Culture collections — their services to biotechnology

The need to recruit qualified and experienced employees is a major concern of many organizations engaged in biotechnology. Universities and industry are scoured to find the right people, but where are we to find promising new candidates for the most numerous, and arguably most talented section of the biotechnological workforce — microorganisms and other types of cells? The international network of culture collections offers the best answer, and a proper understanding of their roles and capabilities can save biotechnologists an immense amount of time and money.Among the millions of microbial, plant and animal cells stored in the world's culture collections it is often possible to find one that exactly suits a biotechnologist's particular requirements or one that can be developed to perform a specific task. This article outlines the services offered by culture collections, generally using the UK collections as examples of the main activities throughout the world.     

Nootkatone—a biotechnological challenge

Due to its pleasant grapefruit-like aroma and various further interesting molecular characteristics, (+)-nootkatone represents a highly sought-after specialty chemical. (+)-Nootkatone is accumulated in its producer plants in trace amounts only, and the demand of the food, cosmetics and pharmaceutical industry is currently predominantly met by chemical syntheses. These typically require environmentally critical reagents, catalysts and solvents, and the final product must not be marketed as a “natural flavour” compound. Both the market pull and the technological push have thus inspired biotechnologists to open up more attractive routes towards natural (+)-nootkatone. The multifaceted approaches for the de novo biosynthesis or the biotransformation of the precursor (+)-valencene to (+)-nootkatone are reviewed. Whole-cell systems of bacteria, filamentous fungi and plants, cell extracts or purified enzymes have been employed. A prominent biocatalytic route is the allylic oxidation of (+)-valencene. It allows the production of natural (+)-nootkatone in high yields under mild reaction conditions. The first sequence data of (+)-valencene-converting activities have just become known.

Fusarium species—a promising tool box for industrial biotechnology

Applied Microbiology and Biotechnology - Global demand for biotechnological products has increased steadily over the years. Thus, need for optimized processes and reduced costs appear as a key...     

The roots—a short history of industrial microbiology and biotechnology

Early biotechnology (BT) had its roots in fascinating discoveries, such as yeast as living matter being responsible for the fermentation of beer and wine. Serious controversies arose between vitalists and chemists, resulting in the reversal of theories and paradigms, but prompting continuing research and progress. Pasteur’s work led to the establishment of the science of microbiology by developing pure monoculture in sterile medium, and together with the work of Robert Koch to the recognition that a single pathogenic organism is the causative agent for a particular disease. Pasteur also achieved innovations for industrial processes of high economic relevance, including beer, wine and alcohol. Several decades later Buchner, disproved the hypothesis that processes in living cells required a metaphysical ‘vis vitalis’ in addition to pure chemical laws. Enzymes were shown to be the chemical basis of bioconversions. Studies on the formation of products in microbial fermentations, resulted in the manufacture of citric acid, and chemical components required for explosives particularly in war time, acetone and butanol, and further products through fermentation. The requirements for penicillin during the Second World War lead to the industrial manufacture of penicillin, and to the era of antibiotics with further antibiotics, like streptomycin, becoming available. This was followed by a new class of high value-added products, mainly secondary metabolites, e.g. steroids obtained by biotransformation. By the mid-twentieth century, biotechnology was becoming an accepted specialty with courses being established in the life sciences departments of several universities. Starting in the 1970s and 1980s, BT gained the attention of governmental agencies in Germany, the UK, Japan, the USA, and others as a field of innovative potential and economic growth, leading to expansion of the field. Basic research in Biochemistry and Molecular Biology dramatically widened the field of life sciences and at the same time unified them considerably by the study of genes and their relatedness throughout the evolutionary process. The scope of accessible products and services expanded significantly. Economic input accelerated research and development, by encouraging and financing the development of new methods, tools, machines and the foundation of new companies. The discipline of ‘New Biotechnology’ became one of the lead sciences. Although biotechnology has historical roots, it continues to influence diverse industrial fields of activity, including food, feed and other commodities, for example polymer manufacture, biofuels and energy production, providing services such as environmental protection, and the development and production of many of the most effective drugs. The understanding of biology down to the molecular level opens the way to create novel products and efficient environmentally acceptable methods for their production.     

What to do in marine biotechnology?

