ABSTRACT
Excelzyme, an enzyme engineering platform located at the Zurich University of Applied Sciences, is dedicated to accelerating the development of tailored biocatalysts for large-scale industrial applications. Leveraging automation and advanced computational techniques, including machine learning, efficient biocatalysts can be generated in short timeframes. Toward this goal, Excelzyme systematically selects suitable protein scaffolds as the foundation for constructing complex enzyme libraries, thereby enhancing sequence and structural biocatalyst diversity. Here, we describe applied workflows and technologies as well as an industrial case study that exemplifies the successful application of the workflow.
Subject(s)
Protein Engineering , Proteins , Humans , Switzerland , Universities , Biocatalysis , Proteins/chemistry , Protein Engineering/methodsABSTRACT
Semi-rational enzyme engineering is a powerful method to develop industrial biocatalysts. Profiting from advances in molecular biology and bioinformatics, semi-rational approaches can effectively accelerate enzyme engineering campaigns. Here, we present the optimization of a ketoreductase from Sporidiobolus salmonicolor for the chemo-enzymatic synthesis of ipatasertib, a potent protein kinase B inhibitor. Harnessing the power of mutational scanning and structure-guided rational design, we created a 10-amino acid substituted variant exhibiting a 64-fold higher apparent kcat and improved robustness under process conditions compared to the wild-type enzyme. In addition, the benefit of algorithm-aided enzyme engineering was studied to derive correlations in protein sequence-function data, and it was found that the applied Gaussian processes allowed us to reduce enzyme library size. The final scalable and high performing biocatalytic process yielded the alcohol intermediate with ≥ 98% conversion and a diastereomeric excess of 99.7% (R,R-trans) from 100 g L-1 ketone after 30 h. Modelling and kinetic studies shed light on the mechanistic factors governing the improved reaction outcome, with mutations T134V, A238K, M242W and Q245S exerting the most beneficial effect on reduction activity towards the target ketone.
ABSTRACT
The communication of changes in the extracellular matrix to the interior of the cell is crucial for a cell's function. The extracellular peptides of the RAPID ALKALINIZATION FACTOR (RALF) family have been identified as ligands of receptor-like kinases of the CrRLK1L subclass, but the exact mechanism of their perception is unclear. We found that Arabidopsis RALF4 and RALF19 redundantly regulate pollen tube integrity and growth, and that their function depends on pollen-expressed proteins of the LEUCINE-RICH REPEAT EXTENSIN (LRX) family, which play a role in cell wall development but whose mode of action is not understood. The LRX proteins interact with RALFs, monitoring cell wall changes, which are communicated to the interior of the pollen tube via the CrRLK1L pathway to sustain normal growth.