Effects of the disruption of repressors and the constitutive expression of V821F mutated Xyr1
The parental strain was the high-producing cellulase strain T. reesei E1AB1 [14], and the strains generated in this study are listed in Table 1.
Table 1 Trichoderma reesei strains used in this study
Strains
|
Genotypes
|
Parents
|
PC-3-7
|
-
|
-
|
E1AB1
|
amdS+, Peg1-aabg1
|
PC-3-7
|
E1AB1-Δpyr4
|
amdS+, Peg1-aabg1, Δpyr4
|
E1AB1
|
E1AB1-da
|
amdS+, Peg1-aabg1, Δace1
|
E1AB1-Δpyr4
|
E1AB1-dr
|
amdS+, Peg1-aabg1, Δrce1
|
E1AB1-Δpyr4
|
E1AB1-daX821F
|
amdS+, Peg1-aabg1, Δace1::Pact1-xyr1 (V821F)
|
E1AB1-Δpyr4
|
E1AB1-daX821F-dr
|
amdS+, Peg1-aabg1, Δace1::Pact1-xyr1 (V821F), Δrce1
|
E1AB1-Δpyr4
|
E1AB1-daX821F-Δpyr4
|
amdS+, Peg1-aabg1, Δace1::Pact1-xyr1 (V821F), Δpyr4
|
E1AB1-daX821F
|
E1AB1-daX821F-drC
|
amdS+, Peg1-aabg1, Δace1::Pact1-xyr1 (V821F), Δrce1::Pact1-crt1
|
E1AB1-daX821F-Δpyr4
|
E1AB1-daX821F-drB
|
amdS+, Peg1-aabg1, Δace1::Pact1-xyr1 (V821F), Δrce1::Pact1-bglr
|
E1AB1-daX821F-Δpyr4
|
E1AB1-daX821F-drV
|
amdS+, Peg1-aabg1, Δace1::Pact1-xyr1 (V821F), Δrce1::Pact1-vib1
|
E1AB1-daX821F-Δpyr4
|
E1AB1-daX821F-drA2
|
amdS+, Peg1-aabg1, Δace1::Pact1-xyr1 (V821F), Δrce1::Pact1-ace2
|
E1AB1-daX821F-Δpyr4
|
E1AB1-daX821F-drA3
|
amdS+, Peg1-aabg1, Δace1::Pact1-xyr1 (V821F), Δrce1::Pact1-ace3 (nominal)
|
E1AB1-daX821F-Δpyr4
|
E1AB1-daX821F-drA3(complete)
|
amdS+, Peg1-aabg1, Δace1::Pact1-xyr1 (V821F), Δrce1::Pact1-ace3 (complete)
|
E1AB1-daX821F-Δpyr4
|
E1AB1-nhA3
|
amdS+, Peg1-aabg1, Pact1-ace3 (nominal)
|
E1AB1-Δpyr4
|
E1AB1-nhA3-Δpyr4
|
amdS+, Peg1-aabg1, Pact1-ace3 (nominal), Δpyr4
|
E1AB1-nhA3
|
E1AB1-nhX821F-nhA3
|
amdS+, Peg1-aabg1, Pact1-xyr1 (V821F), Pact1-ace3 (nominal)
|
E1AB1-nhA3-Δpyr4
|
E1AB1-nhX824V-nhA3
|
amdS+, Peg1-aabg1, Pact1-xyr1 (A824V), Pact1-ace3 (nominal)
|
E1AB1-nhA3-Δpyr4
|
E1AB1-nhXWT-nhA3
|
amdS+, Peg1-aabg1, Pact1-xyr1 (WT), Pact1-ace3 (nominal)
|
E1AB1-nhA3-Δpyr4
|
E1AB1-drA3
|
amdS+, Peg1-aabg1, Δrce1::Pact1-ace3 (nominal)
|
E1AB1-Δpyr4
|
For inducer-free enzyme production, we initially confirmed the effects of the disruption of the repressors and the constitutive expression of the Xyr1 mutation. To do this, we evaluated two repressors, ace1 [22] and rce1 [23], as it has previously been reported that cellulase transcription is increased by the disruption of these repressor genes. The ace1 or rce1 disrupted strains were generated and cultivated in shake flasks on a non-inducing medium containing 3% glucose. However, in cellulase non-inducing conditions when glucose was used as a carbon source, protein productivity after 120 h was extremely low for the E1AB1, Δace1, and Δrce1 strains (0.40, 0.55, and 0.44 g/L, respectively) when compared to that of inducing conditions with cellulose (5.55 g/L; Fig. 1a). Furthermore, cellulases/hemicellulases were not detected in the culture supernatant (Fig. 1b, lane 2-4).
