Search

Additional file 1: Figure S1. (A) DNA region of the plasmid pEC-XT99A-gabTDP134L with the 21 bp sequence deleted in ALE strain GluA T7 (boxed in red). (B) Determination of the minimum inhibitory concentration for tetracycline for strain GluA T0 with the orginal plasmid pEC-XT99A-gabTDP134L and GluA RG1 carrying this plasmid with the 21 bp deletion. Cells were grown in Duetz cultivation system using BHIS supplemented with increasing concentrations of tetracycline (0–80 µg µL−1). The ΔOD600 values were determined after 48 hours and are shown as means with standard deviations (n = 3 cultivations). Figure S2. Dependence of the specific acitvity of GOGAT on the glutamine concentration determined for GRLys1ΔgltB overexpressing the native version of gltBD (A) or the mutated gltBE686QD (B). Crude extracts of GRLys1ΔgltB (pEKEx3-gltBD) and GRLys1ΔgltB (pEKEx3-gltBE686QD) were assayed for specific activity of the glutamate synthase (GOGAT) in the presence of increasing glutamine concentrations (0 mM, 0.1 mM, 0.25 mM, 0.5 mM, 0.75 mM, 1 mM, 2 mM, 5 mM, 10 mM). Values represent means and standard deviations of triplicate measurements. Figure S3. Glutarate production by GluA T7 in chloride-free medium. Cells were grown in Duetz cultivation system using either standard CGXII minimal medium or CGXII minimal medium without chloride supplemented with 40 g L−1 glucose, induced with 1 mM IPTG, and harvested after 48 h. Values and error bars represent means and standard deviations of glutarate (green) and 5AVA concentrations (red) in the culture supernatants (n = 3 cultivations).

The formation of stable emulsions in biphasic biotransformations catalyzed by microbial cells turned out to be a major hurdle for industrial implementation. Recently, a cost-effective and efficient downstream processing approach, using supercritical carbon dioxide (scCO2 ) for both irreversible emulsion destabilization (enabling complete phase separation within minutes of emulsion treatment) and product purification via extraction has been proposed by Brandenbusch et al. (2010). One of the key factors for a further development and scale-up of the approach is the understanding of the mechanism underlying scCO2 -assisted phase separation. A systematic approach was applied within this work to investigate the various factors influencing phase separation during scCO2 treatment (that is pressure, exposure of the cells to CO2 , and changes of cell surface properties). It was shown that cell toxification and cell disrupture are not responsible for emulsion destabilization. Proteins from the aqueous phase partially adsorb to cells present at the aqueous-organic interface, causing hydrophobic cell surface characteristics, and thus contribute to emulsion stabilization. By investigating the change in cell-surface hydrophobicity of these cells during CO2 treatment, it was found that a combination of catastrophic phase inversion and desorption of proteins from the cell surface is responsible for irreversible scCO2 mediated phase separation. These findings are essential for the definition of process windows for scCO2 -assisted phase separation in biphasic whole-cell biocatalysis.