Selective and Sustainable Production of Sub‐terminal Hydroxy Fatty Acids by a Self‐Sufficient CYP102 Enzyme from Bacillus Amyloliquefaciens

Abstract

AbstractEnzymatic hydroxylation of fatty acids by Cytochrome P450s (CYPs) offers an eco‐friendly route to hydroxy fatty acids (HFAs), high‐value oleochemicals with various applications in materials industry and with potential as bioactive compounds. However, instability and poor regioselectivity of CYPs are their main drawbacks. A newly discovered self‐sufficient CYP102 enzyme, BAMF0695 from Bacillus amyloliquefaciens DSM 7, exhibits preference for hydroxylation of sub‐terminal positions (ω‐1, ω‐2, and ω‐3) of fatty acids. Our studies show that BAMF0695 has a broad temperature optimum (over 70 % of maximal enzymatic activity retained between 20 to 50 °C) and is highly thermostable (T50 >50 °C), affording excellent adaptive compatibility for bioprocesses. We further demonstrate that BAMF0695 can utilize renewable microalgae lipid as a substrate feedstock for HFA production. Moreover, through extensive site‐directed and site‐saturation mutagenesis, we isolated variants with high regioselectivity, a rare property for CYPs that usually generate complex regioisomer mixtures. BAMF0695 mutants were able to generate a single HFA regiosiomer (ω‐1 or ω‐2) with selectivities from 75 % up to 91 %, using C12 to C18 fatty acids. Overall, our results demonstrate the potential of a recent CYP and its variants for sustainable and green production of high‐value HFAs.