Liu Tan, Huang Zhiyong, Gui Xi, Xiang Wei, Jin Yubo, Chen Jun, Zhao Jing
College of Ocean and Earth Science, Xiamen University, Xiamen, China.
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
Front Microbiol. 2021 Jan 28;11:630309. doi: 10.3389/fmicb.2020.630309. eCollection 2020.
Sponges, the most primitive multicellular animals, contain a large number of unique microbial communities. Sponge-associated microorganisms, particularly actinomyces, have the potential to produce diverse active natural products. However, a large number of silent secondary metabolic gene clusters have failed to be revived under laboratory culture conditions. In this study, iterative atmospheric room-temperature plasma. (ARTP) mutagenesis coupled with multi-omics conjoint analysis was adopted to activate the inactive wild strain. The desirable exposure time employed in this study was 75 s to obtain the appropriate lethality rate (94%) and mutation positive rate (40.94%). After three iterations of ARTP mutagenesis, the proportion of mutants exhibiting antibacterial activities significantly increased by 75%. Transcriptome analysis further demonstrated that the differential gene expression levels of encoding type I lasso peptide aborycin had a significant upward trend in active mutants compared with wild-type strains, which was confirmed by LC-MS results with a relative molecular mass of 1082.43 ([M + 2H] at = 2164.86). Moreover, metabolome comparative analysis of the mutant and wild-type strains showed that four spectra or mass peaks presented obvious differences in terms of the total ion count or extracting ion current profiles with each peak corresponding to a specific compound exhibiting moderate antibacterial activity against Gram-positive indicators. Taken together, our data suggest that the ARTP treatment method coupled with multi-omics profiling analysis could be used to estimate the valid active molecules of metabolites from microbial crudes without requiring a time-consuming isolation process.
海绵是最原始的多细胞动物，含有大量独特的微生物群落。与海绵相关的微生物，尤其是放线菌，有产生多种活性天然产物的潜力。然而，大量沉默的次生代谢基因簇在实验室培养条件下未能被激活。在本研究中，采用迭代常压室温等离子体（ARTP）诱变结合多组学联合分析来激活无活性的野生菌株。本研究采用的理想暴露时间为75秒，以获得合适的致死率（94%）和突变阳性率（40.94%）。经过三轮ARTP诱变后，表现出抗菌活性的突变体比例显著增加了75%。转录组分析进一步表明，与野生型菌株相比，编码I型套索肽阿博霉素的差异基因表达水平在活性突变体中呈显著上升趋势，液相色谱-质谱结果证实了这一点，其相对分子质量为1082.43（[M + 2H]+ = 2164.86）。此外，对突变体和野生型菌株的代谢组比较分析表明，四个光谱或质谱峰在总离子计数或提取离子电流图谱方面呈现出明显差异，每个峰对应一种对革兰氏阳性指示菌具有中等抗菌活性的特定化合物。综上所述，我们的数据表明，ARTP处理方法与多组学分析相结合可用于评估微生物粗提物中代谢产物的有效活性分子，而无需耗时的分离过程。