During the symposium "Marine Biotechnology: Basics and Applications", held 25 February-1 March, 2003 in Matalasca?as, Spain, a special brainstorm session was organized. Two questions were addressed: 1, "What is the most desirable development in marine biotechnology"?; 2, "What is the most spectacular development in this field in your 'wildest' dreams"? The outcome of this session is reported in this paper. From the more than 250 ideas generated, concern for the environment and human health emerged as the most significant issues.     

Human dignity: a guide to policy making in the biotechnology era?

This article explores the ways in which human dignity is used in debates about controversial biotechnologies, including biobanks, human gene patents, stem cell research and human cloning. Increasingly, human dignity is used as a form of general condemnation and as blanket justification for regulatory restraint. However, this use of human dignity marks a significant departure from the traditional, human-rights informed view of human dignity that has dominated bioethics debates for decades. In addition, on its own, it stands as dubious justification for policies that are aimed at constraining controversial biotechnologies.     

Traversing a wormhole to combat Parkinson’s disease

Human movement disorders represent a significant and unresolved societal burden. Among these, the most prevalent is Parkinson’s disease (PD), a disorder afflicting millions worldwide. Despite major advances, stemming primarily from human genetics, there remains a significant gap in our understanding of what factors underlie disease susceptibility, onset, and progression. Innovative strategies to discern specific intracellular targets for subsequent drug development are needed to more rapidly translate basic findings to the clinic. Here we briefly review the recent contributions of research using the nematode roundworm Caenorhabditis elegans as a model system for identifying and characterizing gene products associated with PD. As a microscopic but multicellular and genetically tractable animal with a well-defined nervous system and an experimentally tenable lifespan, C. elegans affords significant advantages to researchers attempting to determine causative and therapeutic factors that influence neuronal dysfunction and age-associated neurodegeneration. The rapidity with which traditional genetic, large-scale genomic, and pharmacological screening can be applied to C. elegans epitomizes the utility of this animal for disease research. Moreover, with mature bioinformatic and functional genomic data readily available, the nematode is well positioned to play an increasingly important role in PD-associated discoveries.Physicists and astronomers have long posited the concept of a ‘wormhole’ as a means of rapid interstellar travel by analogy with how a worm could eat a hole from one end of an apple, through the center to the other end, and create a shortcut through the intervening space. Unfortunately, bending the fabric of space and time is not typically considered a viable option to more rapidly explore cures for neurodegenerative diseases (and would likely result in a poorly received grant application). The biological equivalent of the space exploration program, the human genome project, has unleashed an age of genomic, proteomic, metabolomic, and bioinformatic analyses that has generated a wealth of datasets primed for subsequent discovery. This exponential growth demands the development of functional means for exploiting this treasure trove of biological information. In this regard, biomedical researchers are literally turning to a worm to accelerate the path toward therapeutic advances and get to the core of mechanisms underlying Parkinson’s disease (PD).While drugs to treat the symptoms of PD have been prescribed for decades (e.g. L-DOPA), an unmet need for innovative strategies to discern disease etiology and treatments that either halt or reverse progression remains. Application of a microscopic nematode roundworm toward gaining insights into a human neurodegenerative disorder may seem impractical; yet, C. elegans affords many advantages for such research, as it has a defined cell lineage, completed genome sequence, and lifespan of only 2 weeks. As opposed to the human brain, which is estimated to have over 100 billion nerve cells, this nematode contains precisely 302 neurons for which a defined neuronal connectivity map has been determined. In this regard, C. elegans is ideal for investigation of diseases associated with neuronal dysfunction and ageing, and represents a model system that is poised to bridge the gap between basic and translational research.Interpretation of disease-associated data obtained in invertebrate systems requires downstream validation in mammals prior to establishing the therapeutic significance of any findings. The likelihood of a positive outcome is significant, however, due