We have shown here that ASR can be used to obtain a panel of biocatalysts, each with a unique pattern of regioselectivity towards fatty acid hydroxylation. Notably, ancestor N374 would be a good starting point for engineering for efficient w-2 hydroxylation, having shown ~ 70 % regioselectivity and 42% product conversion here, as well as relatively high 15 T50, although its longer term stability was not clear. Such predominant w-2 hydroxylation had not been observed previously with other P450s and as such constitutes a new enzyme selectivity. Similarly, N70, N106, and N115 would be useful templates for engineering a higher efficiency biocatalyst for w-5 hydroxylation, since they showed high regioselectivity, almost quantitative conversion of decanoic acid under the conditions used here, along with an overall expression yield that was comparable to or higher than the extant w-5 hydroxylase, P450-TT. Directed evolution by recombination of these ancestors by CLADE may be particularly productive .