Efficient utilisation of abundant renewable sugars and aromatic chemicals by natural or recombinant microorganisms is a key prerequisite for the economic viability of biotechnological valorisation of lignocellulosic residues. But biotechnologically relevant microorganisms rarely utilise aromatics, many lack xylose metabolism, and some prefer glucose or other available organic substrates over pentoses. To establish the utilisation or co-utilisation of renewable substrates by a cell factory, rational genetic interventions are often combined with adaptive laboratory evolution. However, comprehensive studies enabling a holistic understanding of adaptation processes primed by rational metabolic engineering and introduced synthetic genetic devices remain scarce.

The laboratory and industrial workhorse Pseudomonas putida, intensively studied for its potential to up-cycle waste lignin, was previously engineered to utilise and valorise the non-native sugars D-xylose and D-cellobiose, a glucose dimer. However, the assimilation of these substrates into the bacterial biochemical network via the exogenous pathways remained unresolved and inefficient. Here, we describe the elucidation and improvement of the semi-synthetic xylose metabolism and glucose-cellobiose co-metabolism in P. putida using computational and experimental tools. Using the multi-omics analyses, enzyme and cell growth assays, and reverse engineering, we demonstrate how the knowledge-directed metabolic engineering primed further adaptation of P. putida to xylose during laboratory evolution. Using the example of improved cellobiose and glucose co-consumption and valorisation, we also show the descriptive and predictive power of an upgraded genome-scale metabolic model constrained by kinetic and proteomic data. The findings presented here are instrumental for exploiting the semi-synthetic metabolism of renewable sugars in P. putida as well as for the understanding of bacterial adaptation to new substrates.

Learning Objectives: 

1. Review how systems metabolic engineering and laboratory evolution can be used to adapt bacteria to non-native substrates.

2. Summarize how directed engineering cuts can promote parallel evolutionary pathways towards the same phenotype.

3. Demonstrate knowledge on a new "carbon push" strategy enabling the aerobic overproduction of pyruvate from cellulosic sugars by a bacterial catalyst.