Promiscuous Yet Specific: A Methionine-Aromatic Interaction Drives the Reaction Scope of the Family 1 Glycosyltransferase GmUGT88E3 from Soybean.

Family 1 glycosyltransferases (GT1s, UGTs) catalyze the regioselective glycosylation of natural products in a single step. We identified GmUGT88E3 as a particularly promising biocatalyst able to produce a variety of pure, single glycosidic products from polyphenols with high chemical yields. We investigated this particularly desirable duality toward specificity, i.e., promiscuous toward acceptors while regiospecific. Using high-field NMR, kinetic characterization, molecular dynamics simulations, and mutagenesis studies, we uncovered that the main molecular determinant of GmUGT88E3 specificity is a methionine-aromatic bridge, an interaction often present in protein structures but never reported for enzyme-substrate interactions. Here, mutating Met127 led to inactive proteins or 100-fold reduced activity.

Structure-guided engineering of key amino acids in UGT85B1 controlling substrate and stereo-specificity in aromatic cyanogenic glucoside biosynthesis.

Cyanogenic glucosides are important defense molecules in plants with useful biological activities in animals. Their last biosynthetic step consists of a glycosylation reaction that confers stability and increases structural diversity and is catalyzed by the UDP-dependent glycosyltransferases (UGTs) of glycosyltransferase family 1. These versatile enzymes have large and varied substrate scopes, and the structure-function relationships controlling scope and specificity remain poorly understood. Here, we report substrate-bound crystal structures and rational engineering of substrate and stereo-specificities of UGT85B1 from Sorghum bicolor involved in biosynthesis of the cyanogenic glucoside dhurrin. Substrate specificity was shifted from the natural substrate (S)-p-hydroxymandelonitrile to (S)-mandelonitrile by combining a mutation to abolish hydrogen bonding to the p-hydroxyl group with a mutation to provide steric hindrance at the p-hydroxyl group binding site (V132A/Q225W). Further, stereo-specificity was shifted from (S) to (R) by substituting four rationally chosen residues within 6 Å of the nitrile group (M312T/A313T/H408F/G409A). These activities were compared to two other UGTs involved in the biosynthesis of aromatic cyanogenic glucosides in Prunus dulcis (almond) and Eucalyptus cladocalyx. Together, these studies enabled us to pinpoint factors that drive substrate and stereo-specificities in the cyanogenic glucoside biosynthetic UGTs. The structure-guided engineering of the functional properties of UGT85B1 enhances our understanding of the evolution of UGTs involved in the biosynthesis of cyanogenic glucosides and will enable future engineering efforts towards new biotechnological applications.

Evolution-guided engineering of small-molecule biosensors.

Allosteric transcription factors (aTFs) have proven widely applicable for biotechnology and synthetic biology as ligand-specific biosensors enabling real-time monitoring, selection and regulation of cellular metabolism. However, both the biosensor specificity and the correlation between ligand concentration and biosensor output signal, also known as the transfer function, often needs to be optimized before meeting application needs. Here, we present a versatile and high-throughput method to evolve prokaryotic aTF specificity and transfer functions in a eukaryote chassis, namely baker's yeast Saccharomyces cerevisiae. From a single round of mutagenesis of the effector-binding domain (EBD) coupled with various toggled selection regimes, we robustly select aTF variants of the cis,cis-muconic acid-inducible transcription factor BenM evolved for change in ligand specificity, increased dynamic output range, shifts in operational range, and a complete inversion-of-function from activation to repression. Importantly, by targeting only the EBD, the evolved biosensors display DNA-binding affinities similar to BenM, and are functional when ported back into a prokaryotic chassis. The developed platform technology thus leverages aTF evolvability for the development of new host-agnostic biosensors with user-defined small-molecule specificities and transfer functions.

Natural product C-glycosyltransferases - a scarcely characterised enzymatic activity with biotechnological potential.

Covering: up to 2020C-Glycosyltransferases are enzymes that catalyse the transfer of sugar molecules to carbon atoms in substituted aromatic rings of a variety of natural products. The resulting ß-C-glycosidic bond is more stable in vivo than most O-glycosidic bonds, hence offering an attractive modulation of a variety of compounds with multiple biological activities. While C-glycosylated natural products have been known for centuries, our knowledge of corresponding C-glycosyltransferases is scarce. Here, we discuss commonalities and differences in the known C-glycosyltransferases, review attempts to leverage them as synthetic biocatalysts, and discuss current challenges and limitations in their research and application.

