Newly identified maltol derivatives in Korean Red Ginseng and their biological influence as antioxidant and anti-inflammatory agents.

Background: Korean Red Ginseng is a major source of bioactive substances such as ginsenosides. Efficacy of red ginseng extract (RGE), which contains not only saponins but also various non-saponins, has long been studied. In the water-soluble component-rich fraction of RGE (WS), a byproduct generated in the process of extracting saponins from the RGE, we identified previously unidentified molecules and confirmed their efficacy. Methods: The RGE was prepared and used to produce WS, whose components were isolated sequentially according to their water affinity. The new compounds from WS were fractionized and structurally analyzed using nuclear magnetic resonance spectroscopy. Physiological applicability was evaluated by verifying the antioxidant and anti-inflammatory efficacies of these compounds in vitro. Results: High-performance liquid chromatography confirmed that the obtained WS comprised 11 phenolic acid and flavonoid substances. Among four major compounds from fractions 1-4 (F1-4) of WS, two compounds from F3 and F4 were newly identified in red ginseng. The analysis results show that these compound molecules are member of the maltol-structure-based glucopyranose series, and F1 and F4 are particularly effective for decreasing oxidative stress levels and inhibiting nitric oxide secretion, interleukin (IL)-1ß and IL-6, and tumor necrosis factor-α. Conclusion: Our findings suggest that a few newly identified maltol derivatives, such as red ginseng-derived non-saponin in the WS, exhibit antioxidant and anti-inflammatory effects, making them viable candidates for application to pharmaceutical, cosmetic, and functional food materials.

Effects of Phenolic-Rich Pinus densiflora Extract on Learning, Memory, and Hippocampal Long-Term Potentiation in Scopolamine-Induced Amnesic Rats.

Alzheimer's disease is the most common type of dementia with cognitive impairment. Various plant-derived phenolics are known to alleviate cognitive impairment in Alzheimer's disease by radical scavenging and strengthening synaptic plasticity activities. Here, we examined the cognition-improving effect of Pinus densiflora Sieb. et Zucc. bark extract (PBE). We identified and quantified phenolics in the PBE using a UHPLC-Orbitrap mass spectrometer. To evaluate the cognition-enhancing effects of PBE, scopolamine-induced amnesic Sprague-Dawley (SD) rats (5 weeks old) and ion channel antagonist-induced organotypic hippocampal slices of SD rats (7 days old) were used. Twenty-three phenolics were tentatively identified in PBE, 10 of which were quantified. Oral administration of PBE to the scopolamine-induced SD rats improved cognitive impairment in behavioral tests. PBE-fed SD rats showed significantly improved antioxidant indices (superoxide dismutase and catalase activities, and malondialdehyde content) and reduced acetylcholinesterase activity in hippocampal lysate compared with the scopolamine group. PBE increased the long-term potentiation (LTP) induction and rescued LTP from blockades by the muscarinic cholinergic receptor antagonist (scopolamine) and N-methyl-D-aspartate channel antagonist (2-amino-5-phosphonovaleric acid) in the organotypic hippocampal slices. These results suggest that polyphenol-rich PBE is applicable as a cognition-improving agent due to its antioxidant properties and enhancement of LTP induction.

Whole-genome sequencing and analysis of Streptomyces strains producing multiple antinematode drugs.

BACKGROUND: Nematodes are parasitic animals that cause over 100 billion US dollars loss in agricultural business. The whole-genomes of two Streptomyces strains, Streptomyces spectabilis KCTC9218T and Streptomyces sp. AN091965, were sequenced. Both strains produce spectinabilin, an antinematode drug. Its secondary metabolism was examined to aid the development of an efficient nematicidal drug-producing host strain. RESULTS: The whole-genome sequences of S. spectabilis KCTC9218T and Streptomyces sp. AN091965 were analyzed using PacBio and Illumina sequencing platforms, and assembled using hybrid methodology. The total contig lengths for KCTC9218T and AN091965 were 9.97 Mb and 9.84 Mb, respectively. A total of 8,374 and 8,054 protein-coding genes, as well as 39 and 45 secondary metabolite biosynthetic gene clusters were identified in KCTC9218T and AN091965, respectively. 18.4 ± 6.45 mg/L and 213.89 ± 21.30 mg/L of spectinabilin were produced by S. spectabilis KCTC9218T and Streptomyces sp. AN091965, respectively. Pine wilt disease caused by nematode was successfully prevented by lower concentration of spectinabilin injection than that of abamectin recommended by its manufacturer. Production of multiple antinematode drugs, including spectinabilin, streptorubin B, and undecylprodigiosin was observed in both strains using high-resolution liquid chromatography mass spectrometry (LC-MS) analysis. CONCLUSIONS: Whole-genome sequencing of spectinabilin-producing strains, coupled with bioinformatics and mass spectrometry analyses, revealed the production of multiple nematicidal drugs in the KCTC9218T and AN091965 strains. Especially, Streptomyces sp. AN091965 showed high production level of spectinabilin, and this study provides crucial information for the development of potential nematicidal drug producers.

