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

Abstract

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.

Genomic and metabolic plasticity drive alternative scenarios for adaptingPseudomonas putidato non-native substrate D-xylose

“…The selection of a suitable microbial host is a critical first step for the success of any bioengineering project employing ALE (Sandberg et al, 2019). In agreement with other recently published works, our results suggest that P. putida KT2440 and its derivatives are attractive templates for evolutionary experiments that can be directed by knowledgeguided design (Ling et al, 2022;Wirth et al, 2022). Such an approach adopted here provided P. putida strains with a doubled growth rate and substantially reduced growth lag on xylose, which make them promising templates for the biotechnological valorization of xylose or its co-valorization with glucose.…”

“…Transposable elements and gene duplication events are key factors in the genome evolution and environmental adaptability of Pseudomonas species (Silby et al, 2011). Our study, in agreement with several other recent works (Ling et al, 2022;Wirth et al, 2022), highlights the contribution of these types of genome rearrangements in the evolution of biotechnologically relevant genotypes/phenotypes of P.…”