Transcriptomic analysis reveals the early body wall regeneration mechanism of the sea cucumber Holothuria leucospilota after artificially induced transverse fission.

BACKGROUND: Sea cucumbers exhibit a remarkable ability to regenerate damaged or lost tissues and organs, making them an outstanding model system for investigating processes and mechanisms of regeneration. They can also reproduce asexually by transverse fission, whereby the anterior and posterior bodies can regenerate independently. Despite the recent focus on intestinal regeneration, the molecular mechanisms underlying body wall regeneration in sea cucumbers still remain unclear. RESULTS: In this study, transverse fission was induced in the tropical sea cucumber, Holothuria leucospilota, through constrainment using rubber bands. Histological examination revealed the degradation and loosening of collagen fibers on day-3, followed by increased density but disorganization of the connective tissue on day-7 of regeneration. An Illumina transcriptome analysis was performed on the H. leucospilota at 0-, 3- and 7-days after artificially induced fission. The differential expression genes were classified and enriched by GO terms and KEGG database, respectively. An upregulation of genes associated with extracellular matrix remodeling was observed, while a downregulation of pluripotency factors Myc, Klf2 and Oct1 was detected, although Sox2 showed an upregulation in expression. In addition, this study also identified progressively declining expression of transcription factors in the Wnt, Hippo, TGF-ß, and MAPK signaling pathways. Moreover, changes in genes related to development, stress response, apoptosis, and cytoskeleton formation were observed. The localization of the related genes was further confirmed through in situ hybridization. CONCLUSION: The early regeneration of H. leucospilota body wall is associated with the degradation and subsequent reconstruction of the extracellular matrix. Pluripotency factors participate in the regenerative process. Multiple transcription factors involved in regulating cell proliferation were found to be gradually downregulated, indicating reduced cell proliferation. Moreover, genes related to development, stress response, apoptosis, and cell cytoskeleton formation were also involved in this process. Overall, this study provides new insights into the mechanisms of whole-body regeneration and uncover potential cross-species regenerative-related genes.

Heptose-containing bacterial natural products: structures, bioactivities, and biosyntheses.

Covering: up to 2020Glycosylated natural products hold great potential as drugs for the treatment of human and animal diseases. Heptoses, known as seven-carbon-chain-containing sugars, are a group of saccharides that are rarely observed in natural products. Based on the structures of the heptoses, the heptose-containing natural products can be divided into four groups, characterized by heptofuranose, highly-reduced heptopyranose, D-heptopyranose, and L-heptopyranose. Many of them possess remarkable biological properties, including antibacterial, antifungal, antitumor, and pain relief activities, thereby attracting great interest in biosynthesis and chemical synthesis studies to understand their construction mechanisms and structure-activity relationships. In this review, we summarize the structural properties, biological activities, and recent progress in the biosynthesis of bacterial natural products featuring seven-carbon-chain-containing sugars. The biosynthetic origins of the heptose moieties are emphasized.

d-Sedoheptulose-7-phosphate is a common precursor for the heptoses of septacidin and hygromycin B.

Seven-carbon-chain-containing sugars exist in several groups of important bacterial natural products. Septacidin represents a group of l-heptopyranoses containing nucleoside antibiotics with antitumor, antifungal, and pain-relief activities. Hygromycin B, an aminoglycoside anthelmintic agent used in swine and poultry farming, represents a group of d-heptopyranoses-containing antibiotics. To date, very little is known about the biosynthesis of these compounds. Here we sequenced the genome of the septacidin producer and identified the septacidin gene cluster by heterologous expression. After determining the boundaries of the septacidin gene cluster, we studied septacidin biosynthesis by in vivo and in vitro experiments and discovered that SepB, SepL, and SepC can convert d-sedoheptulose-7-phosphate (S-7-P) to ADP-l-glycero-ß-d-manno-heptose, exemplifying the involvement of ADP-sugar in microbial natural product biosynthesis. Interestingly, septacidin, a secondary metabolite from a gram-positive bacterium, shares the same ADP-heptose biosynthesis pathway with the gram-negative bacterium LPS. In addition, two acyltransferase-encoding genes sepD and sepH, were proposed to be involved in septacidin side-chain formation according to the intermediates accumulated in their mutants. In hygromycin B biosynthesis, an isomerase HygP can recognize S-7-P and convert it to ADP-d-glycero-ß-d-altro-heptose together with GmhA and HldE, two enzymes from the Escherichia coli LPS heptose biosynthetic pathway, suggesting that the d-heptopyranose moiety of hygromycin B is also derived from S-7-P. Unlike the other S-7-P isomerases, HygP catalyzes consecutive isomerizations and controls the stereochemistry of both C2 and C3 positions.

