- Open Access
Disruption of alpha-tubulin releases carbon catabolite repression and enhances enzyme production in Trichoderma reesei even in the presence of glucose
Biotechnology for Biofuels volume 14, Article number: 39 (2021)
Trichoderma reesei is a filamentous fungus that is important as an industrial producer of cellulases and hemicellulases due to its high secretion of these enzymes and outstanding performance in industrial fermenters. However, the reduction of enzyme production caused by carbon catabolite repression (CCR) has long been a problem. Disruption of a typical transcriptional regulator, Cre1, does not sufficiently suppress this reduction in the presence of glucose.
We found that deletion of an α-tubulin (tubB) in T. reesei enhanced both the amount and rate of secretory protein production. Also, the tubulin-disrupted (ΔtubB) strain had high enzyme production and the same enzyme profile even if the strain was cultured in a glucose-containing medium. From transcriptome analysis, the ΔtubB strain exhibited upregulation of both cellulase and hemicellulase genes including some that were not originally induced by cellulose. Moreover, cellobiose transporter genes and the other sugar transporter genes were highly upregulated, and simultaneous uptake of glucose and cellobiose was also observed in the ΔtubB strain. These results suggested that the ΔtubB strain was released from CCR.
Trichoderma reesei α-tubulin is involved in the transcription of cellulase and hemicellulase genes, as well as in CCR. This is the first report of overcoming CCR by disrupting α-tubulin gene in T. reesei. The disruption of α-tubulin is a promising approach for creating next-generation enzyme-producing strains of T. reesei.
Cellulose and hemicellulose are naturally produced in higher plants as cell wall components from CO2 and are the two most abundant polysaccharides on Earth. Using this biomass is important to break the social structure of mass consumption and mass disposal that depends on petrochemicals, allowing us to build a sustainable society. Hydrolysis of these polysaccharides with enzymes such as cellulase and hemicellulase provides sugars that can be converted by microbial fermentation into biofuels, bio-based commodity chemicals, and value-added products . Trichoderma reesei, an anamorph of Hypocrea jecorina, is a very important filamentous fungus used to produce industrial cellulases and hemicellulases. The cellulase market is projected to reach about 0.8 billion USD in 2025 , and there have been many studies on enzyme production by T. reesei [3,4,5,6].
Enzyme production is a major focus of research and development. Cellulosic biomass has a strong and complex structure, with hemicellulose and lignin surrounding crystalline cellulose . It is therefore necessary to increase cellulase production and improve its enzymatic hydrolysis efficiency .
The saccharification of cellulosic biomass requires the synergistic effects of various enzymes, such as cellobiohydrolases (CBH, EC 188.8.131.52), which release cellobiose from the ends of crystalline cellulose, endo-β-1,4-glucanases (EG, EC 184.108.40.206), which cleave internal bonds of amorphous cellulose, and β-glucosidases (BGL, EC 220.127.116.11), which convert cellobiose and cello-oligosaccharides into glucose to prevent product inhibition of CBH by cellobiose. These enzymes are collectively referred to as cellulases. Hemicellullose is also synergistically degraded by endo-β-1,4-xylanases (XYN, EC 18.104.22.168), β-xylosidases (BXL, 22.214.171.124), and other accessory enzymes. These enzymes involved in the degradation of hemicellulose are collectively referred to as hemicellulases.
The well-studied fungus Trichoderma reesei produces a complete set of cellulases in abundance when cultivated with inducer such as cellulose, cellobiose and sophorose as reviewed in [9, 10]. The CBH and EG activities of T. reesei are higher than those of other microorganisms, but its BGL activity is lower than that of cellulase mixes from other organisms  and results in cellobiose accumulation and subsequent product inhibition of CBH, reducing enzymatic hydrolysis efficiency . This problem has been addressed by characterizing the BGLs and overexpressing the BGL genes in T. reesei [13,14,15]. In a previous study, we conducted the heterologous production of AaBGL1 (BGL1 from Aspergillus aculeatus) by T. reesei to enhance the degradation of NaOH-pretreated rice straw, thereby reducing the amount of cellulase required [15, 16].
The cellulase and hemicellulase genes in T. reesei are regulated by transcriptional activators (Xyr1, Ace2 and Ace3) and repressors (Ace1 and Cre1) [17,18,19,20,21]. Cre1 is a regulator of CCR. When T. reesei is cultivated in medium containing glucose, Cre1 represses cellulase and hemicellulase gene expression to prioritize the uptake of glucose, resulting in little cellulase production. The hyper-secreting T. reesei mutant RUT-C30 contains a cre1 gene truncated by mutagenesis and has higher cellulase and hemicellulase production in glucose-containing media than its parent strain QM6a [22, 23]. Similarly, the strain PC-3-7, obtained as a mutant capable of producing cellulase on agar plates containing glucose, has a mutation in cre1 [24, 25]. Although cellulase genes such as cbh1 are expressed in the presence of glucose in this cre1-deficient strain , cellulase production is sharply decreased in a glucose concentration-dependent manner , suggesting that even if cre1 is knocked out, enzyme production is repressed under high glucose concentration. Continuous but low glucose feed levels in fed-batch cultures using sugar solutions containing glucose prevent the repression of enzyme production by glucose [28, 29]. However, this approach requires accurate control of the sugar flow rate to avoid glucose accumulation and thus new strains resistant to CCR are desirable.
