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. Herein, 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 by using a panel of genetically programmed, mutually orthogonal conjugation domains. We further demonstrate the utility of the technique in a bienzymatic stereoselective reduction coupled with a cofactor recycling scheme. The domains allow for several cycles of selective removal and replacement of enzymes under mild conditions to regenerate the catalyst system.

Coupled chemistry kinetics demonstrate the utility of functionalized Sup35 amyloid nanofibrils in biocatalytic cascades

“…However, in most cases, the functionality is demonstrated on single-enzyme systems only. An exception is recently developed bacterial amyloid, which was used to assemble a two-enzyme cascade (24). Furthermore, the type of characterizations reported for enzyme functionalized amyloid have often a qualitative nature.…”

Engineered Living Materials: Prospects and Challenges for Using Biological Systems to Direct the Assembly of Smart Materials

“…Thus, the biologically fabricated curli fiber matrix was transformed into a versatile scaffold for multistep in vitro biocatalysis. Furthermore, because the conjugation domains exhibit different stabilities under different conditions, one enzyme can be removed from a multienzyme system without affecting the others . Other recently published papers have focused on the ability to create engineered curli‐based biofilms that are electrically conductive.…”

“…Furthermore, because the conjugation domains exhibit different stabilities under different conditions, one enzyme can be removed from a multi-enzyme system without affecting the others. [41] Other recently published papers have focused on the ability to create engineered curli-based biofilms that are electrically conductive. This was first accomplished by displaying conductive gold nanoparticle binding domains on CsgA in order to create a bio-inorganic hybrid material (Figure 5f).…”

Developing Gram-negative bacteria for the secretion of heterologous proteins

“…In contrast to other bacterial secretion systems, T8SS engineering efforts have focused primarily on engineering the secreted curli fibers to make nanofibrous meshes with a range of properties. CsgA is secreted with C-terminal fusions of a wide range of peptide tags [120], which provides myriad options for functionalizing the amyloid fiber network, from enzyme scaffolding [121] to nanoparticle patterning [122]. This suggests the machinery is robust with respect to protein cargo identity, though the putative size limit remains an issue.…”