Systems Analysis of NADH Dehydrogenase Mutants Reveals Flexibility and Limits of Pseudomonas taiwanensis VLB120’s Metabolism

Abstract

Obligate aerobic organisms rely on a functional electron transport chain for energy conservation 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 single or double knockout mutant strains displayed no, or only a weak, phenotype. Only the mutant deficient in both type 2 dehydrogenases showed a clear phenotype with biphasic growth behavior and a 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 2 and type 1 dehydrogenase deletions. An overall high metabolic flexibility enables P. taiwanensis to cope with the introduced genetic perturbations and maintain stable phenotypes, likely 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, the possible versatile redirecting of carbon fluxes upon genetic interventions can thwart 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. This high degree of metabolic flexibility needs to be taken into account for rational engineering of this promising biotechnological workhorse toward a host with a controlled and efficient supply of redox cofactors for product synthesis.

Cytochrome c Reductase is a Key Enzyme Involved in the Extracellular Electron Transfer Pathway towards Transition Metal Complexes in Pseudomonas Putida

“…P. putida has both NDH‐I and NDH‐II, while the NDH‐I is typically the major complex . This was observed for Arabidopsis , where the respiration rate was inhibited by rotenone for the first 4 h and then progressively recovered to initial levels within 32 h. Similar internal compensation of NADH dehydrogenase activity was also confirmed for P. taiwanensis VLB120, a strain that shares 98.9 % genome similarity with P. putida . Nevertheless, the results confirmed the inhibition of rotenone on NADH dehydrogenase activity and consequently confirmed the active role of NADH dehydrogenase in the EET route.…”

High titer methyl ketone production with tailoredPseudomonas taiwanensisVLB120

“…The highest reported glucose uptake rate for P. taiwanensis VLB120 of 10 mmol gCDW -1 h -1 (Rühl et al, 2009) translates into a maximal product synthesis rate of 0.6 gMK gCDW -1 h -1 and a NAD(P)H turnover of 52 mmol gCDW -1 h -1 . While this is about 2-fold the rate under standard growth conditions (at a specific growth rate of 0.68 h -1 (Nies et al, 2020)), this demand does not challenge the redox cofactor regeneration capacity of P. taiwanensis VLB120 reported to reach 149 mmol gCDW -1 h -1 (Rühl et al, 2009). These in silico analyses indicate a substantial room for improvement of methyl ketone synthesis in P. taiwanensis VLB120 by metabolic engineering.…”

High titer methyl ketone production with tailored Pseudomonas taiwanensis VLB120

“…Optimization of the redox metabolism, such as redox cofactor engineering, shown to be important for methyl ketone production in E. coli, was not pursued. We argue that the high flexibility of Pseudomonas to balance redox cofactors (Kohlstedt and Wittmann, 2019;Nies et al, 2020;Nikel et al, 2016) renders such elaborate engineering efforts superfluous. Moreover, the performed in silico analysis showed that the capacity of the strain for methyl ketone production has not been fully exhausted and can be increased further by metabolic engineering.…”

“…While this is about 2-fold the rate under standard growth conditions (at a specific growth rate of 0.68 h -1 (Nies et al, 2020)), this demand does not challenge the redox cofactor regeneration capacity of P. taiwanensis VLB120 reported to reach 149 mmol gCDW -1 h -1 (Rühl et al, 2009). These in silico analyses indicate a substantial room for improvement of methyl ketone synthesis in P. taiwanensis VLB120 by metabolic engineering.…”