Bifunctional peptides as alternatives to copper-based formulations to control citrus canker.

Citrus canker is an infectious bacterial disease and one of the major threats to the orange juice industry, a multibillion-dollar market that generates hundreds of thousands of jobs worldwide. This disease is caused by the Gram-negative bacterium Xanthomonas citri subsp. citri. In Brazil, the largest producer and exporter of concentrate orange juice, the control of citrus canker is exerted by integrated management practices, in which cupric solutions are intensively used in the orchards to refrain bacterial spreading. Copper ions accumulate and are as heavy metals toxic to the environment. Therefore, the aim of the present work was to evaluate bifunctional fusion proteins (BiFuProts) as novel and bio-/peptide-based alternatives to copper formulations to control citrus canker. BiFuProts are composed of an anchor peptide able to bind to citrus leaves, and an antimicrobial "killer" peptide to protect against bacterial infections of plants. The selected BiFuProt (Mel-CgDEF) was bactericidal against X. citri at 125 µg mL-1, targeting the bacterial cytoplasmic membrane within the first minutes of contact. The results in the greenhouse assays proved that Mel-CgDEF at 250 µg mL-1 provided protection against X. citri infection on the leaves, significantly reducing the number of lesions by area when compared with the controls. Overall, the present work showed that the BiFuProt Mel-CgDEF is a biobased and biodegradable possible alternative for substitute cupric formulations. KEY POINTS: • The bifunctional fusion protein Mel-CgDEF was effective against Xanthomonas citri. • Mel-CgDEF action mechanism was the disruption of the cytoplasmic membrane. • Mel-CgDEF protected citrus leaves against citrus canker disease.

Adhesion Peptide-Functionalized Biobased Microgels for Controlled Delivery of Pesticides.

Widespread use of plant protection agents in agriculture is a major cause of pollution. Apart from active ingredients, the environmental impact of auxiliary synthetic polymers should be minimized if they are highly persistent. An alternative to synthetic polymers is the use of natural polysaccharides, which are abundant and biodegradable. In this study, we explore pectin microgels functionalized with anchor peptides (P-MAPs) to be used as an alternative biobased pesticide delivery system. Using copper as the active ingredient, P-MAPs effectively prevented infection of grapevine plants with downy mildew under semi-field conditions on par with commercial copper pesticides. By using anchor peptides, the microgels tightly bind to the leaf surface, exhibiting excellent rain fastness and prolonged fungicidal activity. Finally, P-MAPs are shown to be easily degradable by enzymes found in nature, demonstrating their negligible long-term impact on the environment.

A peptide-based coating toolbox to enable click chemistry on polymers, metals, and silicon through sortagging.

A versatile peptide-based toolbox for surface functionalization was established by a combination of a universal material binding peptide (LCI-anchor peptide) and sortase-mediated bioconjugation (sortagging). This toolbox facilitates surface functionalization either as a one- or a two-step strategy. In the case of the one-step strategy, the desired functionality was directly introduced to LCI. For the two-step strategy, LCI was modified with a reactive group, which can be further functionalized (e.g., employing "click" chemistry). Sortagging of LCI, employing sortase A from Staphylococcus aureus, was achieved with six different amine compounds: dibenzocyclooctyne amine, biotin-polyethylene glycol amine, Cyanine-3 amine, kanamycin, methoxypolyethylene glycol amine (Mn = 5000 Da), and 2,2,3,3,4,4,4-Heptafluorobutylamine. The purification of LCI-amine sortagging products was performed by a negative purification using Strep-tag II affinity chromatography, resulting in LCI-amine conjugates with purities >90%. For the two-step strategy, the LCI-dibenzocyclooctyne sortagging product was purified and enabled, through copper-free azide-alkyne "click" chemistry, universal surface functionalization of material surfaces such as polypropylene, polyethylene terephthalate, stainless steel, gold, and silicon. The click reaction was performed before or after surface binding of LCI-dibenzocyclooctyne. Finally, in the case of the one-step strategy, polypropylene was directly functionalized with Cyanine-3 and biotin-polyethylene glycol amine.

