Cellulase production and efficient saccharification of biomass by a new mutant Trichoderma afroharzianum MEA-12.

BACKGROUND: Cellulase plays a key role in converting cellulosic biomass into fermentable sugar to produce chemicals and fuels, which is generally produced by filamentous fungi. However, most of the filamentous fungi obtained by natural breeding have low secretory capacity in cellulase production, which are far from meeting the requirements of industrial production. Random mutagenesis combined with adaptive laboratory evolution (ALE) strategy is an effective method to increase the production of fungal enzymes. RESULTS: This study obtained a mutant of Trichoderma afroharzianum by exposures to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), Ethyl Methanesulfonate (EMS), Atmospheric and Room Temperature Plasma (ARTP) and ALE with high sugar stress. The T. afroharzianum mutant MEA-12 produced 0.60, 5.47, 0.31 and 2.17 IU/mL FPase, CMCase, pNPCase and pNPGase, respectively. These levels were 4.33, 6.37, 4.92 and 4.15 times higher than those of the parental strain, respectively. Also, it was found that T. afroharzianum had the same carbon catabolite repression (CCR) effect as other Trichoderma in liquid submerged fermentation. In contrast, the mutant MEA-12 can tolerate the inhibition of glucose (up to 20 mM) without affecting enzyme production under inducing conditions. Interestingly, crude enzyme from MEA-12 showed high enzymatic hydrolysis efficiency against three different biomasses (cornstalk, bamboo and reed), when combined with cellulase from T. reesei Rut-C30. In addition, the factors that improved cellulase production by MEA-12 were clarified. CONCLUSIONS: Overall, compound mutagenesis combined with ALE effectively increased the production of fungal cellulase. A super-producing mutant MEA-12 was obtained, and its cellulase could hydrolyze common biomasses efficiently, in combination with enzymes derived from model strain T. reesei, which provides a new choice for processing of bioresources in the future.

Effects of the Transcription Factor Ace2 from Trichoderma reesei on Cellulase and Hemicellulase Expression in Trichoderma orientalis EU7-22.

Trichoderma orientalis (T. orientalis) EU7-22 has a complete cellulase system and shows a remarkable enzyme activity with high potential in the industry. Ace2 is an important transcriptional factor for cellulase and hemicellulase expression in Trichoderma reesei (T. reesei). However, the ace2 gene cannot be found in the genome of T. orientalis. Researches show that the mechanism of cellulase transcriptional regulation in T. orientalis keeps high similarity with T. reesei up till now. So, in this study, the ace2 of Trichoderma reesei QM9414 was heterologous expressed in T. orientalis EU7-22. As a result, xylanase activity and ß-glucosidase activity of ace2 heterogeneous expression strains are improved and total cellulase activity is decreased. The result of qPCR is in accordance with enzyme activities. This study provides a reference for an in-depth study on transcriptional regulation mechanisms of T. orientalis.

Effect of VIB Gene on Cellulase Production of Trichoderma orientalis EU7-22.

The cellulolytic enzymes from filamentous fungi are widely used for production of biofuels. Molecular biological engineering of fungal strains has been applied to improve the cellulase production in the recent years. The VIB gene affects production of cellulase and known as a new transcription factor. Based on a fast-growing strain Trichoderma orientalis EU7-22, we constructed two recombinant strains that knockout the VIB gene and overexpressed the VIB gene and the effects of the VIB gene on cellulase production under induction conditions were also investigated. Under the condition of induction by avicel and wheat bran, the cellulase activity of the recombinant -VIB strain was almost undetectable, while that of the +VIB strain was greatly improved. FPAase, CMCase, pNPCase, and pNPGase in the crude enzyme produced by the +VIB strain are 1.51, 41.10, 0.86, and 3.47 IU ml-1, respectively, which increased 92, 34, 87, and 38% compared to the parent strain. Therefore, we believe that the VIB gene has an important effect on the cellulase of T. orientalis and overexpression of the VIB gene will increase the cellulase production. This study provided guidance for improving the cellulase production of T. orientalis.

