cbhI Gene Research Articles

Nitrogen deposition-induced stimulation of soil heterotrophic respiration is counteracted by biochar in a subtropical forest

Both atmospheric nitrogen (N) deposition and biochar application can markedly impact soil heterotrophic respiration (RH), an important component of the global carbon cycle. However, the interactive effects of N deposition and biochar application on soil RH in subtropical forest ecosystems remain unclear. Here, we conducted a three-year (2019–2022) field trial within a bamboo forest in subtropical China to examine the responses of soil physicochemical and microbial properties to N deposition and biochar application, and to elucidate how biochar regulates N deposition-induced change in soil RH. Nitrogen deposition stimulated soil RH by 8.1–9.8 % annually over three years compared to the control, and this stimulation was mitigated (by 8.1–8.9 % annually) with biochar addition. In the context of N deposition, the decrease of soil RH by biochar application was not through changing soil temperature, moisture or labile organic carbon content. Biochar treatment reduced the abundances of bacterial glycoside hydrolase family 48 gene (GH48) and fungal glycoside hydrolase family 7 cellobiohydrolase I gene (cbhI) and the activities of β-glucosidase and cellobiohydrolase (CBH), but increased the abundance of cbbL gene and activity of RubisCO enzyme. Furthermore, the RH was correlated positively (P < 0.01) with β-glucosidase and CBH activities and negatively (P < 0.01) with RubisCO enzyme activity. Structural equation modeling revealed that the biochar-induced reduction of soil RH under N deposition was associated with decreases in the abundance of cbhI gene and the activity of CBH in soils. We highlight that management practices can mitigate soil carbon loss in forests through modulating soil microbial functions under atmospheric N deposition, and that biochar application in Moso bamboo forests has the potential to reduce RH by approximately 7.6 × 106 t CO2 yr−1 under atmospheric N deposition.

Effects of maize straw and its biochar application on soil organic carbon chemical composition and carbon degradation genes in a Moso bamboo forest.

We investigated the effects of maize straw and its biochar application on soil organic carbon chemical composition, the abundance of carbon degradation genes (cbhI) and the composition of cbhI gene community in a Moso bamboo forest, to provide the theoretical and scientific basis for enhancing carbon sequestration. We conducted a one-year field experiment in a subtropical Moso bamboo forest with three treatments: control (0 t C·hm-2), maize straw (5 t C·hm-2), and maize straw biochar (5 t C·hm-2). Soil samples were collected at the 3rd and 12th months after the treatment. Soil organic carbon chemical composition, the abundance and community composition of cbhI gene were determined by solid-state 13C NMR, real-time fluorescence quantitative PCR, and high-throughput sequencing, respectively. The results showed that compared with the control, maize straw treatment significantly increased the content of O-alkyl C and decreased aromatic C content, while maize straw biochar treatment showed an opposite effect. Maize straw treatment significantly increased the abundance of cbhI gene and the relative abundance of Penicillium, Gaeumannomyces and Marasmius. However, maize straw biochar treatment reduced the abundance of this gene. The relative abundance of dominant cbhI in soils was positively correlated with the content of O-alkyl C and negatively correlated with the content of aromatic C. Results of redundancy analysis showed that maize straw treatment had a significant effect on the microbial community composition of cbhI gene by changing soil O-alkyl C content, while maize straw biochar affected the microbial community composition of cbhI gene by changing soil pH, organic carbon, and aromatic C content. Maize straw biochar treatment was more effective in increasing soil organic carbon stability and reducing microbial activity associated with carbon degradation in the subtropical Moso bamboo forest ecosystem compared with maize straw treatment. Therefore, the application of biochar has positive significance for maintaining soil carbon storage in subtropical forest ecosystems.

