GDPF: a data resource for the distribution of prokaryotic protein families across the global biosphere.

Microorganisms encode most of the functions of life on Earth. However, conventional research has primarily focused on specific environments such as humans, soil and oceans, leaving the distribution of functional families throughout the global biosphere poorly comprehended. Here, we present the database of the global distribution of prokaryotic protein families (GDPF, http://bioinfo.qd.sdu.edu.cn/GDPF/), a data resource on the distribution of functional families across the global biosphere. GDPF provides global distribution information for 36 334 protein families, 19 734 superfamilies and 12 089 KEGG (Kyoto Encyclopedia of Genes and Genomes) orthologs from multiple source databases, covering typical environments such as soil, oceans, animals, plants and sediments. Users can browse, search and download the distribution data of each entry in 10 000 global microbial communities, as well as conduct comparative analysis of distribution disparities among multiple entries across various environments. The GDPF data resource contributes to uncovering the geographical distribution patterns, key influencing factors and macroecological principles of microbial functions at a global level, thereby promoting research in Earth ecology and human health.

PAT: a comprehensive database of prokaryotic antimicrobial toxins.

Antimicrobial toxins help prokaryotes win competitive advantages in intraspecific or interspecific conflicts and are also a critical factor affecting the pathogenicity of many pathogens that threaten human health. Although many studies have revealed that antagonism based on antimicrobial toxins plays a central role in prokaryotic life, a database on antimicrobial toxins remains lacking. Here, we present the prokaryotic antimicrobial toxin database (PAT, http://bioinfo.qd.sdu.edu.cn/PAT/), a comprehensive data resource collection on experimentally validated antimicrobial toxins. PAT has organized information, derived from the reported literature, on antimicrobial toxins, as well as the corresponding immunity proteins, delivery mechanisms, toxin activities, structural characteristics, sequences, etc. Moreover, we also predict potential antimicrobial toxins in prokaryotic reference genomes and show the taxonomic information and environmental distribution of typical antimicrobial toxins. These details have been fully incorporated into the PAT database, where users can browse, search, download, analyse and view informative statistics and detailed information. PAT resources have already been used in our prediction and identification of prokaryotic antimicrobial toxins and may contribute to promoting the efficient investigation of antimicrobial toxin functions, the discovery of novel antimicrobial toxins, and an improved understanding of the biological roles and significance of these toxins.

Recent advances in discovery and biosynthesis of natural products from myxobacteria: an overview from 2017 to 2023.

Covering: 2017.01 to 2023.11Natural products biosynthesized by myxobacteria are appealing due to their sophisticated chemical skeletons, remarkable biological activities, and intriguing biosynthetic enzymology. This review aims to systematically summarize the advances in the discovery methods, new structures, and bioactivities of myxobacterial NPs reported in the period of 2017-2023. In addition, the peculiar biosynthetic pathways of several structural families are also highlighted.

Three novel species of the genus Flavobacterium, Flavobacterium odoriferum sp. nov., Flavobacterium fragile sp. nov. and Flavobacterium luminosum sp. nov., isolated from activated sludge.

Three Gram-stain-negative, rod-shaped, non-spore-forming bacteria were isolated from activated sludge samples. The results of phylogenetic analysis based on the 16S rRNA gene sequences indicated that the three strains, designated HXWNR29T, HXWNR69T and HXWNR70T, had the highest sequence similarity to the type strains Flavobacterium cheniae NJ-26T, Flavobacterium channae KSM-R2A30T and Flavobacterium amniphilum KYPY10T with similarities of 97.66 %, 98.66 and 98.14 %, respectively. The draft genomes of these three strains were 2.93 Mbp (HXWNR29T), 2.69 Mbp (HXWNR69T) and 2.65 Mbp (HXWNR70T) long with DNA G+C contents of 31.84 %, 32.83 % and 34.66 %, respectively. These genomes contained many genes responsible for carbohydrate degradation and antibiotic resistance. The major fatty acids (>5 %) included iso-C15 : 0, iso-C15 : 0 3-OH and iso-C17 : 0 3-OH. The major menaquinone was MK-6 for all the three strains. The average nucleotide identity (ANI; 72.7-88.5 %) and digital DNA-DNA hybridization (dDDH; 19.6-35.3 %) results further indicated that these three strains represented three novel species within the genus Flavobacterium, for which the names Flavobacterium odoriferum sp. nov. (type strain HXWNR29T = KCTC 92446T = CGMCC 1.61821T), Flavobacterium fragile sp. nov. (type strain HXWNR69T = KCTC 92468T = CGMCC 1.61442T) and Flavobacterium luminosum sp. nov. (type strain HXWNR70T = KCTC 92447T = CGMCC 1.61443T) are proposed.

