Two hundred thirteen cytochrome P450 (P450) genes were collected from bacteria and expressed based on an Escherichia coli expression system to test their hydroxylation ability to testosterone. Twenty-four P450s stereoselectively monohydroxylated testosterone at the 2alpha-, 2beta-, 6beta-, 7beta-, 11beta-, 12beta-, 15beta-, 16alpha-, and 17-positions (17-hydroxylation yields 17-ketoproduct). The hydroxylation site usage of the P450s is not the same as that of human P450s, while the 2alpha-, 2beta-, 6beta-, 11beta-, 15beta-, 16alpha-, and 17-hydroxylation are reactions common to both human and bacterial P450s. Most of the testosterone hydroxylation catalyzed by bacterial P450s is on the beta face.
Two vectors, pT7NScamAB and pRED, have been used for the functional expression of bacterial class I cytochrome P450 (P450) genes in Escherichia coli, which utilize putidaredoxin reductase (CamA) and putidaredoxin (CamB), and the reductase domain of a self-sufficient P450RhF respectively, for electron transfer from NAD(P)H to a P450 protein. We here compared the efficiency of bioconversion with the two vectors towards n-octane, cyclohexane, n-butylbenzene, and 2-n-butylbenzofuran using two well-characterized CYP153A genes, aciA and CYP153A13a (P450balk). As for n-octane bioconversion, aciA and pT7camAB was the best combination for the production of 1-octanol and 1,8-octanediol. As for the bioconversion of cyclohexane, n-butylbenzene and 2-n-butylbenzofuran, CYP153A13a with pRED achieved the most efficient bioconversion, as compared by conversion ratio per active CYP153A protein content. It was also found that 2-n-butylbenzofuran is biotransformed into 4-benzofuran-2-yl-butyric acid via 4-benzofuran-2-yl-butan-1-ol with E. coli cells expressing CYP153A.
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