康宁木霉K801纤维素酶cbh2基因的克隆及序列分析

通过聚合酶链式反应（PCR）技术扩增得到纤维素高产菌株康宁木霉Trichoderma k■ningii K801纤维二糖水解酶（CBHII）基因全序列,并克隆到pGEM-Teasy Vector。序列分析表明,所克隆的cbh2基因长1611bp,包含了纤维二糖水解酶基因的完整编码区序列,并含有三个真核生物典型的内含子序列,其中四个外显子序列共同编码一个471aa的蛋白质。该序列是国内首次克隆得到的cbh2编码区全序列,与国外报道的T. reesei已知序列的同源性达到99.89％,只有两个碱基差别,而推测的氨基酸序列只有一个位置疏水性氨基酸之间发生替代,在推测的氨基酸序列上发现3个潜在的N-糖基化位点。     

Carbon Source Control of Cellobiohydrolase I and II Formation by Trichoderma reesei

Regulation of the formation and secretion of two cellulase components from Trichoderma reesei QM 9414, cellobiohydrolases I and II (CBH I and CBH II, respectively), by the carbon source was investigated. With monoclonal antibodies against CBH I and CBH II it was found that during cultivation on carbon sources which enable fast growth (glucose, glycerol, and fructose), no formation of CBH I occurred, whereas low levels of CBH II were formed. Lactose and cellulose, which allow comparably slower growth, promoted the formation of both CBH I and CBH II. However, noncarbohydrate carbon sources as citrate or acetate, which also enable only slow growth, did not promote the formation of CBH I or CBH II. The addition of glucose or glycerol to lactose- or cellulose-pregrown mycelia, on the other hand, only partially reduced the formation of CBH I. This reduction was also achieved by several other metabolizable and nonmetabolizable carbon compounds, e.g., fructose, galactose, β-methylglucoside, 2-deoxyglucose, and rhamnose, as well as by transfer to no carbon source at all. This result indicates that the control of CBH I synthesis by the carbon source is due to induction and not to repression. The use of cycloheximide and 5-fluorouracil as inhibitors at and before translation, respectively, revealed a half-life for CBH I mRNA of at least several hours, which may, at least in part, account for the prolonged synthesis of some CBH I under these conditions. Northern (RNA) hybridization with full copies of cbh1 and cbh2 genes indicated that the control of CBH I and CBH II biosyntheses by the carbon source operates mainly at the pretranslational level. We conclude that the low rate of cellulase synthesis on glucose and some other carbon sources is due to the lack of an inducer and not to carbon source repression.     

A kinetic model for quantitative evaluation of the effect of hydrogen and osmolarity on hydrogen production by Caldicellulosiruptor saccharolyticus

Background

The main technological impediment to widespread utilization of lignocellulose for the production of fuels and chemicals is the lack of low-cost technologies to overcome its recalcitrance. Organisms that hydrolyze lignocellulose and produce a valuable product such as ethanol at a high rate and titer could significantly reduce the costs of biomass conversion technologies, and will allow separate conversion steps to be combined in a consolidated bioprocess (CBP). Development of Saccharomyces cerevisiae for CBP requires the high level secretion of cellulases, particularly cellobiohydrolases.

Results

We expressed various cellobiohydrolases to identify enzymes that were efficiently secreted by S. cerevisiae. For enhanced cellulose hydrolysis, we engineered bimodular derivatives of a well secreted enzyme that naturally lacks the carbohydrate-binding module, and constructed strains expressing combinations of cbh1 and cbh2 genes. Though there was significant variability in the enzyme levels produced, up to approximately 0.3 g/L CBH1 and approximately 1 g/L CBH2 could be produced in high cell density fermentations. Furthermore, we could show activation of the unfolded protein response as a result of cellobiohydrolase production. Finally, we report fermentation of microcrystalline cellulose (Avicel?) to ethanol by CBH-producing S. cerevisiae strains with the addition of beta-glucosidase.

Conclusions

Gene or protein specific features and compatibility with the host are important for efficient cellobiohydrolase secretion in yeast. The present work demonstrated that production of both CBH1 and CBH2 could be improved to levels where the barrier to CBH sufficiency in the hydrolysis of cellulose was overcome.     

