Data_Sheet_1_Complete Genomic Analysis of Enterococcus faecium Heat-Resistant Strain Developed by Two-Step Adaptation Laboratory Evolution Method.DOCX

Stress resistance is an important trait expected of lactic acid bacteria used in food manufacturing. Among the various sources of stress, high temperature is a key factor that interrupts bacterial growth. In this regards, constant efforts are made for the development of heat-resistant strains, but few studies were done accompanying genomic analysis to identify the causal factors of the resistance mechanisms. Furthermore, it is also thought that tolerance to multiple stresses are equally important. Herein, we isolated one Enterococcus faecium strain named BIOPOP-3 and completed a full-length genome sequence. Using this strain, a two-step adaptive laboratory evolution (ALE) method was applied to obtain a heat-resistant strain, BIOPOP-3 ALE. After sequencing the whole genome, we compared the two full-length sequences and identified one non-synonymous variant and four indel variants that could potentially confer heat resistance, which were technically validated by resequencing. We experimentally verified that the evolved strain was significantly enhanced in not only heat resistance but also acid and bile resistance. We demonstrated that the developed heat-resistant strain can be applied in animal feed manufacturing processes. The multi-stress-resistant BIOPOP-3 ALE strain developed in this study and the two-step ALE method are expected to be widely applied in industrial and academic fields. In addition, we expect that the identified variants which occurred specifically in heat-resistant strain will enhance molecular biological understanding and be broadly applied to the biological engineering field.

胁迫抗性是食品工业用乳酸菌（lactic acid bacteria）所需具备的重要性状。在各类胁迫因子中，高温是抑制细菌生长的关键因素之一。有鉴于此，学界一直在持续开发耐热菌株，但鲜有研究结合基因组分析来解析抗性机制的因果成因。此外，多重胁迫耐受性同样被认为具有重要价值。本研究分离得到一株名为BIOPOP-3的屎肠球菌（Enterococcus faecium）菌株，并完成了其全长基因组测序。以该菌株为出发菌株，本研究采用两步法适应性实验室进化（adaptive laboratory evolution, ALE）技术，获得了耐热菌株BIOPOP-3 ALE。对两株菌进行全基因组测序后，本研究比对了二者的全长基因组序列，鉴定出1个非同义变异位点与4个插入缺失（insertion-deletion, indel）变异位点，这些变异可能赋予菌株耐热性，且经重测序技术验证属实。实验验证表明，进化后的菌株不仅耐热性显著提升，同时耐酸性与耐胆盐能力也得到增强。本研究证实，所开发的耐热菌株可应用于动物饲料生产工艺中。本研究开发的多重胁迫抗性菌株BIOPOP-3 ALE及两步法适应性实验室进化技术，有望在工业与学术领域得到广泛应用。此外，本研究鉴定得到的耐热菌株特异性变异位点，将有助于加深人们对分子生物学机制的理解，并可广泛应用于生物工程领域。