Acremonium Cellulolyticus Research Articles

Identification of the Talaromyces cellulolyticus Gene Encoding an Extracellular Enzyme with β-galactosidase Activity and Testing it as a Reporter for Gene Expression Assays.

The filamentous fungus Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) is currently being intensively studied as a promising industrial producer of a number of secreted cellulolytic enzymes. In this study, the T. cellulolyticus gene lacA, which encodes a protein orthologous to the fungal extracellular β-galactosidases of family 35, was identified. The substitution of the lacA upstream region with a constitutive promoter demonstrated that the product of this gene is effectively secreted and possesses β-galactosidase activity. The optimal pH and temperature values for the hydrolysis of o-nitrophenyl-β-D-galactopyranoside by this enzyme were determined to be pH 4.5-5.5 and 50°C, respectively. The negligible production of β-galactosidase activity by strains expressing lacA under native regulation raises the possibility of using lacA as a reporter gene. To test this hypothesis, the native promoter of lacA was replaced with the strong inducible promoter of the T. cellulolyticus cellobiohydrolase I gene. The cultivation of the resulting strain in various media showed that the β-galactosidase activity depends on cultivation conditions similar to the cellobiohydrolase activity. Thus, the suitability of lacA as a reporter for evaluating promoters with a wide range of expression profiles was demonstrated.

Acremonium cellulolyticus Y-94由来の細胞壁分解酵素の添加がサイレージの発酵品質,乾物回収率および細胞壁成分に及ぼす影響

Acremonium cellulolyticus Y-94由来の細胞壁分解酵素の添加がサイレージの発酵品質,乾物回収率および細胞壁成分に及ぼす影響

Acremonium cellulolyticus Y-94由来のセルラーゼの添加がアルファルファサイレージの発酵品質に及ぼす影響

Acremonium cellulolyticus Y-94由来のセルラーゼの添加がアルファルファサイレージの発酵品質に及ぼす影響

Acremonium cellulolyticus Y-94由来のセルラーゼ処理によってアルファルファ乾燥粉末から遊離する糖類

Acremonium cellulolyticus Y-94由来のセルラーゼ処理によってアルファルファ乾燥粉末から遊離する糖類

Effects of different cellulases on the release of phenolic acids from rice straw during saccharification

Effects of different cellulases on the release of phenolic acids from rice straw during saccharification were investigated in this study. All cellulases tested increased the contents of phenolic acids during saccharification. However, few free phenolic acids were detected, as they were present in conjugated form after saccharification when the cellulases from Trichoderma reesei, Trichoderma viride and Aspergillus niger were used. On the other hand, phenolic acids were present in free form when the Acremonium cellulolyticus cellulase was used. Assays of enzyme activity showed that, besides high cellulase activity, the A. cellulolyticus cellulase exhibited high feruloyl esterase (FAE) activity. A synergistic interaction between FAE and cellulase led to the increase in free phenolic acids, and thus an increase in antioxidative and antiradical activities of the phenolic acids. Moreover, a cost estimation demonstrated the feasibility of phenolic acids as value-added products to reduce the total production cost of ethanol.

Sixteen New Records of Ascomycetes from Crop Field Soil in Korea

The present study reports 16 species of Ascomycota that were previously unknown in Korea, namely Acremonium cellulolyticus (KNU14-25), Acremonium zonatum (KNU14-15), Chaetomium madrasense (KNU14-9), Cladosporium silenes (KNU 14-18-1), Humicolopsis cephalosporioides (KNU15-3), Leptosphaerulina chartarum (KNU14-16), Paecilomyces marquandii (KNU14- 8), Paecilomyces tenuis (KNU14-18-2), Paraphaeosphaeria sporulosa (KNU15-2), Penicillium rubidurum (KNU14-12), Pochonia suchlasporia (KNU15-6), Sporothrix inflata (KNU15-8), Thielavia hyrcaniae (KNU15-1), Thielavia terricola (KNU14-23-1), Xylogone sphaerospora (KNU15-7), and Zopfiella longicaudata (KNU15-5). These fungal species were isolated from soil samples collected from various regions of Korea and identified based on their morphological characteristics and rDNA internal transcribed spacer sequence data. Full descriptions and illustrations for each species are provided.

Draft Genome Sequence of Talaromyces cellulolyticus Strain Y-94, a Source of Lignocellulosic Biomass-Degrading Enzymes.

Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) is a promising fungus for cellulase production. Here, we present the draft genome sequence of T. cellulolyticus strain Y-94. The genome is 36.4 Mbp long and contains genes for several enzymes involved in the degradation of lignocellulosic biomass, including cellulases, hemicellulases, pectinases, and amylases.

Untreated Chlorella homosphaera biomass allows for high rates of cell wall glucan enzymatic hydrolysis when using exoglucanase-free cellulases.

BackgroundChlorophyte microalgae have a cell wall containing a large quantity of cellulose Iα with a triclinic unit cell hydrogen-bonding pattern that is more susceptible to hydrolysis than that of the cellulose Iβ polymorphic form that is predominant in higher plants. This study addressed the enzymatic hydrolysis of untreated Chlorella homosphaera biomass using selected enzyme preparations, aiming to identify the relevant activity profile for the microalgae cellulose hydrolysis. Enzymes from Acremonium cellulolyticus, which secretes a complete pool of cellulases plus β-glucosidase; Trichoderma reesei, which secretes a complete pool of cellulases with low β-glucosidase; Aspergillus awamori, which secretes endoglucanases and β-glucosidase; blends of T. reesei-A. awamori or A. awamori-A. cellulolyticus enzymes; and a purified A. awamori β-glucosidase were evaluated.ResultsThe highest initial glucan hydrolysis rate of 140.3 mg/g/h was observed for A. awamori enzymes with high β-glucosidase, low endoglucanase, and negligible cellobiohydrolase activities. The initial hydrolysis rates when using A. cellulolyticus or T. reesei enzymes were significantly lower, whereas the results for the T. reesei-A. awamori and A. awamori-A. cellulolyticus blends were similar to that for the A. awamori enzymes. Thus, the hydrolysis of C. homosphaera cellulose was performed exclusively by the endoglucanase and β-glucosidase activities. X-ray diffraction data showing negligible cellulose crystallinity for untreated C. homosphaera biomass corroborate these findings. The A. awamori-A. cellulolyticus blend showed the highest initial polysaccharide hydrolysis rate of 185.6 mg/g/h, as measured by glucose equivalent, in addition to the highest predicted maximum glucan hydrolysis yield of 47% of total glucose (w/w). T. reesei enzymes showed the lowest predicted maximum glucan hydrolysis yield of 25% (w/w), whereas the maximum yields of approximately 31% were observed for the other enzyme preparations. The hydrolysis yields were proportional to the enzyme β-glucosidase load, indicating that the endoglucanase load was not rate-limiting.ConclusionsHigh rates of enzymatic hydrolysis were achieved for untreated C. homosphaera biomass with enzymes containing endoglucanase and β-glucosidase activities and devoid of cellobiohydrolase activity. These findings simplify the complexity of the enzyme pools required for the enzymatic hydrolysis of microalgal biomass decreasing the enzyme cost for the production of microalgae-derived glucose syrups.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0215-1) contains supplementary material, which is available to authorized users.

Decreased cellulase and xylanase production in the fungus Talaromyces cellulolyticus by disruption of tacA and tctA genes, encoding putative zinc finger transcriptional factors.

Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) is one of the important strains for industrial cellulase production. An understanding of the control of cellulase gene expression in T. cellulolyticus is insufficient because only a few transcriptional factors related to cellulase gene expression have been identified. In the present study, we disrupted seven putative transcription regulator genes that showed similarity with cellulase or hemicellulase regulator genes in other filamentous fungi and investigated whether these genes are related to cellulase and xylanase production. Among the seven genes, five (tclA, tbgA, tlaA, tmcA, tclB2) had a smaller effect on cellulase and xylanase activities when culturing with cellulose. On the other hand, disruption of tacA and tctA, which are respectively homologues of ace1 (repressor of cellulase) and ctf1 (inducer of cutinase), led to a decrease in cellulase and hemicellulase production due to effects at both the enzymatic and transcriptional levels, indicating that tacA and tctA have positive roles in cellulase and xylanase production in T. cellulolyticus. These results suggest that cellulase and xylanase gene regulation in T. cellulolyticus differs from that in other filamentous fungi and imply that unknown transcriptional mechanisms function in T. cellulolyticus.

Heterologous expression of hyperthermophilic cellulases of archaea Pyrococcus sp. by fungus Talaromyces cellulolyticus.

Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus) is one of the high cellulolytic enzyme-producing fungi. T. cellulolyticus exhibits the potential ability for high amount production of enzyme proteins. Using the homologous expression system under the control of a glucoamylase promoter, some kinds of cellulases of T. cellulolyticus can be expressed by T. cellulolyticus. On the other hand, hyperthermophilic cellulase has been expected to be useful in the industrial applications to biomass. The hyperthermophilic archaea Pyrococcus horikoshii and P. furiosus have GH family 5 and 12 hyperthermophilic endocellulase, respectively. The two kinds of hyperthermophilic endocellulases were successfully produced by T. cellulolyticus using the above expression system under the control of a glucoamylase promoter of T. cellulolyticus. These recombinant cellulases exhibited the same characteristics as those of the recombinant cellulases prepared in E. coli. The productions of the recombinant enzymes were estimated to be over 100 mg/L. In this study, we first report the overexpression of the hyperthermophilic enzymes of archaea using the fungal expression system.

Identification and characterization of core cellulolytic enzymes from Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) critical for hydrolysis of lignocellulosic biomass.

BackgroundEnzymatic hydrolysis of pretreated lignocellulosic biomass is an essential process for the production of fermentable sugars for industrial use. A better understanding of fungal cellulase systems will provide clues for maximizing the hydrolysis of target biomass. Talaromyces cellulolyticus is a promising fungus for cellulase production and efficient biomass hydrolysis. Several cellulolytic enzymes purified from T. cellulolyticus were characterized in earlier studies, but the core enzymes critical for hydrolysis of lignocellulosic biomass remain unknown.ResultsSix cellulolytic enzymes critical for the hydrolysis of crystalline cellulose were purified from T. cellulolyticus culture supernatant using an enzyme assay based on synergistic hydrolysis of Avicel. The purified enzymes were identified by their substrate specificities and analyses of trypsin-digested peptide fragments and were classified into the following glycosyl hydrolase (GH) families: GH3 (β-glucosidase, Bgl3A), GH5 (endoglucanase, Cel5A), GH6 (cellobiohydrolase II, Cel6A), GH7 (cellobiohydrolase I and endoglucanase, Cel7A and Cel7B, respectively), and GH10 (xylanase, Xyl10A). Hydrolysis of dilute acid-pretreated corn stover (PCS) with mixtures of the purified enzymes showed that Cel5A, Cel7B, and Xyl10A each had synergistic effects with a mixture of Cel6A and Cel7A. Cel5A seemed to be more effective in the synergistic hydrolysis of the PCS than Cel7B. The ratio of Cel5A, Cel6A, Cel7A, and Xyl10A was statistically optimized for the hydrolysis of PCS glucan in the presence of Bgl3A. The resultant mixture achieved higher PCS glucan hydrolysis at lower enzyme loading than a culture filtrate from T. cellulolyticus or a commercial enzyme preparation, demonstrating that the five enzymes play a role as core enzymes in the hydrolysis of PCS glucan.ConclusionsCore cellulolytic enzymes in the T. cellulolyticus cellulase system were identified to Cel5A, Cel6A, Cel7A, Xyl10A, and Bgl3A and characterized. The optimized mixture of these five enzymes was highly effective for the hydrolysis of PCS glucan, providing a foundation for future improvement of the T. cellulolyticus cellulase system.

Characterization of the xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus).

We cloned a putative Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus) xlnR gene and isolated a xlnR disruptant strain. XlnR protein was localized in the nucleus. Xylanase production by the xlnR disruptant was lower than in the control strain at both the enzyme and transcriptional level. These data suggest that the XlnR protein regulates xylanase production in T. cellulolyticus.

Gene targeting by RNAi-mediated knockdown of potent DNA ligase IV homologue in the cellulase-producing fungus Talaromyces cellulolyticus.

The genome of the cellulase-producing fungus Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) was screened for a potent DNA ligase IV gene (ligD homologue). Homologous recombination efficiency in T. cellulolyticus is very low. Therefore, suppression of a non-homologous end-joining system was attempted to enable specific gene knockouts for molecular breeding. The transcript levels of ligD homologue were 0.037 of those of the parental YP-4 strain in the Li20 transformant carrying the RNAi construct targeting the ligD homologue. Transformation of the hairpin-type RNAi vector into T. cellulolyticus could be useful in fungal gene knockdown experiments. Cellulase production and protein secretion were similar in the parental YP-4 strain and the Li20 transformant. Knockout transformation of ligD homologue using the Li20 transformant led to 23.1 % double crossover gene targeting. Our results suggest that the potent DNA ligase IV gene of T. cellulolyticus is related to non-homologous end joining and that the knockdown of the ligD homologue is useful in gene targeting.

