Screening the putative xylanase-specific TFs
Based on bioinformatics analysis, we chose seven putative TFs as candidates (Additional file 1) for screening. All these genes were located in five different chromosomes [20, 21], and six of candidates were located in the D segment duplication resulting in higher xylanase activities as reported by Chen et al. [17]. We overexpressed these genes in T. reesei RUT-C30. After creating monoconidial cultures for genetic stability, we measured the xylanase and cellulase activities of the transformants. The xylanase activity, but not the cellulase activity, of the strain overexpressing the sxlr gene (jgi|TrireRUTC30_1|26638, Osxlr) decreased significantly (t test, P < 0.05) (Fig. 1a, b), and its extracellular protein concentration was decreased (Fig. 1c), which might indicate that this gene affects the xylanase activity alone. In contrast, no changes in xylanase activity, cellulase activity, or protein concentration were detected between RUT-C30 and the other six overexpression strains (Fig. 1).
To investigate how the sxlr gene is involved in regulation of xylanase activity in T. reesei, we deleted sxlr gene from the RUT-C30 strain to obtain Δsxlr transformants (Fig. 2a, b). In contrast to the Osxlr strain, the xylanase activity of the Δsxlr strains was increased significantly (Fig. 2c). However, the cellulase activity of the Δsxlr strains was almost the same as the parent RUT-C30 strain. We also deleted the homologous sxlr gene of T. reesei Qm6a (jgi|Trire2|123881, Δ6a-sxlr) using the CRISPR/Cas9 system to verify the common regulation of xylanase activity in T. reesei strains. As expected, the xylanase activity of Δ6a-sxlr transformants was also dramatically increased, similar to the Δsxlr strain (Fig. 2c), while its cellulase activity was not affected. Taken together, these results suggested that sxlr encoded a TF that negatively regulates xylanase activity.
The deletion of SxlR in T. reesei RUT-C30 results in higher xylanase activity and higher reducing sugar yield
We selected the cellulase hyperproduction strain RUT-C30 to explore the potential regulatory mechanism of SxlR in T. reesei. In addition to the sxlr deletion strain (Δsxlr, transformant 1) and the sxlr overexpression strain (Osxlr), we also constructed the in situ re-complementation strain (Rsxlr) based on the Δsxlr strain (Additional file 2). The xylanase activity of the Δsxlr strain increased relative to that of RUT-C30 by 0.7-fold and 1.4-fold after 3 and 7 days, respectively, of incubation in inducing medium containing wheat bran and Avicel (Fig. 3a). The xylanase activity was also examined when xylan or lactose was used as the inducer (the sole carbon source in the inducing medium). The xylanase activity of Δsxlr was 1.3-, 0.9-, and 0.7-fold higher than RUT-C30 after 1, 2, and 3 days, respectively, of cultivation with xylan as the inducer (Fig. 3b), and 14.2-, 4.7-, and 5.2-fold higher, respectively, with lactose as the inducer (Fig. 3c). The xylanase activity of the sxlr re-complementation control strain Rsxlr reverted to the same level as that of RUT-C30. In contrast, the sxlr overexpression strain Osxlr demonstrated weaker xylanase activity than RUT-C30 (Fig. 3a–c). However, no significant difference in cellulase activity was detected between the four strains (Fig. 3d).
Degradation of lignocellulose is a growth-associated process. The growth rate affects the secretion of enzymes directly [22]. We observed the growth of RUT-C30 and its derivative strains on potato dextrose agar (PDA) and minimal medium (MM) containing 1% xylose, xylan, glucose, lactose, or Avicel as carbon sources, respectively (Fig. 4a). RUT-C30 and its derivative strains did not show significantly different growths and sporulations on PDA and MM containing 1% glucose, lactose or Avicel (Fig. 4a). However, the Δsxlr strain demonstrated rapid growth and the Osxlr strain demonstrated reduced growth on MM containing xylose or xylan when compared to RUT-C30. These results could be explained by differences in xylanase activities.
