Figure 1.
Functional assembly of multiple enzymes on engineered bacterial outer membrane vesicles (OMVs).
A trivalent scaffold containing three orthogonal cohesin domains, DocC (from C. cellulolyticum), DocT (from C. thermocellum) and DocF (from R. flavefaciens), and one cellulose-binding module, is displayed onto OMVs using the ice nucleation protein (INP) anchor. The specific interaction between each cohesin-dockerin pair enables the sequential assembly of three dockerin-tagged cellulases (E1, E2 and E3) onto the OMVs at their corresponding position (C, T or F).
Figure 2.
Expression and localization of INP-Scaf3.
(A) SDS-PAGE analysis of whole cell, membrane and OMV fractions of E. coli JC8031 with (INP-scaf3) or without (C) INP-Scaf3 expression. The INP-Scaf3 band is indicated by an arrow (107 kDa). (B) Electron micrograph of engineered OMVs expressing INP-scaf3. The scale bar represents 100 nm. (C) Surface localization of INP-Scaf3. Cells expressing INP-Scaf3 were incubated with mouse monoclonal anti-his6 antibody and Alexa488-conjugated anti-mouse IgG. The scale bar represents 10 µm.
Figure 3.
Binding of dockerin-tagged cellulases to engineered OMVs.
Binding was verified by measuring the amount of reducing sugar released from cellulose hydrolysis after AT (A) or EC (B) binding. BF binding (C) was confirmed by measuring the fluroescence of 4-methylumbelliferone produced from the hydrolysis of 4-Methylumbelliferyl β-D-glucopyranoside. In all cases, wild type OMVs were used as a control.
Figure 4.
SDS-PAGE analysis of enzyme-assembled OMVs.
For better resolution, binding of AT (A) or EC and BF (B) was confirmed using either 10% or 7% SDS-PAGE. Bands corresponding to either INP-scaf3 (107 kDa), AT (53 kDa), EC (90 kDa) or BF (70 kDa) are shown.
Figure 5.
Production of reducing sugars (AT and AT+EC) or glucose (AT+BC+RF) from PASC by enzyme-assembled OMVs or the same amount of free enzymes.