Generation of phytase chimeras with low sequence identities and improved thermal stability
Congratulations Anna Joelle Ruff and Kevin Herrmann on their recent publication!
Being able to recombine more than two genes with four or more crossover points in a sequence independent manner is still a challenge in protein engineering and limits our capabilities in tailoring enzymes for industrial applications. By computational analysis employing multiple sequence alignments and homology modeling, five fragments of six phytase genes (sequence identities 31-64 %) were identified and efficiently recombined through phosphorothioate-based cloning using the PTRec method. By combinatorial recombination, functional phytase chimeras containing fragments of up to four phytases were obtained. Two variants (PTRec 74 and PTRec 77) with up to 32 % improved residual activity (90 °C, 60 min) and retained specific activities of > 1100 U/mg were identified. Both variants are composed of fragments from the phytases of Citrobacter braakii, Hafnia alvei and Yersinia mollaretii. They exhibit sequence identities of ≤ 80 % to their parental enzymes, highlighting the great potential of DNA recombination strategies to generate new enzymes with low sequences identities.
This work was supported by Ministerium für Wirtschaft, Innovation, Digitalisierung und Energie des Landes Nordrhein-Westfalen (MKW 311-005-1706-0001) and the scientific activities of the Bioeconomy Science Center which were financially supported by the Ministerium für Kultur und Wissenschaft within the framework of the NRW Strategieprojekt BioSC (No. 313/323‐400‐00213).
Kevin R Herrmann, Isabell Hofmann, Dennis Jungherz, Malte Wittwer, Belén Infanzón, Stefanie Nicole Hamer, Mehdi D Davari, Anna Joëlle Ruff, Ulrich Schwaneberg, Generation of phytase chimeras with low sequence identities and improved thermal stability. J Biotechnol (2021), 339:14-21. doi: 10.1016/j.jbiotec.2021.07.005.