Expression of a bacterial xylanase in Trichoderma reesei under the egl2 and cbh2 glycosyl hydrolase gene promoters

Expression vectors were constructed for Trichoderma reesei using the promoters, secretion signals and the modular structure of the efficiently expressed and secreted cellulase enzymes EGL2 (Cel5A) and CBH2 (Cel6A) as a prelude to establishing a platform where a gene of interest can be expressed under several promoters simultaneously. The designs featured (i) EGL2sigpro (egl2 promoter and secretion signal), (ii) EGL2cbmlin (egl2 promoter, secretion signal, EGL2 cellulose binding module and linker), (iii) CBH2sigpro (cbh2 promoter and secretion signal) and (iv) CBH2cbmlin (cbh2 promoter, secretion signal, CBH2 cellulose binding module and linker). Recombinant vectors were introduced individually into the high protein-secreting T. reesei RUT-C30 strain to generate single-promoter transformants expressing the Dictyoglomus thermophilum xynB gene that encodes a thermophilic xylanase enzyme (XynB). Ten transformants producing XynB representing each of the four different types of vectors were selected for further testing and the highest XynB production was achieved from a transformant containing 1–2 copies of the EGL2cbmlin vector. Best xylanase producers did not show any particular pattern in terms of the number of gene copies and their mode of integration into the chromosomal DNA. Transformants generated with the cbmlin-type vectors produced multiple forms of XynB which were decorated with various N- and O-glycans. One of the O-glycans was identified as hexuronic acid, whose presence had not been observed previously in the glycosylation patterns of T. reesei.

Stress effects caused by the expression of a mutant cellobiohydrolase I and proteasome inhibition in Trichoderma reesei Rut-C30

Trichoderma reesei Rut-C30 is used widely as an expression host for various gene products. We have explored cellular effects caused by the expression of a mutant form of cellobiohydrolase I (CBHI), the major secreted protein of T. reesei using biochemical and transcriptomic analyses and confocal laser scanning microscopy. The mutated CBHI was tagged fluorescently with Venus to establish the subcellular location of the fusion protein and its potential association with the proteasome, an organelle assigned for the disposal of misfolded proteins. Expression of the mutant CBHI in the high protein-secreting host Rut-C30 caused physiological changes in the fungal hyphae, affected protein secretion and elicited ER stress. A massive upregulation of UPR- and ERAD-related genes sec61, der1, uba1, bip1, pdi1, prp1, cxl1 and lhs1 was observed by qRT-PCR in the CBHIΔ4-Venus strain with four mutations introduced in the DNA encoding the core domain of CBHI. Further stress was applied to this strain by inhibiting function of the proteasome with MG132 (N-benzoylcarbonyl(Cbz)-Leu-Leu-leucinal). The effect of MG132 was found to be specific to the proteasome-associated genes. There are no earlier reports on the effect of proteasome inhibition on protein quality control in filamentous fungi. Confocal fluorescence microscopy studies suggested that the mutant CBHI accumulated in the ER and colocalized with the fungal proteasome. These results provide an indication that there is a limit to how far T. reesei Rut-C30, already under secretion stress, can be pressed to produce higher protein yields.

Enzyme activities and biotechnological applications of cold-active microfungi

Fungi are eukaryotic organisms and considered to be less adaptable to extreme environments when compared to bacteria. While there are no thermophilic microfungi in a strict sense, some fungi have adapted to life in the cold. Cold-active microfungi have been isolated from the Antarctic and their enzyme activities explored with a view to finding new candidates for industrial use. On another front, environmental pollution by petroleum products in the Antarctic has led to a search for, and the subsequent discovery of, fungal isolates capable of degrading hydrocarbons. The work has paved the way to developing a bioremedial approach to containing this type of contamination in cold climates. Here we discuss our efforts to map the capability of Antarctic microfungi to degrade oil and also introduce a novel cold-active fungal lipase enzyme.

Geminin and Brahma act antagonistically to regulate EGFR–Ras–MAPK signaling in Drosophila

Geminin was identified in Xenopus as a dual function protein involved in the regulation of DNA replication and neural differentiation. In Xenopus, Geminin acts to antagonize the Brahma (Brm) chromatin-remodeling protein, Brg1, during neural differentiation. Here, we investigate the interaction of Geminin with the Brm complex during Drosophila development. We demonstrate that Drosophila Geminin (Gem) interacts antagonistically with the Brm–BAP complex during wing development. Moreover, we show in vivo during wing development and biochemically that Brm acts to promote EGFR–Ras–MAPK signaling, as indicated by its effects on pERK levels, while Gem opposes this. Furthermore, gem and brm alleles modulate the wing phenotype of a Raf gain-of-function mutant and the eye phenotype of a EGFR gain-of-function mutant. Western analysis revealed that Gem over-expression in a background compromised for Brm function reduces Mek (MAPKK/Sor) protein levels, consistent with the decrease in ERK activation observed. Taken together, our results show that Gem and Brm act antagonistically to modulate the EGFR–Ras–MAPK signaling pathway, by affecting Mek levels during Drosophila development.

