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W1567381003 abstract "In this feature, leading researchers in the field of environmental microbiology speculate on the technical and conceptual developments that will drive innovative research and open new vistas over the next few years. No review of the power of synthetic biology is complete without a sage affirmation that the fight for environmental protection will be immeasurably served by synthetic microorganisms. Yet, to state the obvious, environmental protection occurs in the environment and synthetic biology occurs in the test tube. Culture and culturability are the original sin of synthetic biology. The field of microbial ecology has blossomed under the insight that the Petri dish is but a pale and partial shadow of real microbial environments. Yet synthetic biologists, who seek to do better than evolution, use that tiny fraction (lets say 0.0001%) of evolutions handiwork that will grow in the lab. The implicit assumption is that in many cases it will be easier, or indeed absolutely necessary, to meet an environmental need, by creating novel a life form in the lab rather than using some pre-existing organism ‘off the peg’. This is an assumption that is far more likely to be met if you only consider the organisms that grow in the lab and ignore competing philosophies such as microbial resource management. However, this ‘diversity blindness’ is not the biggest issue. Indeed it would require churlish counter-hubris to assume that someone somewhere could not engineer an organism with a useful function not found in nature. The question is how to deploy it. There is little evidence that we can get an organism from the laboratory into the field in any kind of systematic way. Those mindful of the fears of rampant microbes have spoken of forms of genetic containment, sequencing tweaks that would ensure the demise of a released but unwanted organism. They hardly need bother. Genetically manipulated (and I suspect cultured organism in general) seem to simply die away when faced with the rigours of the environment. Containment is problematic. Mark van Loosdrecht has pointed out that a sterile and sterilizable reactor (i.e. a very big test tube) will cost 200 times more than an equivalent open reactor. Moreover, the feedstock for such a reactor, presumably the waste itself, would need to be sterilized before use. Immobilization and protection behind membranes will be no less expensive. The simple truth is that it that virtually no one has successfully introduced a cultured or genetically engineered organism into an environment. Moreover, we have absolutely no idea why this is problematic. Those with the courage to admit this might be a problem speak of the need for more robust organisms. However, there is no evidence that the manipulated organisms some how have flimsy phospholipids or inherently inadequate cell walls. Indeed the focus on the individual organism betrays the mindset of the test tube where a single organism can be considered in isolation. In the real world (and we have known this since RA Fisher coined his fundamental theorem) fitness is a function of both the properties of the organism and the properties of all the other organisms it might encounter. This means that the design of an organism to accomplish a function in an open environment must consider not only the function, but also executing that function in the context of the possibly thousands of other species it might encounter. This is, to put it mildly, a tricky problem and there is, at present, no theoretical framework for doing this. If it is resolvable, and it may be, resolution will come when synthetic biology gets out of the test tube and embraces ecology and evolution. This may require advances in all three domains. Which brings me to my final point. If these problems are well known and the solutions are not. Why do so many people continue to assert that they will solve a problem which they no idea how, or perhaps willingness, to address? In private, this question will be greeted with a cynical shrug and the implication that this form of debasement is the price you pay for funding. But this is dangerous, for what might start as naïve optimism and then fade into grant-getting gamesmanship can transmogrify into a moral and intellectual fraud. Defrauding us of not simply money, but that far more precious resource: time. We really do need to radically change environmental biotechnology and we do not have forever to do it. So if you want to do this using synthetic organisms: get out of the test tube or get out of the way. For the better part of four decades, genetic engineering has relied on a universal toolbox containing three indispensable implements: restriction enzymes, ligases and, last but not least, Escherichia coli. Even those of us who now languish behind laptops and in meetings rather than work at the bench instantaneously recognize the smells of Luria broth and plasmid preps. Since the hallmark paper of Cohen and colleagues (1973), many elegant variations on the theme of restriction and ligation have been introduced to make E. coli-based cloning tremendously versatile. However, especially in the field of metabolic engineering, two major limitations of this trusted approach gradually became apparent. First, as the complexity of the desired DNA constructs increased, design of multi-gene constructs based on restriction and ligation turned into a molecular biologist's equivalent of Rubik's cube and the construction itself into a multi-step, painstaking and time-consuming exercise. Even 3 years ago, most of us would not have contemplated the assembly of, for example, a 20-fragment expression vector, neither as part of a PhD or postdoc project, nor in an industrial research setting. Second, the multi-step nature of DNA assembly via restriction and ligation and its requirement for unique restriction sites complicated the implementation of combinatorial approaches in the assembly of complex constructs, for example