Tailoring the expression of Xyr1 leads to efficient production of lignocellulolytic enzymes in Trichoderma reesei for improved saccharification of corncob residues.

BACKGROUND: The filamentous fungus Trichoderma reesei is extensively used for the industrial-scale cellulase production. It has been well known that the transcription factor Xyr1 plays an important role in the regulatory network controlling cellulase gene expression. However, the role of Xyr1 in the regulation of cellulase expression has not been comprehensively elucidated, which hinders further improvement of lignocellulolytic enzyme production. RESULTS: Here, the expression dosage of xyr1 was tailored in T. reesei by differentially overexpressing the xyr1 gene under the control of three strong promoters (Pegl2, Pcbh1, and Pcdna1), and the transcript abundance of xyr1 was elevated 5.8-, 12.6-, and 47.2-fold, respectively. We found expression of cellulase genes was significantly increased in the Pegl2-driven xyr1 overexpression strain QE2X, whereas relatively low in the Pcbh1- and Pcdna1-driven overexpression strains. We also found that the Pegl2-driven overexpression of xyr1 caused a more significant opening of chromatin in the core promoter region of the prominent cellulase genes. Furthermore, the cellulase activity showed a 3.2-fold increase in the strain QE2X, while insignificant improvement in the Pcbh1- and Pcdna1-driven strains. Finally, the saccharification efficiency toward acid-pretreated corncob residues containing high-content lignin by the crude enzyme from QE2X was increased by 57.2% compared to that from the parental strain. Moreover, LC-MS/MS and RT-qPCR analysis revealed that expression of accessory proteins (Cip1, Cip2, Swo1, and LPMOs) was greatly improved in QE2X, which partly explained the promoting effect of the Pegl2-driven overexpression on enzymatic hydrolysis of lignocellulose biomass. CONCLUSIONS: Our results underpin that the precise tailoring expression of xyr1 is essential for highly efficient cellulase synthesis, which provide new insights into the role of Xyr1 in regulating cellulase expression in T. reesei. Moreover, these results also provides a prospective strategy for strain improvement to enhance the lignocellulolytic enzyme production for use in biorefinery applications.

Pharmacokinetic and Bioequivalence Study of Eldecalcitol Soft Capsules in Healthy Chinese Subjects.

Eldecalcitol is a novel active vitamin D3 1,25(OH)2 D3 derivative used in the treatment of osteoporosis. To investigate and compare the pharmacokinetic and bioequivalent profiles of two eldecalcitol soft capsule formulations at a single dose of 0.75 µg in healthy Chinese volunteers, we conducted a randomized single-dose, open-label, two-period crossover study under fasting and fed conditions. Eligible subjects were randomly assigned to receive reference eldecalcitol soft capsules or test capsules in the first treatment period and to receive another formulation in the second period. Serial blood samples were collected for pharmacokinetic analysis. Adverse events were recorded. In total, 28 healthy subjects were enrolled in the fasting trial and 30 subjects were enrolled in the fed trial. The geometric mean ratios of the test formulation to the reference formulation for Cmax , AUC0-t , and AUC0-∞ were 94.2%, 94.0%, and 103.3%, respectively, under fasting conditions and 100.1%, 97.3%, and 96.0%, respectively, under fed conditions. No severe adverse events were observed. The results showed that the test and reference eldecalcitol formulations were bioequivalent and well tolerated in healthy Chinese subjects under fasting and fed conditions.

Development of a powerful synthetic hybrid promoter to improve the cellulase system of Trichoderma reesei for efficient saccharification of corncob residues.

BACKGROUND: The filamentous fungus Trichoderma reesei is a widely used workhorse for cellulase production in industry due to its prominent secretion capacity of extracellular cellulolytic enzymes. However, some key components are not always sufficient in this cellulase cocktail, making the conversion of cellulose-based biomass costly on the industrial scale. Development of strong and efficient promoters would enable cellulase cocktail to be optimized for bioconversion of biomass. RESULTS: In this study, a synthetic hybrid promoter was constructed and applied to optimize the cellulolytic system of T. reesei for efficient saccharification towards corncob residues. Firstly, a series of 5' truncated promoters in different lengths were established based on the strong constitutive promoter Pcdna1. The strongest promoter amongst them was Pcdna1-3 (- 640 to - 1 bp upstream of the translation initiation codon ATG), exhibiting a 1.4-fold higher activity than that of the native cdna1 promoter. Meanwhile, the activation region (- 821 to - 622 bp upstream of the translation initiation codon ATG and devoid of the Cre1-binding sites) of the strong inducible promoter Pcbh1 was cloned and identified to be an amplifier in initiating gene expression. Finally, this activation region was fused to the strongest promoter Pcdna1-3, generating the novel synthetic hybrid promoter Pcc. This engineered promoter Pcc drove strong gene expression by displaying 1.6- and 1.8-fold stronger fluorescence intensity than Pcbh1 and Pcdna1 under the inducible condition using egfp as the reporter gene, respectively. Furthermore, Pcc was applied to overexpress the Aspergillus niger ß-glucosidase BGLA coding gene bglA and the native endoglucanase EG2 coding gene eg2, achieving 43.5-fold BGL activity and 1.2-fold EG activity increase, respectively. Ultimately, to overcome the defects of the native cellulase system in T. reesei, the bglA and eg2 were co-overexpressed under the control of Pcc promoter. The bglA-eg2 double expression strain QPEB70 exhibited a 178% increase in total cellulase activity, whose cellulase system displayed 2.3- and 2.4-fold higher saccharification efficiency towards acid-pretreated and delignified corncob residues than the parental strain, respectively. CONCLUSIONS: The synthetic hybrid promoter Pcc was generated and employed to improve the cellulase system of T. reesei by expressing specific components. Therefore, construction of synthetic hybrid promoters would allow particular cellulase genes to be expressed at desired levels, which is a viable strategy to optimize the cellulolytic enzyme system for efficient biomass bioconversion.

