Identification of bolegrevilol B and C as novel antioxidant compounds in Suillus grevillei mushroom.

Suillus grevillei is a popular species of mushroom available worldwide. In this study, we isolated compounds, bolegrevilol B and bolegrevilol C, from the mushroom and observed their potent lipid peroxidation-inhibiting activity. The structures of bolegrevilol B and bolegrevilol C were elucidated as 3-geranylgeranyl-1,2,4-trihydroxybenzene and 3-geranylgeranyl-1,2-dihydroxy-4-methoxybenzene, respectively, through high-resolution electrospray ionization mass spectrometry (-) and 1D and 2D nuclear magnetic resonance analyses. Bolegrevilol B and C inhibited lipid peroxidation and exhibited IC50 values of 2.0 ± 0.29 µm and 1.0 ± 0.13 µm, respectively. Furthermore, bolegrevilol B and C demonstrated potent neuroprotective activities in neuronal hybridoma N18-RE-105 cells against L-glutamate toxicity (EC50 of 1.8 ± 1.7 n m and 7.2 ± 6.9 n m, respectively). Bolegrevilol B was found in nature for the first time, and, to the best of our knowledge, this is the first study to report the antioxidant activities of bolegrevilol B and C.

Dihydromaniwamycin E, a Heat-Shock Metabolite from Thermotolerant Streptomyces sp. JA74, Exhibiting Antiviral Activity against Influenza and SARS-CoV-2 Viruses.

Dihydromaniwamycin E (1), a new maniwamycin derivative featuring an azoxy moiety, has been isolated from the culture extract of thermotolerant Streptomyces sp. JA74 along with the known analogue maniwamycin E (2). Compound 1 is produced only by cultivation of strain JA74 at 45 °C, and this type of compound has been previously designated a "heat shock metabolite (HSM)" by our research group. Compound 2 is detected as a production-enhanced metabolite at high temperature. Structures of 1 and 2 are elucidated by NMR and MS spectroscopic analyses. The absolute structure of 1 is determined after the total synthesis of four stereoisomers. Though the absolute structure of 2 has been proposed to be the same as the structure of maniwamycin D, the NMR and the optical rotation value of 2 are in agreement with those of maniwamycin E. Therefore, this study proposes a structural revision of maniwamycins D and E. Compounds 1 and 2 show inhibitory activity against the influenza (H1N1) virus infection of MDCK cells, demonstrating IC50 values of 25.7 and 63.2 µM, respectively. Notably, 1 and 2 display antiviral activity against SARS-CoV-2, the causative agent of COVID-19, when used to infect 293TA and VeroE6T cells, with 1 and 2 showing IC50 values (for infection of 293TA cells) of 19.7 and 9.7 µM, respectively. The two compounds do not exhibit cytotoxicity in these cell lines at those IC50 concentrations.

Anti-melanogenic effect of furanoeremophilanes identified from edible wild plants belonging to the genus Cacalia.

Cacalia delphiniifolia and Cacalia hastata are edible wild plants in Japan. We found that these plants have anti-melanogenic activity in B16F10 mouse melanoma cells. Three furanoeremophilanes, cacalol (from C. delphiniifolia), dehydrocacalohastin, and cacalohastin (from C. hastata), were identified as the main active components. The genus Cacalia may be a good source of beneficial materials with anti-melanogenic effects.

Heterologous production of novel and rare C30-carotenoids using Planococcus carotenoid biosynthesis genes.

