An enzymatic continuous-flow reactor based on a pore-size matching nano- and isoporous block copolymer membrane.

Continuous-flow biocatalysis utilizing immobilized enzymes emerged as a sustainable route for chemical synthesis. However, inadequate biocatalytic efficiency from current flow reactors, caused by non-productive enzyme immobilization or enzyme-carrier mismatches in size, hampers its widespread application. Here, we demonstrate a general-applicable and robust approach for the fabrication of a high-performance enzymatic continuous-flow reactor via integrating well-designed scalable isoporous block copolymer (BCP) membranes as carriers with an oriented and productive immobilization employing material binding peptides (MBP). Densely packed uniform enzyme-matched nanochannels of well-designed BCP membranes endow the desired nanoconfined environments towards a productive immobilized phytase. Tuning nanochannel properties can further regulate the complex reaction process and fortify the catalytic performance. The synergistic design of enzyme-matched carriers and efficient enzyme immobilization empowers an excellent catalytic performance with >1 month operational stability, superior productivity, and a high space-time yield (1.05 × 105 g L-1 d-1) via a single-pass continuous-flow process. The obtained performance makes the designed nano- and isoporous block copolymer membrane reactor highly attractive for industrial applications.

Engineering diaryl alcohol dehydrogenase KpADH reveals importance of retaining hydration shell in organic solvent tolerance.

Alcohol dehydrogenases (ADHs) are synthetically important biocatalysts for the asymmetric synthesis of chiral alcohols. The catalytic performance of ADHs in the presence of organic solvents is often important since most prochiral ketones are highly hydrophobic. Here, the organic solvent tolerance of KpADH from Kluyveromyces polyspora was semi-rationally evolved. Using tolerant variants obtained, meticulous experiments and computational studies were conducted to explore properties including stability, activity and kinetics in the presence of various organic solvents. Compared with WT, variant V231D exhibited 1.9-fold improvement in ethanol tolerance, while S237G showed a 6-fold increase in catalytic efficiency, a higher T 50 15 $$ {\mathrm{T}}_{50}^{15} $$ , as well as 15% higher tolerance in 7.5% (v/v) ethanol. Based on 3 × 100 ns MD simulations, the increased tolerance of V231D and S237G against ethanol may be ascribed to their enhanced ability in retaining water molecules and repelling ethanol molecules. Moreover, 6.3-fold decreased KM value of V231D toward hydrophilic ketone substrate confirmed its capability of retaining hydration shell. Our results suggest that retaining hydration shell surrounding KpADH is critical for its tolerance to organic solvents, as well as catalytic performance. This study provides useful guidance for engineering organic solvent tolerance of KpADH and other ADHs.

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.

Engineered Anchor Peptide LCI with a Cobalt Cofactor Enhances Oxidation Efficiency of Polystyrene Microparticles.

A typical component of polymer waste is polystyrene (PS) used in numerous applications, but degraded only slowly in the environment due to its hydrophobic properties. To increase the reactivity of polystyrene, polar groups need to be introduced. Here, biohybrid catalysts based on the engineered anchor peptide LCI_F16C are presented, which are capable of attaching to polystyrene microparticles and hydroxylating benzylic C-H bonds in polystyrene microparticles using commercially available oxone as oxidant. LCI peptides achieve a dense surface coverage of PS through monolayer formation within minutes in aqueous solutions at ambient temperature. The catalytically active cobalt cofactor Co-L1 or Co-L2 with a modified NNNN macrocyclic TACD ligand (TACD=1,4,7,10-tetraazacyclododecane) is covalently bound to the anchor peptide LCI through a maleimide linker. Compared to the free cofactors, a 12- to 15-fold improvement in catalytic activity using biohybrid catalysts based on LCI_F16C was observed.

Benzylic C(sp3 )-H Bond Oxidation with Ketone Selectivity by a Cobalt(IV)-Oxo Embedded in a ß-Barrel Protein.

