DeoR家族转录因子PsrB调控黏质沙雷氏菌合成灵菌红素

DeoR Family Regulator PsrB Regulates Prodigiosin Synthesis in Serratia marcescens

摘要:

灵菌红素（prodigiosin，PG），一种由粘质沙雷氏菌产生的次级代谢产物，因其具有抗细菌、抗疟疾、抗肿瘤和免疫抑制等重要功能而备受关注，然而，对黏质沙雷氏菌合成灵菌红素的调控机制的了解依然有限。作者以黏质沙雷氏菌JNB5-1为出发菌株，通过构建Tn5G转座子插入突变文库，发现并解析了DeoR家族转录调控因子BVG90_04085（PsrB）正调控黏质沙雷氏菌合成灵菌红素的分子机制。结果发现，LB培养基中psrB突变株SK8-61和ΔPsrB产灵菌红素能力仅为出发菌株JNB5-1的0.22倍和0.26倍。RT-qPCR分析菌株JNB5-1及ΔPsrB中pig基因簇的表达水平发现，相比于出发菌株JNB5-1，在psrB缺失突变株ΔPsrB中，灵菌红素合成途径关键基因pigABCDEFGHIJKLMN的表达水平下调了5.81~22.93倍。凝胶阻滞EMSA等实验证实转录因子PsrB可直接与pig基因簇启动子区域结合，表明转录因子PsrB正调控黏质沙雷氏菌合成灵菌红素的分子机制可能为：PsrB与灵菌红素合成基因簇启动子区域直接结合，正向调控pig基因簇的转录水平，进而影响黏质沙雷氏菌合成灵菌红素。最后，发酵培养基中psrB过表达菌株JNB5-1/pXW1906可合成8.61 g/L的灵菌红素，为出发菌株JNB5-1（5.53 g/L）的1.56倍。

Abstract:

Prodigiosin(PG), a secondary metabolite produced by Serratia marcescens, has attracted attention owing to its anti-microbial, anti-malaria, anti-tumor and immunosuppressive activities. However, there is still limited understanding of the regulatory mechanism for prodigiosin biosynthesis in S. marcescens. In this study, a Tn5G transposon insertion mutant library using the strain S. marcescens JNB5-1 as the parental strain was constructed, and BVG90_04085(PsrB), a DeoR family transcription regulator, was identified and elucidated to positively regulate prodigiosin production in strain JNB5-1. Further, the molecular mechanism behind PsrB in regulating prodigiosin production in this strain was investigated. Results showed that when fermentation was done in LB medium, the prodigiosin productivity of psrB mutants SK8-61 and ΔPsrB was only 0.22 and 0.26 times as high as that of the parental strain JNB5-1. The expression level of pig gene cluster analyzed by RT-qPCR showed that the psrB deletion mutant ΔPsrB comparing with the parental strain JNB5-1, and the expression level of a key gene in the prodigiosin pathway, pigABCDEFGHIJKLMN, was down-regulated by 5.81 to 22.93 times. Further, electrophoretic mobility shift assay (EMSA) and other experiments confirmed that regulator PsrB could directly bind to the promoter region of the pig gene cluster, suggesting that the molecular mechanism by which PsrB regulated prodigiosin production in S.marcescens was probably through direct binding to the pig gene cluster, and thus positively regulated the transcriptional level of pig gene cluster, affecting the synthesis of prodigiosin in S. marcescens. Finally, the psrB overexpressed strain JNB5-1/pXW1906 found during prodigiosin fermented in the fermentation medium could synthesize 8.61 g/L prodigiosin, which was 1.56 times of that produced by the parental strain JNB5-1 (5.53 g/L).