In search of alpha-galactosidases with improved kinetic properties for removal of the immunodominant alpha1,3-linked galactose residues of blood group B antigens, we recently identified a novel prokaryotic family of alpha-galactosidases (CAZy GH110) with highly restricted substrate specificity and neutral pH optimum (Liu, Q. P., Sulzenbacher, G., Yuan, H., Bennett, E. P., Pietz, G., Saunders, K., Spence, J., Nudelman, E., Levery, S. B., White, T., Neveu, J. M., Lane, W. S., Bourne, Y., Olsson, M. L., Henrissat, B., and Clausen, H. (2007) Nat. Biotechnol. 25, 454-464). One member of this family from Bacteroides fragilis had exquisite substrate specificity for the branched blood group B structure Galalpha1-3(Fucalpha1-2)Gal, whereas linear oligosaccharides terminated by alpha1,3-linked galactose such as the immunodominant xenotransplantation epitope Galalpha1-3Galbeta1-4GlcNAc did not serve as substrates. Here we demonstrate the existence of two distinct subfamilies of GH110 in B. fragilis and thetaiotaomicron strains. Members of one subfamily have exclusive specificity for the branched blood group B structures, whereas members of a newly identified subfamily represent linkage specific alpha1,3-galactosidases that act equally well on both branched blood group B and linear alpha1,3Gal structures. We determined by one-dimensional (1)H NMR spectroscopy that GH110 enzymes function with an inverting mechanism, which is in striking contrast to all other known alpha-galactosidases that use a retaining mechanism. The novel GH110 subfamily offers enzymes with highly improved performance in enzymatic removal of the immunodominant alpha3Gal xenotransplantation epitope.

A combined approach, comprising PCR screening and genome mining, was used to unravel the diversity and phylogeny of genes encoding 5-aminolevulinic acid synthases (ALASs, hemA gene products) in streptomycetes-related strains. In actinomycetes, these genes were believed to be directly connected with the production of secondary metabolites carrying the C5N unit, 2-amino-3-hydroxycyclopent-2-enone, with biological activities making them attractive for future use in medicine and agriculture. Unlike "classical" primary metabolism ALAS, the C5N unit-forming cyclizing ALAS (cALAS) catalyses intramolecular cyclization of nascent 5-aminolevulinate. Specific amino acid sequence changes can be traced by comparison of "classical" ALASs against cALASs. PCR screening revealed 226 hemA gene-carrying strains from 1,500 tested, with 87% putatively encoding cALAS. Phylogenetic analysis of the hemA homologs revealed strain clustering according to putative type of metabolic product, which could be used to select producers of specific C5N compound classes. Supporting information was acquired through analysis of actinomycete genomic sequence data available in GenBank and further genetic or metabolic characterization of selected strains. Comparison of 16S rRNA taxonomic identification and BOX-PCR profiles provided evidence for numerous horizontal gene transfers of biosynthetic genes or gene clusters within actinomycete populations and even from non-actinomycete organisms. Our results underline the importance of environmental and evolutionary data in the design of efficient techniques for identification of novel producers.

Quinocarcin and SF-1739, potent antitumor antibiotics, share a common tetracyclic tetrahydroisoquinoline (THIQ)-pyrrolidine core scaffold. Herein, we describe the identification of their biosynthetic gene clusters and biochemical analysis of Qcn18/Cya18 generating the previously unidentified extender unit dehydroarginine, which is a component of the pyrrolidine ring. ATP-inorganic pyrophosphate exchange experiments with five nonribosomal peptide synthetases (NRPSs) enabled us to identify their substrates. On the basis of these data, we propose that a biosynthetic pathway comprising a three-component NRPS/MbtH family protein complex, Qcn16/17/19, plays a key role in the construction of tetracyclic THIQ-pyrrolidine core scaffold involving sequential Pictet-Spengler and intramolecular Mannich reactions. Furthermore, data derived from gene inactivation experiments led us to propose late-modification steps of quinocarcin.

A total of 210 Streptomyces were isolated from the soil samples of Tawang, India where temperature varied from 5 �XC during daytime to -2 �XC during the night. Based on antifungal activity, a total of 33 strains, putatively Streptomyces spp., were selected. Optimal growth temperature for the 33 strains was 16 �XC, with growth occurring down to 6 �XC but not above 30 �XC. Phylogenetic analysis based on 16S rDNA sequences revealed the taxonomic affiliation of the 33 strains as species of Streptomyces. To examine the relatedness of the chitinase genes from six strong antifungal Streptomyces strains, a phylogenetic tree was constructed using the catalytic domain nucleotide sequences and resulted in seven distinct monophyletic groups. A quantitative PCR study for chitinase expressing ability revealed that of the six antifungal strains tested, the strain Streptomyces roseochromogenus TSR12 was the most active producer of family 18 chitinase genes. Streptomyces strains with enhanced inhibitory potential usually encode a family 19 chitinase gene; however, our present study did not show expression of this family in the six strains tested.

The Streptomyces phylogroup pratensis (Doroghazi and Buckley, 2010) contains isolates obtained from grassy fields, as well as Streptomyces flavogriseus ATCC 33331 and strain CGMCC 4.1868. This latter strain was received as Streptomyces griseoplanus but was subsequently found to be mislabeled, and S. flavogriseus ATCC 33331 (=IAF-45-CD) was shown to be clearly distinct from the type strain S. flavogriseus ATCC 25452(T) (=CGMCC 4.1884(T)). In order to evaluate the taxonomic position of phylogroup pratensis further, sequences of the 16S rRNA gene and five protein-coding housekeeping genes (atpD, gyrB, recA, rpoB and trpB) were determined for six strains of the phylogroup and type strains of 19 related species, which were selected by a BLAST search based on the sequences of the phylogroup. The 16S rRNA gene sequences for the phylogroup were identical to those of eight species belonging to cluster I of the S. griseus clade. However, in all the individual protein-coding gene and MLSA phylogenies, the phylogroup strains without exception formed an obviously distinct cluster that could be equated with a new species status. The phylogenetic evidence for the new species assignment was also supported by corresponding DNA-DNA hybridization values and by phenotypic characteristics. It is therefore proposed that the phylogroup should be classified as Streptomyces pratensis sp. nov., and the type strain is ch24(T) (=CGMCC 4.6829(T)=NRRL B-24916(T)).