WO2018005493A2 - Regeneratable curli nanofiber-based catalysts for multistep biotransformations - Google Patents

Regeneratable curli nanofiber-based catalysts for multistep biotransformations Download PDF

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WO2018005493A2
WO2018005493A2 PCT/US2017/039505 US2017039505W WO2018005493A2 WO 2018005493 A2 WO2018005493 A2 WO 2018005493A2 US 2017039505 W US2017039505 W US 2017039505W WO 2018005493 A2 WO2018005493 A2 WO 2018005493A2
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polypeptide
conjugation domain
curli
functionalizing
domain
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WO2018005493A3 (en )
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Neel Satish JOSHI
Martin G. NUSSBAUMER
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President And Fellows Of Harvard College
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells being immobilised on or in an organic carrier
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00Biological treatment of water, waste water, or sewage
    • C02F3/02Aerobic processes
    • C02F3/10Packings; Fillings; Grids
    • C02F3/103Textile-type packing
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00Biological treatment of water, waste water, or sewage
    • C02F3/02Aerobic processes
    • C02F3/10Packings; Fillings; Grids
    • C02F3/105Characterized by the chemical composition
    • C02F3/108Immobilising gels, polymers or the like
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245Escherichia (G)
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00Nature of the contaminant
    • C02F2101/10Inorganic compounds
    • C02F2101/20Heavy metals or heavy metal compounds
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00Nature of the contaminant
    • C02F2101/30Organic compounds
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2305/00Use of specific compounds during water treatment
    • C02F2305/08Nanoparticles or nanotubes
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02WCLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W10/00Technologies for wastewater treatment
    • Y02W10/10Biological treatment of water, waste water, or sewage
    • Y02W10/15Aerobic processes

Abstract

Disclosed herein are engineered bacteria that manufacture biofilms from bacterial amyloid structures known as curli fibers. These biofilms and biofilm matrices are capable immobilizing enzymes and generating catalytic surfaces.

Description

REGENERATABLE CURLI NANOFIBER-BASED CATALYSTS FOR MULTISTEP

BIOTRANSFORMATIONS

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No.

62/354,843, filed on June 27, 2016 and to U.S. Provisional Patent Application No.

62/506,722, filed on May 16, 2017. The entire contents of each of the foregoing applications is expressly incorporated herein by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant No. 1410751 awarded by the National Science Foundation. The government has certain rights in the invention.

FIELD

The technology described herein relates to engineered bacteria that manufacture biofilms from bacterial amyloid structures, known as curli fibers, which are capable of immobilizing enzymes and generating catalytic surfaces.

BACKGROUND

Biocatalytic reactions identified and generated by rational design are normally absent in nature. Rational design is a strategy of synthesizing new molecules with a particular functionality, based upon the ability to predict how the molecule's structure will affect its behavior through physical models. Rational design can be used to identify and develop enzymes that are highly specific catalysts for a desired reaction. Stereoselective biocatalytic transformations are of great interest in the synthesis of pharmaceutical intermediates and other high- value chemicals. Existing approaches to biocatalysis rely on purified enzymes or whole cells. These systems, however, have inherent disadvantages, such as high costs of enzyme purification, low mass transport, and the potential toxicity of substrate or product in whole cell systems. Accordingly, there is a need for improved biocatalytic systems.

SUMMARY

Provided herein is a system which overcomes the known problems with biocatalysis, based on rationally designed biocatalytic surfaces that are self-generated by engineered bacteria which produce protective biofilms. Bacterial biofilms are constructed from protective nanoscale scaffolds of proteins, sugars, lipids, and extracellular DNA, and biofilms are self-generated protective structures that allow bacteria to adhere to both natural and man- made surfaces. See U.S. Patent Application Publication No. 2016/0185828, the entire contents of which are expressly incorporated herein by reference in their entirety.

The present disclosure provides engineered or genetically altered microorganisms, such as E. coli, that produce amyloid-based structures called curli fibers that include moieties suited to the immobilization of enzymes (or other functionalizing polypeptides) to produce a bio film having a catalytic surface. The curli protein nanofibers disclosed herein enable one or more enzymes to be immobilized site-specifically on the biofilm, without the need for purification or chemical modification. Specifically, the steps of curli nanofiber production and enzyme immobilization are based on self-assembly, and there is no need for chemical conjugation reagents. Moreover, the methods disclosed herein allow immobilization of multiple enzymes (or other functionalizing polypeptides) simultaneously onto a curli fiber or onto a biofilm comprising a curli fiber described herein. Advantageously, the enzymes that are immobilized onto the curli fibers described herein may be removed from the curli fiber, and optionally additional enzymes may be immobilized onto the curli fiber allowing for the regeneration of the catalytic ability of the curli fiber. Further, when two or more enzymes are immobilized onto the curli fibers described herein, surprisingly one or more of the enzymes may be selectively removed from the curli fiber without affecting the activity of the other immobilized enzymes. For example, by disrupting different protein-protein interactions under different conditions, the selective release of one or more enzymes (or other functionalizing polypeptides) may be accomplished. The curli fibers or biofilms comprising the curli fibers may further be regenerated by attaching one or more desired enzymes (or other functionalizing polypeptides) onto the curli fiber.

The catalytic biofilms disclosed herein are useful for biotransformations that are commonly carried out in industry. For example, dehydrogenases, which are enzymes that are useful in redox transformations that require cofactor regeneration, remain active after immobilization on the biofilms disclosed herein. As another example, different

ketoreductases (KRED) can be co-immobilized with a co-factor recycling dehydrogenase. This enzyme cascade transforms acetophenones to stereo-selective benzyl alcohols, which are desired building blocks for pharmaceutical active ingredients. In another embodiment, enzymes and palladium nanoparticles are co-immobilized on biofilms disclosed herein to create catalytic surfaces that are potentially useful for chemoenzymatic cascades. In one aspect, the invention provides a bio film matrix having a biocatalytic surface, wherein the biofilm matrix comprises a plurality of curli fibers, wherein the plurality of curli fibers comprises a first engineered CsgA polypeptide comprising a first CsgA protein and a first conjugation domain, wherein the first conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMAN11, and DnaEAC35, wherein the first conjugation domain is bound to a first partner conjugation domain which is linked to a first functionalizing polypeptide, and wherein the first partner conjugation domain is selected from the group consisting of a PDZ domain, Tipl, InaD, M13, SZ16, VMAANll, and DnaEC35; a second engineered CsgA polypeptide comprising a second CsgA protein and a second conjugation domain, wherein the second conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMAN11, and DnaEAC35, and wherein the second conjugation domain is bound to a second partner conjugation domain which is linked to a second functionalizing polypeptide, wherein the second partner conjugation domain is selected from the group consisting of a PDZ domain, Tipl, InaD, M13, SZ16, VMAANll, and DnaEC35; and wherein the first functionalizing polypeptide and the second functionalizing polypeptide form an enzymatic cascade. In some embodiments, the curli fiber further comprises a third, a fourth, a fifth, a sixth, a seventh, an eighth or more engineered CsgA polypeptides, wherein each engineered CsgA polypeptide comprises a CsgA protein and a conjugation domain, wherein the conjugation domain is bound to a partner conjugation domain. In some embodiments, each engineered CsgA polypeptide comprises the same type or kind of conjugation domain. In some embodiments, each engineered CsgA polypeptide comprises a different type or kind of conjugation domain. In some embodiments, each type or kind of conjugation domain is bound to a distinct functionalizing peptide via a partner conjugation domain.

In some embodiments, the plurality of curli fibers comprises one type of curli fiber comprising the first engineered CsgA polypeptide and the second engineered CsgA polypeptide. In another embodiment, the plurality of curli fibers comprises a first curli fiber comprising the first engineered CsgA polypeptide and a second curli fiber comprising the second engineered CsgA polypeptide.

In some embodiments, the first engineered CsgA polypeptide and the second engineered CsgA polypeptide are disposed on the same curli fiber within the plurality of curli fibers. In some embodiments, the first engineered CsgA polypeptide is disposed on a first curli fiber within the plurality of curli fibers, and wherein the second engineered CsgA polypeptide is disposed on a second curli fiber within the plurality of curli fibers. In one aspect, the invention provides a bio film matrix having a biocatalytic surface, wherein the biofilm matrix comprises a plurality of curli fibers, wherein the plurality of curli fibers comprises a first engineered CsgA polypeptide comprising a first CsgA protein and a first conjugation domain, wherein the first conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMAN11, and DnaEAC35, wherein the first conjugation domain is bound to a first partner conjugation domain which is linked to a first functionalizing polypeptide, and wherein the first partner conjugation domain is selected from the group consisting of a PDZ domain, Tipl, InaD, M13, SZ16, VMAANll, and DnaEC35; and a second engineered CsgA polypeptide comprising a second CsgA protein and a second conjugation domain, wherein the second conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMAN11, and DnaEAC35, and wherein the second conjugation domain is bound to a second partner conjugation domain which is linked to a second functionalizing polypeptide, wherein the second partner conjugation domain is selected from the group consisting of a PDZ domain, Tipl, InaD, M13, SZ16, VMAANll, and DnaEC35; and wherein the biocatalytic surface is capable of regeneration.

In one aspect, the invention provides a biofilm matrix having a biocatalytic surface, wherein the biofilm matrix comprises a plurality of curli fibers, wherein the plurality of curli fibers comprises a curli fiber comprising a first engineered CsgA polypeptide comprising a first CsgA protein and a first conjugation domain, wherein the first conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMAN11, and DnaEAC35, wherein the first conjugation domain is bound to a first partner conjugation domain which is linked to a first functionalizing polypeptide, and wherein the first partner conjugation domain is selected from the group consisting of a PDZ domain, Tipl, InaD, M13, SZ16, VMAANll, and DnaEC35; and a second engineered CsgA polypeptide comprising a second CsgA protein and a second conjugation domain, wherein the second conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMAN11, and DnaEAC35, and wherein the second conjugation domain is bound to a second partner conjugation domain which is linked to a second functionalizing polypeptide, wherein the second partner conjugation domain is selected from the group consisting of a PDZ domain, Tipl, InaD, M13, SZ16, VMAANll, and DnaEC35; wherein the first functionalizing polypeptide and the second functionalizing polypeptide form an enzymatic cascade.

In another aspect, the invention provides a biofilm matrix having a biocatalytic surface, wherein the biofilm matrix comprises a plurality of curli fibers, wherein the plurality of curli fibers comprises a first curli fiber and a second curli fiber; wherein the first curli fiber comprises a first engineered CsgA polypeptide comprising a first CsgA protein and a first conjugation domain, wherein the first conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMAN11, and DnaEAC35, wherein the first conjugation domain is bound to a first partner conjugation domain which is linked to a first functionalizmg polypeptide, and wherein the first partner conjugation domain is selected from the group consisting of a PDZ domain, Tipl, InaD, M13, SZ16, VMAANll, and DnaEC35; and wherein the second curli fiber comprises a second engineered CsgA polypeptide comprising a CsgA protein and a second conjugation domain, wherein the second conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMAN11, and DnaEAC35, and wherein the second conjugation domain is bound to a second partner conjugation domain which is linked to a second functionalizmg polypeptide, wherein the second partner conjugation domain is selected from the group consisting of a PDZ domain, Tipl, InaD, M13, SZ16, VMAANll, and DnaEC35; and wherein the first functionalizmg polypeptide and the second functionalizing polypeptide form an enzymatic cascade.

In some embodiments, either the first functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionalizing polypeptides are capable of being removed from the plurality of curli fibers.

In some embodiments, the first functionalizing polypeptide, the second

functionalizing polypeptide, or the first and second functionalizing polypeptides are capable of being removed from, and re-immobilized on, the plurality of curli fibers.

In some embodiments, the first functionalizing polypeptide, the second

functionalizing polypeptide, or the first and second functionalizing polypeptide are covalently bound to the plurality of curli fibers. In some embodiments, the first functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionalizing polypeptide are non-covalently bound to the plurality of curli fibers. In some embodiments, the first functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionalizing polypeptides are capable of being removed from the plurality of curli fibers by disruption of a protein-protein interaction. In some embodiments, the protein- protein interaction is disrupted with guanidine, a chelating agent, a pH change, a redox reagent, urea, or other denaturing agent. In some embodiments, the chelating agent is EGTA or EDTA. In some embodiments, the redox reagent is dithiothreitol (DTT).

In some embodiments, the PDZ domain is PDZ1 or ePDZ-bl . In some embodiments, the first conjugation domain and the second conjugation domain are different types of conjugation domains.

In some embodiments, the first conjugation domain and the second conjugation domain are the same type of conjugation domain.

In some embodiments, the first partner conjugation domain and the second partner conjugation domain are different types of partner conjugation domains.

In some embodiments, the first partner conjugation domain and the second partner conjugation domain are the same type of partner conjugation domain.

In some embodiments, the first functionalizing polypeptide is a first enzyme. In some embodiments, the second functionalizing polypeptide is a second enzyme. In some embodiments, the first enzyme is selected form the group consisting of an amylase, a dehydrogenase, and a ketoreductase. In some embodiments, the second enzyme is selected form the group consisting of an amylase, a dehydrogenase, and a ketoreductase. In some embodiments, the first enzyme and the second enzyme are selected from the group consisting of phosphite dehydrogenase (PTDH), RADH, and CtXR.

In some embodiments, the first enzyme catalyzes the regeneration of a co-factor for use by the second enzyme.

In some embodiments, the first enzyme catalyzes the generation of a substrate for use by the second enzyme.

In one aspect, the invention provides a biofilm comprising a biofilm matrix described herein and an engineered microbial cell.

In another aspect, the invention provides a method of producing a biofilm having a biocatalytic surface, comprising culturing a first genetically engineered bacterium in a culture medium, wherein the first genetically engineered bacterium expresses a first engineered CsgA polypeptide and a second engineered CsgA polypeptide, wherein the first engineered CsgA polypeptide comprises a first CsgA protein and a first conjugation domain, wherein the first conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMAN11, and DnaEAC35; and wherein the second engineered CsgA polypeptide comprises a second CsgA protein and a second conjugation domain, wherein the second conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMAN11, and DnaEAC35; thereby forming a plurality of curli fibers which form a biofilm, contacting the biofilm with a first functionalizing polypeptide which is linked to a first partner conjugation domain, wherein the first partner conjugation domain is selected from the group consisting of PDZ domain, Tipl, InaD, M13, SZ16, VMAANl l, and DnaEC35, and wherein the first partner conjugation domain binds to the first conjugation domain, and contacting the biofilm with a second functionalizing polypeptide which is linked to a second partner conjugation domain, wherein the second partner conjugation domain is selected from the group consisting of PDZ domain, Tipl, InaD, M13, SZ16, VMAANH, and DnaEC35, and wherein the second partner conjugation domain binds to the second conjugation domain, wherein the first functionalizing polypeptide and the second functionalizing polypeptide form an enzymatic cascade, thereby producing a biofilm having a biocatalytic surface.

In another aspect, the invention provides a method of producing a biofilm having a biocatalytic surface, comprising culturing a first genetically engineered bacterium and a second genetically engineered bacterium in a culture medium, wherein the first genetically engineered bacterium expresses a first engineered CsgA polypeptide, wherein the first engineered CsgA polypeptide comprises a CsgA protein and a first conjugation domain, wherein the first conjugation domain is selected from the group consisting of EFC A, WRESAI, ARVCF, CaM, SZ21, VMAN11, and DnaEAC35; and wherein the second genetically engineered bacterium expresses a second engineered CsgA polypeptide, wherein the second engineered CsgA polypeptide comprises a CsgA protein and a second conjugation domain, wherein the second conjugation domain is selected from the group consisting of EFC A, WRESAI, ARVCF, CaM, SZ21, VMAN11, and DnaEAC35; thereby forming a plurality of curli fibers which form a biofilm, contacting the biofilm with a first

functionalizing polypeptide which is hnked to a first partner conjugation domain, wherein the first partner conjugation domain is selected from the group consisting of PDZ domain, Tipl, InaD, M13, SZ16, VMAANl l, and DnaEC35, and wherein the first partner conjugation domain binds to the first conjugation domain, and contacting the biofilm with a second functionalizing polypeptide which is hnked to a second partner conjugation domain, wherein the second partner conjugation domain is selected from the group consisting of PDZ domain, Tipl, InaD, M13, SZ16, VMAANl l, and DnaEC35, and wherein the second partner conjugation domain binds to the second conjugation domain, wherein the first functionalizing polypeptide and the second functionalizing polypeptide form an enzymatic cascade, thereby producing a biofilm having a biocatalytic surface.

In yet another aspect, the invention provides a method of producing a biofilm having a biocatalytic surface capable of regeneration, comprising culturing a first genetically engineered bacterium in a culture medium, wherein the genetically engineered bacterium expresses a first engineered CsgA polypeptide and a second engineered CsgA polypeptide, wherein the first engineered CsgA polypeptide comprises a CsgA protein and a first conjugation domain, and wherein the second engineered CsgA polypeptide comprises a CsgA protein and a second conjugation domain, thereby forming a plurality of curli fibers which form a bio film, contacting the bio film with a first functionalizing polypeptide which is hnked to a first partner conjugation domain, wherein the first partner conjugation domain binds to the first conjugation domain, and contacting the biofilm with a second functionalizing polypeptide which is linked to a second partner conjugation domain, wherein the second partner conjugation domain binds to the second conjugation domain, wherein the first functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionalizing polypeptides are capable of being removed from the plurality of curli fibers, thereby producing a biofilm having a biocatalytic surface capable of regeneration.

In another aspect, the invention provides a method of producing a biofilm having a biocatalytic surface capable of regeneration, comprising culturing a first genetically engineered bacterium and a second genetically engineered bacterium in a culture medium, wherein the first genetically engineered bacterium expresses a first engineered CsgA polypeptide, wherein the first engineered CsgA polypeptide comprises a CsgA protein and a first conjugation domain, wherein the second genetically engineered bacterium expresses a second engineered CsgA polypeptide, wherein the second engineered CsgA polypeptide comprises a CsgA protein and a second conjugation domain, thereby forming a plurality of curli fibers which form a biofilm, contacting the biofilm with a first functionalizing polypeptide which is linked to a first partner conjugation domain, wherein the first partner conjugation domain binds to the first conjugation domain, and contacting the biofilm with a second functionalizing polypeptide which is linked to a second partner conjugation domain, wherein the second partner conjugation domain binds to the second conjugation domain, wherein the first functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionalizing polypeptides are capable of being removed from the plurality of curli fibers, thereby producing a biofilm having a biocatalytic surface capable of regeneration.

In some embodiments, the contacting step comprises contacting the biofilm with an exogenous functionalizing polypeptide. In some embodiments, the contacting step comprises contacting the biofilm with a functionalizing polypeptide that is expressed by a genetically engineered bacterium (e.g. , expressed by the first and/or second genetically engineered bacterium).

In some embodiments, the first functionalizing polypeptide and the second functionalizing polypeptide form an enzymatic cascade.

In some embodiments, the first conjugation domain, the second conjugation domain, or both the first conjugation and the second conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMAN11, and DnaEAC35. In some embodiments, the first conjugation domain, the second conjugation domain, or both the first and the second partner conjugation domain is selected from the group consisting of PDZ domain, Tipl, InaD, M13, SZ16, VMAANl l, and DnaEC35.

In some embodiments, either the first functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionalizing polypeptides are capable of being removed from the plurality of curli fibers.

In some embodiments, the first functionalizing polypeptide, the second

functionalizing polypeptide, or the first and second functionalizing polypeptides are capable of being removed from, and re-immobilized on, the plurality of curli fibers.

In some embodiments, the first conjugation domain covalently binds to the first partner conjugation domain, and wherein the second conjugation domain covalently binds to the second partner conjugation domain.

In some embodiments, the first conjugation domain covalently binds to the first partner conjugation domain, and wherein the second conjugation domain non-covalently binds to the second partner conjugation domain.

In some embodiments, the first conjugation domain non-covalently binds to the first partner conjugation domain, and wherein the second conjugation domain covalently binds to the second partner conjugation domain.

In some embodiments, the first conjugation domain non-covalently binds to the first partner conjugation domain, and wherein the second conjugation domain non-covalently binds to the second partner conjugation domain.

In some embodiments, the first functionalizing polypeptide is expressed by the first genetically engineered bacterium.

In some embodiments, the first functionalizing polypeptide is expressed by the second genetically engineered bacterium.

In some embodiments, the second functionalizing polypeptide is expressed by the first genetically engineered bacterium.

In some embodiments, the second functionalizing polypeptide is expressed by the second genetically engineered bacterium.

In some embodiments, the first genetically engineered bacterium is a genetically engineered E. coli bacterium.

In some embodiments, the second genetically engineered bacterium is a genetically engineered E. coli bacterium.

In some embodiments, the methods further comprise a step of removing the genetically engineered bacterium from the biofilm before the contacting steps. In some embodiments, the removing comprises washing the genetically engineered bacterium from the biofilm. In some embodiments, the first functionalizing polypeptide and the second functionalizing polypeptide maintain their catalytic activity after the step of removing the genetically engineered bacterium and/or the step of killing the genetically engineered bacterium.

