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

Abstract

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).

Modular (de)construction of complex bacterial phenotypes by CRISPR/nCas9-assisted, multiplex cytidine base-editing

“…Genomic DNA was purified using the PureLink TM Genomic DNA purification kit (Invitrogen, Waltham, MA, USA) from 2 mL of overnight LB cultures of isolated clones as described previously 98 – 101 . DNA was randomly sheared into short fragments of ca.…”

The pAblo·pCasso self-curing vector toolset for unconstrained cytidine and adenine base-editing in Gram-negative bacteria

“…A 2-ml aliquot of these cultures was used to isolate genomic DNA (gDNA) with the PureLink™ gDNA mini kit (ThermoFisher Scientific). Library construction, sequencing, data analysis and subsequent data quality control were performed by Novogene Co. Ltd (Cambridge, United Kingdom) as previously described ( 41 ). Briefly, gDNA was randomly sheared into short fragments, and the obtained fragments were end-repaired, A-tailed and further ligated with Illumina adapters.…”

The pAblo·pCasso self-curing vector toolset for unconstrained cytidine and adenine base-editing in Gram-negative bacteria

“…A 2-mL aliquot of these cultures was used to isolate genomic DNA (gDNA) with the PureLink ™ gDNA mini kit (ThermoFisher Scientific). Library construction, sequencing, data analysis and subsequent data quality control were performed by Novogene Co. Ltd. (Cambridge, United Kingdom) as previously described (Fernández-Cabezón et al ., 2021). Briefly, gDNA was randomly sheared into short fragments, and the obtained fragments were end-repaired, A-tailed and further ligated with Illumina adapters.…”