Abstract：
Androst-4-ene-3,17-dione (AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) are important drug intermediates that can be biosynthesized from phytosterols. However, the C9 hydroxylation of steroids via 3-ketosteroid 9α-hydroxylase (KSH) limits AD and 4-HBC accumulation. Five active KshAs, the oxidation component of KSH, were identified in Mycobacterium fortuitum ATCC 35855 for the first time. The deletion of kshAs indicated that the five KshA genes were jointly responsible for C9 hydroxylation during phytosterol biotransformation. MFKDΔkshA, the five KshAs deficient strain, blocked C9 hydroxylation and produced 5.37 g/L AD and 0.55 g/L 4-HBC. The dual function reductase Opccr knockout and 17β-hydroxysteroid dehydrogenase Hsd4A enhancement reduced 4-HBC content from 8.75 to 1.72% and increased AD content from 84.13 to 91.34%, with 8.24 g/L AD being accumulated from 15 g/L phytosterol. In contrast, hsd4A and thioesterase fadA5 knockout resulted in the accumulation of 5.36 g/L 4-HBC from 10 g/L phytosterol. We constructed efficient AD (MFKDΔkshAΔopccr_hsd4A) and 4-HBC (MFKDΔkshAΔhsd4AΔfadA5) producers and provided insights for further metabolic engineering of the M. fortuitum ATCC 35855 strain for steroid productions. KEY POINTS: • Five active KshAs were first identified in M. fortuitum ATCC 35855. • Deactivation of all five KshAs blocks the steroid C9 hydroxylation reaction. • AD or 4-HBC production was improved by Hsd4A, FadA5, and Opccr modification.
摘要：
Androst-4-ene-3,17-二酮（AD）和22-羟基-23,24-bisnorchol-4-ene-3-one（4-HBC）是重要的药物中间体，可以从植物甾醇生物合成。然而,类固醇通过3-酮类固醇9α-羟化酶（KSH）的C9羟化限制了AD和4-HBC的积累。五个活跃的Ksha，KSH的氧化成分，首次在偶然分枝杆菌ATCC35855中鉴定。kshAs的缺失表明，在植物甾醇生物转化过程中，五个KshA基因共同负责C9的羟基化。MFKDΔkshA,五种缺乏KshAs的菌株，阻断C9羟基化，产生5.37g/LAD和0.55g/L4-HBC。双功能还原酶Opccr敲除和17β-羟基类固醇脱氢酶Hsd4A增强将4-HBC含量从8.75降低到1.72％，将AD含量从84.13提高到91.34％，从15g/L植物甾醇中积累8.24g/LAD。相比之下,hsd4A和硫酯酶fadA5敲除导致10g/L植物甾醇积累5.36g/L4-HBC。我们构建了有效的AD（MFKDΔkshAΔopccr_hsd4A）和4-HBC（MFKDΔkshAΔhsd4AΔfadA5）生产者，并为进一步代谢工程M.futuitumATCC35855菌株的类固醇生产提供了见解。关键点:•五个活跃的KshA首先在偶然分枝杆菌ATCC35855中被鉴定。•所有五个KshAs的失活阻断类固醇C9羟基化反应。•Hsd4A改善了AD或4-HBC的产生，FadA5和Opccr修改。
