P-LinK: A Method for Generating Multicomponent Cytochrome P450 Fusions with Variable Linker Length

Abstract

Fusion protein construction is a widely employed biochemical technique, especially when it comes to multi-component enzymes such as cytochrome P450s. Here we describe a novel method for generating fusion proteins with variable linker lengths, protein fusion with variable linker insertion (P-LinK), which was validated by fusing P450cin monooxygenase (CinA) to the flavodoxin shuttle protein (CinC). CinC was fused to the C terminus of CinA through a series of 16 amino acid linkers of different lengths in a single experiment employing 3 PCR amplifications. Screening for 2-β-hydroxy-1,8-cineole production by CinA-CinC fusion proteins revealed that enzymatically active variants possessed linker lengths of more than 5 amino acids, reaching optimum enzyme activity at a linker length of 10 amino acids. Our P-LinK method not only minimizes experimental effort and significantly reduces time demands but also requires only a single cloning and transformation step in order to generate multiple linker variants (1 to 16 amino acids long), making the approach technically simple and robust.

Enzyme Fusions in Biocatalysis: Coupling Reactions by Pairing Enzymes

“…In another study, in which a P450 was fused to a flavodoxin shuttle protein, a library of linkers was designed, ranging from one to 16 amino acids. The variants were compared based on whole‐cell conversion, and the optimal linker was found to contain ten amino acids . Strong differences were also found in a β‐glucanase–xylanase fusion with different linker lengths, with greatly improved activities for both enzyme activities .…”

The Impact of Linker Length on P450 Fusion Constructs: Activity, Stability and Coupling

“…In this way, improvements in expression rates, activities and coupling have been observed. The impact of fusing the reductase and haem domain, however, was smaller than expected . To improve the efficiency of P450s for whole cell applications, several aspects have to be considered.…”

“…Another approach towards linker length variation was used by Schwaneberg and colleagues . They studied the influence of the linker length between the P450cin monooxygenase (CinA) and the flavodoxin shuttle protein (CinC), whereas the reductase (Fpr) remained in solution.…”

Aromatic Halogenation by Using Bifunctional Flavin Reductase–Halogenase Fusion Enzymes

“…Previous work demonstrated that Baeyer–Villiger monooxygenases could be genetically fused with NADP + reductases, which simplified cofactor regeneration . Ferrodoxin and flavodoxin reductase‐type domains could also be fused to cytochrome P450 heme domains to generate self‐sufficient hydroxylation catalysts . We therefore envisioned that an FDH‐FR fusion enzyme could be useful for a wide range of in vitro and in vivo applications.…”

“…The genes encoding wild‐type RebH and RebF were genetically fused by using three linkers based on sequences used to create the functional P450‐reductase fusion enzymes noted above . These linkers consisted of 10, 16, and 22 residues (Figure A), and the corresponding fusion enzymes are referred to as H‐10‐F, H‐16‐F, and H‐22‐F.…”