Designed Streptococcus pyogenes Sortase A Accepts Branched Amines as Nucleophiles in Sortagging.

Sortase-mediated ligation (sortagging) is commonly performed using the Staphylococcus aureus sortase A (SaSrtA) that strictly recognizes the N-terminal glycine residue. In this work, a rational design of Streptococcus pyogenes sortase A (SpSrtA) for improved transpeptidase activity toward different N-terminal amino acid residues was conducted. The generated variant SpSrtA M3 (E189H/V206I/E215A) showed up to 6.6-fold (vs SpSrtA wild-type) enhanced catalytic efficiency. Additionally, M3 retains the specificity toward N-terminal alanine, glycine, serine residues, as well as branched (at α-carbon) primary amines as wild-type parent. Furthermore, M3 was applied for head-to-tail backbone cyclization of proteins.

Enhancing Robustness of Sortase A by Loop Engineering and Backbone Cyclization.

Invited for the cover of this issue is Ulrich Schwaneberg and co-workers at RWTH Aachen University and DWI Leibniz-Institut für Interaktive Materialien. The image depicts a loop engineered, and backbone cyclized Staphylococcus aureus sortase A which shows enhanced robustness in site-specific protein and peptide modifications. Read the full text of the article at 10.1002/chem.202002740.

Enhancing Robustness of Sortase A by Loop Engineering and Backbone Cyclization.

Staphylococcus aureus sortase A (SaSrtA) is widely used for site-specific protein modifications, but it lacks the robustness for performing bioconjugation reactions at elevated temperatures or in presence of denaturing agents. Loop engineering and subsequent head-to-tail backbone cyclization of SaSrtA yielded the cyclized variant CyM6 that has a 7.5 °C increased melting temperature and up to 4.6-fold increased resistance towards denaturants when compared to the parent rM4. CyM6 gained up to 2.6-fold (vs. parent rM4) yield of conjugate in ligation of peptide and primary amine under denaturing conditions.

Gas-Phase Transformation of Fluorinated Benzoporphyrins to Porphyrin-Embedded Conical Nanocarbons.

Geodesic nitrogen-containing graphene fragments are interesting candidates for various material applications, but the available synthetic protocols, which need to overcome intrinsic strain energy during the formation of the bowl-shaped skeletons, are often incompatible with heteroatom-embedded structures. Through this mass spectrometry-based gas-phase study, we show by means of collision-induced dissociation experiments and supported by density functional theory calculations, the first evidence for the formation of a porphyrin-embedded conical nanocarbon. The influences of metalation and functionalization of the used tetrabenzoporphyrins have been investigated, which revealed different cyclization efficiencies, different ionization possibilities, and a variation of the dissociation pathway. Our results suggest a stepwise process for HF elimination from the fjord region, which supports a selective pathway towards bent nitrogen-containing graphene fragments.

Matter-tag: A universal immobilization platform for enzymes on polymers, metals, and silicon-based materials.

Enzyme immobilization is extensively studied to improve enzyme properties in catalysis and analytical applications. Here, we introduce a simple and versatile enzyme immobilization platform based on adhesion-promoting peptides, namely Matter-tags. Matter-tags immobilize enzymes in an oriented way as a dense monolayer. The immobilization platform was established with three adhesion-promoting peptides; Cecropin A (CecA), liquid chromatography peak I (LCI), and Tachystatin A2 (TA2), that were genetically fused to enhanced green fluorescent protein and to two industrially important enzymes: a phytase (from Yersinia mollaretii) and a cellulase (CelA2 from a metagenomic library). Here, we report a universal and simple Matter-tag-based immobilization platform for enzymes on various materials including polymers (polystyrene, polypropylene, and polyethylene terephthalate), metals (stainless steel and gold), and silicon-based materials (silicon wafer). The Matter-tag-based enzyme immobilization is performed at ambient temperature within minutes (<10 min) in an aqueous solution harboring the phytase or cellulase by immersing the targeted material. The peptide LCI was identified as universal adhesion promoter; LCI immobilized both enzymes on all investigated materials. The attachment of phytase-LCI onto gold was characterized with surface plasmon resonance spectroscopy obtaining a dissociation constant value (KD ) of 2.9·10-8 M and a maximal surface coverage of 504 ng/cm².

