Genome replication engineering assisted continuous evolution (GREACE) to improve microbial tolerance for biofuels production

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.

In vivo continuous directed evolution

“…Indeed, in vivo continuous evolution methods can enable > 10 11 protein variants to be generated and subjected to selection over > 100 generations of evolution in less than a week [20]. The efficiency of continuous evolution methods can enable long evolutionary trajectories [14,23,32,33] or access to highly evolved biomolecules with new properties that would otherwise require impractical time scales [1,2,18,24,27,31,38]. In addition, some continuous evolution platforms, including several described above, can avoid genetic bottlenecks from modest population sizes, modest screening throughput, or modest mutation rates that commonly constrain some traditional laboratory evolution methods.…”

“…In addition, some continuous evolution platforms, including several described above, can avoid genetic bottlenecks from modest population sizes, modest screening throughput, or modest mutation rates that commonly constrain some traditional laboratory evolution methods. The minimal reliance on researcher intervention during continuous evolution can also make performing multiple parallel evolution experiments more accessible than using traditional directed evolution platforms [12–14,18–20,22–24,27,31,39]. …”

“…Dominant-negative variants of DnaQ provided in trans are known to have dramatic consequences on replication fidelity [30]. Integrating these principles, Ma and coworkers constructed a library of dnaQ mutants using error-prone PCR to be expressed constitutively in continuously growing bacterial cultures [31]. Selection pressures in the form of increasing concentrations of n -butanol or acetic acid were applied to the growing cultures.…”

“…Within 24 to 36 days of continuous evolution, the E. coli populations became adapted to the high solvent concentrations. As the researchers predicted, all the dnaQ variants that evolved in surviving cells were more mutagenic than the wildtype dnaQ , increased the mutation rate by up to 2,800-fold, and contained mutations known to dramatically enhance mutagenesis [30,31]. …”

Biological conversion of methanol by evolved Escherichia coli carrying a linear methanol assimilation pathway

“…To further improve the microbial performance in methanol medium and screen methanol-utilizing mutants, adaptive evolution based on GREACE (genome replication engineering assisted continuous evolution) was conducted (Luan et al 2013). A proofreading-defective element of the DNA polymerase of E. coli (ε subunit encoded by dnaQ gene) was expressed in strain Ec-ΔfrmA-mdh2 PB1 -fls to introduce random mutations into the genomic DNA during continuous passage cultivation in LB medium.…”