Artificial metalloenzymes have emerged as biohybrid catalysts that allow to combine the reactivity of a metal catalyst with the flexibility of protein scaffolds. This work reports the artificial metalloenzymes based on the ß-barrel protein nitrobindin NB4, in which a cofactor [CoII X(Me3 TACD-Mal)]+ X- (X=Cl, Br; Me3 TACD=N,N' ,N''-trimethyl-1,4,7,10-tetraazacyclododecane, Mal=CH2 CH2 CH2 NC4 H2 O2 ) was covalently anchored via a Michael addition reaction. These biohybrid catalysts showed higher efficiency than the free cobalt complexes for the oxidation of benzylic C(sp3 )-H bonds in aqueous media. Using commercially available oxone (2KHSO5 ⋅ KHSO4 ⋅ K2 SO4 ) as oxidant, a total turnover number of up to 220 and 97 % ketone selectivity were achieved for tetralin. As catalytically active intermediate, a mononuclear terminal cobalt(IV)-oxo species [Co(IV)=O]2+ was generated by reacting the cobalt(II) cofactor with oxone in aqueous solution and characterized by ESI-TOF MS.

Empowering Site-Specific Bioconjugations In Vitro and In Vivo: Advances in Sortase Engineering and Sortase-Mediated Ligation.

Sortase-mediated ligation (SML) has emerged as a powerful and versatile methodology for site-specific protein conjugation, functionalization/labeling, immobilization, and design of biohybrid molecules and systems. However, the broader application of SML faces several challenges, such as limited activity and stability, dependence on calcium ions, and reversible reactions caused by nucleophilic side-products. Over the past decade, protein engineering campaigns and particularly directed evolution, have been extensively employed to overcome sortase limitations, thereby expanding the potential application of SML in multiple directions, including therapeutics, biorthogonal chemistry, biomaterials, and biosensors. This review provides an overview of achieved advancements in sortase engineering and highlights recent progress in utilizing SML in combination with other state-of-the-art chemical and biological methodologies. The aim is to encourage scientists to employ sortases in their conjugation experiments.

Machine Learning-Assisted Engineering of Light, Oxygen, Voltage Photoreceptor Adduct Lifetime.

Naturally occurring and engineered flavin-binding, blue-light-sensing, light, oxygen, voltage (LOV) photoreceptor domains have been used widely to design fluorescent reporters, optogenetic tools, and photosensitizers for the visualization and control of biological processes. In addition, natural LOV photoreceptors with engineered properties were recently employed for optimizing plant biomass production in the framework of a plant-based bioeconomy. Here, the understanding and fine-tuning of LOV photoreceptor (kinetic) properties is instrumental for application. In response to blue-light illumination, LOV domains undergo a cascade of photophysical and photochemical events that yield a transient covalent FMN-cysteine adduct, allowing for signaling. The rate-limiting step of the LOV photocycle is the dark-recovery process, which involves adduct scission and can take between seconds and days. Rational engineering of LOV domains with fine-tuned dark recovery has been challenging due to the lack of a mechanistic model, the long time scale of the process, which hampers atomistic simulations, and a gigantic protein sequence space covering known mutations (combinatorial challenge). To address these issues, we used machine learning (ML) trained on scarce literature data and iteratively generated and implemented experimental data to design LOV variants with faster and slower dark recovery. Over the three prediction-validation cycles, LOV domain variants were successfully predicted, whose adduct-state lifetimes spanned 7 orders of magnitude, yielding optimized tools for synthetic (opto)biology. In summary, our results demonstrate ML as a viable method to guide the design of proteins even with limited experimental data and when no mechanistic model of the underlying physical principles is available.

Influences of the Carbohydrate-Binding Module on a Fungal Starch-Active Lytic Polysaccharide Monooxygenase.

