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.“