Enzyme activities are also influenced by the concentration and composition of lignocellulose preparations. The extracellular protein concentrations of the Δsxlr, Osxlr, and RUT-C30 strains were 15.275 ± 0.096, 10.943 ± 0.399, and 13.378 ± 0.240 mg/mL, respectively. The Δsxlr strain had the highest extracellular protein concentration, and Osxlr had the lowest (Fig. 4b). According to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), the band corresponding to ~20 kDa, which was identified as the xylanase protein XYN2 by MALDI-TOF/TOF, was enhanced dramatically in the Δsxlr strain but decreased in the Osxlr strain (Fig. 4c). This indicates that SxlR affects the amount of secreted XYN2 in the lignocellulose preparations produced by T. reesei.
To determine whether the deletion of SxlR in T. reesei RUT-C30 could improve the strain’s efficiency to hydrolyze pretreated lignocellulose, steam-exploded rice straw and steam-exploded rice straw mixed with corn straw were used as saccharification substrates. The crude enzyme complex of the mutant Δsxlr strain produced more reducing sugar than that of RUT-C30 (Fig. 4d). The straw hydrolysis increased to 21% by the supernatant of the Δsxlr strain, while 14.1% by the supernatant of RUT-C30 strain after 3 days of hydrolysis from the pretreated rice and corn straw. Similarly, the straw hydrolysis increased to 11.9% by the supernatant of the Δsxlr strain, while 7.6% by the supernatant of RUT-C30 strain after 3 days of hydrolysis from the pretreated rice straw.
SxlR regulates the GH11 xylanase genes
To further identify the regulation mechanism of SxlR on xylanase activity, we examined the expression levels of five xylanase-encoding genes using qPCR; the xylanases encoded by these genes included three members of GH11 family (XYN1, XYN2, and newly discovered XYN5), a GH10 family member XYN3 and a GH30 family member XYN4 [23]. The transcription levels of the genes encoding the three GH11 members showed significant differences in the Δsxlr strain (upregulated) and the Osxlr strain (downregulated) at all sampling time points using wheat bran and Avicel were used as inducers (Fig. 5a).
The transcription level of the genes encoding XYN3 and XYN4 were also downregulated in the Osxlr strain at all sampling time points (Fig. 5a). However, their transcription levels in the Δsxlr strain changed differently (Fig. 5a).
In the Δsxlr strain, the relative expression level of xyn3 was upregulated compared to the wild-type stain in the first 8 h of incubation in the inducing medium. The relative expression level of xyn4 was upregulated after 4 h of induction, but downregulated after 8 and 12 h of induction (Fig. 5a). When using xylan as the inducer, the transcription level variation tendency of the encoding genes of XYN1, XYN2, XYN5, and XYN3 was similar to that using wheat bran and Avicel as the inducer (Fig. 5b). In contrast, the relative expression level of xyn4 was upregulated in the Osxlr strain after 4 h of induction (Fig. 5b).
To further confirm the qRT-PCR results, qRT-PCR experiments were re-carried out using rpl6e (a ribosomal protein encoding gene) [24] and sar1 (a small GTPase encoding gene) [25] as the reference gene. The transcription level variation tendency of the five xylanase-encoding genes was similar to that using β-actin gene as the reference gene (Additional file 3).
Additionally, we examined the relative expression levels of other genes coding (hemi-) cellulases or their regulators, including three major cellulase genes (cbh1, cbh2, egl1), the key transcriptional activator (xyr1), and two hemi-cellulase genes (β-mannanase, man1 and α-l-arabinofuranosidase, abf1). There were no significant differences in the relative expression levels of these genes between RUT-C30 strain and Δsxlr strain (Additional file 3). These results provide the first experimental evidence that SxlR is a negative and specific regulator of GH11 family xylanases.
