Towards Preparative Scale Steroid Hydroxylation with Cytochrome P450 Monooxygenase CYP106A2

Abstract

Cytochrome P450 monooxygenases are of outstanding interest for the synthesis of pharmaceuticals and fine chemicals, due to their ability to hydroxylate C--H bonds mainly in a stereo- and regioselective manner. CYP106A2 from Bacillus megaterium ATCC 13368, one of only a few known bacterial steroid hydroxylases, enables the oxidation of 3-keto-4-ene steroids mainly at position 15. We expressed this enzyme together with the electron-transfer partners bovine adrenodoxin and adrenodoxin reductase in Escherichia coli. Additionally an enzyme-coupled cofactor regeneration system was implemented by expressing alcohol dehydrogenase from Lactobacillus brevis. By studying the conversion of progesterone and testosterone, the bottlenecks of these P450-catalyzed hydroxylations were identified. Substrate transport into the cell and substrate solubility turned out to be crucial for the overall performance. Based on these investigations we developed a new concept for CYP106A2-catalyzed steroid hydroxylations by which the productivity of progesterone and testosterone conversion could be increased up to 18-fold to yield an absolute productivity up to 5.5 g L(-1) d(-1). Product extraction with absorber resins allowed the recovery of quantitative amounts of 15beta-OH-progesterone and 15beta-OH-testosterone and also the reuse of the biocatalyst.

CYP267A1 and CYP267B1 from Sorangium cellulosum So ce56 are Highly Versatile Drug Metabolizers

“…Optimizations such as changing expression and reaction conditions or engineering P450 enzymes could be potential topics of interest. Several approaches for increasing the performance of the whole-cell system have been published, describing that an increased number of ferredoxin gene copies (Schiffer et al, 2015) or coexpressing a NADPH regenerating system (Zehentgruber et al, 2010) could enhance product formation. In a recent review, numerous approaches and examples were presented to enhance the catalytic activity of P450 enzymes toward potential practical purposes (Gillam, 2008).…”

Renewable Hydrogen Carrier — Carbohydrate: Constructing the Carbon-Neutral Carbohydrate Economy

“…One of the most important features of SyPaB can reach nearly theoretical product yields because selecting only required enzymes eliminates unnecessary pathways that consume a significant fraction of carbon and energy sources [35,36]. In addition, cell-free systems has usually much higher reaction rates than microbial fermentations because of faster mass transfer without cellular membrane and higher biocatalyst density without dilution effects from other biomacromolecules and their side reactions [2,3,31,37,38]. Figure 2.…”

“…An alternative approach for stabilizing highly-active enzymes from mesophilic sources is to use directed evolution or rational design [104][105][106][107]. Also, it is possible to let one host co-express several enzymes [37]. As a result, a combination of the enzyme cocktails from several hosts would greatly decrease fermentation burden and purification requirement.…”

Identification of CYP106A2 as a Regioselective Allylic Bacterial Diterpene Hydroxylase

“…It hydroxylates 3-oxo-D 4 -steroids such as testosterone and progesterone mainly in the b orientation at the 15-position. [8,9,10] Additionally, the production of 6b-, [11] 9a-, and 11a-hydroxyprogesterone has been reported. [12] These hydroxylation products or their derivatives are known as pharmaceuticals or useful precursors in the synthesis of anti-inflammatory, diuretic, anabolic, contraceptive, antiandrogenic, progestational, and antitumor compounds.…”