Use of the biolistic particle delivery system to transform fungal genomes

Biolistic delivery of transforming DNA into fungal genomes, especially when performed on uninucleate haploid conidia, has proven successful in bypassing the time-consuming repetitive purification of protoplasts used for the widely applied polyethylene glycol-mediated method. Biolistic transformation is also relatively quick compared to other available methods and provides a high percentage of stable transformants.

Comparative analysis of the Metarhizium anisopliae secretome in response to exposure to the greyback cane grub and grub cuticles

Metarhizium anisopliae is a well-characterized biocontrol agent of a wide range of insects including cane grubs. In this study, a two-dimensional (2D) electrophoresis was used to display secreted proteins of M. anisopliae strain FI-1045 growing on the whole greyback cane grubs and their isolated cuticles. Hydrolytic enzymes secreted by M. anisopliae play a key role in insect cuticle-degradation and initiation of the infection process. We have identified all the 101 protein spots displayed by cross-species identification (CSI) from the fungal kingdom. Among the identified proteins were 64-kDa serine carboxypeptidase, 1,3 beta-exoglucanase, Dynamin GTPase, THZ kinase, calcineurin like phosphoesterase, and phosphatidylinositol kinase secreted by M. ansiopliae (FI-1045) in response to exposure to the greyback cane grubs and their isolated cuticles. These proteins have not been previously identified from the culture supernatant of M. anisopliae during infection. To our knowledge, this the first proteomic map established to study the extracellular proteins secreted by M. ansiopliae (FI-1045) during infection of greyback cane grubs and its cuticles.

A proteomic view into infection of greyback canegrubs (Dermolepida albohirtum) by Metarhizium anisopliae

Metarhizium anisopliae is a naturally occurring cosmopolitan fungus infecting greyback canegrubs (Dermolepida albohirtum). The main molecular factors involved in the complex interactions occurring between the greyback canegrubs and M. anisopliae (FI-1045) were investigated by comparing the proteomes of healthy canegrubs, canegrubs infected with Metarhizium and fungus only. Differentially expressed proteins from the infected canegrubs were subjected to mass spectrometry to search for pathogenicity related proteins. Immune-related proteins of canegrubs identified in this study include cytoskeletal proteins (actin), cell communication proteins, proteases and peptidases. Fungal proteins identified include metalloproteins, acyl-CoA, cyclin proteins and chorismate mutase. Comparative proteome analysis provided a view into the cellular reactions triggered in the canegrub in response to the fungal infection at the onset of biological control.

Teaching peer review reflective processes in accounting

A well designed peer review process in higher education subjects can lead to more confident and reflective learners who become skilled at making independent judgements of their own and others’ work; essential requirements for successful lifelong learning. The challenge for educators is to ensure their students gain these important graduate attributes within the constraints of a range of internal and external tensions currently facing higher education systems, including, respectively, the realities of large undergraduate Accounting subjects, culturally diverse and time-poor academics and students, and increased calls for public accountability of the Higher Education sector by groups such as the OECD. Innovative curriculum and assessment design and collaborative technologies have the capacity to simultaneously provide some measure of relief from these internal and external tensions and to position students as responsible partners in their own learning. This chapter reports on a two phase implementation of an online peer review process as part of the assessment in a large, under-graduate, International Accounting class. Phase One did not include explicit reflective strategies within the process, and anonymous and voluntary student views served to clearly highlight that students were ‘confused’ and ‘hesitant’ about moving away from their own ideas; often mistrusting the conflicting advice received from multiple peer reviewers. A significant number of students also felt that they did not have the skills to constructively review the work of their peers. Phase Two consequently utilised the combined power of e-Technology, peer review feedback and carefully scaffolded and supported reflective practices from Ryan and Ryan’s Teaching and Assessing Reflective Learning (TARL) model (see Chap. 2). Students found the reflective skills support workshop introduced in Phase Two to be highly useful in maximising the benefits of the peer review process, with 83 % reporting it supported them in writing peer reviews, while 90 % of the respondents reporting the workshop supported them in utilising peer and staff feedback.

Teaching reflection for service-learning

In this chapter, we discuss an approach for teaching pre-service teachers how to critically reflect on their experiences in a Service-learning program in an advanced subject about inclusive education. The approach was informed by critical social theory, with the expectation that students would engage in transformational learning. By explicitly teaching the students to engage in critical reflective thinking (Fishbowl discussions) and examine the depth of their critical reflection against a heuristic (the 4Rs reflection framework), the final-year Bachelor of Education students were able to gain a deeper understanding of the subject and experience transformational learning. We provide contextual information about the Service-learning program and discuss critical social theory for transformational learning, as well as how the teaching team taught critical reflection. Based on the evidence gathered from the students, we consider lessons learned by the teaching team and provide recommendations for teaching reflection in Service-learning programs.

