So far, to improve enzyme efficiency only the active site has been taken into account. However, parts that are far away from the active site can still affect the reaction. The further away from the active site, the less predictable it is what will happen by modifying that part and therefore the rational thinking alone won’t help much anymore. The FutureAgriculture team is now using high-throughput mutagenesis to expand the modifications and cover the entire structure of the enzyme.
Lately, some 10.000 variants of the same enzyme have been created and are currently analysed thanks to a novel high throughput method established in collaboration with external partners in Bordeaux that are not part of the consortium. The method consists in creating very small droplets, in the order of 50 picoliters, containing several thousand copies of a modified enzyme. The enzyme activity in the droplets is then tested very quickly with lasers: 200 droplets are processed per second – which means that in only 5 minutes 60.000 enzyme variants can be tested!
This droplet screening method developed some years ago, relies mainly on sensitive instruments with custom setups adapted to each system. To that end the partners at MPI-TM teamed up with Jean-Christoph Baret at the University of Bordeaux who is an expert in placing the enzymes in small droplets, sorting the good drops from the bad ones and assaying the enzyme activity.
As explains Tobias Erb “Among the variants that we are currently testing we are hoping to find a faster and more specific variant of the key-enzyme of the carbon positive pathway, a new CO2 fixing enzyme. Once we find an improved version, we are going to test it again in the in vitro metabolism and hopefully, it will improve the plant metabolism even more.“
Once identified the best enzymes, it’s time to study them in their context: the carbon positive CO2 fixation pathway. “We want to make sure the enzyme can work well as a team. It’s like in football, you might have great individual players but to score you need a good teamwork, too. Sometimes it makes sense to take out one of the best players and substitute with another option because it interacts better with the rest of the team” explains Tobias Erb (MPI-MP).
To test the interplay of the individual enzymes, the CO2 fixation pathway is first recreated in vitro: a small vessel containing all the ingredients necessary to the pathway i.e. the individual enzymes, the chemical energy (ATP) and its reducing equivalents (NADPH), other cofactors that help the enzyme to exert its function, and a buffer that mimics the cell environment.
The pathway performance is then quantified by measuring the variations in the concentration of the ingredients. This is done by using highly sensitive mass spectrometers that can accurately quantify the amounts of substrates, intermediates and other metabolites for all the reactions that are happening in the vessel.
The team of FutureAgriculture has already tested in vitro the effect of the newly designed pathway onto the CO2 fixing-machinery of the plant. The outcome was extremely encouraging as the pathway showed a beneficial effect on the plant metabolism. This is the very first proof of principle that the pathway is able to improve plant metabolism in the lab condition, and that the pathway if transplanted in vivo has the potential to positively affect plant growth.
“It’s not enough to find an enzyme that can perform a reaction; the enzyme needs to be sufficiently active too” explains Tobias Erb, group leader and director of the MPI-TM in Marburg.
That’s why the partners at the Weizmann Institute and MPI-TM have worked hard to improve the identified enzymes and make them more efficient.
The method used by Erb’s lab starts by looking at the crystal structure of the enzyme – a 3D dimensional model with the position of the atoms. On that model point mutations can be made, usually by modifying the amino acids in active site – where the reaction takes place – to have a positive effect on the enzyme’s efficiency. The changes are not random but according to the scientist’s chemical logic and rational thinking. Two outcomes are important to be achieved:
- A fast enzyme: The faster the enzymes the higher will be the impact on plant growth. If the speed of the enzyme reactions is high then the metabolism of the plant will be very active and can operate with high CO2 fixation efficiency. Thanks to the computer modelling of the photosynthesis developed by the partner at MPI-MP (LINK), the threshold of activity that needs to be reached to ensure high efficiency is well-known.
- An accurate enzyme: The enzyme needs also to be as accurate as possible, which means that it should not have side-reactions. Such side-reaction might be toxic or slow down the metabolism and ultimately affect the plant growth negatively.
Once a set of mutation is decided, the mutations are implemented on the enzyme and their performance is tested in the lab. In Tobias Erb’s words “we try to make sure that the modelling is actually happening, that it becomes real” and therefore there is a lot of trial and error and back and forth, from the model to the experiments. Following this process, the FutureAgriculture team has found all the efficient enzymes necessary to complete a new pathway – the so-called “carbon positive pathway” – that should stimulate CO2 fixation in the plant and increase growth yield. Now it’s time to see how the individual enzymes work together.
Our partner, Tobias Erb from the Max Planck Institute for Terrestrial Microbiology have been awarded the prestigious Otto Bayer Award for his outstanding contributions in the field of “Synthetic biology, especially the application on artificial photosynthesis”. Part of this research is contributing to FutureAgriculture – the group of Tobias Erb is responsible for the in vitro tests of the enzymes. You can read more in the official press release.
After some months of work, we are finally ready to share the official video of FutureAgriculture! The video has been enriched by some hand-drawings made by the fantastic team of MedioMix. The video has also been listed for the ESOF2018 Video Competition “Showcase your project” – don’t forget to put a thumb up and like our video!
We are currently collaborating with MedioMix to create the official video for FutureAgriculture! The first shooting day was at the MPI-MP in Potsdam – it has been a long day among laboratories, greenhouses and finding the right location for interviews. The official video for FutureAgriculture will be released soon, in the meantime, you can check our other videos on the dedicated YouTube Channel.