Structural and functional aspects of mannuronic acid-specific PL6 alginate lyase from the human gut microbe Bacteroides cellulosilyticus.

Alginate is a linear polysaccharide from brown algae consisting of 1,4-linked ß-d-mannuronic acid (M) and α-l-guluronic acid (G) arranged in M, G, and mixed MG blocks. Alginate was assumed to be indigestible in humans, but bacteria isolated from fecal samples can utilize alginate. Moreover, genomes of some human gut microbiome-associated bacteria encode putative alginate-degrading enzymes. Here, we genome-mined a polysaccharide lyase family 6 alginate lyase from the gut bacterium Bacteroides cellulosilyticus (BcelPL6). The structure of recombinant BcelPL6 was solved by X-ray crystallography to 1.3 Å resolution, revealing a single-domain, monomeric parallel ß-helix containing a 10-step asparagine ladder characteristic of alginate-converting parallel ß-helix enzymes. Substitutions of the conserved catalytic site residues Lys-249, Arg-270, and His-271 resulted in activity loss. However, imidazole restored the activity of BcelPL6-H271N to 2.5% that of the native enzyme. Molecular docking oriented tetra-mannuronic acid for syn attack correlated with M specificity. Using biochemical analyses, we found that BcelPL6 initially releases unsaturated oligosaccharides of a degree of polymerization of 2-7 from alginate and polyM, which were further degraded to di- and trisaccharides. Unlike other PL6 members, BcelPL6 had low activity on polyMG and none on polyG. Surprisingly, polyG increased BcelPL6 activity on alginate 7-fold. LC-electrospray ionization-MS quantification of products and lack of activity on NaBH4-reduced octa-mannuronic acid indicated that BcelPL6 is an endolyase that further degrades the oligosaccharide products with an intact reducing end. We anticipate that our results advance predictions of the specificity and mode of action of PL6 enzymes.

A carbohydrate-binding family 48 module enables feruloyl esterase action on polymeric arabinoxylan.

Feruloyl esterases (EC 3.1.1.73), belonging to carbohydrate esterase family 1 (CE1), hydrolyze ester bonds between ferulic acid (FA) and arabinose moieties in arabinoxylans. Recently, some CE1 enzymes identified in metagenomics studies have been predicted to contain a family 48 carbohydrate-binding module (CBM48), a CBM family associated with starch binding. Two of these CE1s, wastewater treatment sludge (wts) Fae1A and wtsFae1B isolated from wastewater treatment surplus sludge, have a cognate CBM48 domain and are feruloyl esterases, and wtsFae1A binds arabinoxylan. Here, we show that wtsFae1B also binds to arabinoxylan and that neither binds starch. Surface plasmon resonance analysis revealed that wtsFae1B's Kd for xylohexaose is 14.8 µm and that it does not bind to starch mimics, ß-cyclodextrin, or maltohexaose. Interestingly, in the absence of CBM48 domains, the CE1 regions from wtsFae1A and wtsFae1B did not bind arabinoxylan and were also unable to catalyze FA release from arabinoxylan. Pretreatment with a ß-d-1,4-xylanase did enable CE1 domain-mediated FA release from arabinoxylan in the absence of CBM48, indicating that CBM48 is essential for the CE1 activity on the polysaccharide. Crystal structures of wtsFae1A (at 1.63 Å resolution) and wtsFae1B (1.98 Å) revealed that both are folded proteins comprising structurally-conserved hydrogen bonds that lock the CBM48 position relative to that of the CE1 domain. wtsFae1A docking indicated that both enzymes accommodate the arabinoxylan backbone in a cleft at the CE1-CBM48 domain interface. Binding at this cleft appears to enable CE1 activities on polymeric arabinoxylan, illustrating an unexpected and crucial role of CBM48 domains for accommodating arabinoxylan.

Employing a biochemical protecting group for a sustainable indigo dyeing strategy.