Mass Biosynthesis of Coumestrol Derivatives and Their Isomers via Soybean Adventitious Root Cultivation in Bioreactors.

Coumestrol (CMS) derivatives are unique compounds, which function as phytoalexins; they are derived from soybean roots, following abiotic and biotic stresses. As a phytoalexin, CMS forms a defense system that enables plants to maintain their viability. However, it is still challenging to achieve the mass production of phytoalexins, which exhibit pharmacological values, via plant breeding. Here, the synthesis of CMS derivatives from the seedling, plant, and adventitious root (AR) of Glycine max were investigated under artificial light, as well as via a chemical elicitor treatment. In the presence of constant light, as well as under treatment with methyl jasmonate, the CMS monoglucoside (coumestrin; CMSN) and malonyl CMSN (M-CMSN) contents of the AR culture (4 weeks) increased drastically. The two CMS derivatives, CMSN and M-CMSN, were obtained as a mixture of isomers, which were identified via nuclear magnetic resonance analysis. These derivatives were also observed in a soybean plant that was grown on artificial soil (AS; 5 weeks) and a Petri dish (9 days) although in considerably lesser quantities than those observed in the AR culture. Compared with the two other media (AS and the Petri dish), the AR culture achieved the superior synthesis of CMSN and M-CMSN within a relatively short cultivation period (<1 month) in laboratory-scale (3 L) and pilot-scale (1,000 L) bioreactors. The isoflavone content of AR under the constant light conditions was three-fold that under dark conditions. Significant quantities of malonyl daidzin and malonyl genistin were produced in the root of AS and the seedling of Petri dish, respectively. Flavonol glycosides were not produced in the AR culture under the dark and light conditions, as well as in AS under the dark condition. However, significant contents of kaempferol glycosides were produced in the leaves of AS and seedling of Petri dish, following the light treatment. Thus, we proposed that the established soybean AR-cultivation approach represented a better method for biosynthesizing phytoalexins, such as the CMS derivatives, as plant-derived functional materials.

Taeanamides A and B, Nonribosomal Lipo-Decapeptides Isolated from an Intertidal-Mudflat-Derived Streptomyces sp.

Two new lipo-decapeptides, namely taeanamides A and B (1 and 2), were discovered from the Gram-positive bacterium Streptomyces sp. AMD43, which was isolated from a mudflat sample from Anmyeondo, Korea. The exact molecular masses of 1 and 2 were revealed by high-resolution mass spectrometry, and the planar structures of 1 and 2 were elucidated using NMR spectroscopy. The absolute configurations of 1 and 2 were determined using a combined analysis of 1H-1H coupling constants and ROESY correlations, the advanced Marfey's method, and bioinformatics. The putative nonribosomal peptide synthetase pathway for the taeanamides was identified by analyzing the full genome sequence data of Streptomyces sp. AMD43. We also found that taeanamide A exhibited mild anti-tuberculosis bioactivity, whereas taeanamide B showed significant bioactivity against several cancer cell lines.

Cyclodimerization of Mohangamide A by Thioesterase Domain Is Directed by Substrates.

Mohangamide A is a pseudo-dimeric nonribosomal peptide biosynthesized along with its monomer, WS9326A, and is expected to be formed by the head-to-tail cyclodimerization of linear WS9326A and another identical peptide chain with a different acyl side chain. In vitro experiments with the N-acetylcysteamine thioesters of the corresponding monomeric intermediates and thioesterase domains of Streptomyces sp. SNM55 and S. calvus showed that this cyclodimerization reaction is directed by the substrate structures and occurs only with both linear intermediates.

Microbial Enzymatic Synthesis of Amikacin Analogs With Antibacterial Activity Against Multidrug-Resistant Pathogens.