Promoting cellulase and hemicellulase production from Trichoderma orientalis EU7-22 by overexpression of transcription factors Xyr1 and Ace3.

The construction of hyper-production strains of cellulase is the prerequisite for the production of biofuels or biochemicals. Trichoderma orientalis EU7-22 with complete cellulase system shows the potential for cellulase production in industrial scale. To improve the cellulase production, two crucial transcription activators Xyr1 and Ace3 were constitutively overexpressed in EU7-22 strain. Cellulase, xylanase and protein secretion were significantly improved in the recombinant strain dxyA-8 under inducing culture, which were 2.34, 0.68 and 1.06 folds higher than those of EU7-22, respectively. Moreover, the FPase and CMCase activities were up to 2.55 IU/mL and 90.38 IU/mL with glucose as carbon source, which were 2.12 and 1.95 folds higher than those of EU7-22 under inducing condition, respectively. Reducing sugar released from pretreated spartina that hydrolyzed by crude enzyme from dxyA-8 had achieved 24% improvement. Therefore, overexpression of these transcription factors effectively promotes the production of cellulase and hemicellulase of Trichoderma orientalis EU7-22.

Stretchable and Wearable Triboelectric Nanogenerator Based on Kinesio Tape for Self-Powered Human Motion Sensing.

Recently, wearable, self-powered, active human motion sensors have attracted a great deal of attention for biomechanics, physiology, kinesiology, and entertainment. Although some progress has been achieved, new types of stretchable and wearable devices are urgently required to promote the practical application. In this article, targeted at self-powered active human motion sensing, a stretchable, flexible, and wearable triboelectric nanogenerator based on kinesio tapes (KT-TENG) haven been designed and investigated systematically. The device can effectively work during stretching or bending. Both the short-circuit transferred charge and open-circuit voltage exhibit an excellent linear relationship with the stretched displacements and bending angles, enabling its application as a wearable self-powered sensor for real-time human motion monitoring, like knee joint bending and human gestures. Moreover, the KT-TENG shows good stability and durability for long-term operation. Compared with the previous works, the KT-TENG without a macro-scale air gap inside, or stretchable triboelectric layers, possesses various advantages, such as simple fabrication, compact structure, superior flexibility and stability, excellent conformable contact with skin, and wide-range selection of triboelectric materials. This work provides a new prospect for a wearable, self-powered, active human motion sensor and has numerous potential applications in the fields of healthcare monitoring, human-machine interfacing, and prosthesis developing.

Rational Design of Si@SiO2/C Composites Using Sustainable Cellulose as a Carbon Resource for Anodes in Lithium-Ion Batteries.

In this work, we propose a novel and facile route for the rational design of Si@SiO2/C anode materials by using sustainable and environment-friendly cellulose as a carbon resource. To simultaneously obtain a SiO2 layer and a carbon scaffold, a specially designed homogeneous cellulose solution and commercial Si nanopowder are used as the starting materials, and the cellulose/Si composite is directly assembled by an in situ regenerating method. Subsequently, Si@SiO2/C composite is obtained after carbonization. As expected, Si@SiO2 is homogeneously encapsulated in the cellulose-derived carbon network. The obtained Si@SiO2/C composite shows a high reversible capacity of 1071 mA h g-1 at a current density of 420 mA g-1 and 70% capacity retention after 200 cycles. This novel, sustainable, and effective design is a promising approach to obtain high-performance and cost-effective composite anodes for practical applications.

Improvement in xylooligosaccharides production by knockout of the ß-xyl1 gene in Trichoderma orientalis EU7-22.