Indigo is an ancient dye uniquely capable of producing the signature tones in blue denim; however, the dyeing process requires chemical steps that are environmentally damaging. We describe a sustainable dyeing strategy that not only circumvents the use of toxic reagents for indigo chemical synthesis but also removes the need for a reducing agent for dye solubilization. This strategy utilizes a glucose moiety as a biochemical protecting group to stabilize the reactive indigo precursor indoxyl to form indican, preventing spontaneous oxidation to crystalline indigo during microbial fermentation. Application of a ß-glucosidase removes the protecting group from indican, resulting in indigo crystal formation in the cotton fibers. We identified the gene coding for the glucosyltransferase PtUGT1 from the indigo plant Polygonum tinctorium and solved the structure of PtUGT1. Heterologous expression of PtUGT1 in Escherichia coli supported high indican conversion, and biosynthesized indican was used to dye cotton swatches and a garment.

Molecular characteristics of plant UDP-arabinopyranose mutases.

l-arabinofuranose is a ubiquitous component of the cell wall and various natural products in plants, where it is synthesized from cytosolic UDP-arabinopyranose (UDP-Arap). The biosynthetic machinery long remained enigmatic in terms of responsible enzymes and subcellular localization. With the discovery of UDP-Arap mutase in plant cytosol, the demonstration of its role in cell-wall arabinose incorporation and the identification of UDP-arabinofuranose transporters in the Golgi membrane, it is clear that the cytosolic UDP-Arap mutases are the key enzymes converting UDP-Arap to UDP-arabinofuranose for cell wall and natural product biosynthesis. This has recently been confirmed by several genotype/phenotype studies. In contrast to the solid evidence pertaining to UDP-Arap mutase function in vivo, the molecular features, including enzymatic mechanism and oligomeric state, remain unknown. However, these enzymes belong to the small family of proteins originally identified as reversibly glycosylated polypeptides (RGPs), which has been studied for >20 years. Here, we review the UDP-Arap mutase and RGP literature together, to summarize and systemize reported molecular characteristics and relations to other proteins.

A Novel Fic (Filamentation Induced by cAMP) Protein from Clostridium difficile Reveals an Inhibitory Motif-independent Adenylylation/AMPylation Mechanism.

Filamentation induced by cAMP (Fic) domain proteins have been shown to catalyze the transfer of the AMP moiety from ATP onto a protein target. This type of post-translational modification was recently shown to play a crucial role in pathogenicity mediated by two bacterial virulence factors. Herein we characterize a novel Fic domain protein that we identified from the human pathogen Clostridium difficile The crystal structure shows that the protein adopts a classical all-helical Fic fold, which belongs to class II of Fic domain proteins characterized by an intrinsic N-terminal autoinhibitory α-helix. A conserved glutamate residue in the inhibitory helix motif was previously shown in other Fic domain proteins to prevent proper binding of the ATP γ-phosphate. However, here we demonstrate that both ATP binding and autoadenylylation activity of the C. difficile Fic domain protein are independent of the inhibitory motif. In support of this, the crystal structure of a mutant of this Fic protein in complex with ATP reveals that the γ-phosphate adopts a conformation unique among Fic domains that seems to override the effect of the inhibitory helix. These results provide important structural insight into the adenylylation reaction mechanism catalyzed by Fic domains. Our findings reveal the presence of a class II Fic domain protein in the human pathogen C. difficile that is not regulated by autoinhibition and challenge the current dogma that all class I-III Fic domain proteins are inhibited by the inhibitory α-helix.

The sugar donor specificity of plant family 1 glycosyltransferases.

Plant family 1 glycosyltransferases (UGTs) represent a formidable tool to produce valuable natural and novel glycosides. Their regio- and stereo-specific one-step glycosylation mechanism along with their inherent wide acceptor scope are desirable traits in biotechnology. However, their donor scope and specificity are not well understood. Since different sugars have different properties in vivo and in vitro, the ability to easily glycodiversify target acceptors is desired, and this depends on our improved understanding of the donor binding site. In the aim to unlock the full potential of UGTs, studies have attempted to elucidate the structure-function relationship governing their donor specificity. These efforts have revealed a complex phenomenon, and general principles valid for multiple enzymes are elusive. Here, we review the studies of UGT donor specificity, and attempt to group the information into key concepts which can help shape future research. We zoom in on the family-defining PSPG motif, on two loop residues reported to interact with the C6 position of the sugar, and on the role of active site arginines in donor specificity. We continue to discuss attempts to alter and expand the donor specificity by enzyme engineering, and finally discuss future research directions.

