Altering the Regioselectivity of Cytochrome P450 CYP102A3 of Bacillus subtilis by Using a New Versatile Assay System

Graphical Abstract

Getting a different alcohol. By using error-prone PCR in combination with a novel high-throughput screening specific for primary alcohols, a mutant of the self-sufficient P450 monooxygenase CYP102A3 from Bacillus subtilis with altered regioselectivity was created. While the wild-type enzyme produced a mixture of three secondary alcohols from n-octane and not even a trace of octan-1-ol, a new double mutant formed 48 % octan-1-ol, 52 % of octan-2- and -3-ol and no octan-4-ol.

Abstract

A novel monooxygenase (CYP102A3) has been discovered within the Bacillus subtilis genome that reveals a similarity of 76 % to the well-known cytochrome P450 BM-3 of B. megaterium (CYP102A1). Both enzymes are natural fusion proteins consisting of a heme domain and a FAD/FMN-reductase domain. Because of their high turnover rates, these biocatalysts are of special interest for industrial applications, but show only limited regioselectivity. In this work, the regioselectivity of CYP102A3 was changed by directed evolution and protein design to hydroxylate substrates not only in different subterminal, but also to a high extent, in terminal carbon chain positions. To enable a high-throughput screening procedure, a very versatile assay was developed that is capable of discriminating between terminal and subterminal hydroxylation of carbon chains. A double mutant of CYP102A3 was obtained that produces 48 % octan-1-ol as the main product of the reaction.