Estimate of the degradation potentials of cellulose, xylan, and chitin across global prokaryotic communities.

Complex polysaccharides (e.g. cellulose, xylan, and chitin), the most abundant renewable biomass resources available on Earth, are mainly degraded by microorganisms in nature. However, little is known about the global distribution of the enzymes and microorganisms responsible for the degradation of cellulose, xylan, and chitin in natural environments. Through large-scale alignments between the sequences released by the Earth Microbiome Project and sequenced prokaryotic genomes, we determined that almost all prokaryotic communities have the functional potentials to degrade cellulose, xylan, and chitin. The median abundances of genes encoding putative cellulases, xylanases, and chitinases in global prokaryotic communities are 0.51 (0.17-1.01), 0.24 (0.05-0.57), and 0.33 (0.11-0.71) genes/cell, respectively, and the composition and abundance of these enzyme systems are environmentally varied. The taxonomic sources of the three enzymes are highly diverse within prokaryotic communities, and the main factor influencing the diversity is the community's alpha diversity index rather than gene abundance. Moreover, there are obvious differences in taxonomic sources among different communities, and most genera with degradation potentials are narrowly distributed. In conclusion, our analysis preliminarily depicts a panorama of cellulose-, xylan-, and chitin-degrading enzymatic systems across global prokaryotic communities.

Constructing a Myxobacterial Natural Product Database to Facilitate NMR-Based Metabolomics Bioprospecting of Myxobacteria.

Myxobacteria are fascinating prokaryotes featuring a potent capacity for producing a wealth of bioactive molecules with intricate chemical topology as well as intriguing enzymology, and thus it is critical to developing an efficient pipeline for bioprospecting. Herein, we construct the database MyxoDB, the first public compendium solely dedicated to myxobacteria, which enabled us to provide an overview of the structural diversity and taxonomic distribution of known myxobacterial natural products. Moreover, we demonstrated that the cutting-edge NMR-based metabolomics was effective to differentiate the biosynthetic priority of myxobacteria, whereby MyxoDB could greatly streamline the dereplication of multifarious known compounds and accordingly speed up the discovery of new compounds. This led to the rapid identification of a class of linear di-lipopeptides (archangimins) and a rare rearranged sterol (corasterol) that were endowed with unique chemical architectures and/or biosynthetic enzymology. We also showcased that NMR-based metabolomics, MyxoDB, and genomics can also work concertedly to accelerate the targeted discovery of a polyketidic compound pyxipyrrolone C. All in all, this study sets the stage for the discovery of many more novel natural products from underexplored myxobacterial resources.

Discovery and Biosynthesis of Glycosylated Cycloheximide from a Millipede-Associated Actinomycete.

Chemical redundancy of microbial natural products (NPs) underscores the importance to exploit new resources of microorganisms. Insect-associated microbes are prolific but largely underexplored sources of diverse NPs. Herein, we discovered the new compound α-l-rhamnosyl-actiphenol (1) from a millipede-associated Streptomyces sp. ML6, which is the first glycosylated cycloheximide-class natural product. Interestingly, bioinformatics analysis of the ML6 genome revealed that the biosynthesis of 1 involves a cooperation between two gene clusters (chx and rml) located distantly on the genome of ML6. We also carried out in vitro enzymatic glycosylation of cycloheximide using an exotic promiscuous glycosyltransferase BsGT-1, which resulted in the production of an additional cycloheximide glycoside cycloheximide 7-O-ß-d-glucoside (5). Although the antifungal and cytotoxic activities of the new compounds 1 and 5 were attenuated relative to those of cycloheximide, our work not only enriches the chemical repertoire of the cycloheximide family but also provides new insights into the structure-activity relationship optimization and ecological roles of cycloheximide.

