Unlocking nature’s glycosylation potential : characterization and engineering of novel sucrose/trehalose synthases

Glycosylation - the addition of a sugar molecule onto an acceptor substrate - is a promising strategy to improve the activity, solubility, stability, flavor and/or pharmacokinetic behavior of chemicals such as pharmaceuticals, neutraceuticals or cosmetics. Glycosylation can efficiently be performed in an aqueous environment under mild reaction conditions by enzymes called GlycosylTransferases (GTs). However, the industrial application of these enzymes in vitro is mainly hampered by their need for nucleotide-activated sugars (e.g. UDP-glucose) as donor substrates, which are highly expensive and rarely available in large quantities. In this doctoral thesis, two strategies to make in vitro reactions with GTs more cost-efficient were evaluated: the use of Sucrose Synthase (SuSy) as intermediate enzyme to produce UDP-glucose from the cheap substrate sucrose and the engineering of GTs to alter the sugar donor specificity towards cheaper glycosyl-phosphates. To this end, several new bacterial SuSy enzymes were cloned, expressed, purified and characterized. In addition, one of them was subjected to extensive mutagenesis to improve or change properties such as substrate affinity, substrate specificity and stability. The enzyme Trehalose glycosylTransferring synthase, on the other hand, was used as a test-case to scrutinize the possibility of changing the donor specificity of GTs from nucleotide sugars towards glycosyl-phosphates through mutagenesis

Sequence determinants of nucleotide binding in Sucrose Synthase : improving the affinity of a bacterial Sucrose Synthase for UDP by introducing plant residues

Sucrose Synthase (SuSy) catalyzes the reversible conversion of sucrose and a nucleoside diphosphate (NDP) into NDP-glucose and fructose. Biochemical characterization of several plant and bacterial SuSys has revealed that the eukaryotic enzymes preferentially use UDP whereas prokaryotic SuSys prefer ADP as acceptor. In this study, SuSy from the bacterium Acidithiobacillus caldus, which has a higher affinity for ADP as reflected by the 25-fold lower K-m value compared to UDP, was used as a test case to scrutinize the effect of introducing plant residues at positions in a putative nucleotide binding motif surrounding the nucleobase ring of NDP. All eight single to sextuple mutants had similar activities as the wild-type enzyme but significantly reduced K-m values for UDP (up to 60 times). In addition, we recognized that substrate inhibition by UDP is introduced by a methionine at position 637. The affinity for ADP also increased for all but one variant, although the improvement was much smaller compared to UDP. Further characterization of a double mutant also revealed more than 2-fold reduction in K-m values for CDP and GDP. This demonstrates the general impact of the motif on nucleotide binding. Furthermore, this research also led to the establishment of a bacterial SuSy variant that is suitable for the recycling of UDP during glycosylation reactions. The latter was successfully demonstrated by combining this variant with a glycosyltransferase in a one-pot reaction for the production of the C-glucoside nothofagin, a health-promoting flavonoid naturally found in rooibos (tea)

The phylogenetic landscape and nosocomial spread of the multidrug-resistant opportunist Stenotrophomonas maltophilia

Recent studies portend a rising global spread and adaptation of human- or healthcare-associated pathogens. Here, we analyse an international collection of the emerging, multidrug-resistant, opportunistic pathogen Stenotrophomonas maltophilia from 22 countries to infer population structure and clonality at a global level. We show that the S. maltophilia complex is divided into 23 monophyletic lineages, most of which harbour strains of all degrees of human virulence. Lineage Sm6 comprises the highest rate of human-associated strains, linked to key virulence and resistance genes. Transmission analysis identifies potential outbreak events of genetically closely related strains isolated within days or weeks in the same hospitals

Analysis of the global population structure of Paenibacillus larvae and outbreak investigation of American foulbrood using a stable wgMLST scheme

Paenibacillus larvae causes the American foulbrood (AFB), a highly contagious and devastating disease of honeybees. Whole-genome sequencing (WGS) has been increasingly used in bacterial pathogen typing, but rarely applied to study the epidemiology of P. larvae. To this end, we used 125 P. larvae genomes representative of a species-wide diversity to construct a stable whole-genome multilocus sequence typing (wgMLST) scheme consisting of 5745 loci. A total of 51 P. larvae isolates originating from AFB outbreaks in Slovenia were used to assess the epidemiological applicability of the developed wgMLST scheme. In addition, wgMLST was compared with the core-genome MLST (cgMLST) and whole-genome single nucleotide polymorphism (wgSNP) analyses. All three approaches successfully identified clusters of outbreak-associated strains, which were clearly separated from the epidemiologically unlinked isolates. High levels of backward comparability of WGS-based analyses with conventional typing methods (ERIC-PCR and MLST) were revealedhowever, both conventional methods lacked sufficient discriminatory power to separate the outbreak clusters. The developed wgMLST scheme provides an improved understanding of the intra- and inter-outbreak genetic diversity of P. larvae and represents an important progress in unraveling the genomic epidemiology of this important honeybee pathogen