Deciphering the Biosynthesis of TDP-ß-l-oleandrose in Avermectin.

Avermectin (AVM) refers to eight macrolides containing a common l-oleandrosyl disaccharide chain indispensable to their antiparasitic bioactivities. We delineated the biosynthetic pathway of TDP-ß-l-oleandrose (1), the sugar donor of AVM, by characterizing AveBVIII, AveBV, and AveBVII as TDP-sugar 3-ketoreductase, 5-epimerase, and 3-O-methyltransferase, respectively. On the basis of this pathway, we successfully reconstituted the biosynthesis of 1 in Escherichia coli. Our work completes the biosynthetic pathway of AVM and lays a solid foundation for further studies.

Activation of paulomycin production by exogenous γ-butyrolactone signaling molecules in Streptomyces albidoflavus J1074.

The interspecies communication roles of γ-butyrolactones (GBLs) have been described for a long time but are still poorly understood. Herein, we analyzed more than 1000 Streptomyces strains and noticed a big quantitative gap between the strains with GBL biosynthetic genes and the strains with GBL receptor genes, which implies the wide-spread of GBLs as interspecies signals in Streptomyces and their great potential in the activation of silent natural product gene clusters. Streptomyces albidoflavus J1074, which has one GBL receptor gene but no GBL biosynthetic gene, was chosen as a target to study the possible interspecies communication roles of GBLs. At first, the GBL biosynthetic genes from Streptomyces coelicolor M145 were expressed in S. albidoflavus J1074, which enabled the S. albidoflavus strains to synthesize Streptomyces coelicolor butanolides (SCBs) and activated the production of paulomycins. Further studies showed that this activation process requires the participation of the GBL receptor gene XNR_4681. The results suggest that the expression of exogenous GBL biosynthetic genes can modulate the metabolisms of GBL non-producing strains, and this regulation role might be meaningful for silent gene cluster activation in Streptomyces. At final, we synthesized racemic-SCB2 and tried to simplify the activation process by adding SCB2 directly to S. albidoflavus J1074, which unfortunately failed to induce paulomycin production.

Design and Biosynthesis of Dimeric Alboflavusins with Biaryl Linkages via Regiospecific C-C Bond Coupling.

Alboflavusins (AFNs) are a group of cyclohexapeptides with moderate antibacterial and antitumor activities from Streptomyces alboflavus sp. 313. In vivo and in vitro studies proposed that AFNs are biosynthesized by a nonribosomal peptide synthetase machinery, and the 6-Cl-L-Trp precursor is supplied by a tryptophan halogenase gene located outside the afn gene cluster. Guided by the structure-activity relationship knowledge about the AFN-like cyclohexapeptides, two dimeric AFNs (di-AFNs) with regiospecific biaryl linkages were designed and generated biotechnologically by expressing the P450 gene hmtS or clpS in S. alboflavus wild-type and mutant strains. The di-AFNs displayed much better antibacterial and antitumor activities than their monomers as anticipated, exemplifying a rational strategy to generate natural product congeners with improved bioactivities.

Hexaricins, Pradimicin-like Polyketides from a Marine Sediment-Derived Streptosporangium sp. and Their Antioxidant Effects.