Microvirga roseola sp. nov. and Microvirga lenta sp. nov., isolated from Taklamakan Desert soil.

Two Gram-negative, rod-shaped, non-spore-forming bacteria, designated SM9T and SM2T, were isolated from Taklamakan Desert soil samples. Phylogenetic analysis based on the 16S rRNA gene sequences showed that strains SM9T and SM2T had the highest sequence similarity to the type strains Microvirga indica BCRC 80972T and Microvirga soli NBRC 112417T with similarity values of 98.2 and 97.7 %, respectively, and Microvirga was among the predominant genera in the desert soil. The draft genomes of these two strains were 4.56 Mbp (SM9T) and 5.08 Mbp (SM2T) long with 65.1 mol% (SM9T) and 63.5 mol% (SM2T) G+C content. To adapt to the desert environment, these two strains possessed pathways for the synthesis of stress metabolite trehalose. The major fatty acids (>5 %) included C18 : 1 ω9c in SM2T, but C16 : 0, C18 : 0 and C19 : 0 cyclo ω8c in SM9T, while the major menaquinone was ubiquinone 10 in both strains. The major polar lipids of SM9T and SM2T were phosphatidylglycerol, phosphatidylethanolamine and phospholipid. The average nucleotide identity and digital DNA-DNA hybridization results further indicated that strains SM9T and SM2T were distinguished from phylogenetically related species and represented two novel species within the genus Microvirga, for which the names Microvirga roseola sp. nov. (type strain SM2T=KCTC 72792T=CGMCC 1.17776T) and Microvirga lenta sp. nov. (type strain SM9T=KCTC 82729T=CCTCC AB 2021131T) are proposed.

Halalkalibacterium roseum gen. nov., sp. nov., a new member of the family Balneolaceae isolated from soil.

A Gram-stain-negative, non-motile, moderately halophilic and facultatively anaerobic bacterium, designated YR4-1T, was isolated from a saline-alkali and sorghum-planting soil sample collected in Dongying, Shandong Province, PR China. Growth occurred at 28-45 °C with the presence of 4.0-20.0 % (w/v) NaCl and pH 6.0-9.0. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that YR4-1T shared the highest similarity of 92.1-92.4 % with the valid published species of Aliifodinibius. The isolate formed a separate clade at the genus level in recently described family Balneolaceae. The draft genome of strain YR4-1T is 3.83 Mbp long with 44.0 mol% G+C content. The strain possesses several genes involved in the osmotic stress response mechanism and diverse metabolic pathways, probably for the living in saline environment. This may lead to a better understanding of the underrepresented Balneolaceae lineage. The major menaquinone was MK-7. The main polar lipid profile was composed of diphosphatidylglycerol, phosphatidylglycerol, phosphoglycolipids, aminophosphoglycolipid, one glycolipid, and four unidentified lipids. The predominant cellular fatty acids were iso-C15 : 0 (35.7 %) and anteiso-C15 : 0 (33.5 %). On the basis of its phenotypic, chemotaxonomic and phylogenetic features, strain YR4-1T represents a novel species of a new genus, for which the name Halalkalibacterium roseum gen. nov., sp. nov. is proposed. The type strain is YR4-1T (=CGMCC 1.17777T=KCTC 72795T).

Challenges and potential for improving the druggability of podophyllotoxin-derived drugs in cancer chemotherapy.

