Enhancing cellulase production by overexpression of xylanase regulator protein gene,xlnR, in Talaromyces cellulolyticus cellulase hyperproducing mutant strain

We obtained strains with the xylanase regulator gene, xlnR, overexpressed (HXlnR) and disrupted (DXlnR) derived from Talaromyces cellulolyticus strain C-1, which is a cellulase hyperproducing mutant. Filter paper degrading enzyme activity and cellobiohydrolase I gene expression was the highest in HXlnR, followed by C-1 and DXlnR. These results indicate that the enhancement of cellulase productivity was succeeded by xlnR overexpression.     

Switchgrass leaching requirements for solid‐state fermentation by Acidothermus cellulolyticus

Growth of Acidothermus cellulolyticus in solid‐state fermentation and its required growth conditions were investigated in this study. Extraction of switchgrass was required for growth. Under the experimental conditions, extraction ratio had the most significant effect on the growth of A. cellulolyticus. Heat treatment (in the form of autoclaving) of switchgrass did not have a significant effect on the growth rate; however, longer heat treatment times had a negative effect on the total growth. Moisture content adjustment had no effect on the release of inhibitors into extracts. Our results showed that leaching at a minimum 40:1 (gram water: gram dry biomass) removed inhibitory compound(s) from switchgrass. Upon extraction A. cellulolyticus colonized switchgrass in solid fermentation without exogenous addition of carbon and nitrogen sources. It is the first demonstration of growth of A. cellulolyticus in solid fermentation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

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 80 h of saccharification of potato pulp.     

Disruption of the L-arabitol dehydrogenase encoding gene in Aspergillus tubingensis results in increased xylanase production

Fungal xylanases are of major importance to many industrial sectors, such as food and feed, paper and pulp, and biofuels. Improving their production is therefore highly relevant. We determined the molecular basis of an improved xylanase-producing strain of Aspergillus tubingensis that was generated by UV mutagenesis in an industrial strain improvement program. Using enzyme assays, gene expression, sequencing of the ladA locus in the parent and mutant, and complementation of the mutation, we were able to show that improved xylanase production was mainly caused by a chromosomal translocation that occurred between a subtilisin-like protease pepD gene and the L -arabitol dehydrogenase encoding gene (ladA), which is part of the L -arabinose catabolic pathway. This genomic rearrangement resulted in disruption of both genes and, as a consequence, the inability of the mutant to use L -arabinose as a carbon source, while growth on D -xylose was unaffected. Complementation with constitutively expressed ladA confirmed that the xylanase overproducing phenotype was mainly caused by loss of ladA function, while a knockout of xlnR in the UV mutant demonstrated that improved xylanase production was mediated by XlnR. This study demonstrates the potential of metabolic manipulation for increased production of fungal enzymes.     

Characterization of a feruloyl esterase B from Talaromyces cellulolyticus

A feruloyl esterase catalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl) group from esterified sugars in plant cell walls. Talaromyces cellulolyticus is a high cellulolytic-enzyme producing fungus. However, there is no report for feruloyl esterase activity of T. cellulolyticus. Analysis of the genome database of T. cellulolyticus identified a gene encoding a putative feruloyl esterase B. The recombinant enzyme was prepared using a T. cellulolyticus homologous expression system and characterized. The purified enzyme exhibited hydrolytic activity toward p-nitrophenyl acetate, p-nitrophenyl trans-ferulate, methyl ferulate, rice husk, and bagasse. HPLC assays showed that the enzyme released ferulic acid and p-coumaric acid from hydrothermal-treated rice husk and bagasse. Trichoderma sp. is well-known high cellulolytic-enzyme producing fungus useful for the lignocellulosic biomass saccharification. Interestingly, no feruloyl esterase has been reported from Trichoderma sp. The results show that this enzyme is expected to be industrially useful for biomass saccharification.     

Morphological,physiological, and molecular characterization of actinomycetes isolated from dry soil,rocks, and monument surfaces

In an extended study on the biodiversity of rock-dwelling bacteria, the colony and cell morphology, physiology, protein patterns, and 16S rDNA sequences of 17 bacterial strains isolated from different surfaces of rocks, stones, and monuments and from various geographical locations were characterized. All except one strain, which was found to be a Bacillus, were members of the order Actinomycetales. The majority of the strains either were closely related to Geodermatophilus obscurus, which was also analyzed in this study, or formed a closely related sister taxon. All of these strains were isolated from the surface of marble in Namibia and Greece and from limestone from the Negev desert, Israel. One strain, G10, of Namibia origin was equidistantly related to Geodermatophilus obscurus, Frankia alni, Sporichthya polymorpha, and Acidothermus cellulolyticus. Three strains from rock varnish in the Mojave desert, California, were found to be highly related to Arthrobacter (formerly Micrococcus) agilis. All clusters could be confirmed from results of studies on morphological and physiological properties and from banding patterns of whole cell proteins. Based on the results of tests, four additional strains were assigned to the lineage defined by strain G10. Received: 16 October 1995 / Accepted: 4 April 1996     

