Flavin Mononucleotide-Dependent l-Lactate Dehydrogenases: Expanding the Toolbox of Enzymes for l-Lactate Biosensors

[Image: see text] The development of L-lactate biosensors has been hampered in recent years by the lack of availability and knowledge about a wider range and diversity of L-lactate-oxidizing enzymes that can be used as bioelements in these sensors. For decades, L-lactate oxidase of Aerococcus viridans (AvLOx) has been used almost exclusively in the field of L-lactate biosensor development and has achieved somewhat like a monopoly status as a biocatalyst for these applications. Studies on other L-lactate-oxidizing enzymes are sparse and are often missing biochemical data. In this work, we made use of the vast amount of sequence information that is currently available on protein databases to investigate the naturally occurring diversity of L-lactate-utilizing enzymes of the flavin mononucleotide (FMN)-dependent α-hydroxy acid oxidoreductase (HAOx) family. We identified the HAOx sequence space specific for L-lactate oxidation and additionally discovered a not-yet described class of soluble and FMN-dependent L-lactate dehydrogenases, which are promising for the construction of second-generation biosensors or other biotechnological applications. Our work paves the way for new studies on α-hydroxy acid biosensors and proves that there is more to the HAOx family than AvLOx.