Putative sugar transporters involved in cellulase production in T. reesei Rut-C30 with lac1 being essential
To find sugar transporters related to cellulase production, transcriptome sequencing analysis was performed by comparing Rut-C30 grown on cellulose to the one on glucose. A total of 49 Differentially Expressed Genes (DEGs) were predicted to be sugar transporters for maltose, fucose, xylose, lactose, d-galactonate, and unknown sugars (Fig. 1A and Additional file 1: Table S1). 33 DEGs were upregulated, of which the fold change of gene M419DRAFT_127980 (lac1) was highest followed by M419DRAFT_138519 (gst), while 16 DEGs were downregulated. In these DEGs, the top three sugar transporter genes with the highest expression level under cellulose condition were M419DRAFT_109243 (crt1), M419DRAFT_137795 (mfs), and M419DRAFT_138519 (gst) according to Fragments Per Kilobase of exon model per Million mapped fragments (FPKM) (Additional file 1: Table S1). Therefore, the expression levels of these four genes (crt1, mfs, gst, and lac1) were measured by qRT-PCR in T. reesei Rut-C30 cultivated on cellulose (Fig. 1B). Gene crt1 possessed the highest mRNA expression level, followed by mfs, gst, and lac1 in a decreasing order. All these four transporter genes exhibited higher mRNA levels in T. reesei grown on cellulose than on glucose, marching well the transcriptome data and demonstrating that their increased expression might benefit the cellulase production. As shown by structure analysis, all four sugar transporters belong to MFS transporters. There are 12 transmembrane domains in CRT1 and GST, and 11 in MFS and LAC1 (Fig. 1C) [26]. Sugar transporters CRT1, MFS, and GST have the n-glycosylation motifs, while LAC1 does not. The transporting sugars of MFS and GST are unknown, and CRT1 and LAC1 are putative lactose permeases. Since gene crt1 essential for cellulase production has been well studied [16, 22, 24, 27], we focused on the other three sugar transporters mfs, gst, and lac1 in further study.
Next, sugar transporter genes mfs, gst, and lac1 were deleted individually using Ku70 as the parental strain, leading to the knockout strains ΔMFS, ΔGST, and ΔLAC1. The cellulase-producing ability of these recombinant strains were assayed (Fig. 1D and Additional file 2: Fig. S1). The individual deletion of the three sugar transporter genes notably inhibited cellulase production. The FPase activity, CMCase activity, pNPCase activity, pNPGase activity, pNPxase activity, and secreted protein concentration in strain ΔMFS were 1.42 IU/mL, 1.69 IU/mL, 0.29 IU/mL, 0.43 IU/mL, 0.26 IU/mL, and 3.74 mg/mL, only 39.75%, 38.66%, 37.19%, 51.57%, 36.30%, and 54.65% of that in strain Ku70, respectively. The decreased degree of cellulase production in strain ΔGST was similar to strain ΔMFS, while higher inhibition effect was found in strain ΔLAC1 than in strain ΔMFS. The cellulase production was almost abolished in strain ΔLAC1, indicating that it is essential for cellulase production. This is very interesting given that the mRNA abundance of lac1 was lowest among the four tested sugar transporters (Fig. 1B). It seems that although its mRNA was very low, lac1 is still indispensable to cellulase production.
In addition, the phenotype of the deletion strains was profiled in terms of cell growth and sporulation ability. The growth of strain Ku70 and deletion strains was investigated by measuring colony diameters on TMM + 2% cellulose plates (Fig. 1E). The colony diameters of the recombinant strains were smaller than that of Ku70 with strain ΔLAC1 possessing the smallest colony diameter. The sporulation ability of the three deletion strains was decreased notably (Fig. 1F). The lowest sporulation amount was found in strain ΔLAC1, only 26.1% of that in Ku70. Taken together, knocking-out genes encoding sugar transporters mfs, gst, and lac1 impaired cellulase production, cell growth, and the sporulation ability in T. reesei Ku70 with lac1 deletion being the worse. Particularly, it seems that LAC1 is required for cellulase production in Ku70 on cellulose.