to optimize expression of a heterologous pathway by testing various combinations of enzymes from different donor organisms or by testing different combinations of promoter fragments in a multi-enzyme, heterologously expressed pathway. During the past 4 years, fast developments in synthetic biology led to the (re)discovery and optimization of a powerful alternative approach for assembling DNA fragments that is entirely independent of restriction and ligation. This approach is based on homologous recombination of short, shared terminal sequences of the linear DNA fragments that need to be assembled and can be subdivided into in vitro and the in vivo methods. In vitro methods for DNA assembly by recombination depend on cell-free systems, in which recombinase enzyme(s) take care of the assembly (see e.g. Gibson et al., 2010a). The major advantage of this method over restriction and ligation is that it enables one-step assembly of multi-fragment constructs, completely independent of the availability of unique restriction sites. The required short stretches of sequence overlap can easily be introduced, either by PCR amplification of target sequences with ‘overhanging’ primers or by the increasingly cost-efficient process of DNA synthesis. After in vitro assembly of a construct, it is transformed to a suitable microbial host, in many cases still E. coli. The in vivo methods are based on the same principles of generation and recombination of homologous termini as the in vitro methods. However, they go one important step further by eliminating the need for separate assembly and transformation steps. The in vivo methods rely on the high efficiency of the cellular homologous recombination machinery of some microorganisms by simply transforming the required cocktail of linear DNA fragments into the host cell. When a suitable origin of replication and selection marker are included among the fragments, the transformed microorganism then faithfully recombines the fragments into an autonomously replicating episome, without any prior enzymatic treatment. In vivo recombination-based DNA-assembly platforms have been developed in Bacillus subtilis (Itaya et al., 2008) and in specific E. coli rec mutants (Datsenko and Wanner, 2000; Li and Elledge, 2005). However, these bacterial systems have a limited capacity to efficiently assemble multiple fragments and are outperformed by the incredible recombination performance of the star player of in vivo assembly, the yeast Saccharomyces cerevisiae. In vivo assembly of DNA fragments in yeast was already proposed and demonstrated 30 years ago (Orr-Weaver et al., 1981; Kunes et al., 1987; Ma et al., 1987). However, it failed to really take off as a mainstay DNA construction method, perhaps mainly due to the absence of efficient techniques to generate the required homologous sequences. For a long time, its application was largely limited to the cloning of large DNA fragments that were difficult to manipulate by restriction and ligation (Larionov et al., 1996). In vivo assembly in S. cerevisiae, also known as transformation-associated recombination (TAR), only really caught the spotlight and took off when it was elegantly applied in the complete chemical synthesis, assembly, and cloning of a synthetic Mycoplasma genome by Gibson and colleagues (2010b). This milestone achievement was accompanied by a thorough evaluation of the high-fidelity assembly properties of S. cerevisiae, which convinced many metabolic engineers, especially those already in the yeast field, to use this new molecular biology platform for the construction of large and complex metabolic pathways (Merryman and Gibson, 2012). As yeast biologists, we have been amazed by the power and simplicity of recombination-based assembly in our ‘pet’ organism when we introduced the technique into our lab. In addition to accelerating construction of unique, multi-gene constructs, the technology facilitates combinatorial approaches in metabolic engineering and, due to its one-step simplicity, is highly compatible with automated, high-throughput strain construction. Gazing in the crystal ball, we would never dare to predict the complete demise of good old E. coli as a molecular biology workhorse. However, we hope and predict that, in the coming years, this simple technique for DNA assembly will find its way into many labs and especially into those that do not have a tradition in yeast molecular genetics. A significant amount of proteins and high-quality lipids in human nutrition originates from fish and shellfish. As a result of the increasing human population and its improving health and welfare, most wild fish species have become overexploited by overfishing. The world's fish populations therefore suffer from dramatic declines. Breeding fish in analogy to breeding domestic ruminants, pigs and poultry will significantly protect endangered wild fish populations and can eventually re-establish an equilibrium of aquatic life. However, this can only be achieved by cultivating more non-carnivorous species and by changing the food requirements of carnivorous species by supplementing their nutrition with correctly processed slaughter waste materials and vegetable products. Furthermore, fish farming must not adversely affect the aquatic and terrestrial equilibrium by emissions of waste products and other pollutants. Worldwide aquaculture has increased at an average of 9.5% per year over the past 30 years, compared with 2.6% for terrestrial meat production, freshwater production representing the highest increase. Salmon production in Norway by aquaculture, for example, has, in less than three decades, increased from virtually zero to over one million tons in 2011. In 2011 the world fisheries production of freshwater fish amounted to 11.5 megatons captured versus 44.3 megatons cultured; of marine fish 78.9 megatons captured versus 19.3 megatons cultured; and of crustaceans 6.1 megatons captured versus 5.3 megatons cultured (FAO, 2012). This leaves a vast potential for future