Crystal structure of Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus) GH family 11 xylanase.

Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus) is one of the mesophilic fungi that can produce high levels of cellulose-related enzymes and are expected to be used for the degradation of polysaccharide biomass. In silico analysis of the genome sequence of T. cellulolyticus detected seven open reading frames (ORFs) showing homology to xylanases from glycoside hydrolase (GH) family 11. The gene encoding the GH11 xylanase C (TcXylC) with the highest activity was used for overproduction and purification of the recombinant enzyme, permitting solving of the crystal structure to a resolution of 1.98 Å. In the asymmetric unit, two kinds of the crystal structures of the xylanase were identified. The main structure of the protein showed a β-jelly roll structure. We hypothesize that one of the two structures represents the open form and the other shows the close form. The changing of the flexible region between the two structures is presumed to induce and accelerate the enzyme reaction. The specificity of xylanase toward the branched xylan is discussed in the context of this structural data and by comparison with the other published structures of xylanases.

Xylanase (GH11) from Acremonium cellulolyticus: homologous expression and characterization.

Cellulosic materials constitute most of the biomass on earth, and can be converted into biofuel or bio-based materials if fermentable sugars can be released using cellulose-related enzymes. Acremonium cellulolyticus is a mesophilic fungus which produces a high amount of cellulose-related enzymes. In the genome sequence data of A. cellulolyticus, ORFs showing homology to GH10 and GH11 xylanases were found. The xylanases of A. cellulolyticus play an important role in cellulolytic biomass degradation. Search of a draft genome sequence of A. cellulolyticus for xylanase coding regions identified seven ORFs showing homology to GH 11 xylanase genes (xylA, xylB, xylC, xylD, xylE, xylF and xylG). These genes were cloned and their enzymes were prepared with a homologous expression system under the control of a glucoamylase promoter. Six of the seven recombinant enzymes were successfully expressed, prepared, and characterized. These enzymes exhibited optimal xylanase activity at pH 4.0 – 4.5. But this time, we found that only XylC had enormously higher relative activity (2947 U•mg −1) than the other xylanases at optimum pH. This result is surprising because XylC does not retain a carbohydrate-binding module 1 (CBM-1) that is necessary to bind tightly own substrate such as xylan. In this study, we discuss the relationship between activity, pH and sequence of seven xylanases in A. cellulolyticus.

RNAi Knockdown of Potent Sugar Sensor in Cellulase-Producing Fungus Acremonium cellulolyticus

A potent sugar sensor gene (g9105) was screened from the genomic data of the cellulase-producing fungus Acremonium cellulolyticus. The transcriptional level of g9105 in the SA49 transformant, which carried the knockdown RNA interference (RNAi) construct, was less than 10% compared with the parental YP-4 strain. The hairpin-type RNAi construct could be useful for this fungal gene knockdown. Changes in cellulase productivity and protein secretion between these two strains were not observed. The numbers of hyphal tips at subapical branching site were counted for the SA49 and YP-4 strains incubated with potato-dextrose medium at 30 °C for 4 days. The hyphal branching ratio of the SA49 strain was higher than that of the YP-4 strain. The present results suggest that the potent sugar sensor gene in A. cellulolyticus could be related with hyphal branch formation.

Taxonomic revision of the cellulose-degrading fungusAcremonium cellulolyticusnomen nudum toTalaromycesbased on phylogenetic analysis

The cellulase-producing fungal strain Y-94, isolated in Japan and invalidly described as Acremonium cellulolyticus nom. nud. strain Y-94, seldom forms enteroarthric conidia under nutrient starvation conditions. Phylogenetic analysis using ITS1-5.8S-ITS2 and RNA polymerase II large subunit gene sequences revealed that strain Y-94 is closely related to Talaromyces, given that these Y-94 sequences showed 100% identity with those of Talaromyces pinophilus NBRC 100533T . By contrast, the identity between β-tubulin-encoding genes from strain Y-94 and T.pinophilus NBRC 100533T was 98.1%. Morphological and phenotypic differences between these strains in colony color, conidiophore formation, and cellulase productivity were observed. Together, these data indicated that strain Y-94 belonged to the genus Talaromyces. We propose that strain Y-94 is a new species, Talaromyces cellulolyticus, on the basis of morphology and molecular evidence. The ex-holotype is Y-94 (=FERM BP-5826, CBS 136886 [holotype] TNS-F-48752).

