Mind the Porins: Differential Effects of Porin Knockouts and Overexpression on Glucose and Xylose Uptake and Utilization in Pseudomonas putida.

Porins serve as the primary transport channels for substrate molecules across the outer membrane of Gram-negative bacteria. Despite their potential to influence substrate uptake in microbial cell factories, porins are often overlooked in metabolic engineering approaches. In this study, we investigate the impact of modulation of sugar porin expression using laboratory and industrial workhorse Pseudomonas putida. We first examined the P. putida porin repertoire through bioinformatic analysis. Among the two selected porin sets, only the one comprising OprB-I, OprB-II and OprB-III was found to be relevant for glucose catabolism in two biotechnologically important P. putida strains. Functional studies involving gene knockouts, complementation and overexpression revealed that the substrate specificity of P. putida OprB porins extends beyond glucose and includes the non-native substrate xylose. Overexpression of oprB-I alone was sufficient to restore sugar utilization in strains with all three oprB genes knocked out. Notably, when Gcd was active in P. putida, oprB-I overexpression accelerated the utilization of glucose and xylose in mixed sugar conditions through altered sugar uptake and oxidation dynamics. This work exposes the relevance of porins in shaping the uptake of major lignocellulosic sugars and highlights the importance of incorporating outer membrane transport considerations into metabolic engineering strategies for Gram-negative bacteria.