A UDP-glycosyltransferase from Bacillus licheniformis was exploited for the glycosylation of phloretin. The in vitro glycosylation reaction confirmed the production of five phloretin glucosides, including three novel glucosides. Consequently, we demonstrated the application of the same glycosyltransferase for the efficient whole-cell biocatalysis of phloretin in engineered Escherichia coli. P hloretin is a dihydrochalcone, an intermediate of the biosynthetic pathway of flavonoids in plants, which is abundantly present in the peel of apple (1, 2) and in strawberries (3). They occur in different glycosidic forms, such as naringin dihydrochalcone, phlorizin, and phloretin-4=-O-glucoside, in the different parts of the plants, contributing to various physiological properties of the plants, as well as to their color. Phloretin and its glycosides have been determined to have beneficial biological activities. Studies have uncovered that phloretin has inhibitory activity against glucose cotransporter 1 (4, 5), antioxidant activity (6), and activity to suppress the tumor necrosis factor alpha-induced inflammatory response, ameliorate inflammation of the colon, positively affect body weight loss (7), modulate Ca 2ϩ -activated K ϩ channels, and increase endothelial nitric oxide production, which might help to protect against atherosclerosis (8). Importantly, phloretin has other biological functions, like anticarcinogenic (9) and estrogenic activities (10) and inhibition of cardiovascular disease (11, 12). Irrespective of their diverse physiological and pharmacological activities, the use of most of the natural polyphenols as drugs and food additives has been limited because of their water insolubility and low absorbability. Glycosylation enhances the bioavailability and pharmacological properties of compounds by increasing their solubility and stability (13,14). Importantly, the sugar moieties of the glycosides often participate in the specific recognition of their biological targets and help to determine their efficacy in drug development (14, 15). According to the CAZy database (http://www .cazy.org/) (16,17,18), glycosyltransferase family 1 (GT1) proteins contain the UDP-glycosyltransferases that are common in all domains of life (19) and predominantly recognize small molecules as the sugar acceptors. A recent report showed that YjiC, a Bacillus licheniformis UDP-glycosyltransferase that falls in the GT1 family of proteins, can glycosylate at different hydroxyl positions of geldanamycin analogs (20). Here, we report the use of this glycosyltransferase for the biosynthesis of diverse phloretin glucosides in vitro and the subsequent application of YjiC for in vivo production of phloretin glucosides in an Escherichia coli mutant generating a cytoplasmic pool of UDP-glucose, since the YjiC-homologous glycosyltransferases from other Bacillus species were found to have flexible glycosyltransferase activities toward different flavonoid groups of compounds. Moreover, we found that by reversing the glycosylation reaction, the enz...

No abstract

Culture broth of an actinomycete isolate, Nocardia sp. CS682 showed specifically higher antibacterial activity against methicillin resistant Staphylococcus aureus (MRSA). Purified substance from the organism, CS-682, which is active against MRSA and Micrococcus leuteus, is a C(28)H(37)NO(8) (M+H(+), observed: 516.83) and identified as an unusual macrolide antibiotic, nargenicin. The chemical structure of CS-682 was identified by FT-IR, (1)H-NMR, (13)C-NMR, and ((1)H-(1)H and (1)H-(13)H) COSY. The anti-MRSA activity of CS-682 was stronger than that of oxacillin, vancomycin, monensin, erythromycin, and spiramycin. Phylogenetic analysis showed that strain CS682 is closely related to Nocardia tenerifensis DSM 44704(T) (98.7% sequence similarity), followed by N. brasiliensis ATCC 19296(T) (98.4% sequence similarity). The ability of Nocardia sp. CS682 to produce nargenicin was unique.