Novel cellulase profile of Trichoderma reesei strains constructed by cbh1 gene replacement with eg3 gene expression cassette

To construct strains of the filamentous fungus Trichoderma reesei with low cellobiohydrolases while high endoglucanase activity, the Pcbh1-eg3-Tcbh1 cassette was constructed and the coding sequence of the cellobiohydrolase I (CBHI) gene was replaced with the coding sequence of the eg3 gene by homologous recombination. Disruption of the cbhl gene was confirmed by PCR, Southern dot blot and Western hybridization analysis in two transforments denoted as L 13 and L29. The filter paper-hydrolyzing activity of strain L29 was 60% of the parent strain Rut C30, and the CMCase activity was increased by 33%. This relatively modest increase suggested that the eg3 cDNA under the control of the cbhl promoter was not efficiently transcribed as the wild type cbhl gene. However our results confirmed that homologous recombination could be used to construct strains of the filamentous fungus Trichoderma reesei with novel cellulase profile. Such strains are of interest from the basic science perspective and also have potential industrial applications.     

Studies of the cellulolytic system of Trichoderma reesei QM 9414. Analysis of domain function in two cellobiohydrolases by limited proteolysis

Limited action of papain on the native forms of two cellobiohydrolases (CBH) from Trichoderma reesei (CBH I, 65 kDa, and CBH II, 58 kDa) leads to the isolation of the respective core fragments (56 kDa and 45 kDa) which are fully active on small, soluble substrates, but have a strongly reduced activity (respectively 10% and 50% of the initial value) on microcrystalline cellulose (Avicel). By partial sequencing at the C terminus of the CBH I core and at the N terminus of the CBH II core the papain cleavage sites have been assigned in the primary structures (at about residue 431 and 82 respectively). This limited action of papain on the native enzymes indicates the presence of hinge regions linking the core to these terminal glycopeptides. The latter conserved sequences appear either at the C or N terminus of several cellulolytic enzymes from Trichoderma reesei [Teeri et al. (1987) Gene 51, 43-52]. The specific activities of the intact enzymes and their cores on two forms of insoluble cellulose (crystalline, amorphous) differentiate the CBH I and CBH II in terms of adsorption and catalytic properties. Distinct functions can be attributed to the terminal peptides: for intact CBH II the N-terminal region contributes in the binding onto both cellulose types; the homologous C-terminal peptide in CBH I, however, only affects the interaction with microcrystalline cellulose. It could be inferred that CBH I and its core bind preferentially to crystalline regions. This seems to be corroborated by the results of CBH I/CBH II synergism experiments.     

The 1,4-beta-D-glucan cellobiohydrolases from Phanerochaete chrysosporium. I. A system of synergistically acting enzymes homologous to Trichoderma reesei.

A physico-chemical and structural characterization of three 1,4-beta-D-glucan cellobiohydrolases (EC. 3.2.1.91), isolated from a culture filtrate of the white-rot fungus Phanerochaete chrysosporium, reveals that the cellulolytic enzyme secretion pattern and thus the general degradation strategy for P. chrysosporium is similar to that of Trichoderma reesei. Partial sequence data show that two of the isolated enzymes, i.e., CBHI, pI 3.82 and CBH62, pI 4.85, are homologous with CBHI and EGI from T. reesei; while, the third, i.e., CBH50, pI 4.87, is homologous to T. reesei CBHII. Limited proteolysis with papain cleaved each of the three enzymes into two domains: a core protein which retained full catalytic activity against low molecular weight substrates and a peptide fragment corresponding to the cellulose binding domain, in striking similarity to the structural organization of T. reesei. CBHI and CBH62 have their binding domain located at the C-terminus, whereas in CBH50 it is located at the N-terminus. It is evident that synergistically acting cellobiohydrolases is a general requirement for efficient hydrolysis of crystalline cellulose by cellulolytic fungi.     