Noncatalytic carbohydrate-binding modules (CBMs) play important roles in the function of lytic polysaccharide monooxygenases (LPMOs) but have not been well demonstrated for starch-active AA13 LPMO. In this study, four new CBMs were investigated systematically for their influence on MtLPMO toward starch in terms of substrate binding, H2O2 production activity, oxidative product yields, and the degradation effect with α-amylase and glucoamylase toward different starch substrates. Among the four MtLPMO-CBM chimeras, MtLPMO-CnCBM harboring the CBM fromColletotrichum nymphaeae showed the highest substrate binding toward different types of starch compared to MtLPMO without CBM. MtLPMO-PvCBM harboring the CBM from Pseudogymnoascus verrucosus and MtLPMO-CnCBM showed dramatically enhanced H2O2 production activity of 4.6-fold and 3.6-fold, respectively, than MtLPMO without CBM. More importantly, MtLPMO-CBM generated more oxidative products from starch polysaccharides degradation than MtLPMO alone, with 6.0-fold and 4.6-fold enhancement obtained from the oxidation of amylopectin and corn starch with MtLPMO-CnCBM, and a 5.2-fold improvement obtained with MtLPMO-AcCBM for amylose. MtLPMO-AcCBM significantly boosted the yields of reducing sugar with α-amylase upon degrading amylopectin and corn starch. These findings demonstrate that CBMs greatly influence the performance of starch-active AA13 LPMOs due to their enhanced binding and H2O2 production activity.

Brush-Like Coatings Provide a Cloak of Invisibility to Titanium Implants.

Orthopedic implants such as knee and hip implants are one of the most important types of medical devices. Currently, the surface of the most advanced implants consists of titanium or titanium-alloys with high porosity at the bone-contacting surface leading to superior mechanical properties, excellent biocompatibility, and the capability of inducing osseointegration. However, the increased surface area of porous titanium provides a nidus for bacteria colonization leading to implant-related infections, one of the main reasons for implant failure. Here, two readily applicable titanium-coatings based on hydrophilic carboxybetaine polymers that turn the surface stealth thereby preventing bacterial adhesion and colonization are developed. These coatings are biocompatible, do not affect cell functionality, exhibit great antifouling properties, and do not cause additional inflammation during the healing process. In this way, the coatings can prevent implant-related infections, while at the same time being completely innocuous to its biological environment. Thus, these coating strategies are a promising route to enhance the biocompatibility of orthopedic implants and have a high potential for clinical use, while being easy to implement in the implant manufacturing process.

Directed Evolution of Material Binding Peptide for Polylactic Acid-specific Degradation in Mixed Plastic Wastes.

In order to preserve our livelihood for future generations, responsible use of plastics in a climate-neutral and circular economy has to be developed so that plastics can be used in an environmentally friendly way by future generations. The prerequisite is that bioplastic polymers such as polylactic acid (PLA) can be efficiently recycled from petrochemical based plastic. Here, a concept in which accelerated PLA degradation in the mixed suspension of PLA and polystyrene (PS) nanoparticles has been achieved through an engineered material binding peptide. After comparison of twenty material binding peptides, Cg-Def is selected due to its PLA binding specificity. Finally, a suitable high-throughput screening system is developed for enhancing material-specific binding toward PLA in presence of PS. Through KnowVolution campaign, a variant Cg-Def YH (L9Y/S19H) with 2.0-fold improved PLA binding specificity compared to PS is generated. Contact angle and surface plasmon resonance measurements validated higher surface coverage of Cg-Def YH on PLA surface and the fusion of Cg-Def YH with PLA degrading enzyme confirmed the accelerated PLA depolymerization (two times higher than only enzyme) in mixed PLA/PS plastics.

A Competitive High-Throughput Screening Platform for Designing Polylactic Acid-Specific Binding Peptides.

Among biobased polymers, polylactic acid (PLA) is recognized as one of the most promising bioplastics to replace petrochemical-based polymers. PLA is typically blended with other polymers such as polypropylene (PP) for improved melt processability, thermal stability, and stiffness. A technical challenge in recycling of PLA/PP blends is the sorting/separation of PLA from PP. Material binding peptides (MBPs) can bind to various materials. Engineered MBPs that can bind in a material-specific manner have a high potential for material-specific detection or enhanced degradation of PLA in mixed PLA/PP plastics. To obtain a material-specific MBP for PLA binding (termed PLAbodies ), protein engineering of MBP Cg-Def for improved PLA binding specificity is reported in this work. In detail, a 96-well microtiter plate based high-throughput screening system for PLA specific binding (PLABS) was developed and validated in a protein engineering (KnowVolution) campaign. Finally, the Cg-Def variant V2 (Cg-Def S19K/K10L/N13H) with a 2.3-fold improved PLA binding specificity compared to PP was obtained. Contact angle and surface plasmon resonance measurements confirmed improved material-specific binding of V2 to PLA (1.30-fold improved PLA surface coverage). The established PLABS screening platform represents a general methodology for designing PLAbodies for applications in detection, sorting, and material-specific degradation of PLA in mixed plastics.

