Synthetic Protein Scaffolds Research Articles

Engineering of itaconic acid pathway via co-localization of CadA and AcnA in recombinant Escherichia coli.

Itaconic acid is an excellent polymeric precursor with a wide range of industrial applications. The efficient production of itaconate from various renewable substrates was demonstrated by engineered Escherichia coli. However, limitation in the itaconic acid precursor supply was revealed by finding out the key intermediate of the tricarboxylic acid in the itaconic acid pathway. Efforts of enhancing the cis-aconitate flux and preserving the isocitrate pool to increase itaconic acid productivity are required. In this study, we introduce a synthetic protein scaffold system between CadA and AcnA to physically combine the two enzymes. Through the introduction of a synthetic protein scaffold, 2.1g L-1 of itaconic acid was produced at pH 7 and 37°C. By fermentation, 20.1g L-1 for 48h of itaconic acid was produced with a yield of 0.34gg-1 glycerol. These results suggest that carbon flux was successfully increased itaconic acid productivity.

“Affimer” synthetic protein scaffolds block oxidized LDL binding to the LOX-1 scavenger receptor and inhibit ERK1/2 activation

In multicellular organisms, a variety of lipid-protein particles control the systemic flow of triacylglycerides, cholesterol, and fatty acids between cells in different tissues. The chemical modification by oxidation of these particles can trigger pathological responses, mediated by a group of membrane proteins termed scavenger receptors. The lectin-like oxidized low-density lipoprotein (LOX-1) scavenger receptor binds to oxidized low-density lipoprotein (oxLDL) and mediates both signaling and trafficking outcomes. Here, we identified five synthetic proteins termed Affimers from a phage display library, each capable of binding recombinant LOX-1 extracellular (oxLDL-binding) domain with high specificity. These Affimers, based on a phytocystatin scaffold with loop regions of variable sequence, were able to bind to the plasma membrane of HEK293T cells exclusively when human LOX-1 was expressed. Binding and uptake of fluorescently labeled oxLDL by the LOX-1-expressing cell model was inhibited with subnanomolar potency by all 5 Affimers. ERK1/2 activation, stimulated by oxLDL binding to LOX-1, was also significantly inhibited (p< 0.01) by preincubation with LOX-1-specific Affimers, but these Affimers had no direct agonistic effect. Molecular modeling indicated that the LOX-1-specific Affimers bound predominantly via their variable loop regions to the surface of the LOX-1 lectin-like domain that contains a distinctive arrangement of arginine residues previously implicated in oxLDL binding, involving interactions with both subunits of the native, stable scavenger receptor homodimer. These data provide a new class of synthetic tools to probe and potentially modulate the oxLDL/LOX-1 interaction that plays an important role in vascular disease.

Using the inner membrane of Escherichia coli as a scaffold to anchor enzymes for metabolic flux enhancement.

Clustering enzymes in the same metabolic pathway is a natural strategy to enhance productivity. Synthetic protein, RNA and DNA scaffolds have been designed to artificially cluster multiple enzymes in the cell, which require complex construction processes and possess limited slots for target enzymes. We utilized the Escherichia coli inner cell membrane as a native scaffold to cluster four fatty acid synthases (FAS) and achieved to improve the efficiency of fatty acid synthesis in vivo. The construction strategy is as simple as fusing target enzymes to the N-terminus or C-terminus of the membrane anchor protein (Lgt), and the number of anchored enzymes is not restricted. This novel device not only presents a similar efficiency in clustering multiple enzymes to that of other artificial scaffolds but also promotes the product secretion, driving the entire metabolic flux forward and further increasing the gross yield compared with that in a cytoplasmic scaffold system.

Novel Split Intein-Mediated Enzymatic Channeling Accelerates the Multimeric Bioconversion Pathway of Ginsenoside.

Cascade reaction systems, such as protein fusion and synthetic protein scaffold systems, can both spatially control the metabolic flux and boost the productivity of multistep enzymatic reactions. Despite many efforts to generate fusion proteins, this task remains challenging due to the limited expression of complex enzymes. Therefore, we developed a novel fusion system that bypasses the limited expression of complex enzymes via a post-translational linkage. Here, we report a split intein-mediated cascade system wherein orthogonal split inteins serve as adapters for protein ligation. A genetically programmable, self-assembled, and traceless split intein was utilized to generate a biocatalytic cascade to produce the ginsenoside compound K (CK) with various pharmacological activities, including anticarcinogenic, anti-inflammatory, and antidiabetic effects. We used two types of split inteins, consensus atypical (Cat) and Rma DnaB, to form a covalent scaffold with the three enzymes involved in the CK conversion pathway. The multienzymatic complex with a size greater than 240 kDa was successfully assembled in a soluble form and exhibited specific activity toward ginsenoside conversion. Furthermore, our split intein cascade system significantly increased the CK conversion rate and reduced the production time by more than 2-fold. Our multienzymatic cascade system that uses split inteins can be utilized as a platform for regulating multimeric bioconversion pathways and boosting the production of various high-value substances.