In some embodiments, the methods further comprise a step of killing the genetically engineered bacterium in the biofilm before the contacting steps.

In some embodiments, the genetically engineered bacterium is cultured in a bioreactor. In some embodiments, the bioreactor is a batch bioreactor or a continuous flow bioreactor.

In another aspect, the present invention provides a method of regenerating a biocatalytic surface on a biofilm matrix produced by a genetically modified bacterium, comprising removing a first functionalizing polypeptide which is linked to a first partner conjugation domain from a plurality of curli fibers of the biofilm matrix, wherein the plurality of curli fibers comprises a first engineered CsgA polypeptide, wherein the first engineered CsgA polypeptide comprises a CsgA protein and a first conjugation domain; and contacting the biofilm matrix with a third functionalizing polypeptide which is linked to a third partner conjugation domain, thereby binding the third partner conjugation domain to the first conjugation domain, wherein the biofilm matrix also comprises a second engineered CsgA polypeptide, wherein the second engineered CsgA polypeptide comprises a second CsgA protein and a second conjugation domain, wherein the second conjugation domain is bound to a second partner conjugation domain which is linked to a second functionalizing polypeptide; thereby regenerating the biocatalytic surface of the biofilm matrix.

In some embodiments, the method further comprises removing the second functionalizing polypeptide which is linked to the second conjugation domain from the plurality of curli fibers; and contacting the biofilm matrix with a fourth functionalizing polypeptide which is linked to a fourth partner conjugation domain, thereby binding the fourth partner conjugation domain to the second conjugation domain.

In some embodiments, the first functionalizing polypeptide, the second functionalizmg polypeptide, or the first and second functionalizmg polypeptides are removed from the biofilm matrix by disrupting a protein-protein interaction. In some embodiments, the protein-protein interaction is disrupted with guanidine, a chelating agent, a pH change, a redox reagent, urea, or other denaturing agent.

In some embodiments, the removal of the first functionalizing polypeptide does not affect the binding of the second conjugation domain to the second partner conjugation domain and does not decrease the function of the second functionalizing polypeptide.

In some embodiments, the removal of the second functionalizing polypeptide does not affect the binding of the first conjugation domain to the third partner conjugation domain and does not decrease the function of the third functionalizing polypeptide.

In some embodiments, the third functionalizing polypeptide is the same type of functionalizing polypeptide as the first functionalizing polypeptide.

In some embodiments, the fourth functionalizing polypeptide is the same type of functionalizing polypeptide as the second functionalizing polypeptide.

In some embodiments, the first engineered CsgA polypeptide and the second engineered CsgA polypeptide are disposed on the same curli fiber within the plurality of curh fibers.

In some embodiments, the first engineered CsgA polypeptide is disposed on a first curli fiber within the plurality of curh fibers, and wherein the second engineered CsgA polypeptide is disposed on a second curli fiber within the plurality of curli fibers.

In yet another aspect, the present invention provides a curli fiber comprising a first engineered CsgA polypeptide and a second engineered CsgA polypeptide, wherein the first engineered CsgA polypeptide comprises a first CsgA protein and a first conjugation domain, wherein the first conjugation domain is selected from the group consisting of EFC A, WRESAI, ARVCF, CaM, SZ21, VMAN11, and DnaEAC35, wherein the first conjugation domain is bound to a first partner conjugation domain linked to a first functionalizing polypeptide, and wherein the first partner conjugation domain is selected from the group consisting of a PDZ domain, Tipl, InaD, M13, SZ16, VMAANl l, and DnaEC35; wherein the second engineered CsgA polypeptide comprises a second CsgA protein and a second conjugation domain, wherein the second conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMAN11, and DnaEAC35, and wherein the second conjugation domain is bound to a second partner conjugation domain linked to a second functionalizing polypeptide, and wherein the second partner conjugation domain is selected from the group consisting of a PDZ domain, Tipl, InaD, M13, SZ16, VMAANl l, and DnaEC35; and wherein the first functionalizing polypeptide and the second functionalizing polypeptide form an enzymatic cascade. In some embodiments, the curli fiber further comprises a third, a fourth, a fifth, a sixth, a seventh, an eighth or more engineered CsgA polypeptides, wherein each engineered CsgA polypeptide comprises a CsgA protein and a conjugation domain, wherein the conjugation domain is bound to a partner conjugation domain. In some embodiments, each engineered CsgA polypeptide comprises the same type or kind of conjugation domain. In some embodiments, each engineered CsgA polypeptide comprises a different type or kind of conjugation domain. In some embodiments, each type or kind of conjugation domain is bound to a distinct functionalizing peptide via a partner conjugation domain.

In yet another aspect, the invention provides a plurality of curli fibers comprising a first engineered CsgA polypeptide and a second engineered CsgA polypeptide, wherein the first engineered CsgA polypeptide comprises a first CsgA protein and a first conjugation domain, wherein the first conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMAN11, and DnaEAC35, wherein the first conjugation domain is bound to a first partner conjugation domain linked to a first functionalizing polypeptide, and wherein the first partner conjugation domain is selected from the group consisting of a PDZ domain, Tipl, InaD, M13, SZ16, VMAANl l, and DnaEC35; wherein the second engineered CsgA polypeptide comprises a second CsgA protein and a second conjugation domain, wherein the second conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMAN11, and DnaEAC35, and wherein the second conjugation domain is bound to a second partner conjugation domain linked to a second functionalizing polypeptide, and wherein the second partner conjugation domain is selected from the group consisting of a PDZ domain, Tipl, InaD, M13, SZ16, VMAANl l, and DnaEC35; and wherein the first functionalizing polypeptide and the second functionalizing polypeptide form an enzymatic cascade. In some embodiments, the plurality of curli fibers further comprises a third, a fourth, a fifth, a sixth, a seventh, an eighth or more engineered CsgA polypeptides, wherein each engineered CsgA polypeptide comprises a CsgA protein and a conjugation domain, wherein the conjugation domain is bound to a partner conjugation domain. In some embodiments, each engineered CsgA polypeptide comprises the same type or kind of conjugation domain. In some embodiments, each engineered CsgA polypeptide comprises a different type or kind of conjugation domain. In some embodiments, each type or kind of conjugation domain is bound to a distinct functionalizing peptide via a partner conjugation domain. In some embodiments, the first engineered CsgA polypeptide and the second engineered CsgA polypeptide are disposed on the same curli fiber within the plurality of curli fibers. In some embodiments, the first engineered CsgA polypeptide is disposed on a first curli fiber within the plurality of curli fibers, and wherein the second engineered CsgA polypeptide is disposed on a second curli fiber within the plurality of curli fibers.

In some embodiments, either the first functionalizing polypeptide, the second functionalizmg polypeptide, or the first and second functionalizing polypeptides are capable of being removed from the curli fiber or the plurality of curli fibers.

In some embodiments, the first functionalizing polypeptide, the second

functionalizing polypeptide, or the first and second functionalizing polypeptides are capable of being removed from, and re-immobilized on, the curli fiber or the plurality of curli fibers.

In some embodiments, the first functionalizing polypeptide, the second

functionalizing polypeptide, or the first and second functionalizing polypeptide are covalently bound to the curli fiber or to the plurality of curli fibers.

In some embodiments, the first functionalizing polypeptide, the second

functionalizing polypeptide, or the first and second functionalizing polypeptide are non- covalently bound to the curli fiber or to the plurality of curli fibers.

In some embodiments, the first functionalizing polypeptide, the second

functionalizing polypeptide, or the first and second functionalizing polypeptides are capable of being removed from the curli fiber or from the plurality of curli fibers by disruption of a protein-protein interaction. In some embodiments, the protein-protein interaction is disrupted with guanidine, a chelating agent, a pH change, a redox reagent, urea, or other denaturing agent. In some embodiments, the chelating agent is EGTA or EDTA. In some embodiments, the redox reagent is DTT.

In some embodiments, the PDZ domain is either PDZ1 or ePDZ-bl.

In some embodiments, the first conjugation domain and the second conjugation domain are different types of conjugation domains.

In some embodiments, the first conjugation domain and the second conjugation domain are the same type of conjugation domain.

In some embodiments, the first functionalizing polypeptide is a first enzyme. In some embodiments, the second functionalizing polypeptide is a second enzyme. In some embodiments, the first enzyme is selected form the group consisting of an amylase, a dehydrogenase, and a ketoreductase. In some embodiments, the second enzyme is selected form the group consisting of an amylase, a dehydrogenase, and a ketoreductase. In some embodiments, the first enzyme and the second enzyme are selected from the group consisting of phosphite dehydrogenase (PTDH), RADH, and CtXR.

In some embodiments, the first enzyme catalyzes the regeneration of a co-factor for use by the second enzyme.

In some embodiments, the first enzyme catalyzes the generation of a substrate for use by the second enzyme.

In one aspect, the invention provides a bio film comprising a curli fiber or a plurality of curli fibers described herein.

In another aspect, the invention provides an article of manufacture comprising a bio film matrix, curli fiber, or plurality of curli fibers described herein. In some embodiments, the article is a bioreactor or a cloth. In some embodiments, the cloth is a cotton cloth. In some embodiments, the bioreactor is a batch reactor or a continuous flow bioreactor.

In yet another aspect, the invention provides a method of producing a chemical entity using a biofilm matrix, curli fiber, or plurality of curli fibers described herein.

In another aspect, the invention provides a method of purifying water using a biofilm matrix, curli fiber, or plurality of curli fibers described herein.

In another aspect, the invention provides a method of cleaning a chemical spill using a biofilm matrix, curli fiber, or plurality of curli fibers described herein.

In one aspect, the invention provides a curli fiber comprising a first CsgA protein and a first conjugation domain, wherein the first conjugation domain is bound to a first partner conjugation domain which is linked to a first functionalizing polypeptide; and a second CsgA protein and a second conjugation domain, wherein the second conjugation domain is bound to a second partner conjugation domain which is linked to a second functionalizing polypeptide. In some embodiments, the first CsgA protein and the second CsgA protein are of the same type (i.e., CsgA proteins having identical amino acid sequences). In some embodiments, the first CsgA protein and the second CsgA protein are different (i.e., CsgA proteins having different amino acid sequences).

In another aspect, the invention provides a plurality of curli fibers comprising a first curli fiber and a second curh fiber, wherein the first curli fiber comprises a first CsgA protein and a first conjugation domain, wherein the first conjugation domain is bound to a first partner conjugation domain which is linked to a first functionalizing polypeptide; and wherein the second curh fiber comprises a second CsgA protein and a second conjugation domain, wherein the second conjugation domain is bound to a second partner conjugation domain which is linked to a second functionalizing polypeptide. In some embodiments, the first CsgA protein and the second CsgA protein are of the same type (i.e., CsgA proteins having identical amino acid sequences). In some embodiments, the first CsgA protein and the second CsgA protein are different (i. e. , CsgA proteins having different amino acid sequences).

In one embodiment, the first conjugation domain of the curli fiber or plurality of curli fibers is selected from the group consisting of SpyTag, EFCA, WRESAI, ARVCF, CaM, SZ21, VMANl 1, and DnaEAC35. In one embodiment, the second conjugation domain of the curli fiber or plurality of curli fibers is selected from the group consisting of SpyTag, EFCA, WRESAI, ARVCF, CaM, SZ21, VMANl 1, and DnaEAC35. In one embodiment, the first conjugation domain and the second conjugation domain of the curli fiber or plurality of curli fibers are different conjugation domains.

In one embodiment, the first partner conjugation domain of the curli fiber or plurality of curli fibers is selected from the group consisting of SpyCatcher, a PDZ domain, Tip 1 , InaD, M13, SZ16, VMAANl 1, and DnaEC35. In one embodiment, the second partner conjugation domain of the curli fiber or plurality of curli fibers is selected from the group consisting of SpyCatcher, a PDZ domain, Tipl, InaD, M13, SZ16, VMAANl l, and

DnaEC35. In one embodiment, the PDZ domain is PDZ1 or ePDZ-bl. In one embodiment, the first partner conjugation domain and the second partner conjugation domain of the curli fiber or plurality of curli fibers are different partner conjugation domains.

In one embodiment, the first functionalizing polypeptide of the curli fiber or plurality of curli fibers is a first enzyme. In one embodiment, the second functionalizing polypeptide of the curli fiber or plurality of curli fibers is a second enzyme. In one embodiment, the first enzyme and the second enzyme of the curli fiber or plurality of curli fibers form an enzymatic cascade.

In one embodiment, the first enzyme of the curli fiber or plurality of curli fibers is selected form the group consisting of amylase, a dehydrogenase, and a ketoreductase. In one embodiment, the second enzyme of the curli fiber or plurality of curli fibers is selected form the group consisting of amylase, a dehydrogenase, and a ketoreductase. In one embodiment, the first enzyme or the second enzyme of the curli fiber or plurality of curli fibers is selected from the group consisting of phosphite dehydrogenase (PTDH), RADH, and CtXR.

In one embodiment, the first partner conjugation domain is non-covalently bound to the first conjugation domain, and the second partner conjugation domain is non-covalently bound to the second conjugation domain, or the first partner conjugation domain is covalently bound to the first conjugation domain, and the second partner conjugation domain is covalently bound to the second conjugation domain.

In one embodiment, the curli fiber is present on a cloth, e.g., a cotton cloth, or a textile.

In another aspect, the invention provides an engineered microbial cell comprising any of the curli fibers or pluralities of curli fibers of the invention. In one embodiment, the engineered microbial cell is an E. coli cell. In one embodiment, the engineered microbial cell is cultured together with a second engineered microbial cell comprising any of the curli fibers or pluralities of curli fibers of the invention, wherein the engineered microbial cells are different cells.

In another aspect, the invention provides a bio film matrix comprising any of the engineered microbial cells of the invention. In another aspect, the invention provides a bio film matrix comprising any of the curli fibers or pluralities of curh fibers of the invention. In one embodiment, the bio film matrix is present on a cloth, e.g., a cotton cloth, or a textile.

In another aspect, the invention provides a bio film matrix having a biocatalytic surface, wherein the biofilm matrix comprises a plurality of curli fibers, wherein each curli fiber within the plurality of curli fibers is bound to a first functionalizing polypeptide and a second functionalizing polypeptide, wherein the first functionalizing polypeptide and the second functionalizing polypeptide form an enzymatic cascade.

In another aspect, the invention provides a biofilm matrix having a biocatalytic surface, wherein the biofilm matrix comprises a plurality of curli fibers comprising a first curli fiber and a second curh fiber, wherein the first curli fiber is bound to a first

functionalizing polypeptide and the second curli fiber is bound to a second functionalizing polypeptide, wherein the first functionalizing polypeptide and the second functionalizing polypeptide form an enzymatic cascade.

In one embodiment, the first functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionalizing polypeptides of the biofilm matrix are capable of being removed from the plurality of curli fibers.

In another aspect, the invention provides a biofilm matrix having a biocatalytic surface capable of regeneration, wherein the biofilm matrix comprises a plurality of curli fibers, wherein each curli fiber within the plurality of curli fibers is bound to a first functionalizing polypeptide and a second functionalizing polypeptide, wherein the first functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionalizing polypeptides are capable of being removed from, and re-immobilized on, the plurality of curli fibers. In another aspect, the invention provides a bio film matrix having a biocatalytic surface capable of regeneration, wherein the biofilm matrix comprises a plurality of curli fibers comprising a first curli fiber and a second curli fiber, wherein the first curli fiber is bound to a first functionalizing polypeptide and the second curli fiber is bound to a second functionalizing polypeptide, wherein the first functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionalizing polypeptides are capable of being removed from, and re-immobilized on, the plurality of curli fibers.

In one embodiment, the first functionalizing polypeptide and the second

functionalizing polypeptide of the biofilm matrix form an enzymatic cascade. In one embodiment, the first functionalizing polypeptide is covalently bound to the curli fiber, and the second functionalizing polypeptide is covalently bound to the curli fiber. In one embodiment, the first functionalizing polypeptide is non-covalently bound to the curli fiber, and the second functionalizing polypeptide is non-covalently bound to the curli fiber. In one embodiment, the first functionalizing polypeptide is covalently bound to the curli fiber, and the second functionalizing polypeptide is non-covalently bound to the curli fiber. In one embodiment, the first functionalizing polypeptide is non-covalently bound to the curli fiber, and the second functionalizing polypeptide is covalently bound to the curli fiber.

In one embodiment, the first functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionalizing polypeptides of the biofilm matrix are capable of being removed from the plurality of curli fibers by disruption of a protein-protein interaction. In one embodiment, the protein-protein interaction is disrupted with guanidine, a chelating agent (e.g., ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or ethylenediaminetetraacetic acid (EDTA)), pH change, a redox reagent (e.g., dithiothreitol (DTT)), urea, or other denaturing agent.

In one embodiment, each curli fiber within the plurality of curli fibers of the biofilm matrix comprises a first CsgA protein and a first conjugation domain, wherein the first conjugation domain is bound to a first partner conjugation domain which is linked to the first functionalizing polypeptide; and a second CsgA protein and a second conjugation domain, wherein the second conjugation domain is bound to a second partner conjugation domain which is linked to the second functionalizing polypeptide. In some embodiments, the first CsgA protein and the second CsgA protein are of the same type (i.e., CsgA proteins having identical amino acid sequences). In some embodiments, the first CsgA protein and the second CsgA protein are different (i.e., CsgA proteins having different amino acid sequences). In one embodiment, the first curli fiber of the bio film matrix comprises a first CsgA protein and a first conjugation domain, wherein the first conjugation domain is bound to a first partner conjugation domain which is linked to the first functionalizing polypeptide; and wherein the second curli fiber of the bio film matrix comprises a second CsgA protein and a second conjugation domain, wherein the second conjugation domain is bound to a first partner conjugation domain which is linked to the second functionalizing polypeptide. In some embodiments, the first CsgA protein and the second CsgA protein are of the same type (i.e., CsgA proteins having identical amino acid sequences). In some embodiments, the first CsgA protein and the second CsgA protein are different (i. e. , CsgA proteins having different amino acid sequences).

In one embodiment, the first conjugation domain of the biofilm matrix is selected from the group consisting of SpyTag, EFCA, WRESAI, ARVCF, CaM, SZ21, VMAN11, and DnaEAC35. In one embodiment, the second conjugation domain of the biofilm matrix is selected from the group consisting of SpyTag, EFCA, WRESAI, ARVCF, CaM, SZ21, VMANU, and DnaEAC35. In one embodiment, the first conjugation domain and the second conjugation domain of the biofilm matrix are different conjugation domains.

In one embodiment, the first partner conjugation domain of the biofilm matrix is selected from the group consisting of SpyCatcher, a PDZ domain, Tipl, InaD, M13, SZ16, VMAANll, and DnaEC35. In one embodiment, the second partner conjugation domain of the biofilm matrix is selected from the group consisting of SpyCatcher, a PDZ domain, Tipl, InaD, M13, SZ16, VMAANll, and DnaEC35. In one embodiment, the PDZ domain is PDZ1 or ePDZ-bl. In one embodiment, the first partner conjugation domain and the second partner conjugation domain of the biofilm matrix are different partner conjugation domains.

In one embodiment, the first functionalizing polypeptide of the biofilm matrix is a first enzyme. In one embodiment, the second functionalizing polypeptide of the biofilm matrix is a second enzyme. In one embodiment, the first enzyme of the biofilm matrix is selected form the group consisting of an amylase, a dehydrogenase, and a ketoreductase. In one embodiment, the second enzyme of the biofilm matrix is selected form the group consisting of an amylase, a dehydrogenase, and a ketoreductase. In one embodiment, the first enzyme and the second enzyme of the biofilm matrix are selected from the group consisting of phosphite dehydrogenase (PTDH), RADH, and CtXR.

In one embodiment, the biofilm matrix further comprises an engineered microbial cell. In one embodiment, the engineered microbial cell of the biofilm matrix is any one of the engineered microbial cells of the invention. In another aspect, the invention provides a method of producing a biofilm having a biocatalytic surface, comprising culturing a genetically engineered bacterium in culture media, wherein the genetically engineered bacterium expresses a first CsgA protein fused to a first conjugation domain, and a second CsgA protein fused to a second conjugation domain, thereby forming a plurality of curli fibers which form a biofilm, contacting the biofilm with a first functionalizing polypeptide which is linked to a first partner conjugation domain, wherein the first partner conjugation domain binds to the first conjugation domain, and contacting the biofilm with a second functionalizing polypeptide which is linked to a second partner conjugation domain, wherein the second partner conjugation domain binds to the second conjugation domain, wherein the first functionalizing polypeptide and the second functionalizing polypeptide form an enzymatic cascade, thereby producing a biofilm having a biocatalytic surface. In some embodiments, the first CsgA protein and the second CsgA protein are of the same type (i.e., CsgA proteins having identical amino acid sequences). In some embodiments, the first CsgA protein and the second CsgA protein are different {i.e., CsgA proteins having different amino acid sequences). In some embodiments the biofilm is contacted with the first functionalizing polypeptide linked to the first partner conjugation domain and with the second functionalizing polypeptide linked to the second partner conjugation domain concurrently. In some embodiments the biofilm is contacted with the first functionalizing polypeptide linked to the first partner conjugation domain and with the second functionalizing polypeptide linked to the second partner conjugation domain sequentially. In one embodiment, the method further comprises a step of processing, e.g., filtering, the culture media onto a cloth, e.g., a cotton cloth, or a textile.