The goal of this study was to enhance the production of xylooligosaccharides (XOs) and reduce the production of xylose. We investigated ß-xylosidases, which were key enzymes in the hydrolysis of xylan into xylose, in Trichoderma orientalis EU7-22. The binary vector pUR5750G/bxl::hph was constructed to knock out the ß-xyl1 gene (encoding ß-xylosidases) in T. orientalis EU7-22 by homologous integration, producing the mutant strain T. orientalis Bxyl-1. Xylanase activity for strain Bxyl-1 was 452.42 IU/mL, which increased by only 0.07% compared to that of parental strain EU7-22, whereas ß-xylosidase activity was 0.06 IU/mL, representing a 91.89% decrease. When xylanase (200 IU/g xylan), produced by T. orientalis EU7-22 and T. orientalis Bxyl-1, was used to hydrolyze beechwood xylan, in contrast to the parental strain, the XOs were enhanced by 83.27%, whereas xylose decreased by 45.80% after 36 h in T. orientalis Bxyl-1. Based on these results, T. orientalis Bxyl-1 has great potential for application in the production of XOs from lignocellulosic biomass.

Enhancing Cellulase and Hemicellulase Production in Trichoderma orientalis EU7-22 via Knockout of the creA.

The role of the transcription factor creA-mediating carbon catabolite repression in Trichoderma orientalis EU7-22 was investigated for cellulase and hemicellulase production. The binary vector pUR5750G/creA::hph was constructed to knock out creA by homologous integration, generating the ΔcreA mutant Trichoderma orientalis CF1D. For strain CF1D, the filter paper activities (FPA), endoglucanase activities (CMC), cellobiohydrolase activity(CBH), ß-glucosidase activity (BG), xylanase activity (XYN), and extracellular protein concentration were 1.45-, 1.15-, 1.71-, 2.51-, 2.72, and 1.95-fold higher in inducing medium and were 6.41-, 7.50-, 10.27-, 11.79-, 9.25-, and 3.77-fold higher in glucose repressing medium, respectively, than those in the parent strain after 4 days. SDS-PAGE demonstrated that the extracellular proteins were largely secreted in the mutant CF1D. Quantitative reverse-transcription polymerase chain reaction indicated that the expressions of cbh1, cbh2, eg1, eg2, bgl1, xyn1, and xyn2 were significantly increasing for the mutant CF1D not only in the inducing medium but also in the repressing medium. Those results indicated that creA was a valid target gene in strain engineering for improved enzyme production in T. orientalis.

Heterologous Expression and Characterization of an Acidic GH11 Family Xylanase from Hypocrea orientalis.

A gene encoding glycoside hydrolase family 11 xylanase (HoXyn11B) from Hypocrea orientalis EU7-22 was expressed in Pichia pastoris with a high activity (413 IU/ml). HoXyn11B was partly N-glycosylated and appeared two protein bands (19-29 kDa) on SDS-PAGE. The recombinant enzyme exhibited optimal activity at pH 4.5 and 55 °C, and retained more than 90% of the original activity after incubation at 50 °C for 60 min. The determined apparent K m and V max values using beechwood xylan were 10.43 mg/ml and 3246.75 IU/mg, respectively. The modes of action of recombinant HoXyn11B on xylo-oligosaccharides (XOSs) and beechwood xylan were investigated by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), which indicated that the modes of action of HoXyn11B are different from HoXyn11A since it is able to release a significant amount of xylose from various substrates. This study provides an opportunity to better understand the hydrolysis mechanisms of xylan by xylanases from Trichoderma.

Time and energy-efficient homogeneous transesterification of cellulose under mild reaction conditions.

In this research, cellulose aliphatic esters (CEs) were synthesized efficiently in an N, N-dimethylacetamide/lithium chloride system (DMAc/LiCl) with vinyl esters (VEs, number of carbon atoms ranging from 4 to 12) as acylation reagent and 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU) as catalyst. The structures and properties of the products have been characterized by various analytical techniques. The key concept in this cellulose modification was the achievement the degree of substitution (DS) >2.75 at much milder temperature and shorter reaction time (under 30°C within 15min-30min), other than previous research. Generally, the DS of obtained CEs showed a reverse trend with the length of the aliphatic chain increasing. The priority of reactions in three positions of the CEs followed the same order of C6>C2>C3 for a homogeneous conversion. There was no decrease of molecule weight under this mild reaction. This research provides a novel homogeneous technology to synthesize various CEs efficiently.