Seven pradimicin-like polyketides were isolated from the dichloromethane extract of the marine sediment-derived Streptosporangium sp. CGMCC 4.7309, including five new hexaricins, D-H (1-5), and known hexaricins A (6) and C (7). Their structures were determined by HRESIMS, 1D and 2D NMR, and other spectroscopic analyses. The absolute configurations of compounds 1-5 were determined on the basis of circular dichroism and specific rotation data. All isolated compounds 1-7 were tested for their antioxidant capacities by DPPH• scavenging, •OH scavenging, and •O2̅ scavenging assays. Compounds 3 and 4 displayed stronger antioxidant activities than the positive control ( tert-butylhydroquinone). The relationship between structure and antioxidant activity is discussed. These compounds could be effective natural antioxidants with considerable pharmaceutical value.

Discovery of pentangular polyphenols hexaricins A-C from marine Streptosporangium sp. CGMCC 4.7309 by genome mining.

Many novel microbial nature products were discovered from Actinobacteria by genome mining methods. However, only a few number of genome mining works were carried out in rare actinomycetes. An important reason precluding the genome mining efforts in rare actinomycetes is that most of them are recalcitrant to genetic manipulation. Herein, we chose the rare marine actinomycete Streptosporangium sp. CGMCC 4.7309 to explore its secondary metabolite diversity by genome mining. The genetic manipulation method has never been established for Streptosporangium strains. At first, we set up the genetic system of Streptosporangium sp. CGMCC 4.7309 unprecedentedly. The draft genome sequencing of Streptosporangium sp. CGMCC 4.7309 revealed that it contains more than 20 cryptic secondary metabolite biosynthetic clusters. A type II polyketide synthases-containing cluster (the hex cluster) was predicted to encode compounds with a pentangular polyphenol scaffold by in silico analysis. The products of the hex cluster were uncovered by comparing the metabolic profile of Streptosporangium sp. CGMCC 4.7309 with that of the hex30 inactivated mutant, in which a key ketoreductase gene was disrupted. Finally, three pentangular polyphenols were isolated and named as hexaricins A (1), B (2), and C (3). The inconsistency of the stereochemistry of C-15 in hexaricins A, B, and C indicates a branch point in their biosynthesis. Finally, the biosynthetic pathway of the hexaricins was proposed based on bioinformatics analysis.

An efficient blue-white screening based gene inactivation system for Streptomyces.

Streptomyces is studied intensively for its outstanding ability to produce bioactive secondary metabolites and for its complicated morphological differentiation process. A classical genetic manipulation system for Streptomyces has been developed and widely used in the community for a long time, using antibiotic resistance markers to select for double-crossover mutants. The screening process is always laborious and time-consuming. However, the lack of a suitable chromogenic reporter for Streptomyces has limited the use of color-based screening system to simplify the selection process for double-crossover mutants. In this study, a blue reporter system for Streptomyces has been established by mining an indigoidine synthetase gene (idgS) from Streptomyces lavendulae CGMCC 4.1386, leading to the development of a time-saving gene inactivation system for Streptomyces by simple blue-white screening. A series of Streptomyces suicide and temperature-sensitive plasmids containing the idgS reporter cassette were constructed and used successfully to inactivate genes in Streptomyces, allowing a simple and efficient screening method to differentiate the colonies for double-crossover (white) and single-crossover (blue) mutants. Inactivation of the putative γ-butyrolactone synthase gene afsA-y via the idgS-based blue-white screening method revealed that the paulomycin production is negatively controlled by afsA-y in Streptomyces sp. YN86.

Biosynthesis of the carbamoylated D-gulosamine moiety of streptothricins: involvement of a guanidino-N-glycosyltransferase and an N-acetyl-D-gulosamine deacetylase.

Streptothricins (STNs) are atypical aminoglycosides containing a rare carbamoylated D-gulosamine (D-GulN) moiety, and the antimicrobial activity of STNs has been exploited for crop protection. Herein, the biosynthetic pathway of the carbamoylated D-GulN moiety was delineated. An N-acetyl-D-galactosamine is first attached to the streptolidine lactam by the glycosyltransferse StnG and then epimerized to N-acetyl-D-gulosamine by the putative epimerase StnJ. After carbamoylation by the carbamoyltransferase StnQ, N-acetyl-D-GulN is deacetylated by StnI to furnish the carbamoylated D-GulN moiety. In vitro studies characterized two novel enzymes: StnG is an unprecedented GT-A fold N-glycosyltransferase that glycosylates the imine nitrogen atom of guanidine, and StnI is the first reported N-acetyl-D-GulN deacetylase.

