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

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.

Industrializing a Bacterial Strain for l-Serine Production through Translation Initiation Optimization.

Turning a proof-of-concept synthetic biology design into a robust, high performing cell factory is a major time and money consuming task, which severely limits the growth of the white biotechnology sector. Here, we extend the use of tunable antibiotic resistance markers for synthetic evolution (TARSyn), a workflow for screening translation initiation region (TIR) libraries with antibiotic selection, to generic pathway engineering, and transform a proof-of-concept synbio design into a process that performs at industrially relevant levels. Using a combination of rational design and adaptive evolution, we recently engineered a high-performing bacterial strain for production of the important building block biochemical l-serine, based on two high-copy pET vectors facilitating expression of the serine biosynthetic genes serA, serC, and serB from three independent transcriptional units. Here, we prepare the bacterial strain for industrial scale up by transferring and reconfiguring the three genes into an operon encoded on a single low-copy plasmid. Not surprisingly, this initially reduces production titers considerably. We use TARSyn to screen both experimental and computational optimization designs resulting in high-performing synthetic serine operons and reach industrially relevant production levels of 50 g/L in fed-batch fermentations, the highest reported so far for serine production.

Tolerance and metabolic response of Pseudomonas taiwanensis VLB120 towards biomass hydrolysate-derived inhibitors.

BACKGROUND: Bio-conversion of lignocellulosic biomass to high-value products offers numerous benefits; however, its development is hampered by chemical inhibitors generated during the pretreatment process. A better understanding of how microbes naturally respond to those inhibitors is valuable in the process of designing microorganisms with improved tolerance. Pseudomonas taiwanensis VLB120 is a natively tolerant strain that utilizes a wide range of carbon sources including pentose and hexose sugars. To this end, we investigated the tolerance and metabolic response of P. taiwanensis VLB120 towards biomass hydrolysate-derived inhibitors including organic acids (acetic acid, formic acid, and levulinic acid), furans (furfural, 5-hydroxymethylfurfural), and phenols (vanillin). RESULTS: The inhibitory effect of the tested compounds varied with respect to lag phase, specific growth rate, and biomass yield compared to the control cultures grown under the same conditions without addition of inhibitors. However, P. taiwanensis was able to oxidize vanillin and furfural to vanillic acid and 2-furoic acid, respectively. Vanillic acid was further metabolized, whereas 2-furoic acid was secreted outside the cells and remained in the fermentation broth without further conversion. Acetic acid and formic acid were completely consumed from the fermentation broth, while concentration of levulinic acid remained constant throughout the fermentation process. Analysis of free intracellular metabolites revealed varying levels when P. taiwanensis VLB120 was exposed to inhibitory compounds. This resulted in increased levels of ATP to export inhibitors from the cell and NADPH/NADP ratio that provides reducing power to deal with the oxidative stress caused by the inhibitors. Thus, adequate supply of these metabolites is essential for the survival and reproduction of P. taiwanensis in the presence of biomass-derived inhibitors. CONCLUSIONS: In this study, the tolerance and metabolic response of P. taiwanensis VLB120 to biomass hydrolysate-derived inhibitors was investigated. P. taiwanensis VLB120 showed high tolerance towards biomass hydrolysate-derived inhibitors compared to most wild-type microbes reported in the literature. It adopts different resistance mechanisms, including detoxification, efflux, and repair, which require additional energy and resources. Thus, targeting redox and energy metabolism in strain engineering may be a successful strategy to overcome inhibition during biomass hydrolysate conversion and lead to development of more robust strains.

Quantifying the Metabolome of Pseudomonas taiwanensis VLB120: Evaluation of Hot and Cold Combined Quenching/Extraction Approaches.

Absolute quantification of free intracellular metabolites is a valuable tool in both pathway discovery and metabolic engineering. In this study, we conducted a comprehensive examination of different hot and cold combined quenching/extraction approaches to extract and quantify intracellular metabolites of Pseudomonas taiwanensis (P. taiwanensis) VLB120 to provide a useful reference data set of absolute intracellular metabolite concentrations. The suitability of commonly used metabolomics tools including a pressure driven fast filtration system followed by combined quenching/extraction techniques (such as cold methanol/acetonitrile/water, hot water, and boiling ethanol/water, as well as cold ethanol/water) were tested and evaluated for P. taiwanensis VLB120 metabolome analysis. In total 94 out of 107 detected intracellular metabolites were quantified using an isotope-ratio-based approach. The quantified metabolites include amino acids, nucleotides, central carbon metabolism intermediates, redox cofactors, and others. The acquired data demonstrate that the pressure driven fast filtration approach followed by boiling ethanol quenching/extraction is the most adequate technique for P. taiwanensis VLB120 metabolome analysis based on quenching efficiency, extraction yields of metabolites, and experimental reproducibility.