In 1980, A. E. Walsby described a square halophilic archaeon. This archaeon is of specific interest because of its unique shape and its abundance in hypersaline ecosystems, which suggests an important ecophysiological role. Ever since its discovery, the isolation and cultivation of 'Walsby's square archaeon' has been a holy grail for many microbiologists working on halophiles. Despite their abundance and easy recognition by microscopy, all cultivation attempts have failed up to now, marking the organism as one of the unculturables. Cultivation of the square archaeon is essential to understand their ecophysiological role and the nature of their unique morphologically features. Here, we report the isolation and cultivation of the enigmatic square archaeon that we propose to name Haloquadratum walsbyi. Pure cultures are easily maintained in simple artificial hypersaline media. Initial growth experiments revealed a tolerance to high concentrations of MgCl(2) (>2 M) in the presence of 3.3 M NaCl. Fresh cultures contained extraordinary large cells (>40 x 40 microm) without any visible division structures, ranking them among the largest prokaryotes known to date. The genome was estimated to contain approximately three million basepairs.

This paper reviews current knowledge on actinomycete integrative and conjugative elements (AICEs). The best characterised AICEs, pSAM2 of Streptomyces ambofaciens (10.9 kb), SLP1 (17.3 kb) of Streptomyces coelicolor and pMEA300 of Amycolatopsis methanolica (13.3 kb), are present as integrative elements in specific tRNA genes, and are capable of conjugative transfer. These AICEs have a highly conserved structural organisation, with functional modules for excision/integration, replication, conjugative transfer, and regulation. Recently, it has been shown that pMEA300 and the related elements pMEA100 of Amycolatopsis mediterranei and pSE211 of Saccharopolyspora erythraea form a novel group of AICEs, the pMEA-elements, based on the unique characteristics of their replication initiator protein RepAM. Evaluation of a large collection of Amycolatopsis isolates has allowed identification of multiple pMEA-like elements. Our data show that, as AICEs, they mainly coevolved with their natural host in an integrated form, rather than being dispersed via horizontal gene transfer. The pMEA-like elements could be separated into two distinct populations from different geographical origins. One group was most closely related to pMEA300 and was found in isolates from Australia and Asia and pMEA100-related sequences were present in European isolates. Genome sequence data have enormously contributed to the recent insight that AICEs are present in many actinomycete genera. The sequence data also provide more insight into their evolutionary relationships, revealing their modular composition and their likely combined descent from bacterial plasmids and bacteriophages. Evidence is accumulating that AICEs act as modulators of host genome diversity and are also involved in the acquisition of secondary metabolite clusters and foreign DNA via horizontal gene transfer. Although still speculative, these AICEs may play a role in the spread of antibiotic resistance factors into pathogenic bacteria. The novel insights on AICE characteristics presented in this review may be used for the effective construction of new vectors that allows us to engineer and optimise strains for the production of commercially and medically interesting secondary metabolites, and bioactive proteins.

Human milk is considered the golden standard in infant nutrition. Free oligosaccharides in human milk provide important health benefits. These oligosaccharides function as prebiotics, immune modulators, and pathogen inhibitors and were found to improve barrier function in the gut. Infant formulas nowadays often contain prebiotics but lack the specific functions of human milk oligosaccharides (hMOS). Milk from domesticated animals also contains milk oligosaccharides but at much lower levels and with less diversity. Goat milk contains significantly more oligosaccharides (gMOS) than bovine (bMOS) or sheep (sMOS) milk and also has a larger diversity of structures. This review summarizes structural studies, revealing a diversity of up to 77 annotated gMOS structures with almost 40 structures fully characterized. Quantitative studies of goat milk oligosaccharides range from 60 to 350 mg/L in mature milk and from 200 to 650 mg/L in colostrum. These levels are clearly lower than in human milk (5–20 g/L) but higher than in other domesticated dairy animals, e.g., bovine (30–60 mg/L) and sheep (20–40 mg/L). Finally, the review focuses on demonstrated and potential functionalities of gMOS. Some studies have shown anti-inflammatory effects of mixtures enriched in gMOS. Goat MOS also display prebiotic potential, particularly in stimulating growth of bifidobacteria preferentially. Although functional studies of gMOS are still limited, several structures are also found in human milk and have known functions as immune modulators and pathogen inhibitors. In conclusion, goat milk constitutes a promising alternative source for milk oligosaccharides, which can be used in infant formula.

GtfB-type α-glucanotransferase enzymes from glycoside hydrolase family 70 (GH70) convert starch substrates into α-glucans that are of interest as food ingredients with a low glycemic index. Characterization of several GtfBs showed that they differ in product- and substrate specificity, especially with regard to branching, but structural information is limited to a single GtfB, preferring mostly linear starches and featuring a tunneled binding groove. Here, we present the second crystal structure of a 4,6-α-glucanotransferase (Limosilactobacillus reuteri NCC 2613) and an improved homology model of a 4,3-α-glucanotransferase GtfB (L. fermentum NCC 2970) and show that they are able to convert both linear and branched starch substrates. Compared to the previously described GtfB structure, these two enzymes feature a much more open binding groove, reminiscent of and evolutionary closer to starch-converting GH13 α-amylases. Sequence analysis of 287 putative GtfBs suggests that only 20% of them are similarly “open” and thus suitable as broad-specificity starch-converting enzymes.