Digitoxose as powerful glycosyls for building multifarious glycoconjugates of natural products and un-natural products.

Digitoxose, a significant 2,6-dideoxyhexose found in nature, exists in many small-molecule natural products. These digitoxose-containing natural products can be divided into steroids, macrolides, macrolactams, anthracyclines, quinones, enediynes, acyclic polyene, indoles and oligosaccharides, that exhibit antibacterial, anti-viral, antiarrhythmic, and antitumor activities respectively. As most of digitoxose-containing natural products for clinical application or preclinical tests, this review also summarizes the biosynthesis of digitoxose, and application of compound diversification by introducing sugar plasmids. It may provide a practical approach to expanding the diversity of digitoxose-containing products.

Design, synthesis and evaluation of antioxidant and anti-inflammatory activities of novel resveratrol derivatives as potential multifunctional drugs.

Oxidative stress and inflammation responses are closely related to the occurrence and development of many diseases. Therefore, anti-oxidation and anti-inflammation have become hot spots in the treatment of diseases. A series of novel resveratrol derivatives which hybrid with benzoylhydrazines were designed, synthesized and assessed for their in vitro antioxidant and anti-inflammatory activity. Initially, the antioxidant abilities of resveratrol derivatives were investigated by DPPH, ABTS radical scavenging and FRAP assays. RAW 264.7 macrophages are routinely used to evaluate the antioxidant and anti-inflammatory activities of drugs, so we used it to construct cell models of oxidative stress and inflammation. Among all the derivatives, compound 5 exhibited superior ROS- and NO-inhibitory activities. The molecular mechanism detected by Western blotting showed that compound 5 could significantly activate the Nrf2 signaling pathway and up-regulate the expression of HO-1 to resist oxidative stress stimulated by H2O2. At the same time, it could down-regulate the expression of apoptosis-related proteins Caspase3 and PARP, alleviating cells damage and apoptosis. In addition, compound 5 dose-dependently inhibited the activation of NF-κB p65/iNOS and MAPKs signaling pathway.

Fungicide-tolerant persister formation during cryptococcal pulmonary infection.

Bacterial persisters, a subpopulation of genetically susceptible cells that are normally dormant and tolerant to bactericides, have been studied extensively because of their clinical importance. In comparison, much less is known about the determinants underlying fungicide-tolerant fungal persister formation in vivo. Here, we report that during mouse lung infection, Cryptococcus neoformans forms persisters that are highly tolerant to amphotericin B (AmB), the standard of care for treating cryptococcosis. By exploring stationary-phase indicator molecules and developing single-cell tracking strategies, we show that in the lung, AmB persisters are enriched in cryptococcal cells that abundantly produce stationary-phase molecules. The antioxidant ergothioneine plays a specific and key role in AmB persistence, which is conserved in phylogenetically distant fungi. Furthermore, the antidepressant sertraline (SRT) shows potent activity specifically against cryptococcal AmB persisters. Our results provide evidence for and the determinant of AmB-tolerant persister formation in pulmonary cryptococcosis, which has potential clinical significance.

Insights into the biosynthesis of septacidin l-heptosamine moiety unveils a VOC family sugar epimerase.

l-Heptopyranoses are important components of bacterial polysaccharides and biological active secondary metabolites like septacidin (SEP), which represents a group of nucleoside antibiotics with antitumor, antifungal, and pain-relief activities. However, little is known about the formation mechanisms of those l-heptose moieties. In this study, we deciphered the biosynthetic pathway of the l,l-gluco-heptosamine moiety in SEPs by functional characterizing four genes and proposed that SepI initiates the process by oxidizing the 4'-hydroxyl of l-glycero-α-d-manno-heptose moiety of SEP-328 (2) to a keto group. Subsequently, SepJ (C5 epimerase) and SepA (C3 epimerase) shape the 4'-keto-l-heptopyranose moiety by sequential epimerization reactions. At the last step, an aminotransferase SepG installs the 4'-amino group of the l,l-gluco-heptosamine moiety to generate SEP-327 (3). An interesting phenomenon is that the SEP intermediates with 4'-keto-l-heptopyranose moieties exist as special bicyclic sugars with hemiacetal-hemiketal structures. Notably, l-pyranose is usually converted from d-pyranose by bifunctional C3/C5 epimerase. SepA is an unprecedented monofunctional l-pyranose C3 epimerase. Further in silico and experimental studies revealed that it represents an overlooked metal dependent-sugar epimerase family bearing vicinal oxygen chelate (VOC) architecture.

