A novel major facilitator transporter TrSTR1 is essential for pentose utilization and involved in xylanase induction in Trichoderma reesei.

Trichoderma reesei (teleomorph Hypocrea jecorina) is an industrially important filamentous fungus for glycoside hydrolases production, with its xylanolytic enzymes widely applied in many areas. However, the molecular mechanisms underlying xylanase expression are still insufficiently understood. In particular, the effect of sugar transporter on the induction of xylanase expression is unclear. In this work, we identified a novel major facilitator transporter TrSTR1 that is capable of transporting xylose by using a xylose utilization system in Saccharomyces cerevisiae. In T. reesei, TrSTR1 is essential for the utilization of d-xylose, l-arabinose, and even their downstream metabolites D-xylitol and L-arabitol. TrSTR1 is also involved in the induction of xylanase expression since both the xylanase activity and extracellular protein concentration in the Tu6△str1 strain were decreased, which further confirmed by a qRT-PCR analysis of the transcript levels of the key transcriptional regulators. Our observations provide new insights into connections between pentose utilization and xylanase production in T. reesei.

An Ime2-like mitogen-activated protein kinase is involved in cellulase expression in the filamentous fungus Trichoderma reesei.

OBJECTIVES: Eukaryotic mitogen-activated protein kinases (MAPKs) play crucial roles in transducing environmental and developmental signals inside the cell and regulating gene expression, however, the roles of MAPKs remain largely unknown in Trichoderma reesei. RESULTS: T. reesei ime2 (TrIme2) encodes an Ime2-like MAPK in T. reesei. The deletion of the TrIme2 gene led to 90% increase in cellulase activity against filter paper during earlier period time of cellulase induction as well as the extracellular protein production. Compared to the parent strain, the transcriptional levels of the three major cellulase genes cbh1,cbh2, egl1 were increased by about 9 times, 4 times, 2 times, respectively, at 8 h after cellulase induction in the ΔTrIme2 mutant. In addition, the disruption of TrIme2 caused over 50% reduction of the transcript levels of cellulase transcriptional regulators cre1 and xyr1. CONCLUSION: TrIme2 functions in regulation of the expression of cellulase gene in T.reesei, and is a good candidate for genetically engineering of T. reesei for higher cellulase production.

Construction and validation a nomogram to predict overall survival for colorectal signet ring cell carcinoma.

To construct and validate a nomogram to predict the overall survival (OS) of colorectal signet ring cell carcinoma (SRCC). The potentially eligible cases were obtained against the SEER database from 2004 to 2015. Log-rank test and Cox analysis were conducted to identify the independent prognostic factors for predicting OS. The identified prognostic factors were later integrated for the construction of an OS prediction nomogram. Altogether 2904 eligible cases were identified, and the median survival time was 18 (range: 0-155) months. As suggested by multivariate analysis, age, primary site, grade, tumor size, T stage, N stage, M stage, surgery, lymph node dissection and chemotherapy were identified as the independent factors for predicting OS. Afterwards, the above variables were incorporated into the nomogram. The C-index indicated better discriminatory ability of the nomogram than AJCC 8th TNM staging and SEER summary stage systems (both P < 0.001). Calibration plots further showed good consistency between the nomogram prediction and actual observation. The time independent area under the curves (tAUCs) for 3-year and 5-year OS in nomogram were larger than AJCC and SEER summary stage system. The constructed nomogram could potentially predict the survival of colorectal SRCC individuals.

Improvement in catalytic activity and thermostability of a GH10 xylanase and its synergistic degradation of biomass with cellulase.

