Bacterial cytochrome P450-catalyzed regio- and stereoselective steroid hydroxylation enabled by directed evolution and rational design

Bioresources and Bioprocessing

Table 2 Selection of CYP106A2 mutants with different profiles for progesterone hydroxylation

Number	Mutation	15β [%]	11α [%]	9α [%]	6β [%]	Progesterone
1	WT-CYP106A2	67.9	18.0	5.4	8.7	n.d.*
2	A395T/G397K	11.2	20.0	45.1	23.7	n.d.*
3	A395H/G397Y	22.9	15.2	38.6	23.3	n.d.*
4	A395I	28.7	50.9	10.2	10.2	n.d.*
5	A395E/G397V	9.4	15.9	48.6	26.1	n.d.*
6	A395R/G397K	9.1	12.9	44.8	33.2	n.d.*
7	A395M/G397S	13.1	65.6	14.4	6.9	n.d.*
8	A395R/G397I	13.2	12.7	47.0	27.2	n.d.*
9	A395K	16.5	37.2	42.4	4.0	n.d.*
10	G397W	27.3	36.4	27.3	9.1	n.d.*
11	A395F	32.5	38.3	11.5	17.7	n.d.*
12	A243V	78.0	8.2	3.6	10.2	n.d.*
13	A395W/G397K	10.5	19.2	55.9	14.3	n.d.*
14	A395E/G397Stop	13.4	15.5	45.4	25.8	n.d.*
15	A395S/G397Y	18.5	13.7	41.1	26.8	n.d.*
16	A395R/G397R	8.8	14.9	45.8	30.6	n.d.*
17	D217V/A395E/G397V	7.5	23.2	55.9	7.9	5.4
18	F165L/A395E/G397V	11.3	17.2	59.7	11.0	0.9
19	A106T/A395R/G397K	13.0	22.4	57.4	6.8	0.4
20	A243V/A395R/G397R	11.8	17.6	49.7	8.3	12.7
21	A106T/A395R/G397R	12.9	14.9	56.2	13.3	2.7
22	F173A	9.7	32.5	44.1	5.0	8.6
23	A243S	3.4	4.6	2.4	82.7	6.9
24	T247A	11.7	13.1	45.7	24.3	4.3
25	A243S/T247A	14.6	13.2	48.0	20.7	20.7
26	F173A/A243S/T247A	13.6	6.5	8.7	57.2	19.1

Relative values in % of progesterone and its monohydroxylated products 15β-, 11α-, 9α- and 6β-hydroxyprogesterone after 24 h in vivo conversion of 200 μM progesterone catalyzed by WT CYP106A2 and mutant proteins thereof. Values show percentages with the sum of the substrate progesterone and all monohydroxylated products set as 100% (n.d.*: not detectable) (Nikolaus et al. 2017)