Bioengineering Nitrogen-Fixing Bacteria and Staple Crops: Genetically Modified Sustainability

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Author: Christine Hirschberger Edited by: Inês Barreiros

Protecting the planet, promoting personal and public health, supporting rural communities and agricultural workers or helping to improve animal welfare - there are many good reasons to buy and eat sustainable food. Sustainable agriculture attempts to develop a method of growing food in an ecologically and ethically responsible manner, and the often-quoted expectation of the world population rising to 9.6 billion people by 2050 makes this even more urgent: sustainable agriculture is often argued to be the key to feeding the growing world population.

Traditionally, organic farmers have often been opposed to genetically modified (GM) crop varieties, mostly because of concerns regarding the safety of both rising and consuming GM plants (although the WHO, the American Medical Association, the National Academy of Sciences, and the American Association for the Advancement of Science have all noted that they are safe). However, GM technology has much to offer to further sustainability in agriculture. One such avenue in which tweaking plant genetics can provide new economically as well as environmentally friendly crops is targeting the levels of nitrogen intake that modern plants need to grow. Nitrogen is the most important nutrient for plants, essential for proteins, nucleic acids, and many other biomolecules. Together with water it is also the greatest global constraint to agricultural productivity. For example, certain areas in sub-Saharan Africa where nitrogen is particularly limited produce yields that reach only 20-40% of those in the US. However, on the flip side, excessive nitrogen fertiliser use is toxic and the European Nitrogen Assessment program, launched in 2011, estimates that the annual cost of the damage inflicted by nitrogen fertilisers across Europe lies between £60 and £280 billion, which is more than twice the gains from using them in European agriculture in the first place. Since sustainable agriculture is seeking for approaches that are the least toxic and require the least energy, and yet keep or even increase productivity and profitability, scientists have long been investigating how to optimise nitrogen intake in crops.

The form in which all staple crops require nitrogen is ammonia, a compound of nitrogen and hydrogen. Nitrogen fixation refers to the process by which nitrogen, taken from the air, is converted to ammonia. However, only a select number of plants are able to do so. These include legumes such as peas, beans, alfalfa and clovers, which explains their protein content – for example, soybeans are capable of producing twice as much protein per acre as any other crop, five to ten times more protein per acre than any acre of land that is used for grazing animals for milk production, and fifteen times more protein per acre than land for meat production. All other crops – the vast majority of all plants – have to take ammonia from the soil, where nitrogen is much more limited than in the atmosphere. This is also why farming crops that cannot fix nitrogen rely so much on synthetic nitrogen fertiliser use, which in turn has contributed to modern day levels of nitrate pollution in the atmosphere by ammonia and oxides of nitrogen.

In organic agriculture, techniques such as intercropping, crop rotation and mulching are used to replenish soils with nutrients. In particular, intercropping and crop rotation help increase the nitrogen content of the soil by introducing nitrogen fixing plants or plants that will be turned under for adding further nutrients. However, these methods are time intensive, may yield less and cost more. Farming legumes does not require the same levels of intercropping or crop rotation. In fact, legumes are often the very plants that are used to replenish the soil with nitrogen, due to their nitrogen fixing activity.

The evolutionary trick behind this legume characteristic is hidden away inside small nodules protruding from their roots: nitrogen fixing bacteria, or rhizobia, which have established a symbiotic relationship with the plants that they infect. The nitrogen fixing microbes live in the soil, but when they encounter legumes they use complex biomolecules to engage in a molecular dialogue with the plants. This process leads to a close association between the root and bacteria, and eventually the colonisation of the root hairs by the microorganisms. Once inside the legume roots, the rhizobia multiply and cause the growth of nodules, aggregations of legume cells and nitrogen-fixing bacteria. Inside these enlargements the bacteria convert nitrogen to ammonia, which the host plant then utilises. In exchange, the plants provide the bacteria with amino acids and protection against the elements or other potentially harmful microbes in the soil. Overall, this relationship between legumes and bacteria is a highly successful one, introducing 40 million tons of nitrogen into the global agricultural system every year. This dynamic has also fascinated scientists for decades: the idea of introducing this pathway into non-legume crops to boost their nitrogen uptake was first proposed in a 1981 report by the US Office of Technology Assessment. Since then, the interest in exploring whether this system can be established in non-legumes has only grown; and our scientific grasp on nitrogen fixation has reached a level at which we can think about engineering this symbiosis to boost sustainable agriculture, using synthetic biology approaches.

