The metabolic response of Pseudomonas taiwanensis to NADH dehydrogenase deficiency

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Abstract

Abstract Obligate aerobic organisms rely on a functional electron transport chain for energy generation and NADH oxidation. Because of this essential requirement, the genes of this pathway are likely constitutively and highly expressed to avoid a cofactor imbalance and energy shortage under fluctuating environmental conditions. We here investigated the essentiality of the three NADH dehydrogenases of the respiratory chain of the obligate aerobe Pseudomonas taiwanensis VLB120 and the impact of the knockouts of corresponding genes on its physiology and metabolism. While a mutant lacking all three NADH dehydrogenases seemed to be nonviable, the generated single or double knockout strains displayed none or only a marginal phenotype. Only the mutant deficient in both type 2 dehydrogenases showed a clear phenotype with biphasic growth behavior and strongly reduced growth rate in the second phase. In-depth analyses of the metabolism of the generated mutants including quantitative physiological experiments, transcript analysis, proteomics and enzyme activity assays revealed distinct responses to type II and type I dehydrogenase deletions. An overall high metabolic flexibility enables P. taiwanensis to cope with the introduced genetic perturbations and maintain stable phenotypes by rerouting of metabolic fluxes. This metabolic adaptability has implications for biotechnological applications. While the phenotypic robustness is favorable in large-scale applications with inhomogeneous conditions, versatile redirecting of carbon fluxes upon genetic interventions can frustrate metabolic engineering efforts. Importance While Pseudomonas has the capability for high metabolic activity and the provision of reduced redox cofactors important for biocatalytic applications, exploitation of this characteristic might be hindered by high, constitutive activity of and consequently competition with the NADH dehydrogenases of the respiratory chain. The in-depth analysis of NADH dehydrogenase mutants of Pseudomonas taiwanensis VLB120 presented here, provides insight into the phenotypic and metabolic response of this strain to these redox metabolism perturbations. The observed great metabolic flexibility needs to be taken into account for rational engineering of this promising biotechnological workhorse towards a host with controlled and efficient supply of redox cofactors for product synthesis.

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