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Australasian Biotechnology, Vol. 11 No. 3, 2001, pp. 27-28 BIOPROCESSING PERSPECTIVE ON BIOPROCESSING D.J. Dall, P.D. East & J.G. Oakeshott Code Number: au01041 Insects as Sources of Novel Bioprocessing Acitvities The insects are a large and diverse group of eukaryotes that have successfully colonised most of the environmental niches on the Earth. In doing so, they have evolved physical tolerances that adapt them to various extremes of moisture availability, temperature stress, nutritional balance and so on. It has long been assumed that these tolerances imply the existence of a corresponding breadth of molecular pathways and capabilities that derive either from the insects themselves, from their microbial associates, or from a combination of both; investigations using methodologies of functional genomics and environmental genomics are now providing increasing evidence to support this view. As yet, these metabolic strategies and capabilities remain both poorly understood, and essentially untapped for human purposes. Nevertheless, there are strong grounds for believing that in the future they will provide means to significantly expand our capabilities in the field of bioprocessing. This report focuses on some specific areas in which insect-associated metabolic processes and pathways are likely to be of interest for bioprocessing purposes. In passing, it should be noted that Australia is particularly well positioned to capitalise on such developments, given that it possesses a unique assemblage of insects (comprising some 10% of the world's total species, with an endemism of 80-90%), as well as other capacities necessary for their effective assessment and use. With widespread adoption of the United Nations Convention on Biological Diversity, strict operating standards now apply to commercial exploitation of biological resources. The legitimate collection, accurate identification, and appropriate preparation and processing of specimens and materials now requires considerable administrative infrastructure, as well as scientific and technical expertise. Involvement of organisations such as the CSIRO, which integrate both commercial and specialist scientific expertise, is likely to maximise effective returns from the industrial applications of these natural national assets. CSIRO Entomology, which is one of the world's largest public sector specialist entomological agencies, is now actively working across several of the areas described below. In overview, the most valuable contributions of insects (and/or their microbial associates) to industrial bioprocessing are likely to derive from utilisation of insect-specific metabolic pathways with unique molecular mechanisms, intermediates and/or products, or insect-derived variants of known molecules that possess unusual physical or structural tolerances. In fact, examples of both such possibilities can be cited from a single aspect of insect metabolism (see below), and many more will emerge as focused attention is brought to bear on this group. As an illustration, it is of considerable interest to note that pathways of lipid transport and metabolism in insects appear to differ quite markedly from those used by vertebrates. While many aspects of these functions in insects remain to be clarified, it is now recognised that diacylglycerol is the main form of lipid transported into and within the insect body cavity (Arrese et al., 2001), implying the existence of significant differences (cf vertebrates) in the enzymes (such as lipases) and other proteins involved in these processes. Given the pivotal role of lipids in human health in the developed world, a comprehensive understanding of these processes is likely to have applications that extend beyond the scope of discussion here. Nevertheless, these insect metabolic mechanisms also have potential to expand industrial bioprocessing capabilities. Although lipases are eponymously named for their ability to catalyse modification of the complex glycerides known collectively as lipids, they are also commonly able to interact, in a stereo-selective manner, with other types of molecules containing ester linkages. Given that a key attribute of useful bioprocessing reactions commonly involves the chiral selectivity involved, enzymes of this type are of immediate and intrinsic interest. In fact, it is reasonable to assert that these and other members of the large 'carboxylic ester hydrolase' group (which also includes esterase enzymes) underpin most bioprocess-based activities currently used in industrial settings. A further example of the novel lipid-processing capabilities of insects serves to reinforce the assertion of their potential in this area of biotech endeavour. For example, most metazoans are unable to modify unsaturated fatty acid molecules to produce linoleic acid; because of the importance of this molecule in metabolic processes it then constitutes an essential plant-derived dietary component for these organisms. In contrast, many species of insects have now been shown to possess ∆12 desaturase capabilities (Batcabe et al., 2000) that appear likely to support synthesis of this metabolite under conditions of dietary stress. Insect-derived sequences encoding metazoan-active forms of this enzyme could be of considerable scientific and industrial significance. Consideration of insect diet leads inevitably to one of the most obvious insect-associated capabilities, namely, biomass