Design of highly efficient cellulase mixtures for enzymatic hydrolysis of cellulose

An extremely highly active cellobiohydrolase (CBH IIb or Cel6B) was isolated from Chrysosporium lucknowense UV18-25 culture filtrate. The CBH IIb demonstrated the highest ability for a deep degradation of crystalline cellulose amongst a few cellobiohydrolases tested, including C. lucknowense CBH Ia, Ib, IIa, and Trichoderma reesei CBH I and II. Using purified C. lucknowense enzymes (CBH Ia, Ib, and IIb; endoglucanases II and V; beta-glucosidase, xylanase II), artificial multienzyme mixtures were reconstituted, displaying an extremely high performance in a conversion of different cellulosic substrates (Avicel, cotton, pretreated Douglas fir wood) to glucose. These mixtures were much or notably more effective in hydrolysis of the cellulosic substrates than the crude multienzyme C. lucknowense preparation and other crude cellulase samples produced by T. reesei and Penicillium verruculosum. Highly active cellulases are a key factor in bioconversion of plant lignocellulosic biomass to ethanol as an alternative to fossil fuels.     

Kinetic parameters and mode of action of the cellobiohydrolases produced by Talaromyces emersonii

Three forms of cellobiohydrolase (EC 3.2.1.91), CBH IA, CBH IB and CBH II, were isolated to apparent homogeneity from culture filtrates of the aerobic fungus Talaromyces emersonii. The three enzymes are single sub-unit glycoproteins, and unlike most other fungal cellobiohydrolases are characterised by noteworthy thermostability. The kinetic properties and mode of action of each enzyme against polymeric and small soluble oligomeric substrates were investigated in detail. CBH IA, CBH IB and CBH II catalyse the hydrolysis of microcrystalline cellulose, albeit to varying extents. Hydrolysis of a soluble cellulose derivative (CMC) and barley 1,3;1,4-beta-D-glucan was not observed. Cellobiose (G2) is the main reaction product released by CBH IA, CBH IB, and CBH II from microcrystalline cellulose. All three CBHs are competitively inhibited by G2; inhibition constant values (K(i)) of 2.5 and 0.18 mM were obtained for CBH IA and CBH IB, respectively (4-nitrophenyl-beta-cellobioside as substrate), while a K(i) of 0.16 mM was determined for CBH II (2-chloro-4-nitrophenyl-beta-cellotrioside as substrate). Bond cleavage patterns were determined for each CBH on 4-methylumbelliferyl derivatives of beta-cellobioside and beta-cellotrioside (MeUmbG(n)). While the Tal. emersonii CBHs share certain properties with their counterparts from Trichoderma reesei, Humicola insolens and other fungal sources, distinct differences were noted.     

Domain structure of cellobiohydrolase II as studied by small angle X-ray scattering: close resemblance to cellobiohydrolase I

Evidence for a domain structure of cellobiohydrolase II (CBH II, 58 kDa) from Trichoderma reesei (Teeri et al., 1987; Tomme et al., 1988) is corroborated by results from SAXS experiments. They indicate a 'tadpole' structure for the intact CBH II in solution (Dmax = 21.5 +/- 0.5 nm; Rg = 5.4 +/- 0.1 nm) and a more isotropic, ellipsoid shape for the core protein (Dmax = 6.0 +/- 0.3 nm; Rg = 2.1 +/- 0.1 nm). The latter was obtained by partial proteolysis with papain which cleaves the native CBH II to give two fragments (Tomme et al., 1988): the core (45 kDa) with the active (hydrolytic) domain and a smaller fragment (11 kDa) coinciding with the tail part of the model and containing the binding domain for unsoluble cellulose. This peptide fragment is conserved in most cellulolytic enzymes from Trichoderma reesei (Teeri et al., 1987). It contains a conserved region (block A) and glycosylated parts (blocks B and B' duplicated and located N-terminally in CBH II). In spite of different domain arrangements in CBH I (blocks B-A at C-terminals) SAXS measurements (Abuja et al., 1988) indicate similar tertiary structures for both cellobiohydrolases although discrete differences in the tail parts exist.     

Characterization of commercial Trichoderma reesei cellulase preparations by denaturing electrophoresis (SDS-PAGE) and immunostaining using monoclonal antibodies.