In another aspect, the invention provides a method of producing a biofilm having a biocatalytic surface capable of regeneration, comprising culturing a genetically engineered bacterium in culture media, wherein the genetically engineered bacterium expresses a first CsgA protein fused to a first conjugation domain, and a second CsgA protein fused to a second conjugation domain, thereby forming a plurality of curli fibers which form a biofilm, contacting the biofilm with a first functionalizing polypeptide which is linked to a first partner conjugation domain, wherein the first partner conjugation domain binds to the first conjugation domain, and contacting the biofilm with a second functionalizing polypeptide which is linked to a second partner conjugation domain, wherein the second partner conjugation domain binds to the second conjugation domain, wherein the first functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionalizing polypeptides are capable of being removed from the plurality of curli fibers, thereby producing a bio film having a biocatalytic surface capable of regeneration. In some embodiments the biofilm is contacted with the first functionalizing polypeptide linked to the first partner conjugation domain and with the second functionalizing polypeptide linked to the second partner conjugation domain concurrently. In some embodiments the biofilm is contacted with the first functionalizing polypeptide linked to the first partner conjugation domain and with the second functionalizing polypeptide linked to the second partner conjugation domain sequentially. In one embodiment, the method further comprises a step of processing, e.g., filtering, the culture media onto a cloth, e.g., a cotton cloth, or a textile.

In one embodiment of the methods, each curli fiber within the plurality of curli fibers comprises the first CsgA protein fused to the first conjugation domain and the second CsgA protein fused to the second conjugation domain. In one embodiment, the plurality of curli fibers comprises a first curli fiber and a second curli fiber, wherein the first curli fiber comprises the first CsgA protein and the first conjugation domain, and wherein the second curli fiber comprises the second CsgA protein and the second conjugation domain. In some embodiments, the first CsgA protein and the second CsgA protein are of the same type (i.e., CsgA proteins having identical amino acid sequences). In some embodiments, the first CsgA protein and the second CsgA protein are different (i.e., CsgA proteins having different amino acid sequences).

In one embodiment of the methods, the first conjugation domain covalently binds to the first partner conjugation domain, and the second conjugation domain covalently binds to the second partner conjugation domain. In one embodiment of the methods, the first conjugation domain covalently binds to the first partner conjugation domain, and the second conjugation domain non-covalently binds to the second partner conjugation domain In one embodiment of the methods, the first conjugation domain non-covalently binds to the first partner conjugation domain, and the second conjugation domain covalently binds to the second partner conjugation domain. In one embodiment of the methods, the first conjugation domain non-covalently binds to the first partner conjugation domain, and the second conjugation domain non-covalently binds to the second partner conjugation domain

In one embodiment of the methods, the first functionalizing polypeptide linked to the first partner conjugation domain, and the second functionalizing polypeptide linked to the second partner conjugation domain are co-expressed by the genetically engineered bacterium. In one embodiment of the methods, the genetically engineered bacterium is a genetically engineered E. coli bacterium.

In one embodiment of the methods, the method further comprises a step of removing the genetically engineered bacterium from the bio film before the contacting steps. In one embodiment of the methods, the removing comprises washing the genetically engineered bacterium from the biofilm. In one embodiment of the methods, the method further comprises a step of killing the genetically engineered bacterium in the biofilm before the contacting steps. In one embodiment of the methods, the first functionalizing polypeptide and the second functionalizing polypeptide maintain their catalytic activity after the step of removing or the step of killing the genetically engineered bacterium. In one embodiment, the method further comprises a step of processing, e.g., filtering, the culture media onto a cloth, e.g., a cotton cloth, or a textile.

In one embodiment of the methods, the genetically engineered bacterium is cultured in a bioreactor. In one embodiment of the methods, the bioreactor is a batch bioreactor or a continuous flow bioreactor. In one embodiment, the method further comprises a step of processing, e.g., filtering, the culture media onto a cloth, e.g., a cotton cloth, or a textile.

In another aspect, the invention provides a method of regenerating a biocatalytic surface on a biofilm matrix produced by a genetically modified bacterium, comprising removing a first functionalizing polypeptide which is linked to a first partner conjugation domain, from a first curli fiber comprising a first CsgA protein and a first conjugation domain; and contacting the biofilm matrix with a third functionalizing polypeptide which is linked to a third partner conjugation domain, thereby binding the third partner conjugation domain to the first conjugation domain, wherein the biofilm matrix also comprises a second curli fiber comprising a second CsgA protein and a second conjugation domain, wherein the second conjugation domain is bound to a second partner conjugation domain which is linked to a second functionalizing polypeptide; thereby regenerating the biocatalytic surface of the biofilm matrix produced by the genetically modified bacterium. In some embodiments, the removal of the first functionalizing polypeptide does not affect the binding of the second conjugation domain to the second partner conjugation domain. In some embodiments, the removal of the first functionalizing polypeptide does not decrease the function of the second functionalizing polypeptide. In some embodiments, the removal of the first functionalizing polypeptide does not affect the binding of the second conjugation domain to the second partner conjugation domain and does not decrease the function of the second functionalizing polypeptide. In some embodiments, the third functionalizing polypeptide is the same type of functionalizing polypeptide as the first functionalizing polypeptide. In some embodiments, the third functionalizing polypeptide is a different type of functionalizing polypeptide as the first functionalizing polypeptide. In one embodiment of the methods, the method further comprises removing the second functionalizmg polypeptide which is linked to the second conjugation domain, from the second curli fiber comprising the second CsgA protein and the second conjugation domain; and contacting the biofilm matrix with a fourth functionalizing polypeptide which is linked to a fourth partner conjugation domain, thereby binding the fourth partner conjugation domain to the second conjugation domain. In some embodiment, the removal of the second functionalizing polypeptide does not affect the binding of the first conjugation domain to the third partner conjugation domain. In some embodiments, the removal of the second functionalizing polypeptide does not decrease the function of the third functionalizing polypeptide. In some embodiment, the removal of the second functionalizing polypeptide does not affect the binding of the first conjugation domain to the third partner conjugation domain and does not decrease the function of the third functionalizing polypeptide. In some embodiments, the fourth functionalizing polypeptide is the same type of functionalizing polypeptide as the second functionalizing polypeptide. In some embodiments, the fourth functionalizing polypeptide is a different type of functionalizing polypeptide as the second functionalizing polypeptide.

In one embodiment of the methods, the first functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionalizing polypeptides are removed from the biofilm matrix by disrupting a protein-protein interaction. In one embodiment of the methods, the protein-protein interaction is disrupted with guanidine, a chelating agent (e.g., EDTA or EGTA), pH change, a redox reagent (e.g., dithiothreitol (DTT)), urea, or other denaturing agent.

In another aspect, the invention provides a method of producing a chemical using any of the curli fibers or pluralities of curli fibers of the invention and/or any of the biofilm matrixes of the invention.

In another aspect, the invention provides a method of purifying water using the any of the curli fibers or pluralities of curli fibers of the invention and/or any of the biofilm matrixes of the invention.

In another aspect, the invention provides a method of cleaning a chemical spill using any of the curli fibers or pluralities of curli fibers of the invention and/or any of the biofilm matrixes of the invention.

Genetically modified bacteria disclosed herein comprise a nucleic acid sequence that encodes a CsgA fusion, which is composed of CsgA protein fused to a first conjugation domain. The CsgA protein unit assembles the curli fibers that make up a biofilm. The CsgA fusions of the bio film then function as a conjugation system to immobilize functionalizmg polypeptides (such as enzymes or metal binding proteins) on the curli fiber. For example, the first conjugation domain that is fused to the CsgA protein unit that makes up the curli fiber, can capture and immobilize a functionalizing peptide, which is linked to a first partner conjugation domain, on the curli fibers. The functionalizing polypeptides then form a surface coating on the biofilm, which has catalytic properties provided by the immobilized enzymes that are attached to the curli fibers.

In one aspect, a curli fiber may comprise a first CsgA fusion bound to a first partner conjugation domain which is liked to a first functionalizing polypeptide, and a second CsgA fusion bound to a second partner conjugation domain which is liked to a second

functionalizing polypeptide. In another aspect, disclosed herein is a plurality of curli fibers comprising a first curli fiber and a second curli fiber, wherein the first curli fiber comprises a first CsgA protein and a first conjugation domain, wherein the first conjugation domain is bound to a first partner conjugation domain which is linked to a first functionalizing polypeptide; and wherein the second curli fiber comprises a second CsgA protein and a second conjugation domain, wherein the second conjugation domain is bound to a second partner conjugation domain which is linked to a second functionalizing polypeptide. The first functionalizing polypeptide and the second functionalizing polypeptide may form an enzymatic cascade. Additionally, the functionalizing polypeptides may be capable of regeneration.

Further features and advantages of certain embodiments of the present invention will become more fully apparent in the following description of embodiments and drawings thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present embodiments will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:

Figure 1 schematically depicts the Biofilm Integrated Nanofiber Display (BIND).

Figure 2A graphically depicts the results of Congo Red pull down assay with CsgA and different tags fused to CsgA.

Figure 2B graphically depicts enzyme activity of amylase and amylase with fused conjugation-domains.

Figure 3A graphically depicts enzyme activities of amylase-tags conjugated to curli fibers presenting different tags. Different groups of conjugation and partner conjugation domains were tested. Complementary conjugation pairs are in black, control with wt-CsgA and wt-Amyl are in white and mismatched conjugation domains are striped.

Figure 3B depicts the enzyme activities of amylase-tags conjugated to curli fibers presenting different tags. Different groups of conjugation domain and partner conjugation domain were tested. Complementary conjugation pairs are boxed.

Figure 4A schematically depicts enzyme removal from BIND and regeneration with new enzymes.

Figure 4B graphically depicts the enzyme removal and new enzyme regeneration results from BIND.

Figure 4C graphically depicts the results of enzyme removal and regeneration in the control.

Figure 4D graphically depicts the results of enzyme removal.

Figure 4E graphically depicts the results of enzyme regeneration.

Figure 5A graphically depicts denaturation yields for additional conditions for enzyme removal and regeneration.

Figure 5B graphically depicts regeneration yields for additional conditions for enzyme removal and regeneration.

Figure 6A schematically depicts bi-enzymatic curli fibers catalyzing the formation of stereoselective alcohol by ketoreductases in a NADH dependent manner.

Figure 6B graphically depicts the activity of PTDH immobilized on BIND depending on immobilization time after one day, seven days, or 17 days.

Figure 6C graphically depicts Michaelis-Menten kinetics of PTDH with NAD+ and NADP+ as substrate in presence of 2 mM phosphite.

Figure 7A graphically depicts co-immobilization of SC-KRED and PTDH-SC on CsgA-ST.

Figure 7B graphically depicts co-immobilization of M13-KRED and PTDH-InaD on mixed CsgA-CaM, CsgA-EFCA fibers.

Figure 7C graphically depicts co-immobilization of M13-KRED and PTDH-InaD on CsgA-CaM-CsgA-EFCA co-fibers.

Figures 8A and 8B depict the flow-reactor assembly with cotton cloth as curli support material (Figure 8A) and the time series of flowing different food coloring through the flow- reactor (flow speed: ~5 ml/min, time between pictures: ~2 minutes).

Figures 9A, 9B, 9C, and 9D demonstrate that ST-CsgA was deposited on different filter materials to evaluate their potential for the use in a flow-based reactor. Figure 9A shows that, after incubation with phosphite dehydrogenase (PTDH), the thick cotton cloth showed the highest enzyme activity, but also a lot of unspecific bound enzyme was present. In contrast, thin cotton cloth showed almost only specific enzyme binding. In contrast to that, enzyme bound non-specific to nylon filter (pore size: 10 pm). Due to the small thickness and the good performance of the thin cotton cloth, we continued with this material. OD 340 nm corresponds to the absorbance of formed NADH. Figure 9B is a SEM picture of CsgA-ST deposited on thin cotton cloth. Figure 9C is a SEM picture of CsgA-ST deposited on thick cotton cloth. Figure 9D is a SEM picture of CsgA-ST deposited on nylon filter.

Figure 10 depicts results using different blocking agents that were tested to optimize the blocking and the washing of unspecific bound PTDH on CsgA-ST. The best ratio of specific bound enzyme (PTDH-SC) versus unspecific bound enzyme (PTDH) was achieved by using 5% milk as a blocking agent. The unspecific binding after blocking and washing with 5% milk on thin cotton cloth was only 13.4% ± 0.4% of the specific binding. OD 340 nm corresponds to the absorbance of formed NADH.

Figure 11 depicts a graph summarizing the results from optimizing the procedure of curli deposition on thin cotton cloth. Five layers of cloth were stacked and curli was deposited on them. Subsequently, enzyme was individually immobilized on them and the activity was measured. The top layer (1) showed clearly the highest activity of all the layers. This implicates that the most effective curli binding occur on the first layer and therefore, we cannot simultaneously deposit curli on multiple cotton cloth. OD 340 nm corresponds to the absorbance of formed NADH.

Figures 12A, 12B, and 12C depict results from testing the stability of deposited CsgA on thin cotton cloth in different ways. Figure 12A: The cloth was incubated for 30 minutes in different organic solvents, acidic and basic conditions. Subsequently, enzyme (amylase) was immobilized and the activity was measured. While the acidic and basic conditions impair the deposited curli only negligible, acetonitrile, ethanol, and DMSO lowers the activity below 50% . Dichloromethane has the least effect of the organic solvents on the curli-cloth interaction. Figure 12B: The impact of washing and drying was tested on the curli fibers and enzymes. To test the stability of the curli fibers on cellulose, the enzymes were immobilized after washing and drying. For the testing of the impact on enzyme activity, the enzyme was immobilized before washing and drying. Only the condition where the cloth with curli fibers was dried before enzyme immobilization the enzyme activity decreased. Figure 12C:

Stability of amylase in the reactor compared to free enzyme. DETAILED DESCRIPTION

The present disclosure is directed to the use of a genetically modified bacterium, which is capable of producing one or more curli fibers comprising CsgA protein units linked to one or more heterologous functionalizing polypeptides through a conjugation domain and a partner conjugation domain. The curli protein nanofibers disclosed herein enable one or more enzymes to be immobilized site- specifically on the bio film, without the need for purification or chemical modification. Moreover, the methods disclosed herein allow immobilization of multiple enzymes (or other functionalizing polypeptides) simultaneously on a biofilm. This allows for the disruption of different protein-peptide interactions under different conditions, thereby allowing for specific release and regeneration of one or more enzymes (or other functionalizing polypeptides) from the surface of the bio films. Aspects of the present disclosure use principles of Biofilm Integrated Nanofiber Display (BIND).

In order that the disclosure may be more readily understood, certain terms are first defined. These definitions should be read in light of the remainder of the disclosure and as understood by a person of ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Additional definitions are set forth throughout the detailed description.

Definitions

As used herein, the term "curli fiber" refers to the primary proteinaceous structural component of E. coli biofilms. Curli fibers are highly robust functional amyloid nanofibers with a diameter of -4-7 nm that exist as extended tangled networks encapsulating the cells. Curli fibers are formed from the extracellular self-assembly of CsgA, a small secreted 13-kDa protein

As used herein, "CsgA" refers to the major structural subunit of the curli fiber. The sequences of CsgA and its homo logs are known in a number of species. For example, the sequence of Escherichia coli CsgA is known (NCBI Gene ID NO: 949055; (polypeptide)). CsgA polypeptide (NCBI Ref Seq: NP_415560):

mkllkvaaiaaivfsgsalagvvpqyggggnhggggnnsgpnselniyqygggnsalalqtdarnsdltitqhgggngadvgqg sddssidltqrgfgnsatldqwngknsemtvkqfgggngaavdqtasnssvnvtqvgfgnnatahqy (SEQ ID NO: 1). A CsgA protein may include naturally occurring mutations or variants of CsgA, homologs of CsgA, or engineered mutations or variants of CsgA. In some embodiments, "CsgA" refers to E. coli CsgA. In some embodiments, "CsgA" refers to a polypeptide having at least 80% homology to SEQ ID NO: 1 (e.g., 80% or greater homology, 90% or greater homology, or 95% or greater homology). In some embodiments, CsgA refers to a polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, CsgA refers to a polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the amino acid sequence of SEQ ID NO: 1.

The terms "protein" and "polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha- amino and carboxy groups of adjacent residues. The terms "protein," and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and "polypeptide" are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

A "nucleic acid" or "nucleic acid sequence" may be any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single- stranded or double-stranded. A single- stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double- stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including mRNA.

As used herein, the term "gene" refers to a nucleic acid fragment that encodes a protein or fragment thereof, optionally including regulatory sequences preceding (5' non- coding sequences) and following (3' non-coding sequences) the coding sequence. In one embodiment, a "gene" does not include regulatory sequences preceding and following the coding sequence. Each gene may be present on a plasmid or bacterial chromosome. In addition, multiple copies of any gene may be present in the bacterium, wherein one or more copies of the gene may be altered as described herein.

A "native gene" refers to a gene as found in nature, optionally with its own regulatory sequences preceding and following the coding sequence. A "chimeric gene" refers to any gene that is not a native gene, optionally comprising regulatory sequences preceding and following the coding sequence, wherein the coding sequences and/or the regulatory sequences, in whole or in part, are not found together in nature. Thus, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory and coding sequences that are derived from the same source, but arranged differently than is found in nature.

As used herein, the term "endogenous gene" refers to a native gene in its natural location in the genome of an organism.

As used herein, a "heterologous" gene or "heterologous sequence" refers to a nucleotide sequence that is not normally found in a given cell in nature. As used herein, a heterologous sequence encompasses a nucleic acid sequence that is exogenously introduced into a given cell. "Heterologous gene" includes a native gene, or fragment thereof, that has been introduced into the host cell in a form that is different from the corresponding native gene. For example, a heterologous gene may include a native coding sequence that is a portion of a chimeric gene to include a native coding sequence that is a portion of a chimeric gene to include non-native regulatory regions that is reintroduced into the host cell. A heterologous gene may also include a native gene, or fragment thereof, introduced into a non- native host cell. Thus, a heterologous gene may be foreign or native to the recipient cell; a nucleic acid sequence that is naturally found in a given cell but expresses an unnatural amount of the nucleic acid and/or the polypeptide which it encodes; and/or two or more nucleic acid sequences that are not found in the same relationship to each other in nature.

As used herein, the term "expression" refers to the transcription and stable accumulation of sense (mRNA) or anti-sense RNA derived from a nucleic acid, and/or to translation of an mRNA into a polypeptide.

As used herein, the term "plasmid" or "vector" refers to an extrachromosomal nucleic acid, e.g., DNA, construct that is not integrated into a bacterial cell' s genome. Plasmids are usually circular and capable of autonomous replication. Plasmids may be low-copy, medium- copy, or high-copy, as is well known in the art. Plasmids may optionally comprise a selectable marker, such as an antibiotic resistance gene, which helps select for bacterial cells containing the plasmid and which ensures that the plasmid is retained in the bacterial cell. A plasmid may comprise a nucleic acid sequence encoding a heterologous gene or gene cassette.

As used herein, the term "transform" or "transformation" refers to the transfer of a nucleic acid fragment into a host bacterial cell, resulting in genetically-stable inheritance. Host bacterial cells comprising the transformed nucleic acid fragment are referred to as "recombinant" or "transgenic" or "transformed" organisms.

As used herein, the term "engineered microbial cell" or "engineered bacterial cell" refers to a bacterial cell or bacteria that have been genetically modified from their native state. For instance, an engineered bacterial cell may have nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications introduced into their DNA. These genetic modifications may be present in the chromosome of the bacteria or bacterial cell, or on a plasmid in the bacteria or bacterial cell. Engineered bacterial cells disclosed herein may comprise exogenous nucleotide sequences on plasmids. Alternatively, engineered bacterial cells may comprise exogenous nucleotide sequences stably incorporated into their chromosome.

As used herein, a "CsgA fusion" or an "engineered CsgA polypeptide" refers to a CsgA polypeptide comprising a heterologous conjugation domain attached to the CsgA at either the C-terminus or the N terminus or both, but without interrupting the sequence of the CsgA polypeptide. In one aspect, a CsgA fusion self- assembles into a curli fiber and is used to capture and immobilize a functionalizing polypeptide which comprises a partner conjugation domain.

As used herein, the term "fusion" or "protein fusion" refers to a chimeric protein created through the joining of two or more genes that originally encoded separate proteins. A protein fusion is created artifically using recombinant DNA technology. Disclosed herein are CsgA proteins fused, or linked, to conjugation domains. Also disclosed herein are functionalizing polypeptides fused, or linked, to partner conjugation domains.