INFLUENCE OF THE TACA TRANSCRIPTION FACTOR KNOCKOUT ON THE TRANSCRIPTION AND EXPRESSION OF THE CBHI GENE IN THE PENICILLIUM VERRUCULOSUM STRAIN

The tacA gene, encoding the TacA repressor protein, was cloned by “walking the uncloned DNA” method from the genomic DNA of the fungus Penicillium verruculosum B1-221-151. Knockout of the tacA and niaD genes by the CRIS-PR/CAS9 led to the production of a new host strain P. verruculosum ΔniaDΔtacA, characterized by a higher rate of extracellular protein biosynthesis. Analysis of the transcription and expression of the cbhI gene in the original P. verruculosum B1-221-151 strain and in the P. verruculosum ΔniaDΔtacA strain showed a sharp increase in the level of cbhI gene transcription 2 h after the start of induction with cellobiose, cellotriose, gentiobiose, and a mixture of di- and trisaccharides in comparison with the transcription of the cbhI gene in the original strain. The speci c activity of cellobiohydrolase I, the main enzyme of the cellulolytic complex of the fungus P. verruculosum, by 96 h of fermentation of the ΔtacA strain increased 3 times compared to the original strain.

Mining activities accelerate the decomposition of organic matter from aquatic ecosystems through soil microbes

AbstractMining activities have been shown to affect the rate of carbon decomposition in aquatic ecosystems by altering the traits of the organic matter (e.g., leaf litter). However, the ecological function of soil microbes that enter aquatic ecosystems in association with organic matter is not known. Here, standardized cotton strip assays (CSA) were used to quantify the effects of soil microbial colonization location (mine vs. forest riverbank) and time (7 days vs. 14 days) on cellulose decomposition in aquatic ecosystems. The CSA results showed that the mine riverbank location and microbial colonization time had significant effects on the microbial community structure, microbial biomass, and functional genes of cellulose decomposition. Compared with the forest riparian zone, the fungal biomass and bacterial cellulose‐decomposing gene (GH48) abundance of cotton strips colonized in the mine riparian zone for 14 days were 12.5‐times and 54.93‐times higher than that of the cotton strips colonized in the forest riparian zone, respectively, while the fungal cellulose‐decomposing gene (cbhI) was detected only after 14 days of colonization. After 90 days of stream incubation, the decomposition rate and GH48 gene abundance of cotton strips colonized in the mine riparian zone for 14 days were significantly higher than those of cotton strips colonized in the forest riparian zone. However, the abundance of the cbhI gene in cotton strips colonized for 14 days was significantly higher than that in cotton strips colonized for 7 days. These results show that bacteria were mainly affected by the microbial colonization environment, while fungi were mainly affected by colonization time. Mining activities accelerate the decomposition of organic matter in aquatic ecosystems by increasing soil fungal biomass and bacterial cellulose decomposition genes. To reduce the carbon loss of aquatic ecosystems, it is necessary to study the potential influence of soil microbes on the decomposition of organic carbon in riparian zones.

Land-use-driven change in soil labile carbon affects microbial community composition and function

The conversion of natural forests to plantations can lead to a substantial loss of soil organic carbon (SOC) and threaten the function of soil microbial communities. However, the effect of forest conversion on the relationship between SOC dynamics and shifts in the composition and function of the microbial community remain poorly understood, although such information is critical for predicting the long-term effect of land-use change on global SOC dynamics and storage. To fill this knowledge gap, our study investigated the influence of two decades of converting natural forest to a tea plantation on SOC storage, microbial community composition, C-degrading enzyme activity and the abundance of C-degrading microbial functional genes in a subtropical region in China. Two decades after converting natural forest to tea plantation, SOC storage decreased by 30%, and labile SOC [including easily mineralized C (Cmin), readily oxidizable C (ROC), dissolved organic C (DOC), and microbial biomass C (MBC)] decreased by 21–71%. Land-use change decreased the relative abundance of copiotrophic bacteria but increased the relative abundance of oligotrophic bacteria and lignin-degrading fungi. This resulted in a decrease in cellulase activity and cbhI gene abundance involved in cellulose degradation and conversely an increase in ligninase activity involved in lignin degradation after the conversion. Such shifts in microbial community composition and function associated with land-use change were mediated by the decrease in the labile C pool rather than the total SOC. Our study provided new insights into not only shifts in microbial communities but also specific C functional genes and related enzymes after forest conversion. These findings highlight that labile SOC, as an important functional soil component for microbial communities, significantly affects SOC dynamics and storage.

