氨肽酶生产菌株<i>Bacillus subtilis</i> LBJ4-5的全基因组测序及基因功能分析

郭宝松; 张玉姣; 代艺伟; 陈映羲; 董亮; 张素芳

doi:10.13386/j.issn1002-0306.2024030345

氨肽酶生产菌株Bacillus subtilis LBJ4-5的全基因组测序及基因功能分析

Whole-genome Sequencing and Sequence Analysis of an Aminopeptidase-producing Bacillus subtilis LBJ4-5

摘要

摘要: 本研究从郫县豆瓣中分离获得一株高产氨肽酶菌株B. subtilis LBJ4-5，通过溶血性试验及抗生素敏感性试验评估了其安全性。同时，采用Illumina二代测序技术和第三代高通量Pacbio测序平台对B. subtilis LBJ4-5进行全基因组测序，获得其基因组特征信息，并通过COG、GO、KEGG、CAZY、CARD和VFDB等数据库进行基因预测及功能注释，以及评价其氨肽酶生产性能和安全性。结果显示，B. subtilis LBJ4-5经48 h摇瓶培养后发酵上清中的氨肽酶活达到179.23 U/mL，菌株无溶血活性且对头孢唑林、万古霉素、青霉素G、头孢氨苄等10种抗生素均高度敏感（直径≥20 mm），显示出良好的安全性。B. subtilis LBJ4-5的基因组由一条环状闭合DNA及一个环状质粒组成，大小为4053926 bp，GC含量为43.66%，共预测到4285个蛋白质编码基因、85个tRNA基因、33个rRNA基因、119个sRNA。在COG、GO、KEGG、CAZY、CARD和VFDB数据库中分别注释到3386、2581、2552、144、256和401个功能基因。进一步的信息挖掘分析表明，B. subtilis LBJ4-5的基因组中含有31个氨肽酶编码基因。而毒力基因与耐药性基因的预测分析，进一步证实了B. subtilis LBJ4-5菌株的安全性。综上，B. subtilis LBJ4-5可用作发酵食品加工的发酵菌剂。

Abstract: The genomic characteristics and gene functional annotation of Bacillus subtilis LBJ4-5, an aminopeptidase-producing strain were studied to predict the presence of aminopeptidase-related genes, resistance, and virulence genes. This strain was isolated from Pixian Douban, and its aminopeptidase activity was measured using the LNA method. The safety of the strain was evaluated through hemolysis and antibiotic sensitivity tests. Simultaneously, using the Illumina second-generation sequencing technology and the third-generation high-throughput Pacbio sequencing platform, the whole genome of B. subtilis LBJ4-5 was sequenced to obtain its genomic characteristic. Gene prediction and functional annotations were performed using COG, GO, KEGG, CAZY, CARD, and VFDB databases. The results showed that after 48 hours of shake flask cultivation, the aminopeptidase activity in the fermentation supernatant of B. subtilis LBJ4-5 reached 179.23 U/mL. The strain showed no hemolytic activity and had high sensitivity to 10 antibiotics, including cefazolin, vancomycin, erythromycin, penicillin G, cephalosporin, streptomycin, amikacin, tetracycline, minocycline, and gentamicin (diameter≥20 mm). The genome of B. subtilis LBJ4-5 consisted of a circular closed DNA and a circular plasmid, with a size of 4053926 bp and a GC content of 43.66%. A total of 4285 protein-coding genes, 85 tRNA genes, 33 rRNA genes, and 119 sRNAs were predicted. Functional gene annotations in the COG, GO, KEGG, CAZY, CARD, and VFDB databases identified 3386, 2581, 2552, 144, 256 and 401 functional genes, respectively. Further analysis indicated the presence of 31 genes encoding aminopeptidases in the genome of B. subtilis LBJ4-5 predictive analysis of virulence revealed that B. subtilis LBJ4-5 possessed only one virulence factor. In conclusion, the experimental results and the predicted aminopeptidase gene information offer a preliminary explanation for the high activity of aminopeptidases in B. subtilis LBJ4-5. Moreover, safety evaluations and analysis of virulence and resistance gene carriage confirmed the safety of B. subtilis LBJ4-5. This strain would be served as a fermentation agent in fermented food processing, enriching the aminopeptidase-producing strain pool.