TY - JOUR
T1 - Selective whole-cell biosynthesis of the designer drug metabolites 15- or 16-betahydroxynorethisterone by engineered Cytochrome P450 BM3 mutants
AU - Reinen, Jelle
AU - Vredenburg, Galvin
AU - Klaering, Karoline
AU - Vermeulen, Nico P E
AU - Commandeur, Jan N M
AU - Honing, Maarten
AU - Vos, J. Chris
PY - 2015/11/1
Y1 - 2015/11/1
N2 - In the present study, the whole-cell biotransformation using strains expressing CYP BM3 mutants has been evaluated for the stereoselective hydroxylation of the steroid norethisterone (NET), a widely used contraceptive. First, an in vitro CYP BM3 mutant library screen was performed to identify mutants with high activity, as well asstereoselectivity. Subsequently, two different whole-cell setups (resting and growing cells) were tested in two different host organisms (Escherichia coli and Saccharomyces cerevisiae) expressing CYP BM3. It was found that resting E. coli whole cells produced the highest amounts of products and therefore this biocatalytic setup was further optimized for application in a laboratory-scale fermentor. In the optimized fermentor setup, high product yields (0.3 g/L 15β-OH-NET and 0.16 g/L 16β-OH-NET) were achieved while it was also shown that the regio- and stereoselectivities of the steroid hydroxylations, as determined during the in vitro library screen, were preserved in the whole-cell system. The combination of a mutant CYP BM3 library and the optimized whole-cell oxidation system represents a promising and cost-effective alternative to a wide range of in vitro biosynthetic routes.
AB - In the present study, the whole-cell biotransformation using strains expressing CYP BM3 mutants has been evaluated for the stereoselective hydroxylation of the steroid norethisterone (NET), a widely used contraceptive. First, an in vitro CYP BM3 mutant library screen was performed to identify mutants with high activity, as well asstereoselectivity. Subsequently, two different whole-cell setups (resting and growing cells) were tested in two different host organisms (Escherichia coli and Saccharomyces cerevisiae) expressing CYP BM3. It was found that resting E. coli whole cells produced the highest amounts of products and therefore this biocatalytic setup was further optimized for application in a laboratory-scale fermentor. In the optimized fermentor setup, high product yields (0.3 g/L 15β-OH-NET and 0.16 g/L 16β-OH-NET) were achieved while it was also shown that the regio- and stereoselectivities of the steroid hydroxylations, as determined during the in vitro library screen, were preserved in the whole-cell system. The combination of a mutant CYP BM3 library and the optimized whole-cell oxidation system represents a promising and cost-effective alternative to a wide range of in vitro biosynthetic routes.
KW - Biotechnology
KW - Cytochrome P450 BM3
KW - Norethisterone
KW - Stereo- and regioselectivity
KW - Whole-cell biocatalysis
UR - http://www.scopus.com/inward/record.url?scp=84939799920&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84939799920&partnerID=8YFLogxK
U2 - 10.1016/j.molcatb.2015.08.003
DO - 10.1016/j.molcatb.2015.08.003
M3 - Article
AN - SCOPUS:84939799920
SN - 1381-1177
VL - 121
SP - 64
EP - 74
JO - Journal of Molecular Catalysis B. Enzymatic
JF - Journal of Molecular Catalysis B. Enzymatic
M1 - 3212
ER -