Land-Use Change Enhanced SOC Mineralization but Did Not Significantly Affect Its Storage in the Surface Layer.

To achieve carbon (C) neutrality and mitigate climate change, it is crucial to understand how converting natural forests to agricultural plantations influences soil organic C (SOC) mineralization. In this study, we investigated the impact of converting evergreen broadleaf forests (EBF) to extensively managed Moso bamboo (Phyllostachys edulis (Carriere) J. Houzeau) plantations (MBP) in subtropical China on SOC mineralization rate; the concentrations of labile SOC fractions such as dissolved organic C (DOC), microbial biomass C (MBC), and readily oxidizable C (ROC); the activities of C-degrading enzymes (cellobiohydrolase and phenol oxidase); and the abundance of C-degrading enzyme-encoding genes (cbhI and lcc). Three paired soil samples were taken from the surface layer (0–20 cm) of adjacent EBF-MBP sites in Anji County, Zhejiang province. Results showed that converting EBF to MBP significantly increased the SOC mineralization rate as well as soil pH, MBC, cellobiohydrolase, and phenol oxidase activities, and cbhI gene abundance, but did not change other soil properties described above. In addition, structural equation modelling (SEM) showed that the conversion increased SOC mineralization rate through increasing soil pH, cbhI gene abundance, MBC, and cellobiohydrolase and phenol oxidase activities. Our novel finding that converting EBF to extensively managed MBP enhanced SOC mineralization via increasing the activities of C-degrading enzymes suggests that C-degrading enzymes were a key factor regulating SOC mineralization in the extensively managed subtropical bamboo plantations.

N, P and straw return influence the accrual of organic carbon fractions and microbial traits in a Mollisol

Soil organic carbon (SOC) is the key to soil fertility and quality. However, SOC has severely declined in the past several decades due in part to inappropriate nitrogen (N) and phosphorus (P) input levels. Thus far, the influences of N and P input levels on the accrual of different SOC fractions and relevant microbial traits at aggregate scales remain uncertain. Here, a 3-year field experiment was used to reveal the effects of N and P input levels on SOC fractions in a Mollisol. Treatments included straw return (SR) and combinations of high nitrogen (N) and high phosphorus (P) inputs (N + P + SR), high N and low P inputs (N + P1/2 + SR), and low N and high P inputs (N1/2 + P + SR). Macroaggregates (>0.25 mm), microaggregates (0.25–0.053 mm), and clay and silt (<0.053 mm) were separated, and then particulate organic carbon (POC) and mineral-associated organic carbon (MOC) were determined after aggregate density fractionation. C-cycling genes (cbhI, GH48 and cbbL genes) were quantified by quantitative PCR, and aggregate bacterial community compositions were analyzed by amplicon sequencing of the 16S rRNA genes. The results showed that the free POC (fPOC) content increased after SR, and SR with N input (irrespective of N level) increased bacterial C-cycling gene abundance (GH48 and cbbL genes) and MOC content; however, only combinations with high N inputs increased the fungal cbhI gene abundance and occluded POC (oPOC) content, while P input levels had little impact on SOC fractions. Structural equation modelling revealed that pH was a determinant of GH48 and cbbL gene abundances, which were positively associated with MOC, while N:P was positively related to cbhI gene abundance and oPOC. Network analysis revealed that SOC fractions increased with Nitrosomonadaceae, Xanthobacteraceae, Gaiellaceae and Subgroup_6 and decreased with Gemmataceae and Betaproteobacteriales, suggesting a functional role in SOC sequestration. Our study suggests that moderate high N input and reduced P input can promote the accrual of SOC fractions in a Mollisol with straw return.