MicroGelzymes: pH-Independent Immobilization of Cytochrome P450 BM3 in Microgels.

Microgels are an emerging class of "ideal" enzyme carriers because of their chemical and process stability, biocompatibility, and high enzyme loading capability. In this work, we synthesized a new type of permanently positively charged poly(N-vinylcaprolactam) (PVCL) microgel with 1-vinyl-3-methylimidazolium (quaternization of nitrogen by methylation of N-vinylimidazole moieties) as a comonomer (PVCL/VimQ) through precipitation polymerization. The PVCL/VimQ microgels were characterized with respect to their size, charge, swelling degree, and temperature responsiveness in aqueous solutions. P450 monooxygenases are usually challenging to immobilize, and often, high activity losses occur after the immobilization (in the case of P450 BM3 from Bacillus megaterium up to 100% loss of activity). The electrostatic immobilization of P450 BM3 in permanently positively charged PVCL/VimQ microgels was achieved without the loss of catalytic activity at the pH optimum of P450 BM3 (pH 8; ∼9.4 nmol 7-hydroxy-3-carboxy coumarin ethyl ester/min for free and immobilized P450 BM3); the resulting P450-microgel systems were termed P450 MicroGelzymes (P450 µ-Gelzymes). In addition, P450 µ-Gelzymes offer the possibility of reversible ionic strength-triggered release and re-entrapment of the biocatalyst in processes (e.g., for catalyst reuse). Finally, a characterization of the potential of P450 µ-Gelzymes to provide resistance against cosolvents (acetonitrile, dimethyl sulfoxide, and 2-propanol) was performed to evaluate the biocatalytic application potential.

KnowVolution of a Fungal Laccase toward Alkaline pH.

To date, commercial laccase preparations are used in the food, textile, and paper and pulp industries (mild pH). Laccases are attractive in the synthesis of dye molecules or oxidative lignin treatment, which take place at high pH (≥8.0). So far, one fungal laccase has been reported to be active at alkaline pH. Herein, engineering of the fungal laccase from Melanocarpus albomyces (MaL) for increased activity toward the substrate 2,6-dimethoxyphenol at pH (≥9.0) is reported. Through a knowledge-gaining directed evolution (KnowVolution) campaign, the key positions Leu365 and Leu513 were identified to increase alkaline tolerance. Both positions are located in close proximity of the T1Cu site. Molecular docking and simulations studies reveal that both substitutions act in a synergic way to stabilize and improve laccase activity at higher pH. Kinetic characterization of the final variant MaL-M1 (L365E/L513M) revealed at pH 9.8 a threefold improved kcat (kcat =(6.0±0.2) s-1 ) compared with that of wild-type M. albomyces laccase (kcat =(2.11±0.07) s-1 ).

Selective Functionalization of Microgels with Enzymes by Sortagging.

Enzyme immobilization has been widely used to improve the stability and recyclability of enzymes in industrial processes. In this work, a sortase-mediated and therefore selective covalent immobilization strategy (sortagging) for enzymes on microgels (GelZyms) was investigated. Aqueous microgels were synthesized from poly(N-vinylcaprolactam)/glycidyl methacrylate (PVCL/GMA) and tagged with the sortase A recognition peptide sequence (LPETG) or its nucleophilic counterpart-tag (GGG). General applicability and selective immobilization were confirmed by subsequent sortagging of five different enzymes (Bacillus subtilis lipase A (BSLA), Yersinia mollaretii phytase (Ym-phytase), Escherichia coli copper efflux oxidase (CueO laccase), cellulase A2, and Bacillus megaterium monooxygenase P450 BM3). The latter was performed directly from the cell lysate to ensure cost-effective immobilization. All five immobilized enzymes were catalytically active and could be recycled (e.g., laccase CueO and monooxygenase P450 BM3 F87A; >55% residual activity after six cycles). Application potential was demonstrated by using CueO decorated microgels for bleaching of the synthetic dye indigo carmine.