It is hoped that this process will allow crops to fix atmospheric nitrogen on their own, independently from nitrogen in the soil or from fertilisers. The development of new molecular techniques for manipulating plants and bacteria, and the advancement of our understanding of the process underlying biological nitrogen fixation have led to various biotechnological methods aimed at developing novel nitrogen fixing crops. These involve approaches to allow non-legume plants to nodulate and give rise to symbiotic nitrogen fixation. There are several ways of going about this and one of them has recently been developed by researchers based in the University of Nottingham: In this case, the breakthrough was the discovery of a strain of nitrogen-fixers in sugar cane, which are capable of intracellularly colonising all major crop plants. From this, it was possible to start the development of a bacterial strain that can confer nitrogen-fixing abilities to every plant cell to which they are applied. In turn, this pioneering nitrogen technology has swiftly been commercialised by a new biotech company and provides an economically friendly, sustainable solution to fertiliser overuse.

Another more invasive strategy is directly introducing relevant rhizobia genes into non-legume crops. The genes encoding the enzyme machinery for nitrogen fixation can be transferred into plants, although the sensitivity and complexity of this molecular process raise challenges for the efficiency of this approach. For example, the most important enzyme of biological nitrogen fixation, nitrogenase, does not tolerate any oxygen, and establishing a suitable environment inside plant cells for the activity of this biomolecule can be tricky. To avoid this problem researchers are now working on transferring the entire nitrogen fixer instead of just its genes.  Engineering non-legume crops to be colonised by nitrogen fixing bacteria would circumvent the issue of the suitable oxygen deprived environment. This is in the hope that the nitrogen fixation will be unaffected by the switch to a different host that is more useful for human food production. Nodulation can be achieved by hijacking a process that is usually reserved for interactions between non-legumes and fungi. This pathway can then be manipulated to be switched on by rhizobial bacteria and to lead to root protrusions akin to nodules, which provide the right environment for nitrogenase. The main issue for this approach lies in the fact that bacteria and plants communicate extensively through chemicals before the symbiosis is established, and the sheer number of the genes that direct this and that are required for nitrogen fixation, as well as their regulation and sensitivity to environmental conditions. While engineering a synthetic symbiosis between two organisms does not present a huge challenge nowadays, this process is hindered in nitrogen fixers and staple crops because all relevant genes are from nature and as such encoded in a complicated, often overlapping manner. This makes it more difficult to change single parts. Nonetheless, techniques such as refactoring are currently being used to slowly substitute large-scale gene clusters from legumes and nitrogen fixers into new hosts. While this presents highly complex bioengineering problems, there is a substantive body of work showing the feasibility of this approach.

One of the objectives of sustainable agriculture consists of fulfilling society’s present needs without endangering future generations’ ability to meet theirs. Nitrogen fixing staple crops are very promising in pointing in that direction, while also offering contributions to a safe and healthy environment with less synthetic nitrogen fertilisers and more economic profitability. Much of the scientific groundwork in this area has already been done and new GM methods are being developed at astonishing speeds. Reducing greenhouse gas emissions and nitrate pollution, growing crops in an ecologically and ethically friendly manner, and feeding the growing world population are highly complex challenges. But complexity can also be framed as an opportunity: it allows the opening of many different avenues for critical changes to our agriculture. If the results of the last few decades of research on biological nitrogen fixation and GM technology can be trusted, one such innovative approach may be found in bioengineering nitrogen fixers.