Heterogeneous Expression and Functional Characterization of Cellulose-Degrading Enzymes from Aspergillus niger for Enzymatic Hydrolysis of Alkali Pretreated Bamboo Biomass.

Enzymatic hydrolysis of cellulosic biomass has caught much attention because of modest reaction conditions and environment friendly conditions. To reduce the cost and to achieve good quantity of cellulases, a heterologous expression system is highly favored. In this study, cellulose-degrading enzymes, GH3 family ß-glucosidase (BGL), GH7 family-related cellobiohydrolases (CBHs), and endoglucanase (EG) from a newly isolated Aspergillus niger BE-2 are highly expressed in Pichia pastoris GS115. The strain produced EG, CBHs, and BGL enzymatic concentration of 0.56, 0.11, and 22 IU/mL, respectively. Mode of actions of the recombinant enzymes for substrate specificity and end product analysis are verified and found specific for cellulose degradation. Bamboo biomass saccharification with A. niger cellulase released a high level of fermentable sugars. Hydrolysis parameters are optimized to obtain reducing sugars level of 3.18 g/L. To obtain reducing sugars from a cellulosic biomass, A. niger could be a good candidate for enzymes resource of cellulase to produce reducing sugars from a cellulosic biomass. This study also facilitates the development of highly efficient enzyme cocktails for the bioconversion of lignocellulosic biomass into monosaccharides and oligosaccharides.

Identification of a genomic region containing a novel promoter resistant to glucose repression and over-expression of ß-glucosidase gene in Hypocrea orientalis EU7-22.

A high concentration of glucose in the medium could greatly inhibit the expression of cellulase in filamentous fungi. The aspartic protease from fungus Hypocrea orientalis EU7-22 could efficiently express under both induction condition and glucose repression condition. Based on the sequence of structure gene of aspartic protease, the upstream sequence harboring the putative promoter proA for driving the expression of aspartic protease was obtained by genome walking. The upstream sequence contained the typical promoter motifs "TATA" and "CAAT". The ß-glucosidase gene (Bgl1) from H. orientalis was cloned and recombined with promoter proA and terminator trpC. The expression cassette was ligated to the binary vector to form pUR5750-Bgl1, and then transferred into the host strain EU7-22 via Agrobacterium tumefaciens mediated transformation (ATMT), using hygromycin B resistance gene as the screening marker. Four transformants Bgl-1, Bgl-2, Bgl-3 and Bgl-4 were screened. Compared with the host strain EU7-22, the enzyme activities of filter paper (FPA) and ß-glucosidase (BG) of transformant Bgl-2 increased by 10.6% and 19.1% under induction condition, respectively. The FPA and BG activities were enhanced by 22.2% and 700% under 2% glucose repression condition, respectively, compared with the host strain. The results showed that the putative promoter proA has successfully driven the over-expression of Bgl1 gene in H. orientalis under glucose repression condition.

Increased biological hydrogen production by deletion of hydrogen-uptake system in photosynthetic bacteria.

Hydrogenases are the key enzymes for the biological hydrogen production, which can be classified as H(2)-uptake hydrogenase and H(2)-production hydrogenase. The genes encoding a membrane-bound [NiFe]-hydrogenase (MBH), which is mainly responsible for hydrogen uptake, from the photosynthetic bacterium Allochromatium vinosum was cloned and sequenced. It consist of two structural genes (hydS, hydL) and two intergenic genes (isp1, isp2), which are therefore organized as hydS-isp1-isp2-hydL. This is different from the arrangement of other typical hydrogenase gene clusters. A deletion mutant-strain PhihydSL, lacking isp1, isp2, partial hydS and hydL genes, was constructed by marker-exchange mutagenesis. Under dark fermentative conditions, the hydrogen production yield by this mutant increased by 62%. The result suggests that the disruption of MBH could greatly improve the hydrogen production in the cells by decreasing the hydrogen uptake.