development in aquaculture. As with intensive animal breeding, aquaculture is strongly hampered by infectious diseases which cause massive economic losses and ecological damage. In shrimp production, a mere 10% of the juvenile shrimps leaving the hatchery reach the age of breeding maturity whereas 90% succumb mainly to infections. This leads to mostly uncontrolled use of huge amounts of antibiotics which represent a high risk to human and animal health and cause enormous environmental damage. In fish culture, antibiotics are primarily administered as medicated food whereby 70–80% of the active substances end up in the environment via food surplus and residual substances excreted from fish. Although there are no clear data on the worldwide use of antibiotics in aquaculture, estimations vary from 300 g to 1.5 kg per ton of fish or shellfish produced. This causes a significant pollution of surface waters leading to ecotoxic effects, impact on natural microbial communities and a large-scale emergence of antibiotic resistant and multi-resistant human and animal bacterial pathogens, in particular of the groups of Vibrio, Aeromonas and various Enterobacteriaceae. Antibiotic resistant pathogens are expected to be the main cause of unsuccessful disease therapy in human medicine in the near future, and this will probably throw back the current successful high public health status in most countries for decades. Hence a major goal that must be reached in agriculture and in aquaculture, in particular, is a drastic cutback or preferentially a ban of the use of antibiotics. The development of adequate vaccines and vaccine application procedures for aquaculture will represent a major challenge in the next two decades in order to achieve the goal of a sustainable fish production. The feasibility of controlling furunculosis, the major infectious disease in farmed salmon, caused by the bacterium Aeromonas salmonicida subsp. salmonicida, by means of vaccines, has been demonstrated in Norvegian salmon production. In 1988, with an annual production of 50 000 tons of salmon, 50 000 kg of antibiotics were used per annum. In 2008 with a production of 850 000 tons of salmon – a 17-fold increase – the use of antibiotics was reduced by a factor of 50 to around 1000 kg per year after the successive introduction of four generations of vaccines between 1989 and 1999 (Stevens, 2011). This example shows the efficacy of vaccines in attempts to reduce or ban altogether the use of antibiotics in aquaculture. While vaccines against furunculosis of salmon present a most successful example of preventive medicine of farmed fish, vaccines and vaccine application methods for most other fish or against most other diseases are still lacking or have low efficacy. The complex nature of the aquatic microbial world, the relatively poor knowledge of the fish immune system and of molecular mechanisms of pathogen–host interaction of many fish diseases require considerable efforts in basic research in order to yield new concepts for fish vaccines. Whereas basic research mostly relies on financing by the public sector, investments by the private sector for the development of new vaccines, novel vaccine formulae, more efficient and less nocent adjuvants as well as for novel rational vaccination procedures such as oral vaccination or immersion vaccination are essential to attain the aim of improved fish health and consequently a more sustainable aquaculture. In order to attract such important investments in the relatively small economic sector of animal health, regulatory procedures that govern registration and licensing of animal vaccines must be simplified and revised to make them affordable for a market with low profit margins. Currently, maximal production costs for vaccines for farm animals and large fish (where individual vaccination is applied) is around €0.10 per dose, whereas that for small fish is estimated to be 100 times lower. In comparison, costs for the registration of a new animal vaccine in Europe currently amount to around one to two million Euro, not including the costs for the preparation of the registration documentation. High costs of administration and regulatory measures often discourage the private sector from investing in the development of novel and more efficient animal vaccines, leaving old, often rather inefficient vaccines on the market or offering no solutions to combating emerging animal epidemics, even though, in many cases, public sector research has provided the necessary basic molecular knowledge. In order to attain the goals needed for sustainable fish production in the future, significant inputs into exciting projects on fish immunology and pathogen–host interaction of fish disease are expected from microbiologists, valuable investments are expected from the private sector and unbureaucratic and efficient procedures are expected from the regulatory authorities. Microbial ecologists are currently being inundated with increasing amounts of omics data, including millions of phylogenetic sequence reads (i.e. pyrotag or I-tag sequences), billions of base pairs of total nucleic acid sequences (i.e. metagenomes and metatranscriptomes), as well as thousands of peptides and metabolites from several types of environmental samples. Although this is a fantastic resource, the current challenge has been to glean relevant information from the data. Soil omics has proven to be particularly challenging due to the high microbial diversity inherent to most soils, combined with the difficulty in extraction of all macromolecules from the soil matrix. That being said, I predict that within the next decade the bioinformatics and computing resources will have caught up to handle mega- and multi-omics datasets from soil. For metagenomics and metatranscriptomics datasets, the three major current limitations are the assembly, annotation and data mining steps. No soil metagenome has been sequenced with sufficient depth to enable assembly of more than a handful of draft