Engineering the endoplasmic reticulum secretory pathway in Trichoderma reesei for improved cellulase production.

The filamentous fungus Trichoderma reesei is an extraordinarily efficient cell factory of industrial cellulase for production of biofuels and other bio-based products because of its excellent potential to secrete cellulolytic enzymes. Engineering the protein secretory pathway may be a powerful means for efficient protein production. However, it is uncertain whether this engineering approach could improve cellulase production in T. reesei. Herein, the endoplasmic reticulum (ER) secretory pathway was engineered for the production of cellulolytic enzymes by multiple strategies, including: (I) overexpression of the key components of protein folding (Pdi1, Ero1 and BiP); (II) overexpression of the glycosylation-related elements (Gpt1 and Gls2); (III) knockout of the ER mannosidase I (Mns1) encoding gene mns1. By utilizing these ER engineering strategies, the secretion of ß-glucosidase was remarkably elevated in the engineered strains, ranging from 29.2 % to 112.5 %. Furthermore, it was found that engineering these components also regulated the ER stress resistance. More importantly, the total cellulase production was increased with varying degrees, which reached a maximum of 149.4 %, using the filter paper assay (FPA) as a characterization method. These results demonstrated that engineering the ER secretory pathway can enhance protein secretion, particularly for cellulase production, which shed light for the development of high-efficient cellulolytic enzymes for economically feasible bioethanol production from lignocellulosic biomass.

Dysregulation of MicroRNAs and PIWI-Interacting RNAs in a Caenorhabditis elegans Parkinson's Disease Model Overexpressing Human α-Synuclein and Influence of tdp-1.

MicroRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs) regulate gene expression and biological processes through specific genetic and epigenetic mechanisms. Recent studies have described a dysregulation of small non-coding RNAs in Parkinson's disease (PD) tissues but have been limited in scope. Here, we extend these studies by comparing the dysregulation of both miRNAs and piRNAs from transgenic Caenorhabditis elegans (C. elegans) nematodes overexpressing pan-neuronally human α-synuclein wild-type (WT) (HASNWT OX) or mutant (HASNA53T OX). We observed 32 miRNAs and 112 piRNAs dysregulated in HASNA53T OX compared with WT. Genetic crosses of HASNA53T OX PD animal models with tdp-1 null mutants, the C. elegans ortholog of TDP-43, an RNA-binding protein aggregated in frontal temporal lobar degeneration, improved their behavioral deficits and changed the number of dysregulated miRNAs to 11 and piRNAs to none. Neuronal function-related genes T28F4.5, C34F6.1, C05C10.3, camt-1, and F54D10.3 were predicted to be targeted by cel-miR-1018, cel-miR-355-5p (C34F6.1 and C05C10.3), cel-miR-800-3p, and 21ur-1581 accordingly. This study provides a molecular landscape of small non-coding RNA dysregulation in an animal model that provides insight into the epigenetic changes, molecular processes, and interactions that occur during PD-associated neurodegenerative disorders.

TDP-1/TDP-43 potentiates human α-Synuclein (HASN) neurodegeneration in Caenorhabditis elegans.

TAR DNA binding protein (TDP-43) is a DNA/RNA binding protein whose pathological role in amyotrophic lateral sclerosis (ALS) and frontal temporal lobe dementia (FTLD) via formation of protein aggregates is well established. In contrast, knowledge concerning its interactions with other neuropathological aggregating proteins is poorly understood. Human α-synuclein (HASN) elicits dopaminergic neuron degeneration via protein aggregation in Parkinson's disease. HASN protein aggregates are also found in TDP-43 lesions and colocalize in Lewy Body Dementia (LBD). To better understand the interactions of TDP-43 and HASN, we investigated the effects of genetic deletion of tdp-1, the Caenorhabditis elegans ortholog of human TDP-43, as well as overexpression of TDP-43, in transgenic models overexpressing HASNWT and HASNA53T. Tdp-1 deletion improved the posture, movement, and developmental delay observed in transgenic animals pan-neuronally overexpressing HASNA53T, and attenuated the loss and impairment of dopaminergic neurons caused by HASNA53T or HASNWT overexpression. Tdp-1 deletion also led to a decrease in protein level, mRNA level and aggregate formation of HASN in living animals. RNA-seq studies suggested that tdp-1 supports expression of lysosomal genes and decreases expression of genes involved in heat shock. RNAi demonstrated that heat shock proteins can mediate HASN neuropathology. Co-overexpression of both human TDP-43 and HASNWT resulted in locomotion deficits, shorter lifespan, and more severe dopaminergic neuron impairments compared to single transgenes. Our results suggest TDP-1/TDP-43 potentiates HASN mediated neurodegeneration in C. elegans. This study indicates a multifunctional role for TDP-1/TDP-43 in neurodegeneration involving HASN.

DNA Sequential Logic Gate Using Two-Ring DNA.

Sequential DNA detection is a fundamental issue for elucidating the interactive relationships among complex gene systems. Here, a sequential logic DNA gate was achieved by utilizing the two-ring DNA structure, with the ability to recognize "before" and "after" triggering sequences of DNA signals. By taking advantage of a "loop-open" mechanism, separations of two-ring DNAs were controlled. Three triggering pathways with different sequential DNA treatments were distinguished by comparing fluorescent outputs. Programmed nanoparticle arrangement guided by "interlocked" two-ring DNA was also constructed to demonstrate the achievement of designed nanostrucutres. Such sequential logic DNA operation may guide future molecular sensors to monitor more complex gene network in biological systems.