As used herein, the term "bound to" refers to an interaction between to molecules or proteins. A protein may be covalently or non-covalently bound to another protein or molecule. As used herein, a "covalent bond" refers to a chemical bond that involves the sharing of electron pairs between atoms. In contrast, a "non-covalent bond" does not involve the sharing of electrons, but involves more dispersed variations of electrmagnetic interactions between molecules. Non-covalent bonds include, but are not limited to, electrostatic, van der Walls forces, and hydrophobic effects.

As used herein, the term "conjugation domain" refers to a polypeptide that can specifically bind to a partner conjugation domain. A conjugation domain may bind to a partner conjugation domain covalently or non-covalently. A conjugation domain can be, e.g., about 100 amino acids or less in size, about 75 amino acids or less in size, about 50 amino acids or less in size, about 40 amino acids or less in size or smaller. Conjugation domains described herein are linked, or fused, to a CsgA protein. Examples of conjugation domains are well known in the art and are described in more detail, below.

As used herein, the term "partner conjugation domain" refers to a polypeptide that can specifically bind to a conjugation domain. A partner conjugation domain may bind to a conjugation domain covalently or non-covalently and may be about the same size as the conjugation domain or larger. For example, a partner conjugation domain can be about 4000 amino acids or less in size, about 3000 amino acids or less in size, about 2000 amino acids or less in size, about 1000 amino acids or less in size, about 500 amino acids or less in size, about 200 amino acids or less in size, about 100 amino acids or less in size, about 75 amino acids or less in size, about 50 amino acids or less in size, about 40 amino acids or less in size, or smaller. Partner conjugation domains described herein are linked, or fused, to a functionalizing polypeptide. Examples of conjugation domains are well known in the art and are described in more detail, below.

As used herein, the term "functionalizing polypeptide" refers to a polypeptide having an activity or function, such that when it is present on a curli fiber and/or in a bio film, it confers upon the curli fiber and/or biofilm a property, function, or activity which it did not have in the absence of the polypeptide. Such functions include catalytic function, recognition function, or structural function. A functionalizing polypeptide can be of any size and include, e.g., an enzyme, a polypeptide that binds another molecule, an antibody, a therapeutic agent, a diagnostic agent, a metal, an antimicrobial agent, an anti-inflammatory agent, an anticancer agent, etc.

As used herein, the term "biofilm matrix" refers to a matrix of extracellular polymeric substances, including, but not limited to extracellular DNA, proteins, glyopeptides, and polysaccharides, which was produced by a mass of microorganisms, such as bacteria, but wherein the microorganisms have been completely or almost completely killed or removed. Accordingly, in one embodiment, a "biofilm matrix" does not comprise any microorganisms, such as bacteria. In one embodiment, a "biofilm matrix" does not comprise any live microorganisms, such as bacteria.

As used herein, the term "biofilm" refers to a matrix of extracellular polymeric substances, including, but not limited to extracellular DNA, proteins, glyopeptides, and polysaccharides, which are produced by a mass of microorganisms, such as bacteria. In one embodiment, a biofilm comprises a biofilm matrix and bacteria. In one embodiment, the bacteria are live bacteria.

As used herein, the term "enzyme" refers to a polypeptide that can act as a catalyst to accelerate or catalyze a chemical reaction. As used herein, the term "enzymatic cascade" refers to two or more polypeptides which are involved in a series of successive chemical reactions.

A "biofilm having a catalytic surface" refers to a biofilm which comprises one or more types of curli fibers which have different properties, functions, and/or activities as a result of the two or more specific functionalizmg polypeptides which are bound to the one or more different types of curli fibers. In one aspect, a curli fiber may comprise a first CsgA fusion polypeptide bound to a first partner conjugation domain which is liked to a first functionalizmg polypeptide, and a second CsgA fusion polypeptide bound to a second partner conjugation domain which is liked to a second functionalizmg polypeptide. In another aspect, disclosed herein is a plurality of curli fibers comprising a first curli fiber and a second curli fiber, wherein the first curli fiber comprises a first CsgA protein and a first conjugation domain, wherein the first conjugation domain is bound to a first partner conjugation domain which is linked to a first functionalizmg polypeptide; and wherein the second curli fiber comprises a second CsgA protein and a second conjugation domain, wherein the second conjugation domain is bound to a second partner conjugation domain which is linked to a second functionalizmg polypeptide. The first functionalizmg polypeptide and the second functionalizmg polypeptide form an enzymatic cascade, and a biofilm comprising such curli fibers has a catalytic surface. The terms "biocatalytic" and "catalytic" are used

interchangeably in this disclosure.

The term "remove" or "removal" refers to the separation, or ehmination of a polypeptide. In one embodiment, a polypeptide may be "removed" from a curli fiber of a biofilm disclosed herein by disruption of the binding between a conjugation domain and a partner conjugation domain. Such "removal" can occur, for example, by the disruption of the protein-protein itneractions between the conjugation domain and the partner conjugation domain.

The term "regenerate," "regeneration," or "regenerated" refers to the process of replacing a new or different functionalizmg polypeptide on a CsgA polypeptide, curli fiber, and/or biofilm after the removal of a first functionalizmg polypeptide, thereby re- immobilizing the functionalizmg polypeptide on the CsgA polypeptide, curli fiber and/or biofilm. For example, when a functionalizing polypeptide is an enzyme, the enzyme may be removed once it loses its enzymatic activity and then regenerated, e.g., replaced, with a new enzyme having enzymatic activity.

Definitions of common terms in cell biology and molecular biology can be found in The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN- 10: 0763766321); Kendrew et al. (eds.), , Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081- 569-8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al, eds.

The articles "a" and "an," as used herein, should be understood to mean "at least one," unless clearly indicated to the contrary.

The phrase "and/or," when used between elements in a list, is intended to mean either (1) that only a single listed element is present, or (2) that more than one element of the list is present. For example, "A, B, and/or C" indicates that the selection may be A alone; B alone; C alone; A and B; A and C; B and C; or A, B, and C. The phrase "and/or" may be used interchangeably with "at least one of or "one or more of the elements in a list.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Curli Fibers

Curli fibers are the primary proteinaceous structural component of bio films. They are highly robust functional amyloid nano fibers with a diameter of -4-7 nm that exist as extended tangled networks encapsulating the cells. Curli fibers are formed from the extracellular self-assembly of CsgA, a small secreted 13-kDa protein, see Chapman, M. R. et al. Role of Escherichia coli curli operons in directing amyloid fiber formation. Science 295, 851-855 (2002), hereby incorporated by reference in its entirety. A homologous outer- membrane protein, CsgB, nucleates CsgA assembly and also anchors the nano fibers to the bacterial surface. Detached curli fibers can also exist as non-cell associated structural components of the extra-cellular membrane (ECM). The curli genes exist as two divergently transcribed operons (csgBAC and csgDEFG), whose seven products mediate the structure (CsgA), nucleation (CsgB), processing (CsgE, F), secretion (CsgC, G), and direct transcriptional regulation (CsgD) of curli nanofibers. This curli secretion system is considered a distinct secretion system of its own in gram-negative bacterium and is named the Type- VIII secretion system (T8SS). See Desvaux et ah , Trends Microbiol. 17, 139-45 (2009) hereby incorporated by reference in its entirety.

In one aspect, E. coli expressing curli fibers may be used for the methods disclosed herein. In another aspect, other useful bacteria with suitable secretions systems known to those of skill in the art may be used to produce the electrically conductive curli fibers of the present disclosure. The bacterium can be non-pathogenic.

As used herein, a "CsgA fusion" or an "engineered CsgA polypeptide" refers to a CsgA polypeptide comprising a heterologous conjugation domain attached to the CsgA at either the C-terminus or the N terminus or both, but without interrupting the sequence of the CsgA polypeptide. In one aspect, a CsgA fusion self- assembles into a curli fiber and is used to capture and immobilize a functionalizing polypeptide comprising a partner conjugation domain on the generated curli fibers. Specifically, the conjugation domain of the CsgA fusion can bind to a partner conjugation domain present as part of the functionalizing polypeptide, thereby capturing, or immobilizing, the functionalizing polypeptide on a curli fiber of a bio film.

As used herein the term "plurality" refers to more than one. A "plurality of curli fibers" refers to more than one particular unit of a curli fiber (e.g., more than one unit of the same kind or type of a particular curli fiber or a combination of two or more units of different kinds or types of curli fiber). In some embodiments, a plurality of curli fibers may include more than one type of curli fiber, wherein each type of curli fiber has a different property, function and/or activity. For example, a plurality of curli fibers can comprise two types of curli fibers, wherein the first curli fiber has a first functionalizing polypeptide, and the second curli fiber has a second functionalizing polypeptide, wherein the first and second

functionalizing polypeptides are different and confer different properties, functions and/or activities to the curli fibers. In some embodiments, a plurality of curli fibers comprises two or more different types of curli fiber, wherein each type of curli fiber comprises different engineered CsgA polypeptides. For example, in some embodiments, the plurality of curli fibers comprises a first type of curli fiber and a second type of curli fiber, wherein the first type of curli fiber comprises a first engineered CsgA polypeptide comprising a first CsgA protein and a first conjugation domain, and the second type of curli fiber comprises a second engineered CsgA polypeptide comprising a second CsgA protein and a second conjugation domain. In some embodiments, the first and second conjugation domains are of the same kind or type. In some embodiments, the first and second conjugation domains are of a different kind or type. In other embodiments, a plurality of curli fibers refers to multiple types of curli fibers, e.g., three types of curli fibers, four types of curli fibers, five types of curli fibers, six types of curli fibers, seven types of curli fibers, eight types of curli fibers, nine types of curli fibers, ten types of curli fibers, etc., wherein each curli fiber comprises a different functionalizing polypeptide. In other embodiments, a plurality of curli fibers includes multiple copies of one curli fiber of a particular type, wherein each curli fiber comprises a first functionalizing polypeptide and a second functionalizing polypeptide.

In some embodiments, a plurality of curli fibers comprises 2 different types of curli fibers. In some embodiments, a plurality of curli fibers comprises 3 different types of curli fibers. In some embodiments, a plurality of curli fibers comprises 4 different types of curli fibers. In some embodiments, a plurality of curli fibers comprises 5 different types of curli fibers. In some embodiments, a plurality of curli fibers comprises 6 different types of curli fibers. In some embodiments, a plurality of curli fibers comprises 7 different types of curli fibers. In some embodiments, a plurality of curli fibers comprises 8 different types of curli fibers. In some embodiments, a plurality of curli fibers comprises 9 different types of curli fibers. In some embodiments, a plurality of curli fibers comprises 10 different types of curli fibers. In some embodiments, a plurality of curli fibers comprises more than 10 different types of curli fibers.

In exemplary embodiments, a plurality of curli fibers comprises 2 or more types of curli fibers, wherein each type of curli fiber is bound to a different enzyme. A plurality of curli fibers can be created by using two or more genetically engineered microorganisms, each capable of producing a non-naturally occurring curli fiber capable of binding to one functionalizing polypeptide through a conjugation domain and partner conjugation domain, to separately produce homogenous curli fibers comprising a particular functionalizing polypeptide, and then mixing the homogenous groups of curli fibers together, thereby forming a plurality of curli fibers comprising different functionalizing polypeptides.

Alternatively, a plurality of curli fibers can be generated using one genetically engineered microorganism which is capable of producing two or more types of curli fibers capable of binding to two or more functionalizing polypeptides through two or more different conjugation domains and partner conjugation domains. "Mixed fibers" refers to a plurality of curli fibers with two or more different functionalizing polypeptides, wherein each curli fiber in the plurality comprises one particular functionalizing polypeptide.

"Co-fibers" refers to a plurality of curli fibers with two or more different functionalizing polypeptides, wherein each curli fiber in the plurality comprises two or more functionalizing polypeptides. Co-fibers can be produced by mixing two or more genetically engineered microorganisms, each capable of producing one or more engineered CsgA polypeptide fused to a different heterologous functional protein domain, e.g., a conjugation domain, capable of binding to a different functionalizing polypeptide, and then inducing CsgA expression, thereby producing curli fibers comprising two or more CsgA polypeptides capable of binding to two or more different functionalizing polypeptides.

Conjugation Domains and Partner Conjugation Domains

A "conjugation domain" refers to a polypeptide that can specifically bind to a partner conjugation domain, either covalently or non-covalently. A conjugation domain can be, e.g., about 100 amino acids or less in size, about 75 amino acids or less in size, about 50 amino acids or less in size, about 40 amino acids or less in size or smaller. Conjugation domains described herein are linked, or fused, to a CsgA protein. Examples of conjugation domains are well known in the art and include, but are not limited to, SpyTag; biotin acceptor peptide (BAP); biotin carboxyl carrier protein (BCCP); and a peptide comprising a LPXTG motif. In exemplary embodiments, the conjugation domain is SpyTag, EFCA, WRESAI, ARVCF, CaM, SZ21, VMAN11, or DnaEA35. Table 1 lists some examples of conjugation domains and their partners.

Table 1: Conjugation Domains

Figure imgf000036_0001

Figure imgf000037_0002

A "partner conjugation domain" refers to a polypeptide that can specifically bind to a conjugation domain, either covalently or non-covalently, and may be about the same size as the conjugation domain or larger. For example, a partner conjugation domain can be about 4000 amino acids or less in size, about 3000 amino acids or less in size, about 2000 amino acids or less in size, about 1000 amino acids or less in size, about 500 amino acids or less in size, about 200 amino acids or less in size, about 100 amino acids or less in size, about 75 amino acids or less in size, about 50 amino acids or less in size, about 40 amino acids or less in size, or smaller. Partner conjugation domains described herein are linked, or fused, to a functionalizing polypeptide. Examples of partner conjugation domains are also well known in the art and include, but are not limited to, SpyCatcher; streptavidin; streptavidin; and peptides comprising aminoglycine. In exemplary embodiments, the partner conjugation domain is SpyCatcher, a PDZ domain (e.g., PDZ1 or ePDZ-bl), Tipl, InaD, M13, SZ16, ΥΜΑΔΝΠ , ΟΓ DnaEC35.

Table 2: Partner Conjugation Domains

Figure imgf000037_0001

Figure imgf000038_0001

Further discussion of conjugation systems comprising a conjugation domain and a partner conjugation domain can be found, e.g., in Mao et al. J Am Chem Soc 2004 126:2670- 1 ; Zakeri et al. PNAS 2012 109:E690-E697; Maeda et al. Appl Environ Microbiol 2008 74:5139-5145; and U.S. Patent Application No. 14/786304, Genetic Reprogramming of Bacterial Biofilms, each of which is incorporated by reference herein in its entirety.

Functionalizing Polypeptides

A "functionalizing polypeptide" refers to a polypeptide having an activity or function, such that when it is immobilized on, or bound to, a curli fiber and/or in a bio film, it confers upon the curli fiber and/or biofilm a property, function, or activity which it did not have in the absence of the polypeptide. Such functions include catalytic functions, recognition functions, or structural functions. A functionalizing polypeptide can be of any size and include, e.g., an enzyme, a polypeptide that binds another molecule, an antibody, a therapeutic agent, a diagnostic agent, a metal, an antimicrobial agent, an anti- inflammatory agent, an anticancer agent, etc. Exemplary functionalizing polypeptides of the current disclosure are enzymes. In one aspect, the functionalizing polypeptide is amylase. In one aspect, the functionalizing polypeptide is dehydrogenase. In one embodiment, the functionalizing polypeptide is phosphite dehydrogenase (PTDH). In one embodiment, the functionalizing polypeptide is a ketoreductase. In one embodiment, the ketoreductase is RADH. In another embodiment, the ketoreductase is CtXR.

In some embodiments, a polypeptide fusion comprising a functionalizing polypeptide and a partner conjugation domain can further comprise an extracellular localization tag, e.g., a sequence which will cause a cell expressing the polypeptide to secrete the polypeptide. The partner conjugation domain can be located on the N-terminus or on the C-terminus of the functionalizing polypeptide.

In some embodiments, a functionalizing polypeptide is linked to a partner conjugation domain through a spacer sequence. In some embodiments, the partner conjugation domain, spacer sequence, and functionalizing polypeptide are attached in series, for example, having the structure, from N-terminus to C-terminus, partner conjugation domain- spacer- functionalizing polypeptide or functionalizing polypeptide- spacer-partner conjugation domain. In some embodiments, the spacer sequence can be 5 or more amino acids in length. In some embodiments, the spacer sequence is more than 5 amino acids in length, but less than 50 amino acids in length. In some embodiments, the spacer sequence is set forth in Table 4, below.

Biofilms

A "bio film" refers to a matrix of extracellular polymeric substances, including, but not limited to extracellular DNA, proteins, glyopeptides, and polysaccharides, which are produced by a mass of microorganisms, such as bacteria. The nature of a biofilm, such as its structure and composition, can depend on the particular species of bacteria present in the biofilm. Bacteria present in a biofilm are commonly genetically or phenotypically different than corresponding bacteria not in a biofilm, such as isolated bacteria or bacteria in a colony. The biofilms disclosed herein are generally produced by culturing an engineered microbial cell comprising a CsgA fusion (and/or comprising a vector or nucleic acid encoding such a polypeptide) under conditions suitable for the production of curli fibers.

Conditions suitable for the production of a biofilm can include, but are not limited to, conditions under which a microbial cell is capable of logarithmic growth and/or polypeptide synthesis. Conditions may vary depending upon the species and strain of microbial cell selected. Conditions for the culture of microbial cells are well known in the art. Biofilm production can also be induced and/or enhanced by methods well known in the art, e.g., contacting cells with sub-inhibitory concentrations of beta-lactam or aminoglycoside antibiotics, exposing cells to fluid flow, contacting cells with exogenous poly-N- acetylglucosamine (PNAG), or contacting cells with quorum sensing signal molecules. In some embodiments, conditions suitable for the production of a biofilm can also include conditions which increase the expression and secretion of CsgA, e.g., by exogenously expressing CsgD.

In one embodiment, a biofilm refers to the matrix of extracellular polymeric substances that is produced by a mass of microorganisms, wherein the bacteria have been killed or removed. In one embodiment, a biofilm does not comprise any bacteria. In another embodiment, a biofilm does not comprise any live bacteria. In some embodiments, the biofilm can further include the bacterium which produced the biofilm. In one aspect, the biofilm further includes a bacterium expressing a plurality of nucleic acids, wherein each nucleic acid encodes a CsgA fusion protein comprising a CsgA polypeptide and a conjugation domain.

Biofilms disclosed herein may be produced by genetically engineering or modifying bacteria to comprise a nucleic acid encoding a CsgA fusion, consisting of a CsgA protein linked to conjugation domain, and growing the engineered bacteria in situ or in culture media. The nucleic acid encoding a CsgA protein fused to the conjugation domain may be heterologous and introduced into the bacterium using methods known to those of skill in the art. The nucleic acid encoding a fusion CsgA protein may result from mutation of the endogenous nucleic acid encoding CsgA using methods known to those of skill in the art.

A "vector" includes a nucleic acid construct designed for delivery to a host cell or transfer between different host cells. A vector can be viral or non- viral. Many vectors useful for transferring genes into target cells are available, e.g., the vectors may be episomal, e.g., plasmids, virus derived vectors or may be integrated into the target cell genome, through homologous recombination or random integration. In some embodiments, a vector can be an expression vector. An "expression vector" can be a vector that has the ability to incorporate and express heterologous nucleic acid fragments in a cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms. The nucleic acid incorporated into the vector can be operatively linked to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that polynucleotide sequence.

In some embodiments, a nucleic acid encoding a CsgA fusion can be present within a portion of a plasmid. Plasmid vectors can include, but are not limited to, pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKClOl, SV 40, pBluescript II SK +/- or KS +/- (see "Stratagene Cloning Systems" Catalog (1993) from Stratagene, La Jolla, Calif, which is hereby incorporated by reference), pQE, pIH821, pGEX, pET series (see Studier et. al., "Use of T7 RNA Polymerase to Direct Expression of Cloned Genes," Gene Expression Technology , vol. 185 (1990), which is hereby incorporated by reference in its entirety).

A "viral vector" may be a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain a transgenic gene in place of non-essential viral genes. The vector and/or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous viral vectors are known in the art and can be used as carriers of a nucleic acid into a cell, e.g., lambda vector system gtl l, gt WES.tB, Charon 4. In some embodiments, the nucleic acid encoding a CsgA fusion, comprising of a CsgA protein fused to a conjugation domain, can be constitutively expressed.

The engineered bacteria can secrete the CsgA fusion, which results in curli fiber production, followed by biofilm formation. Specifically, after secretion, the CsgA fusion is nucleated to form a self-assembling amyloid at the cell surface, and then continues to polymerize into long fibers that eventually encapsulate the cells and provide the biofilm with structural support. See Figure 1.

A bacterial cell described herein can be any of any species. Preferably, the bacterial cells are of a species and/or strain which is amenable to culture and genetic manipulation. In some embodiments, the bacterial cell can be a gram-positive bacterial cell. In some embodiments, the bacterial cell can be a gram- negative bacterial cell. In some embodiments, the parental strain of the bacterial cell of the technology described herein can be a strain optimized for protein expression. Non-limiting examples of bacterial species and strains suitable for use in the present technologies include Escherichia coli, E. coli BL21, E. coli Tuner, E. coli Rosetta, E. coli JM101, and derivatives of any of the foregoing. Bacterial strains for protein expression are commercially available, e.g., EXPRESS™ Competent E. coli (Cat. No. C2523; New England Biosciences; Ipswich, MA). In one embodiment, the curli fibers are produced by engineered or non-naturally occurring bacterium. In one embodiment, the bacterium is E. coli. In one embodiment, the bacterium is non-pathogenic.