With the constant emergence of multidrug-resistant gram-negative bacteria, interest in the development of new aminoglycoside (AG) antibiotics for clinical use has increased. The regioselective modification of AG scaffolds could be an efficient approach for the development of new antibiotics with improved therapeutic potency. We enzymatically synthesized three amikacin analogs containing structural modifications in the amino groups and evaluated their antibacterial activity and cytotoxicity. Among them, 6'-N-acyl-3″-N-methylated analogs showed improved antibacterial activity against the multidrug-resistant gram-negative bacteria tested, while exhibiting reduced in vitro nephrotoxicity compared to amikacin. This study demonstrated that the modifications of the 6'-amino group as well as the 3″-amino group have noteworthy advantages for circumventing the AG-resistance mechanism. The regiospecific enzymatic modification could be exploited to develop novel antibacterial agents with improved pharmacological potential.

Unprecedented Noncanonical Features of the Nonlinear Nonribosomal Peptide Synthetase Assembly Line for WS9326A Biosynthesis.

Systematic inactivation of nonribosomal peptide synthetase (NRPS) domains and translocation of the thioesterase (TE) domain revealed several unprecedented nonlinear NRPS assembly processes during the biosynthesis of the cyclodepsipeptide WS9326A in Streptomyces sp. SNM55. First, two sets of type ΙΙ TE (TEΙΙ)-like enzymes mediate the shuttling of activated amino acids between two sets of stand-alone adenylation (A)-thiolation (T) didomain modules and an "A-less" condensation (C)-T module with distinctive specificities and flexibilities. This was confirmed by the elucidation of the affinities of the A-T didomains for the TEΙΙs and its structure. Second, the C-T didomain module operates iteratively and independently from other modules in the same protein to catalyze two chain elongation cycles. Third, this biosynthetic pathway includes the first example of module skipping, where the interpolated C and T domains are required for chain transfer.

Biosynthesis of Nonimmunosuppressive ProlylFK506 Analogues with Neurite Outgrowth and Synaptogenic Activity.

Separating the immunosuppressive activity of FK506 (1) from its neurotrophic activity is required to develop FK506 analogues as drugs for the treatment of neuronal diseases. Two new FK506 analogues, 9-deoxo-36,37-dihydro-prolylFK506 (2) and 9-deoxo-31-O-demethyl-36,37-dihydro-prolylFK506 (3) containing a proline moiety instead of the pipecolate ring at C-1 and modifications at the C-9/C-31 and C-36-C-37 positions, respectively, were biosynthesized, and their biological activities were evaluated. The proline substitution in 9-deoxo-36,37-dihydroFK506 and 9-deoxo-31-O-demethyl-36,37-dihydroFK506 reduced immunosuppressive activity by more than 120-fold, as previously observed. Compared with FK506 (1), 2 and 3 exhibited ∼1.2 × 105- and 2.2 × 105-fold reductions in immunosuppressive activity, respectively, whereas they retained almost identical neurite outgrowth activity. Furthermore, these compounds significantly increased the strength of synaptic transmission, confirming that replacement of the pipecolate ring with a proline is critical to reduce the strong immunosuppressive activity of FK506 (1) while enhancing its neurotrophic activity.

Anti-Inflammatory Effect of Immature Sword Bean Pod (Canavalia gladiata) in Lipopolysaccharide-Induced RAW264.7 Cells.

Sword bean has been known as a traditional medicinal plant to treat cancer, sinus infection, and suppurative disease. It also possesses hypertension-relieving, antioxidation, and antibacterial effects. However, studies on the efficacy of sword bean are limited to mature beans. Few studies have focused on immature sword bean pod (ISBP). Therefore, this study aimed to investigate the anti-inflammatory effect of ISBP in RAW264.7 cells stimulated with lipopolysaccharide (LPS). After LPS-induced RAW264.7 cells were treated with ISBP at concentrations (0.5, 1, 2, and 5 mg/mL), levels of nitrite oxide (NO) and prostaglandin E2 (PGE2) production, protein, and mRNA levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), inflammatory cytokine secretion level, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity were determined. Under inflammatory conditions induced by LPS, ISBP reduced levels of inflammatory mediators NO and PGE2 by 60% and 23%, respectively. It also decreased protein and mRNA expression levels of iNOS and COX-2 known to synthesize inflammatory mediators. Inflammatory cytokines, interleukin (IL)-6, and IL-1ß, levels were decreased, while interferon gamma level was increased by ISBP based on enzyme-linked immunosorbent assay (ELISA) and real time-polymerase chain reaction results. Finally, ISBP showed the ability to inhibit NF-κB activity. In conclusion, ISBP can alleviate inflammation by controlling inflammation-related substances, and may have efficacy as a healthful functional food and natural anti-inflammatory drug.

Publisher Correction: Complete reconstitution of the diverse pathways of gentamicin B biosynthesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Development of 6'-N-Acylated Isepamicin Analogs with Improved Antibacterial Activity Against Isepamicin-Resistant Pathogens.