The impact of the three sugar transporters on sugar consumption and biomass formation
We assessed the sugar uptake ability of the knockout strains ΔMFS, ΔGST, and ΔLAC1 on disaccharides (cellobiose and lactose) and monosaccharides (glucose, galactose, and mannose). As shown in Fig. 2A, we found that the consumption of all the tested sugars by strains ΔMFS, ΔGST, and ΔLAC1 was delayed to varying degrees compared with strain Ku70. Cellobiose, galactose, and lactose were not fully absorbed within 48 h in all T. reesei strains. The utilization of cellobiose was noticeably repressed in all the three deletion strains. Deletion of gene lac1 inhibited most the utilization of lactose, followed by genes mfs and gst, which is reasonable given that LAC1 is predicted to be lactose permease. The residual glucose in the supernatant of Ku70 was decreased gradually to zero in 36 h, while that in strains ΔMFS, ΔGST, and ΔLAC1 was not reduced significantly in 24 h, but fell sharply from 24 to 48 h. On mannose, a similar pattern to that on cellobiose or glucose was observed in strains ΔMFS and ΔGST but not in ΔLAC1. The mannose uptake of strain ΔLAC1 was comparable to strain Ku70. It seems that the transporters MFS, GST, and LAC1 are involved in the cellular uptake and/or utilization of all the tested sugars (cellobiose, lactose, glucose, galactose, and mannose), except that LAC1 might not be related to that of mannose. The recovery of sugar internalization after 24 h inhibition indicated that there might be functional redundancy proteins for these sugar transporters in T. reesei. The presence of these sugar transporters enables T. reesei to use sugar in a rapid way.
Moreover, the colony diameters of strains T. reesei Ku70, ΔMFS, ΔGST, and ΔLAC1 were calculated on solid medium containing 1% (w/v) cellobiose, lactose, glucose, galactose, mannose, or cellulose (Fig. 2B and C). The radial growth of the mutant strains ΔMFS and ΔGST did not exhibit significant difference in the presence of respective tested sugar. Nevertheless, the colony diameter of strain ΔLAC1 was noticeably decreased in comparison to that of Ku70 on all tested sugar, indicating that the absence of lac1 severely retarded the biomass accumulation in T. reesei.
To further investigate their sugar transport function, genes mfs, gst, and lac1 were heterologously expressed in S. cerevisiae EBY.VW4000 to obtain recombinant strains 4000-MFS, 4000-GST, and 4000-LAC1, respectively. The empty plasmid pRS426 was also transformed into S. cerevisiae EBY.VW4000 to get the control strain 4000-pRS426. The mutant strains were spotted on SC-Ura− plates supplemented with 1% lactose, 1% glucose, 1% galactose, or 1% mannose (Fig. 2D). Maltose is the only carbon source that S. cerevisiae EBY.VW4000 can used for growth; thus, the mutant strains grew best on the plate containing maltose and showed insignificant difference between each other. Meanwhile, all the strains did not grow out on glucose-containing plates. Strains 4000-pRS426 were unable to grow on lactose, galactose, and mannose, while strains 4000-MFS, 4000-GST, and 4000-LAC1 could utilize these sugars to grow in varying degrees. However, the mutant strains were not grown very well, indicating the transport ability of MFS, GST, and LAC1 for these sugars were very weak. Moreover, since S. cerevisiae cannot assimilate cellobiose, β-glucosidase gene gh1-1 and genes mfs, gst, and lac1 were co-expressed in S. cerevisiae W303 to obtain recombinant strains 303-gh1-MFS, 303-gh1-GST, and 303-gh1-LAC1, respectively. In the same way, we also obtained control strain 303-gh1-pRS426 by transforming the empty plasmid pRS426 into S. cerevisiae W303. Both strain 303-gh1-pRS426 and strain 303-gh1-LAC1 were not grown out on cellobiose, while strains 303-gh1-MFS and 303-gh1-GST were grown well on cellobiose. Altogether, sugar transporters MFS, GST, and LAC1 did not transport glucose or maltose, and might weakly transport lactose, galactose, and mannose. Only sugar transporters MFS and GST can transport cellobiose, while sugar transporter LAC1 cannot.