BACKGROUND: Members of the genus Planococcus have been revealed to utilize and degrade solvents such as aromatic hydrocarbons and alkanes, and likely to acquire tolerance to solvents. A yellow marine bacterium Planococcus maritimus strain iso-3 was isolated from an intertidal sediment that looked industrially polluted, from the Clyde estuary in the UK. This bacterium was found to produce a yellow acyclic carotenoid with a basic carbon 30 (C30) structure, which was determined to be methyl 5-glucosyl-5,6-dihydro-4,4'-diapolycopenoate. In the present study, we tried to isolate and identify genes involved in carotenoid biosynthesis from this marine bacterium, and to produce novel or rare C30-carotenoids with anti-oxidative activity in Escherichia coli by combinations of the isolated genes. RESULTS: A carotenoid biosynthesis gene cluster was found out through sequence analysis of the P. maritimus genomic DNA. This cluster consisted of seven carotenoid biosynthesis candidate genes (orf1-7). Then, we isolated the individual genes and analyzed the functions of these genes by expressing them in E. coli. The results indicated that orf2 and orf1 encoded 4,4'-diapophytoene synthase (CrtM) and 4,4'-diapophytoene desaturase (CrtNa), respectively. Furthermore, orf4 and orf5 were revealed to code for hydroxydiaponeurosporene desaturase (CrtNb) and glucosyltransferase (GT), respectively. By utilizing these carotenoid biosynthesis genes, we produced five intermediate C30-carotenoids. Their structural determination showed that two of them were novel compounds, 5-hydroxy-5,6-dihydro-4,4'-diaponeurosporene and 5-glucosyl-5,6-dihydro-4,4'-diapolycopene, and that one rare carotenoid 5-hydroxy-5,6-dihydro-4,4'-diapolycopene is included there. Moderate singlet oxygen-quenching activities were observed in the five C30-carotenoids including the two novel and one rare compounds. CONCLUSIONS: The carotenoid biosynthesis genes from P. maritimus strain iso-3, were isolated and functionally identified. Furthermore, we were able to produce two novel and one rare C30-carotenoids in E. coli, followed by positive evaluations of their singlet oxygen-quenching activities.

A modern purification method for volatile sesquiterpenes produced by recombinant Escherichia coli carrying terpene synthase genes.

Most volatile sesquiterpenes had been purified from plants using distillation and preparative gas chromatography, which is not applicable to many laboratories that do not possess a needed facility. Thus, this review focuses on a modern purification method for volatile sesquiterpenes using Escherichia coli cells that functionally express terpene synthase (Tps) genes. It was recently developed that recombinant E. coli cells carrying Tps genes were cultured in two-layer media (n-octane/TB medium) without harming the cells, and the volatile hydrophobic compounds trapped in the n-octane were purified by two-phase partition (alkane/alkaline 50% MeOH), silica gel column chromatography, and reversed-phase preparative high-performance liquid chromatography (if necessary). Consequently, it was found that the volatile sesquiterpenes are easily purified, the structures of which can then be determined by nuclear magnetic resonance, [α]D and gas chromatography-mass spectrometry analyses. The antioxidant activities of several volatile sesquiterpenes are also presented in this review.

Structural and functional analysis of the carotenoid biosynthesis genes of a Pseudomonas strain isolated from the excrement of Autumn Darter.

There are many reports about carotenoid-producing bacteria and carotenoid biosynthesis genes. In databases for Pseudomonas genome sequences, there are genes homologous to carotenoid biosynthesis genes, but the function of these genes in Pseudomonas has not been elucidated. In this study, we cloned the carotenoid biosynthesis genes from a Pseudomonas sp. strain, named Akiakane, which was isolated from the excrement of the Autumn Darter dragonfly. Using an Escherichia coli functional expression system, we confirmed that the idi, crtE, crtB, crtI, and crtY gene products of the Akiakane strain show predictable catalytic activities. A cluster of six genes was also found, which was comparable to other carotenoid-producing bacteria that belong to the α-Proteobacteria or γ-Proteobacteria class.

Purification and structural analysis of volatile sesquiterpenes produced by Escherichia coli carrying unidentified terpene synthase genes from edible plants of the family Araliaceae.

A simple method to purify volatile sesquiterpenes from recombinant Escherichia coli was developed using the cells that carried known sesquiterpene synthase (Tps) genes ZzZss2 (ZSS2) and ZoTps1. This method was applied for the purification and structural analyses of volatile sesquiterpenes produced by E. coli cells that carried unidentified Tps genes, which were isolated from the Aralia-genus edible plants belonging to the family Araliaceae. Recombinant cells carrying each Tps gene were cultured in the two-layer medium (n-octane/TB medium), and volatile sesquiterpenes trapped in n-octane were purified through two-phase partition, silica gel column chromatography, and reversed-phase preparative high-performance liquid chromatography, if necessary. Further, their structures were confirmed by nuclear magnetic resonance, [α]D, and gas chromatography-mass spectrometry analyses. Herein, the products of E. coli cells that carried two Tps gene (named AcTps1 and AcTps2) in Araria cordata "Udo" and a Tps gene (named AeTps1) in Aralia elata "Taranoki" were studied resulting in identifying functionalities of these cryptic Tps genes.