We then constructed a strain with mutated Xyr1 (V821F) that was constitutively expressed using the constitutive promoter act1 in the ace1 locus (named E1AB1-daX821F strain). This new mutant strain showed a significant increase in extracellular protein production under non-inducing conditions (maximum 2.08 g/L at 96 h; Fig. 1a), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed distinct bands (Fig. 1b, closed arrowheads). Based on the estimated molecular weights, the protein bands likely corresponded to the main xylanases (XYN1, XYN2) and β-xylosidase (BXL1; Fig. 1b, lane 5), as was previously reported [14]; however, the composition was different from that of proteins produced by the parental strain E1AB1 in inducing condition (Fig. 1b, lanes 1 and 5). As shown by these results, hemicellulases were produced sufficiently but cellulases, such as CBH1, were not. Further rce1 gene disruption to the E1AB1-daX821F strain resulted in increased protein production (Fig. 1a), but no significant changes in the enzyme composition compared with the original E1AB1-daX821F strain (Fig. 1b, lane 6). Since the effect of repressor disruption to productivity was confirmed, subsequent constitutive expression of the transcription factors in E1AB1-daX821F was verified using the rce1 gene locus.
Co-expression of various factors related to cellulase transcription in the E1AB1-daX821F strain
The enzymes produced by the E1AB1-daX821F strain under non-inducing conditions were mostly hemicellulases; hence, we examined a combination of transcription factors that were co-expressed to enhance cellulase production. Candidates known as cellulase activators or transcriptionally essential factors, including Crt1, BglR, Vib1, Ace2, and Ace3, were constitutively expressed using the act1 promoter by homologous recombination of rce1 locus in the E1AB1-daX821F strain.
Constitutive expression of Crt1, BglR, Vib1, and Ace2 in E1AB1-daX821F resulted in no significant changes in protein production (Fig. 2a) and enzyme composition (Fig. 2b, lanes 2-5). In contrast, the strains with constitutively expressed Ace3 showed a 1.5-fold increase in protein production when compared to E1AB1-daX821F (Fig. 2a). The enzyme composition analysis revealed that the band ratios that corresponded to hemicellulases (XYN1, XYN2, and BXL1) decreased and those corresponding to cellulases (CBH1, CBH2, and EG1) increased (Fig. 2b, lane 6). The culture supernatant of the Ace3 constitutively expressed strain (named E1AB1-daX821F-drA3) showed a 4.9-fold higher pNPLase (CBH1 activity) and a 5.5-fold higher pNPGase (BGL activity) activities when compared with the E1AB1-daX821F strain. The egl1 promoter has been used to express β-glucosidase AaBGL1 derived from A. aculeatus in the E1AB1 strain, suggesting that the increase in pNPGase activity reflects improved transcription of egl1. Cellulase activities (pNPGase and pNPLase) was improved, but the changes in hemicellulase activities (pNPXase and pNPX2ase) relative to that of the E1AB1-daX821F strain were minor (1.8- and 0.8-fold, respectively). Consequently, it was considered that Ace3 is the main contributor to the specific improvements in cellulase expression. In addition, the unknown protein band observed under non-inducible conditions (Fig. 1b, lanes 2-4 with open arrowhead) was disappeared in the supernatant of Ace3 constitutive expression strain. Thus, it is inferred that cellulases are induced in the E1AB1-daX821F-drA3 strain even in the absence of inducers, and the strain resemble the state in which cellulases are inductively produced.
Consequently, we found that the E1AB1-daX821F-drA3 strain, which combines the constitutive expression of the V821F Xyr1 mutation and Ace3, makes it possible to produce not only hemicellulases but also high yield of cellulases, even under inducer-free conditions.
Confirmation of the genetic combinations required for high cellulase production under inducer-free conditions
As described above, it was revealed that the E1AB1-daX821F-drA3 strain exhibited high cellulase production without inducers, but the strain had multiple genetic modifications: two repressor disruptions (ace1 and rce1) and constitutive expression of the V821F mutated Xyr1 and Ace3. Therefore, we confirmed whether the repressor disruption was essential and whether the effectiveness of Xyr1 was specific to the V821F mutation or not. For co-expressed Ace3, the amino acid sequence of Ace3 used in above study was referenced from the JGI genome database (http://genome.jgi.doe.gov/Trire 2/Trire 2.home.html) (651 or 629 amino acids), but this sequence was proposed to be wrong and the correct translation initiation site and intron were different; actually it was proposed that the complete sequence is 734 amino acids [37]. Consequently, both putative translation initiation sites were cloned from genomic DNA and the constitutive expression of the nominal and complete Ace3 were examined (Fig. 3a).
