The metabolic response ofPseudomonas taiwanensisto NADH dehydrogenase deficiency

Abstract

30Obligate aerobic organisms rely on a functional electron transport chain for energy 31 generation and NADH oxidation. Because of this essential requirement, the genes of 32 this pathway are likely constitutively and highly expressed to avoid a cofactor imbalance 33 and energy shortage under fluctuating environmental conditions. 34We here investigated the essentiality of the three NADH dehydrogenases of the 35 respiratory chain of the obligate aerobe Pseudomonas taiwanensis VLB120 and the 36 impact of the knockouts of corresponding genes on its physiology and metabolism. 37While a mutant lacking all three NADH dehydrogenases seemed to be nonviable, the 38 generated single or double knockout strains displayed none or only a marginal 39 phenotype. Only the mutant deficient in both type 2 dehydrogenases showed a clear 40 phenotype with biphasic growth behavior and strongly reduced growth rate in the 41 second phase. In-depth analyses of the metabolism of the generated mutants including 42 quantitative physiological experiments, transcript analysis, proteomics and enzyme 43 activity assays revealed distinct responses to type II and type I dehydrogenase 44deletions. An overall high metabolic flexibility enables P. taiwanensis to cope with the 45 introduced genetic perturbations and maintain stable phenotypes by rerouting of 46 metabolic fluxes. 47 This metabolic adaptability has implications for biotechnological applications. While the 48 phenotypic robustness is favorable in large-scale applications with inhomogeneous 49 conditions, versatile redirecting of carbon fluxes upon genetic interventions can frustrate 50 metabolic engineering efforts. 51 52 Importance 53While Pseudomonas has the capability for high metabolic activity and the provision of 54 reduced redox cofactors important for biocatalytic applications, exploitation of this 55 3 characteristic might be hindered by high, constitutive activity of and consequently 56 competition with the NADH dehydrogenases of the respiratory chain. The in-depth 57 analysis of NADH dehydrogenase mutants of Pseudomonas taiwanensis VLB120 58 presented here, provides insight into the phenotypic and metabolic response of this 59 strain to these redox metabolism perturbations. The observed great metabolic flexibility 60 needs to be taken into account for rational engineering of this promising 61 biotechnological workhorse towards a host with controlled and efficient supply of redox 62 cofactors for product synthesis. 63 64 65Many industrially relevant molecules, e. g., ethanol, butanediol or isoprene, are more 66 reduced than the industrially-used sugars glucose and sucrose or alternative, upcoming 67 carbon sources such as xylose or glycerol (1-3). The microbial production of those 68 favored compounds hence is inherently redox limited, i.e. by the supply of reduced 69 redox cofactors, generally NADH or NADPH. This bottleneck has been overcome in 70 some cases, e.g., 1,4 butanediol and 1,3-propanediol production in Escherichia coli (4, 71 5) or L-lysine synthesis in Corynebacter...

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.…”

Improved production of 2,3‐butanediol and isobutanol by engineering electron transport chain in Escherichia coli

“…Further studies utilizing external energy sources can be helpful to resolve the problems related to growth retardation and acetate accumulation in ETC mutants. It has been also documented that the inner membrane space is filled with various membrane proteins such as transporters and components of the respiration machinery (Bernsel and Daley, 2009; Papanastasiou et al ., 2013). Thus, the removal of ETC components, which secures considerable volume of the membrane, could increase allocation of sugar transporters in the inner membrane space (Szenk et al ., 2017).…”

“…The metabolic changes owing to mutations of ETC components have been demonstrated in various bacteria such as E. coli, Pseudomonas sp., Corynebacterium glutamicum, Bacillus subtilis, Lactococcus lactis and Zymomonas mobilis (Nies et al, 2019;Koch-Koerfges et al, 2013;Zhu et al, 2011;Kalnenieks et al, 2019;Wu et al, 2015). In most of the cases, the manipulation of respiration level resulted in retardation of growth, decreased oxygen uptake and improved production of organic compounds, such as lactate, acetate, succinate and ethanol, under aerobic condition.…”