Anchor Peptide-Mediated Surface Immobilization of a Grubbs-Hoveyda-Type Catalyst for Ring-Opening Metathesis Polymerization.

Adhesion promoting peptides have been reported to enable efficient enzyme immobilization on various material surfaces. Here we report the first immobilization of a synthetic Grubbs-Hoveyda (GH) type catalyst on two different materials (silica and polypropylene). To this end, the GH catalyst was coupled to an engineered (F16C) variant of the adhesion promoting peptide LCI through thiol-maleimide "click" reaction. Immobilization was performed in an oriented manner through the adhesion promoting peptide by simple incubation with the materials in water and subsequent washing with water and tetrahydrofuran. The immobilized GH catalyst was probed in ring-opening metathesis polymerization of a norbornene derivative to alter the surface properties in a layer-by-layer fashion.

Ultrahigh-throughput screening system for directed polymer binding peptide evolution.

Accumulation of plastics in the environment became a geological indicator of the Anthropocene era. An effective reduction of long-lasting plastics requires a treatment with micro-organisms that release polymer-degrading enzymes. Polymer binding peptides function as adhesion promoters and enable a targeted binding of whole cells to polymer surfaces. An esterase A-based Escherichia coli cell surface display screening system was developed, that enabled directed evolution of polymer binding peptides for improved binding strength to polymers. The E. coli cell surface screening system facilitates an enrichment of improved binding peptides from a culture broth through immobilization of whole cells on polymer beads. The polypropylene (PP)-binding peptide liquid chromatography peak I (LCI) was simultaneously saturated at five positions (Y29, D31, G35, E42, and D45; 3.2 million variants) and screened for improved PP-binding in the presence of the anionic surfactant sodium dodecylbenzenesulfonate (LAS; 0.25 mM). The cell surface system enabled efficient screening of the generated LCI diversity (in total ~10 million clones were screened). Characterization of identified LCI binders revealed an up to 12-fold improvement (eGFP-LCI-CSD-3: E42V/D45H) in PP-binding strength in the presence of the surfactant LAS (0.125 mM). The latter represents a first whole cell display screening system to improve adhesion peptides which can be used to direct and to immobilize organisms specifically to polymer surfaces (e.g., PP) and novel applications (e.g., in targeted plastic degradation).

Stimuli-Responsive Poly( N-Vinyllactams) with Glycidyl Side Groups: Synthesis, Characterization, and Conjugation with Enzymes.

Herein we report the synthesis of new reactive stimuli-responsive polymers by RAFT copolymerization of glycidyl methacrylate and three cyclic N-vinyllactam derivatives. The copolymerization process was thoroughly investigated and the influence of the steric hindrance originating from the monomer structure of cyclic N-vinyllactams on the polymerization process and the properties of obtained copolymers were studied. A series of water-soluble copolymers with variable chemical composition, controlled molecular weight and narrow dispersity ( D) were synthesized and their properties are systematically investigated. Experimentally determined cloud points for different copolymers in aqueous solutions indicate shift of lower critical solution temperature (LCST) to lower values with the increase of GMA content in copolymers and increase of the lactam ring size. The obtained reactive stimuli-responsive copolymers can be efficiently used for encapsulation of cellulase in water-in-oil emulsions forming biohybrid nanogels. The enzymes entrapped in nanogels demonstrated significantly improved resistance against harsh store conditions, chaotropic agents, and organic solvents.

Directed aryl sulfotransferase evolution toward improved sulfation stoichiometry on the example of catechols.