Covering: up to 2020As a main bioactive component of the Chinese, Indian, and American Podophyllum species, the herbal medicine, podophyllotoxin (PTOX) exhibits broad spectrum pharmacological activity, such as superior antitumor activity and against multiple viruses. PTOX derivatives (PTOXs) could arrest the cell cycle, block the transitorily generated DNA/RNA breaks, and blunt the growth-stimulation by targeting topoisomerase II, tubulin, or insulin-like growth factor 1 receptor. Since 1983, etoposide (VP-16) is being used in frontline cancer therapy against various cancer types, such as small cell lung cancer and testicular cancer. Surprisingly, VP-16 (ClinicalTrials NTC04356690) was also redeveloped to treat the cytokine storm in coronavirus disease 2019 (COVID-19) in phase II in April 2020. The treatment aims at dampening the cytokine storm and is based on etoposide in the case of central nervous system. However, the initial version of PTOX was far from perfect. Almost all podophyllotoxin derivatives, including the FDA-approved drugs VP-16 and teniposide, were seriously limited in clinical therapy due to systemic toxicity, drug resistance, and low bioavailability. To meet this challenge, scientists have devoted continuous efforts to discover new candidate drugs and have developed drug strategies. This review focuses on the current clinical treatment of PTOXs and the prospective analysis for improving druggability in the rational design of new generation PTOX-derived drugs.

Assembly processes and source tracking of planktonic and benthic bacterial communities in the Yellow River estuary.

Estuaries connect rivers with the ocean and are considered transition regions due to the continuous inputs from rivers. Microbiota from different sources converge and undergo succession in these transition regions, but their assembly mechanisms along environmental gradients remain unclear. Here, we found that salinity had a stronger effect on planktonic than on benthic microbial communities, and the dominant planktonic bacteria changed more distinctly than the dominant benthic bacteria with changes in salinity. The planktonic bacteria in the brackish water came mainly from seawater, which was confirmed in the laboratory, whereas the benthic bacteria were weakly affected by salinity, which appeared to be a mixture of the bacteria from riverine and oceanic sediments. Benthic bacterial community assembly in the sediments was mainly controlled by homogeneous selection and almost unaffected by changes in salinity, the dominant assemblage processes for planktonic bacteria changed dramatically along the salinity gradient, from homogeneous selection in freshwater to drift in seawater. Our results highlight that salinity is the key driver of estuarine microbial succession and that salinity is more important in shaping planktonic than benthic bacterial communities in the Yellow River estuary.

Two PAAR Proteins with Different C-Terminal Extended Domains Have Distinct Ecological Functions in Myxococcus xanthus.

Bacterial proline-alanine-alanine-arginine (PAAR) proteins are located at the top of the type VI secretion system (T6SS) nanomachine and carry and deliver effectors into neighboring cells. Many PAAR proteins are fused with a variable C-terminal extended domain (CTD). Here, we report that two paar-ctd genes (MXAN_RS08765 and MXAN_RS36995) located in two homologous operons are involved in different ecological functions of Myxococcus xanthusMXAN_RS08765 inhibited the growth of plant-pathogenic fungi, while MXAN_RS36995 was associated with the colony-merger incompatibility of M. xanthus cells. These two PAAR-CTD proteins were both toxic to Escherichia coli cells, while MXAN_RS08765, but not MXAN_RS36995, was also toxic to Saccharomyces cerevisiae cells. Their downstream adjacent genes, i.e., MXAN_RS08760 and MXAN_RS24590, protected against the toxicities. The MXAN_RS36995 protein was demonstrated to have nuclease activity, and the activity was inhibited by the presence of MXAN_RS24590. Our results highlight that the PAAR proteins diversify the CTDs to play divergent roles in M. xanthusIMPORTANCE The type VI secretion system (T6SS) is a bacterial cell contact-dependent weapon capable of delivering protein effectors into neighboring cells. The PAAR protein is located at the top of the nanomachine and carries an effector for delivery. Many PAAR proteins are extended with a diverse C-terminal sequence with an unknown structure and function. Here, we report two paar-ctd genes located in two homologous operons involved in different ecological functions of Myxococcus xanthus; one has antifungal activity, and the other is associated with the kin discrimination phenotype. The PAAR-CTD proteins and the proteins encoded by their downstream genes form two toxin-immunity protein pairs. We demonstrated that the C-terminal diversification of the PAAR-CTD proteins enriches the ecological functions of bacterial cells.

