Comparative genome analysis of a thermotolerant Escherichia coli obtained by Genome Replication Engineering Assisted Continuous Evolution (GREACE) and its parent strain provides new understanding of microbial heat tolerance.
Heat tolerance of microbes is of great importance for efficient biorefinery and bioconversion. However, engineering and understanding of microbial heat tolerance are difficult and insufficient because it is a complex physiological trait which probably correlates with all gene functions, genetic regulations, and cellular metabolisms and activities. In this work, a novel strain engineering approach named Genome Replication Engineering Assisted Continuous Evolution (GREACE) was employed to improve the heat tolerance of Escherichia coli. When the E. coli strain carrying a mutator was cultivated under gradually increasing temperature, genome-wide mutations were continuously generated during genome replication and the mutated strains with improved thermotolerance were autonomously selected. A thermotolerant strain HR50 capable of growing at 50°C on LB agar plate was obtained within two months, demonstrating the efficiency of GREACE in improving such a complex physiological trait. To understand the improved heat tolerance, genomes of HR50 and its wildtype strain DH5α were sequenced. Evenly distributed 361 mutations covering all mutation types were found in HR50. Closed material transportations, loose genome conformation, and possibly altered cell wall structure and transcription pattern were the main differences of HR50 compared with DH5α, which were speculated to be responsible for the improved heat tolerance. This work not only expanding our understanding of microbial heat tolerance, but also emphasizing that the in vivo continuous genome mutagenesis method, GREACE, is efficient in improving microbial complex physiological trait.
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
Guodong
G
Luan
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
Guanhui
G
Bao
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
Zhao
Z
Lin
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
Yang
Y
Li
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; Department of Human Genetics, School of Medicine, University of California, Los Angeles, CA 90095, United States.
Zugen
Z
Chen
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
Yin
Y
Li
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: caiz@im.ac.cn.
Zhen
Z
Cai
D000222:Adaptation, Physiological / Q000235:genetics*; D055028:Comparative Genomic Hybridization; D004261:DNA Replication / Q000235:genetics*; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D024861:Genetic Enhancement / Q000379:methods; D016680:Genome, Bacterial / Q000235:genetics*; D018869:Heat-Shock Response / Q000235:genetics*; D006358:Hot Temperature