Glucansucrases have a broad acceptor substrate specificity and receive increased attention as biocatalysts for the glycosylation of small non-carbohydrate molecules using sucrose as donor substrate. However, the main glucansucrase-catalyzed reaction results in synthesis of α-glucan polysaccharides from sucrose, and this strongly impedes the efficient glycosylation of non-carbohydrate molecules and complicates downstream processing of glucosylated products. This paper reports that suppressing α-glucan synthesis by mutational engineering of the Gtf180-ΔN enzyme of Lactobacillus reuteri 180 results in the construction of more efficient glycosylation biocatalysts. Gtf180-ΔN mutants (L938F, L981A, and N1029M) with an impaired α-glucan synthesis displayed a substantial increase in monoglycosylation yields for several phenolic and alcoholic compounds. Kinetic analysis revealed that these mutants possess a higher affinity for the model acceptor substrate catechol but a lower affinity for its mono-α-d-glucoside product, explaining the improved monoglycosylation yields. Analysis of the available high resolution 3D crystal structure of the Gtf180-ΔN protein provided a clear understanding of how mutagenesis of residues L938, L981, and N1029 impaired α-glucan synthesis, thus yielding mutants with an improved glycosylation potential.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7476-x) contains supplementary material, which is available to authorized users.

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Structural analysis of rebaudioside A derivatives obtained by Lactobacillus reuteri 180 glucansucrase-catalyzed trans-α-glucosylation Gerwig, Gerrit; te Poele, Evelien; Dijkhuizen, Lubbert; Kamerling, Johannis P a b s t r a c tThe wild-type Gtf180-DN glucansucrase enzyme from Lactobacillus reuteri 180 was found to catalyze the a-glucosylation of the steviol glycoside rebaudioside A, using sucrose as glucosyl donor in a transglucosylation process. Structural analysis of the formed products by MALDI-TOF mass spectrometry, methylation analysis and NMR spectroscopy showed that rebaudioside A is specifically a-D-glucosylated at the steviol C-19 b-D-glucosyl moiety (55% conversion). The main product is a mono-(a1 / 6)-glucosylated derivative (RebA-G1). A series of minor products, up to the incorporation of eight glucose residues, comprise elongations of RebA-G1 with mainly alternating (a1 / 3)-and (a1 / 6)-linked glucopyranose residues. These studies were carried out in the context of a program directed to the improvement of the taste of steviol glycosides via enzymatic modification of their naturally occurring carbohydrate moieties. IntroductionThe leaves of the Stevia rebaudiana BERTONI plant contain a high variety of sweet substances, called steviol glycosides [1,2], and so far more than 40 different structures have been elucidated (see review Ref. [3]). Stevioside (~5e20% w/w of dried leaves) and rebaudioside A (~2e5% w/w of dried leaves) are the most abundant components (Fig. 1), tasting about 200e300 times sweeter than sucrose (0.4% aqueous solution). Structurally, steviol glycosides have ent-13-hydroxykaur-16-en-19-oic acid as aglycone, called steviol, but are differing in carbohydrate composition at the C-13-tert-hydroxyl and C-19-carboxyl functions.Due to the growing awareness and concerns for human health related to excessive consumption of sugar (sucrose), the application of steviol glycosides as non-caloric bio-alternatives for sucrose and as substitutes for artificial (synthetic) sweeteners is strongly promoted nowadays [4e8]. Since a couple of years, steviol glycosides have been permitted for use as food additive and sweetener in the USA [9] and in Europe (E960) [10,11]. However, despite their intense sweetness and diverse beneficial pharmacological properties [12e15], the main drawback for successful commercialization of Stevia sweeteners is their slight bitterness and unpleasant (metallic) aftertaste, experienced by more than half of the human population.For natural steviol glycosides with b-D-glucopyranosyl units as constituents, it has been reported that the ratio of the number of glucose units at the C-13 site to that at the C-19 site of the steviol core has a relationship with the sweetness as well as with the quality of taste of the steviol glycosides [16,17]. To improve the Abbreviations: FID, free induction decay; FWHM, full width at half maximum; GLC-EI-MS, gas-liquid chromatography electron impact mass spectrometry; HPAEC, high-pH anion-exchange chromatography; HSQC, 1 H-detected hetero-nucl...

Steviol glycosides from the leaves of the plant Stevia rebaudiana are high-potency natural sweeteners but suffer from a lingering bitterness. The Lactobacillus reuteri 180 wild-type glucansucrase Gtf180-ΔN, and in particular its Q1140E-mutant, efficiently α-glucosylated rebaudioside A (RebA), using sucrose as donor substrate. Structural analysis of the products by MALDI-TOF mass spectrometry, methylation analysis and NMR spectroscopy showed that both enzymes exclusively glucosylate the Glc(β1→C-19 residue of RebA, with the initial formation of an (α1→6) linkage. Docking of RebA in the active site of the enzyme revealed that only the steviol C-19 β-D-glucosyl moiety is available for glucosylation. Response surface methodology was applied to optimize the Gtf180-ΔN-Q1140E-catalyzed α-glucosylation of RebA, resulting in a highly productive process with a RebA conversion of 95% and a production of 115 g/L α-glucosylated products within 3 h. Development of a fed-batch reaction allowed further suppression of α-glucan synthesis which improved the product yield to 270 g/L. Sensory analysis by a trained panel revealed that glucosylated RebA products show a significant reduction in bitterness, resulting in a superior taste profile compared to RebA. The Gtf180-ΔN-Q1140E glucansucrase mutant enzyme thus is an efficient biocatalyst for generating α-glucosylated RebA variants with improved edulcorant/organoleptic properties.