Unleashing the technical present / Nov 30 – Dec 02. Researchers & artists met for this experimental conference in order to explore the meaning and implications of the technosphere. Arren Bar-Even was invited to the SEEDS session – watch the full performance here.
Our project coordinator got the chance to interact with artists and scientists on the topic “ESTHETICS get SYNTHETIC: Knowledge Link Through Art & Science” during the workshop organized by KLAS at the Max Planck Campus in Potsdam-Golm on the 27th – 28th of November 2017.
The workshop was an opportunity to bring together scholars and practitioners to jointly discuss and reflect on contents, approaches and methodologies that draw the link between synthetic biology and artistic research and how those can synergically interact by mutually interrogating and reconsidering their methodologies and modes of operation in an Artist in Residence program like KLAS, in which Arren Bar-Even’s team participate as hosting laboratory.
We just released a new video – this time instead of the typical interview we decided to capture in few minutes the multiple voices of the FutureAgriculture team. Each of us condensed the concept of FutureAgriculture in few words and the result is an extraordinary puzzle of points of view. A special thank to the greenhouse facility at the MPI in Marburg that hosted us for the shooting. We hope that you enjoy the video!
Our ability to test promising pathways in vitro and in vivo is quite limited. The testing of every candidate pathway would become an endless quest – worth of decades and billions of investments. To select only a few promising pathways we need the support of computational models that predict how each pathway will affect the carbon fixation rate in plants. We have generated a model of plant photosynthesis that takes into account both the central carbon fixation pathway (the Calvin cycle) as well as the photorespiration pathway – either the natural pathway or a synthetic alternative route.
This model enables us to estimate which synthetic pathway will result in the highest enhancement of carbon fixation rate, under different conditions such high/low illumination or high/low CO2-availability due to the opening and closing of the stomata. Only the most promising pathways will undergo a more extensive testing in vitro and in vivo.
The model also feeds both the in vivo and in vitro testing by setting important parameters thresholds and by giving suggestions on how to reach them. For example, the model can estimate the needed quantity of each pathway component to reach the best performance possible. Therefore it directs the enzyme engineering phase by specifying the minimal activity that an engineered enzyme needs to reach, or it warns us against toxic or reactive compounds that might accumulate during the activity of the pathway within the cells. We then can fine-tune the expression of the pathway components to avoid such deleterious accumulations.
The model is also continuously improved by integrating the data from the in vivo and in vitro testing i.e. the enzymatic activity, the growth rate, etc. Models are not completely finished yet but they are already well productive in giving us valuable information regarding the expected pathway. They are expected to be fully operative during the 3rd year.
After the identification of the pathways, we need to make sure that all the pathways’ components are available. Our pathways involve both existing and novel reaction – reactions that are not known to be catalysed by any enzyme in nature. We engineer existing enzymes to catalyze such novel reactions in order to sustain the activity of the pathway.
First, we need to find existing enzymes in nature that catalyze similar reactions or that can catalyze the novel reactions promiscuously. The concept of promiscuous enzymes is fundamental for this phase: enzymes generally have evolved to catalyze one primary reaction that represents their main task, but promiscuous enzymes can also catalyze, besides the primary reaction, side-reactions at a lower rate. During the 1st year of the project, we were able to identify the promiscuous enzymes that catalyze all the novel reactions of four of our candidate pathways, although the catalysis rate is quite low as expected. However, they represent the starting seed for the second stage where we are going to enhance the low-rate reactions of interest by three methods:
- Rational design – by applying biochemical knowledge while looking at the enzyme active site and its structure, we can predict amino-acid residues substitutions in order for the enzyme to accept our substrate and support the novel reactions.
- Library of changes in protein sequences – By using a large collection of alteration in the protein sequence, we can systematically screen the changes that result in better activity. To easily identify the best change within the library we have designed in vitro assays that couples the target activity to a measurable property i.e. fluoresce.
- In vivo selection – by creating E. coli strains whose growth depends on the novel reactions, we can directly select for enzymes that catalyze it efficiently. Thanks to this method, we can directly select for higher enzymatic activity by simply selecting the cells that grow faster.
The backbone of our project is the identifications of novel pathways that increase agricultural productivity by enhancing carbon fixation rate and efficiency in plants. The pathway design was completed in the first year, during which we identified more than 100 candidate pathways that can potentially bypass the natural photorespiration without releasing CO2.
We have considered all known enzymes and all known enzymatic mechanism to systematically search for all the possible routes that recycle 2-phosphoglycolate, the product of Rubisco oxygenation, back to the Calvin cycles (that supports carbon fixation). Our candidate pathways contained both reactions catalyzed by existing enzymes as well as plausible reactions, i.e. reactions that potentially can be catalyzed by well-characterized enzymes or that follow a well-known mechanism. We compared the candidate pathways according to various physicochemical properties including thermodynamics, kinetics, resources consumptions, and overlap with endogenous metabolism. Our analysis takes into account also how easy it will be to evolve the novel reactions from existing enzyme and mechanism (i.e. the number of novel enzymes required in the candidate pathway and hints in the scientific literature on existing enzymes that could support such new activity). This approach enabled us to select the most promising pathways in terms of such properties and test them in vitro, reconstructing the pathway from its enzymatic components. We are now implementing the pathways in E. coli, using it as a platform for the pathway selection, before finally moving to cyanobacteria and plants.