Synthetic Protein Scaffolds for Improving R-(-)-Linalool Production in Escherichia coli.

R-(-)-Linalool is widely used in the pharmaceutical, agrochemical, and fragrance industries; however, its applications are limited owing to low yield and high cost of production. To improve the production efficiency of R-(-)-linalool in Escherichia coli, three enzymes [E. coli-derived isopentenyl diphosphate isomerase, Abies grandis-derived geranyl diphosphate synthase, and Streptomyces clavuligerus-derived (3R)-linalool synthases] were physically colocalized to synthetic complexes using synthetic protein scaffolds of GTPase-binding domain, Src homology 3, and PSD95/DlgA/Zo-1. R-(-)-Linalool was produced at the highest concentration in the strain IGL114 containing a scaffold ratio of 1:1:4. By further optimizing the inducer, temperature, and glycerol concentration, the production titer of R-(-)-linalool in the shake flask was increased by approximately 10 times compared with that of the scaffold-free control and was 2.78 times the previously reported yield. The production in the fermenter was about 1.5 times the previous highest production. In general, the final strain accumulated 277.8 and 1523.2 mg/L R-(-)-linalool under the conditions of shake-flask and fed-batch fermentation, respectively. This study provides a foundation for the assembly of bacterial intracellular protein scaffolds.

O16: TARGETING THE LOX-1 SCAVENGER RECEPTOR ATTENUATES ATHEROSCLEROSIS AND NEOINTIMAL HYPERPLASIA IN APO-E NULL TRANSGENIC MICE

Abstract Introduction The clinical consequences of atherosclerosis and post-procedure neointimal hyperplasia (NIH) represent a significant proportion of the vascular surgeon's workload. Lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) is a class E scavenger receptor expressed in endothelial cells implicated in atherosclerosis and NIH. This study aims to demonstrate the therapeutic potential of LOX-1 targeting using a transgenic mouse model and LOX-1-specific non-antibody synthetic protein scaffolds called affimers. Method Apolipoprotein-E (APO-E) null and APO-E/LOX-1 double-null transgenic mice were fed a high cholesterol western diet for 12 weeks. One group of APO-E null mice was treated subcutaneously with LOX-1 affimer. All mice underwent unilateral common femoral artery wire injury at week 8. Mice were culled and tissues harvested for evaluation of atherosclerotic burden and femoral artery NIH. Studies were carried out with Home Office approval. Result Compared with APO-E null controls, carotid and femoral bifurcation atherosclerosis (p&amp;lt;0.05) and total aortic plaque coverage (p&amp;lt;0.01) were significantly reduced in APO-E/LOX-1 double-null mice. Reductions were observed in APO-E null mice treated with LOX-1 affimer, however these were non-significant. Femoral artery NIH was reduced by 16% in APO-E/LOX-1 double null and 11% in LOX-1 affimer treated APO-E null mice compared with untreated APO-E controls, however these reductions were non-significant. Conclusion This study demonstrates the therapeutic potential of LOX-1 targeting in large vessel atherosclerotic disease and NIH. Knockout of the LOX-1 transgene produced promising results, while results following LOX-1 affimer therapy were less impressive. Improvements in affimer pharmacokinetics and drug delivery optimisation are avenues for future studies. Take-home message The LOX-1 scavenger receptor is a potential novel therapeutic target in large-vessel atherosclerosis and neointimal hyperplasia.

A “push-pull-restrain” strategy to improve citronellol production in Saccharomyces cerevisiae

Microbial production of monoterpenes has attracted increasing attention in recent years. Up to date, there are only few reports on the biosynthesis of the monoterpene alcohol citronellol that is widely used as fragrant and pharmaceutical intermediates. Here, we engineered Saccharomyces cerevisiae by employing a “push-pull-restrain” strategy to improve citronellol production based on the reduction of geraniol. Starting from a engineered geraniol-producing strain, different reductases were investigated and the best performing iridoid synthase from Catharanthus roseus (CrIS) resulted in 285.89 mg/L enantiomerically pure S-citronellol in shake flasks. Geranyl diphosphate (GPP), the most important precursor for monoterpenes, was enhanced by replacing the wild farnesyl diphosphate synthase (Erg20) with the mutant Erg20F96W, increasing the citronellol titer to 406.01 mg/L without negative influence on cell growth. Moreover, we employed synthetic protein scaffolds and protein fusion to colocalize four sequential enzymes to achieve better substrate channeling along with the deletion of an intermediate degradation pathway gene ATF1, which elevated the citronellol titer to 972.02 mg/L with the proportion of 97.8% of total monoterpenes in YPD medium. Finally, the engineered strain with complemented auxotrophic markers produced 8.30 g/L of citronellol by fed-batch fermentation, which was the highest citronellol titer reported to date. The multi-level engineering strategies developed here demonstrate the potential of monoterpenes overproduction in yeast, which can serve as a generally applicable platform for overproduction of other monoterpenes.

