Production and Characterization of Cellulose Nanocrystals from Eucalyptus Dissolving Pulp Using Endoglucanases from Myceliophthora thermophila.

Endoglucanase (EG) is a key enzyme during enzymatic preparation of cellulose nanocrystals (CNCs). Myceliophthora thermophila is a thermophilic fungus that has thermal properties and a high secretion of endoglucanases (EGs), and could serve as potential sources of EGs for the preparation of CNCs. In this work, four different GH families (GH5, GH7, GH12, and GH45) of EGs from M. thermophila were expressed and purified, and their enzymatic characteristics and feasibility of application in CNC preparation were investigated. It was shown that the MtEG5A from M. thermophila has good potential in the enzymatic preparation of CNCs using eucalyptus dissolving pulp as feedstock. It was also observed that there was a synergistic effect between the MtEG5A and other MtEGs in the preparation of CNCs, which improved the yield and properties of CNCs obtained by enzymatic hydrolysis. This study provides a reference for understanding the enzymatic characteristics of different families of EGs from M. thermophile and their potential application in nanocellulose production.

Deletion of the middle region of the transcription factor ClrB in Penicillium oxalicum enables cellulase production in the presence of glucose.

Enzymes that degrade lignocellulose to simple sugars are of great interest in research and for biotechnology because of their role in converting plant biomass to fuels and chemicals. The synthesis of cellulolytic enzymes in filamentous fungi is tightly regulated at the transcriptional level, with the transcriptional activator ClrB/CLR-2 playing a critical role in many species. In Penicillium oxalicum, clrB overexpression could not relieve the dependence of cellulase expression on cellulose as an inducer, suggesting that clrB is controlled post-transcriptionally. In this study, using a reporter gene system in yeast, we identified the C-terminal region of ClrB/CLR-2 as a transcriptional activation domain. Expression of clrBID , encoding a ClrB derivative in which the DNA-binding and transcriptional activation domains are fused together to remove the middle region, led to cellulase production in the absence of cellulose in P. oxalicum Strikingly, the clrBID -expressing strain produced cellulase on carbon sources that normally repress cellulase expression, including glucose and glycerol. Results from deletion of the carbon catabolite repressor gene creA in the clrBID -expressing strain suggested that the effect of clrBID is independent of CreA's repressive function. A similar modification of clrB in Aspergillus niger resulted in the production of a mannanase in glucose medium. Taken together, these results indicate that ClrB suppression under noninducing conditions involves its middle region, suggesting a potential strategy to engineer fungal strains for improved cellulase production on commonly used carbon sources.

The Role of Cross-Pathway Control Regulator CpcA in the Growth and Extracellular Enzyme Production of Penicillium oxalicum.

CpcA is a conserved transcriptional activator for the cross-pathway control of amino acid biosynthetic genes in filamentous fungi. Previous studies of this regulator mainly revealed its function under amino acid starvation condition, where amino acid biosynthetic inhibitors were added in the culture. In this study, the biological function of CpcA in Penicillium oxalicum was investigated under different cultivation conditions. Disruption of cpcA led to decreased cell growth either in the presence or absence of histidine biosynthetic inhibitor, and the phenotype could be rescued by the addition of exogenous amino acid sources. In addition, CpcA was required for the rapid production of cellulase when cells were cultured on cellulose. Transcript abundance measurement showed that a set of amino acid biosynthetic genes as well as two major cellulase genes were significantly down-regulated in cpcA deletion mutant relative to wild type. Taken together, the results revealed the biological role of CpcA in supporting normal growth and extracellular enzyme production of P. oxalicum under amino acid non-starvation condition.

Three glycoside hydrolase family 12 enzymes display diversity in substrate specificities and synergistic action between each other.

