Harnessing Nature-Inspired Catechol Amino Acid to Engineer Sticky Proteins and Bacteria

Abstract

3,4-Dihydroxy-L-phenylalanine (DOPA) serves as a post-translational modification amino acid present in mussel foot proteins. Mussels exploit the exceptional adhesive properties of DOPA to adhere to a wide range of surfaces. This study presents the development of sticky proteins and bacteria through the site-specific incorporation of DOPA using Genetic Code Expansion Technology. Through the optimization of the DOPA incorporation system, proteins containing DOPA demonstrate significantly improved binding abilities to various organic and metallic materials. The material-binding capabilities of DOPA to combat different types of biofoulings are harnessed by integrating it into intrinsically disordered proteins. Beyond the creation of adhesive proteins for anti-biofouling purposes, this highly efficient DOPA incorporation system is also applied to engineer adhesive bacteria, resulting in a remarkable increase in their binding capability to diverse materials including 400 folds of improvement to polyethylene terephthalate (PET). This substantial enhancement in PET binding of these bacteria has allowed to develop a unique approach for PET degradation, showcasing the innovative application of Genetic Code Expansion in cell engineering.

Keywords: anti‐biofouling; genetic code expansion; noncanonical amino acid; sticky protein; unnatural amino acid.

Figure 1.. Optimization of DOPA Incorporation System.

(A) Creation of sticky proteins and bacteria via incorporation of nature-inspired non-canonical amino acid DOPA. (B) Assessing the efficiency of various DOPA integration systems and expression conditions using sfGFP fluorescence analysis. Error bars represent ± standard error of the mean from 3 biologically independent experiments. (C-D) ESI-MS spectra of sfGFP-151DOPA proteins expressed using (C) 12D4-chPheRS/3C11-chPheT and (D) WT-chPheRS/3C11-chPheT pairs (optimized system). (E) SDS-PAGE analysis of sfGFPs expressed by WT-chPheRS/3C11-chPheT pair in LB with different DOPA concentrations.

Figure 2:. Binding Ability of DOPA-Containing Proteins to Different Materials.

(A) SDS-PAGE analysis of WT and DOPA-containing sfGFP and scFv proteins. (B) ESI-MS spectra of sfGFP-151DOPA and scFv-113DOPA proteins. (C) Comparison of binding abilities of WT and DOPA-containing proteins towards various materials quantified by ImageJ grey scale analysis. Error bars represent ± standard error of the mean from 3 biologically independent experiments. Note of p values: ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (D) Representative AFM 3D images (500 nm × 500 nm) of WT and DOPA-containing proteins treated glass. (E) Height distribution histograms of (D).

Figure 3.. Creation of Sticky Proteins for Biofouling Mitigation.

(A) Schematic illustration of biofouling mitigation with DOPA-containing IDP proteins. (B) ESI-MS spectra of WT and DOPA-containing IDPs expressed by the optimized incorporation system. (C) Binding abilities of WT and DOPA-containing IDPs towards various materials quantified by ImageJ grey scale analysis. (D-F) Representative Nikon A1-Rsi confocal images of (D) E. coli (harboring sfGFP-WT), (E) HEK293T, and (F) HeLa cells (stained by Lipo-BODIPY dye) distributions on different surfaces treated by PBS, IDP-WT, or IDP-DOPA proteins. (G) Fluorescent quantification of (D-F) by ImageJ. Error bars represent ± standard error of the mean from (C) 3 and (G) 5 biologically independent experiments. Note of p values: ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Figure 4.. Creation of Sticky Bacteria Utilizing DOPA-Containing Membrane Proteins.

(A) Schematic illustration of sticky bacteria with DOPA-containing membrane protein (OmpA) expression. (B) Zeiss LSM800 Airyscan confocal images of DH10B E. coli with WT/DOPA-containing OmpA (harboring sfGFP-WT) distributions on metal surfaces. (C) SEM images of DH10B E. coli with WT/DOPA-containing OmpA distributions on material surfaces. (D) Bacteria number quantification of (C). Error bars represent ± standard error of the mean from 3 biologically independent experiments. Note of p values: ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Figure 5.. Creation of Sticky Bacteria to Improve PET Biodegradation.

(A) Plasmid constructs and schematic illustration of sticky bacteria with surface-displayed PETase for PET degradation enhancement. (B) SDS-PAGE analysis of the surface-displayed PETase and OmpA expression. Please refer to Fig. S9 for raw data. (C) Verification of PETase surface display by GFP binding assay. (D) Activity of surface-displayed PETase evaluated by NPB assay. (E) PET degradation efficiency of surface-displayed PETase of WT and sticky bacteria tested by fluorogenic assay. (F) ESI-MS spectra of PET degradation product generated from (E). Error bars represent ± standard error of the mean from 3 biologically independent experiments. Note of p values: ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.