Novel approach to improve progesterone hydroxylation selectivity by CYP106A2 via rational design of adrenodoxin binding.

Bacterial P450s have considerable potential for biotechnological applications. The P450 CYP106A2 from Bacillus megaterium ATCC 13368 converts progesterone to several hydroxylated products that are important precursors for pharmaceutical substances. As high yields of monohydroxylated products are required for biotechnological processes, improving this conversion is of considerable interest.

Binding modes of CYP106A2 redox partners determine differences in progesterone hydroxylation product patterns.

Natural redox partners of bacterial cytochrome P450s (P450s) are mostly unknown. Therefore, substrate conversions are performed with heterologous redox partners; in the case of CYP106A2 from ATCC 13368, bovine adrenodoxin (Adx) and adrenodoxin reductase (AdR). Our aim was to optimize the redox system for CYP106A2 for improved product formation by testing 11 different combinations of redox partners.

Engineering of versatile redox partner fusions that support monooxygenase activity of functionally diverse cytochrome P450s.

Most bacterial cytochrome P450 monooxygenases (P450s or CYPs) require two redox partner proteins for activity. To reduce complexity of the redox chain, the Bacillus subtilis flavodoxin YkuN (Y) was fused to the Escherichia coli flavodoxin reductase Fpr (R), and activity was tuned by placing flexible (GGGGS) or rigid ([E/L]PPPP) linkers (n = 1-5) in between. P-linker constructs typically outperformed their G-linker counterparts, with superior performance of YR-P5, which carries linker ([E/L]PPPP).

Engineering of CYP106A2 for steroid 9α- and 6β-hydroxylation.

CYP 106A2 from Bacillus megaterium ATCC 13368 has been described as a 15β-hydroxylase showing also minor 11α-, 9α- and 6β-hydroxylase activity for progesterone conversion. Previously, mutant proteins with a changed selectivity towards 11α-OH-progesterone have already been produced. The challenge of this work was to create mutant proteins with a higher regioselectivity towards hydroxylation at positions 9 and 6 of the steroid molecule.