PoCel12A, PoCel12B, and PoCel12C are genes that encode glycoside hydrolase family 12 (GH12) enzymes in Penicillium oxalicum. PoCel12A and PoCel12B are typical GH12 enzymes that belong to fungal subfamilies 12-1 and 12-2, respectively. PoCel12C contains a low-complexity region (LCR) domain, which is not found in PoCel12A or PoCel12B and independent of fungal subfamily 12-1 or 12-2. Recombinant enzymes (named rCel12A, rCel12B and rCel12C) demonstrate existing diversity in the substrate specificities. Although most members in GH family 12 are typical endoglucanases and preferentially hydrolyze ß-1,4-glucan (e.g., carboxymethylcellulose), recombinant PoCel12A is a non-typical endo-(1-4)-ß-glucanase; it preferentially hydrolyzes mix-linked ß-glucan (barley ß-glucan, ß-1,3-1,4-glucan) and slightly hydrolyzes ß-1,4-glucan (carboxymethylcellulose). Recombinant PoCel12B possesses a significantly high activity against xyloglucan. A specific activity of rCel12B toward xyloglucan (239 µmol/min/mg) is the second-highest value known. Recombinant PoCel12C shows low activity toward ß-glucan, carboxymethylcellulose, or xyloglucan. All three enzymes can degrade phosphoric acid-swollen cellulose (PASC). However, the hydrolysis products toward PASC by enzymes are different: the main hydrolysis products are cellotriose, cellotetraose, and cellobiose for rCel12A, rCel12B, and rCel12C, correspondingly. A synergistic action toward PASC among rCel12A and rCel12B is observed, thereby suggesting a potential application for preparing enzyme cocktails used in lignocellulose hydrolysis.

Introduction of heterologous transcription factors and their target genes into Penicillium oxalicum leads to increased lignocellulolytic enzyme production.

Genetic engineering of transcription factors is an efficient strategy to improve lignocellulolytic enzyme production in fungi. In this study, the xylanase transcriptional regulators of Trichoderma reesei (Xyr1) and Neurospora crassa (XLR-1), as well as their constitutively active mutants (Xyr1A824V and XLR-1A828V), were heterologously expressed in Penicillium oxalicum. The two heterologous regulators were identified to be able to activate lignocellulolytic enzyme gene expression in P. oxalicum. Particularly, expression of T. reesei Xyr1 resulted in a higher cellulase production level compared with the expression of native xylanase transcriptional regulator XlnR using the same promoter. Xyr1A824V and XLR-1A828V were found to be able to confer P. oxalicum more enhanced lignocellulolytic abilities than wild-type regulators Xyr1 and XLR-1. Furthermore, introduction of regulatory modules containing Xyr1A824V/XLR-1A828V and their target cellulase genes resulted in greater increases in cellulase production than alone expression of transcriptional regulators. Through the cumulative introduction of three regulatory modules containing regulator mutants and their corresponding target cellulase genes from P. oxalicum, T. reesei, and N. crassa, a 2.8-fold increase in cellulase production was achieved in P. oxalicum.

Synergistic and Dose-Controlled Regulation of Cellulase Gene Expression in Penicillium oxalicum.

Filamentous fungus Penicillium oxalicum produces diverse lignocellulolytic enzymes, which are regulated by the combinations of many transcription factors. Here, a single-gene disruptant library for 470 transcription factors was constructed and systematically screened for cellulase production. Twenty transcription factors (including ClrB, CreA, XlnR, Ace1, AmyR, and 15 unknown proteins) were identified to play putative roles in the activation or repression of cellulase synthesis. Most of these regulators have not been characterized in any fungi before. We identified the ClrB, CreA, XlnR, and AmyR transcription factors as critical dose-dependent regulators of cellulase expression, the core regulons of which were identified by analyzing several transcriptomes and/or secretomes. Synergistic and additive modes of combinatorial control of each cellulase gene by these regulatory factors were achieved, and cellulase expression was fine-tuned in a proper and controlled manner. With one of these targets, the expression of the major intracellular ß-glucosidase Bgl2 was found to be dependent on ClrB. The Bgl2-deficient background resulted in a substantial gene activation by ClrB and proved to be closely correlated with the relief of repression mediated by CreA and AmyR during cellulase induction. Our results also signify that probing the synergistic and dose-controlled regulation mechanisms of cellulolytic regulators and using it for reconstruction of expression regulation network (RERN) may be a promising strategy for cellulolytic fungi to develop enzyme hyper-producers. Based on our data, ClrB was identified as focal point for the synergistic activation regulation of cellulase expression by integrating cellulolytic regulators and their target genes, which refined our understanding of transcriptional-regulatory network as a "seesaw model" in which the coordinated regulation of cellulolytic genes is established by counteracting activators and repressors.

