title: Engineering of recombinant E. coli cells co-expressing P450pyrTM monooxygenase and glucose dehydrogenase for highly regio- and stereoselective hydroxylation of alicycles with cofactor recycling.
abstract: E. coli (P450pyrTM-GDH) with dual plasmids, pETDuet containing P450pyr triple mutant I83H/M305Q/A77S (P450pyrTM) and ferredoxin reductase (FdR) genes and pRSFDuet containing glucose dehydrogenase (GDH) and ferredoxin (Fdx) genes, was engineered to show a high activity (12.7 U g⁻¹  cdw) for the biohydroxylation of N-benzylpyrrolidine 1 and a GDH activity of 106 U g⁻¹ protein. The E. coli cells were used as efficient biocatalysts for highly regio- and stereoselective hydroxylation of alicyclic substrates at non-activated carbon atom with enhanced productivity via intracellular recycling of NAD(P)H. Hydroxylation of N-benzylpyrrolidine 1 with resting cells in the presence of glucose showed excellent regio- and stereoselectivity, giving (S)-N-benzyl-3-hydroxypyrrolidine 2 in 98% ee as the sole product in 9.8 mM. The productivity is much higher than that of the same biohydroxylation using E. coli (P450pyrTM)b without expressing GDH. E. coli (P450pyrTM-GDH) was found to be highly regio- and stereoselective for the hydroxylation of N-benzylpyrrolidin-2-one 3, improving the regioselectivity from 90% of the wild-type P450pyr to 100% and giving (S)-N-benzyl-4-hydroxylpyrrolidin-2-one 4 in 99% ee as the sole product. A high activity of 15.5 U g⁻¹  cdw was achieved and (S)-4 was obtained in 19.4 mM. E. coli (P450pyrTM-GDH) was also found to be highly regio- and stereoselective for the hydroxylation of N-benzylpiperidin-2-one 5, increasing the ee of the product (S)-N-benzyl-4-hydroxy-piperidin-2-one 6 to 94% from 33% of the wild-type P450pyr. A high activity of 15.8 U g⁻¹  cdw was obtained and (S)-6 was produced in 3.3 mM as the sole product. E. coli (P450pyrTM-GDH) represents the most productive system known thus far for P450-catalyzed hydroxylations with cofactor recycling, and the hydroxylations with E. coli (P450pyrTM-GDH) provide with simple and useful syntheses of (S)-2, (S)-4, and (S)-6 that are valuable pharmaceutical intermediates and difficult to prepare.
authors.affiliation: Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576, Singapore.
authors.forename: Son Q
authors.initials: SQ
authors.lastname: Pham
authors.forename: Pengfei
authors.initials: P
authors.lastname: Gao
authors.forename: Zhi
authors.initials: Z
authors.lastname: Li
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D049109:Cell Proliferation; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D005287:Ferredoxin-NADP Reductase / Q000235:genetics / Q000378:metabolism; D005288:Ferredoxins / Q000235:genetics / Q000378:metabolism; D042843:Glucose 1-Dehydrogenase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006900:Hydroxylation; D009249:NADP / Q000032:analysis / Q000378:metabolism*; D015202:Protein Engineering; D011759:Pyrrolidines / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013237:Stereoisomerism
chemical_list: D001426:Bacterial Proteins; D005288:Ferredoxins; D011759:Pyrrolidines; D011993:Recombinant Fusion Proteins; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D042843:Glucose 1-Dehydrogenase; D005287:Ferredoxin-NADP Reductase