Factors Involved in Hydrolysis of Microcrystalline Cellulose by Acetivibrio cellulolyticus

Acetivibrio cellulolyticus cellulase obtained by the water elution of residual cellulose from the growth medium was compared with the cellulase activity present in culture supernatants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that water elution released most of the protein bands which adhered to undigested cellulose from the culture medium. The enzyme in the culture supernatant and that eluted from residual cellulose had specific activities for Avicel hydrolysis that were 20- to 40-fold greater than that of Trichoderma reesei cellulase. However, Ca²⁺ and a reducing agent such as dithiothreitol were required for maximum Avicel hydrolysis rates by these A. cellulolyticus enzyme preparations. The effect of these agents on p-nitrophenyl lactopyranoside hydrolysis suggested that they were required by an exoglucanase component. Supernatant enzyme preparations contained large amounts of carbohydrate which was separated from most of the cellulase protein by phenyl-Sepharose chromatography. Removal of this carbohydrate, which interfered with protein fractionations, allowed for an activity stain analysis of the supernatant enzyme.     

Enzymatic hydrolyzing performance of Acremonium cellulolyticus and Trichoderma reesei against three lignocellulosic materials

Background  

Bioethanol isolated from lignocellulosic biomass represents one of the most promising renewable and carbon neutral alternative liquid fuel sources. Enzymatic saccharification using cellulase has proven to be a useful method in the production of bioethanol. The filamentous fungi Acremonium cellulolyticus and Trichoderma reesei are known to be potential cellulase producers. In this study, we aimed to reveal the advantages and disadvantages of the cellulase enzymes derived from these fungi.     

Improvement of cellulase production in cultures of Acremonium cellulolyticus using pretreated waste milk pack with cellulase targeting for biorefinery

Cellulase production in cultures of Acremonium cellulolyticus was significantly improved by using waste milk pack (MP) that had been pretreated with cellulase. When MP cellulose pretreated with cellulase (3 FPU/g MP) for 12 h was used as the sole carbon source for A. cellulolyticus culture in a 3-L fermentor, the cellulase activity was 16 FPU/ml. This was 25-fold higher (0.67 FPU/ml) compared with untreated MP cellulose and was comparable to that achieved with pure cellulose (Solka Floc). As the pretreatment progressed, roughness on the surface of untreated MP cellulose became to be smooth, but development of fissures on the surface of pretreated MP cellulose was observed. Cellulase pretreatment of MP increased both the accessibility of A. cellulolyticus to the surface and number of adsorption sites of cellulase on the surface of MP cellulose, leading to improved cellulase production in the A. cellulolyticus.     

Studies on Cellulose Hydrolysis by Acetivibrio cellulolyticus

Acetivibrio cellulolyticus extracellular cellulase extensively hydrolyzed crystalline celluloses such as Avicel (FMC Corp., Food and Pharmaceutical Products Div., Philadelphia, Pa.) but only if it was desalted and supplemented with Ca²⁺. The Ca²⁺ effect was one of increased enzyme stability in the presence of the ion. Although preincubation of the cellulase complex at 40°C for 5 h without added Ca²⁺ had a negligible effect on endoglucanase activity or on the subseqent hydrolysis of amorphous cellulose, the capacity of the enzyme to hydrolyze crystalline cellulose was almost completely lost. Adsorption studies showed that 90% of the Avicel-solubilizing component of the total enzyme preparation bound to 2% Avicel at 40°C. Under these conditions, only 15% of the endoglucanase and 25% of the protein present in the enzyme preparation adsorbed to the substrate. The protein profile of the bound enzyme, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was complex and distinctly different from the profile observed for total cellulase preparations. The specific activity of A. cellulolyticus cellulase with respect to Avicel hydrolysis was compared with that of commercially available Trichoderma reesei cellulase.     

Accumulation of an iodophilic polysaccharide during growth of Acetivibrio cellulolyticus on cellobiose

Maximum growth of Acetivibrio cellulolyticus in 1% cellobiose (w/v, added as filter sterilized solution) medium was observed after about 24h of incubation at 35°C. The metabolic end products of growth were H₂, CO₂, acetic acid, ethanol and glucose. Growth was adversely affected if cellobiose was autoclaved with the rest of the media ingredients. In the presence of an excess of cellobiose, the cells accumulated large quantities of an iodophilic polysaccharide (IPS). The maximum IPS accumulation (about 37% of the cell dry weight) was observed after about 12h growth under nitrogen-limiting conditions. Starvation of these cells anaerobically, in a pH 7.0 phosphate buffer for 10 h at 35°C, resulted in about 50% drop in the IPS. The results also indicated that A. cellulolyticus accumulated this iodophilic polysaccharide during growth on cellobiose but not during cultivation on cellulose.Abbreviation IPS iodophilic polysaccharide Issued as NRCC No. 19386     