A step forward to the optimized HlyA type 1 secretion system through directed evolution.

Secretion of proteins into the extracellular space has great advantages for the production of recombinant proteins. Type 1 secretion systems (T1SS) are attractive candidates to be optimized for biotechnological applications, as they have a relatively simple architecture compared to other classes of secretion systems. A paradigm of T1SS is the hemolysin A type 1 secretion system (HlyA T1SS) from Escherichia coli harboring only three membrane proteins, which makes the plasmid-based expression of the system easy. Although for decades the HlyA T1SS has been successfully applied for secretion of a long list of heterologous proteins from different origins as well as peptides, but its utility at commercial scales is still limited mainly due to low secretion titers of the system. To address this drawback, we engineered the inner membrane complex of the system, consisting of HlyB and HlyD proteins, following KnowVolution strategy. The applied KnowVolution campaign in this study provided a novel HlyB variant containing four substitutions (T36L/F216W/S290C/V421I) with up to 2.5-fold improved secretion for two hydrolases, a lipase and a cutinase. KEY POINTS: • An improvement in protein secretion via the use of T1SS • Reaching almost 400 mg/L of soluble lipase into the supernatant • A step forward to making E. coli cells more competitive for applying as a secretion host.

Engineering All-Round Cellulase for Bioethanol Production.

One strategy to decrease both the consumption of crude oil and environmental damage is through the production of bioethanol from biomass. Cellulolytic enzyme stability and enzymatic hydrolysis play important roles in the bioethanol process. However, the gradually increased ethanol concentration often reduces enzyme activity and leads to inactivation, thereby limiting the final ethanol yield. Herein, we employed an optimized Two-Gene Recombination Process (2GenReP) approach to evolve the exemplary cellulase CBHI for practical bioethanol fermentation. Two all-round CBHI variants (named as R2 and R4) were obtained with simultaneously improved ethanol resistance, organic solvent inhibitor tolerance, and enzymolysis stability in simultaneous saccharification and fermentation (SSF). Notably, CBHI R4 had a 7.0- to 34.5-fold enhanced catalytic efficiency (kcat/KM) in the presence/absence of ethanol. Employing the evolved CBHI R2 and R4 in the 1G bioethanol process resulted in up to 10.27% (6.7 g/L) improved ethanol yield (ethanol concentration) than non-cellulase, which was far more beyond than other optimization strategies. Besides bioenergy fields, this transferable protein engineering routine holds the potential to generate all-round enzymes that meet the requirement in biotransformation and bioenergy fields.

Transformative Materials for Interfacial Drug Delivery.

Drug delivery systems (DDS) are designed to temporally and spatially control drug availability and activity. They assist in improving the balance between on-target therapeutic efficacy and off-target toxic side effects. DDS aid in overcoming biological barriers encountered by drug molecules upon applying them via various routes of administration. They are furthermore increasingly explored for modulating the interface between implanted (bio)medical materials and host tissue. Herein, an overview of the biological barriers and host-material interfaces encountered by DDS upon oral, intravenous, and local administration is provided, and material engineering advances at different time and space scales to exemplify how current and future DDS can contribute to improved disease treatment are highlighted.

FhuA: From Iron-Transporting Transmembrane Protein to Versatile Scaffolds through Protein Engineering.

