Metabolomics Analysis Reveals Global Metabolic Changes in the Evolved E. coli Strain with Improved Growth and 1-Butanol Production in Minimal Medium

Laviña, Walter A.; Sakurai, Sana Subhan Memon; Pontrelli, Sammy; Putri, Sastia Prama; Fukusaki, Eiichiro

doi:10.3390/metabo10050192

Cited by 4 publications

(2 citation statements)

References 26 publications

(52 reference statements)

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“…, mutL and mutS ) or in proofreading DNA polymerases ( e.g. , dnaQ ), as well as the overexpression of certain dominant-negative mutator alleles of the same genes, are known to result in mutator phenotypes. , Conditional mutator phenotypes have been applied to the phenotypic optimization of MCFs. − However, a major problem of these conditional phenotypes is the relatively low control of temporal activity afforded by the cognate devices. A typical drawback of these systems is that the ability to effectively halt DNA mutagenesis is limited, and the cells will continue to mutate even after a desired phenotype is achieved …”

Section: Introductionmentioning

confidence: 99%

Spatiotemporal Manipulation of the Mismatch Repair System of Pseudomonas putida Accelerates Phenotype Emergence

2021

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The development of complex phenotypes in industrially relevant bacteria is a major goal of metabolic engineering, which encompasses the implementation of both rational and random approaches. In the latter case, several tools have been developed toward increasing mutation frequencies, yet the precise control of mutagenesis processes in cell factories continues to represent a significant technical challenge. Pseudomonas species are endowed with one of the most efficient DNA mismatch repair (MMR) systems found in the bacterial domain.Here, we investigated if the endogenous MMR system could be manipulated as a general strategy to artificially alter mutation rates in Pseudomonas species. To bestow a conditional mutator phenotype in the platform bacterium Pseudomonas putida, we constructed inducible mutator devices to modulate the expression of the dominant-negative mutL E36K allele. Regulatable overexpression of mutL E36K in a broad-host-range, easy-to-cure plasmid format resulted in a transitory inhibition of the MMR machinery, leading to a significant increase (up to 438-fold) in DNA mutation frequencies and a heritable fixation of mutations in the genome. Following such an accelerated mutagenesis-followed by selection approach, three phenotypes were successfully evolved: resistance to antibiotics streptomycin and rifampicin (either individually or combined) and reversion of a synthetic uracil auxotrophy. Thus, these mutator devices could be applied to accelerate the evolution of metabolic pathways in long-term evolutionary experiments, alternating cycles of (inducible) mutagenesis coupled to selection schemes toward the desired phenotype(s).

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Section: Introductionmentioning

confidence: 99%

Spatiotemporal Manipulation of the Mismatch Repair System of Pseudomonas putida Accelerates Phenotype Emergence

2021

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“…We are convinced that quantitative approaches in metabolite analysis will help to reduce the time required to establish an efficient whole-cell biocatalyst. In this Special Issue, Laviña, et al demonstrated how metabolomics was utilized to characterize adaptive laboratory evolution Escherichia coli strains [ 1 ]. With the targeted strategy, metabolites in the central carbon metabolic pathways were quantified to understand the overall effect of the mutations acquired through evolution to improve 1-butanol production.…”

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confidence: 99%