Tubulin is a protein that forms microtubules and centrosomes in eukaryotic cells. There are two types of tubulin, alpha-tubulin and beta-tubulin, and these heterodimers polymerize to form the protofilaments of microtubules. Microtubules are formed when these protofilaments are joined into tubular structures and function in many essential cellular processes such as mitosis . Fungal alpha- and beta-tubulins are classified as α1, α2, α3, β1 and β2 by phylogenetic analysis . α1-Tubulins are found in all fungi examined to date, but α2-tubulins are found only in Ascomycota (except for Saccharomycetes and Taphrinomycotina) and α3-tubulins are found only in some Basidiomycetes. β1-Tubulins are found in all fungi but β2-tubulins are found only in Basidiomycetes and Yarrowia lipolytica. The two alpha-tubulins of A. nidulans, TubA (NCBI Reference Sequence: XP_657920.1) and TubB (NCBI Reference Sequence: XP_680839.1), belong to the α1 and α2-tubulin classes, respectively, and their functions were previously analyzed . TubA is involved in nuclear division and TubB may be involved in germination. Deletion of the tubB gene of A. nidulans had no detectable effect on vegetative growth or asexual reproduction but the tubB gene is essential for sexual development . In addition, genomic analysis of A. nidulans revealed two other α2-tubulin gene copies, and these tubulins likely have distinct functions. Genomic analysis of T. reesei showed that it has one α1-tubulin, one α2-tubulin, and two β1-tubulins.
In this study, we evaluated the protein production of the three recombinant strains of T. reesei PC-3-7 expressing BGL from A. aculeatus, which has been constructed in previous studies, and found that the protein production was decreased in two strains. But one strain, E1AB1, led to an unexpectedly phenotype of improvement of secretory protein. We hence conducted in-depth analysis of this strain and found that gene encoding alpha-tubulin was disrupted.
Sugar consumption during mixed-substrate fermentation and transcriptome analysis of the tubulin-disrupted strain clearly indicate a release from CCR. This is the first report of overcoming CCR by disrupting the tubulin-encoding gene to increase cellulase production in T. reesei.
Protein production in aabgl1-expressing recombinants
We investigated the effect of overexpression of BGL on cellulase production in T. reesei by culturing the recombinant strains X3AB1, C1AB1 and E1AB1 expressing A. aculeatus bgl1 (aabgl1) under the control of the xyn3, cbh1 and egl1 promoters, respectively (Table 1). In the X3AB1 strain, the aabgl1 expression cassette was integrated into the xyn3 locus homologously, while the C1AB1 and E1AB1 strains were constructed by non-homologous insertion of the aabgl1 cassette. The strains were cultured in 2-L jar fermenters using 10% (w/v) cellulose and 2% (w/v) xylan as carbon sources. Protein production stopped when the ammonia solution, which is a nitrogen source and is used to keep the pH constant, was no longer supplied. So, the endpoint of the cultivation was the sampling point after the ammonia solution was no longer supplied. As shown in Fig. 1, X1AB1 and C1AB1 produced 14.7 g/L and 15.1 g/L protein, respectively, which are lower than the protein production levels of PC-3-7 (16.0 g/L, p < 0.05). E1AB1 produced 17.4 g/L protein, which is significantly higher than PC-3-7 (p < 0.05).
Identification of the expression cassette integration site in E1AB1
We determined the reason for increased protein production in E1AB1 by identifying the locus of the gene insertion in E1AB1 using inverse PCR. As shown in Additional file 1: Fig. S1a, the aabgl1-expressing cassette was inserted in the middle of the DNA encoding alpha-tubulin B (gene ID: 120830, hereinafter called tubB). PCR amplification from the start codon to the stop codon of tubB showed that the full length of the open reading frame (ORF) was amplified in PC-3-7, whereas a PCR fragment of tubB ORF with inserted aabgl1-expressing cassette was amplified in E1AB1 (Additional file 1: Fig. S1b). In addition, PCR amplification from amdS in the aabgl1-expressing cassette to upstream of tubB in the genome was observed only in E1AB1. These results indicated that the aabgl1-expressing cassette was inserted in tubB locus. Thus, higher protein production by E1AB1 may be related to the disruption of tubB. Alpha-tubulin polymerizes with beta-tubulin into microtubules and genome analysis has shown that there are two alpha-tubulin genes (gene ID: 120789 and 120830) in T. reesei . Although microtubules are a major component of the eukaryotic cytoskeleton, and microtubules function in many essential cellular processes such as mitosis , no decrease in growth rate or glucose consumption was observed in E1AB1 (Additional file 2: Fig. S2).
Construction of a tubB-deleted strain
To examine the effect of disruption of tubB gene, we constructed tubB-deleted strains. In one strain, PC-3-7 was transformed with linearized pUC-ΔtubB-amdS, in which the tubB ORF was replaced by the amdS marker. In the other strain, PC-3-7 was transformed with pUC-ΔtubB-Pegl1-aabgl1-amdS containing the 5′ and 3′ flanking regions of the tubB ORF linked with the aabgl1 ORF under the control of the egl1 promoter and amdS marker. In each case, a homologous recombination transformant was identified by colony PCR analysis, providing the transformant of pUC-ΔtubB-amdS, designated PC-3-7ΔtubB, and the transformant of pUC-ΔtubB-Pegl1-aabgl1-amdS, designated PC-3-7ΔtubB::Pegl1-aabgl1.
To observe the effect of tubB disruption on morphology, a growth assay of PC-3-7ΔtubB and PC-3-7 on PDA plates was performed. The mycelial morphology on PDA plate is shown in Fig. 2a and the change in colony size over time is shown in Fig. 2b. The growth speed of PC-3-7ΔtubB on PDA plate was obviously lower than that of PC-3-7. However, this decrease of growth rate was not observed when PC-3-7ΔtubB was cultured in liquid medium (Additional file 2: Fig. S2).
The hyphae of PC-3-7ΔtubB cultured in liquid medium containing glucose or cellulose were compared with PC-3-7 using scanning electron microscopy (SEM) (Fig. 3). There was no difference in the SEM images of these strains when cultivated in glucose medium (Fig. 3a, b). However, when cultivated in cellulose medium, rounded hyphae were frequently observed in the SEM image of PC-3-7ΔtubB (Fig. 3c, d). The mean cell length and diameter values for 70 cells randomly selected from each SEM image are shown in Table 2. PC-3-7ΔtubB cells cultivated in glucose were longer than PC-3-7 cells under the same conditions. When the cells were cultivated with cellulose, the cell diameters of PC-3-7ΔtubB were significantly larger than those of PC-3-7. The cell ratio (length/diameter) of PC-3-7ΔtubB was significantly larger than that of PC-3-7 when cultured with glucose and significantly smaller when cultured with cellulose. These results indicate that PC-3-7ΔtubB cells are more elongated when cultured with glucose and more rounded when cultured with cellulose, suggesting that the disruption of tubB affects cell morphology cultured in liquid medium.