Metabolomic, functional, and ecologic responses of the common freshwater fungus Neonectria lugdunensis to mine drainage stress

Metal contamination of watersheds is a global problem. Here, we conducted litter decomposition studies with Neonectria lugdunensis, a cosmopolitan aquatic fungus. Fungal isolates from four reference (non-impacted) and six metal-contaminated streams (due to mine drainage) were exposed to mine drainage and reference stream waters in Central Portugal. Impact of mine drainage waters on N. lugdunensis hyphae was investigated by performing metabolomic profiling of 200 lipids and 25 amino acids (AA) with ultra-high performance liquid chromatography-mass spectrometry. In parallel, functional response of N. lugdunensis isolates was assessed through expression profiles of a functional gene, cellobiohydrolase I (CbhI). Ecological performance via leaf mass loss was also determined. Exposure to mine drainage waters altered the concentration of numerous AA and lipids. Most strikingly, a gradual increase in the concentration of the triacylglycerols (TAG) with shorter acyl chains and lesser unsaturation was observed after the exposure to mine drainage waters. In addition, the changes in the concentration of numerous TAG, lysophosphatidylcholines, and AA were more significant in the isolates from the metal-contaminated streams after exposure to mine drainage water. CbhI gene of the isolates from reference streams was down-regulated by metal stress, while those from metal-contaminated streams remained unaffected. Finally, leaf mass loss was influenced by both exposure to mine drainage waters and the origin of isolates. Overall, our study demonstrates unique functional signatures displayed by fungi under metal stress and the relevant role that fungal AA and lipids play to cope with metal toxicity.

Functional characterisation of cellobiohydrolase I (Cbh1) from Trichoderma virens UKM1 expressed in Aspergillus niger

Cellobiohydrolases catalyze the processive hydrolysis of cellulose into cellobiose. Here, a Trichoderma virens cDNA predicted to encode for cellobiohydrolase (cbhI) was cloned and expressed heterologously in Aspergillus niger. The cbhI gene has an open reading frame of 1518 bp, encoding for a putative protein of 505 amino acid residues with a calculated molecular mass of approximately 54 kDa. The predicted CbhI amino acid sequence has a fungal type carbohydrate binding module separated from a catalytic domain by a threonine rich linker region and showed high sequence homology with glycoside hydrolase family 7 proteins. The partially purified enzyme has an optimum pH of 4.0 with stability ranging from pH 3.0 to 6.0 and an optimum temperature of 60 °C. The partially purified CbhI has a specific activity of 4.195 Umg−1 and a low Km value of 1.88 mM when p-nitrophenyl-β-D-cellobioside (pNPC) is used as the substrate. The catalytic efficiency (kcat/Km) was 5.68 × 10−4 mM−1s−1, which is comparable to the CbhI enzymes from Trichoderma viridae and Phanaerochaete chrysosporium. CbhI also showed activity towards complex substrates such as Avicel (0.011 Umg−1), which could be useful in complex biomass degradation. Interestingly, CbhI also exhibited a relatively high inhibition constant (Ki) for cellobiose with a value of 8.65 mM, making this enzyme more resistant to end-product inhibition compared to other fungal cellobiohydrolases.

Contrasting soil fungal community responses to experimental nitrogen addition using the large subunit rRNA taxonomic marker and cellobiohydrolase I functional marker.