BACKGROUND: Xylanase is one of the most extensively used biocatalysts for biomass degradation. However, its low catalytic efficiency and poor thermostability limit its applications. Therefore, improving the properties of xylanases to enable synergistic degradation of lignocellulosic biomass with cellulase is of considerable significance in the field of bioenergy. RESULTS: Using fragment replacement, we improved the catalytic performance and thermostability of a GH10 xylanase, XylE. Of the ten hybrid enzymes obtained, seven showed xylanase activity. Substitution of fragments, M3, M6, M9, and their combinations enhanced the catalytic efficiency (by 2.4- to fourfold) as well as the specific activity (by 1.2- to 3.3-fold) of XylE. The hybrids, XylE-M3, XylE-M3/M6, XylE-M3/M9, and XylE-M3/M6/M9, showed enhanced thermostability, as observed by the increase in the T 50 (3-4.7 °C) and T m (1.1-4.7 °C), and extended t 1/2 (by 1.8-2.3 h). In addition, the synergistic effect of the mutant xylanase and cellulase on the degradation of mulberry bark showed that treatment with both XylE-M3/M6 and cellulase exhibited the highest synergistic effect. In this case, the degree of synergy reached 1.3, and the reducing sugar production and dry matter reduction increased by 148% and 185%, respectively, compared to treatment with only cellulase. CONCLUSIONS: This study provides a successful strategy to improve the catalytic properties and thermostability of enzymes. We identified several xylanase candidates for applications in bioenergy and biorefinery. Synergistic degradation experiments elucidated a possible mechanism of cellulase inhibition by xylan and xylo-oligomers.

Functional Analysis of a Highly Active ß-Glucanase from Bispora sp. MEY-1 Using Its C-terminally Truncated Mutant.

A ß-1,3-1,4-glucanase-encoding gene, Bisglu16B, was identified in Bispora sp. MEY-1. The deduced BisGlu16B consists of an N-terminal signal peptide, a catalytic module of glycoside hydrolase family 16 (GH16), and a C-terminal serine/proline-rich module. After expression in Pichia pastoris GS115, the purified recombinant BisGlu16B showed maximal activity at pH 4.0 and 55 °C and had broad substrate specificity (ß-1,3-/ß-1,4-mixed, ß-1,3-, ß-1,4-, and ß-1,6-linked glucan, and ß-1,4-mannan). The enzyme possessed high specific activities toward barley ß-glucan (34 700 U·mg-1), lichenan (23 900 U·mg-1), and laminarin (9 000 U·mg-1). After removing the C-terminal module, the truncated mutant, BisGlu16B-ΔC, retained similar enzymatic properties to the wild type but displayed significantly enhanced activities (up to 2.5-fold). Functional and structural analyses indicated that the C-terminal module plays a key role in the substrate binding of BisGlu16B. This study provided an excellent candidate glucanase for industrial purposes and revealed the functions of a C-terminal serine/proline-rich region.

Gene microarray assessment of multiple genes and signal pathways involved in androgen-dependent prostate cancer becoming androgen independent.

To study the gene expression change and possible signal pathway during androgen-dependent prostate cancer (ADPC) becoming androgen-independent prostate cancer (AIPC), an LNCaP cell model of AIPC was established using flutamide in combination with androgen-free environment inducement, and differential expression genes were screened by microarray. Then the biological process, molecular function and KEGG pathway of differential expression genes are analyzed by Molecule Annotation System (MAS). By comparison of 12,207 expression genes, 347 expression genes were acquired, of which 156 were up-ragulated and 191 down-regulated. After analyzing the biological process and molecule function of differential expression genes, these genes are found to play crucial roles in cell proliferation, differntiation, cell cycle control, protein metabolism and modification and other biological process, serve as signal molecules, enzymes, peptide hormones, cytokines, cytoskeletal proteins and adhesion molecules. The analysis of KEGG show that the relevant genes of AIPC transformation participate in glutathione metabolism, cell cycle, P53 signal pathway, cytochrome P450 metabolism, Hedgehog signal pathway, MAPK signal pathway, adipocytokines signal pathway, PPAR signal pathway, TGF-ß signal pathway and JAK-STAT signal pathway. In conclusion, during the process of ADPC becoming AIPC, it is not only one specific gene or pathway, but multiple genes and pathways that change. The findings above lay the foundation for study of AIPC mechanism and development of AIPC targeting drugs.