Differential reinforcement of enzymatic hydrolysis by adding chemicals and accessory proteins to high solid loading substrates with different pretreatments.

High dosage of enzyme is required to achieve effective lignocellulose hydrolysis, especially at high-solid loadings, which is a significant barrier to large-scale bioconversion of lignocellulose. Here, we screened four chemical additives and three accessory proteins for their effects on the enzymatic hydrolysis of various lignocellulosic materials. The effects were found to be highly dependent on the composition and solid loadings of substrates. For xylan-extracted lignin-rich corncob residue, the enhancing effect of PEG 6000 was most pronounced and negligibly affected by solid content, which reduced more than half of enzyme demand at 20% dry matter (DM). Lytic polysaccharide monooxygenase enhanced the hydrolysis of ammonium sulfite wheat straw pulp, and its addition reduced about half of protein demand at the solid loading of 20% DM. Supplementation of the additives in the hydrolysis of pure cellulose and complex lignocellulosic materials revealed that their effects are tightly linked to pretreatment strategies.

An aldonolactonase AltA from Penicillium oxalicum mitigates the inhibition of ß-glucosidase during lignocellulose biodegradation.

Efficient deconstruction of lignocellulose is achieved by the synergistic action of various hydrolytic and oxidative enzymes. However, the aldonolactones generated by oxidative enzymes have inhibitory effects on some cellulolytic enzymes. In this work, D-glucono-1,5-lactone was shown to have a much stronger inhibitory effect than D-glucose and D-gluconate on ß-glucosidase, a vital enzyme during cellulose degradation. AltA, a secreted enzyme from Penicillium oxalicum, was identified as an aldonolactonase which can catalyze the hydrolysis of D-glucono-1,5-lactone to D-gluconic acid. In the course of lignocellulose saccharification conducted by cellulases from P. oxalicum or Trichoderma reesei, supplementation of AltA was able to relieve the decrease of ß-glucosidase activity obviously with a stimulation of glucose yield. This boosting effect disappeared when sodium azide and ethylenediaminetetraacetic acid (EDTA) were added to the saccharification system to inhibit the activities of oxidative enzymes. In summary, we describe the first heterologous expression of a fungal secreted aldonolactonase and its application as an efficient supplement of cellulolytic enzyme system for lignocellulose biodegradation.

Efficient production and evaluation of lignocellulolytic enzymes using a constitutive protein expression system in Penicillium oxalicum.

Native lignocellulolytic enzyme systems secreted by filamentous fungi can be further optimized by protein engineering or supplementation of exogenous enzyme components. We developed a protein production and evaluation system in cellulase-producing fungus Penicillium oxalicum. First, by deleting the major amylase gene amy15A, a strain Δ15A producing few extracellular proteins on starch was constructed. Then, three lignocellulolytic enzymes (BGL4, Xyn10B, and Cel12A) with originally low expression levels were successfully expressed with selected constitutive promoters in strain Δ15A. BGL4 and Cel12A overexpression resulted in increased specific filter paper activity (FPA), while the overexpression of Xyn10B improved volumetric FPA but not specific FPA. By switching the culture medium, this platform is convenient to produce originally low-expressed lignocellulolytic enzymes in relatively high purities on starch and to evaluate the effect of their supplementation on the performance of a complex cellulase system on cellulose.

