SO4 resulted in a drop in pH after 3 days from the initial of pH 5.5 to 2.5, while growth on soy meal peptone after 3 days resulted in pH of 6. The poor growth on (NH4)2SO4 was attributed to the drop in pH, which is consistent with previous studies demonstrating optimal growth and enzyme production by T. aurantiacus at pH ≥5 (12). To prevent the drop in pH during cultivation, the (NH4)2SO4-containing medium was buffered with 25 mM sodium citrate. The citrate buffer maintained the pH around 5 for the submerged T. aurantiacus culture and doubled the fungal growth compared to adding (NH4)2SO4 alone. This minimal medium was tested in a fed-batch experiment with D-xylose, cellobiose and L-arabinose as described before (Fig. 2). Although the overall protein production was lower in the minimal medium than in medium amended with soy meal peptone, the enzyme activity patterns were consistent between both tests. Therefore this improved minimal medium was used to assess gene expression patterns during induction.
Differential gene expression analysis of T. aurantiacus
The RNA-Seq study was performed using the fed-batch system described above. T. aurantiacus was grown in D-glucose minimal medium and then shifted to shake flasks containing minimal medium, where D-xylose, cellobiose and L-arabinose were added through continuous feeding. Additionally, a starvation condition (only minimal medium without a carbon source) and a CAZyme-repressing condition (high D-glucose minimal medium) were added as controls. RNA-Seq was performed using 3 biological replicates for each of those conditions.
A Venn diagram was generated to uncover genes differently expressed in D-glucose medium and the sugar fed-batch conditions compared to no carbohydrate medium to investigate the effects of those sugars on gene expression (Fig. 3). All up-regulated genes of the D-xylose, cellobiose, L-arabinose and D-glucose treatments compared to no carbohydrate medium (logFC > 1, pval < 0.05) were used for this analysis. All numbers in parenthesis are T. aurantiacus protein IDs from the JGI Mycocosm database unless otherwise stated.
Genes specific for the L-arabinose fed-batch condition were a putative xylulokinase (Prot. ID: 63574) an arabinofuranosidase (Prot. ID: 51505) and an alpha-xylosidase (Prot. ID: 58732) together with a sugar transporter (Prot. ID: 44260) that is most closely related to the D-xylose transporter xtrD (AN0250) of A. nidulans (33).
For the D-xylose feed condition, no genes specific for xylan and C5 sugar metabolism were found except for a putative alpha-glucuronidase (36875) and an F-box protein orthologue (Prot. ID: 43769) of the A. nidulans gene fbxA. Deletion of fbxA caused reduced secretion of xylanases in its native host and impaired CCR, which was indicated by resistance of the fbxA mutant to the D-glucose analog 2-deoxy-D-glucose (34). Additionally, an unknown sugar transporter (Prot. ID: 55223) was up-regulated. A putative transcriptional regulator with highest similarity to the A. nidulans sexual development regulator nsdD was found here, which was recently found to be vital for cellulase and xylanase in P. oxalicum (35).
When the genes specific for both C5 sugar feeds were investigated, we found two of the most highly secreted enzymes of T. aurantiacus: the xylanase (Prot. ID: 1236) and endoglucanase (Prot. ID: 65156) which have been identified earlier by proteomics (3). Also, a putative beta-xylosidase (Prot. ID: 64461), xylithol dehydrogenase (Prot. ID: 66263) and D-xylose dehydrogenase (Prot. ID: 63693) were identified, indicating expression of a variety of genes related to xylan hydrolysis and the D-xylose and L-arabinose catabolism of T. aurantiacus. The metabolism of those two C5 sugars has been extensively investigated for the related fungus A. niger (36, 37). Orthologous of all necessary enzymes for D-xylose and L-arabinose catabolism were identified in T. aurantiacus, except for the L-arabinose reductase, the first enzyme needed for L-arabinose assimilation (Supplement Fig. 1). All the genes in the T. aurantiacus D-xylose/L-arabinose assimilation pathway were highly up-regulated during L-arabinose feed, while D-xylose caused up-regulation of the same genes except the putative L-xylulose reductase orthologue. Another pathway that appeared to be up-regulated during the C5 sugar feeds was the unfolded protein response (UPR) (Supplement Fig. 2). Several components of this pathway, namely a bipA (38) orthologue (Prot. ID: 46710) was up-regulated in the L-arabinose condition and clxA (39) (Prot. ID: 65748) was highly expressed in the L-arabinose and D-xylose condition. We found that other UPR related genes, such as the orthologue of the regulator that activates UPR, hacA (40) (Prot. ID: 7916), and the protein disulfide-isomerase pdiA (41) (Prot. ID: 64656), were highly expressed during D-xylose and L-arabinose feeding relative to all other conditions (Supplement Fig. 2). UPR genes are often expressed during fungal enzyme secretion to counteract protein folding stress (42).
Intriguingly, the cellobiose feed condition did not display any plant cell wall degradation related CAZymes that were specific for this condition. However, genes up-regulated during D-xylose, cellobiose and L-arabinose feed were the main secreted cellobiohydrolase (Prot. ID: 41785) and a beta-glucosidase (Prot. ID: 38776). Therefore, the expression of the main secreted xylanase, endoglucanase and cellobiohydrolase together with several xylan degrading enzymes happened in the C5 sugar conditions, while the fungus in the cellobiose condition appeared to only express the cellobiohydrolase at high levels this time point. Lastly, a serine carboxypeptidase (Prot. ID: 57314) was identified up-regulated in all sugar feeds, which had highest sequence similarity to protH of A. niger. This enzyme is predicted to be secreted, which could thus be involved in degradation of secreted CAZymes.
