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Carbohydrate active enzymes (CAZymes) are vital for the lignocellulose-based biorefinery. The development of hypersecreting fungal protein production hosts is therefore a major aim for both academia and industry. However, despite advances in our understanding of their regulation, the number of promising candidate genes for targeted strain engineering remains limited. Here, we resequenced the genome of the classical hypersecreting Neurospora crassa mutant exo-1 and identified the causative point of mutation to reside in the F-box protein-encoding gene, NCU09899. The corresponding deletion strain displayed amylase and invertase activities exceeding those of the carbon catabolite derepressed strain Δcre-1, while glucose repression was still mostly functional in Δexo-1 Surprisingly, RNA sequencing revealed that while plant cell wall degradation genes are broadly misexpressed in Δexo-1, only a small fraction of CAZyme genes and sugar transporters are up-regulated, indicating that EXO-1 affects specific regulatory factors. Aiming to elucidate the underlying mechanism of enzyme hypersecretion, we found the high secretion of amylases and invertase in Δexo-1 to be completely dependent on the transcriptional regulator COL-26. Furthermore, misregulation of COL-26, CRE-1, and cellular carbon and nitrogen metabolism was confirmed by proteomics. Finally, we successfully transferred the hypersecretion trait of the exo-1 disruption by reverse engineering into the industrially deployed fungus Myceliophthora thermophila using CRISPR-Cas9. Our identification of an important F-box protein demonstrates the strength of classical mutants combined with next-generation sequencing to uncover unanticipated candidates for engineering. These data contribute to a more complete understanding of CAZyme regulation and will facilitate targeted engineering of hypersecretion in further organisms of interest.

BackgroundFungal enzymes are vital for industrial biotechnology, including the conversion of plant biomass to biofuels and bio-based chemicals. In recent years, there is increasing interest in using enzymes from thermophilic fungi, which often have higher reaction rates and thermal tolerance compared to currently used fungal enzymes. The thermophilic filamentous fungus Thermoascus aurantiacus produces large amounts of highly thermostable plant cell wall-degrading enzymes. However, no genetic tools have yet been developed for this fungus, which prevents strain engineering efforts. The goal of this study was to develop strain engineering tools such as a transformation system, a CRISPR/Cas9 gene editing system and a sexual crossing protocol to improve the enzyme production.ResultsHere, we report Agrobacterium tumefaciens-mediated transformation (ATMT) of T. aurantiacus using the hph marker gene, conferring resistance to hygromycin B. The newly developed transformation protocol was optimized and used to integrate an expression cassette of the transcriptional xylanase regulator xlnR, which led to up to 500% increased xylanase activity. Furthermore, a CRISPR/Cas9 gene editing system was established in this fungus, and two different gRNAs were tested to delete the pyrG orthologue with 10% and 35% deletion efficiency, respectively. Lastly, a sexual crossing protocol was established using a hygromycin B- and a 5-fluoroorotic acid-resistant parent strain. Crossing and isolation of progeny on selective media were completed in a week.ConclusionThe genetic tools developed for T. aurantiacus can now be used individually or in combination to further improve thermostable enzyme production by this fungus.

Lytic polysaccharide monooxygenases (LPMOs) have emerged as key proteins for depolymerization of cellulose. These copper-containing enzymes oxidize C-1 and/or C-4 bonds in cellulose, promoting increased hydrolysis of the oxidized cellulose chains. The LPMO from Thermoascus aurantiacus, a thermophilic ascomycete fungus, has been extensively studied and has served as a model LPMO. A method was developed to purify the LPMO from culture filtrates of T. aurantiacus along with its native cellobiohydrolase and endoglucanase. The activity of the purified LPMO was measured with a colorimetric assay that established the Topt of the native LPMO at 60 °C. Purification of the components of the T. aurantiacus cellulase mixture also enabled quantification of the amounts of cellobiohydrolase, endoglucanase and LPMO present in the T. aurantiacus culture filtrate, establishing that the LPMO was the most abundant protein in the culture supernatants. The importance of the LPMO to activity of the mixture was demonstrated by saccharifications with Avicel and acid-pretreated corn stover.

Researchers have explored many pedagogical approaches in an effort to assist students in finding understanding and comfort in required statistics courses. This study investigates the impact of mnemonic aids used during tests on students' statistics course performance in particular. In addition, the present study explores several hypotheses that have been proposed to explain the potential benefit of using these aids during examinations. These include the student engagement hypothesis, the perception of control hypothesis, the dependency hypothesis, and a placebo effect hypothesis. The results indicate that student-generated testing aids are clearly superior to the other forms of in-test aids examined, however they were not superior to conditions where mnemonic aids are not allowed. Results are discussed in terms of their implications for the four hypotheses regarding the beneficial effects of mnemonic aids used during examinations.

Testing aids, including student-prepared testing aids (a.k.a., cheat sheets or crib notes) and open-textbook exams, are common practice in post-secondary assessment. There is a considerable amount of published research that discusses and investigates the impact of these testing aids. However, the findings of this research are contradictory and inconclusive. The current meta-analytic investigation provides a general measure of the impact of both student-prepared testing aids and the use of open-textbook exams on student exam performance in post-secondary education, while examining variables that may moderate the effects of testing aids on student exam performance. The results indicate that, overall, testing aids can produce a moderate impact on student exam performance, with student-prepared testing aids associated with a larger effect (d = 0.402) relative to open-textbook exams (d = 0.257). The results are discussed in terms of their implications for college course instructors and for informing the broader debate about the role of testing aids in long-term student learning and mastery of course material.

This article reports a study that examines the efforts of one school--Duquesne University in Pittsburgh, Pennsylvania--to improve student writing in first-year learning communities by promoting so-called process-based writing pedagogy outside of writing classes. Administrators encouraged instructors of subject-matter classes to integrate the writing pedagogy used in the first-year writing classes, thus improving both the writing and the teaching of writing across the community. Several surveys assessed how Duquesne's learning community program integrated the "process-based" writing pedagogy utilized in the first-year writing classes across all of the courses in each community--and how students and instructors evaluated the resultant improvement in student writing. Results suggest that these efforts were largely unsuccessful. Reasons included faculty overwork, too little attention to writing pedagogy, communication difficulties, and status differences between writing teachers and other faculty members.

The drug Enbrel may be effective for treating a spinal disease called ankylosing spondylitis, according to a study of 40 patients. This drug blocks the activity of tumor necrosis factor alpha, which is produced in greater than normal amounts in patients with arthritis.