First, we performed constitutive expression of Xyr1V821F and nominal Ace3 using non-homologous recombination (E1AB1-nhX821F-nhA3 strain). The protein productivity of the E1AB1-nhX821F-nhA3 strain of the two undisrupted repressors (ace1, rce1) was lower (2.48 g/L) than that of the E1AB1-daX821F-drA3 strain (3.17 g/L) but higher than that of the E1AB1-daX821F strain (2.08 g/L; Fig. 3b). Enzymes produced by the E1AB1-nhX821F-nhA3 strain showed a decrease in the composition of hemicellulase and an increase in the composition of cellulase (Fig. 3c lane 3). In other words, disruptions of ace1 and rce1 are not essential for the inducer-free production of cellulases, but the disruption of these repressors contributes to the alleviation of inhibition and enables efficient protein production.
We also examined whether cellulases could be efficiently produced only by the constitutive expression of nominal Ace3 without V821F mutated Xyr1 (E1AB1-nhA3 strain). The intensity of protein bands corresponding to cellulases was increased (Fig. 3c, lane 4), indicating that cellulases could be slightly induced with the constitutive expression of nominal Ace3 under non-inducible conditions. However, protein bands corresponding to hemicellulases were not observed following SDS-PAGE, and the total protein concentration was significantly lower when compared to that of the E1AB1-daX821F strain (Fig. 3b). This suggests that the constitutive expression of nominal Ace3 alone did not affect protein productivity and hemicellulase production, but slightly affected cellulase production.
Furthermore, when the constitutive expression of wild-type Xyr1 was introduced in the nominal Ace3 expressed strain (E1AB1-nhXWT-nhA3 strain), the protein concentration and SDS-PAGE band pattern (Fig. 3b: 0.51 g/L; Fig. 3c lane 5) showed almost the same phenotype as the strain of the nominal Ace3 alone (E1AB1-nhA3 strain) (Fig. 3b: 0.61 g/L; Fig. 3c lane 4). In other words, Co-expression of wild-type Xyr1 and Ace3 did not have no effect on protein production in non-inducing condition, so the effective mutation of Xyr1 such as V821F was essential for inducer free production.
We then confirmed whether the V821F mutated Xyr1 was the specific effective mutant or not. In addition to V821F, the A824V mutation was also previously identified as an effective mutation [30], so a strain co-expressed A824V mutated Xyr1 and Ace3 (E1AB1-nhX824V-nhA3 strain) was constructed and the protein productivity and its composition of the produced enzyme was evaluated (Fig. 3a). The protein productivity of the A824V mutation co-expressed strain (2.17 g/L) was significantly higher than that of Ace3 alone (0.61 g/L) or when co-expressed with Xyr1WT (0.51 g/L; Fig. 3a and 3d), but the productivity tended to be lower than that of V821F (2.54 g/L; p = 0.07). The enzyme composition of E1AB1-nhX824V-nhA3 strain was mainly composed of cellulase and this was similar to that of E1AB1-nhX821F-nhA3 strain, so the mutation of A824V and V821F in Xyr1 seemed to be almost the same effect on inducer free production. However, when Xyr1A824V was used, the band intensities for the hemicellulases (BXL1, XYN1, and XYN2) were slightly decreased compared to those when Xyr1V821F was used. Therefore, although there may be slight differences in protein production and hemicellulase expression, in this system it is important to co-express Ace3 and Xyr1, which are effective glucose-blind phenotypes, and the effect is not unique to the V821F mutation.
The nominal and complete Ace3 sequences were then examined (Fig. 3a). Improvement in cellulase production was also confirmed in the complete Ace3, but protein productivity of Xyr1V821F-complete Ace3 decreased by 14% and the cellulase band intensity also decreased compared with that of the Xyr1V821F-nominal Ace3 (Fig. 3d, 3e lanes 3, 4). Thus, it was concluded that it is not necessary to have the complete N-terminal sequence for inducer-free production; partial truncation of the DNA-binding domain of Ace3 is preferable.
These results demonstrated that although the disruption of the ace1 and rce1 repressors was not essential for inducer-free production, it was preferable for productivity. As for the transcriptional activators, it is effective to constitutively express both the mutated Xyr1 exhibiting a glucose-blind phenotype (V821F, A824V, and so on, but not the wild-type) and the DNA-binding domain truncated nominal Ace3.