Expression analysis of glycoside hydrolases, auxiliary family enzymes and carbohydrate esterases
The analysis of expression patterns was extended to compare expression of related CAZyme proteins. The carbohydrate active enzymes (CAZy) database classifies enzymes relevant for the degradation, modification or creation of glycosidic bonds (43). The JGI mycocosm protein portal contains those CAZyme annotations for T. aurantiacus, where 323 CAZy genes are annotated in the T. aurantiacus genome (44). Enzymes belonging to the glycoside hydrolases (GH), auxiliary activity (AA) and carbohydrate esterases (CE) have been found to be most important for plant cell wall deconstruction (15). Five CAZymes (Prot. ID: 3070, AA 9; 41785, GH7; 65156, GH5; 1236, GH10; 46699, GH3) have been previously identified in T. aurantiacus ATCC 26904 from liquid cultures grown on plant biomass by proteomics analysis (3). Virtually nothing is known about what other genes of this fungus respond to plant polysaccharides and their breakdown products. To uncover the expression trends of those unknown genes, we generated a heat map of the expression trends of the T. aurantiacus GH, AA and CE families.
The GH heat map (Fig. 2a) contained two main clusters: cluster 1 showed GHs that were highly up-regulated on D-glucose or D-glucose and other conditions, while cluster 2 contained GHs that exhibited low expression on D-glucose. Cluster 1 contained diverse types of GHs with only few predicted types of GH related to cellulose, xylan and pectin deconstruction. Conversely, the main four secreted GH described above were all found in cluster 2 (Fig. 4a). Cluster 2 also contained most of the cellulase, xylanase and pectinase genes and was divided into 2 sub-clusters (Cluster 2.1 and 2.2). Cluster 2.1 contained GHs that were highly up-regulated in no carbohydrate medium and to different degrees up-regulated during the sugar feed conditions. Thus, cluster 2.1 was comprised of GH that most likely resemble starvation responsive genes. The beta-glucosidase that was previously identified in the T. aurantiacus supernatant (3) was also found to be strongly expressed during starvation and to a lesser extend during the sugar feed conditions. However, Cluster 2.2 contained GHs that were highly expressed under the sugar feed conditions and showed lower expression during starvation. This cluster appeared to resemble the genes induced by the sugar feed conditions and contains the previously identified endoglucanase, cellobiohydrolase and xylanase (marked). Also, most of cluster 2.2 genes are putative orthologues of A. niger GHs linked to plant cell wall deconstruction (Fig. 2a). A. niger orthologues of the GH51 arabinofuranosidase afbA, was found to released L-arabinose from arabinan and an unknown putative GH5 (T.ID: 44708) with highest similarity to exgC in A. nidulans were found highly expressed on D-xylose. Genes highly expressed on L-arabinose showed high similarity to the alpha-glucuronidase aguA, alpha-xylosidase axlB and the beta-xylosidase xlnD. In addition to the high expression of the main secreted GH5, 7 and 10, we also found another putative GH5 that had highest similarity to exgA and was highly expressed on both C5 sugars. A putative beta-glucosidase with highest similarity to bgl4 was the only GH highly expressed under all three inducing conditions. Few GHs showed elevated expression on cellobiose with only one gene exclusively expressed under this condition predicted beta-glucuronidase (T-ID: 61003) that had the highest similarity to two uncharacterized A. nidulans genes: AN7089 and AN3992. Genes highly expressed during cellobiose feed in cluster 2.2 were mostly cellulases (GH1, GH3, GH5 and GH7). This is in accordance with our finding that cellobiose feed led to only weak xylanase activity in the culture supernatant while cellulases were confirmed to be produced by SDS-PAGE and activity was high for glucose release from Avicel (Fig. 1).
Similar trends were found for the AA heat map (Fig. 4b), where cluster 1 contained AAs that were highly expressed on D-glucose with almost no expression on all other conditions. Cluster 2 was divided in 2 sub-clusters. Cluster 2.1 contained AA that were highly expressed during starvation including 4 predicted laccases, whose expression is often associated with starvation also in other fungi, and one putative unknown LPMO (T.ID: 2027). Cluster 2.2 contained genes that displayed low expression on D-glucose and no carbohydrate medium, and high expression during the sugar feed conditions. Here, the well-studied T. aurantiacus LPMO (marked) was found highly expressed during D-xylose and L-arabinose and to a less extent on cellobiose. Two predicted laccases showed a similar trend. Interestingly, one further laccase (T.ID: 39797) was highly expressed on D-glucose. This gene has a high similarity to A. niger mcoH and the Trichophyton benhamiae conidial pigment biosynthesis oxidase. Therefore, this laccase might be required for pigmentation and could serve as a color locus for strain engineering efforts to identify targeted integrations of DNA constructs, using the newly developed transformation procedure for T. aurantiacus (6, 7).
The CE expression heat map (Fig. 2c) revealed that all genes displayed virtually no expression on D-glucose, with the exception of a putative chitin-deacetylase (T.ID: 45413). Most CEs showed high expression on both D-xylose and L-arabinose and low expression on cellobiose, consistent with their deconstruction of bonds in hemicellulose. None of these genes had high similarities to genes characterized in Aspergilli. The CEs that might likely be of interest for lignocellulose deconstruction are those four that showed low expression on D-glucose and no carbon and thus seemed to be specifically expressed on one or both C5 sugars (Fig. 3c, black frame).