Generation of phytase variants for improved phosphorus mobilization
Congratulations to Kevin Herrmann, Christin Brethauer, Niklas Siedhoff, Mehdi Davari and Anna Joelle Ruff on their recent publication!
Phytases are hydrolytic enzymes capable of releasing phosphate stepwise from phytate, the major phosphorus sink in seeds, cereals and legumes. Limitations such as low enzyme activity or incomplete phytate hydrolysis to inositol constitute a major challenge in the application of phytase in food and feed. In this article, we report the first successful KnowVolution campaign of E. coli phytase for improved hydrolysis of lower phosphorylated reaction intermediates (InsP4 and InsP3). Screening of in total ten thousand clones resulted in a dataset of 46 variants for all three isomers (Ins(2,4,5)P3, Ins(2,3,4,5)P4, Ins(1,2,5,6)P4). This generated dataset was used to train machine learning models with PyPEF to predict recombinants. Finally, screening of random mutagenesis and saturation mutagenesis libraries identified six variants with more than 2.5-fold improved InsP4 hydrolysis, wherein the variant T23L/K24S exhibited 3.7-fold improved relative activity for Ins(1,2,5,6)P4 and simultaneously showed 3.2-fold improved hydrolysis of Ins(2,3,4,5)P4. The reported substitutions are the first published Ec phy variants with improved hydrolysis of InsP4 and InsP3.
We acknowledge the support of the Ministry of Economy, Innovation, Digitalization and Energy of North Rhine-Westphalia (MKW 311-005-1706-0001) and the Bioeconomy Science Center funded by the Ministry of Culture and Science within the NRW Strategy Project BioSC (No. 313/323-400-00213).
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Herrmann, R. K., Brethauer, C., Siedhoff, E. N., Hofmann, I., Eyll, J., Davari, D. M., Schwaneberg, U. and Ruff, J. A. (2022). Evolution of E. coli Phytase Toward Improved Hydrolysis of Inositol Tetraphosphate. Frontiers in Chemical Engineering. https://doi.org/10.3389/fceng.2022.838056