Simultaneous expression of the bacterial Dictyoglomus thermophilum xynB gene under three different Trichoderma reesei promoters

Multiple copies of expression cassettes driven by the Trichoderma reesei xylanase 2 (xyn2) and cellobiohydrolase 2 (cbh2) promoters were introduced into the recombinant T. reesei EC-21 generated to express a thermostable Dictyoglomus thermophilum xylanase (XynB) under the egl2 promoter for further improvement of the enzyme yield. The transformants were screened based on increased XynB activity only. Multiple promoter transformant MPP-4 expressing the xynB gene under all the three promoters was found to be the highest producer of XynB, giving a 65% increase in yield compared to the parental single-promoter recombinant EC-21. The multiple-promoter transformant strains harboured six to nine copies of the xynB gene. Amongst the three promoters, egl2 seemed to have the strongest effect on XynB expression. The shotgun approach we used proved to be effective for rapid enhancement of protein expression using three promoters active at the near-neutral pH of the cultivation medium.

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.

Regulating small things : genes, gametes and nanotechnology

Biotechnology and nanotechnology both intersect with other technologies in ways that open new possibilities for further technological progress. The potential for increased convergence between technological fields highlights the need for regulatory frameworks to be integrated, flexible and responsive. Within a federal legal system such as Australia’s, there is a need to ensure that we adopt a coordinated national approach to the crafting of regulatory solutions. In addition, there is a need for global cooperation in the development of international standards and regulatory harmonisation. Finally, this article considers the role that law plays in negotiating risk in relation to new technologies.

Ecological risk analysis of transgenic plants

Public concern about the safety of many forms of industrial technology are known to be linked to a range of factors including a perceived lack of confidence in regulatory decision making.1 The use of transgenic plants in agriculture may be seen as an issue that could generate similar concern. Criticism has been made about the completeness of knowledge on the potential for aberrant behaviour of genetically manipulated organisms (GMO's) in release environments, and the adequacy of existing pre­‐release screening and assessment methodologies (Goldberg & Tjaden, 1990). Such comments are important because any perceived shortcomings in the pre-release assessment of GMO safety may lead to decreased public support of the technology -­‐and the industry itself...

Entrepreneurship education in non-business schools : best practice for Australian contexts of knowledge and innovation communities (Final Report)

In the current era of global economic instability, business and industry have already identified a widening gap between graduate skills and employability. An important element of this is the lack of entrepreneurial skills in graduates. This Teaching Fellowship investigated two sides of a story about entrepreneurial skills and their teaching. Senior players in the innovation commercialisation industry, a high profile entrepreneurial sector, were surveyed to gauge their needs and experiences of graduates they employ. International contexts of entrepreneurship education were investigated to explore how their teaching programs impart the skills of entrepreneurship. Such knowledge is an essential for the design of education programs that can deliver the entrepreneurial skills deemed important by industry for future sustainability. Two programs of entrepreneurship education are being implemented at QUT that draw on the best practice exemplars investigated during this Fellowship. The QUT Innovation Space (QIS) focuses on capturing the innovation and creativity of students, staff and others. The QIS is a physical and virtual meeting and networking space; a connected community enhancing the engagement of participants. The Q_Hatchery is still embryonic; but it is intended to be an innovation community that brings together nascent entrepreneurial businesses to collaborate, train and support each other. There is a niche between concept product and business incubator where an experiential learning environment for otherwise isolated ‘garage-at-home’ businesses could improve success rates. The QIS and the Q_Hatchery serve as living research laboratories to trial the concepts emerging from the skills survey. The survey of skills requirements of the innovation commercialisation industry has produced a large and high quality data set still being explored. Work experience as an employability factor has already emerged as an industry requirement that provides employee maturity. Exploratory factor analysis of the skills topics surveyed has led to a process-based conceptual model for teaching and learning higher-order entrepreneurial skills. Two foundational skills domains (Knowledge, Awareness) are proposed as prerequisites which allow individuals with a suite of early stage entrepreneurial and behavioural skills (Pre-leadership) to further leverage their careers into a leadership role in industry with development of skills around higher order elements of entrepreneurship, management in new business ventures and progressing winning technologies to market. The next stage of the analysis is to test the proposed model through structured equation modelling. Another factor that emerged quickly from the survey analysis broadens the generic concept of team skills currently voiced in Australian policy documents discussing the employability agenda. While there was recognition of the role of sharing, creating and using knowledge in a team-based interdisciplinary context, the adoption and adaptation of behaviours and attitudes of other team members of different disciplinary backgrounds (interprofessionalism) featured as an issue. Most undergraduates are taught and undertake teamwork in silos and, thus, seldom experience a true real-world interdisciplinary environment. Enhancing the entrepreneurial capacity of Australian industry is essential for the economic health of the country and can only be achieved by addressing the lack of entrepreneurial skills in graduates from the higher education system. This Fellowship has attempted to address this deficiency by identifying the skills requirements and providing frameworks for their teaching.

Nucleic acid hybridizations as radioactive tool for rapid detection of banana bunchy top virus

Banana bunchy top disease (BBTD) caused by banana bunchy top virus (BBTV) was radioactively detected by nucleic acid hybridization techniques. Results showed that, 32P-labelled insert of pBT338 was hybridized with nucleic acid extracts from BBTV-infected plants from Egypt and Australia but not with those from CMV-infected plants from Egypt. Results revealed that BBTV was greatly detected in midrib, roots, meristem, corm, leaves and pseudostem respectively. BBTV was also detected in symptomless young plants prepared from diseased plant materials grown under tissue culture conditions but was not present in those performed from healthy plant materials. The sensitivity of dot blot and Southern blot hybridizations for the detection of BBTV was also performed for the detection of BBTV.

