Introduction

For decades, scientific breakthroughs and technological progress in biotechnology have empowered the notion of the bioeconomy, in which biomass is used to produce society’s energy, chemicals and materials. More than that, the bioeconomy has been postulated as a crucial component in the much-needed transition to a more sustainable world - one that is, above all, less dependent on fossil fuels. But despite the promises and progress of biotech, the bioeconomy has far from realised its full potential. In part, the biorefineries that were supposed to provide the foundation of a sustainable bioeconomy simply don’t exist yet. In our iGEM project, we aim to tackle this issue by developing an enabling technology for truly self-sustaining biorefineries, which can be used for the production of virtually any product for which microbes can be engineered. This technology, based on a synthetic consortium of cyanobacteria and chemotrophs, can be used in wide variety of applications to empower (find synonym) all facets of the bioeconomy. In the following application scenarios, we contextualize several such applications and discuss in detail the design aspects required for their real-world implementations.

The problem

Climate Change

Today, 82% of the world’s energy supply is derived from fossil resources. According to many, this reliance on fossil fuels drives global warming and climate change. Yet the notion of climate change itself is shrouded in controversy. Surveys show that a majority of Americans think climate change is a topic of significant scientific disagreement. Yet despite popular consensus, overwhelming evidence suggests that human-caused climate change is real and poses tremendous costs and future risks to society and natural systems. In a recent report on the issue, the American Association for the Advancement of Science (AAAS) documented what we actually know about climate change. Their three most important conclusions were as follows:

Climate scientists agree that climate change is happening. Based on well-established evidence, about 97% of climate scientists conclude that climate change is real, happening now, and caused by humans. This notion is not supported by a single study, but, as AAAS reports, by a “a converging stream of evidence over the past two decades from surveys of scientists, content analyses of peer-reviewed studies, and public statements issued by virtually every membership organization of experts in this field.”
We’re making fast progress to “pushing our climate system toward abrupt, unpredictable, and potentially irreversible changes with highly damaging impacts.” The earth’s average temperature is increasing to a level beyond anything that has been experienced over the past millions of years. Although this means that there is massive uncertainty regarding the exact consequences this increase will have, the fact that there will be consequences is certain. It is a real possibility that as temperatures rise, critical elements of the earth’s ecosystem will experience sudden and irreversible change with massively disruptive consequences to societies and natural systems.
The sooner we act to prevent further climate change, the lower the risk and costs. Fortunately, there are still many things we can do to change the path we’re on. Yet sitting idle will only increase risk and decrease the range of options. Carbon dioxide accumulate in our atmosphere for decades or centuries, which means the effects of CO₂ emission cannot be fixed unless there is some large-scale, cost-effective way to remove CO₂ from the atmosphere.

This last note prevents a shimmer of hope: if preventive action is taken now, the worst consequences of climate change might be averted. Indeed, with the recent verdict by the Dutch court of justice, obligating the state government to reduce its CO₂ output with 25% by 2020, fighting climate change has become not only a moral and humanitarian necessity, but even a legal imperative. What is needed now are the tools to actually do so. Enter the bioeconomy.

Bioeconomy 1.0

The use of biomass to produce products isn’t new: as far back as the first world war, Germany, searching for alternative ways to sustain its war efforts, attempted to convert sugars to fuels. Ever since, technology has modernised, but not changed, the underlying premise: to convert microbial or plant-based biomass to useful products. Corn has been a prime example of this. It can be used, through a process of fermentation, to produce bio-ethanol, which can be added to gasoline to create more carbon-neutral fuels for vehicles. There is one major downside: production that depends directly on plant-based biomass competes with arable land. Mexico experienced the consequences of such competition firsthand when, in 2008, subsidy-fueled US ethanol production from mexican corn pushed up prices, which in turn led to an increase in prices for staple foods like tortillas of 25%, increasing hunger in Mexico and causing tens of thousands of people to march the streets of Mexico City. Obviously, these solutions, although initially hailed for their potential for carbon neutral bioproduction, are ultimately unsustainable.

Bioeconomy 2.0

There are other ways to use biomass for sustainable production though. Ways in which biomass doesn’t function as the primary source of energy, but as a vehicle to capture and convert the most freely abundant source of energy available: sunlight. Algae and cyanobacteria are organisms that do exactly that. They use photosynthesis fix CO₂ as their primary carbon source in order to grow and, if genetically engineered to do so, produce useful products. With oxygen as the only side-product, this sounds like the perfect solution to the problem of CO₂-dependent climate change and sustainable bioproduction - a promise that was quickly acted upon by the second wave of bioeconomy startups like Photanol and Algenol. Yet there are still limits, not with regards to the availability of land, but regarding the technology itself. Simply put, production yields for many products, including biofuels, one the most important potential products derived from photosynthetic organisms, are too low to be cost-effective -- by far the most significant obstacle when it comes to widespread production. And although there are several commodity chemicals and products, like fragrances, that can already benefit from the sustainable production capacity of cyanobacteria, there is another, mostly unacknowledged problem that is preventing widespread implementation of photosynthesis-based biorefineries: genetic instability.