Engineering the acyltransferase domain of epothilone polyketide synthase to alter the substrate specificity.

BACKGROUND: Polyketide synthases (PKSs) include ketone synthase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains to catalyse the elongation of polyketide chains. Some PKSs also contain ketoreductase (KR), dehydratase (DH) and enoylreductase (ER) domains as modification domains. Insertion, deletion or substitution of the catalytic domains may lead to the production of novel polyketide derivatives or to the accumulation of desired products. Epothilones are 16-membered macrolides that have been used as anticancer drugs. The substrate promiscuity of the module 4 AT domain of the epothilone PKS (EPOAT4) results in production of epothilone mixtures; substitution of this domain may change the ratios of epothilones. In addition, there are two dormant domains in module 9 of the epothilone PKS. Removing these redundant domains to generate a simpler and more efficient assembly line is a desirable goal. RESULTS: The substitution of module 4 drastically diminished the activity of epothilone PKS. However, with careful design of the KS-AT linker and the post-AT linker, replacing EPOAT4 with EPOAT2, EPOAT6, EPOAT7 or EPOAT8 (specifically incorporating methylmalonyl-CoA (MMCoA)) significantly increased the ratio of epothilone D (4) to epothilone C (3) (the highest ratio of 4:3 = 4.6:1), whereas the ratio of 4:3 in the parental strain Schlegelella brevitalea 104-1 was 1.4:1. We also obtained three strains by swapping EPOAT4 with EPOAT3, EPOAT5, or EPOAT9, which specifically incorporate malonyl-CoA (MCoA). These strains produced only epothilone C, and the yield was increased by a factor of 1.8 compared to that of parental strain 104-1. Furthermore, mutations of five residues in the AT domain identified Ser310 as the critical factor for MMCoA recognition in EPOAT4. Then, the mutation of His308 to valine or tyrosine combined with the mutation of Phe310 to serine further altered the product ratios. At the same time, we successfully deleted the inactive module 9 DH and ER domains and fused the ΨKR domain with the KR domain through an ~ 25-residue linker to generate a productive and simplified epothilone PKS. CONCLUSIONS: These results suggested that the substitution and deletion of catalytic domains effectively produces desirable compounds and that selection of the linkers between domains is crucial for maintaining intact PKS catalytic activity.

The Discovery and Biosynthesis of Nicotinic Myxochelins from an Archangium sp. SDU34.

Myxobacteria are a prolific source of structurally diverse natural products, and one of the best-studied myxobacterial products is the siderophore myxochelin. Herein, we report two new compounds, myxochelins N (1) and O (2), that are nicotinic paralogs of myxochelin A, from the terrestrial myxobacterium Archangium sp. SDU34; 2 is functionalized with a rare 2-oxazolidinone. A precursor-feeding experiment implied that the biosynthesis of 1 or 2 was due to altered substrate specificity of the loading module of MxcE, which likely accepts nicotinic acid and benzoic acid instead of more conventional 2,3-dihydroxybenzoic acid. We also employed a phylogenomic approach to map the evolutionary relationships of the myxochelin biosynthetic gene clusters (BGCs) in all the available myxobacterial genomes, to pave the way for the future discovery of potentially hidden myxochelin derivatives. Although the biological function of 1 and 2 is unclear yet, this work underpins that even extensively studied BGCs in myxobacteria can still produce new chemistry.

Discovery of recombinases enables genome mining of cryptic biosynthetic gene clusters in Burkholderiales species.

Bacterial genomes encode numerous cryptic biosynthetic gene clusters (BGCs) that represent a largely untapped source of drugs or pesticides. Mining of the cryptic products is limited by the unavailability of streamlined genetic tools in native producers. Precise genome engineering using bacteriophage recombinases is particularly useful for genome mining. However, recombinases are usually host-specific. The genome-guided discovery of novel recombinases and their transient expression could boost cryptic BGC mining. Herein, we reported a genetic system employing Red recombinases from Burkholderiales strain DSM 7029 for efficient genome engineering in several Burkholderiales species that currently lack effective genetic tools. Using specialized recombinases-assisted in situ insertion of functional promoters, we successfully mined five cryptic nonribosomal peptide synthetase/polyketide synthase BGCs, two of which were silent. Two classes of lipopeptides, glidopeptins and rhizomides, were identified through extensive spectroscopic characterization. This recombinase expression strategy offers utility within other bacteria species, allowing bioprospecting for potentially scalable discovery of novel metabolites with attractive bioactivities.

