The application of constitutively solvent‐tolerant P. taiwanensis VLB120ΔCΔttgV for stereospecific epoxidation of toxic styrene alleviates carrier solvent use

Abstract

For whole-cell biotransformations involving toxic organic compounds, two-liquid phase setups are typically applied employing an apolar extractive phase. Bis(2-ethylhexyl)phthalate (BEHP) has proven to be an ideal solvent for stereospecific styrene epoxidation with recombinant E. coli, providing excellent extractive properties and a high biocompatibility. In eco-efficiency evaluations, BEHP, however, has been identified as a critical factor regarding costs and environmental impact. In this study, the constitutive solvent tolerance of Pseudomonas taiwanensis VLB120ΔCΔttgV is shown to enable high specific activities (up to 180 U g ) and extensive reduction of the BEHP amount in two-liquid phase setups and thus to constitute an excellent tool to improve the environmental and economic efficiency of such processes. At a 90% reduction of carrier solvent use and accordingly increased aqueous styrene concentrations, this strain still showed reasonably high specific styrene epoxidation activities (100 U g ), while the solvent-adaptable wildtype strain immediately was toxified. A moderate 55% reduction of BEHP enabled a specific activity of 150 U g and thus represents a good trade-off between volumetric productivity maximization and environmental impact minimization. These results for the first time show a clear benefit of a solvent-tolerant compared to solvent-sensitive host strains and how such a benefit can be achieved.

Constitutively solvent‐tolerant Pseudomonas taiwanensis VLB120∆C∆ttgV supports particularly high‐styrene epoxidation activities when grown under glucose excess conditions

“…Under these conditions, P. taiwanensis VLB120Δ C Δ ttgV_AeGFP did not show higher epoxidation activities than the VLB120Δ C_AeGFP strain (Figure ), which was in contrast to their behavior in resting‐cell assays (Supporting Information Figure S2) and in two‐liquid phase batch cultures of the Δ C and Δ C Δ ttgV strains (Volmer et al, ). After 3 hr, both strains reached a maximal activity of 30–40 U/g CDW , which decreased toward the end of the biotransformation.…”

“…Constitutively solvent‐tolerant P. taiwanensis VLB120∆ C ∆ ttgV has been shown to exhibit two‐fold improved styrene epoxidation activities compared with P. taiwanensis VLB120∆ C (Volmer et al, , ). The substitution of the styA gene with a styA‐eGFP fusion construct allowed determining the intracellular oxygenase amount and revealed that increased oxygenase levels in the constitutively solvent‐tolerant mutant constitute one, but not the only reason for the observed elevated activities.…”

“…On the other hand, solvent‐tolerant Pseudomonas strains have been reported to boost their metabolic capacity upon adaptation to the presence of toxic solvents, to provide the energy required to operate solvent tolerance mechanisms (Blank, Ionidis, et al, ; Segura et al, ; Volkers et al, ; Volkers, Snoek, Ruijssenaars, & de Winde, ). Thus, the regulator TtgV, of which the knockout enabled constitutive solvent tolerance (Volmer et al, , ), may, besides the inhibition of efflux pump gene expression, also be involved in the regulation of the energy metabolism. As a consequence, its knockout enabled a higher metabolic capacity and thus higher epoxidation activities under glucose excess conditions.…”

“…Two‐liquid phase biotransformations were carried out in a 3.1 L stirred‐tank KLF2000 bioreactor (Bioengineering, Wald, Switzerland) as described previously (Kuhn, Bühler, & Schmid, ; Kuhn, Julsing, et al, ; Volmer et al, ). In short, biotransformations based on batch cultures (1 L RB medium; 30°C, pH = 7.2, 20 g/L glucose, 1 vvm) were started, when a biomass concentration of 0.5 g CDW /L was reached, by addition of well‐mixed 920 ml bis(2‐ethylhexyl) phthalate (BEHP) and 80 ml styrene and continued until glucose was depleted.…”

The biological mechanisms of butanol tolerance and the application of solvent‐tolerant bacteria for environmental protection

“…At present, titanium silicate molecular sieves, heteropoly compounds and metal complexes are often used to catalyze the epoxidation of styrene to form styrene oxide, but they are limited by expense and low activity and stability respectively. Volmer et al . used a whole‐cell biocatalytic method to facilitate the catalytic reaction of Pseudomonas taiwanensis VLB120ΔCΔttgV with organic solvent tolerance to diphenyl phthalate environments, showing relatively high styrene epoxidation activity.…”