Sulfation is an important way for detoxifying xenobiotics and endobiotics including catechols. Enzymatic sulfation occurs usually with high chemo- and/or regioselectivity under mild reaction conditions. In this study, a two-step p-NPS-4-AAP screening system for laboratory evolution of aryl sulfotransferase B (ASTB) was developed in 96-well microtiter plates to improve the sulfate transfer efficiency toward catechols. Increased transfer efficiency and improved sulfation stoichiometry are achieved through the two-step screening procedure in a one-pot reaction. In the first step, the p-NPS assay is used (detection of the colorimetric by-product, p-nitrophenol) to determine the apparent ASTB activity. The sulfated product, 3-chlorocatechol-1-monosulfate, is quantified by the 4-aminoantipyrine (4-AAP) assay in the second step. Comparison of product formation to p-NPS consumption ensures successful directed evolution campaigns of ASTB. Optimization yielded a coefficient of variation below 15% for the two-step screening system (p-NPS-4-AAP). In total, 1760 clones from an ASTB-SeSaM library were screened toward the improved sulfation activity of 3-chlorocatechol. The turnover number (kcat = 41 ± 2 s-1) and catalytic efficiency (kcat/KM = 0.41 µM-1 s-1) of the final variant ASTB-M5 were improved 2.4- and 2.3-fold compared with ASTB-WT. HPLC analysis confirmed the improved sulfate stoichiometry of ASTB-M5 with a conversion of 58% (ASTB-WT 29%; two-fold improvement). Mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) confirmed the chemo- and regioselectivity, which yielded exclusively 3-chlorocatechol-1-monosulfate. For all five additionally investigated catechols, the variant ASTB-M5 achieved an improved kcat value of up to 4.5-fold and sulfate transfer efficiency was also increased (up to 2.3-fold).

Directed Evolution of Hyaluronic Acid Synthase from Pasteurella multocida towards High-Molecular-Weight Hyaluronic Acid.

Hyaluronic acid (HA), with diverse cosmetic and medical applications, is the natural glycosaminoglycan product of HA synthases. Although process and/or metabolic engineering are used for industrial HA production, the potential of protein engineering has barely been realised. Herein, knowledge-gaining directed evolution (KnowVolution) was employed to generate an HA synthase variant from Pasteurella multocida (pmHAS) with improved chain-length specificity and a twofold increase in mass-based turnover number. Seven improved pmHAS variants out of 1392 generated by error-prone PCR were identified; eight prospective positions were saturated and the most beneficial amino acid substitutions were recombined. After one round of KnowVolution, the longest HA polymer (<4.7 MDa), through an engineered pmHAS variant in a cell-free system, was synthesised. Computational studies showed that substitutions from the best variant (T40L, V59M and T104A) are distant from the glycosyltransferase sites and increase the flexibility of the N-terminal region of pmHAS. Taken together, these findings suggest that the N terminus may be involved in HA synthesis and demonstrate the potential of protein engineering towards improved HA synthase activity.

KnowVolution Campaign of an Aryl Sulfotransferase Increases Activity toward Cellobiose.

Sulfated polysaccharides such as cellulose can mimic the functionalities of pathophysiologically important glycosaminoglycans. Enzymatic sulfation offers a green chemistry route to selective (mono)sulfation of oligosaccharides (e.g., cellobiose as a building block of cellulose) in aqueous solution, at ambient temperature, and high chemoselectivity. Here, we report the first KnowVolution campaign for the aryl sulfotransferase B (ASTB) from Desulfitobacterium hafniense to advance ASTB toward a synthetically attractive biocatalyst. The generated final recombination variant (ASTB-M5) carries two amino acid substitutions (Leu446Pro and Val579Lys) leading to an up to 7.6-fold increase in specific activity (6.15 U mg-1 ) that was obtained with one round of KnowVolution. Mass spectrometry analysis confirmed a monosulfated product of cellobiose and structure elucidation by NMR confirmed the sulfation at the positions C-3 or C-4 of GlcNAc-linker-tBoc as opposed to the preferred C-6 by chemical means. Computational analysis suggested an important role of Leu446Pro in substrate-binding and recognized Val579Lys as a distal substitution.

A robust protocol for directed aryl sulfotransferase evolution toward the carbohydrate building block GlcNAc.