Evaluation of secretory protein production of tubB-deleted strains
We compared the amount of secretory protein produced by the constructed tubB-deleted strains by cultivating in 2-L jar fermenters using 10% (w/v) cellulose and 2% (w/v) xylan as carbon sources (Fig. 4). The endpoint of the cultivation was the sampling point after the ammonia solution, which is a nitrogen source and used to keep the pH constant, was no longer supplied, and the changes in the amount of secreted protein are shown in Fig. 4a, and the amount of secreted protein at the endpoint is shown in Fig. 4b. The PC-3-7ΔtubB and E1AB1 showed significantly higher secretory protein production (17.4—19.5 g/L) than the parent PC-3-7 (16.0 g/L), with PC-3-7ΔtubB showing the highest secretory protein production. The cultivations of E1AB1 and PC-3-7ΔtubB::Pegl1-aabgl1 were complete in 120 h while PC-3-7 and PC-3-7ΔtubB required 144 h. Initial protein production rates by the ΔtubB strains were especially higher than that of PC-3-7 (Fig. 4a). These results indicate that disruption of the tubB gene enhances both the amount and rate of protein secretion.
Effects of glucose on protein production by tubB-deficient strains
Since high expression of BGL improves the release of glucose from cellulose, we investigated the effect of glucose on tubB-deficient strains by culturing PC-3-7ΔtubB and PC-3-7 in 2-L jar fermenters using 10% (w/v) cellulose with and without 2.5% (w/v) glucose for 120 h. When cultured using only cellulose (Fig. 5a), PC-3-7ΔtubB showed higher protein secretion (14.4 g/L) than PC-3-7 (7.5 g/L). When cultured using cellulose and glucose (Fig. 5b), protein secretion by PC-3-7 was extremely low (2.8 g/L) whereas PC-3-7ΔtubB produced more protein (15.4 g/L) than when cultured with only cellulose. To confirm whether the amount of cellulase and hemicellulase increased along with the increase in the amount of secreted protein, the cellulase (pNPGase, pNPLase, and CMCase) and hemicellulase (pNPXase, pNPX2ase, and xylanase) activities in the culture supernatant were measured after 120 h of cultivation. The results of enzymatic assay are shown in Fig. 5c, and specific activity and the SDS-PAGE of the culture broth are shown in Additional file 3: Fig. S3. Each enzyme activity (Fig. 5c) was consistent with the amount of secreted protein (Fig. 5a, b), suggesting that enzyme production was enhanced in PC-3-7ΔtubB compared to PC-3-7. There was no difference in each specific activity, except for the pNPXase activity of PC-3-7 when cultured with only cellulose (Additional file 3: Fig. S3a). SDS-PAGE of PC-3-7ΔtubB showed no difference in protein composition and amount with and without glucose (Additional file 3: Fig. S3b, c). In contrast, PC-3-7 exhibited decreased amount of secretory protein and production of an unknown protein of about 75 kDa following the addition of glucose.
Figure 5d, e shows the concentration of the exhaust CO2 during fermentation. As shown in Fig. 5d, PC-3-7ΔtubB generated more CO2 by respiration than PC-3-7 even at the early stage of culture, indicating that PC-3-7ΔtubB can immediately utilize cellulose as a carbon source. When cultured in medium containing both cellulose and glucose, PC-3-7 generated more CO2 than PC-3-7ΔtubB up to 20 h, then a sharp drop was observed at 20–23 h. PC-3-7ΔtubB cultured in the same medium exhibited a slight reduction in CO2 generation at 28 h (Fig. 5e).
Figure 5f, g shows the glucose and cellobiose concentration in the culture broth. The cultivation of PC-3-7 and PC-3-7ΔtubB in medium containing only cellulose (Fig. 5f) resulted in barely detectable levels of glucose whereas cellobiose was detectable in the culture broth. Cellobiose accumulated in the medium between 24 and 30 h, and PC-3-7 accumulated more cellobiose than PC-3-7ΔtubB. When cultivated in medium containing both cellulose and glucose (Fig. 5g), PC-3-7 showed a higher glucose consumption rate than PC-3-7ΔtubB, and the glucose was depleted between 18 and 24 h. The sharp drop in exhaust CO2 was likely caused by switching the assimilating carbon source from glucose to cellulose (Fig. 5e).
RNA-seq analysis of the tubB-deficient strain PC-3-7ΔtubB
PC-3-7ΔtubB shows high protein production even in the presence of glucose. We investigated the underlying mechanism caused by the deletion of tubB by determining the transcription profile of PC-3-7ΔtubB by RNA-sequencing (RNA-seq) analysis. PC-3-7 and PC-3-7ΔtubB were cultured in 2-L jar fermenters using 10% (w/v) cellulose with and without 2.5% (w/v) glucose. Cells cultured in medium with only cellulose were collected after 24 h (PC-3-7_24h_C and ΔtubB_24h_C) and 48 h (PC-3-7_48h_C and ΔtubB_48h_C). Cells cultivated in medium containing both cellulose and glucose were collected after the CO2 concentration decreased to adjust the growth phase (24 h for PC-3-7; PC-3-7_24h_C + G, 30 h for PC-3-7ΔtubB; ΔtubB_30h_C + G), and 48 h (PC-3-7_48h_C + G and ΔtubB_48h_C + G). RNA-seq was performed on the extracted RNA samples and normalized gene expression (RPKM, reads per kilobase per million) was calculated for all 9,115 coding sequences.
The RPKM value of the tubB gene was 0 in all PC-3-7ΔtubB samples, clearly indicating that tubB was deleted in PC-3-7ΔtubB. The RPKM values of the other tubulin genes were unaffected by deletion of the tubB gene (Additional file 4: Table S1).