Enhanced production of a novel cyclic hexapeptide antibiotic (NW-G01) by Streptomyces alboflavus 313 using response surface methodology.

NW-G01, produced by Streptomyces alboflavus 313, is a novel cyclic hexapeptide antibiotic with many potential applications, including antimicrobial activity and antitumor agents. This study developed a system for optimizing medium components in order to enhance NW-G01 production. In this study, Plackett-Burman design (PBD) was used to find the key ingredients of medium components, and then response surface methodology (RSM) was implemented to determine their optimal concentrations. The results of PBD revealed that the crucial ingredients related to the production of NW-G01 were (NH(4))(2)SO(4), peptone and CaCO(3). A prediction model has been built in the experiments of central composite design and response surface methodology, and its validation has been further verified. The optimal medium composition was determined (g/L): corn starch 15, glucose 15, peptone 3.80, (NH(4))(2)SO(4) 0.06, NaCl 1.5, CaCO(3) 1.30, MgSO(4)·7H(2)O 0.015, K(2)HPO(4)·3H(2)O 0.015, MnCl(2)·4H(2)O 0.015, FeSO(4)·7H(2)O 0.015, and ZnSO(4)·7H(2)O 0.015. Compared with NW-G01 production (5.707 mg/L) in non-optimized fermentation medium, the production of NW-G01 (15.564 mg/L) in optimized fermentation medium had a 2.73-fold increase.

[Diversity and antimicrobial activities of actinomycetes from pesticide-contaminated spots in Shandong Peninsula].

OBJECTIVE: In order to study diversity and find antimicrobial activities of actinomycetes from pesticide-contaminated spots in Shandong Peninsula. METHODS: The phylogenetic analysis of 154 isolated strains was done based on 16SrDNA sequences. Antimicrobial activities of 10 non-Streptomyces strains were tested by using cylinder-plate method and hypha growth rate method. RESULTS: Among the strains, 154 strains belonged to 7 families, 8 genera: Streptomyces (87.01%), Kocuria, Microbacterium, Nocardiopsis, Knoellia, Pseudonocardia, Micromonospora, Actinoplanes. The fermentation broths of 10 non-Streptomyces strains had inhibitory actives against all tested phytopathogenic fungi (Botrytis cinerea, Fusarium oxysporum f. sp. niveum, Gibberella zeae, Sclerotinia sclerotiorum, Colletotrichum gloesporioides) and bacteria (Staphylococcus aureus, Bacillus subtills, Bacillus cereus, Escherichai coli, Pseudomonas aeruginos) , especially Microbacterium oxydans JN853773 and Kocuria rosea JN192402 had strong inhibitory effects. CONCLUSION: Abundant diversity of actinomycetes existed in pesticide contaminated spots in Shandong Peninsula. Microbacterium oxydans JN853773 and Kocuria rosea JN192402 showed high antimicrobial activities and could be further exploited.

Discovery of novel septacidin congeners from a high yield heterologous expression strain Streptomyces albus 1597.

Septacidin is an adenine nucleoside antibiotic with antifungal and antitumor activities. During the efforts to construct a better septacidin producer, we obtained a high yield strain S. albus 1597 by putting the biosynthetic gene cluster (BGC) of septacidin under the control of the constitutive strong promoter ermE*. S. albus 1597 could produce new septacidin congeners SEP-538 and SEP-552 with shorter fatty acyl chains. Moreover, SEP-624 with an unprecedented hydroxylated fatty acyl chain was also isolated from this titre improved strain, enriching the diversity of septacidins. SEP-552 showed moderate inhibitory effects against Epidermophyton floccosum 57312 with MIC value 62.5 µM, while SEP-538 and SEP-624 only exhibited weak antifungal activities. The structure-activity relationship investigation revealed that the antifungal activity of septacidins is significantly influenced by the length of and the decoration on their fatty acyl chains.

