分枝杆菌HGMS2甾醇降解中新C-23代谢产物的鉴定及其代谢途径分析

Identification of a new C-23 metabolite in sterol degradation of Mycobacterium neoaurum HGMS2 and analysis of its metabolic pathways

摘要:

新金分枝杆菌(Mycobacterium neoaurum)通过敲除羟酰基辅酶A脱氢酶(hydroxyacyl-CoA dehydrogenase, Hsd4A)或酰基辅酶A硫解酶(acyl-CoA thiolase, FadA5)基因来生产22-羟基-23,24-双降甾-4-烯-3-酮(22-hydroxy-23,24-bisnorchol-4-en-3-one, BA)甾体类药物中间体来受到广泛关注。本实验室前期发现，敲除fadA5基因后，发酵产物中出现了一种新的代谢产物。本研究通过对该物质的结构鉴定、fadA5基因的蛋白同源序列比对、M. neoaurum HGMS2的系统发育树分析和基因敲除等手段探究该物质的代谢途径。结果表明该物质为C23类代谢中间体，即24-norchol-4-ene-3,22-dione (将其命名为3-OPD)。该物质是通过硫酯酶(thioesterase, TE)催化3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA (22-O-BNC-CoA)生成侧链为β-酮酸结构的物质后通过生物体的自动脱羧反应生成的。这些结果对开发新的甾类中间体有重要价值。

Abstract:

Mycobacterium neoaurum has the ability to produce steroidal intermediates known as 22-hydroxy-23,24-bisnorchol-4-en-3-one (BA) upon the knockout of the genes for either the hydroxyacyl-CoA dehydrogenase (Hsd4A) or acyl-CoA thiolase (FadA5). In a previous study, we discovered a novel metabolite in the fermentation products when the fadA5 gene was deleted. This research aims to elucidate the metabolic pathway of this metabolite through structural identification, homologous sequence analysis of the fadA5 gene, phylogenetic tree analysis of M. neoaurum HGMS2, and gene knockout. Our findings revealed that the metabolite is a C23 metabolic intermediate, named 24-norchol-4-ene-3,22-dione (designated as 3-OPD). It is formed when a thioesterase (TE) catalyzes the formation of a β-ketonic acid by removing CoA from the side chain of 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA (22-O-BNC-CoA), followed by spontaneously undergoing decarboxylation. These results have the potential to contribute to the development of novel steroid intermediates.