Novel biocatalysts for industrial fructooligosaccharides (FOS) production from fucosyltransferase and mutagenized invertase genes constitutively expressed in <i>Komagataella phaffii</i> (previously <i>Pichia pastoris</i> )

Fructosyltransferases (FTFs) and invertases catalyze the production of short-chain fructo-oligosaccharides (FOS), levan, and inverted syrups from sucrose—compounds that are increasingly sought after in the global food industry. Advancements in protein and enzyme engineering, structure-function analysis, and biocatalyst design have significantly enhanced their availability, efficiency, and application in human and animal nutrition. However, large-scale FOS production remains monopolized by major companies, limiting broader access to these bioprocesses. This research introduces for the first time two novel enzymatic catalysts that enable the industrial-scale production of these enzymes and their respective products. Specifically, the genes encoding β-fructosidase (BfrA, EC 3.2.1.26) from the extremophile Thermotoga maritima and sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99) from Schedonorus arundinaceus were modified—through multimeric cassette expression for 1-SST and site-directed mutagenesis for BfrA. These engineered enzyme variants were constitutively expressed in Komagataella phaffii, paving the way for novel industrial biocatalysts. These novel BfrA and 1-SST enzymatic catalysts offer solutions for converting sucrose into short-chain FOS of the levan- and inulin-type.