Dynamic Protease Activation on a Multimeric Synthetic Protein Scaffold via Adaptable DNA-Based Recruitment Domains.

Hexameric hemoprotein (HTHP) is employed as a scaffold protein for the supramolecular assembly and activation of the apoptotic signalling enzyme caspase‐9, using short DNA elements as modular recruitment domains. Caspase‐9 assembly and activation on the HTHP platform due to enhanced proximity is followed by combinatorial inhibition at high scaffold concentrations. The DNA recruitment domains allow for reversible switching of the caspase‐9 assembly and activity state using short modulatory DNA strands. Tuning of the recruitment domain affinity allows for generating kinetically trapped active enzyme complexes, as well as for dynamic repositioning of caspases over scaffold populations and inhibition using monovalent sink platforms. The conceptual combination of a highly structured multivalent protein platform with modular DNA recruitment domains provides emergent biomimicry properties with advanced levels of control over protein assembly.

Dynamic Protease Activation on a Multimeric Synthetic Protein Scaffold via Adaptable DNA‐Based Recruitment Domains

AbstractHexameric hemoprotein (HTHP) is employed as a scaffold protein for the supramolecular assembly and activation of the apoptotic signalling enzyme caspase‐9, using short DNA elements as modular recruitment domains. Caspase‐9 assembly and activation on the HTHP platform due to enhanced proximity is followed by combinatorial inhibition at high scaffold concentrations. The DNA recruitment domains allow for reversible switching of the caspase‐9 assembly and activity state using short modulatory DNA strands. Tuning of the recruitment domain affinity allows for generating kinetically trapped active enzyme complexes, as well as for dynamic repositioning of caspases over scaffold populations and inhibition using monovalent sink platforms. The conceptual combination of a highly structured multivalent protein platform with modular DNA recruitment domains provides emergent biomimicry properties with advanced levels of control over protein assembly.

Design of Smart Antibody Mimetics with Photosensitive Switches.

As two prominent examples of intracellular single-domain antibodies or antibody mimetics derived from synthetic protein scaffolds, monobodies and nanobodies are gaining wide applications in cell biology, structural biology, synthetic immunology, and theranostics. Herein, a generally applicable method to engineer light-controllable monobodies and nanobodies, designated as moonbody and sunbody, respectively, is introduced. These engineered antibody-like modular domains enable rapid and reversible antibody-antigen recognition by utilizing light. By the paralleled insertion of two light-oxygen-voltage domain 2 modules into a single sunbody and the use of bivalent sunbodies, the range of dynamic changes of photoswitchable sunbodies is substantially enhanced. Furthermore, the use of moonbodies or sunbodies to precisely control protein degradation, gene transcription, and base editing by harnessing the power of light is demonstrated.

Enhanced Production of Malic Acid by Co-localization of Phosphoenolpyruvate Carboxylase and Malate Dehydrogenase Using Synthetic Protein Scaffold in Escherichia coli

To produce malic acid from non-oxidative pathway route in Escherichia coli using two key enzymes and synthetic scaffold complex. E. coli was engineered to produce malic acid from glucose by co-localization of two key enzymes phosphoenolpyruvate carboxylase (Ppc) and malate dehydrogenase (MdhA) with synthetic scaffold complex. Scaffold plasmid has produced the maximum concentration of 3.51 g/L malic acid from 10 g/L glucose in 48 h of culture. pH 5.5 and temperature 30°C were optimum for malic acid production without any engineering of competing metabolic pathways. E. coli mutant strains and different concentrations of glucose also tested. When 50 g/L glucose was used as substrate, 20.4 g/L of malic acid was produced.

Self-Assembled Multienzyme Nanostructures on Synthetic Protein Scaffolds.

