Proteomic analysis of the biomass hydrolytic potentials of Penicillium oxalicum lignocellulolytic enzyme system

Additional file 2: Table S1. The functional annotations of proteins identified in the proteome of SP. Mass spectrometry-based proteomics study was performed to comprehensively dissect the lignocellulolytic enzyme profile of SP. Accession, Protein name, PSM, Calc. MW, CBM, Calc. pI and CAZy family of identified proteins were shown

Konferencija Što činimo po pitanju transparentnosti

Additional file 2: Figure S2. PCR and phenotypic analysis of the Δcre1 strain T. reesei SDC11. a PCR analysis of T. reesei SDC11 with SP4 as control. 1 and 2 represent the fragment (upstream region and open reading frame of gene cre1) amplificated by the prime pair cre1-2426UF/cre1-1069R in T. reesei SDC11 and SP4, respectively; 3 and 4 represent the internal fragment of gene cre1 amplificated by the prime pair cre1-497F/cre1-1069R in T. reesei SDC11 and SP4, respectively. 5 and 6 represent the fragment of gene pyrG amplificated by the prime pair pyrG-UF1/pyrG-2426DR in T. reesei SDC11 and SCP11, respectively. b Southern blot analysis of the genomic DNA isolated from SP4 and SCP11, which were digested with EcoRI/HindIII. A 5.5-kb fragment is present in the parental strain SP4, and a 7.0-kb band is shown in Δcre1 + pyrG strain SCP11. c Growth of T. reesei SN1, Δcre1 + pyrG strain SCP11 and Δcre1 strain SDC11 on MM plate. d Growth of T. reesei SP4, Δcre1 + pyrG strain SCP11 and Δcre1 strain SDC11 on the MM plate containing uracil (0.1%)

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Characterization and strain improvement of a hypercellulytic variant, Trichoderma reesei SN1, by genetic engineering for optimized cellulase production in biomass conversion improvement

The filamentous fungus Trichoderma reesei is a widely used strain for cellulolytic enzyme production. A hypercellulolytic T. reesei variant SN1 was identified in this study and found to be different from the well-known cellulase producers QM9414 and RUT-C30. The cellulose-degrading enzymes of T. reesei SN1 show higher endoglucanase (EG) activity but lower β-glucosidase (BGL) activity than those of QM9414 and RUT-C30. A uracil auxotroph strain, SP4, was constructed by pyr4 deletion in SN1 to improve transformation efficiency. The BGL1-encoding gene bgl1 under the control of a modified cbh1 promoter was overexpressed in SP4. A transformant, SPB2, with four additional copies of bgl1 exhibited a 17.1-fold increase in BGL activity and a 30% increase in filter paper activity. Saccharification of corncob residues with crude enzyme showed that the glucose yield of SPB2 is 65% higher than that of SP4. These results reveal the feasibility of strain improvement through the development of an efficient genetic transformation platform to construct a balanced cellulase system for biomass conversion

The GATA-Type Transcriptional Factor Are1 Modulates the Expression of Extracellular Proteases and Cellulases in Trichoderma reesei

Trichoderma reesei is a biotechnologically important filamentous fungus with the remarkable ability to secrete large amounts of enzymes, whose production is strongly affected by both the carbon and nitrogen sources. While the carbon metabolism regulators are extensively studied, the regulation of enzyme production by the nitrogen metabolism regulators is still poorly understood. In this study, the GATA transcription factor Are1, which is an orthologue of the Aspergillus global nitrogen regulator AREA, was identified and characterized for its functions in regulation of both protease and cellulase production in T. reesei. Deletion of the are1 gene abolished the capability to secrete proteases, and complementation of the are1 gene rescued the ability to produce proteases. Quantitative RT-PCR analysis revealed that the transcripts of protease genes apw1 and apw2 were also significantly reduced in the Δare1 strain when grown in the medium with peptone as the nitrogen source. In addition, deletion of are1 resulted in decreased cellulase production in the presence of (NH4)2SO4. Consistent with the reduction of cellulase production, the transcription levels of the major cellulase genes, including cbh1, cbh2, egl1, and egl2, were dramatically decreased in Δare1. Sequence analysis showed that all promoter regions of the tested protease and cellulase genes contain the consensus GATA elements. However, the expression levels of the major cellulase transcription activator Xyr1 and the repressor Cre1 had no significant difference between Δare1 and the parental strain QM9414, indicating that the regulatory mechanism deserves further investigation. Taken together, these results demonstrate the important role of Are1 in the regulation of protease and cellulase production in T. reesei, although these processes depend on the kind of nitrogen sources. The findings in this study contribute to the understanding of the regulation network of carbon and nitrogen sources in filamentous fungi

