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The present invention concerns glycosyltransferase enzyme mutants having improved half-lives and thermal stability compared to the parent enzyme UDP-glycosyltransferase (PtUGT) from the indigo producing plant Polygonum tinctorium/Persicaria tinctoria; and further provides a composition, kit, and methods employing these mutants for glycosylation of desired compounds, such as indoxyl compounds.

Family 1 glycosyltransferases (GT1s, UGTs) form natural product glycosides with exquisite control over regio- and stereoselectivity, representing attractive biotechnological targets. However, regioselectivity cannot be predicted and large-scale activity assessment efforts of UGTs are commonly performed via mass spectrometry or indirect assays that are blind to regioselectivity. Here, we present a large high performance liquid chromatography screening discriminating between regioisomeric products of 40 diverse UGTs (28.6% average pairwise sequence identity) against 32 polyphenols, identifying enzymes able to reach high glycosylation yields (≥90% in 24 h) in 26/32 cases. In reactions with >50% yield, we observed perfect regioselectivity for 47% (75/158) on polyphenols presenting two hydroxyl groups and for 30% (43/143) on polyphenols presenting ≥3 hydroxyl groups. Moreover, we developed a nuclear magnetic resonance-based procedure to identify the site of glycosylation directly on enzymatic mixtures. We further selected seven regiospecific reactions catalyzed by four enzymes on five dihydroxycoumarins. We characterized the four enzymes, showing that temperature optima are functions of the acceptor substrate, varying by up to 20 °C for the same enzyme. Furthermore, we performed short molecular dynamics simulations of 311 ternary complexes (UGT, UDP-Glc, and glycosyl acceptor) to investigate the molecular basis for regioselectivity. Interestingly, it appeared that most UGTs can accommodate acceptors in configurations favorable to the glycosylation of either hydroxyl. In contrast, evaluation of hydroxyl nucleophilicity appeared to be a strong predictor of the hydroxyl predominantly glycosylated by most enzymes.

The world is currently facing a climate crisis facilitated by greenhouse gas emissions. The chemical industry is a major contributor to this issue, as conventional chemical productions are energy-intensive and mostly rely on petrochemical-based feedstocks. Microbial production provides a promising sustainable alternative, as it requires less energy and can utilise renewable feedstocks. However, the most prominent issue with microbial approaches lies with the economic viability of biochemicals. In the case of bulk chemicals, which have lower profit margins, most large-scale bio-based productions struggle to compete with the higher yields and lower production costs of their fossil fuel-based counterparts. If sustainable bio-based productions are to make an impact on the global market, it is vital to improve their feasibility and competitiveness in large-scale industrial settings. Thermophilic fermentation can provide advantages to the development of the biochemical market, as high-temperature productions offer several benefits. This includes significantly reduced process costs, lower contamination risks, and easier extraction of volatile compounds. Amongst thermophilic species, Parageobacillus thermoglucosidasius is a promising candidate for bulk-chemical production. Here, we present the development of a strain of P. thermoglucosidasius that is optimised for the conversion of acetic acid into acetone. This is accomplished through metabolic engineering, omics-based analysis, and the application of adaptive evolution-based strategies. The aim is to optimise growth, acetate tolerance, and acetone production in P. thermoglucosidasius to facilitate the development of large-scale sustainable acetone production, and to empower the development of other chemical production strategies that intend to take advantage of the thermophilic aspects of this species.

The fluorinase enzyme represents the only biological mechanism capable of forming stable C–F bonds characterized in nature thus far, offering a biotechnological route to the biosynthesis of value-added organofluorines. The fluorinase is known to operate in a hexameric form, but the consequence(s) of the oligomerization status on the enzyme activity and its catalytic properties remain largely unknown. In this work, this aspect was explored by rationally engineering trimeric fluorinase variants that retained the same catalytic rate as the wild-type enzyme. These results ruled out hexamerization as a requisite for the fluorination activity. The Michaelis constant (KM) for S-adenosyl-l-methionine, one of the substrates of the fluorinase, increased by two orders of magnitude upon hexamer disruption. Such a shift in S-adenosyl-l-methionine affinity points to a long-range effect of hexamerization on substrate binding – likely decreasing substrate dissociation and release from the active site. A practical application of trimeric fluorinase is illustrated by establishing in vitro fluorometabolite synthesis in a bacterial cell-free system.

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.

Glycosylation reactions are essential but challenging from a conventional chemistry standpoint. Conversely, they are biotechnologically feasible as glycosyltransferases can transfer sugar to an acceptor with perfect regio- and stereo-selectivity, quantitative yields, in a single reaction and under mild conditions. Low stability is often alleged to be a limitation to the biotechnological application of glycosyltransferases. Here we show that these enzymes are not necessarily intrinsically unstable, but that they present both dilution-induced inactivation and low chemostability towards their own acceptor substrates, and that these two phenomena are synergistic. We assessed 18 distinct GT1 enzymes against three unrelated acceptors (apigenin, resveratrol, and scopoletin—respectively a flavone, a stilbene, and a coumarin), resulting in a total of 54 enzymes: substrate pairs. For each pair, we varied catalyst and acceptor concentrations to obtain 16 different reaction conditions. Fifteen of the assayed enzymes (83%) displayed both low chemostability against at least one of the assayed acceptors at submillimolar concentrations, and dilution-induced inactivation. Furthermore, sensitivity to reaction conditions seems to be related to the thermal stability of the enzymes, the three unaffected enzymes having melting temperatures above 55°C, whereas the full enzyme panel ranged from 37.4 to 61.7°C. These results are important for GT1 understanding and engineering, as well as for discovery efforts and biotechnological use.

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 β-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.

The eobiotic compound indican lends itself to a compelling biocatalytic dyeing strategy for denim, in which the formation of corrosive byproducts is avoided. However, the efficient and scalable production of indican remains a key bottleneck. This work focuses on the in vitro characterization of PtUGT1, a glycosyltransferase from Polygonum tinctorium that catalyzes the formation of indican via the glycosylation of indoxyl. Here, the buffer composition and enzyme concentration were identified as key parameters for enzyme activity and stability. The short lifetime of the enzyme under reaction conditions initiated an immobilization study. As a consequence, an amino-functionalized methacrylate resin was identified as a highly functional option for efficient immobilization of PtUGT1, allowing immobilization yields of >98% for enzyme loadings up to 7.6 wt %. We further report a stabilization factor of 47 and significantly improved overall biocatalytic productivity. The straightforward handling and reuse of the described heterogeneous biocatalyst is demonstrated.

Glycosyltransferases (GTs) catalyze the formation of glycosidic bonds in carbohydrates and glycoconjugates, with various outcomes depending not only on the acceptor molecules they bind but also on the type of glycosidic bond they form (C−O, C−N, C−S, or C−C). Here we show that the glucosyltransferase UGT1 from the indigo plant Polygonum tinctorium catalyzes either N-, O-, or S-glycosylation with similar rates. We solve the structure of the enzyme in complex with its donor and acceptor substrates and elucidate the molecular basis of N-, O-, and S-specificities using experimental mutagenesis and QM/MM simulations, revealing distinct mechanisms for N-, O-, and S-glycosylation. We also show that the active site can be engineered to increase or favor one of the three glycosylation activities over another. These results will foster the design of more active and specific enzyme variants for production of glycosides.