Human activities have resulted in increased nitrogen inputs into terrestrial ecosystems, but the impact of nitrogen on ecosystem function, such as nutrient cycling, will depend at least in part on the response of soil fungal communities. We examined the response of soil fungi to experimental nitrogen addition in a loblolly pine forest (North Carolina, USA) using a taxonomic marker (large subunit rDNA, LSU) and a functional marker involved in a critical step of cellulose degradation (cellobiohydrolase, cbhI) at five time points that spanned fourteen months. Sampling date had no impact on fungal community richness or composition for either gene. Based on the LSU, nitrogen addition led to increased fungal community richness, reduced relative abundance of fungi in the phylum Basidiomycota and altered community composition; however, similar shifts were not observed with cbhI. Fungal community dissimilarity of the LSU and cbhI genes was significantly correlated in the ambient plots, but not in nitrogen-amended plots, suggesting either functional redundancy of fungi with the cbhI gene or shifts in other functional groups in response to nitrogen addition. To determine whether sequence similarity of cbhI could be predicted based on taxonomic relatedness of fungi, we conducted a phylogenetic analysis of publically available cbhI sequences from known isolates and found that for a subset of isolates, similar cbhI genes were found within distantly related fungal taxa. Together, these findings suggest that taxonomic shifts in the total fungal community do not necessarily result in changes in the functional diversity of fungi.

Cloning and Sequence Analysis of the Cellobiohydrolase I Genes from Some Basidiomycetes

Genes encoding the cellobiohydrolase enzyme (CBHI), designated as cbhI, were isolated from the basidiomycetes Auricularia fuscosuccinea, Pleurotus giganteus, P. eryngii, P. ostreatus, and P. sajor-caju. Initially, the fungal genomic DNA was extracted using a modified cetyltrimethyl ammonium bromide (CTAB) protocol and used as a DNA template. The cbhI genes were then amplified and cloned using the pGEM-T Easy Vector Systems. The sizes of these PCR amplicons were between 700~800 bp. The DNA sequences obtained were similar showing high identity to the cbhI gene family. These cbhI genes were partial consisting of three coding regions and two introns. The deduced amino acid sequences exhibited significant similarity to those of fungal CBHI enzymes belonging to glycosyl hydrolase family 7.

Responses of soil cellulolytic fungal communities to elevated atmospheric CO2 are complex and variable across five ecosystems

Elevated atmospheric CO(2) generally increases plant productivity and subsequently increases the availability of cellulose in soil to microbial decomposers. As key cellulose degraders, soil fungi are likely to be one of the most impacted and responsive microbial groups to elevated atmospheric CO(2). To investigate the impacts of ecosystem type and elevated atmospheric CO(2) on cellulolytic fungal communities, we sequenced 10,677 cbhI gene fragments encoding the catalytic subunit of cellobiohydrolase I, across five distinct terrestrial ecosystem experiments after a decade of exposure to elevated CO(2). The cbhI composition of each ecosystem was distinct, as supported by weighted Unifrac analyses (all P-values; < 0.001), with few operational taxonomic units (OTUs) being shared across ecosystems. Using a 114-member cbhI sequence database compiled from known fungi, less than 1% of the environmental sequences could be classified at the family level indicating that cellulolytic fungi in situ are likely dominated by novel fungi or known fungi that are not yet recognized as cellulose degraders. Shifts in fungal cbhI composition and richness that were correlated with elevated CO(2) exposure varied across the ecosystems. In aspen plantation and desert creosote bush soils, cbhI gene richness was significantly higher after exposure to elevated CO(2) (550 µmol mol(-1)) than under ambient CO(2) (360 µmol mol(-1) CO(2)). In contrast, while the richness was not altered, the relative abundance of dominant OTUs in desert soil crusts was significantly shifted. This suggests that responses are complex, vary across different ecosystems and, in at least one case, are OTU-specific. Collectively, our results document the complexity of cellulolytic fungal communities in multiple terrestrial ecosystems and the variability of their responses to long-term exposure to elevated atmospheric CO(2).

Active and total microbial communities in forest soil are largely different and highly stratified during decomposition.