Production of single cell protein from brewer's spent grain through enzymatic saccharification and fermentation enhanced by ammoniation pretreatment.

Brewer's spent grain (BSG) is a major low-value by-product of beer industry. To realize the high value application of BSG, this work proposed a strategy to produce single cell protein (SCP) with oligosaccharide prebiotics from BSG, via ammoniation pretreatment, enzymatic hydrolysis, and fermentation. The optimum conditions of ammoniation pretreatment obtained by response surface method were 11 % ammonia dosage (w/w), 63 °C for 26 h. Suitable enzyme and yeast were screened to enhance the conversion of cellulose and hemicellulose in BSG into sugars and maximize the SCP yield. It was shown that using lignocellulolytic enzyme SP from Penicillium oxalicum and Trichosporon cutaneum, about 310 g of SCP with 80 g of arabinoxylo-oligosaccharides were obtained from 1000 g of BSG. This process is low cost, high efficiency, and easy to implement, which has good industrial application prospects.

N-glycoform diversity of cellobiohydrolase I from Penicillium decumbens and synergism of nonhydrolytic glycoform in cellulose degradation.

Four cellobiohydrolase I (CBHI) glycoforms, namely, CBHI-A, CBHI-B, CBHI-C, and CBHI-D, were purified from the cultured broth of Penicillium decumbens JU-A10. All glycoforms had the same amino acid sequence but displayed different characteristics and biological functions. The effects of the N-glycans of the glycoforms on CBH activity were analyzed using mass spectrum data. Longer N-glycan chains at the Asn-137 of CBHI increased CBH activity. After the N-glycans were removed using site-directed mutagenesis and homologous expression in P. decumbens, the specific CBH activity of the recombinant CBHI without N-glycosylation increased by 65% compared with the wild-type CBHI with the highest specific activity. However, the activity was not stable. Only the N-glycosylation at Asn-137 can improve CBH activity by 40%. rCBHI with N-glycosylation only at Asn-470 exhibited no enzymatic activity. CBH activity was affected whether or not the protein was glycosylated, together with the N-glycosylation site and N-glycan structure. N-Glycosylation not only affects CBH activity but may also bring a new feature to a nonhydrolytic CBHI glycoform (CBHI-A). By supplementing CBHI-A to different commercial cellulase preparations, the glucose yield of lignocellulose hydrolysis increased by >20%. After treatment with a low dose (5 mg/g substrate) of CBHI-A at 50 °C for 7 days, the hydrogen-bond intensity and crystalline degree of cotton fibers decreased by 17 and 34%, respectively. These results may provide new guidelines for cellulase engineering.

Cellodextrin transporters play important roles in cellulase induction in the cellulolytic fungus Penicillium oxalicum.

Cellodextrin transporters (cellodextrin permeases) have been identified in fungi in recent years. However, the functions of these transporters in cellulose utilization and cellulase expression have not been well studied. In this study, three cellodextrin transporters, namely, CdtC, CdtD, and CdtG, in the cellulolytic fungus Penicillium oxalicum (formally was classified as P. decumbens) were identified, and their functions were analyzed. The deletion of a single cellodextrin transporter gene slightly decreased cellobiose consumption, but no observable effect on cellulase expression was observed, which was attributed to the overlapping activity of isozymes. Further simultaneous deletion of cdtC and cdtD resulted in significantly decreased cellobiose consumption and poor growth on cellulose. The extracellular activity and transcription level of cellulases in the mutant without cdtC and cdtD were significantly lower than those in the wild-type strain when grown on cellulose. This result provides direct evidence of the crucial function of cellodextrin transporters in the induction of cellulase expression by insoluble cellulose.

Preparation of nanocellulose crystal from bleached pulp with an engineering cellulase and co-production of ethanol.

