Abstact

Flavoproteins are involved in a wide array of biological processes. These proteins contain one or more flavins as their cofactor, bound either noncovalently or covalently, and catalyze a wide breadth of redox reactions. In commonly used expression strains, flavoproteins are typically produced as holo (flavin-bound) flavoproteins. The ability to produce their apo form (flavoprotein devoid of flavins) will facilitate dedicated structural and mechanistic studies, while it also allows the incorporation of new-to-nature flavin-like cofactors. To facilitate this, we constructed, using the CRISPR/Cas9 system, an Escherichia coli strain that is impaired in producing the canonical FMN and FAD cofactors, due to a deletion in the ribB gene. This riboflavin auxotrophic strain is able to produce apoproteins of FMN- and FAD-dependent flavoproteins. We demonstrate that it can also be used for the incorporation of flavin derivatives by supplementing the medium with the respective riboflavin derivative. In parallel, we constructed, by genomic integration, a strain expressing an FAD synthetase from a T7 promoter and a flavin transferase from a lac promoter. This strain facilitates employing the newly developed methodology of flavin-tagging and flavin-fixing of target proteins, resulting in proteins carrying a covalently tethered FMN. It eliminates the need for two or more plasmids to generate covalently flavinylated flavoproteins. A third strain was prepared in which the features of riboflavin auxotrophy and flavin transferase activity were combined. This strain is perfectly suited for generating flavoproteins carrying a covalently anchored flavin derivative. These newly engineered strains, derivatives of E. coli BL21-AI, represent powerful tools for producing, investigating, and applying flavoproteins.