Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie gory, h. 1, b. 40, Moscow, Russian Federation, 119991.
Skolkovo Institute of Science and Technology, Nobelya str., 3, Moscow, Russian Federation, 121205.
BMC Biotechnol. 2021 Jan 13;21(1):7. doi: 10.1186/s12896-021-00668-9.
Bacterial degradation/transformation of steroids is widely investigated to create biotechnologically relevant strains for industrial application. The strain of Nocardioides simplex VKM Ac-2033D is well known mainly for its superior 3-ketosteroid Δ-dehydrogenase activity towards various 3-oxosteroids and other important reactions of sterol degradation. However, its biocatalytic capacities and the molecular fundamentals of its activity towards natural sterols and synthetic steroids were not fully understood. In this study, a comparative investigation of the genome-wide transcriptome profiling of the N. simplex VKM Ac-2033D grown on phytosterol, or in the presence of cortisone 21-acetate was performed with RNA-seq.
Although the gene patterns induced by phytosterol generally resemble the gene sets involved in phytosterol degradation pathways in mycolic acid rich actinobacteria such as Mycolicibacterium, Mycobacterium and Rhodococcus species, the differences in gene organization and previously unreported genes with high expression level were revealed. Transcription of the genes related to KstR- and KstR2-regulons was mainly enhanced in response to phytosterol, and the role in steroid catabolism is predicted for some dozens of the genes in N. simplex. New transcription factors binding motifs and new candidate transcription regulators of steroid catabolism were predicted in N. simplex. Unlike phytosterol, cortisone 21-acetate does not provide induction of the genes with predicted KstR and KstR2 sites. Superior 3-ketosteroid-Δ-dehydrogenase activity of N. simplex VKM Ac-2033D is due to the kstDs redundancy in the genome, with the highest expression level of the gene KR76_27125 orthologous to kstD2, in response to cortisone 21-acetate. The substrate spectrum of N. simplex 3-ketosteroid-Δ-dehydrogenase was expanded in this study with progesterone and its 17α-hydroxylated and 11α,17α-dihydroxylated derivatives, that effectively were 1(2)-dehydrogenated in vivo by the whole cells of the N. simplex VKM Ac-2033D.
The results contribute to the knowledge of biocatalytic features and diversity of steroid modification capabilities of actinobacteria, defining targets for further bioengineering manipulations with the purpose of expansion of their biotechnological applications.
细菌对甾体的降解/转化被广泛研究，以创造具有工业应用价值的生物技术相关菌株。放线菌诺卡氏菌（Nocardioides simplex）VKM Ac-2033D 菌株以其优越的 3-酮甾体 Δ-脱氢酶活性而闻名，可作用于各种 3-氧代甾体和固醇降解的其他重要反应。然而，其生物催化能力以及对天然甾体和合成甾体的活性的分子基础尚未完全了解。在这项研究中，我们使用 RNA-seq 对 N. simplex VKM Ac-2033D 在植物甾醇或可的松 21-醋酸酯存在下生长时的全基因组转录组进行了比较研究。
尽管植物甾醇诱导的基因模式通常类似于富含分枝菌酸的放线菌（如分枝杆菌属、分枝杆菌属和红球菌属）中涉及植物甾醇降解途径的基因集，但基因组织的差异和以前未报道的高表达水平的基因被揭示出来。与 KstR 和 KstR2 调节子相关的基因转录主要是对植物甾醇的响应而增强的，并且预测了几十种 N. simplex 中的基因在甾体代谢中的作用。在 N. simplex 中预测到了新的转录因子结合基序和新的候选甾体代谢转录调节剂。与植物甾醇不同，可的松 21-醋酸酯不会诱导具有预测 KstR 和 KstR2 位点的基因。N. simplex VKM Ac-2033D 的优越 3-酮甾体-Δ-脱氢酶活性归因于基因组中 kstDs 的冗余，其中基因 KR76_27125 与 kstD2 同源，对可的松 21-醋酸酯的表达水平最高。在这项研究中，通过 N. simplex VKM Ac-2033D 的全细胞，扩展了 N. simplex 3-酮甾体-Δ-脱氢酶的底物谱，包括孕激素及其 17α-羟基化和 11α、17α-二羟基化衍生物，这些衍生物在体内有效地被 1(2)-脱氢。
这些结果有助于了解放线菌生物催化甾体修饰能力的多样性，并确定了进一步生物工程改造的目标，以扩大其生物技术应用。