The development of new aminoglycoside (AG) antibiotics has been required to overcome the resistance mechanism of AG-modifying enzymes (AMEs) of AG-resistant pathogens. The AG acetyltransferase, AAC(6')-APH(2″), one of the most typical AMEs, exhibiting substrate promiscuity towards a variety of AGs and acyl-CoAs, was employed to enzymatically synthesize new 6'-N-acylated isepamicin (ISP) analogs, 6'-N-acetyl/-propionyl/-malonyl ISPs. They were all active against the ISP-resistant Gram-negative bacteria tested, and the 6'-N-acetyl ISP displayed reduced toxicity compared to ISP in vitro. This study demonstrated the importance of the modification of the 6'-amino group in circumventing AG-resistance and the potential of regioselective enzymatic modification of AG scaffolds for the development of more robust AG antibiotics.

Minor components of aminoglycosides: recent advances in their biosynthesis and therapeutic potential.

Covering: up to 2019 There is significant demand for new aminoglycoside antibiotics due to the widespread emergence of multidrug-resistant Gram-negative bacteria and their high toxicity, but these are not easily accessible in nature because their biosynthetic gene clusters are less commonly found in actinomycetes than are other natural products. Mining minor aminoglycoside components whose pharmacological activity has not yet been assessed could be an alternative approach for the development of next-generation antibiotics for use in the post-antibiotic era. Here, we review the biosynthetic steps responsible for the structural diversity of aminoglycosides and highlight current developments regarding the use of natural minor and semi-synthetic aminoglycosides as promising therapeutic leads or candidates.

Characterization of the Ohmyungsamycin Biosynthetic Pathway and Generation of Derivatives with Improved Antituberculosis Activity.

The cyclic depsipeptides ohmyungsamycin (OMS) A (1) and B (2), isolated from the marine-derived Streptomyces sp. SNJ042, contain two non-proteinogenic amino acid residues, ß-hydroxy-l-phenylalanine (ß-hydroxy-l-Phe) and 4-methoxy-l-tryptophan (4-methoxy-l-Trp). Draft genome sequencing of Streptomyces sp. SNJ042 revealed the OMS biosynthetic gene cluster consisting of a nonribosomal peptide synthetase (NRPS) gene and three genes for amino acid modification. By gene inactivation and analysis of the accumulated products, we found that OhmL, encoding a P450 gene, is an l-Phe ß-hydroxylase. Furthermore, OhmK, encoding a Trp 2,3-dioxygenase homolog, and OhmJ, encoding an O-methyltransferase, are suggested to be involved in hydroxylation and O-methylation reactions, respectively, in the biosynthesis of 4-methoxy-l-Trp. In addition, the antiproliferative and antituberculosis activities of the OMS derivatives dehydroxy-OMS A (4) and demethoxy-OMS A (6) obtained from the mutant strains were evaluated in vitro. Interestingly, dehydroxy-OMS A (4) displayed significantly improved antituberculosis activity and decreased cytotoxicity compared to wild-type OMS A.

Microbial production of O-methylated flavanones from methylated phenylpropanoic acids in engineered Escherichia coli.

Methylated flavonoids possess improved bioactivities compared to their unmethylated counterparts. In this study, for the efficient production of O-methylated flavonoids from simple methylated phenylpropanoic acids, a recombinant Escherichia coli strain expressing 4-coumarate:coenzyme A ligase (4CL) from Oryza sativa and chalcone synthase (CHS) from Hordeum vulgare was constructed; this strain produced significant amount of homoeriodictyol (~ 52 mg/L) as well as a few amount of hesperetin (0.4 mg/L), respectively, from ferulic acid and 4-methylcaffeic acid. This demonstrates, for the first time, that the scarce but valuable methylated flavanones can be successfully produced from methylated phenylpropanoic acids in a microbial host via an artificial biosynthetic pathway consisting of 4CL and CHS that can accept O-methylated precursors.

A Review of the Microbial Production of Bioactive Natural Products and Biologics.

A variety of organisms, such as bacteria, fungi, and plants, produce secondary metabolites, also known as natural products. Natural products have been a prolific source and an inspiration for numerous medical agents with widely divergent chemical structures and biological activities, including antimicrobial, immunosuppressive, anticancer, and anti-inflammatory activities, many of which have been developed as treatments and have potential therapeutic applications for human diseases. Aside from natural products, the recent development of recombinant DNA technology has sparked the development of a wide array of biopharmaceutical products, such as recombinant proteins, offering significant advances in treating a broad spectrum of medical illnesses and conditions. Herein, we will introduce the structures and diverse biological activities of natural products and recombinant proteins that have been exploited as valuable molecules in medicine, agriculture and insect control. In addition, we will explore past and ongoing efforts along with achievements in the development of robust and promising microorganisms as cell factories to produce biologically active molecules. Furthermore, we will review multi-disciplinary and comprehensive engineering approaches directed at improving yields of microbial production of natural products and proteins and generating novel molecules. Throughout this article, we will suggest ways in which microbial-derived biologically active molecular entities and their analogs could continue to inspire the development of new therapeutic agents in academia and industry.