consumption. As most people are aware, the natural 'bioprocessing' activities of insects are of considerable importance to global nutrient cycles, albeit sometimes to the detriment of human intentions and constructions. Indeed, much of the research done on insects has, as its specific aim, the development of strategies to limit these activities by one means or another. Once again, however, better understanding might deliver bigger benefit. In particular, insects' capacity to grow on exceedingly nutrient-poor and/or 'unbalanced' dietary sources - be they woollen sweaters, bird feathers, or dry wood - points to the existence of potentially useful catabolic and anabolic capabilities. Although elements of the digestive processes of some insects are almost certainly conferred by their microbial associates, the contribution of insects themselves to the enzymes required for processes such as cellulose digestion are becoming increasingly apparent. While records of cellulase enzymes from insects are at present limited to termites and cockroaches, this quite likely reflects the dearth of information currently available for groups such as the xylophagous weevils and long-horn beetles. Given the restricted diets of some of these latter groups it is likely that many examples of other enzymes with biomass degradation properties will ultimately be found. Their microbial associates are also likely to provide activities of interest; continuing the theme of termite digestion, several studies suggest that xylanase enzymes, which are important in effective digestion of wood, are derived largely, if not exclusively, from their symbiotic gut bacteria. It is worth noting that in almost all the examples noted here, the insect-derived materials can usefully be viewed as molecular 'start points', rather than unalterably predetermined products. Well-established procedures of molecular biology now allow techniques such as 'enzyme engineering' or 'directed evolution' to be iteratively applied once a requisite cDNA has been cloned, in order to produce molecules with highly desirable functionalities. Insects themselves are presumed to use just these same processes when circumstances demand. For example, long-term exposure of insect populations to insecticidal compounds has repeatedly resulted in evolution of diminished sensitivity to those compounds. Because insecticidal toxins often have complex structures, mechanisms with capacity for their selective modification are of interest on several grounds. Thus, insecticidal compounds can be long-lived in the environment, present at unsatisfactorily high levels in production plants, storage containers and product applicators, and can cause user poisoning if not handled correctly. In these circumstances, deployment of molecules evolved by insects to break down those toxins has considerable potential for 'bio-remediation', as well as a pleasing philosophical symmetry. Most of the examples mentioned above relate to mechanisms that insects use to degrade complex materials for their own subsequent purposes. However, insects also produce a variety of highly structured materials; while some of these are intrinsically useful, many of the synthesis processes also involve interesting molecular mechanisms and pathway intermediates. Some of these specialised synthetic pathways relate to production of complex 'secondary' metabolites with functions that include pigmentation, signalling, and defence; these compounds frequently have potential applications as, for example, natural bio-degradable flavourings, dyes and fragrances. Materials derived from the cochineal insect have been used as dyes and colourings for centuries, in contrast, recent adoption of firefly luciferase as a tool for laboratory research highlights the potential of materials from these sources. Other complex molecules of a polymeric nature are commonly used by insects and other invertebrates. Various types of silk fibres are used in structures such as cocoons and webs, and both the materials themselves and the associated processing strategies are of considerable interest (Vollrath & Knight, 2001). Another such material is the skin or 'cuticle' that insects employ as their barrier against the environment. This material is a complex cross-linked mixture of proteins and a polysaccharide biopolymer called chitin; it can range in form from a lightweight flexible material to one that is hard and resilient. The insect enzymes, substrates and processes that are involved in the various phases of cuticle production are being progressively identified and isolated; development of the capacity to synthesise this biodegradable material for human purposes would represent a marvel of bioprocessing! Finally, it is possible to regard insects themselves as miniaturised bioprocessors; indeed, they have been treated as such for centuries, and farmed for products such as honey and silk. The ability to genetically manipulate insects, as well as insect-specific microbes and pathogens, provides an opportunity to benefit from their intrinsic bio-processing abilities, which include the ability to transform low quality food-stuffs into high quality products. Use of silkmoths and their viruses for production of recombinant proteins was first suggested more that 15 years ago (Maeda et al., 1985), and, in the face of concerns about global capacity for in vitro production, remains an option of considerable interest. References
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