Biology subjects

Fernandez-Cabezon, L.

Publications and source records attributed to Fernandez-Cabezon, L..

2 recordsLinked to original sources

bioRxiv2022

A synthetic C2 auxotroph of Pseudomonas putida for evolutionary engineering of alternative sugar catabolic routes

Acetyl-coenzyme A (AcCoA) is a metabolic hub in virtually all living cells, serving as both a key precursor of essential biomass components and a metabolic sink for catabolic pathways of a large variety of substrates. Owing to this dual role, tight growth-production coupling schemes can be implemented around the AcCoA node. Inspired by this concept, a synthetic C2 auxotrophy was implemented in the platform bacterium Pseudomonas putida through an in silico-guided engineering approach. A growth-coupling strategy, driven by AcCoA demand, allowed for direct selection of an alternative sugar assimilation route--the phosphoketolase (PKT) shunt from bifidobacteria. Adaptive laboratory evolution forced the synthetic auxotroph to integrate the PKT shunt to restore C2 prototrophy. Large-scale structural chromosome rearrangements were identified as possible mechanisms for adjusting the network-wide proteome profile, resulting in improved PKT-dependent growth phenotypes. 13C-based metabolic flux analysis revealed an even split between the native Entner-Doudoroff and the synthetic PKT pathway for glucose processing, leading to enhanced carbon conservation. These results demonstrate that the P. putida metabolism can be radically rewired to incorporate a synthetic C2 metabolism, creating novel network connectivities and highlighting the importance of unconventional engineering strategies to support efficient microbial production.

synthetic biology↗

bioRxiv2021

Spatiotemporal manipulation of the mismatch repair system of Pseudomonas putida accelerates phenotype emergence

Developing 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 towards increasing mutation frequencies--yet the precise spatiotemporal control of mutagenesis processes continues to represent a significant technical challenge. Pseudomonas species are endowed with one of the most efficient DNA mismatch repair (MMR) systems found in bacteria. 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 mutLE36K allele. Regulatable overexpression of mutLE36K 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 mutation frequencies and a heritable fixation of genome mutations. Following such accelerated mutagenesis-followed-by selection approach, three phenotypes were successfully evolved: resistance to antibiotics streptomycin and rifampicin and reversion of a synthetic uracil auxotrophy. Thus, these mutator devices could be applied to accelerate evolution of metabolic pathways in long-term evolutionary experiments, alternating cycles of (inducible) mutagenesis coupled to selection schemes.

synthetic biology↗