The significant role of biofilms in pathogenicity has spurred research into preventing their formation and promoting their disruption, resulting in overlooked opportunities to develop biofilms as a synthetic biological platform for self-assembling functional materials. Here we present Biofilm-Integrated Nanofiber Display (BIND) as a strategy for the molecular programming of the bacterial extracellular matrix material by genetically appending peptide domains to the amyloid protein CsgA, the dominant proteinaceous component in Escherichia coli biofilms. These engineered CsgA fusion proteins are successfully secreted and extracellularly self-assemble into amyloid nanofibre networks that retain the functions of the displayed peptide domains. We show the use of BIND to confer diverse artificial functions to the biofilm matrix, such as nanoparticle biotemplating, substrate adhesion, covalent immobilization of proteins or a combination thereof. BIND is a versatile nanobiotechnological platform for developing robust materials with programmable functions, demonstrating the potential of utilizing biofilms as large-scale designable biomaterials.

23Biocatalytic transformations generally rely on purified enzymes or whole cells to perform 24 complex transformations that are used on industrial scales for chemical, drug, and biofuel 25 synthesis, pesticide decontamination and water purification. However, both of these systems 26 have inherent disadvantages related to the costs associated with enzyme purification, the long-27 term stability of immobilized enzymes, catalyst recovery and compatibility with harsh reaction 28 conditions. We developed a novel strategy for producing rationally designed biocatalytic 29 surfaces based on Biofilm Integrated Nanofiber Display (BIND), which exploits the curli system 30 of E. coli to create a functional nanofiber network capable of covalent immobilization of 31 enzymes. This approach is attractive because it is scalable, represents a modular strategy for site-32 specific enzyme immobilization, and has the potential to stabilize enzymes under denaturing 33 environmental conditions. We site-specifically immobilized a recombinant α-amylase, fused to 34 the SpyCatcher attachment domain, onto E. coli curli fibers displaying complementary SpyTag 35 capture domains. We characterized the effectiveness of this immobilization technique on the 36 biofilms and tested the stability of immobilized α-amylase in unfavorable conditions. This 37 enzyme-modified biofilm maintained its activity when exposed to a wide range of pH and 38 organic solvent conditions. In contrast to other biofilm-based catalysts, which rely on cellular 39 metabolism to remain active, the modified curli-based biofilm remained active even after cell 40 death due to organic solvent exposure. This work lays the foundation for a new and versatile 41 method of using the extracellular polymeric matrix of E. coli for creating novel biocatalytic 42 surfaces. 43 44 3

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.

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