Bootstrapped Biocatalysis: Biofilm-Derived Materials as Reversibly Functionalizable Multienzyme Surfaces

Abstract

Cell-free biocatalysis systems offer many benefits for chemical manufacturing, but their widespread applicability is hindered by high costs associated with enzyme purification, modification, and immobilization on solid substrates, in addition to the cost of the material substrates themselves. Here we report a “bootstrapped” biocatalysis substrate material that is produced directly in bacterial culture and is derived from biofilm matrix proteins, which self-assemble into a nanofibrous mesh. We demonstrate that this material can simultaneously purify and immobilize multiple enzymes site-specifically, and directly from crude cell lysates using a panel of genetically programmed, mutually orthogonal conjugation domains. We further demonstrate the utility of the technique in a bi- enzymatic stereoselective reduction coupled with cofactor recycling scheme. The domains allow for several cycles of selective removal and replacement of enzymes under mild conditions to regenerate the catalyst system.