The study investigated the feasibility of co-production of nanocellulose crystal (CNC) and ethanol using the bleached pine kraft pulp (BPKP) as a substrate by enzymatic hydrolysis. An engineering strain Penicillum oxalicum cEES-XM was constructed to produce suitable cellulase used in enzymatic hydrolysis of BPKP for preparing CNC. The cellulase from Trichoderma reesei SCB18 was used for simultaneous saccharification and fermentation of residues and hydrolysates from enzymatic hydrolysis for producing ethanol. The result showed that the CNC yield reached 7.35 % (w/w) by hydrolysis at 10 % solid content, and the final ethanol concentration was 13.27 mg/mL in fermentation liquor. Using SEM, XRD, TGA, and DLS methods, the characteristics of CNC including, morphology, crystallinity, thermal stability and particle size distribution, were also examined. This work provided a reference for realizing high-efficient application of cellulose in the pulp.

Enzymatic saccharification and ethanol fermentation of reed pretreated with liquid hot water.

Reed is a widespread-growing, inexpensive, and readily available lignocellulosic material source in northeast China. The objective of this study is to evaluate the liquid hot water (LHW) pretreatment efficiency of reed based on the enzymatic digestibility and ethanol fermentability of water-insoluble solids (WISs) from reed after the LHW pretreatment. Several variables in the LHW pretreatment and enzymatic hydrolysis process were optimized. The conversion of glucan to glucose and glucose concentrations are considered as response variables in different conditions. The optimum conditions for the LHW pretreatment of reed area temperature of 180°C for 20min and a solid-to-liquid ratio of 1 : 10. These optimum conditions for the LHW pretreatment of reed resulted in a cellulose conversion rate of 82.59% in the subsequent enzymatic hydrolysis at 50°C for 72 h with a cellulase loading of 30 filter paper unit per gram of oven-dried WIS. Increasing the pretreatment temperature resulted in a higher enzymatic digestibility of the WIS from reed. Separate hydrolysis and fermentation of WIS showed that the conversion of glucan to ethanol reached 99.5% of the theoretical yield. The LHW pretreatment of reed is a suitable method to acquire a high recovery of fermentable sugars and high ethanol conversion yield.

Dealloying Ag-Al alloy to prepare nanoporous silver as a substrate for surface-enhanced Raman scattering: effects of structural evolution and surface modification.

Sensitive detection of molecules by using the surface-enhanced Raman scattering (SERS) technique depends on the nanostructured metallic substrate and many efforts have been devoted to the preparation of SERS substrates with high sensitivity, stability, and reproducibility. Herein, we report on the fabrication of stable monolithic nanoporous silver (NPS) by chemical dealloying of Ag-Al precursor alloys with an emphasis on the effect of structural evolution on SERS signals. It was found that the dealloying conditions had great influence on the morphology (the ligament/pore size) and the crystallization status, which determined the SERS signal of rhodamine 6G on the NPS. NPS with small pores, low residual Al, and perfect crystallization gave high SERS signals. A high enhancement factor of 7.5 × 10(5) was observed on bare NPS obtained by dealloying Ag(30)Al(70) in 2.5 wt % HCl at room temperature followed by 15 min aging at around 85 °C. After coating Ag nanoparticles on the NPS surface, the enhancement factor increased to 1.6 × 10(8) owing to strong near-field coupling between the ligaments and nanoparticles.

[Progress in the production of lignocellulolytic enzyme systems using Penicillium species].

The efficient production of lignocellulolytic enzyme systems is an important support for large-scale biorefinery of plant biomass. On-site production of lignocellulolytic enzymes could increase the economic benefits of the process by lowering the cost of enzyme usage. Penicillium species are commonly found lignocellulose-degrading fungi in nature, and have been used for industrial production of cellulase preparations due to their abilities to secrete complete and well-balanced lignocellulolytic enzyme systems. Here, we introduce the reported Penicillium species for cellulase production, summarize the characteristics of their enzymes, and describe the strategies of strain engineering for improving the production and performance of lignocellulolytic enzymes. We also review the progress in fermentation process optimization regarding the on-site production of lignocellulolytic enzymes using Penicillium species, and suggest prospect of future work from the perspective of building a "sugar platform" for the biorefinery of lignocellulosic biomass.