Myxadazoles, Myxobacterium-Derived Isoxazole-Benzimidazole Hybrids with Cardiovascular Activities.

There is a continuous need for novel microbial natural products to fill the drying-up drug development pipeline. Herein, we report myxadazoles from Myxococcus sp. SDU36, a family of novel chimeric small molecules that consist of N-ribityl 5,6-dimethylbenzimidazole and a linear fatty acid chain endowed with an isoxazole ring. The experiments of genome sequencing, gene insertion mutation, isotope labelling, and precursor feeding demonstrated that the fatty acid chain was encoded by a non-canonical PKS/NRPS gene cluster, whereas the origin of N-ribityl 5,6-dimethylbenzimidazole was related to the vitamin B12 metabolism. The convergence of these two distinct biosynthetic pathways through a C-N coupling led to the unique chemical framework of myxadazoles, which is an unprecedented hybridization mode in the paradigm of natural products. Myxadazoles exhibited potent vasculogenesis promotion effect and moderate antithrombotic activity, underscoring their potential usage for the treatment of cardiovascular diseases.

Psychroflexus aurantiacus sp. nov., isolated from soil in the Yellow River Delta wetlands.

A Gram-stain-negative, strictly aerobic, non-motile, orange-coloured bacterium, designated YR1-1T, was isolated from a soil sample collected from the Yellow River Delta wetlands (PR China). Growth was observed at a salinity of 1.0-15.0 % NaCl, 4-45 °C and pH 6.0-9.0. The results of phylogenetic analysis based on the 16S rRNA gene sequences indicated that YR1-1T represented a member of the genus Psychroflexus, with the highest sequence similarity to Psychroflexus sediminis YIM-C238T (97.9 %), followed by Psychroflexus aestuariivivens (97.1 %) and Psychroflexus torquis (96.4 %). The average nucleotide identity and digital DNA-DNA hybridization values between YR1-1T and other closely related type strains of species of the genus Psychroflexus were 68.7-86.3% and 17.8-30.9 %. The genome of the strain was 2 899 374 bp in length with 39.8 % DNA G+C content. The predominant fatty acids (>10 %) were iso-C15 : 0 and anteiso-C15 : 0. The major respiratory quinone was menaquinone-6 (MK-6) and the major polar lipids were phosphatidylethanolamine, phospholipid, diphosphatidylglycerol, two unidentified aminolipids and four unidentified lipids. The combined genotypic and phenotypic data indicate that YR1-1T represents a novel species within the genus Psychroflexus, for which the name Psychroflexus aurantiacus sp. nov., is proposed. The type strain is YR1-1T (=KCTC 72794T=CGMCC 1.17458T).

Deinococcus terrestris sp. nov., a gamma ray- and ultraviolet-resistant bacterium isolated from soil.

Strain SDU3-2T was isolated from a soil sample collected in Shandong Province, PR China. Cells of SDU3-2T were spherical, Gram-stain-positive, aerobic and non-motile. Cellular growth of the strain occurred at 25-45 °C, pH 5.5-8.5 and with 0-1.5 % (w/v) of NaCl. Phylogenetic analysis based on the 16S rRNA gene sequences showed that strain SDU3-2T was closest to the type strain Deinococcus murrayi ALT-1bT with a similarity of 95.2 %. The draft genome was 3.49 Mbp long with 69.2 mol% G+C content. Strain SDU3-2T exhibited high resistance to gamma radiation (D10 >12 kGy) and UV (D10 >900 J m-2). The strain encoded many genes for resistance to radiation and oxidative stress, which were highly conserved with other Deinococcus species, but possessed interspecific properties. The major fatty acids of SDU3-2T cells were C15 : 1 ω6c, C16 : 1 ω7c/C16 : 1 ω6c, and C17 : 1 ω8c, the major menaquinone was menaquinone-8, and the major polar lipids were an unidentified phosphoglycolipid, four unidentified glycolipids and an unidentified phospholipid. The average nucleotide identity and DNA-DNA hybridization results further indicated that strain SDU3-2T represents a new species in the genus Deinococcus, for which the name Deinococcus terrestris sp. nov. is proposed. The type strain is SDU3-2T (=CGMCC 1.17147T=KCTC 43098T).