Stability analysis of a plant glycosyltransferase

Background: Glycosylation can significantly improve physicochemical or biological properties of small molecules such as vitamins, fragrances and antibiotics. Glycosyltransferases (GTs) are carbohydrate-active enzymes that can efficiently catalyse such reactions. We recently discovered a promising biocatalyst (GT-1) for the glycosylation of a wide variety of compounds, but its thermostability will be crucial for industrial applications. Materials and Methods: GT-1 was recombinantly expressed and purified via Ni-NTA chromatography. Thermal half-life time (t50(50°C)) at 50 °C was assessed by sampling at fixed intervals until the residual activity had dropped to 50%, while the melting temperature (Tm) was determined via differential scanning fluorimetry. The bio-informatic tools YASARA and 3DM were used to select target positions for engineering. Results: GT-1 was succesfully expressed and yields up to 40 mg purified protein per liter culture were obtained. Further, t50(50°C) and Tm values of respectively 90 min and 56 °C were found. Discussion: The kinetic stability of GT-1 was rather low. Indeed, after 1 min of incubation at 50 °C, residual activity already dropped to 70%. Clearly, enzyme engineering strategies need to be developed. Therefore, a homology model was constructed and structural analysis revealed a strikingly large amount of open spaces. Filling these cavities with larger amino acids should increase hydrophobic packing, yielding a more stable enzyme. Conclusion: The kinetic and thermal stability of GT-1 was assessed. Engineering the enzyme’s stability by a combination of smart (sequence-based) and rational (structure-based) mutagenesis will be key to biocatalyst development

The quest for a thermostable sucrose phosphorylase reveals sucrose 6'-phosphate phosphorylase as a novel specificity

Sucrose phosphorylase is a promising biocatalyst for the glycosylation of a wide range of compounds, but its industrial application has been hampered by the low thermostability of known representatives. Hence, in this study, the putative sucrose phosphorylase from the thermophile Thermoanaerobacterium thermosaccharolyticum was recombinantly expressed and fully characterised. The enzyme showed significant activity on sucrose (optimum at 55 A degrees C), and with a melting temperature of 79 A degrees C and a half-life of 60 h at the industrially relevant temperature of 60 A degrees C, it is far more stable than known sucrose phosphorylases. Substrate screening and detailed kinetic characterisation revealed however a preference for sucrose 6'-phosphate over sucrose. The enzyme can thus be considered as a sucrose 6'-phosphate phosphorylase, a specificity not yet reported to date. Homology modelling and mutagenesis pointed out particular residues (Arg134 and His344) accounting for the difference in specificity. Moreover, phylogenetic and sequence analysis suggest that glycoside hydrolase 13 subfamily 18 might harbour even more specificities. In addition, the second gene residing in the same operon as sucrose 6'-phosphate phosphorylase was identified as well, and found to be a phosphofructokinase. The concerted action of both these enzymes implies a new pathway for the breakdown of sucrose, in which the reaction products end up at different stages of the glycolysis

Glycosyltransferase cascades for natural product glycosylation : use of plant instead of bacterial sucrose synthases improves the UDP-glucose recycling from sucrose and UDP

Natural product glycosylations by Leloir glycosyltransferases (GTs) require expensive nucleotide-activated sugars as substrates. Sucrose synthase (SuSy) converts sucrose and uridine 5'-diphosphate (UDP) into UDP-glucose. Coupling of SuSy and GT reactions in one-pot cascade transformations creates a UDP cycle, which regenerates the UDP-glucose continuously and so makes it an expedient donor for glucoside production. Here we compare SuSys with divergent kinetic characteristics for UDP-glucose recycling in the synthesis of the natural C-glucoside nothofagin. Development of a fast reversed-phase ion-pairing HPLC method, quantifying all relevant reactants from the coupled conversion in a single run, was key to dissect the main factors of recycling efficiency. Limitations due to high K-M, both for UDP and sucrose, were revealed for the bacterial SuSy from Acidithiobacillus caldus. The L637M-T640V double mutant of this SuSy with a 60-fold reduced KM for UDP substantially improved UDP-glucose recycling. The SuSy from Glycine max (soybean) was nevertheless the most active enzyme at the UDP ( 0.5 mM) and sucrose ( 1 M) concentrations used. It was also unexpectedly stable at up to 50 degrees C where spontaneous decomposition of UDP-glucose started to become problematic. The herein gained in-depth understanding of requirements for UDP-glucose regeneration supports development of efficient GT-SuSy cascades

Identification of sucrose synthase in nonphotosynthetic bacteria and characterization of the recombinant enzymes

Sucrose synthase (SuSy) catalyzes the reversible conversion of sucrose and a nucleoside diphosphate into fructose and nucleotide (NDP)-glucose. To date, only SuSy's from plants and cyanobacteria, both photosynthetic organisms, have been characterized. Here, four prokaryotic SuSy enzymes from the nonphotosynthetic organisms Nitrosomonas Europaea (SuSyNe), Acidithiobacillus caldus (SuSyAc), Denitrovibrio acetiphilus (SusyDa), and Melioribacter roseus (SuSyMr) were recombinantly expressed in Escherichia coli and thoroughly characterized. The purified enzymes were found to display high-temperature optima (up to 80 A degrees C), high activities (up to 125 U/mg), and high thermostability (up to 15 min at 60 A degrees C). Furthermore, SuSyAc, SuSyNe, and SuSyDa showed a clear preference for ADP as nucleotide, as opposed to plant SuSy's which prefer UDP. A structural and mutational analysis was performed to elucidate the difference in NDP preference between eukaryotic and prokaryotic SuSy's. Finally, the physiological relevance of this enzyme specificity is discussed in the context of metabolic pathways and genomic organization