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Transforming the future of agriculture through synthetic photorespiration

Periodic Reporting for period 3 - FutureAgriculture (Transforming the future of agriculture through synthetic photorespiration)

Reporting period: 2018-07-01 to 2019-06-30

Carbon fixation is the most important biological processes on earth, supporting our biosphere by transforming inorganic carbon into organic matter and literally feeding all life forms. The Calvin–Benson–Bassham Cycle (CBBC or Calvin cycle) – operating in higher plants, algae, and many bacteria – is responsible for ≥ 95% of the carbon fixed in the biosphere. Despite being under a strong selective pressure for eons, the Calvin cycle still displays inefficiencies related to the enzymes it employs; especially one of its key enzymes, the carboxylating enzyme RuBisCO, is inefficient. RuBisCO is very slow and cannot fully distinguish between CO2 and molecular oxygen. When oxygen replaces CO2 as substrate for RuBisCO’s activity, a toxic waste product, 2-phosphoglycolate (2PG), is produced. 2PG must be recycled back into the Calvin cycle via a process termed photorespiration. However, plant photorespiration dissipates energy and releases CO2, thereby directly counteracting the function of RuBisCO, reducing the effective rate of carbon fixation, and lowering agricultural productivity.
FutureAgriculture aims to boost agricultural productivity by designing and engineering plants that directly overcome the deficits of natural photorespiration and that support higher photosynthetic rate and yield. Alternative metabolic pathways that can bypass photorespiration without releasing CO2 are screened in silico by taking into account all known enzymes, as well as enzymes that could be easily evolved from them. The synthetic enzymes are integrated with existing ones to obtain entirely new pathways optimized by chemical logic, which will, in turn, be realized in vitro and then in vivo within bacteria and plants. Their implementation in plants is expected to significantly increase plant growth rate and biomass yield under various environmental conditions. This will provide the basis for increasing agricultural productivity of the crops that comprise >60% of agricultural production, including rice, wheat, barley, oat, soybean, cotton, and potato.
During the last two years, the FutureAgriculture Consortium has almost completed the in silico and in vitro phases of the project and is moving towards the third phase in which the synthetic pathways are implemented in photosynthetic living organisms (in vivo).
In the in-silico phase, the Consortium has uncovered dozens of possible metabolic pathways to bypass native photorespiration without releasing CO2. Five such pathways were selected as highly promising, as they succeed in recycling the toxic waste product 2PG back into the Calvin cycle with minimal consumption of cellular resources, minimal overlap with natural metabolism, and using enzymes that are either naturally available or are easy to engineer. The analytical search was possible thanks to a novel software developed by the team at MPI-TM to identify and characterize synthetic pathways.
The teams at MPI-TM and WIZ have identified, engineered and tested all the enzyme candidates that sustain the activity of three of the synthetic pathways, and are now working to fully reconstruct the pathways in vitro and prove the improvement in CO2-fixation rate. The Consortium is further optimizing the enzymes necessary for the pathways, with the aim to choose the most efficient, fast and precise ensemble.
Regarding the in vivo phase, the Consortium is carefully moving towards it one step at the time. The pathways are being implemented in engineered E. coli strains that serve as a platform for pathway testing and evolution towards higher in vivo activity. This innovative platform was selected among the finalist for the Innovation Radar Prize 2017 under the category Excellence Science. The Consortium is still refining details to ensure that the selected enzymes can perform their function in model organisms. The team at ICL is working on highly photorespiration-dependent cyanobacterial strains and has built a custom CO2 monitor to characterize the advantages of the synthetic pathways over their natural counterparts. Meanwhile, partners at EVO are carefully selecting and testing the specification that will allow the correct production of the enzymes in Arabidopsis and Brachypodium model plants.
In today’s world, one in seven people is malnourished. This situation is expected to worsen as human population keeps increasing at a staggering rate. Feeding 10–15 billion people in the year 2100 is a tremendously challenging task that will only be met by the implementation of drastic measures to increase agricultural productivity. Hence, the seed industry is seeking sustainable and economically viable solutions to increase crop yield despite numerous challenges, such as finite arable land and water resources, deleterious environmental conditions (e.g. drought, salinity), reduced availability of fertilizers, climate volatility, and depletion of soil nutrients. A fundamental way to improve plant productivity and performance is through the use of plant genomics. Along these lines, FutureAgriculture offers not an incremental improvement but rather a leap in agricultural productivity. Most other research efforts that aim to improve photosynthetic yield involve enormous implementation barriers. In contrast, FutureAgriculture’s engineering aims can be achieved within a reasonable timeframe as they are strictly genetic/metabolic and do not involve morphological or any other structural modifications. Hence, the development and implementation of synthetic photorespiration routes can transform the future of agriculture.
Furthermore, while the improvement of photosynthetic rate and yield via transgenic approaches has been a hot research topic for many years, these efforts focused on introducing existing pathways into new plant hosts. FutureAgriculture adopts a radically different approach. Rather than reshuffling and grafting existing enzymes in a fashion that resembles natural evolution and is in line with current metabolic-engineering thinking, the project systematically explores novel pathways that cannot be obtained by mixing-and-matching of existing, natural enzymes. FutureAgriculture’s approach demands the de novo engineering of new enzymes to catalyze metabolic transformations that are unknown in nature. These synthetic enzymes are integrated with existing ones to obtain entirely new pathways optimized by chemical logic, which will in turn be realized within bacteria and plants. Given the combinatorial nature of metabolic pathways, the addition of only one novel reaction dramatically expands the solution space of possible pathways, thus fully realizing the potential of synthetic biology. Yet, so far, only a handful of studies implemented synthetic pathways that harbor novel enzymes. FutureAgriculture takes this strategy to a new level by constructing de novo pathways within the very core of carbon metabolism.
Schematic illustration of the beneficial effects in the engineered plants