一株产灵菌红素黏质沙雷氏菌的分离鉴定及其基因组分析

Isolation, identification, and genomic analysis of a prodigiosin-producing strain of Serratia marcescens

摘要:

【目的】 对甘蓝根际微生物进行分离，从中选择具有优良性状的菌株，对其次级代谢产物合成能力进行评估，为甘蓝根际微生物资源的发掘利用奠定基础。【方法】 采集重庆市武隆区仙女山镇仙女山村甘蓝根，分离培养根际微生物菌株，从中选择一株黏质沙雷氏菌(Serratia marcescens)，利用PacBio RS II平台和Illumina Hiseq平台完成全基因组测序，通过antiSMASH分析评估其合成次级代谢产物的潜力，采用Red/ET重组工程技术克隆目的基因簇并进行异源表达。【结果】 从甘蓝根际分离鉴定到一株产灵菌红素的黏质沙雷氏菌，对其进行基因组测序及分析发现，基因组含有11个次级代谢产物生物合成基因簇，其中9个基因簇与已知化合物编码基因簇相似性较低，说明该菌具有产生多种新型次级代谢产物的潜力；HPLC分析和高分辨质谱检测鉴定了灵菌红素；克隆了灵菌红素生物合成基因簇，并在大肠杆菌中实现了异源表达；选择4种大肠杆菌常用启动子驱动灵菌红素基因簇的表达，发现rpoS启动子具有较好的效果。【结论】 本研究从甘蓝根际分离得到一株产灵菌红素的黏质沙雷氏菌，完成了全基因组测序和分析，从中克隆得到灵菌红素生物合成基因簇，并成功在大肠杆菌中异源表达。

关键词:甘蓝;黏质沙雷氏菌;灵菌红素;基因组分析;生物合成基因簇;异源表达

Abstract:

[Objective] To select the elite strains among the microorganisms isolated from the rhizosphere of Brassica oleracea var. capitata and evaluate the secondary metabolism potential of the strains, laying a solid foundation for mining the microbial resources in the rhizosphere of this plant. [Methods] The roots of B. oleracea var. capitata were collected from Xiannüshan Town in Wulong District of Chongqing. The conventional methods were then used to isolate microbial strains, and one strain of Serratia marcescens was selected for genome sequencing (PacBio RS II and Illumina HiSeq) and bioinformatic analysis. The antiSMASH was used for the detection and comparison of biosynthetic gene clusters (BGCs) encoding secondary metabolites. Red/ET recombination was employed to capture the BGC for prodigiosin. [Results] One prodigiosin-producing strain of S. marcescens was isolated from the rhizosphere of B. oleracea var. capitata. The genome of this strain contained 11 putative BGCs (1-11) for secondary metabolites, of which 9 BGCs displayed low similarities to the BGCs encoding known compounds. This result suggested that the strain had great potential of producing novel secondary metabolites. Prodigiosin was identified by HPLC and high-resolution mass spectrometry (HRMS). Heterologous expression of BGC4 resulted in the production of prodigiosin in Escherichia coli. Four common promoters were used to drive the expression of BGC in E. coli, and the highest prodigiosin production was observed with the rpoS promoter. [Conclusion] A prodigiosin-producing strain of S. marcescens was isolated from the rhizosphere of B. oleracea var. capitata. The BGC for prodigiosin was captured from the chromosome of this strain and expressed successfully in the surrogate host E. coli BAP1.

Key words:Brassica oleracea var. capitata;Serratia marcescens;prodigiosin;genomic analysis;biosynthetic gene cluster;heterologous expression