Taken together, the absence of sugar transporters delayed but not compromised sugar uptake in T. reesei. All three sugar transporters displayed weak sugar transporting ability toward cellobiose, lactose, galactose, and mannose, and did not transport glucose and mannose. The deletion of lac1 inhabits significantly biomass formation of T. reesei regardless of sugar type, while the absence of mfs and gst did not. These results demonstrate that the three sugar transporters facilitate sugar consumption, though they possessed low sugar transporting ability, and lac1 plays a highly positive role in biomass formation on different sugars.
Cellular distribution of the sugar transporters
We further studied the cellular distribution of the sugar transporters under the control of their own promoters by integrating fluorescence gene DsRed to their 3′-UTR through homologous recombination in T. reesei Ku70 (Additional file 3: Fig. S2), leading to recombinant strains MFS-DsRed, GST-DsRed, and LAC1-DsRed that expressed DsRed-tagged fusion proteins MFS-DsRed, GST-DsRed, and LAC1-DsRed, respectively. By confocal laser scanning microscopy (CLSM), strong red fluorescence was observed in strain MFS-DsRed grown on cellulose or glucose (Fig. 3A), featuring as dispersed cortical puncta and the characteristic perinuclear ER rings that are often seen in fungi [28,29,30,31,32]. Although it was not that significant, the distribution of MFS-DsRed on the cell membrane was found (Additional file 4: Fig. S3), which is consistent with the structure analysis that MFS contains the signal peptide (Fig. 1C). Red fluorescence was also found in strain GST-DsRed on cellulose with weaker fluorescence intensity than that in strain MFS-DsRed. The fluorescence intensity of strain GST-DsRed grown on glucose was too weak to be observed. Unfortunately, no red fluorescence was detected in strain LAC1-DsRed on cellulose or lactose, possibly owing to the low mRNA level of lac1 (Fig. 1B). Therefore, LAC1-DsRED was overexpressed under the strong promoter CBH to investigate cellular distribution of LAC1, resulting in the recombinant strain LAC1-DsRED-OE that displayed almost the same (hemi)cellulose activities to the parental strain C30 (Additional file 5: Fig. S4). Strain LAC1-DsRed-OE also showed red fluorescence with the structure of ER rings as found in strain MFS-DsRed and GST-DsRed (Fig. 3). All sugar transporters were not accumulated at apical regions (Additional file 6: Fig. S5). No ER ring was found in in the control strain DsRed expressing red fluorescence protein alone. To further verify that all three sugar transporters are localized in ER, mutant strains MFS-DsRed, GST- DsRed, and LAC1-DsRed were treated with ER-tracker (Fig. 3B). The co-localization of all three sugar transporters MFS-DsRed, GST-DsRed, and LAC1-DsRed with ER was observed, as indicated by the observation of yellow florescence. On the contrary, when treated with ER-tracker, no yellow fluorescence was observed strain DsRed. In short, these three sugar transporters are intracellular sugar transporters, being localized in ER.
The effect of gene lac1 on the transcriptome of T. reesei
To explore the role of lac1 in cellulase production, we performed the transcriptome analysis of strain ΔLAC1 cultured on cellulose at 72 h. Compared with strain Ku70, there are 2471 deferentially expressed genes (DEGs) in strain ΔLAC1, of which 1454 genes were downregulated and 1017 were upregulated (Additional file 7: Table S2). Significant gene ontology (GO) functional enrichment analysis of these DEGs in strain ΔLAC1 demonstrated that the most enriched biological process (BP) was “carbohydrate metabolic process,” which includes “carbohydrate catabolic process” and “monocarboxylic acid biosynthetic process” (Fig. 4A). The other enriched biological processes belong to “ribosome biogenesis” and “protein glycosylation.” In the category of cellular components (CC), DEGs were concentrated in “extracellular region” and “intrinsic component of membrane.” For the enriched molecular function, “hydrolase activity” has the maximum DEGs, followed by “acetyltransferase activity” and “carbohydrate binding.” All DEGs in “polysaccharide binding” and “cellulose binding,” which are the subcategories of “carbohydrate binding,” were downregulated. By Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs, we screened the top 5 KEGG pathways with padj-value < 0.05, including “ribosome biogenesis in eukaryotes,” “protein processing in endoplasmic reticulum,” “glycerophospholipid metabolism,” “ether lipid metabolism,” and “steroid biosynthesis” (Fig. 4B). 27 of 30 DEGs in “protein processing in endoplasmic reticulum” were downregulated (Additional file 8: Table S3), and 28 of 30 DEGs in “glycerophospholipid metabolism,” “ether lipid metabolism,” and “steroid biosynthesis” were downregulated except genes M419DRAFT_72858 and M419DRAFT_25484 (Additional file 9: Table S4). On the contrary, the DEGs in “ribosome biogenesis in eukaryotes” were all upregulated (Additional file 10: Table S5). To summarize, the absence of gene lac1 had impact on cellulase synthesis, ribosome biogenesis, protein glycosylation, lipid metabolism, and acetylation.