to the evolutionary conservation of metazoan genomes. For example, human homologs have been identified for at least 50% of C. elegans genes. Remarkably, these include all orthologs of reported genes linked to familial forms of PD (Fig. 1), with the one exception being the gene encoding α-synuclein. Despite this difference, ectopic overexpression of both wildtype and mutant (A53T) human α-synuclein led to motor deficits or DA neuron loss in C. elegans (Lasko et al., 2003). Subsequent reports also demonstrated phenotypic changes associated with driving α-synuclein expression from a variety of pan-neuronal and specific neuronal promoters (Cao et al., 2005; Ved et al., 2005; Kuwahara et al., 2006). The translational utility of C. elegans is perhaps best demonstrated by the characterization of conserved neuroprotective genes that block α-synuclein-associated degeneration in nematodes (Cooper et al., 2006; Gitler et al., 2008; Hamamichi et al., 2008).Open in a separate windowFig. 1PD-associated gene products and their prospective sites of cellular functionPD is hypothesized to be a consequence of the dysfunction of intersecting and compensatory protein degradation components, including those associated with the lysosome and autophagy, as well as those associated with the ubiquitin proteasome system (UPS). Inefficient clearance and enhanced misfolding, or expression, of α-synuclein has been shown to block intracellular trafficking and increase cytotoxicity. Accordingly, mutations in Parkin (an E3 ubiquitin ligase), ubiquitin C-terminal hydrolase-1 (UCHL-1), as well as in a lysosomal ATPase termed ATP13A2 and the Gaucher’s disease-related protein glucocerebrosidase (GBA), have further implicated protein degradation pathways in PD. PD-associated mutations in genes linked with mitochondrial function, such as those encoding DJ-1 and the PTEN-induced kinase (PINK1), presumably affect the production of reactive oxygen species (ROS), which accelerate protein damage and neurodegeneration. While mutations in the leucine-rich repeat kinase 2 (LRRK2) protein now represent the most common heritable cause of PD, the function of this large, 2527 amino acid multidomain-containing protein remains undefined.The rich, 40-year history of C. elegans genetics provides a legacy of behavioral and neuroanatomical information that researchers draw upon to elucidate relationships between gene function and PD-associated mechanisms (Bargmann, 1998). Using traditional genetic suppressor and enhancer screening, investigators have begun to dissect genetic contributors to DA neuron development and function (Chase et al., 2004). A limbless simple invertebrate will certainly never recapitulate the phenotypic behaviors associated with the tremor and dyskinesia of PD, yet this nematode does afford opportunities for accurate quantification of factors that influence DA neuron survival. Loss of DA neurons in C. elegans is not lethal and leads only to a subtle behavioral deficit where affected animals display an inability to discriminate local mechanosensory cues (Sawin et al., 2000). Unfortunately, this behavior, while quantifiable, is not robust enough to be easily used in extensive screening paradigms.Mammalian and primate modeling approaches to PD have traditionally involved use of neurotoxins to induce DA neuron loss and evaluate the consequences of neurodegeneration. Among these toxins are 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), as well as the pesticides rotenone and paraquat. Worms are susceptible to these toxins (Nass et al., 2002; Braungart et al., 2004; Cao et al., 2005); moreover, the defined and transparent neuroanatomy of C. elegans includes precisely eight neurons that produce DA, thereby enabling an unparalleled level of quantitative analysis of neurodegeneration and protection by use of fluorescent protein labeling (Fig. 2). Furthermore, these animals can be evaluated in specific genetic or transgenic backgrounds to screen for factors that confer either neuroprotection or enhancement of degeneration. One neuroprotective gene product termed TOR-2, a human torsinA-related protein with molecular chaperone-like activity (Caldwell et al., 2008), was shown to deter multiple forms of toxic insults to DA neurons, including 6-OHDA, excess intracellular DA production, and α-synuclein overexpression (Cao et al., 2005). The neuroprotective capacity of torsin-like proteins renders them intriguing candidates for subsequent drug targeting and validation in mammalian systems, where human torsinA is endogenously expressed in DA neurons (Augood et al., 1998).Open in a separate windowFig. 