一株高产苯乳酸的古墓土壤葡糖醋杆菌FBFS97的全基因组测序与分析

Genome sequencing and analysis of Gluconacetobacter tumulisoli FBFS97, a high-yield strain of phenyllactic acid

摘要:

【背景】苯乳酸(phenyllactic acid，PLA)是一种应用潜力巨大的天然广谱抑菌物质。本课题组前期分离得到一株高产PLA的醋酸菌(acetic acid bacteria，AAB)——葡糖醋杆菌(Gluconacetobacter sp.) FBFS97，但尚未鉴定到种，而且其产PLA的分子机理尚不清楚。【目的】确定FBFS97的种属关系，解析FBFS97的遗传信息，特别是与PLA产生相关的基因。【方法】采用光学显微镜和扫描电镜对FBFS97的菌体形态进行表征，通过16S rRNA基因序列分析对FBFS97进行分类鉴定，并以高效液相色谱分析苯丙氨酸对其产PLA的影响。在此基础上，对FBFS97进行全基因组测序、拼接和基因预测，并进行GO/COG聚类、KEGG代谢通路和VFDB毒力等分析，以及PLA生物合成途径的预测。【结果】根据16S rRNA基因序列的比对结果，结合形态学分析，该菌被鉴定为古墓土壤葡糖醋杆菌(Gluconacetobacter tumulisoli)。将1 000 mg/L苯丙氨酸添加到FBFS97液体培养基中，发酵液中PLA最高浓度可达400 mg/L，为对照组的8倍。该菌的基因组大小为3 988 308 bp，(G+C)mol%含量为66.62%，编码基因3 500个；KEGG代谢通路分析表明，该菌基因组中存在经莽草酸途径合成PLA的所有基因；VFDB毒力预测结果显示，该菌基因组中不存在产生毒素的相关基因。【结论】首次报道了一株高产PLA的AAB——古墓土壤葡糖醋杆菌FBFS97的全基因组序列信息，并发现该菌株的基因组中含有合成PLA的所有相关基因，为后续进一步研究FBFS97产生PLA的生物合成途径提供了理论依据。

Abstract:

[Background] Phenyllactic acid (PLA) is a natural broad-spectrum antibacterial substance with a great application potential. In the previous work, Gluconacetobacter sp. FBFS97, an acetic acid bacterium (AAB) strain with high-yield PLA, was isolated, but the specific species of this strain and the molecular mechanism of PLA production is unclear. [Objective] To determine the species relationship of FBFS97, explore the genetic information of FBFS97, especially the genes related to PLA biosynthesis. [Methods] The morphology of FBFS97 was characterized by light microscopy and scanning electron microscopy, while the classification was identified by 16S rRNA gene sequence alignment. The effect of phenylalanine on FBFS97 producing PLA was detected by high-performance liquid chromatography (HPLC). On this basis, the complete genome was sequenced by Illumina MiSeq sequencers, and genome assembly, gene prediction, functional annotation, GO/COG cluster, metabolic pathway and virulence were analyzed using the relevant software, and the biosynthetic pathway of PLA is predicted. [Results] The strain was identified as Gluconacetobacter tumulisoli by 16S rRNA gene sequence alignment analysis and morphological analysis. When 1 000 mg/L phenylalanine was added to the liquid medium of FBFS97, the maximum concentration of PLA in the fermentation broth reached 400 mg/L, which was 8 times higher than that of control group. The genome size of FBFS97 is 3 988 308 bp with 66.62% (G+C)mol% and 3 500 encoding genes, and no toxin-related gene was predicted by VFDB database in the genome, and whole genes relative for biosynthetic PLA by the shikimate pathway were found. [Conclusion] This is first attempt to describe the whole genome sequence of Gluconacetobacter tumulisoli sp. FBFS97, a high-yield strain of PLA. The genes related to PLA biosynthesis are found in the FBFS97 genome, which provides a basis for further investigation on the PLA biosynthetic pathway in FBFS97.