The highest expression levels of carbohydrate-active enzymes (CAZymes) were obtained for cbh1, cbh2, egl1, egl2, egl3, egl4, egl5, xyn2, cip1 and swo, with cbh1 being the highest expressed gene under all conditions except PC-3-7_48h_C + G (Fig. 6 and Additional file 5: Table S2). Interestingly, xyn1 was highly expressed by PC-3-7ΔtubB under all conditions but at extremely low levels in PC-3-7. The gene expression of transcription factors of cellulases and hemicellulases is listed in Additional file 6: Table S3; ace1, vel1 and vib1 were highly expressed in PC-3-7, and the expression of cre1 was slightly elevated in PC-3-7ΔtubB.
DEG analysis between PC-3-7 and PC-3-7ΔtubB
We analyzed the transcriptional differences by differentially expressed gene (DEG) analysis using the R and DESeq2 package. We identified 416 DEGs (187 upregulated and 229 downregulated) between PC-3-7_48h_C and PC-3-7_48h_C + G, 13 DEGs (12 upregulated and 1 downregulated) between ΔtubB_48h_C and ΔtubB_48h_C + G, 744 DEGs (478 upregulated and 266 downregulated) between PC-3-7_48h_C and ΔtubB_48h_C, and 803 DEGs (545 upregulated and 258 downregulated) between PC-3-7_48h_C + G and ΔtubB_48h_C + G with adjusted p < 0.01. Scatter plots of gene expression profiles compared above are shown in Fig. 7 and those that were identified to be DEGs were highlighted. Moreover, correlation analyses of the RPKM values of all coding sequences (CDSs) are shown in Additional file 7: Table S4. There was a high correlation (R = 0.988, R2 = 0.976) between ΔtubB_48h_C and ΔtubB_48h_C + G (Fig. 7b) but no correlation (R = 0.597, R2 = 0.357) between PC-3-7_48h_C and PC-3-7_48h_C + G (Fig. 7a). In both strains, cellulase genes are induced when cellulose is the sole carbon source. The addition of glucose totally altered gene expression in PC-3-7 but had almost no effect on PC-3-7ΔtubB cellulase gene expression, showing that cellulase gene expression in PC-3-7ΔtubB was induced even in the presence of glucose.
The 744 DEGs between PC-3-7_48h_C and ΔtubB_48h_C contained 74 CAZymes, 57 of which were highly expressed in PC-3-7ΔtubB (Fig. 7c), and the 803 DEGs between PC-3-7_48h_C + G and ΔtubB_48h_C + G contained 75 CAZymes, 63 of which were highly expressed in PC-3-7ΔtubB (Fig. 7d). Cellulase genes such as cbh1, 2, egl4, 5 and 6 were particularly significantly expressed in PC-3-7ΔtubB in both C and C + G condition (Additional file 8: Table S5). Moreover, other CAZymes, such as alpha-glucuronidase (gene ID: 72526; aguA), alpha-D-galactosidase (72704), acetyl xylan esterases (54219 and 73632; axe1), xylanases (xyn1, 2, 3, 4 and 5) and pectinase (103049) were significantly expressed in PC-3-7ΔtubB in both C and C + G condition. Knockout of tubB thus increased the expression of CAZymes not originally induced by cellulose.
Gene ontology enrichment analysis
We analyzed the effect of deletion of the tubB gene by gene ontology (GO) enrichment analysis of the DEGs between PC-3-7_48h_C and ΔtubB_48h_C. GO enrichment analysis was performed using the R and GOSeq package. For each enriched GOs, the number of genes, the number of DEGs, and the number of DEGs that are upregulated in PC-3-7ΔtubB were listed in Additional file 9: Table S6. The most enriched GOs were GO:0005975 (carbohydrate metabolic process) and GO:0004553 (hydrolase activity, hydrolyzing O-glycosyl compounds) and most of the DEGs corresponding to these GOs were highly expressed in PC-3-7ΔtubB. Since the genes corresponding to these GOs are glycoside hydrolase, the most significant effect of the knockout of tubB was the change in the expression of CAZymes. In addition, GOs related to transporter such as GO:0006810 (transporter), GO:0016020 (membrane), GO:0006865 (amino acid transport), GO:0005351 (carbohydrate:proton symporter activity) and GO:0008643 (carbohydrate transport) were identified in the enriched GOs and where the corresponding DEGs were highly expressed in PC-3-7ΔtubB, indicating that the expression levels of many transporters were simultaneously upregulated due to deletion of the tubB gene. Twenty-three sugar transporter-related genes (GO:0005351 or GO:0008643, or annotated genes encoding sugar transporters) were differentially expressed between PC-3-7_48h_C and ΔtubB_48h_C and 19 genes were upregulated in PC-3-7ΔtubB. The most significantly upregulated gene was gene ID 69957 or Tr69957 (log2 fold change = 6.15), reported as a cellobiose, mannose and xylose transporter . ID 3405 or crt1, reported as a cellobiose and sophorose transporter which plays a key role in the cellulolytic signaling process [35, 36], showed the highest gene expression level of the significantly expressed transporters. ID 50894 or str1, reported as a transporter induced by pentose, ID 121482 or str2, reported as a transporter with broad substrate specificity , and ID 79202 and 77517, reported as a lactose transporter , were all upregulated in PC-3-7ΔtubB.
Analysis of sugar transport
Based on these RNA-seq analyses, knockout of tubB may alter sugar transport ability and thus we analyzed the uptake rates of glucose, xylose, cellobiose, and a mixture of glucose and cellobiose in PC-3-7 and PC-3-7ΔtubB. The cells were cultured in 1% (w/v) glucose medium for 2 days, then each sugar was added at a final concentration of 0.2% (w/v) and the sugar concentration in the medium was monitored (Fig. 8). There was no difference in sugar uptake between PC-3-7 and PC-3-7ΔtubB when glucose (Fig. 8a) or xylose (Fig. 8b) was added. In both strains, glucose was depleted in about 2 h and xylose was consumed in about 4 h. When cellobiose was added, PC-3-7ΔtubB showed a higher consumption rate than PC-3-7. PC-3-7ΔtubB assimilated 0.2% (w/v) cellobiose in about 2 h while PC-3-7 could not consume it in 4 h (Fig. 8c). When glucose and cellobiose were added, PC-3-7 first assimilated glucose and after its depletion, cellobiose was assimilated slowly (Fig. 8d). On the other hand, PC-3-7ΔtubB assimilated glucose and cellobiose simultaneously, with glucose consumed more slowly than when only glucose was added. These results suggested that PC-3-7ΔtubB has a higher cellobiose uptake rate than the parent strain, and can uptake cellobiose even in the presence of glucose.