Characteristics of enzymes from Acremonium cellulolyticus strains and their utilization in the saccharification of potato pulp

In this study, the abilities to produce enzymes by four Acremonium cellulolyticus strains were analyzed. Saccharification of potato pulp was performed to investigate the effects of the enzymes produced by A. cellulolyticus and to confirm the possibility of using A. cellulolyticus in the saccharification of potato pulp. Amylase, pectinase, galactosidase, and cellulase were produced by A. cellulolyticus from several carbon sources. Potato pulp was found to be a suitable substrate for A. cellulolyticus growth. The addition of cellulose not only improved the activity of cellulase but also improved the activity of α-galactosidase. Lactose and galactose induced the production of β-galactosidase and pectinase. Four strains of A. cellulolyticus were cultured in potato pulp to evaluate their abilities to produce cellulase, amylase, pectinase and galactosidase. Among them, A. cellulolyticus strain CF-2612 exhibited the highest production of all the enzymes. By using the crude enzymes from A. cellulolyticus strain CF-2612, 86% yield for glucose and 94% yield for galactose were achieved after 80h of saccharification of potato pulp.

Cellulose-inducible xylanase Xyl10A from Acremonium cellulolyticus: Purification, cloning and homologous expression

Cellulose-inducible endo-β-1,4-xylanase (Xyl10A) from the mesophilic fungus Acremonium cellulolyticus was purified, characterized, and expressed by a homologous expression system. A. cellulolyticus CF-2612 produces a high level of xylanase upon induction by Solka-Floc cellulose. To identify this xylanase, the major fraction showing xylanase activity was purified from the CF-2612 culture supernatant, and its gene was identified from the genome sequence. Amino acid sequence homology of Xyl10A revealed that the purified xylanase, designated Xyl10A, exhibited significant homology to family 10 of the glycoside hydrolases (GH10), possessing a cellulose-binding module 1 in the C-terminal region. The xyl10A gene was cloned and expressed in A. cellulolyticus under the control of a glucoamylase promoter. Two recombinant Xyl10As (rXyl10A-I, 53kDa, and rXyl10A-II, 51kDa) were purified that have slightly different molecular weights based on SDS–PAGE. The rXyl10As had the same physicochemical and enzymatic properties as wtXyl10A: high thermostability (Tm 80.5°C), optimum pH 5.0 and specific activity 232–251U/mg for birchwood xylan. The molecular weights of N-deglycosylated rXyl10As were consistent with that of wild-type Xyl10A (wtXyl10A, 51kDa).

Construction of a starch-inducible homologous expression system to produce cellulolytic enzymes from Acremonium cellulolyticus

A starch-inducible homologous expression system in Acremonium cellulolyticus was constructed to successfully produce recombinant cellulolytic enzymes. A. cellulolyticus Y-94 produced amylolytic enzymes and cellulolytic enzymes as major proteins in the culture supernatant when grown with soluble starch (SS) and Solka-Flock cellulose (SF), respectively. To isolate a strong starch-inducible promoter, glucoamylase (GlaA), which belongs to glycoside hydrolase family 15, was purified from the SS culture of Y-94, and its gene was identified in the genome sequence. The 1.4-kb promoter and 0.4-kb terminator regions of glaA were amplified by polymerase chain reaction (PCR) and used in the construction of a plasmid that drives the expression of the cellobiohydrolase I (Cel7A) gene from A. cellulolyticus. The resultant expression plasmid, containing pyrF as a selection marker, was randomly integrated into the genome of the A. cellulolyticus Y-94 uracil auxotroph. The prototrophic transformant, Y203, produced recombinant Cel7A as an extracellular protein under control of the glaA promoter in the SS culture. Recombinant and wild-type Cel7A were purified from the SS culture of Y203 and the SF culture of A. cellulolyticus CF-2612, respectively. Both enzymes were found to have the same apparent molecular weight (60 kDa), thermostability (T m 67.0 °C), and optimum pH (pH 4.5), and showed similar catalytic properties for soluble and insoluble substrates. These results suggest that the A. cellulolyticus starch-inducible expression system will be helpful for characterization and improvement of fungal cellulolytic enzymes.