Soils of coniferous forest ecosystems are important for the global carbon cycle, and the identification of active microbial decomposers is essential for understanding organic matter transformation in these ecosystems. By the independent analysis of DNA and RNA, whole communities of bacteria and fungi and its active members were compared in topsoil of a Picea abies forest during a period of organic matter decomposition. Fungi quantitatively dominate the microbial community in the litter horizon, while the organic horizon shows comparable amount of fungal and bacterial biomasses. Active microbial populations obtained by RNA analysis exhibit similar diversity as DNA-derived populations, but significantly differ in the composition of microbial taxa. Several highly active taxa, especially fungal ones, show low abundance or even absence in the DNA pool. Bacteria and especially fungi are often distinctly associated with a particular soil horizon. Fungal communities are less even than bacterial ones and show higher relative abundances of dominant species. While dominant bacterial species are distributed across the studied ecosystem, distribution of dominant fungi is often spatially restricted as they are only recovered at some locations. The sequences of cbhI gene encoding for cellobiohydrolase (exocellulase), an essential enzyme for cellulose decomposition, were compared in soil metagenome and metatranscriptome and assigned to their producers. Litter horizon exhibits higher diversity and higher proportion of expressed sequences than organic horizon. Cellulose decomposition is mediated by highly diverse fungal populations largely distinct between soil horizons. The results indicate that low-abundance species make an important contribution to decomposition processes in soils.

Cloning of two cellobiohydrolase genes from Trichoderma viride and heterogenous expression in yeast Saccharomyces cerevisiae

Cellobiohydrolase genes cbhI and cbhII were isolated from Trichoderma viride AS3.3711 and T. viride CICC 13038, respectively, using RT-PCR technique. The cbhI gene from T. viride AS3.3711 contains 1,542 nucleotides and encodes a 514-amino acid protein with a molecular weight of approximately 53.96 kDa. The cbhII gene from T. viride CICC 13038 was 1,413 bp in length encoding 471 amino acid residues with a molecular weight of approximately 49.55 kDa. The CBHI protein showed high homology with enzymes belonging to glycoside hydrolase family 7 and CBHII is a member of Glycoside hydrolase family 6. CBHI and CBHII play a role in the conversion of cellulose to glucose by cutting the disaccharide cellobiose from the non-reducing end of the cellulose polymer chain. The two cellobiohydrolase (CBHI, CBHII) genes were successfully expressed in Saccharomyces cerevisiae H158. Maximal activities of transformants Sc-cbhI and Sc-cbhII were 0.03 and 0.089 units ml(-1) under galactose induction, respectively. The optimal temperatures of the recombinant enzymes (CBHI, CBHII) were 60 and 70 degrees C, respectively. The optimal pHs of recombinant enzymes CBHI and CBHII were at pH 5.8 and 5.0, respectively.

Identification and expression of cellobiohydrolase (CBHI) gene from an endophytic fungus, Fusicoccum sp. (BCC4124) in Pichia pastoris

A gene encoding a cellobiohydrolase (CBHI) was isolated from Fusicoccum sp. (BCC4124), an endophytic fungus belongs in phylum Ascomycota, using 5′ and 3′ rapid amplification of cDNA end (RACE) technique. This CBHI gene contains 1395 nucleotides and encodes a 465-amino acid protein with a molecular weight of approximately 50 kDa. The deduced amino acid sequence showed significant similarity to those of other fungal CBHI belonging to family 7 of glycosyl hydrolase. Interestingly, the result from the amino acid alignment revealed that this CBHI does not contain the cellulose binding domain nor the linker region. The CBHI gene was successfully expressed in Pichia pastoris KM71. The purified recombinant CBHI has ability to hydrolyze Avicel, filter paper and 4-methylumbelliferyl β- d-cellobioside (MUC) but not carboxymethylcellulose (CMC). It showed an optimal working condition at 40 °C, pH 5 with K m and V max toward MUC of 0.57 mM and 3.086 nmol/min/mg protein, respectively. The purified enzyme was stable at pH range of 3–11. The enzyme retained approximately 50% of its maximal activity after incubating at 70–90 °C for 30 min. Due to its stability through wide range of pH, and moderately stable at high temperature, this enzyme has potential in various biotechnology applications.