In one embodiment, disclosed herein are methods of producing a biofilm by culturing an engineered bacteria in culture media, wherein the engineered bacteria comprises one nucleic acid encoding a curli fiber or plurality of curli fibers. In another embodiment, disclosed herein are methods of producing a biofilm by culturing an engineered bacteria in culture media, wherein the engineered bacteria comprises more than one nucleic acid encoding a plurality of curli fibers. Engineered bacteria disclosed herein may comprise one or more different nucleic acid sequences encoding a plurality of CsgA proteins fused to different conjugation domains. Each conjugation domain may include an orthogonal specificity for a specific partner conjugation domain that is linked to a functionalizing polypeptide. The orthogonal specificity enables the simultaneous immobilization of two or more distinct functionalizing polypeptides, e.g., enzymes.

In one aspect, the conjugation domain then captures and immobilizes a

functionalizing polypeptide, which is linked to a partner conjugation domain, to the curli fiber and biofilm. In one embodiment, the functionalizing polypeptide linked to the partner conjugation domain is also expressed and secreted by the engineered bacteria. In another embodiment, the functionalizing polypeptide linked to the partner conjugation domain is expressed and secreted by a different engineered bacteria. In yet another embodiment, the functionalizing polypeptide linked to the partner conjugation domain is isolated or purified, and then added to the curli fibers and biofilm.

The terms "bind", "attach", "bound to" or "attached to" refers to the interaction and binding of two polypeptides or proteins to each other. Binding may result in the immobilization of one or more target proteins, e.g., functionalizing polypeptides, to a CsgA fusion protein, curli fiber, plurality of curli fibers, and/or biofilm. "Immobilization" refers to the binding and attachment of a target protein, e.g., a functionalizing polypeptide, to a CsgA fusion protein, curli fiber, plurality of curli fibers, and/or biofilm. Immobilization can be reversible, e.g., through non-covalent bonding, or irreversible, e.g., through covalent bonding.

In one aspect, a functionalizing polypeptide is immobilized on the curli fibers of the biofilm in a self- assembling manner. In one embodiment, the immobilization in a self- assembling manner does not require chemical conjugation. In one aspect, a functionalizing polypeptide is immobilized site-specifically. In one embodiment, the conjugation domain is non-covalently bound to the partner conjugation domain of the functionalizing polypeptide. In one embodiment, the conjugation domain is covalently bound to the partner conjugation domain of the functionalizing polypeptide. In one embodiment, the functionalizing polypeptide is immobilized without protein purification.

In one embodiment, two or more of the same functionalizing polypeptide are immobilized on the same curli fiber. In another embodiment, two or more functionalizing polypeptides are immobilized on the same curli fiber. In yet another embodiment, a first functionalizing polypeptide is immobilized on a first type of curli fiber, and a second functionalizing polypeptide is immobilized on a second type of curli fiber.

In one embodiment, the first functionalizing polypeptide and the second functionalizing polypeptide form an enzymatic cascade. In one embodiment, the

immobilized functionalizing polypeptides maintain catalytic activity when present on the curli fibers in the biofilm. In one embodiment, the different functionalizing polypeptides do not inhibit the production of the biofilm. In one embodiment, the functionalizing

polypeptides immobilized on the curli fibers of the biofilm maintain catalytic activity after bacterium death. In some embodiments, the first functionalizing polypeptide (e.g., a first enzyme) catalyzes the regeneration of a co-factor for use by the second functionalizing polypeptide (e.g., a second enzyme).

In some embodiments, two, three, four, five, six, seven, eight, nine, ten or more functionalizing polypeptides (e.g., enzymes) are immobilized on a curli fiber described herein, wherein each functionalizing polypeptide catalyzes the regeneration of a co-factor for use by a different functionalizing polypeptide immobilized on the curli fiber. In some embodiments, two, three, four, five, six, seven, eight, nine, ten or more functionalizing polypeptides (e.g., enzymes) are immobilized on a curli fiber described herein, wherein each functionalizing polypeptide catalyzes the generation of a substrate for use by a different functionalizing polypeptide immobilized on the curli fiber.

In some embodiments, a biofilm described herein comprises two different types of curli fibers, wherein a functionalizing polypeptide is immobilized onto each curli fiber, and wherein each functionalizing polypeptide catalyzes the regeneration of a co-factor for use by a different functionalizing polypeptide immobilized on the curli fiber. In some embodiments, a biofilm described herein comprises two different types of curli fibers, wherein a functionalizing polypeptide is immobilized onto each curli fiber, and wherein each functionalizing polypeptide catalyzes the generation of a substrate for use by a different functionalizing polypeptide immobilized on the curli fiber. In one embodiment, the functionalizing polypeptides immobilized on the curli fibers form a biofilm comprising a catalytic surface. In some embodiments, the functionalizing polypeptides, e.g., enzymes, function to form an enzymatic cascade. "Enzymatic cascade", "enzyme cascade", or "multi- enzymatic cascade" refers to a series or sequence of successive chemical reactions or transformations on a substrate catalyzed by a combination or series of different enzymes. Often, the chemical transformations are carried out in series in a single step (e.g., a single step manufacturing process).

In one aspect, a curli fiber, plurality of curli fibers, and/or biofilm disclosed herein comprises at least two functionalizing polypeptides, e.g., enzymes. In some embodiments, the curli fiber, plurality of curli fibers, and/or biofilm has three functionalizing polypeptides. In some embodiments, the curli fiber, plurality of curli fibers, and/or biofilm has four functionalizing polypeptides. In some embodiments, the curli fiber, plurality of curli fibers, and/or biofilm has five functionalizing polypeptides. In some embodiments, the curli fiber, plurality of curli fibers, and/or biofilm has six functionalizing polypeptides. In some embodiments, the curli fiber, plurality of curli fibers, and/or biofilm has seven functionalizing polypeptides. In some embodiments, the curli fiber, plurality of curli fibers, and/or biofilm has eight functionalizing polypeptides. In some embodiments, the curli fiber, plurality of curli fibers, and/or biofilm has nine functionalizing polypeptides. In some embodiments, the curli fiber, plurality of curli fibers, and/or biofilm has 10 functionalizing polypeptides. In some embodiments, the curli fiber, plurality of curli fibers, and/or biofilm has more than 10 functionalizing polypeptides.

For example, a biofilm described herein may comprise either one type of curli fiber comprising at least two CsgA fusion proteins, or at least two types of curli fibers each comprising a different CsgA fusion protein, wherein a first conjugation domain is non- covalently bound to a first partner conjugation domain, and a second conjugation domain is non-covalently bound to a second partner conjugation domain.

In one embodiment, a biofilm described herein comprises either one type of curli fiber comprising at least two CsgA fusion proteins, or at least two types of curli fibers each comprising a different CsgA fusion protein, wherein a first conjugation domain is covalently bound to a first partner conjugation domain, and a second conjugation domain is covalently bound to a second partner conjugation domain.

In one embodiment, a biofilm described herein comprises either one type of curli fiber comprising at least two CsgA fusion proteins, or at least two types of curli fibers each comprising a different CsgA fusion protein, wherein a first conjugation domain is non- covalently bound to a first partner conjugation domain, and a second conjugation domain is covalently bound to a second partner conjugation domain.

In one embodiment, a biofilm described herein comprises either one type of curli fiber comprising at least two CsgA fusion proteins, or at least two types of curli fibers each comprising a different CsgA fusion protein, wherein a first conjugation domain is covalently bound to a first partner conjugation domain, and a second conjugation domain is non- covalently bound to a second partner conjugation domain.

Removal and Regeneration

In one embodiment, one or more of the immobilized functionalizing polypeptides are releasable or removable from the curli fiber(s). The term "remove" or "removal" refers to the separation, or elimination of a polypeptide from the curli fiber, plurality of curli fibers, or the biofilm. In one embodiment, a polypeptide may be "removed" from a curli fiber of a biofilm disclosed herein by disruption of the binding between a conjugation domain and a partner conjugation domain. Such "removal" can occur, for example, by the disruption of the protein-protein itneractions between the conjugation domain and the partner conjugation domain. When a functionalizing polypeptide is an enzyme, the enzyme may be removed once it loses its enzymatic activity.

In one aspect, the immobilized functionalizing polypeptide can be released by disrupting the protein-peptide interaction between the partner conjugation domain of the functionalizing polypeptide and the conjugation domain of the CsgA fusion protein. In one aspect, the protein-peptide interaction is disrupted with guanidine, a chelating agent (e.g., EDTA or EGTA), pH change, a redox reagent (e.g., dithiothreitol (DTT)), urea or other denaturing agent. In one aspect, the protein-peptide interaction is disrupted with guanidine. In some embodiments, the functionalizing polypeptide is removed by denaturation.

The term "regenerate," "regeneration," or "regenerated" refers to the process of replacing a new or different functionalizing polypeptide on a CsgA polypeptide, curli fiber, and/or biofilm after the removal of a first functionalizing polypeptide, thereby re- immobilizing the functionalizing polypeptide on the CsgA polypeptide, curli fiber and/or biofilm. For example, when a functionalizing polypeptide is an enzyme, the enzyme may be removed once it loses its enzymatic activity and then regenerated, e.g., replaced, with a new enzyme having enzymatic activity.

In exemplary embodiments, one or more functionalizing polypeptides can be regenerated on a curli fiber, plurality of curli fibers, and/or biofilm that is capable of binding to two or more functionalizing polypeptides. Each functionalizing polypeptide can be removed sequentially or concurrently. For example, two functionalizing polypeptides can be removed at the same time from the curli fiber, plurality of curli fibers and/or bio film or in sequential steps: wherein a first functionalizing polypeptide is removed and regenerated first, and a second functionalizing polypeptide is removed and regenerated at a later time point. Alternatively, the functionalizing polypeptides of a curli fiber, plurality or curli fibers, and/or bio film can be regenerated concurrently, e.g., two functionalizing polypeptides can be removed and regenerated at the same time. Removal and regeneration steps may be repeated.

In one aspect, the curli fibers maintain catalytic activity upon death of the bacterium. In one embodiment, the catalytic activity of the biofilm can be regenerated when, for example, an enzyme loses catalytic activity, by removing the enzymatically inactive enzyme and re-immobilizing another enzyme having functional activity. According to another aspect, the functionalizing polypeptide can be immobilized and then regenerated after the death of the engineered bacterium.

Specifically, curli fibers and/or bio films having a catalytic surface can be made by contacting a CsgA fusion protein comprising a conjugation domain (or a cell, curli fiber and/or biofilm comprising that polypeptide) with a functionalizing polypeptide comprising a partner conjugation domain. In some embodiments, the CsgA fusion and the functionalizing polypeptide comprising the partner conjugation domain are maintained in contact for a period of time, i.e., the "binding step." In some embodiments, the binding step is followed by a washing step, e.g., to remove excess unbound functionalizing polypeptide.

In some embodiments, a CsgA fusion protein comprising a conjugation domain is bound to (or binds) the partner conjugation domain in the presence of albumin (i.e., the "binding step"). In some embodiments, the albumin is BSA. In some embodiments, the albumin is present at about 0.1% to about 10%. In some embodiments, the albumin is present at about 0.5% to about 5%. In some embodiments, the albumin is present at about 1% to about 2%. In some embodiments, the binding step is allowed to proceed for at least about 2 hours, e.g., about 2 hours or more, about 6 hours or more, about 12 hours or more, or about 24 hours or more. In some embodiments, the binding step is allowed to proceed in the presence of albumin.

In some embodiments, the washing step proceeds for about 10 minutes to about 6 hours.

In some embodiments, the washing step proceeds for about 30 minutes to about 3 hours. In some embodiments, the washing step proceeds for about 90 minutes. In some embodiments, the polypeptides are agitated (e.g., shaken) during the washing step. In some embodiments, the washing step comprises washing the polypeptides in a solution of albumin. In some embodiments, the albumin is BSA. In some embodiments, the albumin is present at about 0.01% to about 3%. In some embodiments, the albumin is present at about 0.1% to about 1%. In some embodiments, the albumin is present at about 0.3%. In some

embodiments, the washing step comprises 2 or more successive washes. In some

embodiments, the washing step comprises 3 successive washes.

Methods and Uses

Disclosed herein is a versatile platform to immobilize enzymes on proteinaceous nanofibers of the extracellular matrix of bacteria, e.g., E. coli. One or more functionalizing polypeptides, e.g., enzymes, are immobilized side-specifically onto a biofilm surface, for example, with no need of any protein purification. Additionally, the functionalizing polypeptides, e.g., enzymes, are immobilized in a self-assembling manner, for example, where no additional chemical conjugation is needed. The functionalizing polypeptides, e.g., enzymes, can be specifically released from the biofilm by disrupting the different tag- conjugation domain systems. After the removal of old enzymes, fresh enzymes can again be immobilized, regenerating the enzymatic activity of the biofilm with no significant loss.

The compositions and methods described herein are useful in various applications, including bioremediation, production of bulk and fine chemicals and enzymes, production of biofuels and biohydrogen, and use in microbial fuel cells. Various applications and methods for growing and using biofilms as biocatalysts in industrial processes have been described in Gross et al. Microbial Biofilms: New Catalysts for Maximizing Productivity for Long-Term Biotransformations. Biotechnol. Bioeng. 98(6), 1123- 1134 (2007); Rosche et al. Microbial biofilms: a concept for industrial catalysis?, Trends Biotechnol. 27(11), 636-643 (2009); Gross et al. Characterization of a Biofilm Membrane Reactor and Its Prospects for Fine Chemical Synthesis. Biotechnol. Bioeng. 105(4), 705-717 (2010); Halan et al. Biofilms as living catalysts in continuous chemical syntheses. Trends Biotechnol. 30(9), 453-465 (2012); Gross et al. Engineered Catalytic Biofilms for Continuous Large Scale Production of n- Octanol and (S)-Styrene Oxide. Biotechnol. Bioeng. 110(2), 424-436 (2013); Karande et al. Segmented Flow is Controlling Growth of Catalytic Biofilms in Continuous Multiphase Microreactors. Biotechnol. Bioeng. 111(9), 1831- 1840 (2014); and Karande et al.

Applications of Multiphasic Microreactors for Biocatalytic Reactions. Org. Process Res. Dev. 20(2), 361-370 (2016), hereby incorporated by reference in their entirety. Methods for purifying amyloid fibers (e.g. , by filtration) have been described, for example, in

International Patent Application No. PCT/US2017/32923, filed on May 16, 2017, the entire contents of which are incorporated by reference herein in its entirety.

In some embodiments, the compositions and methods are used for cofactor recycling. For example, two enzymes are immobilized on a bio film of the invention, wherein the first enzyme is responsible for catalyzing the desired transformation, but uses a stoichiometric amount of a cofactor (e.g., NAD(P)H), and the second is an enzyme responsible for catalyzing the regeneration of the cofactor by re-reduction.

In one aspect, phosphite dehydrogenase (PTDH), which regenerates NAD(P)1" to NAD(P)H, is co -immobilized with a ketoreductase (RADH or CtXR). The stable immobilization of functional PTDH is a step in an enzyme cascade, where NAD(P)H act as co-factor. The in situ recycling of NAD(P)H can lower the costs of such enzyme cascades. In one aspect, PTDH and KRED are co-immobilized onto a bio film surface. The present disclosure contemplates different immobilization strategies and optimal conditions for different enzyme cascades. In one aspect, one or more or a plurality of different enzymes may be immobilized on a biofilm surface as described herein. Three exemplary enzymes include those that are homo-oligomeric proteins (PTDH and CtXR: homo-dimers, RADH: tetramer).

In some embodiments, the compositions and methods are used for multi- transformation cascades, wherein multiple immobilized enzymes on a biofilm carry out chemical transformations on a substrate in series in a single step manufacturing process. In some embodiments, the compositions and methods described herein are used to produce a chemical, e.g., a pharmaceutically useful product. In some embodiments, the compositions and methods described herein are used to clean up or remediate a harmful chemical spill, e.g., by catalyzing chemical reactions that transform the harmful chemical into a more benign chemical.

The compositions and methods described herein can be used in a bioreactor. In some embodiments, a curli fiber, plurality of curli fibers, genetically engineered microorganism, and/or biofilm is used in a bioreactor to catalytically transform a substrate. In some embodiments, the bioreactor is a batch reactor. In batch reactors, microorganism are typically grown in suspended mode, used to carry out a reaction, and then are not reused after completion of the reaction. In some embodiments, the bioreactor is a continuous flow bioreactor. In a continuous flow bioreactor, a substrate flows through the bioreactor at a continuous rate. The microorganisms in the continuous flow bioreactor carry out a reaction on the substrate as it flows through the system, and then the products of the reaction are collected continuously as they flow out of the reactor. In some embodiments, a genetically engineered microorganism is cultured and grown in a continuous flow bioreactor, wherein it produces a curli fiber, plurality of curli fibers and/or biofilm capable with a biocatalytic surface. In some embodiments, the genetically engineered microorganism is killed or removed, but the curli fiber, plurality of curli fibers and/or biofilm retains biocatalytic activity in the continuous flow bioreactor. In some embodiments, the bioreactor can be a known type of bioreactor, or a bioreactor custom-made for the particular application. See Rosche et al. Microbial biofilms: a concept for industrial catalysis? Trends Biotechnol. 27(11), 636-643 (2009); Halan et al. Biofilms as living catalysts in continuous chemical syntheses. Trends Biotechnol. 30(9), 453-465 (2012); and Karande et al. Applications of Multiphasic Microreactors for Biocatalytic Reactions. Org. Process Res. Dev. 20(2), 361-370 (2016), hereby incorporated by reference in their entirety. A packed bed reactor {e.g., a trickle bed reactor or submerged bed reactor), a fluidized bed reactor, an air lift reactor, an expanded granular sludge bed reactor, a microreactor, or a multiphasic microreactor may also be used. In some embodiments, the bioreactor comprises a cloth or textile comprising {e.g., coated) with a curli fiber, plurality of curh fibers and/or biofilm described herein. For example, in some embodiments, the bioreactor comprises a vessel {e.g., a cylinder) containing strips or pieces of cloth comprising a curli fiber, plurality of curli fibers and/or biofilm described herein. In some embodiments, the bioreactor comprises a filter comprising a curli fiber, plurality of curli fibers and/or biofilm described herein.

The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

EXAMPLES

The following examples are set forth as being representative of the present disclosure. These examples are not to be construed as limiting the scope of the present disclosure as these and other equivalent embodiments will be apparent in view of the present disclosure, figures and accompanying claims.

Unless otherwise stated, the present invention was performed using standard procedures, as described, for example in Sambrook et al, Molecular Cloning: A Laboratory Manual (3 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2001); Davis et al, Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1995); or Methods in Enzymology: Guide to Molecular Cloning

Techniques Vol.152, S. L. Berger and A. R. Kimmel Eds., Academic Press Inc., San Diego, USA (1987); and Current Protocols in Protein Science (CPPS) (lohn E. Coligan, et. al, ed., John Wiley and Sons, Inc.), which are all incorporated by reference herein in their entireties.

EXAMPLE I: Materials and Methods Cell Strains, Plasmids, and Reagents

All chemicals were purchased from Sigma- Aldrich, unless stated otherwise. Organic solvents were purchased in >98% purity or HPLC grade. All strains and vectors are listed in Tables 3 and 4. All the tags were cloned to the C-terminus of CsgA with a 39aa linker in between (GS6-Flag-GS4-TEV-GS4-His6-GS4). CsgA, CsgA-SpyTag, CsgA-EFCA, CsgA- WRESAI, CsgA-ARVCF, CsgA-CaM, CsgA-SZ21, CsgA-VMANl l and CsgA-DnaEAC35 genes were cloned into pBbEla vectors. The CsgA deletion mutant PHL628-AcsgA

(MG1655 malA-Kan ompR234 AcsgA) (Vidal et al, 1998) used for biofilm experiments (Toba et al, 2011). CsgA was expressed in YESCA media, containing 10 g/L of casamino acids (Fisher, BP1424) and 1 g/L of yeast extract (Fisher, BP1422). DPBS

(LifeTechnologies, 14190-144) without calcium or magnesium was used as the general buffer for enzymatic reactions with a-amylase (abbr. PBS). TBST (2.4 g Tris base, 8.8 g NaCl, 1 mL Tween-20, per L, pH 7.4-7.6) was used as wash buffer.