Bacterial aryl sulfotransferases (AST) utilize p-nitrophenylsulfate (pNPS) as a phenolic donor to sulfurylate typically a phenolic acceptor. Interest in aryl sulfotransferases is growing because of their broad variety of acceptors and cost-effective sulfuryl-donors. For instance, aryl sulfotransferase A (ASTA) from Desulfitobacterium hafniense was recently reported to sulfurylate d-glucose. In this study, a directed evolution protocol was developed and validated for aryl sulfotransferase B (ASTB). Thereby the well-known pNPS quantification system was advanced to operate efficiently as a continuous screening system in 96-well MTP format with a true coefficient of variation of 14.3%. A random mutagenesis library (SeSaM library) of ASTB was screened (1,760 clones) to improve sulfurylation of the carbohydrate building block N-acetylglucosamine (GlcNAc). The beneficial variant ASTB-V1 (Val579Asp) showed an up to 3.4-fold increased specific activity toward GlcNAc when compared to ASTB-WT. HPLC- and MS-analysis confirmed ASTB-V1's increased GlcNAc monosulfurylation (2.4-fold increased product formation) representing the validation of the first successful directed evolution round of an AST for a saccharide substrate.

Directed evolution of polypropylene and polystyrene binding peptides.

Surface functionalization of biological inert polymers (e.g., polypropylene PP; polystyrene PS) with material binding peptides facilitates an efficient immobilization of enzymes, bioactive peptides or antigens at ambient temperature in water. The developed robust directed evolution protocol enables to tailor polymer binding anchor peptides (PBPs) for efficient binding under application conditions. Key for a successful directed evolution campaign was to develop an epPCR protocol with a very high mutation frequency (60 mutations/kb) to ensure sufficient diversity in PBPs (47 aas LCI: "liquid chromatography peak I"; 44 aas TA2: "Tachystatin A2"). LCI and TA2 were genetically fused to the reporter egfp to quantify peptide binding on PP and PS by fluorescence analysis. The Peptide-Polymer evolution protocol (PePevo protocol) was validated in two directed evolution campaigns for two PBPs and polymers (LCI: PP; TA2: PS). Surfactants were used as selection pressure for improved PBP binders (non-ionic surfactant Triton X-100; 1 mM for LCI-PP // anionic surfactant LAS; 0.5 mM for TA2-PS). PePevo yielded an up to three fold improved PP-binder (LCI-M1-PP: I24T, Y29H, E42 K and LCI-M2-PP: D31V, E42G) and an up to six fold stronger PS-binder (TA2-M1-PS: R3S, L6P, V12 K, S15P, C29R, R30L, F33S, Y44H and TA2-M2-PS: F9C, C24S, G26D, S31G, C41S, Y44Q).

Sortase-Mediated Surface Functionalization of Stimuli-Responsive Microgels.

In this work we explored an enzyme-mediated method for selective and efficient decoration of aqueous microgels with biomolecules. Poly(N-vinylcaprolactam) (VCL) microgels with varied amounts of glycidyl methacrylate (GMA) as comonomer incorporated in the microgel shell were synthesized and characterized in regard to their size, swelling degree, and temperature-responsiveness in aqueous solutions. The surface of the PVCL/GMA microgel containing 5 mol % glycidyl methyacrylate was modified by grafting of a specific recognition peptide sequence (LPETG) for Sortase A from Staphylococcus aureus (Sa-SrtAΔ59). Sortase-mediated conjugation of the enhanced Green Fluorescent Protein (eGFP) carrying a N-terminal triglycine tag to LPETG-modified microgels was successfully performed. Conjugation of eGFP to the microgel surface was qualitatively proven by confocal microscopy and by fluorescence intensity measurements. The developed protocol enables a precise control of the amount of eGFP grafted to the microgel surface as evidenced by the linear increase of fluorescence intensity of modified microgel samples. The kinetic of the sortase-mediated coupling reaction was determined by time-dependent fluorescence intensity measurements. In summary, sortase-mediated coupling reactions are a simple and powerful technique for targeted surface functionalization of stimuli-responsive microgels with biomolecules.