2Anterior dopamine neurons of C. elegansCell bodies and processes of the six anterior DA neurons including two pairs of the CEP (cephalic) neurons and one pair of ADE (anterior deirid) neurons, as illuminated using GFP driven from the dopamine transporter promoter (P_dat-1::GFP). Two additional DA neurons (PDEs) are found in the posterior (not shown).Directed and comprehensive screens have been undertaken to define chemical and genetic effectors of worm DA neuron sensitivity to 6-OHDA (Nass et al., 2005; Maranova and Nichols, 2007). These studies identified a collection of intragenic mutations in the gene sequence encoding the key protein required for DA reuptake into presynaptic neurons, the DA transporter (DAT-1). Structural and functional relationships revealed through this work have implications not only for PD, but also for other disorders associated with DAT function (e.g. depression, ADHD). It has also been shown that worm dat-1 knockout mutants exhibit diminished α-synuclein-dependent degeneration, which indicates a possible role for DAT in maintaining an important balance of intraneuronal levels of DA, the dysregulation of which may contribute to cytotoxicity (Cao et al., 2005).Elegant studies in Drosophila have demonstrated the capacity for model systems to reveal unsuspected relationships between PD gene products, such as PINK1 and Parkin, and their impact on mitochondrial integrity and morphology (Clark et al., 2006; Park et al., 2006; Poole et al., 2008). Likewise, the ability of worm research to link genotype to phenotype in this manner is accelerating our understanding of the underlying nature of PD. Ved et al. investigated the functional consequences of rotenone-induced stress and reported that pan-neuronal overexpression of human αsynuclein (wild-type or A53T mutant), RNAi knockdown of a C. elegans DJ-1 ortholog (B0432.2), or a deletion mutant of the C. elegans parkin ortholog (pdr-1), all produced similar patterns of mitochondrial vulnerability in response to pharmacological challenges associated with complex I inhibition (Ved et al., 2005). Furthermore, the effect of rotenone, which led to mortality in these animals, was more severe in α-synuclein-expressing strains and the pdr-1 deletion mutant than in control animals of wild-type background. Initial work on worm PDR-1 revealed that this Parkin ortholog, an E3 ubiquitin ligase, cooperates with conserved degradation machinery to mediate ubiquitin conjugation (Springer et al., 2005). Co-expression of C. elegans pdr-1 and α-synuclein variants in human cell cultures showed that a truncated protein derived from an in-frame pdr-1 (lg103) deletion allele causes aggregation of α-synuclein, similar to Parkin variants isolated from PD patients. This altered gene product also resulted in proteotoxicity and hypersensitivity to ER stress (Springer et al., 2005). These examples demonstrate how mutant analysis in C. elegans offers a powerful strategy for uncovering functional relationships between gene products with key roles in PD pathogenesis in mammalian cells.C. elegans has been recently used to show a protective role for human LRRK2 (leucine-rich repeat kinase 2) against mitochondrial toxicity induced by rotenone, an effect potentiated by knockdown of an endogenous worm ortholog, lrk-1 (Wolozin et al., 2008). These data are surprising when considering a PD-associated G2019S mutant version of LRRK2 that exhibits increased kinase activity was also found to be protective; kinase activity has been suggested to contribute to the pathogenesis associated with LRRK2 neurotoxicity (West et al., 2007). An initial report on lrk-1 function in C. elegans indicated a role for the product of this gene in establishing neuronal polarity (Sakaguchi-Nakashima et al., 2007). As mutations in LRRK2 now represent the most common genetic cause of PD (Haugarvoll et al., 2008), further insights gleaned from C. elegans on this important, yet relatively uncharacterized, protein family will undoubtedly be informative.The capacity for genomic-scale screening has brought C. elegans to the forefront of modern functional analysis (Kamath and Ahringer, 2003). In contrast to mammals, where individual knockouts of mice are expensive and time consuming, worms are an efficient and economical alternative. Hamamichi et al. took a hypothesis-driven approach toward identification of putative genetic modifiers of PD via a multi-tiered screen for C. elegans genes that impact the misfolding of transgenic human α-synuclein, as well as neuroprotection, in vivo (Hamamichi et al., 2008). This study encompassed RNAi knockdown of nearly 900 bioinformatically prioritized gene targets, comprising components of cellular pathways implicated in protein folding or degradation, as well as gene products that are either co-expressed or interact with worm orthologs of familial PD genes. From this varied, but biased, ‘guilt by association’ list of targets, 20 candidate genes emerged as having the greatest propensity to influence α-synuclein misfolding. Internal validation was evident within this short list, as included were the worm homologs of two established recessive PD genes (DJ-1, PINK1), as well as a gene (ULK2) shown to be one of only six identified in a genome-wide association study of