Synthetically primed adaptation of Pseudomonas putida to a non-native substrate D-xylose

To broaden microbial cell factories´ substrate scope towards renewable substrates, rational genetic interventions are often combined with adaptive laboratory evolution (ALE). However, comprehensive studies enabling a holistic understanding of adaptation processes primed by detailed knowledge of metabolism remain scarce, especially for non-model organisms. The industrial workhorse Pseudomonas putida was engineered to utilize the non-native sugar D-xylose, but its assimilation into the bacterial biochemical network via the exogenous pathway remained unresolved. Here, we elucidated the xylose metabolism and established a foundation for further engineering followed by ALE. By de-repressing native glycolysis, we unlocked the route for xylose-derived carbon and obtained a strain with a substantially reduced lag phase on xylose. We then enhanced the pentose phosphate pathway in two lag-shortened strains and allowed ALE to fine-tune the rewired metabolism. Following the metabolism tuning, we employed multi-level analysis that provided unique insights into the parallel paths of bacterial adaptation to the non-native carbon source.