A Novel 3-Phytosterone-9α-Hydroxylase Oxygenation Component and Its Application in Bioconversion of 4-Androstene-3,17-Dione to 9α-Hydroxy-4-Androstene-3,17-Dione Coupling with A NADH Regeneration Formate Dehydrogenase

Abstract

9α-Hydroxy-4-androstene-3,17-dione (9-OH-AD) is one of the significant intermediates for the preparation of β-methasone, dexamethasone, and other steroids. In general, the key enzyme that enables the biotransformation of 4-androstene-3,17-dione (AD) to 9-OH-AD is 3-phytosterone-9α-hydroxylase (KSH), which consists of two components: a terminal oxygenase (KshA) and ferredoxin reductase (KshB). The reaction is carried out with the concomitant oxidation of NADH to NAD+. In this study, the more efficient 3-phytosterone-9α-hydroxylase oxygenase (KshC) from the Mycobacterium sp. strain VKM Ac-1817D was confirmed and compared with reported KshA. To evaluate the function of KshC on the bioconversion of AD to 9-OH-AD, the characterization of KshC and the compounded system of KshB, KshC, and NADH was constructed. The optimum ratio of KSH oxygenase to reductase content was 1.5:1. An NADH regeneration system was designed by introducing a formate dehydrogenase, further confirming that a more economical process for biological transformation from AD to 9-OH-AD was established. A total of 7.78 g of 9-OH-AD per liter was achieved through a fed-batch process with a 92.11% conversion rate (mol/mol). This enzyme-mediated hydroxylation method provides an environmentally friendly and economical strategy for the production of 9-OH-AD.

Rational development of mycobacteria cell factory for advancing the steroid biomanufacturing

“…VKM, researchers found several key enzymes of steroid catabolism, such as KSH and KstD (Bragin et al 2013). These results not only confirmed the hypotheses raised by previous studies, but also guided the design of experiments to increase production (Rohman and Dijkstra 2021;Zhang et al 2019b).…”

Green Manufacturing of Steroids via Mycolicbacteria: Current Status and Development Trends

“…However, in the construction of production strains for androst-4-ene-3,17-dione (AD) and 9α-hydroxy-androst-4-ene-3,17-dione (9α-OHAD), all KstD isozymes are eliminated to reduce the generation of by-products [48]. Ksh, another key enzyme, is responsible for catalyzing the formation of 9α-hydroxylated products, which can be utilized independently for the preparation of steroid medications [71]. The low activity of Ksh also represents a bottleneck in 9α-OHAD production strains, leading to the formation of by-products like AD.…”

Improving the production of 9α-hydroxy-4-androstene-3,17-dione from phytosterols by 3-ketosteroid-Δ1- dehydrogenase deficiency and multiple gene regulation in Mycobacterium fortuitum

“…For example, the kshAB of M. neoaurum JC-12 and Mycobacterium sp. VKM Ac-1817D were co-expressed in Bacillus subtilis and Escherichia coli, and the effective conversion of AD to 9-OHAD was realized [11,12]. However, due to the low activity of KSH hydroxylase, the hydrophobicity of AD and its toxic effects on cells, etc., the low dosage of AD limits its large-scale application [4].…”