Aryl Polyenes, a Highly Abundant Class of Bacterial Natural Products, Are Functionally Related to Antioxidative Carotenoids.

Bacterial pigments of the aryl polyene type are structurally similar to the well-known carotenoids with respect to their polyene systems. Their biosynthetic gene cluster is widespread in taxonomically distant bacteria, and four classes of such pigments have been found. Here we report the structure elucidation of the aryl polyene/dialkylresorcinol hybrid pigments of Variovorax paradoxus B4 by HPLC-UV-MS, MALDI-MS and NMR. Furthermore, we show for the first time that this pigment class protects the bacterium from reactive oxygen species, similarly to what is known for carotenoids. An analysis of the distribution of biosynthetic genes for aryl polyenes and carotenoids in bacterial genomes is presented; it shows a complementary distribution of these protective pigments in bacteria.

Identification of a novel hedycaryol synthase gene isolated from Camellia brevistyla flowers and floral scent of Camellia cultivars.

MAIN CONCLUSION: A novel terpene synthase (Tps) gene isolated from Camellia brevistyla was identified as hedycaryol synthase, which was shown to be expressed specifically in flowers. Camellia plants are very popular because they bloom in winter when other plants seldom flower. Many ornamental cultivars of Camellia have been bred mainly in Japan, although the fragrance of their flowers has not been studied extensively. We analyzed floral scents of several Camellia cultivars by gas chromatography-mass spectrometry (GC-MS) and found that Camellia brevistyla produced various sesquiterpenes in addition to monoterpenes, whereas Camellia japonica and its cross-lines produced only monoterpenes, including linalool as the main product. From a flower of C. brevistyla, we isolated one cDNA encoding a terpene synthase (TPS) comprised of 554 amino acids, which was phylogenetically positioned to a sole gene clade. The cDNA, designated CbTps1, was expressed in mevalonate-pathway-engineered Escherichia coli, which carried the Streptomyces mevalonate-pathway gene cluster in addition to the acetoacetate-CoA ligase gene. A terpene product was purified from recombinant E. coli cultured with lithium acetoacetate, and analyzed by (1)H-nulcear magnetic resonance spectroscopy ((1)H-NMR) and GC-MS. It was shown that a sesquiterpene hedycaryol was produced, because (1)H-NMR signals of the purified product were very broad, and elemol, a thermal rearrangement product from hedycaryol, was identified by GC-MS analysis. Spectroscopic data of elemol were also determined. These results indicated that the CbTps1 gene encodes hedycaryol synthase. Expression analysis of CbTps1 showed that it was expressed specifically in flowers, and hedycaryol is likely to be one of the terpenes that attract insects for pollination of C. brevistyla. A linalool synthase gene, which was isolated from a flower of Camellia saluenensis, is also described.

Multiple transformation with the crtYB gene of the limiting enzyme increased carotenoid synthesis and generated novel derivatives in Xanthophyllomyces dendrorhous.

Xanthophyllomces dendrorhous (in asexual state named as Phaffia rhodozyma) is a fungus which produces astaxanthin, a high value carotenoid used in aquafarming. Genetic pathway engineering is one of several steps to increase the astaxanthin yield. The limiting enzyme of the carotenoid pathway is phytoene synthase. Integration plasmids were constructed for transformation with up to three copies of the crtYB gene. Upon stepwise transformation, the copy numbers of crtYB was continuously increased leading to an almost saturated level of phytoene synthase as indicated by total carotenoid content. Several carotenoid intermediates accumulated which were absent in the wild type. Some of them are substrates and intermediates of astaxanthin synthase. They could be further converted into astaxanthin by additional transformation with the astaxanthin synthase gene. However, three intermediates exhibited an unusual optical absorbance spectrum not found before. These novel keto carotenoid were identified by HPLC co-chromatography with reference compounds generated in Escherichia coli and one of them 3-HO-4-keto-7',8'-dihydro-ß-carotene additionally by NMR spectroscopy. The others were 4-keto-ß-zeacarotene and 4-keto-7',8'-dihydro-ß-carotene. A biosynthesis pathway with their origin from neurosporene and the reason for their synthesis especially in our transformants has been discussed.