SpyCatcher gene was acquired from Addgene (plasmid # 35044). PDZ1 of the Drosophila INAD protein (abbr. InaD) and Tax- interacting protein- 1 Q43C (abbr. Tipl) genes were a kind gift from Prof. Jian Xia (Department of Chemistry, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China). The ePDZ-bl gene was a kind gift from Prof. Shohei Koide (Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL). The split intein genes were acquired from Addgene Tvo VMAN11 (abbr. VMAN11) (plasmid # 61813), Tvo VMAANl 1 (abbr. VMAANl 1) (plasmid # 61827), Npu DnaEAC35 (abbr. CnaEAC35) (plasmid # 12172), and Npu DnaEC35 (abbr. DnaEC35) (plasmid # 15335). The a-amylase gene was isolated from Bacillus licheniformis ATCC 14580. The Pseudomonas stutzeri phosphite dehydrogenase Opt 13 (abbr. PTDH) gene was acquired from Addgene (plasmid # 61698). The Ralstonia sp. alcohol dehydrogenase (abbr. RADH) gene was a kind gift from Prof. Wolfgang Kroutil (Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Graz, Austria) and Prof. Dorte Rother (Forschungszentrum Jiilich GmbH, Institute of Bio- and Geosciences, IBG-1 :

Biotechnology, Jiilich, Germany). The Candida tenuis xylose reductase (abbr. CtXR) was a kind gift from Prof. Regina Kratzer and Prof. Bernd Nidetzky (Institute of Biotechnology and Biochemical Engineering, University of Technology, Graz, Austria).

SpyCatcher, InaD, Tipl, ePDZ, M13, SZ16, VMAANl l and DnaEC35 were inserted at the N-terminus of a-Amylase with a spacer in between (GS6-cMyc-GS6). SpyCatcher was also inserted at the C-terminus of a-Amylase with a GS4-TEV spacer in between. The constructs were subcloned into a pET28b vector (Novagen, 69865). All the a-Amylase-tag constructs (SC-Amyl, InaD-Amyl, Tipl-Amyl, ePDZ-Amyl, M13-Amyl, SZ16-Amyl, Amyl- SC, Amyl-VMAANl l and Amyl-DnaEC35 were expressed in BL21(DE3) cells grown in lysogeny broth.

SpyCatcher and ePDZ were inserted at the C-terminus of PTDH with a GS6-cMyc- GS6 spacer in between, and His6, His6-SpyCatcher and His6-M13 were inserted at the N- terminus of RADH and CtXR with a GS6-HA-GS6 spacer in between. The constructs were subcloned into a pET15b vector. All these constructs (His6-PTDH, His6-PTDH-SC, His6- PTDH-ePDZ, His6-RADH, His6-SC-RADH, His6-M13-RADH, His6-CtXR, His6-SC-CtXR and His6-M13-CtXR) were expressed in BL21(DE3) cells grown in lysogeny broth.

Cells were lysed using a Misonix Probe Sonicator 4000. Millipore hydrophilic PTFE filter plates (MSRLN0410) and the Millipore MultiScreen vacuum manifold apparatus were used for filter plate assays. For amylase activity, 4-nitrophenyl-α-D-maltopentaoside (pNPMP, Carbosynth, EN05438) was used as a substrate. For PTDH activity, sodium phosphite dibasic pentahydrate (Sigma, 04283), NAD+ (Sigma, N7004), and NADP+ (Sigma, N0505) were used as substrates. For RADH activity, 2-hydroxy-2-methylpropiophenone (abbr. 2-HPP) (Sigma, 405655), and NADPH (Sigma, N7505) were used as substrate. For CtXR activity, ortho-chloroacetophenone (abbr. OCAP) (Sigma, 183709), and NADH (Sigma, 10107735001) were used as substrate. 2-Methyl-l-phenyl- l,2-propanediol (abbr. 2- MPP) (molMall, 1124) and l-(2-chlorophenyl)ethanol (abbr. CPE) (Santa Cruz

Biotechnology, sc-264529) were used as alcohol standards for HPLC analysis. To inhibit protease activity 1 tbl of protease inhibitor complete (Roche, 05892970001) was used dissolved in 50 ml of corresponding buffer.

All the absorbance measurements were performed on a BioTek HI microplate reader. An iBlot 2 Dry Blotting system (LifeTechnologies) was used for transferring gels to PVDF membranes (LifeTechnologies, IB4010). Anti-HA (HRP) (abl l90) and anti-c-Myc (HRP) (abl9312) were purchased from abeam. Western Blots were developed using Clarity ECL Substrate (BioRad, 170-5060). Images of gels and Western blots were taken on a FluorChem M (proteinsimple) imager. pH was measured using a Mettler Toledo FE20-Basic pH meter with an InLablRoutine probe. Scanning Electron Microscope (SEM) images were taken on a Zeiss Ultra Plus FESEM. HPLC measurements were done on an Agilent 1200 system with a dual pump system and an Agilent 1260 auto sampler in reversed phase using a Poroshell 120 EC-C18 column.

Table 3 provides a list of bacterial strains used in the Examples. Table 4 provides a list of plasmids used in the Examples.

Table 3: Bacterial Strains

Figure imgf000052_0001
Figure imgf000053_0002

Table 4: Plasmids

Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001

Figure imgf000058_0001

EXAMPLE II: Curli Expression

PHL628-AcsgA cells were transformed with pBbE1a plasmids with CsgA, CsgA- SpyTag, CsgA-EFCA, CsgA-WRESAI, CsgA-ARVCF, CsgA-CaM, CsgA-SZ21, VMANl l and DnaEAC35. The cells were then streaked onto LB plates with 100μg/mL carbenicillin (abbr. carb.). Transformed PHL628 cells were grown up in YESCA with 100pg/ml carb. until an OD of 0.4-0.6 at 30 C. Curli expression was induced with 0.3 mM IPTG. Cultures were shaken for 18-24 h at 25 C and 220 rpm.

EXAMPLE III: Quantitative Congo Red (CR) Binding Assays

Congo Red (CR) binding assay was adapted from previously published methods, see Chapman, M. R. et al. Role of Escherichia coli curli operons in directing amyloid fiber formation. Science 295, 851-855 (2002). One mL of induced culture was pelleted at 5000 g for 5 min and resuspended gently in 1ml of PBS. 100μΙ of 0.3M Congo Red was added and allowed to incubate at 25 C for lOmin. The cells were then pelleted at 16,000g for 10 min and the absorbance of the supernatant was measured at 490 nm in a BioTek HI microplate reader. The amount of CR binding was determined by subtracting the amount of this measurement from a PBS-CR control.

EXAMPLE IV: Amylase-tag Expression

BL21(DE3) cells transformed with pET28b Amylase-tag constructs were grown up in overnight cultures in LB at 30° C with 100 μg/mL kanamycin. One liter of LB was supplemented with 100 μg/mL kanamycin, inoculated with the overnight culture, and grown up at 30° C until an OD of 0.4. Amylase-tag expression was induced with 0.3 mM IPTG and allowed to express overnight at 18° C. Cells were harvested and lysed in TBST, 2 μl/ 50ml benzonase nuclease, and 1 tablet / 50 ml protease inhibitors, by sonicating the cells using a probe sonicator at 15W 5x (0.5min on, 0.5 min off) cycles. The amylase-tag constructs were purified on a Ni-NTA column and concentrated with Amicon Ultra- 15 centrifugal filters (mwco: 30 kDa).

EXAMPLE V: Amylase-catcher Activity Assay

4-Nitrophenyl-α-D-maltopentaoside (pNPMP) was chosen as the substrate to measure α -amylase activity because hydrolysis of 4-nitrophenol (pNP) from the pentasaccharide can be monitored at 405nm. The activity assays were done in PBS. Note, the absorbance of pNP was dependent on its protonation state. To stop the reaction and to have the pNP in the deprotonated state 5% v/v 1 M NaOH was added.

EXAMPLE VI: PTDH-catcher, RADH-catcher, and CtXR-catcher Expression

BL21(DE3) cells transformed with pET15b PTDH-, RADH-, and CtXR-catcher constructs were grown up in overnight cultures in LB at 30° C with 100 μg/mL carbenicillin. 500 ml r of LB was supplemented with 100 μg/mL carbenicillin, inoculated with the overnight culture, and grown up at 30° C until an OD of 0.5. Enzyme-catcher expression was induced with 0.3 mM IPTG and allowed to express overnight at 20° C. Cells were harvested and lysed in TBST, 2 μl/ 50ml benzonase nuclease, and 1 tablet / 50 ml protease inhibitors, by sonicating the cells using a probe sonicator at 15W 5x (0.5min on, 0.5 min off) cycles. The PTDH-, RADH-, and CtXR-tag constructs were purified on a Ni-NTA column and concentrated with Amicon Ultra-15 centrifugal filters (mwco: 30 kDa).

EXAMPLE VII: PTDH constructs Activity Assay

Sodium phosphite was used as substrate for the PTDH constructs to reduce NAD+ and NADP+. The PTDH construct activity assays were done in lOOmM Tris/HCl pH 7.5. The activity of the PTDH constructs was measured by the absorbance at 340 nm of the reduced form, NADH and NADPH. To stop the reaction 10% v/v 1 M HC1 was added, leading to a pH of 4-5.

EXAMPLE VIII: KRED Constructs Activity Assay

For RADH construct activity, 2-hydroxy-2-methylpropiophenone (abbr. 2-HPP), and NADPH were used as substrates. For CtXR constructs activity, ortho-chloroacetophenone (abbr. OCAP), and NADH were used as substrates. The PTDH construct activity assays were done in lOOmM Tris/HCl pH 7.5. The activities of the KRED constructs were measured by measuring the decrease of absorbance at 340nm, based on the oxidization of the cofactor NADPH (RADH), and NADH (CtXR) in presence of the substrate 2-HPP, and OCAP, respectively. To stop the reaction 10% v/v 1 M HC1 was added, leading to a pH of 4-5.

EXAMPLE IX: Quantification of Alcohol Production with HPLC

The production of MPP and CPE by RADH and CtXR, respectively, was quantified with reversed phase HPLC with a Poroshell 120 EC-C18 column, which was heated to 40° C. For the separation of 2-HPP and its corresponding alcohol MPP, the mobile phase was 20% MeOH in ddH2O with 0.1% acetic acid. For the separation of OCAP and its corresponding alcohol CPE, the mobile phase was 30% MeOH in ddH2O with 0.1% acetic acid. The samples were separated in isocratic mode for 10 min. The HPLC was controlled and the results were analyzed with OpenLAB CDS (ChemStation Edition) (Agilent) software. 2- Methyl-l-phenyl-l,2-propanediol (abbr. 2-MPP) and l-(2-chlorophenyl)ethanol (abbr. CPE) were used as alcohol standards for HPLC analysis.

EXAMPLE X: Curli Biofilm Assays

PHL628 biofilms expressing wild-type CsgA, CsgA-SpyTag, CsgA-EFCA, CsgA- WRESAI, CsgA-ARVCF, CsgA-CaM, CsgA-SZ21, CsgA-VMANl l and CsgA-DnaEAC35 were cultured for 18 h at 25° C at 220 rpm as described above. Curli content was measured using the quantitative CR binding assay. Fifty to hundred microliters of cells (normalized to CR absorption) were transferred onto filter plates, which were previously blocked with 2% BSA for at least 1.5 h. For suspended biofilm assays, the biofilms were distributed into Eppendorf tubes and the same conjugation procedures followed. The media was filtered through using a vacuum manifold. Cells were washed with TBST. Cells were incubated with 10 μΜ of the corresponding Amylase-catcher (CsgA-SpyTag: Amyl-SC, CsgA-EFCA: InaD- Amyl, CsgA-WRESAI: Tipl-Amyl, CsgA-ARVCF: ePDZ-Amyl, CsgA-CaM: M13-Amyl, CsgA-SZ21: SZ16-Amyl, CsgA-VMANl l: VM ΑΔΝ 11 - Amyl and CsgA-DnaEAC35:

DnaEC35-Amyl) constructs in TBST with proteinase inhibitor overnight (for CsgA-CaM: Ml 3- Amyl CaCb. was added to a concentration of 5 mM).

For activity assays on the biofilm, the conjugation mixture was removed using vacuum filtration and the biofilms were washed three times with 2% BSA in TBST over 40 min. 1 mM pNPMP in PBS was added to the biofilm and the activity assays plates were placed on a desktop shaker and shaken at 150 rpm at room temperature for 1.5-2 h. At the end of the experiment, the supernatant was vacuum filtered into a new 96- well plate, 0.5 M NaOH was added to increase pH to 12-14 (to bring pNP to a uniform protonation state), and pNP hydrolyzation was measured at 405 nm. In the data analysis, the reference activity is to pH 7.5 PBS. All data points are averages of reactions done in triplicate with error bars indicating standard deviation.

EXAMPLE XI: Enzyme Removal and Regeneration on Biofilms

For the enzyme removal and regeneration assay, basically the same procedure as the curli bio film assay was done, but with one exception. After depositing curli biofilm on the plates, the biofilms were incubated with the corresponding denaturing agent. 6M GdnCl was used as denaturing agents for CsgA-SpyTag: Amyl-SC, CsgA-WRESAI: Tip 1- Amy 1, CsgA- ARVCF: ePDZ-Amyl, CsgA-CaM: M13-Amyl, and CsgA-SZ21: SZ16-Amyl. For CsgA- EFCA: InaD-Amyl 6 M urea, or 6 M urea and 10 mM DTT was used. Additionally, for CsgA-CaM: M13-Amyl 1 mM EGTA was used. After enzyme activity measurement, the biofilms with immobilized enzymes were washed three times with TBST. ΙΟΟμΙ of denaturing agents were added and incubated for 2h. Subsequently, the plates were washed three times with TBST and the residual enzymatic activity was measured as described above. To regenerate the biofilms with enzymes, the plates were washed three times with TBST and incubate again with 10 μΜ of enzyme. This cycle was five times repeated.

EXAMPLE XII: CsgA Conjugation Gels

PHL628 expressing CsgA-ST were grown to OD 0.6 and induced with 0.3 mM IPTG. Cells were allowed to express curli for 20h. Cells were spun down at 5000g for 5 min and supernatant discarded. The pellets were resuspended in 3 niL PBS with protease inhibitors, and incubate with ΙΟΟμΙ of 713μΜ PTDH-SC, ΙΟΟμΙ of 418μΜ SC-RADH, or 50μ1 718μΜ SC-CtXR over night at RT on a tabletop shaker. The samples were spun down at 5000g for 5 min and redissolved in 5ml of TBST and protease inhibitor. This step was twice repeated to wash away free enzyme-SpyCatcher constructs. The pellet was then resuspended in 25 ml 20 mM Tris/HCl pH 7.5, 0.5 M NaCl and 7 M GdnCl. The samples were frozen and thawed one time and consequently cells were sonicated on ice using a probe sonicator at 15W 5x (0.5min on, 0.5 min off) cycles. The supernatant was purified on a Ni-NTA column and concentrated with Amicon Ultra-15 centrifugal filters (mwco: 30 kDa). Residues were dissolved in 2x Laemmli buffer heated for 5 min 95°C and ran on an SDS-PAGE gel. Gels were stained with Commassie Blue. EXAMPLE XIII: Western Blots

For western blots, 15 μΐ^ of reaction mixture was mixed with 2x Laemmli buffer and heated at 95°C for 5 min and ran on an SDS-PAGE gel. The gel was transferred onto PVDF membrane, blocked with 5% milk powder in TBST, incubated with HRP-conjugated antibodies to detect the specific tags on the enzymes (PTDH: cMyc-tag, RADH and CtXR: HA-tag), washed with TBST and developed using Clarity ECL Western Substrate.

EXAMPLE XIV: Microscopy

Scanning electron microscopy was performed on the Zeiss Ultra Plus FESEM (Harvard University Center for Nanoscale Systems). For SEM imaging, the enzyme-bound biofilms were fixed in 2% glutaraldehyde + 4% paraformaldehyde for 30 min, then washed twice with water. The filter membranes were detached from the filter plate, dehydrated with an increasing ethanol gradient, dried on a critical point dryer, then gold-sputtered and imaged on an FESEM (5kV operating voltage). Confocal microscopy (Leica SP5 X MP Inverted Confocal Microscope, Wyss Institute) was performed on fixed cells from PCF plate samples. Cells were incubated with DAPI for 20 min and washed 4x over 2h with 0.2% BSA in PBS. Membranes from PCF plates were cut out and placed between two cover slips to image with a 63x glycerol lens.

EXAMPLE XV: Fusion of Different Heterologous Functional Protein Domains Do Not Impair the Secretion and Formation of Curli Fibers

From the CR binding assay, it is shown that the fusion of different conjugation domains (i.e., SpyTag, PDZ domain tags: Tipl, InaD and ePDZ, split inteins: Npu DnaE and Tvo VMA, calmodulin and SynZip21) to CsgA do not impair the secretion and formation of curli fibers (Fig. 2A). Only in the case of CaM and SZ21 does the fusion of the conjugation protein domains to amylase slightly impair the activity. The other conjugation domains do not impair the amylase activity at all. They even increase the activity, probably due to enhanced solubility (Fig. 2B).

Different fusions of CsgA fusion proteins to different functionalizing polypeptides comprising a partner conjugation domain show the ability to conjugate amylase onto curli- nanofibers in either a covalent or non-covalent manner. All the functionalizing polypeptides, except the split inteins, are able to immobilize amylase onto BIND. Biofilms with the complementary tag (i.e., partner conjugation domain) show a much higher enzymatic activity compared with the control biofilms (wt-CsgA) (Fig. 3A). Mismatched functionalizing polypeptides (with non-matching partner conjugation domains) show enzyme activity only at the background level. Only CsgA-ePDZ incubated with Tipl-amyl show a higher activity as the background.

Fig. 3B depicts the enzymatic activity of amylase fused to different partner conjugation domains. In most cases, the enzymatic activity is highest in those biofilms where the partner conjugation domains match the conjugation domains. Only ePDZ-Amy showed higher activity when paired with mismatched conjugation domains (CsgA-CaM and CsgA- SZ21).

EXAMPLE XVI: Regeneration of Enzymes on Curli Fiber through Enzyme Removal and Immobilization

An important feature for enzyme active surfaces is the ability to regenerate them with new enzymes. To this end, different conditions were tested to disrupt the curli-enzyme interactions. Many of the functionalizing polypeptides comprising a partner conjugation domain interact with the CsgA fusions based on non-covalent interactions. Only SpyTag- SpyCatcher forms a covalent iso-peptide bond and InaD forms a disulfide bond. The binding of M13-amyl to CsgA-CaM is Ca2+ dependent.

Different conjugation domains were genetically fused to the curli gene (CsgA), cloned into a pBela vector and transformed into a CsgA deletion E. coli strain (PHL628-AcsgA). PHL628 biofilm was expressed ON and curli-tag secretion was tested with a Congo Red binding assay. Different catcher-domains were fused to amylase, and cloned into pET vectors. The amylases with conjugation-domains were then expressed in BL21 cells. E. coli biofilm was deposited onto filter plates. Subsequently, the biofilm was incubated ON with amylase-conjugation domains ON. After rigorous washes the enzyme activity of the biofilms was measured. The enzymes were released by incubating the biofilm for 2h under different conditions (6M guanidine, 6M urea, 6M urea + lOmM DTT, ImM EGTA, pH 3, pH 11). After rigorous washing the enzyme activity was measured and subsequently new enzymes with the complementary partner conjugation-domains were immobilized on the biofilm. Enzyme activity of the regenerated biofilm was measured to calculate the regeneration yield. Besides amylase, PTDH was immobilized on curli for co-factor (NAD(P)H) regeneration.

All pairs, except the two covalent ones (SC and InaD), can be disrupted with 6M guanidine. The InaD conjugation can be disrupted by addition of lOmM DTT to 6M urea. Enzymes immobilized with SpyTag-SpyCatcher were not affected by guanidine treatment. See Figures 4A-4C. Incubating the different biofilms in a pH 3 buffer disrupt the binding of ePDZ-amyl, Tipl-amyl, M13-amyl and SZ16-amyl. The binding of SZ16-amyl to CsgA- SZ21 was also disrupted at pH 5 and pH 11 (Fig. 5A). Additionally, the binding of Ml 3- amyl to CsgA-CaM was also disrupted at pH 11 and by lmM EGTA.

New enzymes were immobilized again onto all the biofilms, where enzymes were released (Figs. 4A-4E & Fig. 5B). The lowest regeneration yield was found at the ePDZ biofilm, which was treated with buffer pH 3.

EXAMPLE XVII: Activity of PTDH Immobilized on BIND

Phosphite dehydrogenase (PTDH), which regenerates NAD+ to NADH, was also immobilized onto BIND, as a proof of principle using a more industrially relevant enzyme (Figs. 6A-6C). The in situ regeneration of the co-factor NAD+ is important for a variety of biotransformations.

PTDH with a fused SpyCatcher domain was immobilized on CsgA-SpyTag. This enzymatic active biofilm showed the ability to generate NADH from phosphite and NAD+. Even after 17 days, the activity only decreased to about 50% (Fig. 6B). PTDH showed the ability to regenerate NADH as well as NADPH (Fig. 6C).

As schematically depicted in Fig. 6A, two different ketoreductases (RADH and CtXR) were also immobilized onto BIND. For the conversion of their substrate (2-hydroxy- 2-methylpropiophenone: 2-HPP and o-chloroacetophenone: OCAP, respectively) the co- factor NADPH or NADH is needed. To have an in-situ co-factor regeneration, phosphite dehydrogenase (PTDH), which regenerates NAD(P)+ to NAD(P)H, was co-immobilized with the ketoreductases onto BIND.