Paucihalobacter ruber gen. nov., sp. nov., isolated from a haloalkaline lake sediment sample.

A Gram-stain-negative, strictly aerobic, non-motile, rod-shaped bacterium, designated CWB-1T, was isolated from a haloalkaline lake sediment sample collected from the bottom of Chaiwopu Lake, Urumchi, Xinjiang Province, PR China. Strain CWB-1T grew at 4-40 °C (optimum, 30-35 °C), pH 6.5-9.0 (optimum, pH 6.5-7.0) and with 0.5-5.5 % (w/v) NaCl (optimum, 2.5-3.0 %). Phylogenetic analyses based on the 16S rRNA gene sequence and the whole genome sequence both revealed that strain CWB-1T belonged to the family Flavobacteriaceae. The strain had the highest similarity of the 16S rRNA gene sequence to Psychroserpens jangbogonensis PAMC 27130T (92.8 %). The genome of strain CWB-1T was 3 548 011 bp long with 36.3 % DNA G+C content. The predominant fatty acids (>10 %) in the CWB-1T cells were iso-C15 : 0, iso-C17 : 0 3-OH and summed feature 1 (iso-C15 : 1 H/C13 : 0 3-OH). The major respiratory quinone was menaquinone-6 and the major polar lipids were phosphatidylethanolamine, an unidentified aminolipid and two unidentified lipids. Based on the phylogenetic analyses, as well as the phenotypic characteristics, a novel genus and species of the family Flavobacteriaceae, Paucihalobacter ruber gen. nov., sp. nov., is proposed. The type strain is CWB-1T (=KCTC 72450T=CGMCC 1.17149T).

Effects of glycosylation on the bioactivity of rapamycin.

The macrolactone rapamycin (RAP) presents a broad range of bioactivities, but its clinical applications are compromised due to the poor water solubility and low bioavailability, which could probably be overcome by glycosylation. In this study, we tested a set of promiscuous glycosyltransferases (GTs) to modify rapamycin with four different sugar donors. BsGT-1 displayed the best glycosylation activity with a preference for UDP-glucose, and the glycosylation happened at C-28 or C-40 of rapamycin, producing rapamycin-40-O-ß-D-glucoside (RG1), and two new compounds rapamycin-28-O-ß-D-glucoside (RG2) and rapamycin-28,40-O-ß-D-diglucoside (RG3). The glycosylation remarkably improved water solubility and almost completely abolished cytotoxicity but simultaneously attenuated the antifungal, antitumor, and immunosuppression bioactivities of rapamycin. We found the glycosylation at C-40 had less effect on the bioactivities than that at C-28. The molecular docking analysis revealed that the glycosylation, especially the glycosylation at C-28, weakened the hydrophobic and hydrogen bonding contacts between the rapamycin glucosides and the binding proteins: the FK506-binding protein (FKBP12) and the FKBP12-rapamycin binding (FRB) domain. This study highlights a succinct approach to expand the chemical diversity of the therapeutically important molecule rapamycin by using promiscuous glycosyltransferases. Moreover, the fact that glycosyl moieties at different positions of rapamycin affect bioactivity to different extents inspires further glycosylation engineering to improve properties of rapamycin. KEY POINTS: • Rapamycin was glycosylated efficiently by some promiscuous GTs. • Glycosylation improved water solubility, attenuated cytotoxicity, and bioactivities. • Glycosylation affected the interactions between ligand and binding proteins.