Construction and heterologous expression of the di-AFN A1 biosynthetic gene cluster in Streptomyces model strains.

Natural cyclohexapeptide AFN A1 fromStreptomyces alboflavus 313 has moderate antibacterial and antitumor activities. An artificial designed AFN A1 homodimer, di-AFN A1, is an antibiotic exhibiting 10 to 150 fold higher biological activities, compared with the monomer. Unfortunately, the yield of di-AFN A1 is very low (0.09 ± 0.03 mg·L-1) in the engineered strain Streptomyces alboflavus 313_hmtS (S. albo/313_hmtS), which is not friendly to be genetically engineered for titer improvement of di-AFN A1 production. In this study, we constructed a biosynthetic gene cluster for di-AFN A1 and increased its production through heterologous expression. During the collection of di-AFN A1 biosynthetic genes, the afn genes were located at three sites of S. alboflavus 313 genome. The di-AFN A1 biosynthetic gene cluster (BGC) was first assembled on one plasmid and introduced into the model strain Streptomyces lividans TK24, which produced di-AFN A1 at a titer of 0.43 ± 0.01 mg·L-1. To further increase the yield of di-AFN A1, the di-AFN A1 BGC was multiplied and split to mimic the natural afn biosynthetic genes, and the production of di-AFN A1 increased to 0.62 ± 0.11 mg·L-1 in S. lividans TK24 by the later strategy. Finally, different Streptomyces hosts were tested and the titer of di-AFN A1 increased to 0.81 ± 0.17 mg·L-1, about 8.0-fold higher than that in S. albo/313_hmtS. Successful heterologous expression of di-AFN A1 with a remarkable increased titer will greatly facilitate the following synthetic biological study and drug development of this dimeric cyclohexapeptide.

Identification of antimicrobial peptides from the human gut microbiome using deep learning.

The human gut microbiome encodes a large variety of antimicrobial peptides (AMPs), but the short lengths of AMPs pose a challenge for computational prediction. Here we combined multiple natural language processing neural network models, including LSTM, Attention and BERT, to form a unified pipeline for candidate AMP identification from human gut microbiome data. Of 2,349 sequences identified as candidate AMPs, 216 were chemically synthesized, with 181 showing antimicrobial activity (a positive rate of >83%). Most of these peptides have less than 40% sequence homology to AMPs in the training set. Further characterization of the 11 most potent AMPs showed high efficacy against antibiotic-resistant, Gram-negative pathogens and demonstrated significant efficacy in lowering bacterial load by more than tenfold against a mouse model of bacterial lung infection. Our study showcases the potential of machine learning approaches for mining functional peptides from metagenome data and accelerating the discovery of promising AMP candidate molecules for in-depth investigations.

Discovery of Brominated Alboflavusins With Anti-MRSA Activities.

As methicillin-resistant Staphylococcus aureus (MRSA) is becoming a serious pathogenic threaten to human health worldwide, there is an urgent need to discover new antibiotics for the treatment of MRSA infections. Alboflavusins (AFNs) are a group of halogenated cyclohexapeptides with anti-MRSA activities. In this study, two novel brominated AFN congeners (compounds 1 and 2) were isolated from the wild-type strain Streptomyces alboflavus sp. 313 that was fermented in the production medium supplemented with NaBr; two new (compounds 3 and 5) and a known (compound 4) dehelogenated AFN congeners were isolated from S. alboflavus ΔafnX, in which the tryptophan halogenase gene afnX was inactivated. The structures of these compounds were assigned by careful NMR and MS analyses. The anti-MRSA activities of varied AFN congeners were assessed against different MRSA strains, which revealed that compounds 1 and 2 with bromine displayed effective activities against the tested MRSA strains. Especially, compound 2 showed good anti-MRSA activity, while compounds 3, 4, and 5 without halogen exhibited weak anti-MRSA activities, outlining the influence of halogen substitution to the bioactivities of AFNs.