Biosynthesis of Nonimmunosuppressive FK506 Analogues with Antifungal Activity.

A reduction in the strong immunosuppressive activity of FK506 (1) is essential for developing this compound as an antifungal agent. Seven new FK506 analogues modified at both the FK506-binding protein 12- and the calcineurin-binding regions were biosynthesized. 9-DeoxoFK520 (7) exhibited a >900-fold reduction in the in vitro immunosuppressive activity but maintained significant antifungal activity, indicating that the C-9 and C-21 positions are critical for separation of immunosuppressive and antifungal activities. 7 exhibited robust synergistic antifungal activity with fluconazole. FK506 (1) is a 23-membered macrolide produced by several Streptomyces species and is used as an immunosuppressive drug to prevent the rejection of transplanted organs. FK506 has also exhibited antifungal, neuroprotective, and neuroregenerative activities. In humans, FK506 binds to FK506-binding protein (FKBP) 12, and the resulting FKBP12-FK506 complex interacts with a Ca2+-calmodulin-dependent phosphatase, calcineurin (CaN). Inactivation of CaN by forming the FKBP12-FK506-CaN ternary complex prevents the activation of nuclear factor of activated T cells (NF-AT), inhibiting the production of interleukin-2 and subsequent T-cell proliferation. This CaN signaling pathway also plays a critical role in the growth and pathogenesis of major fungal pathogens such as Cryptococcus neoformans, Candida albicans, and Aspergillus fumigatus. Therefore, the synthesis of FK506 analogues that can discriminate human FKBP12/CaN from its fungal counterparts may separate antifungal activity from the immunosuppressive activity, thereby allowing the development of a novel antifungal agent.

High-yield production of multiple O-methylated phenylpropanoids by the engineered Escherichia coli-Streptomyces cocultivation system.

BACKGROUND: O-Methylated phenylpropanoids, which are generally present in small amounts in plants, have improved or distinct biological activities and pharmacological properties as opposed to their unmethylated counterparts. Although microbial production could be a useful tool for the efficient and environment-friendly production of methylated phenylpropanoids, a high-yield microbial production of neither tri-methylated stilbenes nor di-/tri-methylated flavonoids has been achieved to date. RESULTS: A methyltransferase from Streptomyces avermitilis (SaOMT2), which has been known to possess 7-O-methylation activity toward several flavonoids, exhibited more diverse regiospecificity and catalyzed mono-, di-, and tri-methylation of stilbene, flavanone, and flavone when it was expressed in Streptomyces venezuelae. For the efficient production of multi-methylated phenylpropanoids, a cocultivation system was developed by employing engineered Escherichia coli strains producing pterostilbene, naringenin, and apigenin, respectively, along with SaOMT2-expressing S. venezuelae mutant. Consequently, high-yield microbial production of tri-methylated stilbenes and di-/tri-methylated flavonoids (including 3,5,4'-trimethoxystilbene, 5-hydroxy-7,4'-dimethoxyflavanone, 4'-hydroxy-5,7-dimethoxyflavanone, 5,7,4'-trimethoxyflavanone, 5-hydroxy-7,4'-dimethoxyflavone, and 5,7,4'-trimethoxyflavone) has been demonstrated for the first time. CONCLUSIONS: This cocultivation system based on the phenylpropanoid-producing E. coli and SaOMT2-expressing S. venezuelae provides an efficient tool for producing scarce and potentially valuable multi-methylated phenylpropanoids and will enable further development of these compounds as pharmaceuticals and nutraceuticals.

Author Correction: Complete reconstitution of the diverse pathways of gentamicin B biosynthesis.

In the version of this article originally published, reference to another structure of GenB1 was omitted (Dow, G. T., Thoden, J. B., & Holden, H. M. The three-dimensional structure of NeoB: an aminotransferase involved in the biosynthesis of neomycin. Protein Sci. 27, 945-956 (2018)). This paper is now cited as reference 32, and "Another structure of GenB1 was also reported independently during the revision of this article32" was added to the text in the Discussion section. This error has been corrected in the PDF and HTML versions of the article.