polymorphisms in PD patients (Fung et al., 2006). Importantly, overexpression of select cDNAs in C. elegans DA neurons revealed that five of seven gene products tested, chosen from the primary effectors of α-synuclein misfolding identified in the RNAi screen, exhibited significant protection from age- and dose-dependent neurodegeneration induced by α-synuclein (Hamamichi et al., 2008). Thus, application of C. elegans in this context uncovered functionally evaluated targets identified by the two primary clinical criteria associated with PD: α-synuclein accumulation and DA neurodegeneration.These genes, which included uncharacterized proteins, as well as regulators of autophagy, lysosomal function, cellular trafficking, and G-protein signaling, now represent outstanding candidates for strategically targeted drug development and validation in mammalian systems. A more recently conducted unbiased genome-wide RNAi screen for genetic factors that influence αsynuclein inclusion formation in C. elegans also yielded an over-representation of genes encoding components of vesicular trafficking, as well as specific age-associated genes (e.g. sir-2.1) (van Ham et al., 2008). Similar validation of the effect that these additional targets may have on DA neuron survival will likely reveal functionally significant relationships as well. Associations between PD and lysosomal degradation are of growing interest following the identification of a lysosomal ATPase, ATP13A2 (PARK9), as a gene product linked to hereditary early-onset PD with dementia (Ramirez et al., 2006), in addition to the discovery of a higher incidence of PD in patients with mutations in the Gaucher’s disease-associated gene, GBA, encoding the lysosomal storage-enzyme glucocerebrosidase. (Aharon-Peretz et al., 2004; Clark et al., 2007). It is easy to envision an expansion of such gene-specific data and the application of C. elegans toward investigating the functional consequences of single-nucleotide polymorphisms (SNPs) found in human populations, as these may potentially lead to genetic biomarkers of disease susceptibility.The burgeoning promise that a microscopic worm may contribute toward the goal of drug discovery for PD has become a tangible reality. Containing a rudimentary nervous system, unlike single-celled organisms, but more amenable to transgenic analysis and drug screening than mammals, C. elegans serves as an excellent intermediary bottleneck in translational research pipelines to characterize therapeutic gene and drug targets across animal models. Concerted efforts toward therapeutic development have been initiated that exploit the high-throughput screening capabilities of yeast cells to define numerous gene targets of interest, followed by subsequent evaluation of their neuroprotective capacity in worms, fruit flies and rat neuronal cultures (Cooper et al., 2006; Gitler et al., 2008). This approach has already revealed that overproduction of αsynuclein leads to a cytotoxic blockage in intracellular vesicular trafficking that can be alleviated by specific members of the Rab protein family (Cooper et al., 2006; Gitler et al., 2008). Thus, through the employment of a combination of powerful model systems, not only has a prospective cellular cause of PD been illuminated, but targeted screens for small molecules that protect against underlying functional aspects of neurodegeneration are now possible.When envisioning future directions whereby C. elegans will benefit PD research, several inescapable advantages of this model system should be considered. The most obvious of these is the well-established use of the worm for investigating mechanisms of aging (Kenyon, 2005). Indeed, the only definitive risk factor for PD is aging, where PD symptomatically affects over 2% of people above age 65. Extensive studies on worm longevity mutants (e.g. daf-2 or age-1) have demonstrated that evolutionarily conserved mechanisms are shared between invertebrates and mammals. Microarray and genomic-scale RNAi analyses conducted in age-extending backgrounds have yielded significant insights into gene sets that implicate dietary restriction and insulin-like signaling pathways as crucial mediators of lifespan (Murphy et al., 2003; Panowski et al., 2007). The vital, but poorly understood, interface between aging and age-associated neurodegenerative disease represents an exciting frontier that is readily explored using C. elegans.Furthermore, in the advent of the microRNA (miRNA) revolution, our nascent understanding of putative regulatory roles for miRNAs in neuron function will likely soon interface with our understanding of neurological disease and PD (Kosik, 2006; Kim et al., 2007). Considering the pioneering role worm research has played in defining miRNA function, a universe of possibilities exist, as worm researchers are well-poised to explore relationships between miRNAs, ageing and neuroprotective gene activity (Ibanez-Ventoso, 2007).