Construction of transplastomic lettuce (Lactuca sativa) dominantly producing astaxanthin fatty acid esters and detailed chemical analysis of generated carotenoids.

The plastid genome of lettuce (Lactuca sativa L.) cv. Berkeley was site-specifically modified with the addition of three transgenes, which encoded ß,ß-carotenoid 3,3'-hydroxylase (CrtZ) and ß,ß-carotenoid 4,4'-ketolase (4,4'-oxygenase; CrtW) from a marine bacterium Brevundimonas sp. strain SD212, and isopentenyl diphosphate isomerase from a marine bacterium Paracoccus sp. strain N81106. Constructed transplastomic lettuce plants were able to grow on soil at a growth rate similar to that of non-transformed lettuce cv. Berkeley and generate flowers and seeds. The germination ratio of the lettuce transformants (T0) (98.8%) was higher than that of non-transformed lettuce (93.1 %). The transplastomic lettuce (T1) leaves produced the astaxanthin fatty acid (myristate or palmitate) diester (49.2% of total carotenoids), astaxanthin monoester (18.2%), and the free forms of astaxanthin (10.0%) and the other ketocarotenoids (17.5%), which indicated that artificial ketocarotenoids corresponded to 94.9% of total carotenoids (230 µg/g fresh weight). Native carotenoids were there lactucaxanthin (3.8%) and lutein (1.3 %) only. This is the first report to structurally identify the astaxanthin esters biosynthesized in transgenic or transplastomic plants producing astaxanthin. The singlet oxygen-quenching activity of the total carotenoids extracted from the transplastomic leaves was similar to that of astaxanthin (mostly esterified) from the green algae Haematococcus pluvialis.

Structural elucidation of humulone autoxidation products and analysis of their occurrence in stored hops.

The transformation of α-acids [in hops (Humulus lupulus L.)] to iso-α-acids (in beer) during the brewing process is well known, but the occurrence and structure of the oxidized α-acids during hop storage are not well documented. Because an understanding of these oxidized compounds is essential to optimize the effects of oxidized hops on the quality of beer, we investigated the autoxidation products of humulone (a representative congener of α-acids) using a simplified autoxidation model. Among the oxidation products, tricyclooxyisohumulones A (1) and B (2), tricycloperoxyisohumulone A (3), deisopropyltricycloisohumulone (4), and the hemiacetal 5 of tricycloperoxyhumulone A (5') were isolated, and their structures were elucidated for the first time. The occurrence of compounds 1-4 in stored hops was verified using LC/MS/MS analysis. We also monitored the levels of compounds 1-4 during hop storage using LC/MS/MS analysis.

New and rare carotenoids isolated from marine bacteria and their antioxidant activities.

Marine bacteria have not been examined as extensively as land bacteria. We screened carotenoids from orange or red pigments-producing marine bacteria belonging to rare or novel species. The new acyclic carotenoids with a C30 aglycone, diapolycopenedioc acid xylosylesters A-C and methyl 5-glucosyl-5,6-dihydro-apo-4,4'-lycopenoate, were isolated from the novel Gram-negative bacterium Rubritalea squalenifaciens, which belongs to phylum Verrucomicrobia, as well as the low-GC Gram-positive bacterium Planococcus maritimus strain iso-3 belonging to the class Bacilli, phylum Firmicutes, respectively. The rare monocyclic C40 carotenoids, (3R)-saproxanthin and (3R,2'S)-myxol, were isolated from novel species of Gram-negative bacteria belonging to the family Flavobacteriaceae, phylum Bacteroidetes. In this review, we report the structures and antioxidant activities of these carotenoids, and consider relationships between bacterial phyla and carotenoid structures.