Production of highly efficient cellulase mixtures by genetically exploiting the potentials of Trichoderma reesei endogenous cellulases for hydrolysis of corncob residues

Abstract Background Trichoderma reesei is one of the most important fungi utilized for cellulase production. However, its cellulase system has been proven to be present in suboptimal ratio for deconstruction of lignocellulosic substrates. Although previous enzymatic optimization studies have acquired different types of in vitro synthetic mixtures for efficient lignocellulose hydrolysis, production of in vivo optimized cellulase mixtures by industrial strains remains one of the obstacles to reduce enzyme cost in the biofuels production from lignocellulosic biomass. Results In this study, we used a systematic genetic strategy based on the pyrG marker to overexpress the major cellulase components in a hypercellulolytic T. reesei strain and produce the highly efficient cellulase mixture for saccharification of corncob residues. We found that overexpression of CBH2 exhibited a 32-fold increase in the transcription level and a comparable protein level to CBH1, the most abundant secreted protein in T. reesei, but did not contribute much to the cellulolytic ability. However, when EG2 was overexpressed with a 46-fold increase in the transcription level and a comparable protein level to CBH2, the engineered strain QPE36 showed a 1.5-fold enhancement in the total cellulase activity (up to 5.8 U/mL FPA) and a significant promotion of saccharification efficiency towards differently pretreated corncob residues. To assist the following genetic manipulations, the marker pyrG was successfully excised by homologous recombination based on resistance to 5-FOA. Furthermore, BGL1 was overexpressed in the EG2 overexpression strain QE51 (pyrG-excised) and a 11.6-fold increase in BGL activity was obtained. The EG2–BGL1 double overexpression strain QEB4 displayed a remarkable enhancement of cellulolytic ability on pretreated corncob residues. Especially, a nearly complete cellulose conversion (94.2%) was found for the delignified corncob residues after 48 h enzymatic saccharification. Conclusions These results demonstrate that genetically exploiting the potentials of T. reesei endogenous cellulases to produce highly efficient cellulase mixtures is a powerful strategy to promote the saccharification efficiency, which will eventually facilitate cost reduction for lignocellulose-based biofuels

A Modified Flexor Tendon Suture Technique Combining Kessler and Loop Lock Flexor Tendon Sutures

OBJECTIVES: In the present study, a novel single knot tenorrhaphy was developed by combining the modified Kessler flexor tendon suture (MK) with the loop lock technique. METHODS: A total of 48 porcine flexor digitorum profundus tendons were collected and randomly divided into six groups. The tendons were transversely cut and then repaired using six different techniques, the MK method, double knot Kessler-loop lock flexor tendon suture (DK), and single knot Kessler-loop lock flexor tendon suture (SK), each in combination with the epitendinous suture (P), and the same three techniques without P. Furthermore, by performing the load-to-failure tests, the biomechanical properties and the time taken to complete a repair, for each tenorrhaphy, were assessed. RESULTS: Compared to the MK+P method, DK+P was more improved, thereby enhancing the ultimate tensile strength. The SK+P method, which required fewer knots than DK+P, was easier to perform. Moreover, the SK+P repair increased the force at a 2-mm gap formation, while requiring lesser knots than DK+P. CONCLUSION: As opposed to the traditional MK+P method, the SK+P method was improved and exhibited better biomechanical properties, which may facilitate early mobilization after the repair