A life cycle assessment of early-stage enzyme manufacturing simulations from sustainable feedstocks.

Enzyme-catalyzed reactions have relatively small environmental footprints. However, enzyme manufacturing significantly impacts the environment through dependence on traditional feedstocks. With the objective of determining the environmental impacts of enzyme production, the sustainability potential of six cradle-to-gate enzyme manufacturing systems focusing on glucose, sea lettuce, acetate, straw, and phototrophic growth, was thoroughly evaluated. Human and ecosystem toxicity categories dominated the overall impacts. Sea lettuce, straw, or phototrophic growth reduces fermentation-based emissions by 51.0, 63.7, and 79.7%, respectively. Substituting glucose-rich media demonstrated great potential to reduce marine eutrophication, land use, and ozone depletion. Replacing organic nitrogen sources with inorganic ones could further lower these impacts. Location-specific differences in electricity result in a 14% and a 27% reduction in the carbon footprint for operation in Denmark compared to the US and China. Low-impact feedstocks can be competitive if they manage to achieve substrate utilization rates and productivity levels of conventional enzyme production processes.

A growth selection system for sucrose synthases (SuSy): design and test.

High throughput screening (HTS) methods of enzyme variants are essential for the development of robust biocatalysts suited for low impact, industrial scale, biobased synthesis of a myriad of compounds. However, for the majority of enzyme classes, current screening methods have limited throughput, or need expensive substrates in combination with sophisticated setups. Here, we present a straightforward, high throughput selection system that couples sucrose synthase activity to growth. Enabling high throughput screening of this enzyme class holds the potential to facilitate the creation of robust variants, which in turn can significantly impact the future of cost effective industrial glycosylation.

Glucuronan lyases from family PL7 use a Tyr/Tyr syn ß-elimination catalytic mechanism for glucuronan breakdown.

TpPL7A and TpPL7B, members of CAZy family PL7, act as ß-glucuronan lyases. TpPL7A diverges by lacking the catalytic histidine, identified as the Brønsted base in PL7 alginate lyases. Our research, including TpPL7A's crystal structure, and mutagenesis studies, reveals a shared syn-ß-elimination mechanism with a single tyrosine serving as both base and acid catalyst. This mechanism may extend to subfamily PL7_4 glucuronan lyases.

Chemoenzymatic indican for light-driven denim dyeing.

Blue denim, a billion-dollar industry, is currently dyed with indigo in an unsustainable process requiring harsh reducing and alkaline chemicals. Forming indigo directly in the yarn through indican (indoxyl-ß-glucoside) is a promising alternative route with mild conditions. Indican eliminates the requirement for reducing agent while still ending as indigo, the only known molecule yielding the unique hue of blue denim. However, a bulk source of indican is missing. Here, we employ enzyme and process engineering guided by techno-economic analyses to develop an economically viable drop-in indican synthesis technology. Rational engineering of PtUGT1, a glycosyltransferase from the indigo plant, alleviated the severe substrate inactivation observed with the wildtype enzyme at the titers needed for bulk production. We further describe a mild, light-driven dyeing process. Finally, we conduct techno-economic, social sustainability, and comparative life-cycle assessments. These indicate that the presented technologies have the potential to significantly reduce environmental impacts from blue denim dyeing with only a modest cost increase.

GASP: A Pan-Specific Predictor of Family 1 Glycosyltransferase Acceptor Specificity Enabled by a Pipeline for Substrate Feature Generation and Large-Scale Experimental Screening.

