Li Yin
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering，CAS-TWAS Centre of Excellence for Biotechnology，Institute of Microbiology, Chinese Academy of Sciences
Microbial production of biofuels requires robust cell growth and metabolism under tough conditions. Conventionally, such tolerance phenotypes were acquired through evolutionary engineering using the principle of “Mutagenesis followed-by Selection”. The iterative rounds of mutagenesis-selection and frequent manual interventions resulted in discontinuous and inefficient strain improvement processes. We developed a continuous and more efficient evolutionary engineering method termed as “Genome Replication Engineering Assisted Continuous Evolution” (GREACE) using “Mutagenesis coupled-with Selection” as its core principle. The core design of GREACE is to introduce an in vivo continuous mutagenesis mechanism into microbial cells by introducing a group of genetically modified proofreading elements of the DNA polymerase complex to accelerate the evolution process under stressful conditions. The genotype stability and phenotype heritability can be stably maintained once the genetically modified proofreading element is removed, thus scarless mutants with desired phenotypes can be obtained. Kanamycin resistance of E. coli was rapidly improved to confirm the concept and feasibility of GREACE. Intrinsic mechanism analysis revealed that during the continuous evolution process, the accumulation of genetically modified proofreading elements with mutator activities endowed the host cells with enhanced adaptation advantages. We further demonstrated that GREACE can be applied to engineer microbial tolerance to n-butanol and acetate. In addition, we discovered that adaptation to specific stresses prefers accumulation of genetically modified elements with specific mutator strengths. Successful isolation of E. coli strains with improved n-butanol and acetate tolerances demonstrated the potential of GREACE as a promising approach for strain improvement in biofuels production.
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