Crystal ball – 2013 (2012)

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. 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