GnuR Represses the Expression of Glucose and Gluconate Catabolism in Pseudomonas putida KT2440

In Pseudomonas putida KT2440, a prime chassis for biotechnology, the clustered distribution of glucose catabolism genes and four related transcription factors (TFs) may facilitate the tight regulation of glucose catabolism. However, the genes under the direct control of these TFs remain unidentified, leaving their regulatory roles elusive. Furthermore, the carbon source gluconate was metabolised similarly to glucose in KT2440, but the responses of these catabolic and TF genes to gluconate were unclear. Here, these mysteries were unravelled through multi-omics analysis integrated with physiological studies. First, we found that the expression of these catabolic and TF genes were significantly induced by both glucose and gluconate in KT2440. The independent responses of these genes to glucose and gluconate were differentiated in the gcd deletion mutant. We then defined the regulon of GnuR, one of the four related TFs, and discovered that GnuR directly repressed the expression of catabolic genes involved in the Entner–Doudoroff and the peripheral glucose and gluconate metabolism pathways. These results were further confirmed by physiological studies. Finally, a regulatory mode of an incoherent feedforward loop involving GnuR is proposed.