Glycosylation represents a major chemical challenge; while it is one of the most common reactions in Nature, conventional chemistry struggles with stereochemistry, regioselectivity, and solubility issues. In contrast, family 1 glycosyltransferase (GT1) enzymes can glycosylate virtually any given nucleophilic group with perfect control over stereochemistry and regioselectivity. However, the appropriate catalyst for a given reaction needs to be identified among the tens of thousands of available sequences. Here, we present the glycosyltransferase acceptor specificity predictor (GASP) model, a data-driven approach to the identification of reactive GT1:acceptor pairs. We trained a random forest-based acceptor predictor on literature data and validated it on independent in-house generated data on 1001 GT1:acceptor pairs, obtaining an AUROC of 0.79 and a balanced accuracy of 72%. The performance was stable even in the case of completely new GT1s and acceptors not present in the training data set, highlighting the pan-specificity of GASP. Moreover, the model is capable of parsing all known GT1 sequences, as well as all chemicals, the latter through a pipeline for the generation of 153 chemical features for a given molecule taking the CID or SMILES as input (freely available at https://github.com/degnbol/GASP). To investigate the power of GASP, the model prediction probability scores were compared to GT1 substrate conversion yields from a newly published data set, with the top 50% of GASP predictions corresponding to reactions with >50% synthetic yields. The model was also tested in two comparative case studies: glycosylation of the antihelminth drug niclosamide and the plant defensive compound DIBOA. In the first study, the model achieved an 83% hit rate, outperforming a hit rate of 53% from a random selection assay. In the second case study, the hit rate of GASP was 50%, and while being lower than the hit rate of 83% using expert-selected enzymes, it provides a reasonable performance for the cases when an expert opinion is unavailable. The hierarchal importance of the generated chemical features was investigated by negative feature selection, revealing properties related to cyclization and atom hybridization status to be the most important characteristics for accurate prediction. Our study provides a GT1:acceptor predictor which can be trained on other data sets enabled by the automated feature generation pipelines. We also release the new in-house generated data set used for testing of GASP to facilitate the future development of GT1 activity predictors and their robust benchmarking.

DNA binding by the plant-specific NAC transcription factors in crystal and solution: a firm link to WRKY and GCM transcription factors.

NAC (NAM/ATAF/CUC) plant transcription factors regulate essential processes in development, stress responses and nutrient distribution in important crop and model plants (rice, Populus, Arabidopsis), which makes them highly relevant in the context of crop optimization and bioenergy production. The structure of the DNA-binding NAC domain of ANAC019 has previously been determined by X-ray crystallography, revealing a dimeric and predominantly ß-fold structure, but the mode of binding to cognate DNA has remained elusive. In the present study, information from low resolution X-ray structures and small angle X-ray scattering on complexes with oligonucleotides, mutagenesis and (DNase I and uranyl photo-) footprinting, is combined to form a structural view of DNA-binding, and for the first time provide experimental evidence for the speculated relationship between plant-specific NAC proteins, WRKY transcription factors and the mammalian GCM (Glial cell missing) transcription factors, which all use a ß-strand motif for DNA-binding. The structure shows that the NAC domain inserts the edge of its core ß-sheet into the major groove, while leaving the DNA largely undistorted. The structure of the NAC-DNA complex and a new crystal form of the unbound NAC also indicate limited flexibility of the NAC dimer arrangement, which could be important in recognizing suboptimal binding sites.

Functional characterization of the phosphotransferase system in Parageobacillus thermoglucosidasius.

Parageobacillus thermoglucosidasius is a thermophilic bacterium characterized by rapid growth, low nutrient requirements, and amenability to genetic manipulation. These characteristics along with its ability to ferment a broad range of carbohydrates make P. thermoglucosidasius a potential workhorse in whole-cell biocatalysis. The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) catalyzes the transport and phosphorylation of carbohydrates and sugar derivatives in bacteria, making it important for their physiological characterization. In this study, the role of PTS elements on the catabolism of PTS and non-PTS substrates was investigated for P. thermoglucosidasius DSM 2542. Knockout of the common enzyme I, part of all PTSs, showed that arbutin, cellobiose, fructose, glucose, glycerol, mannitol, mannose, N-acetylglucosamine, N-acetylmuramic acid, sorbitol, salicin, sucrose, and trehalose were PTS-dependent on translocation and coupled to phosphorylation. The role of each putative PTS was investigated and six PTS-deletion variants could not grow on arbutin, mannitol, N-acetylglucosamine, sorbitol, and trehalose as the main carbon source, or showed diminished growth on N-acetylmuramic acid. We concluded that PTS is a pivotal factor in the sugar metabolism of P. thermoglucosidasius and established six PTS variants important for the translocation of specific carbohydrates. This study lays the groundwork for engineering efforts with P. thermoglucosidasius towards efficient utilization of diverse carbon substrates for whole-cell biocatalysis.

The function of UDP-glycosyltransferases in plants and their possible use in crop protection.

