Abstract
BackgroundMicrobial 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.ResultsThe 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.ConclusionsWe 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.
Highlights
Microbial production of biofuels requires robust cell growth and metabolism under tough conditions
Modified proofreading elements of the DNA polymerase complex (ε subunit encoded by dnaQ gene) were used to trigger perturbations on genome replication for in vivo continuous mutagenesis
Procedure of the “Genome Replication Engineering Assisted Continuous Evolution” The core idea of GREACE is to introduce genetic perturbations into genome replication machinery, so that in vivo continuous mutagenesis can be coupled with simultaneous phenotypes selection
Summary
Microbial production of biofuels requires robust cell growth and metabolism under tough conditions. Efficient microbial production of biofuels from renewable resources requires robust cell growth and stable metabolism under tough industrial conditions, represented by inhibitory components in substrates and toxic products [1,2] Microbial tolerance to these inhibitory environmental factors is a complex phenotype usually controlled by multiple genes [3,4], and is difficult to be engineered by targeted metabolic engineering approaches [5]. Examples of evolutionary engineering include successive passage for metabolic evolution [7,8,9], physical and chemical mutagenesis [10], global transcription machinery engineering [2,11], artificial transcription factors engineering [12,13], and ribosome engineering [14] All these methods use “Mutagenesis followed-by Selection” as core principle, meaning that firstly introducing genetic diversity by spontaneous mutations, exogenous mutagens, or genetic perturbations, followed by selection of desired phenotypes [6].
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