genomes. One example is the recent successful assembly of a draft genome of a novel methanogen from permafrost soil (Mackelprang et al., 2011), but the microbial diversity in permafrost is lower than that in most other types of soil. Another example was the recent assembly of several draft genomes of novel species, including from phyla with no cultivated representatives, from uranium-contaminated sediments (Wrighton et al., 2012). Although a typical soil still has a higher diversity than permafrost or contaminated sediment, with sufficient sequencing depth now approaching terabases of sequence per sample, even soil should be possible to better assemble. It should also be possible to use binning approaches such as emergent self-organizing map analysis (ESOM) (Dick et al., 2009) that take into account read signatures, together with read coverage to bin reads into draft genome bins, as demonstrated for other sample types (Dick et al., 2009; Wrighton et al., 2012). In addition, I predict that we will have better functional gene databases that have been validated for screening the assembled data and raw reads. Also, it will be advantageous to have more omics data collected across time series and across environmental gradients, such as targeted in the Earth Microbiome Project (http://www.earthmicrobome.org). Once we have access to well annotated soil metagenomes, the expression can be further validated by determining which genes are expressed and represented in metatranscriptomes, or translated into proteins and represented in metaproteomes. Having access to multi-omics data from the same samples should greatly increase our ability to gain a better understanding of the key roles that microbes play in different soil habitats and how those functional roles may be perturbed, for example by climate change. Already we are seeing examples of where different omics datasets have been combined and correlated for other types of environments, including marine (Mason et al., 2012) and the human gut (Erickson et al., 2012). Finally, once we have this information more easily accessible and at our fingertips it will be possible to get answers to questions that have been eluding soil microbial ecologists to date including the following: (i) What microbial species and biochemical pathways are most important for cycling of soil nutrients, including carbon and nitrogen under given conditions? (ii) What microbial species and functions are perturbed by climate change, pollutants or other anthropogenic factors? (iii) What are the optimum combinations of bacteria, fungi, archaea, protists and viruses for a healthy soil that supports the growth of plants? and (iv) How are soil microbial community members and functional processes predictive of the potential for greenhouse gas emissions, bioremediation of pollutants and optimal crop growth? Eventually, this information should be relatively easy to obtain as sequencing costs continue to decline, computing facilities continue to increase, and databases continue to be updated. Recent advances in metagenomics, metatranscriptomics and metabolomics techniques have enabled researchers to ask previously intractable questions about the biological world. Looking toward the future, it is clear that ‘multi-omics’ analyses that combine these three approaches, perhaps also including metaproteomics and single-cell or microculture genomics, will lead to important insights throughout microbial ecology. Rapid improvement in high-throughput sequencing technologies facilitates the analysis of larger and more complex microbial assemblages, and there is now great potential for advancements that impact our everyday lives. In this respect, production of fuels from renewable sources will be increasingly important to our security, economy and environment. Microbes will play a central role in the robust synthesis of fuel from plants and algae. In particular, algae are an attractive candidate for mass-scale fuel production because they can grow rapidly on non-arable land. Already there are several companies capitalizing on this promising approach (e.g. Solix Biofuels, Sapphire Energy, Algenol Biofuels), and a more complete understanding of algal genomes, transcriptomes, and metabolomes will lead to higher yields and lower cost. However, monocultures are inherently susceptible to invasion, and this principle also applies to monocultures of algae. Building better models of individual species, and of the interactions of more stable multi-species assemblages, through an understanding of their interactions across expression levels is thus crucial. Manipulation of biofuel strains on the genomic level, including mutants, knockouts and transgenics, is an important component of optimization of fuel production, but it is at present difficult to design a selection for improved biofuel producers, and screening techniques are still laborious. For individual strain improvement, transcriptional data, especially collected over time to assess response to perturbation and to multiple growth conditions, complemented by metabolomics analyses, would provide crucial data for improved systems biology models that seek to predict which alterations are most likely to increase overall efficiency of sunlight conversion, or enhance production of target molecules, e.g. advanced liquid biofuels such as butanol. For example, light to biomass conversion can be improved by making structural changes to reduce the antenna size in order to minimize overshading in liquid culture (Ort and Melis, 2011), but can also be improved by altering the regulation of light harvesting proteins (Beckmann et al., 2009). A better understanding of the regulatory and metabolic networks of target biofuel-producing algae could potentially provide numerous additional targets of the second type, especially when coupled with increasingly advanced metabolic reconstruction techniques that can incorporate multi-omic data (Yizhak et al., 2010). However, an understanding of individual species is unlikely to be