Building a syntrophic Pseudomonas putida consortium with reciprocal substrate processing of lignocellulosic disaccharides

Synthetic microbial consortia can leverage their expanded enzymatic reach to tackle biotechnological challenges too complex for single strains, such as lignocellulose valorisation. The benefit of metabolic cooperation comes with a catch - installing stable interactions between consortium members. We constructed a syntrophic consortium of Pseudomonas putida strains for lignocellulosic disaccharide processing. Two strains were engineered to hydrolyse and metabolise lignocellulosic sugars: one grows on xylose and hydrolyses cellobiose to produce glucose, while the other grows on glucose and cleaves xylobiose to produce xylose. This specialisation allows each strain to provide essential growth substrate to its partner, establishing a stable mutualistic interaction, which we term reciprocal substrate processing. Key enzymes from Escherichia coli (xylose isomerase pathway) and Thermobifida fusca (glycoside hydrolases) were introduced into P. putida to broaden its carbohydrate utilisation capabilities and arranged in a way to install the strain cross-dependency. A mathematical model of the consortium assisted in predicting the effects of substrate composition, strain ratios, and protein expression levels on population dynamics. Our results demonstrated that modulating extrinsic factors such as substrate concentration can optimise growth and balance fitness disparities between the strains, but achieving this by altering intrinsic factors such as glycoside hydrolase expression levels is much more challenging. This study underscores the potential of synthetic microbial consortia to facilitate the bioconversion of lignocellulosic sugars and offers insights into overcoming the challenges of establishing synthetic microbial cooperation.