This study revealed that enzyme production was enhanced in a tubB-disrupted strain. Additionally, the deletion of tubB enabled high enzyme production in the presence of glucose. Cellobiose uptake was enhanced in the tubB-disrupted strain and cellobiose was taken up simultaneously with glucose, suggesting release from CCR. This is the first report that alpha-tubulin in T. reesei is involved in cellulase and hemicellulase gene expression, enzyme production, and CCR, and that CCR can be further released by genetic modification of T. reesei.
Microtubules formed by polymerization of tubulin proteins are not only involved in cell division, but also in vesicular trafficking. In T. reesei, it has been reported that disruption of rho3 encoding ras-type small GTPase involved in cell polarity and vesicle fusion with the plasma membrane caused a reduction of secreted protein production . However, there have been no reports that knockout of tubulin, which is involved in vesicular transport, improves the amount of proteins secreted by vesicular transport. So, this is the first report that deletion of tubulin enhances the secretory protein production in fungi. In this study, we confirmed that disruption of the tubB gene enhanced sugar transporter genes, and these membrane proteins are also transported by vesicular transport. Whether TubB is involved in the transport of extracellular proteins, transport of membrane proteins such as sugar transporters, or some other mechanism will require further molecular biological analysis of TubB.
T. reesei SEU-7, constructed by the overexpression of endogenous BGL in RUT-C30, exhibited a significant decrease in cellulase production by the addition of only 1% (w/v) glucose into medium containing cellulose . This suggests that a high glucose concentration decreases cellulase production even in a Cre1-deficient strain, consistent with the extremely low protein production of PC-3-7 in glucose-containing medium (Fig. 5b). Several studies have investigated this decrease in enzyme production caused by glucose, aiming to further release CCR. Random mutagenesis released CCR in PC-3-7, although the molecular mechanism was not determined . We found that disruption of the tubB gene enabled efficient enzyme production by utilizing cellulose in the presence of glucose. High protein production at a high glucose concentration cannot be achieved by knockout of cre1 alone . Thus, PC-3-7ΔtubB strain may be applicable to various T. reesei industrial enzyme production processes, such as fed-batch culture with insoluble inducers (such as cellulose and lignocellulose) and soluble sugars , fed-batch culture with glucose and soluble inducers , and fed-batch culture with glucose by gene modification of cellulase transcription activators .
High protein production, a characteristic of the tubulin-disrupted strain, was observed when the strain was cultured with only cellulose, cellulose and xylan, or cellulose and glucose. This strain also has a high production rate of enzyme. Figure 4 shows rapid enzyme production by E1AB1, PC-3-7ΔtubB::Pegl1-aabgl1 and PC-3-7ΔtubB. Protein production by E1AB1 and PC-3-7ΔtubB::Pegl1-aabgl1 was complete after 5 days, whereas PC-3-7ΔtubB continued to produce protein for 6 days, resulting in higher protein production than the other strains. The early cessation of protein production by E1AB1 and PC-3-7ΔtubB::Pegl1-aabgl1 may be due to fast degradation of cellulose as a result of the secretion of AaBGL1 into the culture medium. On the other hand, PC-3-7ΔtubB does not produce AaBGL1 but its enzyme production rate is similar to that of E1AB1. Therefore, disruption of the tubB gene causes the increase in the rate of enzyme production regardless of the high expression of BGL. Figure 5d shows that PC-3-7ΔtubB generates more CO2 than PC-3-7 from the early stage of fermentation when cultured with cellulose, indicating that cellulose is efficiently degraded and assimilated by PC-3-7ΔtubB. Since PC-3-7ΔtubB can produce enzyme from the early stage of fermentation due to further release of CCR, PC-3-7ΔtubB can assimilate more sugars released from cellulose than PC-3-7, which may improve the rate of enzyme production.
PC-3-7ΔtubB has a higher uptake rate of cellobiose than PC-3-7 (Fig. 8). Sugar transporter genes are upregulated in PC-3-7ΔtubB, with cellobiose transporter ID 69957 being the most upregulated and cellobiose transporter crt1 being the highest expressed transporter (Additional file 10: Table S7). In addition, when cultivated in a mixture of cellobiose and glucose, PC-3-7 did not decrease the concentration of cellobiose until glucose was depleted whereas PC-3-7ΔtubB decreased the cellobiose concentration (Fig. 8c, d). Moreover, when cultured with cellulose and glucose, no cellobiose could be detected in PC-3-7 until the glucose was depleted, whereas cellobiose was released similarly by PC-3-7ΔtubB as when cultivated with only cellulose (Fig. 5f, g). These results suggest that cellulase was produced even in the presence of glucose by PC-3-7ΔtubB and cellobiose was released from cellulose into the culture broth, but cellulase was not produced by PC-3-7. Furthermore, the high cellobiose uptake ability of PC-3-7ΔtubB even in the presence of glucose is evidence of its further resistance to CCR compared to PC-3-7. The ability of faster and de-repressed cellobiose uptake may eliminates the product inhibition of CBH by keeping the extracellular cellobiose concentration at low level (Fig. 5f), and reduces the excessive generation of glucose by uptake before cellobiose is degraded by BGL, leading to less CCR. In addition, faster uptake of cellobiose may lead to an increase in the intracellular cellobiose concentration, which may help keep cellulase induction condition by cellobiose. These three aspects may explain the high amount and rate of enzyme production by tubB-deleted strain.