Regulation of the xylanase gene, cgxA, from Chaetomium gracile by transcriptional factors, XlnR and AnRP

The roles of XlnR and AnRP in regulating the expression of the xylanase gene, cgxA, from Chaetomium gracile were investigated using Aspergillus nidulansas an intermediate host. The XlnR consensus binding sequence –GGCTAA– in the promoter region was functional in vivo. The cgxA gene was induced when xylan was used as a carbon source but this inducibility was abolished when the XlnR binding sequence was mutated. Furthermore, the induction by xylan was increased when the AnRP binding sequence –TTGACAAAT– was mutated. Electrophoretic mobility shift assays using partially purified AnRP and an Aspergillus oryzae XlnR fusion protein, MalE-AoXlnR, provided evidence that the binding of the two proteins to their respective sites in the cgxA promoter region was mutually exclusive.     

Metabolism of Acetivibrio cellulolyticus during optimized growth on glucose,cellobiose and cellulose

Summary The growth of Acetivibrio cellulolyticus in 2.5 l batch cultures was optimized by controlling the growth pH at 6.7, the dissolved inorganic sulphide concentration at 0.4–0.6 mM, and by constant removal of hydrogen from the cultures by sparging with N₂/CO₂ or N₂ gas. An initial ethanol concentration of 0.15% (w/v) in cellobiose media resulted in specific growth rates which were reduced by about 75% compared to growth rates of 0.17 h^–1 in control cultures. Acetivibrio cellulolyticus had to be adapted for growth on glucose and ¹⁴C-radiotracer studies indicated that glucose was metabolized by the Embden-Meyerhof pathway. The specific growth rate (=0.03h^–1) and molar growth yield (Y_glucose=21.5) were considerably lower than those obtained (=0.17 h^–1, Y_cellobiose=68.9) in cellobiose media. A Y_ATP of 12.8 was obtained during growth on cellobiose. The mol product formed per mol Avicel cellulose fermented (on anhydroglucose equivalent basis) were 3.70 H₂, 2.64 CO₂, 0.73 acetate, 0.39 ethanol and 0.03 total soluble sugars on glucose basis. Maximum cellulase activity was observed in cellulose-grown cultures.National Research council of Canada No. 20826     

Conversion of Cellulose to Methane and Carbon Dioxide by Triculture of Acetivibrio cellulolyticus, Desulfovibrio sp., and Methanosarcina barkeri

The fermentation of cellulose by monocultures of Acetivibrio cellulolyticus and cocultures of A. cellulolyticus-Methanosarcina barkeri, A. cellulolyticus-Desulfovibrio sp., and A. cellulolyticus-M. barkeri-Desulfovibrio sp. was studied. The monoculture produced ethanol, acetate, H₂, and CO₂. More acetate and less ethanol was formed by the cocultures than by the monoculture. Acetate was utilized by M. barkeri in coculture with A. cellulolyticus after a lag period, whereas ethanol was metabolized by the sulfate reducer only under conditions of low H₂ partial pressure, i.e., when cocultured with A. celluloyticus-M. barkeri or when grown together with the methanogen. Only the three-component culture carried out the rapid conversion of cellulose to CO₂ and methane. Furthermore, this culture hydrolyzed the most cellulose—85% of that initially present. This amount was increased to 90% by increasing the population of M. barkeri in the triculture. Methane production was also increased, and a quicker fermentation rate was achieved.     

A transcriptional activator, AoXlnR, controls the expression of genes encoding xylanolytic enzymes in Aspergillus oryzae

By deletion across the promoter region of the xynF1 gene encoding the major Aspergillus oryzae xylanase, a 53-bp DNA fragment containing the XlnR binding sequence GGCTAAA as well as two similar sequences was shown to confer xylan inducibility on the gene. Complementary and genomic DNAs encoding the Aspergillus niger xlnR homologous gene, abbreviated AoxlnR, were cloned from A. oryzae and sequenced. AoXlnR comprised 971 amino acids with a zinc binuclear cluster domain at the N-terminal region and revealed 77.5% identity to the A. niger XlnR. Recombinant AoXlnR protein encompassing the zinc cluster region of the N-terminal part bound to both the consensus binding sequence and its cognate sequence, GGCTGA, with an approximately 10 times lower affinity. GGCTA/GA is more appropriate as the XlnR consensus binding sequence. Both sequences functioned independently in vivo in XlnR-mediating induction of the xynF1 gene. This was further confirmed by using an AoxlnR disruptant. Neither the xynF1 nor the xylA gene was expressed in the disruptant, suggesting that the xylan-inducible genes in A. oryzae may also be controlled in the same manner as described for A. niger.