Accelerating whole-cell biocatalysis by reducing outer membrane permeability barrier.

Whole-cell biocatalysts are preferred in many biocatalysis applications. However, due to permeability barriers imposed by cell envelopes, whole-cell catalyzed reactions are reportedly 10-100-fold slower than reactions catalyzed by free enzymes. In this study, we accelerated whole-cell biocatalysis by reducing the membrane permeability barrier using molecular engineering approaches. Escherichia coli cells with genetically altered outer membrane structures were used. Specifically, a lipopolysaccarides mutant SM101 and a Braun's lipoprotein mutant E609L were used along with two model substrates that differ substantially in size and hydrophobicity, nitrocefin, and a tetrapeptide N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The reduction of the outer membrane permeability by genetic methods led to significant increases (up to 380%) in reaction rates of whole-cell catalyzed reactions. The magnitude of increase in biocatalysis rates was dependent on the substrates and on the nature of mutations introduced in the outer membrane structure. Notably, mutations in outer membrane can render the outer membrane completely permeable to one substrate, a barrierless condition that maximizes the reaction rate. The impact of the mutations introduced on the permeability barrier of the membranes was compared to the impact of polymixin B nonapeptide, a known potent permeabilizer acting on lipopolysaccharides. Our results suggest that genetic modifications to enhance the permeability of hydrophilic molecules should target the Lipid A region. However, strategies other than reduction of Lipid A synthesis should be considered. As we have demonstrated with tetrapeptide, membrane engineering can be much more effective in reducing a permeability barrier than are exogenous permeabilizers. This work, to our knowledge, is the first use of a molecular membrane engineering approach to address substrate permeability limitations encountered in biocatalysis applications.