EXAMPLE XVIII: KRED-PTDH Enzyme Cascade

To prove the functionality of the bi-enzymatic cascade on BIND, PTDH was co- immobilized with each ketoreductase. To do so, different conjugation / partner conjugation domain systems were tested. KREDs with fused SpyCatcher were co-immobilized with PTDH-SpyCatcher on to CsgA-SpyTag. Different ratios of KRED:PTDH were tested during the enzyme immobilization onto biofilm (Fig. 7A). For enzyme cascade on CsgA-ST, PHL628 biofilms expressing CsgA-SpyTag were cultured as described above. To the CsgA- ST biofilm, 100 μl of 10 μΜ SC-KRED and PTDH-SC mixture in a ratio of 9: 1, 8:2, 7:3, and 6:4 were added.

Furthermore, KREDs with an M13-tag and PTDH with an InaD-domain were immobilized on either curli fibers, which were a mix of already expressed CsgA-CaM (binds M13) and CsgA-EFCA (is bound by InaD) (mixed fibers), or on curli fibers, where the cells were mixed before induction of curli expression (co-fibers) (see Figs. 7B and 7C). Mixed fibers are a mixture of fibers, which are homogeneous, where as co-fibers are composed of both, CsgA-CaM and CsgA-EFCA, with a statistically distribution. For mixed fibers, PHL628 biofilms expressing CsgA-CaM and CsgA-EFCA were cultured as described above, and mixed in the desired ratio (9.5:0.5, 9: 1, 7:3, and 5:5) relative to their Congo red binding ability. For co-fibers, bacteria having the CsgA-CaM, and CsgA-EFCA were mixed based on their ODeoo, induced with 0.3 mM IPTG, cultured as the other samples. To the mixed fibers and the co-fibers 50 μl of 5 μΜ M13-KRED and 50 μl of 5 μΜ PTDH-InaD was added. All the enzymes were dissolved in TBST, inhibitor, and 5mM CaCi2.

After the immobilization, the biofilms were extensively washed to get rid of free enzymes. The conjugation mixture was removed using vacuum filtration and the biofilms were washed three times with 2% BSA in TBST over 40 min. The enzymatic active biofilm was then incubated with phosphite, oxidized co-factor (RADH: NADP+, CtXR: NAD+) and ketone (RADH: OCAP, CtXR: 2-HPP). More specifically, lOmM ketone, 50 mM phosphite, 2 mM NAD(P)+, and 5 mM CaCl2 in 100 mM Tris/HCl pH 7.5 was added to the biofilm and the activity assays plates were placed on a desktop shaker and shaken at 150 rpm at room temperature for 8 h. After 8 h of incubation at room temperature, the conversion of the ketone to the alcohol was quantified with HPLC. At the end of the experiment, the supernatant was vacuum filtered into a new 96-well plate, 1 M HC1 was added, leading to a pH of 4-5. The quantification of the produced alcohol was quantified with HPLC, using pure alcohol as standard.

The production of stereospecific alcohol was confirmed for all the different combinations (Figs. 7A-C). The highest yield for RADH and CtXR was found on co-fibers. This seems to be the most favorable setup for both KREDs.

CsgA wt biofilm served as a negative control. All these biofilms were deposited as described above.

EXAMPLE XIX

Whole enzyme cascades with integrated inorganic nanoparticles can be immobilized via methods described herein for the production of a variety of high- value chemicals, degradation of environmental toxins or remediation of wastewater. According to certain aspects, enzyme active biofilms as described herein do not require living microbes, for example, to allow use of such catalytic biofilms under conditions where bacteria do not survive, such as in moving-bed bioreactors under harsh conditions. The self-assembly behavior of the curli fibers described herein provide the ability to regenerate enzyme and the ability to immobilize enzymes on to a surface without the need for enzyme purification and usage of conjugation chemistry.

EXAMPLE XX: Use of Cotton Cloth to Up-Scale the Use of Catalytic Biofilms

Performing chemical transformations in a high-throughput manner is of great interest to the pharmaceutical industry. Continuous flow reactors are often the best choice because they are cost-efficient and offer steady state conditions for constant product qualities.

(Schoemaker, H. E.; Mink, D.; Wubbolts, M. G., Science 2003, 299 (5613), 1694.) However, the challenge of creating a flow-based system that is compatible with a wide range of enzymes and maintains their long stability remains a challenge with conventional enzyme immobilization techniques, because of inefficient immobilization, limited enzyme availability or poor enzyme stability. Provided herein is a modular, multi-enzymatic, self-assembling flow reactor based on enzyme decorated E. coli amyloid fibers (curli). The system reduces the cost and increases the predictability of enzyme immobilization procedures by

autonomously assembling a bio synthetically-produced enzyme modified matrix without the need of any enzyme purification and with the potential to regenerate itself.

The utility of biofilms is already exemplified by their use in applications like wastewater treatment (Kikuchi, T.; Tanaka, S., Critical Reviews in Environmental Science and Technology 2012, 42 (10), 1007; Larsen, M. W.; Bornscheuer, U. T.; Hult, K., Protein Expression and Purification 2008, 62 (1), 90; and Larsen, P.; Nielsen, J. L.; Otzenj, D.; Nielsen, P. H., Applied and Environmental Microbiology 2008, 74 (5), 1517), biocatalysis (Gross, R.; Hauer, B.; Otto, K.; Schmid, A., Biotechnology and Bioengineering 2007, 98 (6), 1123; Li, X. Z.; Hauer, B.; Rosche, B., Applied Microbiology and Biotechnology 2007, 76 (6), 1255; and Tsoligkas, A. N.; Winn, M.; Bowen, J.; Overton, T. W.; Simmons, M. J. H.; Goss, R. J. M., ChemBioChem 2011, 12 (9), 1391), and fermentation (McNamara, C. J.; Anastasiou, C. C; O'Flaherty, V.; Mitchell, R., International Biodeterioration &

Biodegradation 2008, 61 (2), 127; and Morikawa, M., Journal of Bioscience and

Bioengineering 2006, 101 (1), 1). The biofilm extracellular matrix components protect the cells from harsh environmental conditions (extreme pH and temperature, organic solvents, detergents, etc.) and keep them immobilized on a surface. Biofilms exhibit distinct order on the nano and micro-scale that is designed to regulate mass transfer throughout the material. Due to the fibrous nature of the biofilm extracellular matrix components, biofilms have a vast surface area suitable for the immobilization of catalysts or binding agents that are highly stable and compatible with several bioreactor designs for batch processing or continuous flow processes (Gross et al., 2007, Rosche et al, 2009, Tsoligkas et al., 2011 and Karande, R.; Schmid, A.; Buehler, K., Organic Process Research & Development 2016, 20 (2), 361). However, the process of growing up the biofilms is time consuming (several days) and also cost consuming. This is mainly because the vast majority of the cells are in the planktonic state and not a part of the biofilm. Therefore, a majority of the bacteria are washed away during the medium exchange. This loss of biomass makes previous processes uneconomical.

Used herein is a Bio film-Integrated Nanofiber Display (BIND) platform, which allows the genetic fusion of peptides, or even protein, to CsgA, the monomeric subunit of curli fibers. See also Nguyen, P. Q.; Botyanszki, Z.; Tay, P. K. R.; Joshi, N. S., Nature communications 2014, 5, 4945. Curli can be modified to present different substrate binding peptides (e.g., steel, carbon nanotubes, gold surfaces) or peptides for metal sequestration. Additionally, CsgA can be modified with peptides that interact with small protein units forming covalent or non-covalent interaction for enzyme immobilization on curli fibers. See Botyanszki, Z. ; Tay, P. K. R.; Nguyen, P. Q.; Nussbaumer, M. G.; Joshi, N. S.,

Biotechnology and Bioengineering 2015, 112 (10), 2016 and Nussbaumer, M. G.; Nguyen, P. Q.; Tay, P. K. R.; Naydich, A.; Hysi, E.; Botyanszki, Z.; Joshi, N. S., 2017.

The methods described herein overcome the issue of time- and cost-consuming biofilm formation by filtering the modified curli fibers onto cotton cloth. Because the bacterial grow up and the expression of curli fibers is in suspension, the formation of the biomass takes only one day and the whole biomass can be filtered on cotton. During this filtering process, curli is deposited on the cotton fibers. Because the curli fibers bind to cellulose (Blanco, L. P.; Evans, M. L.; Smith, D. R.; Badtke, M. P.; Chapman, M. R., Trends in Microbiology 2012, 20 (2), 66.), this binding process is highly effective, and the vast majority of the curli fibers can be bound onto the cotton cloth. Additionally, cotton cloth is a very cheap supporting material, is flexible and thin. Immobilizing curli on cotton results in a big active surface, which can present a variety of peptides for the interaction with different materials and to immobilize enzyme on the cotton. One big field of use is the

immobilization of enzymes onto the cotton for biocatalysis purposes. This example immobilizes different enzymes on curli fibers (see Figures 8-12). Moreover, multiple enzymes can be immobilized to build enzyme cascades. The immobilization step can be done with crude cell lysate and does not need any cost intensive protein purification step, which also contributes to a cheap production of our enzymatic active surfaces. The cotton cloth with deposited curli on it can be wrapped and put into a flow cartridge. In this cartridge enzyme can be flown through, where it gets immobilized on the curli fibers, resulting in a enzymatic active flow reactor.

However, enzymatic active flow reactor is not the only field of use for this system. Enzymes on curli-modified cotton can be used as filters. For instance for the degradation of organophosphates, which are the active agents in some chemical warfare agents, such as Sarin, Tabun or VX nerve agents. Another field is wastewater treatment, e.g., to degrade hormones. By displaying heavy metal binding peptides on curli, these filters can also be used for the sequestration of toxic metals. Furthermore, textiles with enhanced functionalities can be produced. By immobilizing enzymes such as amylase, proteases or lipases, which are used in laundry detergents, it would be possible to degrade stains immediately when they come into contact with the textile. Curli displaying antimicrobial peptides could also eliminate the emerging of bad odors in clothes.

This system is able to present a big variety of peptides with different functions on a thin and flexible surface. Focusing on enzyme immobilization, this system is a very fast and cheap way to specifically immobilize enzyme on a surface. There is no need for a long surface treatment, nor is an enzyme purification step needed. Because all the interaction is based on peptide-protein interactions, there is no need for any use of conjugation chemistry, which contributes to the environmental compatibility of this system.

By immobilizing curli on cotton cloth, a thin and flexible surface is achieved, which can be used to present a variety of functional peptides. These peptides functionalize the cloth with their inherent properties, and enable to immobilize enzymes on the cotton.

Other examples of applications for these technologies include:

Flow reactor:

multiple enzymes can be simultaneously immobilized on the cloth for enzyme cascades

several cartridges can be connected to get serial enzyme cascades

enzyme can be regenerated on the cloth

the enzymatic conversion is not only restricted to aqueous phase, but can also be done in organic phase or mixed phases

curli with peptides that coordinate catalysts (e.g., Pt, Pd, Au, Cu)

Filters:

waste water treatment o immobilized enzymes for the degradation of hormones, endocrine disrupting compounds (e.g., BPA), fertilizers,...

o curli presenting peptides for the sequestration of metals, especially rare earth metals and heavy metals

gas filters (with enzymes, functional peptides or coordinated catalysts)

o degradation of organophosphates like Sarin or other nerve agents o carbon monoxide converting and eliminating filters

combination of different types of filters (multi-purpose filters)

Detection

enzymes that give amongst others a colorimetric output to detect

Textiles

immobilization of biological detergents (amylases, lipases, proteases) on to textiles for self-cleaning textiles

biological detergents that can be renewed during every washing cycle

curli presenting antimicrobial peptides

textiles with integrated detection batches

Materials and Methods for Example XX

Materials

• 96 well micro test plates (Sarstedt)

• Bacto Casamino Acids (223120, BD)

• Benzonase Nuclease 25 U/μL (EMD Milipore)

• Carbenicillin (C2130, Teknova)

• Casamino acids (223050, BD)

• cOmplete ultra tablets, Mini, EASYpack Protease Inhibitor Cocktail (Sigma Aldrich)

• Falcon Polystyrene Microplates -6 well plates (0877218, Fisher Scientific)

• Guanidine hydrochloride (Sigma- Aldrich)

• IPTG: Isopropyl-β-D-thiogalactopyranoside (156000, Research Products

International)

• LB (Luria-Bertani) Broth Miller (244620, BD)

• NAD+: β-Nicotinamide adenine dinucleotide hydrate (N7004, Sigma Aldrich)

• Sodium phosphite dibasic pentahydrate (04283, Sigma- Aldrich)

• TBS-T: 20X TBS Tween 20 Buffer (28360, ThermoFisher) • Yeast Extract (AC61180, Fisher Scientific)

Assay buffer NADH formation: 100 mM Tris pH 7.5, 20 mM sodium phosphite, 10 mM NAD+

LB Agar Plates for cultivation of PHL267 and BL21: 12.5 g LB agar, 7.5 g Agar. Add dH2O to 500 mL, autoclave for 1 h, cool, add carbenicillin to final concentration of 100 μg/mL

LB Agar Plates for cultivation of PQN4: 12.5 g LB agar, 7.5 g Agar. Add dH2O to 500 mL, autoclave for 1 h, cool, add carbenicillin to final concentration of 100 μg/mL and glucose to a final concentration of 2 % (m/v)

LB M edium:.25.0 g LB agar. Add dH2O to 500 mL, autoclave for 1 h, cool, add carbenicillin to final concentration of 100 μg/mL

Lysis Buffer: 30 mL TBS-T, 1.2 pL benzonase =1 U/mL, 2 mM Mg2, 2 cOmplete Ultra tablets

YESCA Medium: 10 g casamino acids, 1 g yeast extract. Add dH2O to 1000 mL, autoclave for 1 h, cool, add carbenicillin to final concentration of 100 μg/mL

Expression of SpyTag-Curli Nanofibers

Colonies of transformed PQN4 and PHL628- cells were picked for inoculation of 5 mL LB containing 100 μg/mL carbenicillin, and additional 2% (m/v) glucose for PQN4 cultures. Cultures were grown for 6 h at 37°C and shaking at 225 rpm (Multitron Standard Incubator, Infors HT). For curli fiber expression in PQN4, cultures were diluted 100-fold in fresh LB medium with 100 μg/mL carbenicillin and expression was allowed overnight at 37°C and 225 rpm. For expression in PHL628, cultures were diluted 100-fold in YESCA medium and incubated at 37°C and 225 rpm until an optical density (OD) of 0.5-0.6 at 600 nm was reached. Expression was induced by adding IPTG to a final concentration of 3 mM, and the cultures were incubated at 25°C overnight. For relative quantification of curli nanofiber expression, a pulldown assay was performed by spinning down 1 mL of cell culture for 5 min at 8,000 x g and resuspending the pellet in 1 mL 0.0015% (m/v) Congo Red solution, followed by a 10 min incubation and centrifugation for 10 min at 16,900 x g.

Absorbance was measured in triplicate at 490 nm, and absorbance values of the samples were subtracted from absorbance of Congo Red solution.

Preparation of SpyCatcher- Enzyme Cell Lysate

Colonies of transformed BL21 cells grown on LB agar plates with 100 pg/mL carbenicillin were picked for inoculation of 5 mL LB medium with 100 μg/mL carbenicillin. Cultures were grown overnight at 37 °C and 225 rpm, and the overnight cultures were subsequently diluted 100-fold in fresh LB medium with 100 μg/mL carbenicillin. When an OD of 0.5-0.6 at 600 nm was reached, expression was induced with 3 mM IPTG and cultures were incubated overnight at 20°C and 225 rpm. Next, cell cultures were centrifuged for 15 min at 5,000 x g, and the pellet was resuspended in 30 mL lysis buffer per 500 mL cell culture. Resuspended cells were subjected to 6 sonication cycles with 30 s sonication at 40% amplitude each followed by a 30 s break, and centrifuged at 20,000 x g for 30 min. The supernatant was collected and stored on ice.

Enzyme Immobilization on Cotton Pieces

Cotton pieces with 5 cm diameter were cut out of cotton fabric. The cotton pieces were washed by vacuum- filtering 10 mL TBS-T, and 40 mL curli nanofiber culture were applied and filtered. After three rinses with 5 mL TBS-T, the cloth was incubated with 10 mL 8 M GdmCl for 10 min. Next, three rinses with 5 mL TBS-T followed, and the cotton pieces were cut to fit to 6-well plates and each piece was transferred into a well with the upper side of the cotton pieces facing upwards. 3 mL 5% milk in TBS-T were added and incubated for 1 hour with a VWR Standard Orbital shaker at RT, and subsequently a 2 hour incubation in 2- fold diluted enzyme lysate in 5% milk in TBS-T followed under the same conditions. After three rinses with 3 mL TBS-T, 3 mL assay buffer were added and incubated for 30 min under constant shaking.

Amylase Activity Measurement on Cotton Pieces

All experiments were carried out in duplicate, and activity was determined by using 10 mM NAD+ in 100 mM Tris pH 7.5 as assay buffer and measuring NADH absorbance at 340 nm after 30 min shaking at a VWR Standard Orbital shaker at RT. All measurements were performed in triplicate and absorbance of a substrate blank was subtracted.

PTDH Activity Measurement on Cotton Pieces

All experiments were carried out in duplicate, and activity was determined by using 1 mM pNPMP in PBS pH 7.4 as assay buffer. After 60 min shaking with a VWR Standard Orbital shaker at RT, 5% v/v 1 M NaOH was added to stop the reaction and to convert all pNP to the deprotonated state. The pNP absorbance was measured at 405 nm. All measurements were performed in triplicate and absorbance of a substrate blank was subtracted.

Stability Experiments on Cotton Pieces

Stability measurements under varying conditions were performed either solely on curli SpyTag-nanofibers that were treated with GdmCl and washed three times (as described above), or on curli SpyTag-nanofibers with immobilized PTDH SpyCatcher after rinsing them three times. Drying was performed by drying cotton pieces for 10 min under air flow. For imitating washing conditions, the cotton pieces were incubated at 30°C and 0.01% SDS for half an hour under constant stirring.

Bioreactor set-up

A 13 cm diameter circle was cut out of cloth, and 35 mL TBS-T were filtered by vacuum filtration as a washing step. Subsequently, 200 mL of untreated cell culture were applied and another washing step was performed by filtering 35 mL TBS-T. Two strips (5 cm wide each) were cut out of one circle and a total of 6 strips was assembled into an acrylamide cartridge, with the side with curli fibers pointing towards the outside of the cartridge in order to assemble the bioreactor. All subsequent steps were performed in the assembled reactor through a bottom-up liquid flow controlled by a peristaltic pump. 30 mL 8 M GdmCl were pumped through the reactor at a rate of 3 mL/min. Subsequently, a washing step with TBS-T (40 mL at 6mL/min) and a blocking step with 5% milk in TBS-T (180 mL at 3 mL/min) were performed, before applying crude cell lysate of PTDH-SpyCatcher expressing BL21 cells (180 mL at 3 mL/min) for enzyme immobilization. The reactor was washed with milk (40 mL, 6 mL/min) and once with 100 mL TBS-T (6 mL/min) to remove non- specifically bound enzyme. The reactor was stored at 4°C in 0.02% Sodium azide in TBS-T and for every activity measurement, the reactor was washed with 40 mL of TBS-T (3 mL/min) and two 1 mL samples were taken during the subsequent incubation in assay buffer (3 ml/min).

Absorbance measurements at 340 nm were performed in triplicates in 96-well plates at a Synergy HI Hybrid Multi-Mode Microplate reader (BioTek). 100 of a 5-fold diluted PTDH-SC sample in substrate, incubated for 10 min at 750 rpm RT in an Eppendorf thermocycler, served as control.

