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Front. Microbiol. | doi: 10.3389/fmicb.2018.02781

Development of Freeze-Thaw Tolerant Lactobacillus rhamnosus GG by Adaptive Laboratory Evolution

Ye Won Kwon1,  Jae-Han Bae1, Seul-Ah Kim1 and  Nam Soo Han1*
  • 1Chungbuk National University, South Korea

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 create tolerant strains against freezing and thawing stress. 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 cls), 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.

Keywords: Adaptive laboratory evolution, Lactic acid bacteria, Lactobacillus rhamnosus GG, Freeze-thaw stress, Genome sequencing

Received: 01 Aug 2018; Accepted: 30 Oct 2018.

Edited by:

Giuseppe Spano, University of Foggia, Italy

Reviewed by:

Mariagiovanna Fragasso, Council for Agricultural and Economics Research, Italy
Teresa Zotta, Italian National Research Council, Italy  

Copyright: © 2018 Kwon, Bae, Kim and Han. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Prof. Nam Soo Han, Chungbuk National University, Cheongju, South Korea,