sufficient: designing communities that are robust to invasion has the potential to assist with problems scaling biofuels systems from laboratory to production scale. Diseases of algae are common, with pathogens including viruses, bacteria, fungi and other eukaryotes (Gachon et al., 2010). Individual strains of algae have strategies for inhibiting bacterial growth, such as by secreting compounds that interfere with bacterial quorum sensing (Rajamani et al., 2008), and the possibilities for manipulation of this type of inter-kingdom signalling are just beginning to be explored. Some phytoplankton also inhibit the growth of competing species under certain conditions (Prince et al., 2008), or of themselves, (Vardi et al., 2007), and these effects could also be manipulated (e.g. by moving normally autoinhibitory pathways into a different species to create allelopathic interactions). However, our ability to understand these effects in order to design robust synthetic communities are still very much emerging: even in bacteria, predicting mutualistic and antagonistic interactions is still challenging (Freilich et al., 2011), and algal genomes are substantially more complex, especially with the partitioning of metabolism among multiple organelles. The combination of improved modelling techniques that incorporate multiple species and compartments, together with a better multi-omics understanding of individual strains and their responses to pathogens alone or in combination, will thus help us design robust communities for efficient conversion of sunlight into biofuels. Microorganisms are the dominating species on our planet. Their wide metabolic capacity makes them essential for the cycling of nutrients in the nature and vital for the function of plant growth and animal gut ecosystems. Moreover, microorganisms are used in a multitude of traditional and modern biotechnological applications. In a future sustainable society, with a need for a decreased consumption of fossil carbon sources, the use of microorganism needs to be intensified for making a variety of different organic building blocks, e.g. for pharmaceuticals, as well as for the production of different energy sources. The question then arises, how do we find these new microbial resources? Most of the knowledge we have today about microorganisms, their physiological capacities and possibilities to use for biotechnological applications has arisen from traditional microbiological methodologies, i.e. the isolation and cultivation of pure strains and species. For many years these methods represented the main tools for generating knowledge about microbial life. Today we have access to powerful tools providing detailed information about the genomic structure and gene expression of isolated microorganisms, giving valuable insights about microbial metabolism and pointing towards new biotechnological possibilities. These new technologies also allow for metagenomic analyses of various complex environments and with comparative sequence analyses we can answer questions related to evolution and metabolic functions, as well as reveal information about previously unknown microbial life. Furthermore, by screening different environments we find patterns and can see connections between environmental conditions and microbial composition and genetic expressions. This is all great and fantastic and we learn a lot! However, what it also obvious from the results is that a majority of the microbial life that is detected relates to unknown microorganisms. The biogas process is one example of a highly complex microbial environment. This is an amazing microbial process with a great functional capacity. It includes both generalist and specialists, together forming a microbial community that by intricate interactions converts large organic macromolecules into a variety of smaller organic components and finally into methane. The great functional diversity of this process suggests it as a possible source for new organisms to be used for biotechnological applications. I will use this process as a way to exemplify why I think molecular microbial ecologist to a greater extent have to consider also using a traditional route for their research in order to find new and important microbial resources. The biogas process has been used for more than a hundred years for small-scale production of energy for rural households. Today the process is also used in industrial countries for large-scale production of biogas from a variety of different urban and rural waste streams, as well as from dedicated energy crops. In my mind, the breakthrough for the industrial use of this process was a greater understanding of the microbiology behind the process, possible to obtain only after the invention of techniques for cultivation of strict anaerobes (Hungate, 1950). We now have a reasonable understanding of the overall microbial flow in this system and what is required for management to reach sufficient biogas production. Even so, the functionality can still be improved and many questions remain to be answered in order to reach higher stability and efficiency with economic and environmental outcome. Many investigations in this research field, in common with many other fields of microbial ecology, are devoted to different levels of community analysis. We are all fascinated by the new techniques and their possibilities. However, in my opinion there is a great risk that we will get lost in the giant maze of sequences. How can we understand and resolve issues about functional capacity and management and find new organisms for biotechnological applications when a major part of the targeted microbial life is represented by unknown microorganisms (Zakrzewski et al., 2012)! Furthermore, critical functions can also be performed by key players present at low abundances and thus not even targ" @default.
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W1567381003 title "Crystal ball - 2013" @default.
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