The CAZyme genes upregulated by tubB deletion were egl4 (gene ID: 73643), xyn1(74223), xyn2 (123818), xyn4 (111849), and axe1 (73632) (Fig. 6, Additional file 5: Table S2). In addition, other genes such as cip2 (123940), bxl1 (121127), aguA (72526) and glycoside hydrolase family 27 (putative α-galactosidase, 72704) were also highly expressed in PC-3-7ΔtubB (Additional file 8: Table S5). Since all the expressed genes except for egl4 are hemicellulases, deletion of the tubB gene may affect the induction of hemicellulases by cellulose.
In T. reesei QM9414, xyn1 was induced by xylose and xylan but not by cellulose, whereas xyn2 was induced by xylan and cellulose . In PC-3-7, both xyn1 and xyn2 were induced by cellulose, xylose and xylan, but the expression of xyn1 was lower than that of xyn2 . According to previous studies, the response of xyn1 to cellulose changed following the mutation of QM9414 to PC-3-7. In PC-3-7ΔtubB, the expression level of xyn1 induced by cellulose increased to the same level as that of xyn3, which was reported to be highly expressed in the presence of cellulose. The expression of xyn1 was activated by Xyr1 and repressed by the transcription factors Cre1 and Ace1 . However, in the present study, the expression of cre1 and ace1 was not significantly changed by tubB deletion, suggesting that the high expression of xyn1 was not due to changes in the expression levels of these transcription factors. The upregulation of many CAZyme genes by tubB deletion indicates an unknown regulatory mechanism for these genes involving tubB.
This study revealed that α2-tubulin in T. reesei is involved in the transcriptional regulation of cellulases and hemicellulases, CCR, cellobiose uptake, and protein production. Additionally, the α2-tubulin-deficient strain showed further resistance to CCR. This strain has the very useful characteristic of high resistance to CCR and high enzyme production even in the presence of glucose. Therefore, PC-3-7ΔtubB may be applicable to all T. reesei enzyme production processes utilizing glucose or cellulose as a carbon source. This study will help reduce the cost of enzyme production and aid the low cost production of sugar from cellulosic biomass.
Fungal strains and maintenance
The fungal strains are listed in Table 1. The strains were maintained on potato dextrose agar (PDA, Difco Laboratories, Detroit, MI) plates.
For preculture for enzyme production, 107 conidia of each strain were inoculated into 50 mL of basal medium  containing 1% (w/v) glucose in a 500-mL Erlenmeyer flask. Conidia were counted using a TC20 Automated Cell Counter (Bio-Rad, Hercules, CA). The basal medium comprised 0.14% (w/v) (NH4)2SO4, 0.2% (w/v) KH2PO4, 0.03% (w/v) CaCl2·2H2O, 0.03% (w/v) MgSO4·7H2O, 0.1% (w/v) polypeptone, 0.05% (w/v) yeast extract, 0.1% (w/v) Tween 80 and 0.1% (w/v) trace element solution in 50 mM tartarate-Na buffer (pH 4.0). The trace element solution contained 6 mg of H3BO3, 26 mg of (NH4)6Mo7O24·4H2O, 100 mg of FeCl3·6H2O, 40 mg of CuSO4·5H2O, 8 mg of MnCl2·4H2O and 200 mg of ZnCI2 in 100 mL of distilled water. Precultures were carried out with shaking at 220 rpm and 28 °C for 2 days. The main cultures (1 L) of basal medium containing carbon sources such as microcrystalline cellulose (Avicel PH 101, Sigma–Aldrich, St. Louis, MO), xylan (beechwood xylan, Tokyo Chemical Industry Co. Ltd., Tokyo, Japan) and glucose (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in a 2-L jar fermenter (BMZ-01KP2, Biott Co., Ltd., Tokyo, Japan) were inoculated with 100 mL of preculture. The main culture was carried out at 28 °C, with 0.5 volumes of air per volume of liquid per minute aeration and 3 ppm dissolved oxygen controlled by mechanical agitation. The pH was controlled at 4.5 using ammonia solution (5% w/v). An aliquot of the culture broth (2 mL) was collected every 24 h, cells were harvested by centrifugation at 20,000 × g for 5 min, then the supernatant was filtered through a 0.20-μm cellulose acetate membrane filter (13CP020AN; Advantec, Toyo Roshi Kaisha Ltd., Tokyo, Japan). The endpoint of the cultivation was the sampling point after the ammonia solution, which is a nitrogen source and used to keep the pH constant, was no longer supplied. The culture supernatants were analyzed to determine protein concentration using the Bradford protein assay (Bio-Rad) with bovine gamma globulin as the standard. The fermentations were carried out in triplicate. Student's t-test was used to compare the amount of protein production, and a p value less than 0.05 was considered statistically significant.
To study the morphology of the tubB-deficient strains, transformants and parent strain were cultivated on PDA plate. The plates were grown at 30 °C for 4 days and the colony size (diameter) was measured for every day. The colony size was compared using Student's t-test.
Scanning electron microscope (SEM) observation
SEM samples were prepared according to a previously reported method  with minor modifications. T. reesei was grown in 50 mL of basal medium containing 1% (w/v) glucose for 1 day or 1% (w/v) cellulose for 3 days in a 500-mL Erlenmeyer flask at 28 °C with shaking at 250 rpm. Hyphae were fixed in 2% (v/v) glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4. Post-fixation procedures were conducted by Hanaichi Ultrastructure Research Institute Co., Ltd. (Aichi, Japan). For SEM image analysis, 70 cells were randomly chosen and cell length and diameter were measured using ImageJ (http://imagej.nih.gov/ij/). The cell length was measured from septum to septum. The length, diameter and ratio (length/diameter) of the cells were compared using Student's t-test.
Cell growth analysis
For preculture, 107 conidia of each strain were inoculated into 50 mL of basal medium containing 1% (w/v) glucose in a 500-mL Erlenmeyer flask. Main culture was started by inoculating 5 mL of precultures in the 50 mL of basal medium with 1%(w/v) glucose in a 500-mL Erlenmeyer flask. The culture was carried out with shaking at 220 rpm and 28 °C for 24 h. An aliquot of the culture broth (2 mL) was collected as appropriate, and cells were harvested by centrifugation at 20,000×g for 5 min, then the supernatant was filtered through a 0.20-μm cellulose acetate membrane filter (13CP020AN) and used for sugar analysis. Cells were washed twice with 1 mL of distilled water, and then the wet cell weight was measured.
