Metabolic and Regulatory Rearrangements Underlying Efficient d-Xylose Utilization in Engineered Pseudomonas putida S12

Previously, an efficient d-xylose utilizing Pseudomonas putida S12 strain was obtained by introducing the d-xylose isomerase pathway from Escherichia coli, followed by evolutionary selection. In the present study, systemic changes associated with the evolved phenotype were identified by transcriptomics, enzyme activity analysis, and inverse engineering. A key element in improving the initially poor d-xylose utilization was the redistribution of 6-phospho-d-gluconate (6-PG) between the Entner-Doudoroff pathway and the oxidative pentose phosphate (PP) pathway. This redistribution increased the availability of 6-PG for oxidative decarboxylation to d-ribose-5-phosphate, which is essential for the utilization of d-xylose via the nonoxidative PP pathway. The metabolic redistribution of 6-PG was procured by modified HexR regulation, which in addition appeared to control periplasmic sugar oxidation. Because the absence of periplasmic d-xylonate formation was previously demonstrated to be essential for achieving a high biomass yield on d-xylose, the aberrant HexR control appeared to underlie both the improved growth rate and biomass yield of the evolved d-xylose utilizing P. putida strain. The increased oxidative PP pathway activity furthermore resulted in an elevated NADH/NAD+ ratio that caused the metabolic flux to be redirected from the TCA cycle to the glyoxylate shunt, which was also activated transcriptionally. Clearly, these findings may serve as an important case in point to engineer and improve the utilization of non-natural carbon sources in a wide range of industrial microorganisms.