Ultra sensitive label free surface enhanced Raman spectroscopy method for the detection of biomolecules

We present a proof of concept for a novel nanosensor for the detection of ultra-trace amounts of bio-active molecules in complex matrices. The nanosensor is comprised of gold nanoparticles with an ultra-thin silica shell and antibody surface attachment, which allows for the immobilization and direct detection of bio-active molecules by surface enhanced Raman spectroscopy (SERS) without requiring a Raman label. The ultra-thin passive layer (~1.3 nm thickness) prevents competing molecules from binding non-selectively to the gold surface without compromising the signal enhancement. The antibodies attached on the surface of the nanoparticles selectively bind to the target molecule with high affinity. The interaction between the nanosensor and the target analyte result in conformational rearrangements of the antibody binding sites, leading to significant changes in the surface enhanced Raman spectra of the nanoparticles when compared to the spectra of the un-reacted nanoparticles. Nanosensors of this design targeting the bio-active compounds erythropoietin and caffeine were able to detect ultra-trace amounts the analyte to the lower quantification limits of 3.5×10−13 M and 1×10−9 M, respectively.

Enhancing itaconic acid production by Aspergillus terreus

Aspergillus terreus is successfully used for industrial production of itaconic acid. The acid is formed from cis-aconitate, an intermediate of the tricarboxylic (TCA) cycle, by catalytic action of cis-aconitate decarboxylase. It could be assumed that strong anaplerotic reactions that replenish the pool of the TCA cycle intermediates would enhance the synthesis and excretion rate of itaconic acid. In the phylogenetic close relative Aspergillus niger, upregulated metabolic flux through glycolysis has been described that acted as a strong anaplerotic reaction. Deregulated glycolytic flux was caused by posttranslational modification of 6-phosphofructo-1-kinase (PFK1) that resulted in formation of a highly active, citrate inhibition-resistant shorter form of the enzyme. In order to avoid complex posttranslational modification, the native A. niger pfkA gene has been modified to encode for an active shorter PFK1 fragment. By the insertion of the modified A. niger pfkA genes into the A. terreus strain, increased specific productivities of itaconic acid and final yields were documented by transformants in respect to the parental strain. On the other hand, growth rate of all transformants remained suppressed which is due to the low initial pH value of the medium, one of the prerequisites for the accumulation of itaconic acid by A. terreus mycelium. © 2010 Springer-Verlag.

A QUM partnership approach to regulating biosimilars in Australia

Despite the realisation of the potential implications from biosimilars is relatively recent, much has already been written about raising the awareness of differences between biosimilars and originating/ reference listed (innovator) pharmaceuticals. The European Medicines Agency has led the global charge in regulating biosimilars. Regardless of sufficient similarities across international regulations, differences do exist across jurisdictions. The consideration of regulating biosimilars demands a congruent approach across all stages: pre-registration (Australian copyright protection, patent, international obligations), registration (confidential information, international regulators, safety and efficacy), post-registration (Pharmaceutical Benefit Scheme, prescriber and dispenser awareness). Our National Medicines Policy could provide the necessary congruent framework and function for national and international regulation of biosimilars. The Policy concedes that pharmaceuticals will be affected by financial policies and trade considerations, international treaty obligations, industrial policies, education policies and the need for public-private partnerships.

Health Law in Australia [Second Edition]

Health Law in Australia is the country’s leading text in this area and was the first book to deal with health law on a comprehensive national basis. In this important field that continues to give rise to challenges for society Health Law in Australia takes a logical, structured approach to explain the breadth of this area of law across all Australian jurisdictions. By covering all the major areas in this diverse field, Health Law in Australia enhances the understanding of the discipline as a whole. Beginning with an exploration of the general principles of health law, including chapters on “Negligence”, “Children and Consent to Medical Treatment”, and “Medical Confidentiality and Patient Privacy”, the book goes on to consider beginning-of-life and end-of-life issues before concluding with chapters on emerging areas in health law, such as biotechnology, genetic technologies and medical research. The contributing authors are national leaders who are specialists in these areas of health law and who can share with readers the results of their research. Health Law in Australia has been written for both legal and health audiences and is essential reading for undergraduate and postgraduate students, researchers and scholars in the disciplines of law, health and medicine, as well as health and legal practitioners, government departments and bodies in the health area, and private health providers.

Biolistic transformation of elite indica rice (Oryza sativa L.) cultivars through semi-solid and liquid medium selection systems

Following microprojectile mediated delivery of a plasmid construct (pAHC-25) encoding bar (bialophos resistance) gene into five-day-old scutellar calli derived from mature embryos, the effectiveness of selection procedure for bar-gene expressing tissue was compared for two indica rice cultivars (IR-64 and Karnal Local). While IR-64 transformants could be selected through the generally used semi-solid selection medium, the same procedure was not effective in the basmati cultivar Karnal Local. In the latter case, while lower concentrations (2–4 mg 1−1) of the selective agent phosphinothricin (PPT) yielded only escapes, higher concentrations (6–8 mg l−1) inhibited proliferation of transformed as well as untransformed sectors. For Karnal Local, a liquid medium based selection system was successfully utilized for recovering transformed sectors and, eventually, regenerants. The study demonstrates the generation of transformants of two elite indica cultivars using the environment-independent system of mature embryos from seeds.