A total of 47 DEGs were known or predicted to be related to (hemi)cellulase production in T. reesei (Fig. 4C and Additional file 11: Table S6). Most of these DEGs were downregulated, except that genes M419DRAFT_127187, M419DRAFT_12566, vel1, lae1, bglR, and stp1 (Fig. 4C and Additional file 11: Table S6) were upregulated. Particularly, the transcriptional levels of (hemi)cellulase genes, including 2 cellobiohydrolases (cel6a and cel7a), 7 endoglucanases (cel5a, cel7b, cel45a, cel61b, cel12a, cel74a, and cel61a), 6 β-glucosidases (cel3f, cel3a, cel3d, cel3b, cel3j, and cel3g), 6 xylanases (xyn5, xyn6, xyn3, xyn1, xyn2, and xyn4), and 1 β-xylosidase (bxl1), were all reduced markedly, matching well with the decreased (hemi)cellulase activities as observed above. Genes encoding the auxiliary proteins, like the cellulose-induced proteins CIP1 and CIP2 [33], which have been reported to enhance cellulose degradation, were also down-expressed markedly. Furthermore, 7 cellulase transcription factors were significantly downregulated, including the well-known cellulase transcription activators XYR1 [34] and ACE3 [35]. The transcriptional level of MFS sugar transporter gene stp1, the absence of which was found to enhance the cellulase gene induction, was upregulated [22, 36].
The mRNA dynamic of crucial genes related to cellulase production in T. reesei ΔLAC1
We analyzed the relative expression level of genes related to cellulase production, including (hemi)cellulase genes (cel3a, cel7a, cel7b, and bxl1), cellulase transcriptional factors (activators xyr1, ace3, and clr2, repressor xpp1, and clr3), sugar transporter genes (crt1 and stp1), and lactose metabolism-related gene xyl1 in T. reesei Ku70 and ΔLAC1 during the whole fermentation process for cellulase generation (Fig. 5). The mRNA levels of all these genes were reduced significantly at 24 h in strain ΔLAC1, except genes clr3, xpp1, and stp1. The mRNA levels of (hemi)cellulase genes (cel3a, cel7a, cel7b, and bxl1) and the well-known cellulase transcriptional activator XYR1 in ΔLAC1 were all severely reduced at 24 h compared to Ku70, which, however, was recovered to comparable levels to that in Ku70 during the rest fermentation process (Fig. 5A). At 168 h, there was no significant difference in the transcriptional level of genes cel3a, cel7a, bxl1, and xyr1 between strain Ku70 and strain ΔLAC1, while the expression level of gene cel7b of strain ΔLAC1 was much higher than that of strain Ku70. When looking into the data closely, the totally different expression patterns of (hemi)cellulase genes were found in strains Ku70 and ΔLAC1. The relative expression levels of these four genes in strain Ku70 were highest in 24 h and then decreased. However, the expression levels of these four genes of strain ΔLAC1 peaked at 120 h. As compared to strain Ku70, the expression levels of two cellulase transcription activators ACE3 and CLR2 (Fig. 5B), sugar transporter CRT1 (Fig. 5C), and lactose metabolism-related gene xyl1(Fig. 5D) in strain ΔLAC1 were reduced at 24 h, were comparable to Ku70 during the middle fermentation stage, and were decreased again at 168 h. The mRNA abundance of gene clr3 in strain ΔLAC1 was increased at 24 h, but reduced after 24 h. The expression level of sugar transporter STP1 was increased in strain ΔLAC1 during the whole fermentation process, while gene xpp1 stayed constant in strain ΔLAC1 at 24 h and 120 h, but was much higher than Ku70 at 72 h and lower at 168 h, exhibiting a fluctuated change.