Carbon metabolism and product inhibition determine the epoxidation efficiency of solvent-tolerant Pseudomonas sp. strain VLB120ΔC

Utilization of solvent tolerant bacteria as biocatalysts has been suggested to enable or improve bioprocesses for the production of toxic compounds. Here, we studied the relevance of solvent (product) tolerance and inhibition, carbon metabolism, and the stability of biocatalytic activity in such a bioprocess. Styrene degrading Pseudomonas sp. strain VLB120 is shown to be solvent tolerant and was engineered to produce enantiopure (S)-styrene oxide from styrene. Whereas glucose as sole source for carbon and energy allowed efficient styrene epoxidation at rates up to 97 μmol/min/(g cell dry weight), citrate was found to repress epoxidation by the engineered Pseudomonas sp. strain VLB120ΔC emphasizing that carbon source selection and control is critical. In comparison to recombinant Escherichia coli, the VLB120ΔC-strain tolerated higher toxic product levels but showed less stable activities during fed-batch cultivation in a two-liquid phase system. Epoxidation activities of the VLB120ΔC-strain decreased at product concentrations above 130 mM in the organic phase. During continuous two-liquid phase cultivations at organic-phase product concentrations of up to 85 mM, the VLB120ΔC-strain showed stable activities and, as compared to recombinant E. coli, a more efficient glucose metabolism resulting in a 22% higher volumetric productivity. Kinetic analyses indicated that activities were limited by the styrene concentration and not by other factors such as NADH availability or catabolite repression. In conclusion, the stability of activity of the solvent tolerant VLB120ΔC-strain can be considered critical at elevated toxic product levels, whereas the efficient carbon and energy metabolism of this Pseudomonas strain augurs well for productive continuous processing.