Sequential enzymes in a biosynthetic pathway often self-assemble to form nanomachineries known as multienzyme complexes inside cells. Enzyme self-assembly insulates toxic intermediates, increases the efficiency of intermediate transfer, minimizes metabolic crosstalk, streamlines flux, and improves the product yield. Artful structures and superior catalytic functions of these natural nanomachines inspired the development of synthetic multienzyme complexes to expedite biosynthesis. Here we present a versatile self-assembly strategy to construct multienzyme nanostructures based on synthetic protein scaffolds. The protein scaffolds were formed using the spontaneous protein reaction of SpyCatcher and SpyTag. Two types of protein scaffolds were generated: two skeleton proteins cross-linked and hierarchically assembled into heterogeneous nanostructures (the cross-linked scaffold), and head-to-tail cyclization of a dual-reactive skeleton protein gave a homogeneous cyclic scaffold. Sequential enzymes from the menaquinone biosynthetic pathway were assembled on both scaffolds through the docking domain interactions derived from polyketide synthases. Both scaffolded assemblies effectively increased the yield of the final product of the cascade catalytic reaction in menaquinone biosynthesis. Surprisingly, the rate enhancements were driven by different mechanisms: the cross-linked scaffold assembly streamlined the overall flow of the reactants, whereas the cyclic scaffold assembly accelerated the catalytic efficiency of the rate-limiting enzyme. Altogether, self-assembly of sequential enzymes by combining the SpyCatcher/SpyTag reaction and the docking domain interactions yielded protein-based nanostructures with special architecture, exceptional catalytic activity, and unexpected catalytic mechanisms. This work demonstrates a versatile strategy of gaining more powerful biocatalysts by protein self-assembly for efficient bioconversion of valuable chemicals.

Improving L-serine production in Escherichia coli via synthetic protein scaffold of SerB, SerC, and EamA

Although L-Serine has wide applications and great market potential, its application is limited by its high cost and low yields. Microbial factories have many advantages for producing L-serine from renewable carbon resources. To achieve L-serine production with high efficiency, three enzymes (SerB, SerC, and EamA) were physically re-localized by using a scaffold system GBD:SH3:PDZ. Such strategy was highly effective in improving the production of L-serine in Escherichia coli. The highest concentration of L-serine harvested was at 3.8 g/l after 48 h in strain E. coli with the scaffold GBD:SH3:PDZ at ratio of 1:2:1, reaching a high yield of 0.24 mol/mol glucose.

163 Lox-1-specific affimers block oxldl accumulation in vitro

IntroductionLectin-like oxidised low density lipoprotein receptor 1 (LOX-1, SREC-1, OLR1) is a class E scavenger which binds oxidised low density lipoproteins (oxLDL). It is expressed on endothelial cells, macrophages and...

Synthetic Protein Scaffolds for Biosynthetic Pathway Colocalization on Lipid Droplet Membranes.

Eukaryotic biochemistry is organized throughout the cell in and on membrane-bound organelles. When engineering metabolic pathways this organization is often lost, resulting in flux imbalance and a loss of kinetic advantages from enzyme colocalization and substrate channeling. Here, we develop a protein-based scaffold for colocalizing multienzyme pathways on the membranes of intracellular lipid droplets. Scaffolds based on the plant lipid droplet protein oleosin and cohesin-dockerin interaction pairs recruited upstream enzymes in yeast ester biosynthesis to the native localization of the terminal reaction step, alcohol-O-acetyltransferase (Atf1). The native localization is necessary for high activity and pathway assembly in close proximity to Atf1 increased pathway flux. Screening a library of scaffold variants further showed that pathway structure can alter catalysis and revealed an optimized scaffold and pathway expression levels that produced ethyl acetate at a rate nearly 2-fold greater than unstructured pathways. This strategy should prove useful in spatially organizing other metabolic pathways with key lipid droplet-localized and membrane-bound reaction steps.

A novel assay using synthetic protein scaffolds to detect soluble lectin-like Oxidised Low Density Lipoprotein Receptor-1 in vascular disease

Methods: The bacterium E.coli was used to produce recombinant Adhirons that recognised human sLOX-1. Different ELISA-based assays were analysed for the ability of Adhirons to bind and detect sLOX-1 in biological buffers and human plasma. Results: Hybrid antibody-sLOX-1-Adhiron and homogenous AdhironsLOX-1-Adhiron assays could detect sLOX-1 levels down to picogram per millilitre sensitivity. Analysis of sLOX-1 levels using conventional antibody or Adhiron-based assays suggests that (1) sLOX-1 levels are elevated in chronic disease states, (2) Adhirons have high affinity and specificity for sLOX-1, and (3) Adhiron-based assays are a viable alternative for monitoring sLOX-1 biomarker levels. Conclusions: This study demonstrates that Adhirons can act as probes for monitoring sLOX-1 levels in vascular disease states. Further refinement and optimisation is needed for monitoring sLOX-1 levels in human blood samples and relating this to patient disease status. These studies provide a platform for using sLOX-1 as a biomarker in acute and chronic vascular disease.