Genetic Instability

Microbes have been forged by four billion years of evolutionary pressure to grow and thrive in an extremely wide range of habitats. As any organism, mutations that reduce their fitness are quickly filtered out of a population. In this case, fitness refers to the capacity to grow and divide, which is a direct function of the amount of biomass a microbe can produce per unit of time. Needless to say, channeling valuable resources away from the production of biomass reduces growth, which is exactly what happens when we engineer microbes to produce valuable - in the eyes of the beholder, which is us - products. Often, the engineering of such production processes in cyanobacteria involves the insertion of novel genes and pathways. One can easily imagine how a mutation that would render foreign insertions inactive would increase the growth capacity of the sub-population with such mutations, allowing them to quickly overtake the entire population. With mutations thus occurring [to do: insert frequency], losing an engineered function can be matter of time. Indeed, consider an engineered strain that is cultivated at a large scale biorefinery to produce iso-butanol and finding out mid-way through a commercial run that productivity suddenly declines. This might be the result of one or few nucleotide mutations or the loss of a varyingly sized gene fragment, both leading to either a loss of expression or functionality of the RNA or protein it codes for. Although this phenomenon is understood in relatively high detail in, say, E. coli, where known genes involved in the repair and/or mutation of DNA can be exploited to minimize this risk, this is not the case in cyanobacteria.

In fact, research into genetic instability in cyanobacteria is, despite the massive increase in attempts to use phototrophs for biotech purposes, surprisingly sparse -- so much so that Patrik Jones from Imperial College refers to it as an elephant in the room: “important, obvious, yet largely ignored”. Of course, such fate is often reserved for negative results; few people want to spend their time and energy following up on dead ends, which are unlikely to lead to a publications. Although this lack of information makes it hard to draw conclusions on the extent to which genetic instability is a problem in developing photosynthesis-based biorefineries, the issue is certainly pervasive enough to warrant full acknowledgement and attention when it comes to designing cyanobacterial strains for real-world applications.

The project

With our project, we aim to tackle the productivity and instability issues by harnessing the power of microbial consortia. Of course, symbiosis between phototrophs and other organisms are well documented in the natural world, e.g. bacterial mats, and in evolutionary history, e.g. the origins of the chloroplast. In fact, microbes rarely live in isolation outside the lab - they are constantly interacting with other species. We believe these collaborative, multi-species systems can be leveraged to create products in a way that comes closer to true sustainability (although the scrutinization of the belief is of course the aim of these scenario’s). The idea is simple: phototrophs, in our case the model cyanobacterium Synechocystis PCC 6803, normally use CO₂, sunlight and water to create the biomass they need for growth. Through genetic engineering, some of this flux can be redirected to create desirable products. Although complex product formation is still limited by from genetic instability or lack of productivity at present, we can engineer Synechocystis to produce and export relatively simple carbon compounds instead. These compounds, e.g. sugars, can then be used to feed other species, like E. coli, that can use these compounds for their own biomass production, and, if properly engineered, the production of valuable products. Since water, light, and CO₂ are virtually infinite in supply, and absolutely non-harmful to the environment (quite the opposite), the production of carbon-compounds from Synechocystis, and the subsequent product production by E. coli, is essentially a self-sustaining, carbon-neutral process.

Besides the carbon-neutral production potential of cyanobacteria, this approach thus benefits from the fact that genetic engineering practices for production formation is much wider established in chemotrophs like E. coli. Indeed, results obtained in E. coli cannot be translated directly into cyanobacteria. For example, an E. coli strain designed to produce 1-butanol via an engineered pathway produced more than 30 g/L, but the cyanobacterial strain with the same pathway produced only trace amounts (in a later study, the optimized pathway for S. elongatus, another cyanobacterium, reached a maximum titer of 2.8 g/L). By combining the sustainable photosynthetic capacity of cyanobacteria with the fermentative production potential of chemotrophs, we essentially combine the best of both worlds, and, importantly, are not inhibited by the process of re-inventing the many wheels that have been fine-tuned in E. coli in cyanobacteria. Several important questions remain: how to tackle the issue of genetic instability? How should the consortium practically be implemented in a biorefinery? And how to deal with potential safety concerns? These questions will be addressed in the following sections

Genetically stable production in cyanobacteria

As mentioned, genetic stability is a crucial design constraint that must be taken into account if photosynthetic bioproduction is to be used in real-world applications. This constraint has therefore guided our efforts in choosing what carbon compound we want Synechocystis to share with its neighboring species in the biorefinery. The issue is best explained through an example: say there are two options for engineering Synechocystis such that it produces carbon compounds on which a chemotroph like E. coli can thrive: 1) via several inserts of foreign genes that allow, through multiple steps, for the accumulation of a sugar, including a gene encoding for a transporter needed to transport the sugar to the extracellular environment, or 2) via a knock-out that leads to the accumulation of sugars that can naturally diffuse through the cell wall. Both are viable and explored options in Synechocystis, but only the second of these is likely not to lead to scale-up problems in the real world. After all, mutations in the foreign genes, bound to occur, would increase the fitness of those strains, quickly resulting in a population in which the insertions are outcompeted in the relentless struggle for optimal growth. A knockout, however, cannot be undone by a simple mutation, and is therefore likely to be significantly more stable in terms of productive capacity as a function of time.

