茎瘤芥根际一株产紫色杆菌素杜擀氏菌的分离鉴定及其基因组分析

Identification and genome analysis of the violacein- producing Duganella sp. isolated from Brassica juncea var. tumida rhizosphere

摘要:

【目的】对茎瘤芥根际微生物进行分离鉴定，分析微生物菌群构成，选择具有优良特性的菌株，评估其次级代谢产物合成能力，为茎瘤芥根际微生物多样性和菌种资源的挖掘利用奠定基础。【方法】采集重庆市涪陵区二渡村和邓家村的茎瘤芥根，分离培养根际微生物菌株，通过菌株形态观察和看家基因的序列分析，对菌株进行初步鉴定、归类和保存。选择具有优良性状的菌株，利用Pacbio RS II和Illumina HiSeq平台完成全基因组测序，通过antiSMASH分析评估其次级代谢产物合成潜力，克隆目的基因簇并进行异源表达和产物鉴定。【结果】分离得到256株微生物，初步鉴定120株；从中鉴定了一株产紫色杆菌素的杜擀氏菌BjR8，完成了基因组测序及分析，发现该菌基因组为一条环状染色体，全长7 205 593 bp，GC含量为64.67%，含有6 241个编码基因。生物信息学分析发现基因组含有9个次级代谢产物生物合成基因簇，其中7个基因簇与已知化合物编码基因簇同源性较低，说明该菌具有产生多种新型次级代谢产物的潜力；克隆得到紫色杆菌素生物合成基因簇，并在变铅青链霉菌TK23中完成了异源表达。【结论】从茎瘤芥根际分离得到256株微生物，初步分析了茎瘤芥根际的微生物菌群构成；完成了一株产紫色杆菌素杜擀氏菌的基因组测定与分析，从中克隆了紫色杆菌素基因簇，并成功在链霉菌中实现异源表达。

Abstract:

[Objective] Rhizosphere contains a phylogenetically diverse array of microorganisms affecting the growth and health of plants. This study aims to examine the diversity and structure of the microbial community in Brassica juncea var. tumida rhizosphere. The isolated microbial strains can be explored to unleash their chemical potential. [Methods] Roots of B. juncea var. tumida were collected from two villages in Fuling Disctrict of Chongqing. Microbial isolates were then obtained after cultivation on different media. One strain was selected for genome sequencing (Pacbio RS II and Illumina HiSeq) and subsequent bioinformatic analysis. The antiSMASH 6.0 was used for the detection and comparison of biosynthetic gene clusters (BGCs) encoding secondary metabolites. A multiplex amplification-and-ligation strategy was employed to clone the gene cluster responsible for violacein biosynthesis. The gene cluster was then transferred into Streptomyces lividans TK23 by intergeneric conjugation. [Results] A total of 256 microbial isolates were obtained, of which 120 were identified. Duganella sp. BjR8, which produced violacein, was detected. Sequencing of the genome of BjR8 revealed a single circular chromosome of 7 205 593 bp in length with 64.67% G+C content and 6 421 coding sequences. Bioinformatic analysis showed that the strain contained 9 putative secondary metabolite BGCs, of which 7 had low similarities to BGCs encoding known compounds. Heterologous expression of the violacein gene cluster in S. lividans TK23 resulted in the production of deoxyviolacein. [Conclusion] The diversity and structure of the microbial community was examined by phylogenetic analysis of microorganisms isolated from B. juncea var. tumida rhizosphere. Bioinformatic analysis revealed that BjR8 had great biosynthetic potential for novel compounds. The vio gene cluster was cloned from BjR8 and expressed successfully in the surrogate host S. lividans TK23.