Glucose, xylose and cellobiose in the culture supernatants were analyzed according to a previously reported method  with minor modifications. The samples were diluted and analyzed on a LaChrom Elite HPLC System (Hitachi High-Technologies, Ibaraki, Japan) equipped with an ICSep-ION-300 column (Tokyo Chemical Industry Co. Ltd.). Separation was performed by isocratic elution with 5 mM H2SO4 at a flow rate of 0.5 mL/min for 30 min.
SDS-PAGE was carried out with Any kD Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad) for 35 min at 200 V. The gel was activated for 5 min and imaged using the ChemiDoc MP imaging system (Bio-Rad). The molecular weights of protein bands were estimated using Image Lab software (Bio-Rad). The molecular mass marker comprised 5 or 10 μL of Precision Plus Protein Unstained Standard (Bio-Rad).
Identification of the locus of the inserted cassette by inverse PCR
Genomic DNA was extracted and purified using a modified version of the protocol from Seiboth et al. . The genomic DNA was treated with the restriction enzymes XbaI and XhoI (Thermo Fisher Scientific, San Jose, CA) and then self-ligated using Ligation High (Toyobo, Osaka, Japan). Self-ligated DNAs were used as templates for PCR amplifications using the DNA polymerase PrimeSTAR Max (Takara Bio, Shiga, Japan). Amplified fragments were cloned and sequenced.
Construction of plasmids for deletion of the tubB gene
The PCR primers are listed in Additional file 11: Table S8. Restriction sites for vector linearization and vector-specific extensions were introduced into these primers to facilitate cloning as required. The amplified fragments were ligated using an In-Fusion HD cloning kit (Takara Bio). Plasmids, primer pairs and templates used for construction of the plasmids are listed in Additional file 12: Table S9.
Construction of pUC-ΔtubB-amdS
The promoter, ORF, transcription terminator, and 3′ flanking sequences of T. reesei cbh1 were amplified from the genomic DNA of T. reesei strain PC-3-7 using the primers swaI tubB F and swaI tubB R. A vector fragment was amplified by inverse PCR using pUC118 (Takara Bio) as the template with the primers swaI pUC F and swaI pUC R. The amplified fragments were ligated using an In-Fusion HD cloning kit to yield pUC-tubB.
The gene amdS (encoding an A. nidulans acetamidase gene) was amplified from pUC-amdS using the primers amdS F and amdS R . The fragment generated by inverse PCR using pUC-tubB with the primers tubBback-amdS F and tubBfront-amdS R was fused to the amdS fragment to generate pUC-ΔtubB-amdS.
Construction of pUC-ΔtubB-Pegl1-aabgl1-amdS
The aabgl1 expression cassette with the amdS marker containing the egl1 promoter, the ORF of aabgl1, and the transcription terminator and 3′ flanking sequences of egl1 and amdS marker were amplified from the genomic DNA of T. reesei strain E1AB1 using the PCR primers tubBfront-Pegl1 F and amdS R. A vector fragment was amplified by inverse PCR using pUC-tubB with the primers tubBback-amdS F and tubBfront R. The obtained fragments were ligated to yield pUC-ΔtubB-Pegl1-aabgl1-amdS.
Transformation of T. reesei
Plasmids were linearized with SwaI prior to transformation. T. reesei was transformed using the protoplast-PEG method  modified to use Yatalase (Takara Bio) as a protoplasting enzyme instead of Novozyme 234 (Novozymes, Bagsværd, Denmark). Transformed protoplasts were plated on transformation medium [2.0% (w/v) glucose, 18.27% (w/v) sorbitol, 0.06% (w/v) acetamide, 0.2% (w/v) CaCl2, 0.06% (w/v) MgSO4, 0.21% (w/v) CsCl and 0.1% (w/v) trace element solution in 100 mM KH2PO4 buffer (pH 5.5)]. The trace element solution contained 500 mg of FeSO4·7H2O, 200 mg of CoCl2, 160 mg of MnSO4·H2O and 140 mg of ZnSO4·7H2O in 100 mL of distilled water. After 2 weeks’ incubation at 28 °C, candidate transformants were streaked twice on selective plates (transformation medium without sorbitol) for several days at 28 °C to isolate single colonies, then single colonies were transferred to PDA plates to form conidia for 1 week at 28 °C. One transformant was confirmed by colony PCR using KOD FX Neo (Toyobo) according to the manufacturer's protocol.
Cellulase activities were measured as pNPGase for BGL activity, pNPLase for CBH1 activity, and CMCase for EG activity. Hemicellulase activities were measured as pNPXase for BXL activity and pNPX2ase and xylanase for XYN activity. Enzymatic activity against synthetic substrate was measured by assaying the p-nitrophenol released from 1 mM p-nitrophenyl glucopyranoside (pNPG, Sigma-Aldrich), p-nitrophenyl lactoside (pNPL, Sigma-Aldrich), p-nitrophenyl xyloside (pNPX, Sigma-Aldrich), and p-nitrophenyl xylobioside (pNPX2, Megazyme) in 50 mM sodium acetate buffer (pH 5.0) at 50 °C for 10 min. Reactions were performed in a total volume of 0.1 mL and stopped by adding an equal volume of 0.4 M NaHCO3. The released p-nitrophenol was quantified by measuring the absorbance at 420 nm. One unit of activity was defined as the amount of enzyme that produced 1 μmol of p-nitrophenol per minute from the substrate. CMCase and xylanase activities were determined with 1%(w/v) carboxymethyl cellulose (Tokyo Chemical Industry) and 1%(w/v) beechwood xylan (Tokyo Chemical Industry) in 50 mM sodium acetate buffer (pH 5.0) at 50 °C for 10 min. Reactions were performed in a total volume of 0.1 mL and stopped by adding an equal volume of alkaline 3,5-dinitrosalicylic (DNS) and heating for 5 min at 100 °C . Then the absorbance at 540 nm was detected. One unit of activity was defined as the amount of enzyme that produced 1 μmol of reducing sugar per minute in glucose equivalents.
