Effective Generation of Glucosylpiericidins with Selective Cytotoxicities and Insights into Their Biosynthesis.

Exploring unknown glycosyltransferases (GTs) is important for compound structural glycodiversification during the search for drug candidates. Piericidin glycosides have been reported to have potent bioactivities; however, the GT responsible for piericidin glucosylation remains unknown. Herein, BmmGT1, a macrolide GT with broad substrate selectivity and isolated from Bacillus methylotrophicus B-9987, was found to be able to glucosylate piericidin A1 in vitro. Next, the codon-optimized GT gene sbmGT1, which was designed based on BmmGT1, was heterologously expressed in the piericidin producer Streptomyces youssoufiensis OUC6819. Piericidin glycosides thus significantly accumulated, leading to the identification of four new glucopiericidins (compounds 3, 4, 6, and 7). Furthermore, using BmmGT1 as the probe, GT1507 was identified in the genome of S. youssoufiensis OUC6819 and demonstrated to be associated with piericidin glucosylation; the overexpression of this gene led to the identification of another new piericidin glycoside, N-acetylglucosamine-piericidin (compound 8). Compounds 4, 7, and 8 displayed cytotoxic selectivity toward A549, A375, HCT-116, and HT-29 solid cancer cell lines compared to the THP-1 lymphoma cell line. Moreover, database mining of GT1507 homologs revealed their wide distribution in bacteria, mainly in those belonging to the high-GC Gram-positive and Firmicutes clades, thus representing the potential for identification of novel tool enzymes for compound glycodiversification. IMPORTANCE Numerous bioactive natural products are appended with sugar moieties and are often critical for their bioactivities. Glycosyltransferases (GTs) are powerful tools for the glycodiversification of natural products. Although piericidin glycosides display potent bioactivities, the GT involved in glucosylation is unclear. In this study, five new piericidin glycosides (compounds 3, 4, 6, 7, and 8) were generated following the overexpression of GT-coding genes in a piericidin producer. Three of them (compounds 4, 7, and 8) displayed cytotoxic selectivity. Notably, GT1507 was demonstrated to be related to piericidin glucosylation in vivo. Furthermore, mining of GT1507 homologs from the GenBank database revealed their wide distribution across numerous bacteria. Our findings would greatly facilitate the exploration of GTs to glycodiversify small molecules in the search for drug candidates.

Deciphering a Cyclodipeptide Synthase Pathway Encoding Prenylated Indole Alkaloids in Streptomyces leeuwenhoekii.

Cyclodipeptide synthases (CDPSs) catalyze the formation of cyclodipeptides using aminoacylated tRNAs as the substrates and have great potential in the production of diverse 2,5-diketopiperazines (2,5-DKPs). Genome mining of Streptomyces leeuwenhoekii NRRL B-24963 revealed a two-gene locus, saz, encoding CDPS SazA and a unique fused enzyme (SazB) harboring two domains: phytoene synthase-like prenyltransferase (PT) and methyltransferase (MT). Heterologous expression of the saz gene(s) in Streptomyces albus J1074 led to the production of four prenylated indole alkaloids, among which streptoazines A to C (compounds 3 to 5) are new compounds. Expression of different gene combinations showed that the SazA catalyzes the formation of cyclo(l-Trp-l-Trp) (cWW; compound 1), followed by consecutive prenylation and methylation by SazB. Biochemical assays demonstrated that SazB is a bifunctional enzyme, catalyzing sequential C-3/C-3' prenylation(s) by SazB-PT and N-1/N-1' methylation(s) by SazB-MT. Of note, the substrate selectivity of SazB-PT and SazB-MT was probed, revealing the stringent specificity of SazB-PT but relative flexibility of SazB-MT.IMPORTANCE Natural products with a 2,5-diketopiperazine (2,5-DKP) skeleton have long sparked interest in drug discovery and development. Recent advances in microbial genome sequencing have revealed that the potential of cyclodipeptide synthase (CDPS)-dependent pathways encoding new 2,5-DKPs are underexplored. In this study, we report the genome mining of a new CDPS-encoding two-gene operon and activation of this cryptic gene cluster through heterologous expression, leading to the discovery of four indole 2,5-DKP alkaloids. The cyclo(l-Trp-l-Trp) (cWW)-synthesizing CDPS SazA and the unusual prenyltransferase (PT)-methyltransferase (MT) fused enzyme SazB were characterized. Our results expand the repertoire of CDPSs and associated tailoring enzymes, setting the stage for accessing diverse prenylated alkaloids using synthetic biology strategies.

Structure characterization of low molecular weight sulfate Ulva polysaccharide and the effect of its derivative on iron deficiency anemia.

Sulfate Ulva polysaccharide with low molecular weight was prepared by enzymatic method and name SUE. The structural characterization of SUE and the effect of its derivative SUE-iron (III) on iron deficiency anemia were studied. Results showed SUE with molecular weight of 178 kDa were consisted of 57.9% rhamnose, 12.1% glucose, 16.3% glucuronic acid, and 13.7% xylose. The backbone contained (1 → 3, 4)-linked rhamnose, (1 → 4)-linked xylose, (1 → 6)-linked glucose and sulfate substitution was at C-3 of rhamnose. Due to high contents of sulfate group (23.7 ±â€¯1.1%) and uronic acid, SUE-iron (III) with 20.3% iron content was synthesized. In order to evaluate the effects of SUE-iron (III) supplementation, an IDA animal model was created. After iron supplement administration, the SUE­iron (III) showed effective effect on returning hemoglobin, red blood cells, serum iron, and erythropoietin to the normal levels. The hematological index of rats showed no difference from that in positive group. Besides, SUE-iron (III) is beneficial to alleviate inflammatory damage caused by IDA. These suggest that SUE-iron (III) might be exploited as safe and effective new iron supplement.

Heterologous expression of an agarase gene in Bacillus subtilis, and characterization of the agarase.

A ß-agarase was identified from Pseudoalteromonas sp. Q30F and heterologously expressed in Bacillus subtilis WB800n. The ß-agarase, Aga862 encoded by aga862 gene in an open reading frame of 1338 bp is 445 amino acids in length, and has a calculated molecular mass of 50.1 kDa and an estimated isoelectric point of 4.81. Protein sequence analysis showed that Aga862 belongs to family 16 of glycoside hydrolases (GH16) and carbohydrate-binding module family 13 (CBM13). The agarase was expressed in B. subtilis WB800n and purified by precipitation, anion exchange and gel filtration for a specific activity of 4.6 U/mg, a 27.8-fold improvement over the activity of the crude enzyme. Aga862 exhibited optimal activity at 45 °C and pH 6.5, and showed excellent pH stability with retention of over 80% relative activities after preincubation for the pH range of 3.0-10.0 at 4 °C for 3 h. The agarase exhibited a Km value of 14.15 mg/mL toward agarose and a Vmax of 256.41 U/mg. The mass spectrometry analysis revealed that the end products of agar degradation were neoagarotetraose and neoagarohexaose. Recombinant Aga862 has great potential for the manufacture of agaro-oligosaccharides for the non-pathogenic nature and safety of the B. subtilis WB800n.