Fifteen different cellulase preparations from Trichoderma reesei, obtained either commercially or from pilot plants, were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using monoclonal antibodies against two cellobiohydrolases (CBH I, CBH II), an endoglucanase (EG I), and beta-glucosidase. The staining patterns were compared with the activities of the preparations against filter paper (FPU), carboxymethylcellulose (CMC-ase), cellobiose (beta-glucosidase), and azocasein (protease). Variable amounts of proteolytic degradation products of CBH I, CBH II, and EG I were seen in most samples, and only half of them contained intact beta-glucosidase. The degree of proteolysis did not correlate with any significant difference in the respective activities of these preparations against filter paper cellulose or carboxymethylcellulose. In more than 50% of all cases a decreased beta-glucosidase activity and the absence of intact beta-glucosidase protein in Western blots was observed in preparations displaying high proteolytic activity.     

High frequency one-step gene replacement in Trichoderma reesei. II. Effects of deletions of individual cellulase genes

Four cellulase genes of Trichoderma reesei, cbh1, cbh2, egl1 and egl2, have been replaced by the amdS marker gene. When linear DNA fragments and flanking regions of the corresponding cellulase locus of more than 1 kb were used, the replacement frequencies were high, ranging from 32 to 52%. Deletion of the major cellobiohydrolase 1 gene led to a 2-fold increase in the production of cellobiohydrolase II; however, replacement of the cbh2 gene did not affect the final cellulase levels and deletion of egl1 or egl2, slightly increased production of both cellobiohydrolases. Based on our results, endoglucanase II accounts for most of the endoglucanase activity produced by the hypercellulolytic host strain. Furthermore, loss of the egl2, gene causes a significant drop in the filter paper-hydrolysing activity, indicating that endoglucanase II has an important role in the total hydrolysis of cellulose.     

High frequency one-step gene replacement in Trichoderma reesei. II. Effects of deletions of individual cellulase genes

Four cellulase genes of Trichoderma reesei, cbh1, cbh2, egl1 and egl2, have been replaced by the amdS marker gene. When linear DNA fragments and flanking regions of the corresponding cellulase locus of more than 1 kb were used, the replacement frequencies were high, ranging from 32 to 52%. Deletion of the major cellobiohydrolase 1 gene led to a 2-fold increase in the production of cellobiohydrolase II; however, replacement of the cbh2 gene did not affect the final cellulase levels and deletion of egl1 or egl2, slightly increased production of both cellobiohydrolases. Based on our results, endoglucanase II accounts for most of the endoglucanase activity produced by the hypercellulolytic host strain. Furthermore, loss of the egl2, gene causes a significant drop in the filter paper-hydrolysing activity, indicating that endoglucanase II has an important role in the total hydrolysis of cellulose.     

Novel Cellulase Profile of Trichoderma reesei Strains Constructed by cbh1 Gene Replacement with eg3 Gene Expression Cassette

The filamentous fungus Trichoderma reesei is consi-dered to be the most efficient cellulase producer, and hasa long history in the production of hydrolytic enzymes,which was widely used in the food and feed industriesand recently also used in the textile,…     

Cellobiohydrolase II is the main conidial-bound cellulase in Trichoderma reesei and other Trichoderma strains

Monoclonal antibodies have been used to determine the presence of cellobiohydrolases I and II (CBH I and II), and endoglucanase I (EG I) on the surface of conidia from Trichoderma reesei QM 9414 and RUT C-30, and 8 other Trichoderma species. For this purpose, proteins were released from the conidial surface by treatment with a non-ionic detergent (Triton X-100 and -octylglucoside), followed by SDS-PAGE/Western blotting and immunostaining. Both CBH I and II were clearly present, but — unlike in extracellular culture fluids from Trichoderma — CBH II was the predominant cellulase. In T. reesei EG I could not be detected. The higher producer strain T. reesei RUT C-30 exhibited a higher conidial level of CBH II than T. reesei QM 9414. In order to assess the importance of the conidial CBH II level for cellulase induction by cellulose, multiple copies of the chb2 gene were introduced into the T. reesei genome by cotransformation using PyrG as a marker. Stable multicopy transformants secreted the 2- to 4-fold level of CBH II into the culture medium when grown on lactose as a carbon source, but their CBH I secretion was unaltered. Upon growth on cellulose, both CBH I and CBH II secretion was enhanced. Those strain showing highest cellulase activity on cellulose also appeared to contain the highest level of conidial bound CBH II. CBH II was also the predominant conidial cellulase in various other Trichoderma sp. However, roughly the same amount of conidial bound CBH II was detected in all strains, although their cellulase production differed considerably.     