This might sound straightforward, but is an integral part of the aforementioned elephant in the room. And there is a way to take this one step further: by finding target metabolites (i.e. carbon compounds that can be shared and used by E. coli) whose production is directly coupled to biomass formation, we can essentially avoid the fitness pressure that leads to the collapse of other constructs. Another example is in order: the formation of biomass includes several steps in which metabolites are recycled, i.e. they are formed as a side-product of a biomass-formation reaction, and are later converted back to their original form to be used again. If the recycling step of such a process is knocked out, the side-product, which should of course be non-toxic, accumulates. And since the accumulation of side-product is directly coupled to the formation of biomass, there is much less fitness pressure to remove the genetic alterations in order to optimize biomass production, as the genetic alterations are part of biomass formation itself. In fact, any genetic tweaks adopted by Synechocystis in order increase growth rate will, due to this direct coupling, lead to a direct increase in the yield of the desired product as well. These design considerations, and their potential benefits in large-scale biorefinery applications, led us to focus on exactly the type of growth-coupled genetic alterations outlined above, resulting in a stable Synechocystis strain that accumulates and naturally exports acetate, which can in turn be used by E. coli for biomass and product formation.

Safety Considerations

Public concern of outbreak by GMO’s is a real issue. The large-scale industrial application of our consortium of GMO’s would, if anything, only increase such concerns. For that reason, we have decided to implement a safety switch in our genetic designs, allowing the Synechocystis and E. coli to thrive when together, but not separately. In our consortium, E. coli is already dependent on Synechocystis, from which it receives the carbon it requires to grow. In addition to that, we also engineered Synechocystis to be fully dependent on E. coli via an amino acid auxotrophy. This means that we use a Synechocystis knock-out strain that is unable to synthesise one of its essential amino acids, which it requires for growth. Only when supplied with the amino acid externally, which it in this case receives from our E. coli strain, engineered to produce and share the same amino acid, can it grow. This way, we ensure that our consortium functions only in its appropriate context, hopefully alleviating some of the concerns regarding GMO’s.

Consortium organization

Our project focusses on the creation of a stable, self-sustaining enabling technology for biorefineries in the form of a phototrophic-driven consortium. Although we will not be able to actually construct a biorefinery of the type that could be implemented in industrial applications, our consortium would ultimately inhabit the compartments of a biorefinery where conditions are optimized for stable product formation. There are several ways in which to operate such a synthetic consortium in a refiner. Of course, the specifics of the biorefinery and its organisation depend largely on the actual application in which it is used (see application scenarios below). Yet it is worthwhile to consider the two most basic options - a single compartment for both species, our initial idea, versus separate compartments for both species - in order to highlight the way practical imperatives can constrain biorefinery design options. These considerations, which shaped our outlook on the final implementation of our biorefinery in significant ways, are based largely on discussions we had with Professor Atsumi from UC Davis, head of one of the most prominent research groups in the metabolic engineering of cyanobacteria for bioproduction.

Our initial idea was that, in the biorefineries’ ultimate embodiment, Synechocystis and E. coli would simply live together in the same compartment. Although straightforward, there are downsides to this approach: most significantly, each species has its own specific preferences with regards to its optimal environment for growth happiness. Combining both species in the same medium likely results in a suboptimal environment for both (e.g. light blocking by E. coli in the vicinity of Synechocystis). Since the E. coli growth rate and its final product titer will be a direct function of the carbon compound secreted by Synechocystis, such suboptimal conditions would be an impediment to productivity. As an alternative, Atsumi suggested a two-compartment system, one in which Synechocystis and E. coli grow in separate, optimized media. These are then connected via a semipermeable membrane filter and a pump that transfers the acetate-containing media of Synechocystis to the E. coli-containing compartment. This approach would allow for carefully controlled optimization of growth parameters for each species, without light-blocking inhibition otherwise induced by E. coli’s presence near Synechocystis. These considerations reflect the important choices that must be made when it comes to constructing the actual biorefinery. Nevertheless, they are based on the concept of a simple land-based based biorefinery that consists out of relatively simple vessels. Depending on the type and location of application though, the actual biorefinery variations might be even more diverse, as the below scenarios illustrate.

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