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Engineering of Pseudomonas taiwanensis VLB120 for constitutive solvent tolerance and increased specific styrene epoxidation activity.

ABSTRACT: The application of whole cells as biocatalysts is often limited by the toxicity of organic solvents, which constitute interesting substrates/products or can be used as a second phase for in situ product removal and as tools to control multistep biocatalysis. Solvent-tolerant bacteria, especially Pseudomonas strains, are proposed as promising hosts to overcome such limitations due to their inherent solvent tolerance mechanisms. However, potential industrial applications suffer from tedious, unproductive adaptation processes, phenotypic variability, and instable solvent-tolerant phenotypes. In this study, genes described to be involved in solvent tolerance were identified in Pseudomonas taiwanensis VLB120, and adaptive solvent tolerance was proven by cultivation in the presence of 1% (vol/vol) toluene. Deletion of ttgV, coding for the specific transcriptional repressor of solvent efflux pump TtgGHI gene expression, led to constitutively solvent-tolerant mutants of P. taiwanensis VLB120 and VLB120?C. Interestingly, the increased amount of solvent efflux pumps enhanced not only growth in the presence of toluene and styrene but also the biocatalytic performance in terms of stereospecific styrene epoxidation, although proton-driven solvent efflux is expected to compete with the styrene monooxygenase for metabolic energy. Compared to that of the P. taiwanensis VLB120?C parent strain, the maximum specific epoxidation activity of P. taiwanensis VLB120?C?ttgV doubled to 67 U/g of cells (dry weight). This study shows that solvent tolerance mechanisms, e.g., the solvent efflux pump TtgGHI, not only allow for growth in the presence of organic compounds but can also be used as tools to improve redox biocatalysis involving organic solvents.

SUBMITTER: Volmer J

PROVIDER: S-EPMC4178659 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Engineering of Pseudomonas taiwanensis VLB120 for constitutive solvent tolerance and increased specific styrene epoxidation activity.

Volmer Jan J Neumann Christoph C Bühler Bruno B Schmid Andreas A

Applied and environmental microbiology 20140815 20

The application of whole cells as biocatalysts is often limited by the toxicity of organic solvents, which constitute interesting substrates/products or can be used as a second phase for in situ product removal and as tools to control multistep biocatalysis. Solvent-tolerant bacteria, especially Pseudomonas strains, are proposed as promising hosts to overcome such limitations due to their inherent solvent tolerance mechanisms. However, potential industrial applications suffer from tedious, unpro ...[more]

PMID: 25128338

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Project description:BackgroundBio-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).ResultsThe 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.ConclusionsIn 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.

Project description: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.

Dataset Information

Engineering of Pseudomonas taiwanensis VLB120 for constitutive solvent tolerance and increased specific styrene epoxidation activity.

Publications

Engineering of Pseudomonas taiwanensis VLB120 for constitutive solvent tolerance and increased specific styrene epoxidation activity.

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