Protein engineering has emerged as a powerful methodology to tailor the properties of proteins. It empowers the design of biohybrid catalysts and materials, thereby enabling the convergence of materials science, chemistry, and medicine. The choice of a protein scaffold is an important factor for performance and potential applications. In the past two decades, we utilized the ferric hydroxamate uptake protein FhuA. FhuA is, from our point of view, a versatile scaffold due to its comparably large cavity and robustness toward temperature as well as organic cosolvents. FhuA is a natural iron transporter located in the outer membrane of Escherichia coli (E. coli). Wild-type FhuA consists of 714 amino acids and has a ß-barrel structure composed of 22 antiparallel ß-sheets, closed by an internal globular "cork" domain (amino acids 1-160). FhuA is robust in a broad pH range and toward organic cosolvents; therefore, we envisioned FhuA to be a suitable platform for various applications in (i) biocatalysis, (ii) materials science, and (iii) the construction of artificial metalloenzymes.(i) Applications in biocatalysis were achieved by removing the globular cork domain (FhuA_Δ1-160), thereby creating a large pore for the passive transport of otherwise difficult-to-import molecules through diffusion. Introducing this FhuA variant into the outer membrane of E. coli facilitates the uptake of substrates for downstream biocatalytic conversion. Furthermore, removing the globular "cork" domain without structural collapse of the ß-barrel protein allowed the use of FhuA as a membrane filter, exhibiting a preference for d-arginine over l-arginine.(ii) FhuA is a transmembrane protein, which makes it attractive to be used for applications in non-natural polymeric membranes. Inserting FhuA into polymer vesicles yielded so-called synthosomes (i.e., catalytic synthetic vesicles in which the transmembrane protein acted as a switchable gate or filter). Our work in this direction enables polymersomes to be used in biocatalysis, DNA recovery, and the controlled (triggered) release of molecules. Furthermore, FhuA can be used as a building block to create protein-polymer conjugates to generate membranes.(iii) Artificial metalloenzymes (ArMs) are formed by incorporating a non-native metal ion or metal complex into a protein. This combines the best of two worlds: the vast reaction and substrate scope of chemocatalysis and the selectivity and evolvability of enzymes. With its large inner diameter, FhuA can harbor (bulky) metal catalysts. Among others, we covalently attached a Grubbs-Hoveyda-type catalyst for olefin metathesis to FhuA. This artificial metathease was then used in various chemical transformations, ranging from polymerizations (ring-opening metathesis polymerization) to enzymatic cascades involving cross-metathesis. Ultimately, we generated a catalytically active membrane by copolymerizing FhuA and pyrrole. The resulting biohybrid material was then equipped with the Grubbs-Hoveyda-type catalyst and used in ring-closing metathesis.The number of reports on FhuA and its various applications indicates that it is a versatile building block to generate hybrid catalysts and materials. We hope that our research will inspire future research efforts at the interface of biotechnology, catalysis, and material science in order to create biohybrid systems that offer smart solutions for current challenges in catalysis, material science, and medicine.

[Commentary: polymer binding modules accelerate enzymatic degradation of poly(ethylene terephthalate)].

The large scale production and indiscriminate use of plastics led to serious environmental pollution. To reduce the negative effects of plastics waste on the environment, an approach of enzymatic degradation was put forward to catalyze plastics degradation. Protein engineering strategies have been applied to improve the plastics degrading enzyme properties such as activity and thermal stability. In addition, polymer binding modules were found to accelerate the enzymatic degradation of plastics. In this article, we introduced a recent work published in Chem Catalysis, which studied the role of binding modules in enzymatic hydrolysis of poly(ethylene terephthalate) (PET) at high-solids loadings. Graham et al. found that binding modules accelerated PET enzymatic degradation at low PET loading (< 10 wt%) and the enhanced degradation cannot be observed at high PET loading (10 wt%-20 wt%). This work is beneficial for the industrial application of polymer binding modules in plastics degradation.

A Flow Cytometry-Based Ultrahigh-Throughput Screening Method for Directed Evolution of Oxidases.

Oxidases are of interest to chemical and pharmaceutical industries because they catalyze highly selective oxidations. However, oxidases found in nature often need to be re-engineered for synthetic applications. Herein, we developed a versatile and robust flow cytometry-based screening platform "FlOxi" for directed oxidase evolution. FlOxi utilizes hydrogen peroxide produced by oxidases expressed in E. coli to oxidize Fe2+ to Fe3+ (Fenton reaction). Fe3+ mediates the immobilization of a His6 -tagged eGFP (eGFPHis ) on the E. coli cell surface, ensuring the identification of beneficial oxidase variants by flow cytometry. FlOxi was validated with two oxidases-a galactose oxidase (GalOx) and a D-amino acid oxidase (D-AAO)-yielding a GalOx variant (T521A) with a 4.4-fold lower Km value and a D-AAO variant (L86M/G14/A48/T205) with a 4.2-fold higher kcat than their wildtypes. Thus, FlOxi can be used for the evolution of hydrogen peroxide-producing oxidases and applied for non-fluorescent substrates.