Identification and purification of the main components of cellulases from a mutant strain of Trichoderma viride T 100-14

A new mutant strain of fungus Trichoderma viride T 100-14 was cultivated on 1% microcrystalline cellulose (Avicel) for 120h and the resulting culture filtrate was prepared for protein identification and purification. To identify the predominant catalytic components, cellulases were separated by an adapted two-dimensional electrophoresis technique. The apparent major spots were identified by high performance liquid chromatography electrospray ionization mass (HPLC-ESI-MS). Seven of the components were previously known, i.e., the endoglucanases Cel7B (EG I), Cel12A (EG III), Cel61A (EG IV), the cellobiohydrolases Cel7A (CBH I), Cel6A (CBH II), Cel6B (CBH IIb) and the beta-glucosidase. The seven major components in the fermentation broth of T. viride T 100-14 probably constitute the essential enzymes for crystalline cellulose hydrolysis and they were further purified to electrophoretic homogeneity by a series of chromatography column. Hydrolysis studies of the purified elements revealed that three of the cellulases were classified as cellobiohydrolases due to their main activities on p-nitrophenyl-beta-d-cellobioside (pNPC). Three of the cellulases, with the abilities of hydrolyzing both carboxymethyl-cellulose (CMC) and Avicel indicate their endoglucanase activities. It deserved noting that the beta-glucosidase from the T 100-14 displayed an extremely high activity on p-nitrophenyl-beta-D-glycopyranoside (pNPG), which suggested it was a good candidate for the conversion of cellobiose to glucose.     

The conserved terminal region of Trichoderma reesei cellulases forms a strong antigenic epitope for polyclonal antibodies.

The specificity of polyclonal antibodies (Pab) raised against Trichoderma reesei cellulases has been studied. cDNAs lacking regions coding for certain functional domains were produced by preparing series of 3'-end deletions from the cDNAs for two cellobiohydrolases, CBH I and CBH II, and an endoglucanase, EG I. The proteins coded by the full length cDNAs and the truncated proteins coded by the deleted cDNAs were expressed in yeast Saccharomyces cerevisiae, under the control of the ADC1 promoter. Each polyclonal antiserum showed cross-reactivity with other cellulases. Pabs for CBH I and CBH II both recognized EG I. Pab for EG I strongly recognized both CBH I and CBH II. By analyzing the truncated proteins, we found that these antibodies were almost entirely directed against the conserved tail of the cellulase enzymes.     

Enhanced production of cellobiohydrolases in Trichoderma reesei and evaluation of the new preparations in biofinishing of cotton

In the search for suitable cellulase combinations for industrial biofinishing of cotton, five different types of Trichoderma reesei strains were constructed for elevated cellobiohydrolase production: CBHI overproducers with and without endoglucanase I (EGI), CBHII overproducers with and without endoglucanase II (EGII) and strains overproducing both CBHI and CBHII without the major endoglucanases I and II. One additional copy of cbh1 gene increased production of CBHI protein 1.3-fold, and two copies 1.5-fold according to ELISA (enzyme-linked immunosorbent assay). The level of total secreted proteins was increased in CBHI transformants as compared to the host strain. One copy of the cbh2 expression cassette in which the cbh2 was expressed from the cbh1 promoter increased production of CBHII protein three- to four-fold when compared to the host strain. T. reesei strains producing elevated amounts of both CBHI and CBHII without EGI and EGII were constructed by replacing the egl1 locus with the coding region of the cbh1 gene and the egl2 locus with the coding region of cbh2. The cbh1 was expressed from its own promoter and the cbh2 gene using either the cbh1 or cbh2 promoter. Production of CBHI by the CBH-transformants was increased up to 1.6-fold and production of CBHII up to 3.4-fold as compared with the host strain. Approximately similar amounts of CBHII protein were produced by using cbh1 or cbh2 promoters. When the enzyme preparation with elevated CBHII content was used in biofinishing of cotton, better depilling and visual appearance were achieved than with the wild type preparation; however, the improvement was not as pronounced as with preparations with elevated levels of endoglucanases (EG).