Xyr1, a major transcription activator in T. reesei, regulates expression of most cellulase and hemi-cellulase genes directly, including xylanases [26, 27]. To investigate the relationship between Xyr1 and SxlR, we determined the relative expression levels of sxlr in the Δxyr1 strain (an xyr1-deletion strain derived from RUT-C30) and in the Oxyr1 strain (an xyr1-overexpression strain derived from RUT-C30) as well as the relative expression levels of xyr1 in Δsxlr strain and Osxlr strain (Fig. 5c). Comparing with xyr1, the transcription level of sxlr was quite low in RUT-C30. The xyr1 transcription level decreased significantly in the Osxlr strain at all the sampling time points. In contrast, its transcription level in the Δsxlr strain significantly increased at 4-h induction, and then decreased from 8-h induction. It seemed that SxlR might repress xyr1 expression somehow. The transcription level of sxlr in the Oxyr1 strain was nearly the same as that in RUT-C30 strain, and its transcription level in the Δxyr1 strain was lower or similar to that in RUT-C30 strain. The variation tendency of the sxlr transcription level was quite different from that of the downstream genes of Xyr1, of which the transcription were sharply repressed after the deletion of Xyr1 [27].
SxlR binds the promoters of GH11 xylanase genes
According to functional domain analysis, SxlR contains two distinct conserved domains, one GAL4-like Zn2Cys6 binuclear cluster DNA binding domain at the N-terminus (cd00067: residues 282–317) and one fungal transcription factor regulatory middle homology region at the C-terminus (cd12148: residues 436–853). To verify the regulation of the GH11 xylanase genes by SxlR, the GAL4-like Zn2Cys6 binuclear cluster DNA binding domain was expressed in vitro for EMSAs. The nearly 1500-bp upstream regions (nucleotide position −1500 to −1) assumed to be the promoter regions of the xylanase-encoding genes were divided into six parts (for example, xyn2-P1, 268 bp, nucleotide position −268 to −1; xyn2-P2, 268 bp, position −516 to −249; xyn2-P3, 268 bp, position −764 to −497; xyn2-P4, 268 bp, position –1012 to −745; xyn2-P5, 268 bp, position −1260 to −993; xyn2-P6, 268 bp, position −1508 to −1241). Based on the EMSA gel shifts, SxlR could bind the xyn1-P5, xyn2-P4, xyn2-P5, and xyn5-P5 promoter regions of the three GH11 xylanases (Fig. 6a). No specific gel shift was observed for the xyn3 and xyn4 promoters, which belonged to the GH10 and GH30 families, respectively (Fig. 5b; Additional file 4). It means that SxlR plays a critical role in the inhibition of GH11 family genes through binding to their promoter regions directly.
To identify the binding motif of SxlR, three promoter fragments (xyn2-P4, xyn1-P5, and xyn5-P5) were each divided into two 144-bp segments (Additional file 5A). SxlR bound to xyn2-P4-1, xyn1-P5-2, and xyn5-P5-2 (Additional file 5B). Based on MEME Suite (http://meme-suite.org/tools/meme) analysis, three candidate consensus motifs were predicted (Additional file 5C, D). However, the deletion of these three motifs did not affect the binding of SxlR to these DNA fragments (Additional file 5E). The 144 -bp fragments were further shortened for identification of a consensus motif, and two motifs were predicted (Fig. 7a). Based on the disappearance of the SxlR-DNA complex with the deletion of motif 5 (Fig. 7b), the consensus motif of SxlR was determined as 5′-CATCSGSWCWMSA-3′ (Fig. 7c, d). In this motif, one guanine and one cytosine are present in the core region and the consistent nucleotides are located in the flanks. The potential SxlR binding motif were not detected in the promoter regions of Xyr1 and xylanases XYN3 and XYN4. It suggested that SxlR might not regulate the transcription of Xyr1 and the GH10 family member XYN3 as well as the GH30 family member XYN4 directly.