Evolutionary Engineering of a Cp*Rh(III) Complex-Linked Artificial Metalloenzyme with a Chimeric ß-Barrel Protein Scaffold.

Evolutionary engineering of our previously reported Cp*Rh(III)-linked artificial metalloenzyme was performed based on a DNA recombination strategy to improve its catalytic activity toward C(sp2)-H bond functionalization. Improved scaffold design was achieved with α-helical cap domains of fatty acid binding protein (FABP) embedded within the ß-barrel structure of nitrobindin (NB) as a chimeric protein scaffold for the artificial metalloenzyme. After optimization of the amino acid sequence by directed evolution methodology, an engineered variant, designated NBHLH1(Y119A/G149P) with enhanced performance and enhanced stability was obtained. Additional rounds of metalloenzyme evolution provided a Cp*Rh(III)-linked NBHLH1(Y119A/G149P) variant with a >35-fold increase in catalytic efficiency (kcat/KM) for cycloaddition of oxime and alkyne. Kinetic studies and MD simulations revealed that aromatic amino acid residues in the confined active-site form a hydrophobic core which binds to aromatic substrates adjacent to the Cp*Rh(III) complex. The metalloenzyme engineering process based on this DNA recombination strategy will serve as a powerful method for extensive optimization of the active-sites of artificial metalloenzymes.

Plastibodies for multiplexed detection and sorting of microplastic particles in high-throughput.

Sensitive high-throughput analytic methodologies are needed to quantify microplastic particles (MPs) and thereby enable routine monitoring of MPs to ultimately secure animal, human, and environmental health. Here we report a multiplexed analytical and flow cytometry-based high-throughput methodology to quantify MPs in aqueous suspensions. The developed analytic MPs-quantification platform provides a sensitive as well as high-throughput detection of MPs that relies on the material binding peptide Liquid Chromatography Peak I (LCI) conjugated to Alexa-fluorophores (LCIF16C-AF488, LCIF16C-AF594, and LCIF16C-AF647). These fluorescent material-binding peptides (also termed plastibodies) were used to fluorescently label polystyrene MPs, whereas Alexa-fluorophores alone exhibited a negligible background fluorescence. Mixtures of polystyrene MPs that varied in size (500 nm to 5 µm) and varied in labeled populations were analyzed and sorted into distinct populations reaching sorting efficiencies >90 % for 1 × 106 sorted events. Finally, a multiplexed quantification and sorting with up to three plastibodies was successfully achieved to validate that the combination of plastibodies and flow cytometry is a powerful and generally applicable methodology for multiplexed analysis, quantification, and sorting of microplastic particles.

Self-Sufficient In Vitro Multi-Enzyme Cascade for Efficient Synthesis of Danshensu from l-DOPA.

Danshensu (DSS), a traditional Chinese medicine, is widely used for the treatment of cardiovascular and cancer diseases. Here, a one-pot multi-enzyme cascade pathway was designed for DSS synthesis from l-DOPA using tyrosine aminotransferase from Escherichia coli (EcTyrB) and d-isomer-specific 2-hydroxyacid dehydrogenase from Lactobacillus frumenti (LfD2-HDH). Glutamate dehydrogenase from Clostridium difficile (CdgluD) was also introduced for a self-sufficient system of α-ketoglutaric acid and NADH. Under optimal conditions (35 °C, pH 7.0, EcTyrB:LfD2-HDH:CdgluD = 3:2:1, glutamate:NAD+ = 1:1), 98.3% yield (at 20 mM l-DOPA) and space-time yield of 6.61 g L-1 h-1 (at 40 mM l-DOPA) were achieved. Decreased yields of DSS at elevated l-DOPA concentrations (100 mM) could be attributed to an inhibited CdgluD activity caused by NH4+ accumulation. This developed multi-enzyme cascade pathway (including EcTyrB, LfD2-HDH, and CdgluD) provides an efficient and sustainable approach for the production of DSS from l-DOPA.