Analysis of gene expression and produced enzymes
We evaluated the comparative transcription level (ΔΔCt method using pgk1 gene as a normalizer and E1AB1-daX821F strain as calibrator) of the major saccharifying enzyme genes after 48 h of glucose culturing. In the E1AB1-daX821F-drA3 strain, the transcription level of the major cellulases cbh1, cbh2, and egl1, increased to the level of that of the E1AB1-daX821F strain, but the increase in expression of the major hemicellulases xyn1, xyn2, and bxl1 were a maximum of 1.7-fold (Fig. 4a), which is in agreement with the aforementioned enzymatic activity (Fig. 2c). These gene expression results also suggested that Ace3 is a cellulase-specific transcription factor. Owing to the effect of the Xyr1 mutation, the transcription of major cellulases in the E1AB1-daX821F strain was approximately four orders of magnitude higher than that of the parent strain E1AB1 (relative cbh1 expression level: 0.00013), but it was possible to further increase the transcription level of the cellulases by the constitutive expression of the nominal Ace3 (6.4-fold).
Consistent with the gene expression analysis results, the enzymes produced by the E1AB1-daX821F-drA3 strain exhibited a similar amount of hemicellulase and a specific increase in the cellulase (CBH1, CBH2, EG1) components when compared to the E1AB1-daX821F strain (Fig. 4b). The cellulase/hemicellulase composition ratio of the E1AB1-drA3 and the E1AB1-daX821F-drA3 strains in the non-induced culture using glucose was similar to that of the induced culture of the parental E1AB1 strain using cellulose (Fig. 4b; see Additional file Fig. S1). For the E1AB1-daX821F-drA3 strain, the composition ratio of the hemicellulases (XYN1, XYN2, BXL1) was relatively high, as an effect of the mutated Xyr1 phenotype. In the E1AB1-drA3 strain, the hemicellulase composition ratio and activity (pNPX2ase, pNPXase) were extremely low (see Additional file Fig. S2), and the degradative activity of pNPX2ase and pNPXase was only 0.6% and 1.5%, respectively, when compared to that of the E1AB1-daX821F-drA3 strain. That is, the constitutive expression of Ace3 can activate cellulase production, but not hemicellulase; as a result, the produced enzyme cocktail contained extremely low levels of hemicellulase.
Saccharification tests on microcrystalline cellulose were conducted using enzymes produced under cellulose (induced) and glucose (inducer-free) conditions. In the evaluation using the same protein dosage, the amount of released glucose was the highest by the enzyme produced by the parental E1AB1 strain induced with cellulose (268 g-glucose/g-protein), but the enzyme produced by the E1AB1-daX821F-drA3 strain was the most superior under inducer-free conditions (225 g-glucose/g-protein; Fig. 4c). The enzymes from the E1AB1-drA3 strain also showed relatively high saccharification activity per protein (178 g-glucose/g-protein), and significantly higher saccharification activity than the enzymes derived from the E1AB1-daX821F strain (77 g-glucose/g-protein). This is estimated to be due to the high composition ratio of the cellulase components within the added protein. However, as described above, the concentrations of the enzymes produced by the E1AB1-drA3 strain were only 0.57 g/L protein (Fig. 4b; see Additional file Fig. S3); thus, the absolute amount of the cellulase in the enzymes of E1AB-drA3 was approximately 27% of that in E1AB1-daX821F-drA3 (Fig. 4b). In contrast, the enzyme cocktail derived from the E1AB1-daX821F strain contained a high protein concentration, but its composition was mainly hemicellulases; as a result, the absolute amount of cellulases was equivalent to that of the E1AB1-drA3 and E1AB1-daX821F strains (Fig. 4b) and significantly lower than that of the E1AB1-daX821F-drA3 strain. Therefore, when saccharification was performed with the same volume (0.87 mL culture supernatant/g cellulose: approximately 2.0 mg E1AB1-daX821F-drA3 strain derived protein/g cellulose), the enzymes derived from the E1AB1-daX821F and E1AB1-drA3 strains showed almost the same amount of released glucose (6.5 and 7.5 g/L glucose), significantly lower than that of the E1AB1-daX821F-drA3 strain (19.9 g/L glucose; Fig. 4d). As a result, the E1AB1-daX821F-drA3 strain was considered superior to that of the existing inducer-free Xyr1V821F overexpressed strain.
From the above, the E1AB1-daX821F-drA3 strain was found to have two combined important phenotypes: increased cellulase composition ratio by constitutive expression of Ace3 and high protein productivity under non-inducing conditions by the introduction of mutated Xyr1. We achieved the most important and essential property of the cellulolytic enzyme, the high production of cellulase in a non-inducing production system.