Synthetic Protein Scaffolds Based on Peptide Motifs and Cognate Adaptor Domains for Improving Metabolic Productivity.

The efficiency of many cellular processes relies on the defined interaction among different proteins within the same metabolic or signaling pathway. Consequently, a spatial colocalization of functionally interacting proteins has frequently emerged during evolution. This concept has been adapted within the synthetic biology community for the purpose of creating artificial scaffolds. A recent advancement of this concept is the use of peptide motifs and their cognate adaptor domains. SH2, SH3, GBD, and PDZ domains have been used most often in research studies to date. The approach has been successfully applied to the synthesis of a variety of target molecules including catechin, D-glucaric acid, H2, hydrochinone, resveratrol, butyrate, gamma-aminobutyric acid, and mevalonate. Increased production levels of up to 77-fold have been observed compared to non-scaffolded systems. A recent extension of this concept is the creation of a covalent linkage between peptide motifs and adaptor domains, which leads to a more stable association of the scaffolded systems and thus bears the potential to further enhance metabolic productivity.

Simple synthetic protein scaffolds can create adjustable artificial MAPK circuits in yeast and mammalian cells.

As hubs for eukaryotic cell signaling, scaffold proteins are attractive targets for engineering and manipulating signaling circuits. We designed synthetic scaffolds with a repeated PDZ domain that interacted with engineered kinases of the mitogen-activated protein kinase (MAPK) cascade involved in yeast mating to investigate how modular interactions mediate kinase cascades. The synthetic scaffolds functioned as logic gates of signaling circuits. We replaced the endogenous yeast scaffold Ste5 with designer scaffolds with a variable numbers of a PDZ domain that bound kinases or phosphatases engineered with a PDZ-binding motif. Although association with the membrane was necessary for pathway activity, surprisingly, mating responses occurred when the circuit contained a scaffold with only two PDZ domains, which could only bind two of the three kinases simultaneously. Additionally, the three tiers of the MAPK pathway exhibited decreasing positional plasticity from the top [MAPK kinase kinase (MAPKKK)] to the bottom (MAPK) tier such that binding of a MAPKKK, but not a MAPK, from the osmoregulatory pathway or protein kinase C pathway to the synthetic scaffold activated a reporter of the mating response. We also showed that the output duration and intensity could be altered by recruiting phosphatases or varying the affinity of the recruited proteins for the scaffold and that a designer MAPK scaffold functioned in mammalian cells. Thus, this synthetic approach with designer scaffolds should enable the rational manipulation or engineering of signaling pathways and provide insight into the functional roles of scaffold proteins.

Production of gamma-aminobutyric acid from glucose by introduction of synthetic scaffolds between isocitrate dehydrogenase, glutamate synthase and glutamate decarboxylase in recombinant Escherichia coli

Escherichia coli were engineered for the direct production of gamma-aminobutyric acid from glucose by introduction of synthetic protein scaffold. In this study, three enzymes consisting GABA pathway (isocitrate dehydrogenase, glutamate synthase and glutamate decarboxylase) were connected via synthetic protein scaffold. By introduction of scaffold, 0.92g/L of GABA was produced from 10g/L of glucose while no GABA was produced in wild type E. coli. The optimum pH and temperature for GABA production were 4.5 and 30°C, respectively. When competing metabolic network was inactivated by knockout mutation, maximum GABA concentration of 1.3g/L was obtained from 10g/L glucose. The recombinant E. coli strain which produces GABA directly from glucose was successfully constructed by introduction of protein scaffold.

Assembly of cellulases with synthetic protein scaffolds in vitro

Abstract Background Enzymatic cascades in metabolic pathways are spatially organized in such a way as to facilitate the flow of substrates. The construction of artificial cellulase complexes that mimic natural multienzyme assemblies can potentially enhance the capacity for cellulose hydrolysis. In this study, an artificial cellulase complex was constructed by tethering three cellulases to a synthetic protein scaffold. Results Three pairs of interacting proteins were selected and characterized. The artificial protein scaffolds were constructed by fusing three interacting proteins. Cellulases were tethered to these synthetic scaffolds in different orders. The optimal assembly resulted in a 1.5-fold higher hydrolysis of cellulose than that achieved by unassembled cellulases. Conclusions A novel artificial protein scaffold was constructed and used to assemble three cellulases. The resultant increase in enzymatic activity suggests that this can be used as a strategy for enhancing the biocatalytic capacity of enzyme cascades.