Advantages of C. elegans as a model for Parkinson’s disease

• All orthologs of genes linked to familial PD have been identified in C. elegans, except α-synuclein
• Ectopic overexpression of α-synuclein has neurotoxic effects in C. elegans, which are blocked by neuroprotective genes
• C. elegans has a simple nervous system (302 neurons compared with over 100 billion in humans) that is amenable to quantitative analysis of neurodegeneration
• C. elegans is susceptible to toxins commonly used to model neurodegeneration
• C. elegans studies predict relationships between cellular signaling, trafficking and protein degradation pathways, which are being tested for their susceptibility to targeted drug development for PD

Finally, although the primary advances in PD etiology have come through human genetics, the largely idiopathic nature of PD remains linked to inexplicable environmental causes. C. elegans research into innate immunity and cellular stress response has already provided mechanistic insights into organismal defenses and environmental influences on homeostasis (Kim et al., 2002; Mohri-Shiomi and Garsin, 2008). An expansion of neurotoxicity studies conducted in C. elegans is warranted and may yield a greater understanding of the interplay between genetic composition and environmental factors such as heavy metals, pesticides and other untested exposures.The worm is unquestionably a powerful system, yet it has its limitations: the anatomical and functional connectivity of the neuronal circuitry within this simple nematode cannot recapitulate the complex features of mammalian dopamine neurons or mimic the precise behavioral deficits associated with their loss. Likewise, as a cautionary caveat to inferring conservation of function from genetic interaction data, Tischler et al. demonstrated that synthetic lethal gene interactions between yeast and worm genes are not significantly conserved (Tischler et al., 2008). In this context, as the march toward systems biology proceeds and integrated analyses of gene or protein activity continue to lead to an increasing number of complex datasets, our ability to eventually define causes and cures will depend on functional strategies for wading through the emerging ‘information overload’. Science will benefit from the efficient manner by which C. elegans research can contribute to the quest for translational and personalized medical breakthroughs, by boldly going where no worm has gone before.     

Bioinformatics in microbial biotechnology – a mini review

The revolutionary growth in the computation speed and memory storage capability has fueled a new era in the analysis of biological data. Hundreds of microbial genomes and many eukaryotic genomes including a cleaner draft of human genome have been sequenced raising the expectation of better control of microorganisms. The goals are as lofty as the development of rational drugs and antimicrobial agents, development of new enhanced bacterial strains for bioremediation and pollution control, development of better and easy to administer vaccines, the development of protein biomarkers for various bacterial diseases, and better understanding of host-bacteria interaction to prevent bacterial infections. In the last decade the development of many new bioinformatics techniques and integrated databases has facilitated the realization of these goals. Current research in bioinformatics can be classified into: (i) genomics – sequencing and comparative study of genomes to identify gene and genome functionality, (ii) proteomics – identification and characterization of protein related properties and reconstruction of metabolic and regulatory pathways, (iii) cell visualization and simulation to study and model cell behavior, and (iv) application to the development of drugs and anti-microbial agents. In this article, we will focus on the techniques and their limitations in genomics and proteomics. Bioinformatics research can be classified under three major approaches: (1) analysis based upon the available experimental wet-lab data, (2) the use of mathematical modeling to derive new information, and (3) an integrated approach that integrates search techniques with mathematical modeling. The major impact of bioinformatics research has been to automate the genome sequencing, automated development of integrated genomics and proteomics databases, automated genome comparisons to identify the genome function, automated derivation of metabolic pathways, gene expression analysis to derive regulatory pathways, the development of statistical techniques, clustering techniques and data mining techniques to derive protein-protein and protein-DNA interactions, and modeling of 3D structure of proteins and 3D docking between proteins and biochemicals for rational drug design, difference analysis between pathogenic and non-pathogenic strains to identify candidate genes for vaccines and anti-microbial agents, and the whole genome comparison to understand the microbial evolution. The development of bioinformatics techniques has enhanced the pace of biological discovery by automated analysis of large number of microbial genomes. We are on the verge of using all this knowledge to understand cellular mechanisms at the systemic level. The developed bioinformatics techniques have potential to facilitate (i) the discovery of causes of diseases, (ii) vaccine and rational drug design, and (iii) improved cost effective agents for bioremediation by pruning out the dead ends. Despite the fast paced global effort, the current analysis is limited by the lack of available gene-functionality from the wet-lab data, the lack of computer algorithms to explore vast amount of data with unknown functionality, limited availability of protein-protein and protein-DNA interactions, and the lack of knowledge of temporal and transient behavior of genes and pathways.