Quercetin-3-O-glucuronide induces ABCA1 expression by LXRα activation in murine macrophages.

Reverse cholesterol transport (RCT) removes excess cholesterol from macrophages to prevent atherosclerosis. ATP-binding cassette, subfamily A, member 1 (ABCA1) is a crucial cholesterol transporter involved in RCT to produce high density lipoprotein-cholesterol (HDLC), and is transcriptionally regulated by liver X receptor alpha (LXRα), a nuclear receptor. Quercetin is a widely distributed flavonoid in edible plants which prevented atherosclerosis in an animal model. We found that quercetin-3-O-glucuronide (Q3GA), a major quercetin metabolite after absorption from the digestive tract, enhanced ABCA1 expression, in vitro, via LXRα in macrophages. In addition, leaf extracts of a traditional Asian edible plant, Nelumbo nucifera (NNE), which contained abundant amounts of quercetin glycosides, significantly elevated plasma HDLC in mice. We are the first to present experimental evidence that Q3GA induced ABCA1 in macrophages, and to provide an alternative explanation to previous studies on arteriosclerosis prevention by quercetin.

3-ß-Glucosyl-3'-ß-quinovosyl zeaxanthin, a novel carotenoid glycoside synthesized by Escherichia coli cells expressing the Pantoea ananatis carotenoid biosynthesis gene cluster.

Escherichia coli cells that express the full six carotenoid biosynthesis genes (crtE, crtB, crtI, crtY, crtZ, and crtX) of the bacterium Pantoea ananatis have been shown to biosynthesize zeaxanthin 3,3'-ß-D-diglucoside. We found that this recombinant E. coli also produced a novel carotenoid glycoside that contained a rare carbohydrate moiety, quinovose (chinovose; 6-deoxy-D-glucose), which was identified as 3-ß-glucosyl-3'-ß-quinovosyl zeaxanthin by chromatographic and spectroscopic analyses. The chirality of the aglycone of these zeaxanthin glycosides had been shown to be 3R,3'R, in which the hydroxyl groups were formed with the CrtZ enzyme. It was here demonstrated that zeaxanthin synthesized from ß-carotene with CrtR or CYP175A1, the other hydroxylase with similar catalytic function to CrtZ, possessed the same stereochemistry. It was also suggested that the singlet oxygen-quenching activity of zeaxanthin 3,3'-ß-D-diglucoside, which has a chemical structure close to the new carotenoid glycoside, was superior to that of zeaxanthin.

Identification of antioxidants produced by Lactobacillus plantarum.

We identified two compounds that demonstrated 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity from cultures of Lactobacillus plantarum. Spectroscopic analyses proved these compounds to be L-3-(4-hydroxyphenyl) lactic acid (HPLA) and L-indole-3-lactic acid (ILA). The respective EC50 values for HPLA and ILA were 36.6 ± 4.3 mM and 13.4 ± 1.0 mM.

Production of hydroxlated flavonoids with cytochrome P450 BM3 variant F87V and their antioxidative activities.

A variant of P450 BM3 with an F87V substitution [P450 BM3 (F87V)] is a substrate-promiscuous cytochrome P450 monooxygenase. We investigated the bioconversion of various flavonoids (favanones, chalcone, and isoflavone) by using recombinant Escherichia coli cells, which expressed the gene coding for P450 BM3 (F87V), to give their corresponding hydroxylated products. Potent antioxidative activities were observed in some of the products.

Biocatalytic synthesis of flavones and hydroxyl-small molecules by recombinant Escherichia coli cells expressing the cyanobacterial CYP110E1 gene.