Overexpression of Aspergillus aculeatus cellobiohydrolase I in Aspergillus oryzae

To express the cbhI gene, encoding Aspergillus aculeatus cellobiohydrolase I (CBHI), in Aspergillus oryzae, a plasmid was constructed. The strain that displayed the strongest CBHI activity among the transformants produced about 941 mg/ l in liquid culture. It was confirmed by a PCR method that the plasmid was integrated at the niaD locus.

Overexpression of Aspergillus aculeatus Cellobiohydrolase I in Aspergillus oryzae

To express the cbhI gene, encoding Aspergillus aculeatus cellobiohydrolase I (CBHI), in Aspergillus oryzae, a plasmid was constructed. The strain that displayed the strongest CBHI activity among the transformants produced about 941 mg/l in liquid culture. It was confirmed by a PCR method that the plasmid was integrated at the niaD locus.

Cloning of the cbhI and cbhII genes involved in cellulose utilisation by the straw mushroom Volvariella volvacea.

The straw mushroom Volvariella volvacea is cultivated on substrates rich in cellulose and has been shown to produce a family of cellulolytic enzymes. A PCR-based strategy was adopted to clone genes involved in cellulose utilisation, using degenerate primers designed to amplify conserved catalytic domain sequences of cellobiohydrolases (CBHs). PCR with these primers produced two DNA fragments with sequence similarity to the cbhI and cbhII gene families detected in Trichoderma, Phanerochaete and Agaricus species. Full-length clones of these genes were obtained from an EMBL3 genomic library, and RACE-PCR was used to verify the presence of introns. The cbhI homologue has a coding region of 1722 bp, containing two introns, generating a 536 amino acid polypeptide product. The cbhII gene has a coding region of 1693 bp, containing five introns, and gives rise to a 470-amino acid polypeptide product. Northern and PCR analyses were used to study the expression of the genes. These revealed that transcripts of both genes were induced on medium containing cellulose with cbhI being expressed more strongly than cbhII - but were repressed on medium containing glucose.

Expression of Trichoderma reesei exo-cellobiohydrolase I in transgenic tobacco leaves and calli.

Expression of Trichoderma reesei exo-cellobiohydrolase I (CBHI) gene in transgenic tobacco was under the control of CaMV 35S promoter. In transgenic leaf tissues, CBHI activity up to 66.1 micromol/h/g total protein was observed. In transgenic calli, the highest CBHI activity was 83.6 micromol h/g total protein. Protein immunoblot analysis confirms the presence of CBHI enzyme in both transgenic calli and leaf tissues. CBHI expression levels accounted for about 0.11% and 0.082% of total protein in transgenic leaf tissues and calli, respectively. Furthermore, expression of CBHI gene did not affect normal growth and development of transgenic plants.

Cloning, nucleotide sequence, and transcriptional analysis of Aspergillus aculeatus no. F-50 cellobiohydrolase I ( cbhI) gene

The cbhI gene, coding for a major cellobiohydrolase (CBHI) of Aspergillus aculeatus, was cloned and sequenced. The gene consists of 1620-bp and encodes a protein containing 540 amino acids with a calculated molecular mass of 56,723 Da. CBHI, composed of an N-terminal catalytic domain belonging to family 7 of the glycosyl hydrolases, and a C-terminal cellulose-binding domain (CBD) belonging to family I of the CBDs, showed high similarity with other fungal CBHIs, especially with that of Penicillium janthinellum. The cbhI gene transcription start points in A. aculeatus were defined by primer extension, and the putative promoter sequence was analyzed. This sequence was found to be closely related to the consensus sequences of various fungal genes. Transcription analysis by ribonuclease protection assay revealed that the cbhI gene is induced by low-molecular-weight cellooligosaccharide and repressed by glucose. The results emphasize the possibility that in the A. aculeatus cellulase system, cellobiose is the true inducer and the role of the cbhI gene lies within the cascade regulating cellulase induction.