Total RNA extraction and cDNA synthesis were performed using an RNeasy Plant Mini Kit (Qiagen, Crawley, UK) according to the manufacturer's instructions. Total RNA was employed for next-generation sequencing libraries using a TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA). Libraries were sequenced using the Illumina MiSeq platform. The obtained sequences were quality-filtered by CLC Genomics Workbench using the default settings. Filtered reads were mapped to T. reesei QM6a cds sequences downloaded from the Ensembl Fungi database (https://fungi.ensembl.org/Trichoderma_reesei/Info/Index/) by CLC Genomics Workbench using the default settings. The raw count matrix consisting of 9,115 genes × 24 samples (= 2 strains × 2 sampling times × 2 media × 3 biological replicates) was obtained. The RPKM of each gene was calculated by CLC Genomics Workbench using the default settings. The raw read count matrix for every gene and every sample was shown in Additional file 13: Table S10. RPKM values for every gene for each sample was shown in Additional file 14: Table S11.
Differentially expressed gene (DEG) analysis
Normalization of the raw count matrix and identification of the DEGs was performed using R (ver. 3.6.1, R Core Team (2018). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/.) and DESeq2 package . Genes for which the adjusted p < 0.01 were identified as statistically significant DEGs.
Gene ontology (GO) enrichment analysis of DEGs
GO enrichment analysis was implemented by using the R and GOseq packages based on Wallenius non-central hyper-geometric distribution , which can adjust for gene length bias in DEGs. GO annotations of each gene were obtained from the JGI database (http://genome.jgi.doe.gov/Trire2/Trire2.home.html). GO terms with over-represented p < 0.01 were considered significantly enriched in DEGs.
Availability of data and materials
The datasets used and/or analyzed during the current study are included in this published article and its supplemental information files.
Carbon catabolite repression
Differentially expressed gene
Open reading frame
Scanning electron microscope
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We would like to thank Prof. Yasushi Morikawa from Nagaoka University of Technology for valuable comments during this study. This study was partly supported by the New Energy and Industrial Technology Development Organization (NEDO) under the Ministry of Economy, Trade and Industry, Japan.
This study was partly supported by the New Energy and Industrial Technology Development Organization (NEDO) under the Ministry of Economy, Trade and Industry, Japan.
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Maps of the aabgl1-expression cassette integrated in the T. reesei E1AB1 and PC-3-7 genomes. (a) Maps of the genome around the tubB gene (Gene ID: 120830) of PC-3-7 and E1AB1 are shown on the top and bottom, respectively. The arrows indicate the gene and its direction of transcription and the squares indicate the promoter and terminator region. Blue, green, yellow and orange indicate the sequences around tubB, egl1, amdS and aabgl1, respectively. Scale bar indicates 1 kb. The sites where the primers used to determine the inserted locus could anneal are marked with black arrows. (b) Identification of inserted locus by PCR amplification. At the top of each lane, the genome used as template and the primer number used are indicated. M in the lane indicates marker (Gene Ladder Wide 2, Nippon Gene, Toyama, Japan).
. Cell growth and glucose consumption of PC-3-7, E1AB1 and PC-3-7ΔtubB in the basal medium with glucose. Culture was started by inoculating 5 mL of precultures in the 50 mL of basal medium with 1%(w/v) glucose in a 500-mL Erlenmeyer flask. The culture was carried out with shaking at 220 rpm and 28°C for 24 hours. Blue line indicates PC-3-7, orange line indicates E1AB1 and gray color indicates PC-3-7ΔtubB. a Wet cell weight. b Glucose concentration in the culture broth. Data are expressed as mean ± SD of three biological replicates.
Specific enzyme activity and SDS-PAGE analysis of the produced protein. a Specific activity of cellulolytic and hemicellulolytic enzyme of PC-3-7 and PC-3-7ΔtubB when cultivated in medium with 10% (w/v) microcrystalline cellulose or 10% (w/v) microcrystalline cellulose and 2.5% (w/v) glucose. Data are expressed as mean ± SD of three biological replicates. SDS-PAGE analysis loading 1 μL of each culture supernatant after 120 hours of cultivation (b) and loading 5 μg of each protein amount (c). The molecular mass marker comprised 5 μL (c) or 10 μL (d) of Precision Plus Protein Unstained Standard (Bio-Rad). Lanes: Precision Plus Protein Unstained Standard (M); supernatant of PC-3-7 cultivated in the medium with 10% (w/v) microcrystalline cellulose (1) and cultivated in the medium with 10% (w/v) microcrystalline cellulose and 2.5% (w/v) glucose (2); supernatant of PC-3-7ΔtubB cultivated in the medium with 10% (w/v) microcrystalline cellulose (3) and cultivated in the medium with 10% (w/v) microcrystalline cellulose and 2.5% (w/v) glucose (4).
RPKM values of tubulins.
RPKMs of major CAZymes.
RPKM values of transcription factors.
Correlation matrix of RPKMs between all RNA-seq conditions.
CAZymes that were significantly changed in the PC-3-7ΔtubB compared to PC-3-7.
Enriched gene ontology categories of DEGs between PC-3-7 and PC-3-7ΔtubB.
The differentially expressed predicted sugar transporter genes.
Plasmids, PCR templates and primers used for plasmid construction.
Matrix table with raw read counts.
RPKM values for every gene.
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Shibata, N., Kakeshita, H., Igarashi, K. et al. Disruption of alpha-tubulin releases carbon catabolite repression and enhances enzyme production in Trichoderma reesei even in the presence of glucose. Biotechnol Biofuels 14, 39 (2021). https://doi.org/10.1186/s13068-021-01887-0
- Trichoderma reesei
- Biomass saccharification enzyme
- Carbon catabolite repression
- Glucose resistant