Development of Freeze-Thaw Tolerant Lactobacillus rhamnosus GG by Adaptive Laboratory Evolution
Development of Freeze-Thaw Tolerant Lactobacillus rhamnosus GG by Adaptive Laboratory Evolution
复制标题
DOI：
10.3389/fmicb.2018.02781
复制
发表时间：
2018-11-20
影响因子：
5.2
通讯作者：
Han, Nam Soo
中科院分区：
文献类型：
--
作者：
Kwon, Ye Won;Bae, Jae-Han;Han, Nam Soo
微生物作为发酵剂或益生菌的工业应用需要它们的保存以确保存活力和代谢活性。冷冻通常用于此目的，但由冰晶形成引起的冷损伤可能导致微生物活性严重降低。本研究应用适应性实验室进化（ALE）技术对一株乳酸菌进行了耐冻融菌株的筛选。对鼠李糖乳杆菌GG进行冻融生长（FTG）150个循环，重复4次。经过150个周期后，FTG进化的突变体表现出比祖先更好的适应性（存活率），更快的生长速度和更短的滞后期。对两个进化突变体的基因组测序分析显示，六个基因和一个基因间空间的远距离基因座存在遗传变异。功能丧失突变被认为改变了微生物细胞膜（c/s中的一个插入）、肽聚糖（dacA和murQ中的两个错义突变）和荚膜多糖（wze中的一个错义突变）的结构，导致细胞流动性增加。因此，L.利用FTG-ALE技术，在基因组DNA末端位点协同进化，成功地将鼠李糖酵母GG转化为耐逆突变体。这项研究首次报道了dacA和murQ在细胞冻融敏感性中的功能，并证明了适当设计的ALE的简单处理可以导致宿主微生物细胞中多个靶基因的智能遗传变化。
The industrial application of microorganisms as starters or probiotics requires their preservation to assure viability and metabolic activity. Freezing is routinely used for this purpose, but the cold damage caused by ice crystal formation may result in severe decrease in microbial activity. In this study, adaptive laboratory evolution (ALE) technique was applied to a lactic acid bacterium to select tolerant strains against freezing and thawing stresses. Lactobacillus rhamnosus GG was subjected to freeze-thaw-growth (FTG) for 150 cycles with four replicates. After 150 cycles, FTG-evolved mutants showed improved fitness (survival rates), faster growth rate, and shortened lag phase than those of the ancestor. Genome sequencing analysis of two evolved mutants showed genetic variants at distant loci in six genes and one intergenic space. Loss-of-function mutations were thought to alter the structure of the microbial cell membrane (one insertion in c/s), peptidoglycan (two missense mutations in dacA and murQ), and capsular polysaccharides (one missense mutation in wze), resulting in an increase in cellular fluidity. Consequently, L. rhamnosus GG was successfully evolved into stress-tolerant mutants using FTG-ALE in a concerted mode at distal loci of DNA. This study reports for the first time the functioning of dacA and murQ in freeze-thaw sensitivity of cells and demonstrates that simple treatment of ALE designed appropriately can lead to an intelligent genetic changes at multiple target genes in the host microbial cell.