BACKGROUND: Cyanobacteria possess several cytochrome P450s, but very little is known about their catalytic functions. CYP110 genes unique to cyanaobacteria are widely distributed in heterocyst-forming cyanobacteria including nitrogen-fixing genera Nostoc and Anabaena. We screened the biocatalytic functions of all P450s from three cyanobacterial strains of genus Nostoc or Anabaena using a series of small molecules that contain flavonoids, sesquiterpenes, low-molecular-weight drugs, and other aromatic compounds. RESULTS: Escherichia coli cells carrying each P450 gene that was inserted into the pRED vector, containing the RhFRed reductase domain sequence from Rhodococcus sp. NCIMB 9784 P450RhF (CYP116B2), were co-cultured with substrates and products were identified when bioconversion reactions proceeded. Consequently, CYP110E1 of Nostoc sp. strain PCC 7120, located in close proximity to the first branch point in the phylogenetic tree of the CYP110 family, was found to be promiscuous for the substrate range mediating the biotransformation of various small molecules. Naringenin and (hydroxyl) flavanones were respectively converted to apigenin and (hydroxyl) flavones, by functioning as a flavone synthase. Such an activity is reported for the first time in prokaryotic P450s. Additionally, CYP110E1 biotransformed the notable sesquiterpene zerumbone, anti-inflammatory drugs ibuprofen and flurbiprofen (methylester forms), and some aryl compounds such as 1-methoxy and 1-ethoxy naphthalene to produce hydroxylated compounds that are difficult to synthesize chemically, including novel compounds. CONCLUSION: We elucidated that the CYP110E1 gene, C-terminally fused to the P450RhF RhFRed reductase domain sequence, is functionally expressed in E. coli to synthesize a robust monooxygenase, which shows promiscuous substrate specificity (affinity) for various small molecules, allowing the biosynthesis of not only flavones (from flavanones) but also a variety of hydroxyl-small molecules that may span pharmaceutical and nutraceutical industries.

Antioxidant Activities of Pistillarin in the Clustered Coral Mushroom, Ramaria botrytis (Agaricomycetes), and Discovery of Pistillarin B.

Ramaria botrytis is a popular mushroom in Asian countries, particularly known for its crispy texture and rich nutrient content. In this study, we found potent lipid peroxidation-inhibiting activity in this mushroom and identified the active compound associated with this activity as pistillarin. To our knowledge, this is the first report of the presence of pistillarin in R. botrytis. Our study is the first to investigate the inhibitory effects of pistillarin against physiological reactive oxygen species such as 1O2, •OH, and O2- and lipid peroxidation, which damages living tissues. We also clarified its characteristic antioxidant activities, with no 1O2 and •OH quenching activity (IC50 > 100 µÐœ), moderate O2- quenching activity (IC50 of 10.2 µÐœ), and potent lipid peroxidation-inhibiting activity (IC50 of 0.66 µÐœ), for the first time. Furthermore, we found a new pistillarin-related compound (pistillarin B) in salted R. botrytis. We isolated pistillarin B, determined its structure, and examined its lipid peroxidation-inhibiting activity (IC50 of 6.88 µÐœ).

Changes of Crocin and Other Crocetin Glycosides in Saffron Through Cooking Models, and Discovery of Rare Crocetin Glycosides in the Yellow Flowers of Freesia Hybrida.

Crocetin glycosides such as crocin are noted as functional food materials since the preventive effects of crocin have been reported against chronic disease and cancer. However, it is unclear how these apocarotenoids are structurally changed through cooking for our intake. We examined such changes in crocetin glycosides (crocin, tricrocin, and crocin-3) contained in saffron (stigmas of Crocus sativus) through cooking models. These glycosides were almost kept stable in boiling for 20 min (a boiled cooking model), while hydrolysis of the ester linkage between glucose and the crocetin aglycone occurred in a grilled cooking model (180°C, 5 min), along with a 13-cis isomerization reaction in a part of crocetin subsequently generated. We further here revealed that the yellow petals of freesia (Freesia x hybrida) with yellow flowers accumulate two unique crocetin glycosides, which were identified to be crocetin (mono)neapolitanosyl ester and crocetin dineapolitanosyl ester. A similar result as above was obtained on their changes through the cooking models. Utility applications of the freesia flowers as edible flowers are also suggested in this study. Additionally, we evaluated singlet oxygen (1O2)-quenching activities of the crocetin glycosides contained in saffron and freesia, and crocetin and 13-cis crocetin contained in the grilled saffron, indicating that they possessed moderate 1O2-quenching activities (IC50 24-64 µM).