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Claims

1. A bio film matrix having a biocatalytic surface, wherein the bio film matrix comprises a plurality of curli fibers,
wherein the plurality of curli fibers comprises:
a first engineered CsgA polypeptide comprising a first CsgA protein and a first conjugation domain, wherein the first conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMAN11, and DnaEAC35, wherein the first conjugation domain is bound to a first partner conjugation domain which is linked to a first functionalizing polypeptide, and wherein the first partner conjugation domain is selected from the group consisting of a PDZ domain, Tip 1 , InaD, M13, SZ16, VMAANll, and DnaEC35;
a second engineered CsgA polypeptide comprising a second CsgA protein and a second conjugation domain, wherein the second conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMAN11, and DnaEAC35, and wherein the second conjugation domain is bound to a second partner conjugation domain which is linked to a second functionalizing polypeptide, wherein the second partner conjugation domain is selected from the group consisting of a PDZ domain, Tipl, InaD, M13, SZ16, VMAANll, and DnaEC35; and
wherein the first functionalizing polypeptide and the second functionalizing polypeptide form an enzymatic cascade.
2. A bio film matrix having a biocatalytic surface, wherein the bio film matrix comprises a plurality of curli fibers,
wherein the plurality of curli fibers comprises:
a first engineered CsgA polypeptide comprising a first CsgA protein and a first conjugation domain, wherein the first conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMAN11, and DnaEAC35, wherein the first conjugation domain is bound to a first partner conjugation domain which is linked to a first functionalizing polypeptide, and wherein the first partner conjugation domain is selected from the group consisting of a PDZ domain, Tipl, InaD, M13, SZ16, VMAANll, and DnaEC35;
a second engineered CsgA polypeptide comprising a second CsgA protein and a second conjugation domain, wherein the second conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMANll, and DnaEAC35, and wherein the second conjugation domain is bound to a second partner conjugation domain which is linked to a second functionalizing polypeptide, wherein the second partner conjugation domain is selected from the group consisting of a PDZ domain, Tipl, InaD, M13, SZ16, VMAANll, and DnaEC35; and
wherein the biocatalytic surface is capable of regeneration.
3. The bio film matrix of claim 1 or claim 2, wherein the plurality of curli fibers comprises one type of curli fiber comprising the first engineered CsgA polypeptide and the second engineered CsgA polypeptide.
4. The biofilm matrix of claim 1 or claim 2, wherein the plurality of curli fibers comprises a first curli fiber comprising the first engineered CsgA polypeptide and a second curli fiber comprising the second engineered CsgA polypeptide.
5. The biofilm matrix of any one of claims 1-4, wherein either the first functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionalizing polypeptides are capable of being removed from the plurality of curli fibers.
6. The biofilm matrix of any one of claims 1-4, wherein the first functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionalizing polypeptides are capable of being removed from, and re-immobilized on, the plurality of curli fibers.
7. The biofilm matrix of any of claims 1-4, wherein the first functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionalizing polypeptide are covalently bound to the plurality of curli fibers.
8. The biofilm matrix of any of claims 1-6, wherein the first functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionalizing polypeptide are non-covalently bound to the plurality of curli fibers.
9. The biofilm matrix of claim 5 or claim 6, wherein the first functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionalizing polypeptides are capable of being removed from the plurality of curli fibers by disruption of a protein-protein interaction.
10. The biofilm matrix of claim 9, wherein the protein-protein interaction is disrupted with guanidine, a chelating agent, a pH change, a redox reagent, urea, or other denaturing agent.
11. The biofilm matrix of claim 10, wherein the chelating agent is EGTA or
EDTA.
12. The biofilm matrix of any one of claims 1-11, wherein the PDZ domain is PDZ1 or ePDZ-bl.
13. The biofilm matrix of any one of claims 1-12, wherein the first conjugation domain and the second conjugation domain are different types of conjugation domains.
14. The biofilm matrix of any one of claims 1-12, wherein the first conjugation domain and the second conjugation domain are the same type of conjugation domain.
15. The biofilm matrix of any one of claims 1-14, wherein the first partner conjugation domain and the second partner conjugation domain are different types of partner conjugation domains.
16. The biofilm matrix of any one of claims 1-12 or 14, wherein the first partner conjugation domain and the second partner conjugation domain are the same type of partner conjugation domain.
17. The biofilm matrix of any one of claims 1-16, wherein the first functionalizing polypeptide is a first enzyme.
18. The biofilm matrix of any one of claims 1-17, wherein the second functionalizing polypeptide is a second enzyme.
19. The biofilm matrix of claim 17 or claim 18, wherein the first enzyme is selected form the group consisting of an amylase, a dehydrogenase, and a ketoreductase.
20. The bio film matrix of claim 18 or claim 19, wherein the second enzyme is selected form the group consisting of an amylase, a dehydrogenase, and a ketoreductase.
21. The biofilm matrix of any one of claims 17-20, wherein the first enzyme and the second enzyme are selected from the group consisting of phosphite dehydrogenase (PTDH), RADH, and CtXR.
22. The biofilm matrix of claim 18, wherein the first enzyme catalyzes the regeneration of a co-factor for use by the second enzyme.
23. The biofilm matrix of claim 18, wherein the first enzyme catalyzes the generation of a substrate for use by the second enzyme.
24. A biofilm comprising the biofilm matrix of any one of claims 1-23 and an engineered microbial cell.
25. A method of producing a biofilm having a biocatalytic surface, comprising: culturing a first genetically engineered bacterium in a culture medium, wherein the first genetically engineered bacterium expresses a first engineered CsgA polypeptide and a second engineered CsgA polypeptide, wherein the first engineered CsgA polypeptide comprises a first CsgA protein and a first conjugation domain, wherein the first conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMANl l, and DnaEAC35; and wherein the second engineered CsgA polypeptide comprises a second CsgA protein and a second conjugation domain, wherein the second conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMANl l, and DnaEAC35; thereby forming a plurality of curli fibers which form a biofilm, contacting the biofilm with a first functionalizing polypeptide which is linked to a first partner conjugation domain, wherein the first partner conjugation domain is selected from the group consisting of PDZ domain, Tipl, InaD, M13, SZ16, VMAANl l, and DnaEC35, and wherein the first partner conjugation domain binds to the first conjugation domain, and
contacting the biofilm with a second functionalizing polypeptide which is linked to a second partner conjugation domain, wherein the second partner conjugation domain is selected from the group consisting of PDZ domain, Tipl, InaD, M13, SZ16, VMAANH, and DnaEC35, and wherein the second partner conjugation domain binds to the second conjugation domain,
wherein the first functionalizing polypeptide and the second functionalizing polypeptide form an enzymatic cascade,
thereby producing a biofilm having a biocatalytic surface.
26. A method of producing a biofilm having a biocatalytic surface, comprising: culturing a first genetically engineered bacterium and a second genetically engineered bacterium in a culture medium, wherein the first genetically engineered bacterium expresses a first engineered CsgA polypeptide, wherein the first engineered CsgA polypeptide comprises a CsgA protein and a first conjugation domain, wherein the first conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMAN11, and DnaEAC35; and wherein the second genetically engineered bacterium expresses a second engineered CsgA polypeptide, wherein the second engineered CsgA polypeptide comprises a CsgA protein and a second conjugation domain, wherein the second conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMAN11, and DnaEAC35; thereby forming a plurality of curli fibers which form a biofilm,
contacting the biofilm with a first functionalizing polypeptide which is linked to a first partner conjugation domain, wherein the first partner conjugation domain is selected from the group consisting of PDZ domain, Tipl, InaD, M13, SZ16, VMAANH, and DnaEC35, and wherein the first partner conjugation domain binds to the first conjugation domain, and
contacting the biofilm with a second functionalizing polypeptide which is linked to a second partner conjugation domain, wherein the second partner conjugation domain is selected from the group consisting of PDZ domain, Tipl, InaD, M13, SZ16, VMAANl l, and DnaEC35, and wherein the second partner conjugation domain binds to the second conjugation domain,
wherein the first functionalizing polypeptide and the second functionalizing polypeptide form an enzymatic cascade,
thereby producing a biofilm having a biocatalytic surface.
27. A method of producing a biofilm having a biocatalytic surface capable of regeneration, comprising:
culturing a first genetically engineered bacterium in a culture medium, wherein the first genetically engineered bacterium expresses a first engineered CsgA polypeptide and a second engineered CsgA polypeptide, wherein the first engineered CsgA polypeptide comprises a CsgA protein and a first conjugation domain, and wherein the second engineered CsgA polypeptide comprises a CsgA protein and a second conjugation domain, thereby forming a plurality of curli fibers which form a biofilm,
contacting the biofilm with a first functionalizing polypeptide which is linked to a first partner conjugation domain, wherein the first partner conjugation domain binds to the first conjugation domain, and
contacting the biofilm with a second functionalizing polypeptide which is linked to a second partner conjugation domain, wherein the second partner conjugation domain binds to the second conjugation domain,
wherein the first functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionalizing polypeptides are capable of being removed from the plurality of curli fibers,
thereby producing a biofilm having a biocatalytic surface capable of regeneration.
28. A method of producing a biofilm having a biocatalytic surface capable of regeneration, comprising:
culturing a first genetically engineered bacterium and a second genetically engineered bacterium in a culture medium, wherein the first genetically engineered bacterium expresses a first engineered CsgA polypeptide, wherein the first engineered CsgA polypeptide comprises a CsgA protein and a first conjugation domain, wherein the second genetically engineered bacterium expresses a second engineered CsgA polypeptide, wherein the second engineered CsgA polypeptide comprises a CsgA protein and a second conjugation domain, thereby forming a plurality of curli fibers which form a biofilm,
contacting the biofilm with a first functionalizing polypeptide which is linked to a first partner conjugation domain, wherein the first partner conjugation domain binds to the first conjugation domain, and
contacting the biofilm with a second functionalizing polypeptide which is linked to a second partner conjugation domain, wherein the second partner conjugation domain binds to the second conjugation domain, wherein the first functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionalizing polypeptides are capable of being removed from the plurality of curli fibers,
thereby producing a biofilm having a biocatalytic surface capable of regeneration.
29. The method of claim 27 or claim 28, wherein the first functionalizing polypeptide and the second functionalizing polypeptide form an enzymatic cascade.
30. The method of any one of claims 27-29, wherein the first conjugation domain, the second conjugation domain, or both the first conjugation and the second conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMAN11, and DnaEAC35.
31. The method of any one of claims 27-30, wherein the first conjugation domain, the second conjugation domain, or both the first and the second partner conjugation domain is selected from the group consisting of PDZ domain, Tipl, InaD, M13, SZ16, VMAANl l, and DnaEC35.
32. The method of any one of claims 25-31, wherein either the first
functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionalizing polypeptides are capable of being removed from the plurality of curli fibers.
33. The method of any one of claims 25-31, wherein the first functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionalizing polypeptides are capable of being removed from, and re- immobilized on, the plurality of curli fibers.
34. The method any one of claims 25-31, wherein the first conjugation domain covalently binds to the first partner conjugation domain, and wherein the second conjugation domain covalently binds to the second partner conjugation domain.
35. The method any one of claims 25-33, wherein the first conjugation domain covalently binds to the first partner conjugation domain, and wherein the second conjugation domain non-covalently binds to the second partner conjugation domain.
36. The method any one of claims 25-33, wherein the first conjugation domain non-covalently binds to the first partner conjugation domain, and wherein the second conjugation domain covalently binds to the second partner conjugation domain.
37. The method any one of claims 25-33, wherein the first conjugation domain non-covalently binds to the first partner conjugation domain, and wherein the second conjugation domain non-covalently binds to the second partner conjugation domain.
38. The method of claim 27 or claim 28, wherein the first functionalizing polypeptide is expressed by the first genetically engineered bacterium.
39. The method of claim 27, wherein the first functionalizing polypeptide is expressed by a second genetically engineered bacterium.
40. The method of claim 28, wherein the first functionalizing polypeptide is expressed by the second genetically engineered bacterium.
41. The method of any one of claims 27, 28, and 38-40, wherein the second functionalizing polypeptide is expressed by the first genetically engineered bacterium.
42. The method of claim 27, wherein the second functionalizing polypeptide is expressed by a second genetically engineered bacterium.
43. The method of claim 28, wherein the second functionalizing polypeptide is expressed by the second genetically engineered bacterium.
44. The method of any one of claims 25-28, wherein the first genetically engineered bacterium is a genetically engineered E. coli bacterium.
45. The method of claim 26 or 28, wherein the second genetically engineered bacterium is a genetically engineered E. coli bacterium.
46. The method of any one of claims 25-45, further comprising a step of removing the genetically engineered bacterium from the bio film before the contacting steps.
47. The method of claim 42, wherein the removing comprises washing the genetically engineered bacterium from the biofilm.
48. The method of any one of claims 25-47, further comprising a step of killing the genetically engineered bacterium in the biofilm before the contacting steps.
49. The method of any one of claims 46-48, wherein the first functionalizing polypeptide and the second functionalizing polypeptide maintain their catalytic activity after the step of removing the genetically engineered bacterium and/or the step of killing the genetically engineered bacterium.
50. The method of any one of claims 25-49, wherein the genetically engineered bacterium is cultured in a bioreactor.
51. The method of claim 50, wherein the bioreactor is a batch bioreactor or a continuous flow bioreactor.
52. A method of regenerating a biocatalytic surface on a biofilm matrix produced by a genetically modified bacterium, comprising:
removing a first functionalizing polypeptide which is linked to a first partner conjugation domain from a plurality of curli fibers of the biofilm matrix, wherein the plurality of curli fibers comprises a first engineered CsgA polypeptide, wherein the first engineered CsgA polypeptide comprises a CsgA protein and a first conjugation domain; and contacting the biofilm matrix with a third functionalizing polypeptide which is linked to a third partner conjugation domain, thereby binding the third partner conjugation domain to the first conjugation domain,
wherein the biofilm matrix also comprises a second engineered CsgA polypeptide, wherein the second engineered CsgA polypeptide comprises a second CsgA protein and a second conjugation domain, wherein the second conjugation domain is bound to a second partner conjugation domain which is linked to a second functionalizing polypeptide;
thereby regenerating the biocatalytic surface of the biofilm matrix.
53. The method of claim 52, further comprising:
removing the second functionalizing polypeptide which is linked to the second conjugation domain from the plurality of curli fibers; and
contacting the bio film matrix with a fourth functionalizing polypeptide which is linked to a fourth partner conjugation domain, thereby binding the fourth partner conjugation domain to the second conjugation domain.
54. The method of claim 52 or claim 53, wherein the first functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionalizing polypeptides are removed from the bio film matrix by disrupting a protein-protein interaction.
55. The method of claim 54, wherein the protein-protein interaction is disrupted with guanidine, a chelating agent, a pH change, a redox reagent, urea, or other denaturing agent.
56. The method of claim 52, wherein the removal of the first functionalizing polypeptide does not affect the binding of the second conjugation domain to the second partner conjugation domain and does not decrease the function of the second functionalizing polypeptide.
57. The method of claim 53, wherein the removal of the second functionalizing polypeptide does not affect the binding of the first conjugation domain to the third partner conjugation domain and does not decrease the function of the third functionalizing polypeptide.
58. The method of claim 52, wherein the third functionalizing polypeptide is the same type of functionalizing polypeptide as the first functionalizing polypeptide.
59. The method of claim 53, wherein the fourth functionalizing polypeptide is the same type of functionalizing polypeptide as the second functionalizing polypeptide.
60. The method of any one of claims 52-59, wherein the plurality of curli fibers comprises one type of curli fiber comprising the first engineered CsgA polypeptide and the second engineered CsgA polypeptide.
61. The method of any one of claims 52-59, wherein the plurality of curli fibers comprises a first curli fiber comprising the first engineered CsgA polypeptide and a second curli fiber comprising the second engineered CsgA polypeptide.
62. A curli fiber comprising a first engineered CsgA polypeptide and a second engineered CsgA polypeptide,
wherein the first engineered CsgA polypeptide comprises a first CsgA protein and a first conjugation domain, wherein the first conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMANU, and DnaEAC35, wherein the first conjugation domain is bound to a first partner conjugation domain linked to a first functionalizing polypeptide, and wherein the first partner conjugation domain is selected from the group consisting of a PDZ domain, Tipl, InaD, M13, SZ16, VMAANll, and DnaEC35;
wherein the second engineered CsgA polypeptide comprises a second CsgA protein and a second conjugation domain, wherein the second conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMANU, and DnaEAC35, and wherein the second conjugation domain is bound to a second partner conjugation domain linked to a second functionalizing polypeptide, and wherein the second partner conjugation domain is selected from the group consisting of a PDZ domain, Tipl, InaD, M13, SZ16, VMAANll, and DnaEC35; and
wherein the first functionalizing polypeptide and the second functionalizing polypeptide form an enzymatic cascade.
63. A plurality of curli fibers comprising a first engineered CsgA polypeptide and a second engineered CsgA polypeptide,
wherein the first engineered CsgA polypeptide comprises a first CsgA protein and a first conjugation domain, wherein the first conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMANU, and DnaEAC35, wherein the first conjugation domain is bound to a first partner conjugation domain linked to a first functionalizing polypeptide, and wherein the first partner conjugation domain is selected from the group consisting of a PDZ domain, Tipl, InaD, M13, SZ16, VMAANll, and DnaEC35;
wherein the second engineered CsgA polypeptide comprises a second CsgA protein and a second conjugation domain, wherein the second conjugation domain is selected from the group consisting of EFCA, WRESAI, ARVCF, CaM, SZ21, VMANU, and DnaEAC35, and wherein the second conjugation domain is bound to a second partner conjugation domain linked to a second functionalizing polypeptide, and wherein the second partner conjugation domain is selected from the group consisting of a PDZ domain, Tipl, InaD, M13, SZ16, VMAANll, and DnaEC35; and
wherein the first functionalizing polypeptide and the second functionalizing polypeptide form an enzymatic cascade.
64. The plurality of curli fibers of claim 63, wherein the plurality of curli fibers comprises one type of curli fiber comprising the first engineered CsgA polypeptide and the second engineered CsgA polypeptide.
65. The plurality of curli fibers of claim 63, wherein the plurality of curli fibers comprises a first curli fiber comprising the first engineered CsgA polypeptide and a second curli fiber comprising the second engineered CsgA polypeptide.
66. The curli fiber, or the plurality of curli fibers, of any one of claims 62-65, wherein either the first functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionahzmg polypeptides are capable of being removed from the curli fiber, or the plurality of curli fibers.
67. The curli fiber, or the plurality of curli fibers, of any one of claims 62-65, wherein the first functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionalizing polypeptides are capable of being removed from, and re- immobilized on, the curli fiber or the plurality of curli fibers.
68. The curli fiber, or the plurality of curli fibers, of any one of claims 62-65, wherein the first functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionalizing polypeptide are covalently bound to the curli fiber or to the plurality of curli fibers.
69. The curli fiber, or the plurality of curli fibers, of any of claims 62-67, wherein the first functionalizing polypeptide, the second functionalizing polypeptide, or the first and second functionalizing polypeptide are non-covalently bound to the curli fiber or to the plurality of curli fibers.
70. The curli fiber, or the plurality of curli fibers, of any of claims 62-67, wherein the first functionalizmg polypeptide, the second functionalizmg polypeptide, or the first and second functionalizmg polypeptides are capable of being removed from the curli fiber or from the plurality of curli fibers by disruption of a protein-protein interaction.
71. The curli fiber, or the plurality of curli fibers, of claim 70, wherein the protein- protein interaction is disrupted with guanidine, a chelating agent, a pH change, a redox reagent, urea, or other denaturing agent.
72. The curli fiber, or the plurality of curli fibers, of claim 71, wherein the chelating agent is EGTA or EDTA.
73. The curli fiber, or the plurality of curli fibers, of any one of claims 62-72, wherein the PDZ domain is either PDZ1 or ePDZ-bl.
74. The curli fiber, or the plurality of curli fibers, of any one of claims 62-73, wherein the first conjugation domain and the second conjugation domain are different types of conjugation domains.
75. The curli fiber, or the plurality of curli fibers, of any one of claims 62-73, wherein the first conjugation domain and the second conjugation domain are the same type of conjugation domain.
76. The curli fiber, or the plurality of curli fibers, of any one of claims 62-75, wherein the first functionalizmg polypeptide is a first enzyme.
77. The curli fiber, or the plurality of curli fibers, of any one of claims 62-76, wherein the second functionalizmg polypeptide is a second enzyme.
78. The curli fiber, or the plurality of curli fibers, of claim 76 or claim 77, wherein the first enzyme is selected form the group consisting of an amylase, a dehydrogenase, and a ketoreductase.
79. The curli fiber, or the plurality of curli fibers, of claim 77 or claim 78, wherein the second enzyme is selected form the group consisting of an amylase, a dehydrogenase, and a ketoreductase.
80. The curli fiber, or the plurality of curli fibers, of any one of claims 76-79, wherein the first enzyme and the second enzyme are selected from the group consisting of phosphite dehydrogenase (PTDH), RADH, and CtXR.
81. The curli fiber, or the plurality of curli fibers, of any one of claims 76-80, wherein the first enzyme catalyzes the regeneration of a co-factor for use by the second enzyme.
82. The curli fiber, or the plurality of curli fibers, of any one of claims 76-81, wherein the first enzyme catalyzes the generation of a substrate for use by the second enzyme.
83. A biofilm comprising the curli fiber, or the plurality of curli fibers, of any one of claims 62-82.
84. An article of manufacture comprising the biofilm matrix of any one of claims 1-24, or the curli fiber, or the plurality of curli fibers, of any one of claims 62-82.
85. The article of manufacture of claim 84, wherein the article is a bioreactor or a cloth.
86. The article of manufacture of claim 85, wherein the cloth is a cotton cloth.
87. The article of manufacture of claim 85, wherein the bioreactor is a batch reactor or a continuous flow bioreactor.
88. A method of producing a chemical entity using the biofilm matrix of any one of claims 1-24, or the curli fiber, or plurality of curli fibers, of any one of claims 62-82.
89. A method of purifying water using the biofilm matrix of any one of claims 1- 24, or the curli fiber, or plurality of curli fibers, of any one of claims 62-82.
90. A method of cleaning a chemical spill using the biofilm matrix of any one of claims 1-24, or the curli fiber, or plurality of curli fibers, of any one of claims 62-82.
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