A model for reflection in the pedagogic field of higher education

This chapter provides an introduction to the use of pedagogical patterns in capturing and sharing educational design experience. In higher education, helping students to learn to engage in productive reflection presents a complex set of challenges. Delicate balances must be found: too little structure and support for students’ reflective work can leave them floundering; too much, and some will remain dependent. Moreover, this is a dynamic teaching problem – scaffolding needs to be adjusted as students develop confidence and capability, which they will do at different rates. The model presented in this chapter embraces the three main elements that teachers can legitimately design, or help set in place, to support their students’ reflective activity: good tasks, the right tools, and appropriate divisions of labour. It delineates a complex, shifting architecture of tasks, tools and people, activities and outcomes associated with reflective learning. It shows how the designable elements of this complex mix can be described in patterns and pattern languages, which then become design resources for teachers’ own action, reflection and professional development.

Sustainable Pedagogical Change for Embedding Reflective Learning Across Higher Education Programs

For pedagogical change to be sustained over time, and over the span of higher education courses, it needs to be framed widely, rather than ‘tacked on’. The framing includes curriculum reform and resource provision alongside staff pedagogical development. This is especially true for initiatives (such as reflective writing and assessment) that target broad-based, high-level skills and dispositions. For various reasons, such initiatives can easily become lost because of the discipline-specific focus of a syllabus outweighs the initiative, or because lack of resources compromises a desired approach. Course improvement in higher education contexts is typically difficult and episodic. In such circumstances, we argue that a strategic and trustworthy approach is necessary where practitioner-lead pedagogic development is fostered through trust and communication and is purposefully embedded within key dimensions of curriculum integration and resource provision. This chapter describes an approach to pedagogical change where curriculum, pedagogy and resources are simultaneously and collaboratively orchestrated to provide an effective framework for sustainable and effective change. A robust conceptual model is proposed to guide the implementation of such change.

Representation and authenticity of historic landscapes in Australia and China

Histories of past communities are embedded in landscapes around the world but many are suffering from material change or neglect of their fabric. This study was aimed at discovering and representing the authentic intangible experience of two historic landscapes for conservation purposes. A 2500 year old site in Yangzhou, China and a 2000 year old site on St Helena Island in Moreton Bay were found to be managed under two culturally different regimes of authenticity. This research has contributed to challenging the notion that there is only one way to conserve authenticity in historic landscapes of the Asia Pacific.

Using the TARL Model in Psychology: Supporting First and Final Year Students to Compose Reflections

Reflective thinking is an important skill in psychology, both as a tool in the therapeutic process and in professional development. The adapted 4Rs Model provides reflective writing structure and highlights the importance of Reporting & Responding, Relating, Reasoning and Reconstructing to students who are new to writing reflections. This chapter presents a case in which the 4Rs model (modified from the 5Rs model in Chap. 2 of this edition) was adopted to support reflective writing skills of undergraduate psychology students in a first year unit and in a final year unit. Although all students reflected on their learning within the units, the support activities leading up to the reflective tasks were adjusted to account for differences in the abilities of the cohorts and the focus of the units. In an evaluation survey, both groups of students endorsed statements about the importance of reflections and the utility of using the model. First year students also reported some difficulties understanding the 4Rs. This chapter will explore how first and final year students can be supported to develop reflection skills through scaffolding and modification of the same approaches and model.

An ePortfolio Approach: Supporting Critical Reflection for Pedagogic Innovation

This chapter investigates the capacity of a well-supported holistic ePortfolio program, the QUT Student ePortfolio Program (QSeP), to support critical reflection for pedagogic innovation in higher education, by exploring practice examples. The chapter looks across faculty and discipline areas to illustrate a range of ePortfolio learning case studies, which have led pedagogical innovation across a whole institution, to enhance student learning and support academic teaching. The ePortfolio strategies discussed support innovation in learning and teaching where academics use the ePortfolio approach in different ways to develop connectedness (productive pedagogies) within learning. Students are supported to develop awareness of the connections between formal and informal learning opportunities and between their learning and personal and professional goals. Students are guided to understand what they have learned and how they have learned in terms of generic employability skills or graduate attributes and also in relation to professional standards and competencies and personal goals. In essence, the ePortfolio-supported pedagogy creates capstone events enabling students to develop a professional identity and understanding of ongoing professional development. The examples are drawn from distinct discipline areas and illustrate the capacity of ePortfolio to underpin pedagogic innovation across discipline areas: • Bachelor of Information Technology—the ePortfolio approach supports students to explore the IT industry as a means of clarifying personal expectations and goals, thereby enhancing student potential in the course c• Bachelor of Nursing and Master of Nursing Science—students develop a professional ePortfolio to show development of the nursing competencies • Master of Information Technology—Library and Information students compile a Professional Portfolio for assessment in the Professional Practice subject • Bachelor of Laws—Virtual Law Placement (VLP) is a unit of study that challenges students to critically reflect on their performance and development duringthe work placement Each case study illustrates the academic teaching goal and student ePortfolio task in context. Issues, challenges and support strategies are identified. Comments from the students and their lecturers give an indication of the effectiveness of the ePortfolio approach to meet learning and teaching goals.