No abstract

Streptomyces sp. VN1 was isolated from the coastal region of Phu Yen Province (central Viet Nam). Morphological, physiological, and whole genome phylogenetic analyses suggested that strain Streptomyces sp. VN1 belonged to genus Streptomyces. Whole genome sequencing analysis showed its genome was 8,341,703 base pairs in length with GC content of 72.5%. Diverse metabolites, including cinnamamide, spirotetronate antibiotic lobophorin A, diketopiperazines cyclo-L-proline-L-tyrosine, and a unique furan-type compound were isolated from Streptomyces sp. VN1. Structures of these compounds were studied by HR-Q-TOF ESI/MS/MS and 2D NMR analyses. Bioassay-guided purification yielded a furan-type compound which exhibited in vitro anticancer activity against AGS, HCT116, A375M, U87MG, and A549 cell lines with IC 50 values of 40.5, 123.7, 84.67, 50, and 58.64 µM, respectively. In silico genome analysis of the isolated Streptomyces sp. VN1 contained 34 gene clusters responsible for the biosynthesis of known and/or novel secondary metabolites, including different types of terpene, T1PKS, T2PKS, T3PKS, NRPS, and hybrid PKS-NRPS. Genome mining with HR-Q-TOF ESI/MS/MS analysis of the crude extract confirmed the biosynthesis of lobophorin analogs. This study indicates that Streptomyces sp. VN1 is a promising strain for biosynthesis of novel natural products. Natural products (NPs) have been starting points of drug discovery for several decades. Major antimicrobials and chemotherapeutics entering clinical trials are often based on NPs 1,2. Drugs with a natural origin can be produced as primary or secondary metabolites from versatile living organisms. Different microorganisms such as Streptomyces, myxobacteria and uncultured bacteria are major sources of such beneficial NPs 3,4. Moreover, different metabolic engineering approaches and sophisticated techniques employing systems biology or synthetic biology can assist in harnessing the full potential of these bacteria in terms of productivity or creating diverse products 5,6. Hence, there is renewed interest in mining microorganisms for new leads owing to the remarkable success of microbial metabolites as starting points for developing effective antibiotics, anticancer agents, and agrochemicals 7. Streptomyces are Gram-positive, aerobic bacteria in the order of Actinomycetales within the class of Actinobacteria. Genus Streptomyces was first proposed by Waksman and Henrici in 1943. It was classified into the family of Streptomycetaceae based on its morphology and cell wall chemotype 8. Previous studies have shown that more than 74% of current antibiotics are derived from the genus Streptomyces 9. Using integrated approaches of compound screening and drug development, Streptomyces arsenal has been found to be able to combat antibiotic resistance 5. Multiple approaches such as ribosome engineering 10 and genome mining 11 have been used to find new secondary metabolites in old Streptomyces strains. Besides the effort to work on old strains to explore novel biosynthetic gene clusters (BGCs),...

Nargenicin A1, an antibacterial produced by Nocardia sp. CS682 (KCTC 11297BP), demonstrates effective activity against various Gram-positive bacteria. Hence, we attempted to enhance nargenicin A1 production by utilizing the cumulative effect of synthetic biology, metabolic engineering and statistical media optimization strategies. To facilitate the modular assembly of multiple genes for genetic engineering in Nocardia sp. CS682, we constructed a set of multi-monocistronic vectors, pNV18L1 and pNV18L2 containing hybrid promoter (derived from ermE* and promoter region of neo ), ribosome binding sites (RBS), and restriction sites for cloning, so that each cloned gene was under its own promoter and RBS. The multi-monocistronic vector, pNV18L2 containing transcriptional terminator showed better efficiency in reporter gene assay. Thus, multiple genes involved in the biogenesis of pyrrole moiety (ngnN2, ngnN3, ngnN4, and ngnN5 from Nocardia sp. CS682), glucose utilization (glf and glk from Zymomonas mobilis), and malonyl-CoA synthesis (accA2 and accBE from Streptomyces coelicolor A3 (2)), were cloned in pNV18L2. Further statistical optimization of specific precursors (proline and glucose) and their feeding time led to ~84.9 mg/L nargenicin from Nocardia sp. GAP, which is ~24-fold higher than Nocardia sp. CS682 (without feeding). Furthermore, pikC from Streptomyces venezuelae was expressed to generate Nocardia sp. PikC. Nargenicin A1 acid was characterized as novel derivative of nargenicin A1 produced from Nocardia sp. PikC by mass spectrometry (MS) and nuclear magnetic resonance (NMR) analyses. We also performed comparative analysis of the anticancer and antibacterial activities of nargenicin A1 and nargenicin A1 acid, which showed a reduction in antibacterial potential for nargenicin A1 acid. Thus, the development of an efficient synthetic biological platform provided new avenues for enhancing or structurally diversifying nargenicin A1 by means of pathway designing and engineering.

No abstract

Two promiscuous Bacillus licheniformis glycosyltransferases, YdhE and YojK, exhibited prominent stereospecific but nonregiospecific glycosylation activity of 20 different classes of 59 structurally different natural and non-natural products. Both enzymes transferred various sugars at three nucleophilic groups (OH, NH2, SH) of diverse compounds to produce O-, N-, and S-glycosides. The enzymes also displayed a catalytic reversibility potential for a one-pot transglycosylation, thus bestowing a cost-effective application in biosynthesis of glycodiversified natural products in drug discovery.