Optimization and Engineering of a Self-Sufficient CYP102 Enzyme from Bacillus amyloliquefaciens towards Synthesis of In-Chain Hydroxy Fatty Acids

Cytochrome P450 (CYP) mediated enzymatic hydroxylation of fatty acids present a green alternative to chemical synthesis hydroxy (HFAs), which are high-value oleochemicals with various uses in materials industry and medical field. Although many CYPs require the presence additional reductase proteins for catalytic activity, self-sufficient have their partner naturally fused into domain, leading greatly simplified biotransformation process. A recently discovered CYP, BAMF2522 from Bacillus amyloliquefaciens DSM 7, exhibits novel regioselectivity by hydroxylating in-chain positions palmitic acid generating ?-1 ?-7 HFAs, rare regiodiversity profile among CYPs. Besides, F89I mutant expanded up ?-9 position acid. Here, we further characterize this enzyme determining optimum temperature pH as well thermal stability. Moreover, using extensive site-directed site-saturation mutagenesis, obtained variants that demonstrate increased (?-4 ?-9) medium long chain acids. Remarkably, when six-residue was reacted acid, 84% total product content sum ?-7, ?-8 HFA products, highest selectivity observed date CYP. In short, our study demonstrates potential identified CYP its mutants sustainable production variety enriched HFAs.