Glycosyltransferases catalyse the transfer of a glycosyl moiety from a donor to an acceptor. Members of this enzyme class are ubiquitous throughout all kingdoms of life and are involved in the biosynthesis of countless types of glycosides. Family 1 glycosyltransferases, also referred to as uridine diphosphate-dependent glycosyltransferases (UGTs), glycosylate small molecules such as secondary metabolites and xenobiotics. In plants, UGTs are recognised for their multiple functionalities ranging from roles in growth regulation and development, in protection against pathogens and abiotic stresses and in adaptation to changing environments. In this study, we review UGT-mediated glycosylation of phytohormones, endogenous secondary metabolites, and xenobiotics and contextualise the role this chemical modification plays in the response to biotic and abiotic stresses and plant fitness. Here, the potential advantages and drawbacks of altering the expression patterns of specific UGTs along with the heterologous expression of UGTs across plant species to improve stress tolerance in plants are discussed. We conclude that UGT-based genetic modification of plants could potentially enhance agricultural efficiency and take part in controlling the biological activity of xenobiotics in bioremediation strategies. However, more knowledge of the intricate interplay between UGTs in plants is needed to unlock the full potential of UGTs in crop resistance.

Sequence mining yields 18 phloretin C-glycosyltransferases from plants for the efficient biocatalytic synthesis of nothofagin and phloretin-di-C-glycoside.

C-glycosyltransferases (C-GTs) offer selective and efficient synthesis of natural product C-glycosides under mild reaction conditions. In contrast, the chemical synthesis of these C-glycosides is challenging and environmentally harmful. The rare occurrence of C-glycosylated compounds in Nature, despite their stability, suggests that their biosynthetic enzymes, C-GTs, might be scarce. Indeed, the number of characterized C-GTs is remarkably lower than O-GTs. Therefore, discovery efforts are crucial for expanding our knowledge of these enzymes and their efficient application in biocatalytic processes. This study aimed to identify new C-GTs based on their primary sequence. 18 new C-GTs were discovered, 10 of which yielded full conversion of phloretin to its glucosides. Phloretin is a dihydrochalcone natural product, with its mono-C-glucoside, nothofagin, having various health-promoting effects. Several of these enzymes enabled highly selective production of either nothofagin (UGT708A60 and UGT708F2) or phloretin-di-C-glycoside (UGT708D9 and UGT708B8). Molecular docking simulations, based on structural models of selected enzymes, showed productive binding modes for the best phloretin C-GTs, UGT708F2 and UGT708A60. Moreover, we characterized UGT708A60 as a highly efficient phloretin mono-C glycosyltransferase (kcat  = 2.97 s-1 , KM  = 0.1 µM) active in non-buffered, dilute sodium hydroxide (0.1-1 mM). We further investigated UGT708A60 as an efficient biocatalyst for the bioproduction of nothofagin.

An improved integrative GFP-based vector for genetic engineering of Parageobacillus thermoglucosidasius facilitates the identification of a key sporulation regulator.

Parageobacillus thermoglucosidasius is a thermophilic Gram-positive bacterium, which is a promising host organism for sustainable bio-based production processes. However, to take full advantage of the potential of P. thermoglucosidasius, more efficient tools for genetic engineering are required. The present study describes an improved shuttle vector, which speeds up recombination-based genomic modification by incorporating a thermostable sfGFP variant into the vector backbone. This additional selection marker allows for easier identification of recombinants, thereby removing the need for several culturing steps. The novel GFP-based shuttle is therefore capable of facilitating faster metabolic engineering of P. thermoglucosidasius through genomic deletion, integration, or exchange. To demonstrate the efficiency of the new system, the GFP-based vector was utilised for deletion of the spo0A gene in P. thermoglucosidasius DSM2542. This gene is known to be a key regulator of sporulation in Bacillus subtilis, and it was therefore hypothesised that the deletion of spo0A in P. thermoglucosiadius would produce an analogous sporulation-inhibited phenotype. Subsequent analyses of cell morphology and culture heat resistance suggests that the P. thermoglucosidasius ∆spo0A strain is sporulation-deficient. This strain may be an excellent starting point for future cell factory engineering of P. thermoglucosidasius, as the formation of endospores is normally not a desired trait in large-scale production.