Efficient Constitutive Expression of Cellulolytic Enzymes in Penicillium oxalicum for Improved Efficiency of Lignocellulose Degradation.

Efficient cellulolytic enzyme production is important for the development of lignocellulose-degrading enzyme mixtures. However, purification of cellulases from their native hosts is time- and labor-consuming. In this study, a constitutive expression system was developed in Penicillium oxalicum for the secreted production of proteins. Using a constitutive polyubiquitin gene promoter and cultivating with glucose as the sole carbon source, nine cellulolytic enzymes of different origins with relatively high purity were produced within 48 h. When supplemented to a commercial cellulase preparation, cellobiohydrolase I from P. funiculosum and cellobiohydrolase II from Talaromyces verruculosus showed remarkable enhancing effects on the hydrolysis of steam-exploded corn stover. Additionally, a synergistic effect was observed for these two cellobiohydrolases during the hydrolysis. Taken together, the constitutive expression system provides a convenient tool for the production of cellulolytic enzymes, which is expected to be useful in the development of highly efficient lignocellulose-degrading enzyme mixtures.

Optimization of an artificial cellulase cocktail for high-solids enzymatic hydrolysis of cellulosic materials with different pretreatment methods.

Optimization of the composition of cellulase mixtures is an effective strategy to improve their hydrolytic efficiency and reduce protein demand during enzymatic degradation of lignocelluloses. In this study, the mixture design method was used to optimize the ratios of endoglucanase II (EG II), cellobiohydrolase I (CBH I) and ß-glucosidase I (BG I) from Penicillium oxalicum in an artificial cellulase mixture for the hydrolysis of six different cellulosic materials. The optimal composition of enzyme mixture was distinctly different among not only cellulosic materials with different pretreatment methods but hydrolyses at different solids concentrations. CBH I was most critical for the hydrolysis of two acid-pretreated materials, probably due to its strong adsorption on lignin. A higher proportion of EG II was needed for the hydrolysis of ammonium sulfite pretreated wheat straw. The requirements of specific cellulase components were more pronounced at high solids concentrations, highlighting the importance of considering solids loading when optimizing cellulase cocktails.

Engineering of filamentous fungi for efficient conversion of lignocellulose: Tools, recent advances and prospects.

Filamentous fungi, as the main producers of lignocellulolytic enzymes in industry, need to be engineered to improve the economy of large-scale lignocellulose conversion. Investigation of the cellular processes involved in lignocellulolytic enzyme production, as well as optimization of enzyme mixtures for higher hydrolysis efficiency, have provided effective targets for the engineering of lignocellulolytic fungi. Recently, the development of efficient genetic manipulation systems in several lignocellulolytic fungi opens up the possibility of systems engineering of these strains. Here, we review the recent progresses made in the engineering of lignocellulolytic fungi and highlight the research gaps in this area.

Catalytic performance of lignin peroxidase in a novel reverse micelle.

To enhance the catalytic activity of lignin peroxidase (LiP) in a reverse micelle, a synthesized two-tail nonionic surfactant N-gluconyl glutamic acid didecyl ester (GGDE) was used to formulate a novel reverse micelle. Based on the LiP catalyzed oxidation of veratryl alcohol (VA) in this novel GGDE/TritonX-100-cyclohexane-H(2)O reverse micelle, the effects of the size of the reverse micelle, the buffer pH, and the concentration of H(2)O(2) on the catalytic activity of LiP were investigated. Under the optimized conditions, the catalytic efficiency of LiP in the GGDE/TritonX-100 reverse micelle was 40 times higher than that in the AOT reverse micelle. The full expression of catalytic activity of LiP in this medium was mainly due to the lack of electrostatic interaction between LiP and the head group of GGDE and TritonX-100 and to the size fit between LiP and the inner water cavity of the reverse micelle.