Genome Replication Engineering Assisted Continuous Evolution (GREACE) to Improve Microbial Tolerance for Biofuels Production

Abstract

Background: Microbial production of biofuels requires robust cell growth and metabolism under tough conditions. Conventionally, such tolerance phenotypes were engineered 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. This work aimed to develop a more continuous and efficient evolutionary engineering method termed as "Genome Replication Engineering Assisted Continuous Evolution" (GREACE) using "Mutagenesis coupled-with Selection" as its core principle. Results: 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 showed that GREACE can also be applied to engineer n-butanol and acetate tolerances. In less than a month, an E. coli strain capable of growing under an n-butanol concentration of 1.25% was isolated. As for acetate tolerance, cell growth of the evolved E. coli strain increased by 8-fold under 0.1% of acetate. In addition, we discovered that adaptation to specific stresses prefers accumulation of genetically modified elements with specific mutator strengths. Conclusions: We developed a novel GREACE method using "Mutagenesis coupled-with Selection" as core principle. Successful isolation of E. coli strains with improved n-butanol and acetate tolerances demonstrated the potential of GREACE as a promising method for strain improvement in biofuels production.

Advanced strategies and tools to facilitate and streamline microbial adaptive laboratory evolution

“…Recently, the development of modern molecular biology has contributed to the generation of several novel in vivo mutagenesis methods (Table 2). On the one hand, global in vivo mutagenesis methods, like genome replication engineering-assisted continuous evolution (GREACE) [91,92] and mutagenesis plasmid (MP6) [93], are proposed to increase global mutation rates in E. coli and S. cerevisiae by overexpression of mutator genes on plasmid vectors (Figure 3A). It was notable that, by expressing six mutator genes simultaneously, the MP6 mutator plasmid increased mutation rate by 322 000-fold in E. coli, surpassing various traditional in vivo mutagenesis methods.…”

“…ICE was successfully applied in improving a two-enzyme xylose catabolism-related pathway [103]. And GREACE facilitated the improvement of 1-butanol and acetate tolerance after introducing global perturbations [91]. Notably, when targeted to a global transcription factor [105], targeted methods also have the potential to reprogram genome-wide gene expression and unlock complex phenotypes.…”

Strategies for directed and adapted evolution as part of microbial strain engineering

“…In this regard, the approach of growth enrichment is most effective for improving stress tolerance and nutrient usage as these phenotypes easily couple with cell growth . As examples, growth selection linked with gene diversity strategies enable the selection of variants with improved growth rate in extreme environments, such as low pH and high temperature, as well as high concentrations of toxic compounds such as ionic liquids . Catabolic pathways can likewise be improved via directed evolution and growth selection.…”

“…To address this, Luan et al . established a genome replication engineering assisted continuous evolution (GREACE) strategy to improve a strain's tolerance to extreme environments (Fig. (F)).…”

“…Specifically, by introducing a proofreading element library for the E. coli DNA polymerase complex it was possible to select for the ideal mutator gene. This method successfully improved thermotolerance (capable of growth in 50°C), kanamycin resistance (capable of growth in 300 µg L −1 kanamycin), n‐butanol (capable of growth in 1.25% n‐butanol) and acetate (8‐fold improvement in 0.1% acetate) tolerances of E. coli …”