Ikuko Kozone

Engineered Streptomyces avermitilis Host for Heterologous Expression of Biosynthetic Gene Cluster for Secondary Metabolites

An industrial microorganism, Streptomyces avermitilis, which is a producer of anthelmintic macrocyclic lactones, avermectins, has been constructed as a versatile model host for heterologous expression of genes encoding secondary metabolite biosynthesis. Twenty of the entire biosynthetic gene clusters for secondary metabolites were successively cloned and introduced into a versatile model host S. avermitilis SUKA17 or 22. Almost all S. avermitilis transformants carrying the entire gene cluster produced metabolites as a result of the expression of biosynthetic gene clusters introduced. A few transformants were unable to produce metabolites, but their production was restored by the expression of biosynthetic genes using an alternative promoter or the expression of a regulatory gene in the gene cluster that controls the expression of biosynthetic genes in the cluster using an alternative promoter. Production of metabolites in some transformants of the versatile host was higher than that of the original producers, and cryptic biosynthetic gene clusters in the original producer were also expressed in a versatile host.

Streptomyces associated with a marine sponge Haliclona sp.; biosynthetic genes for secondary metabolites and products.

Terrestrial actinobacteria have served as a primary source of bioactive compounds; however, a rapid decrease in the discovery of new compounds strongly necessitates new investigational approaches. One approach is the screening of actinobacteria from marine habitats, especially the members of the genus Streptomyces. Presence of this genus in a marine sponge, Haliclona sp., was investigated using culture-dependent and -independent techniques. 16S rRNA gene clone library analysis showed the presence of diverse Streptomyces in the sponge sample. In addition to the dominant genus Streptomyces, members of six different genera were isolated using four different media. Five phylogenetically new strains, each representing a novel species in the genus Streptomyces were also isolated. Polyphasic study suggesting the classification of two of these strains as novel species is presented. Searching the strains for the production of novel compounds and the presence of biosynthetic genes for secondary metabolites revealed seven novel compounds and biosynthetic genes with unique sequences. In these compounds, JBIR-43 exhibited cytotoxic activity against cancer cell lines. JBIR-34 and -35 were particularly interesting because of their unique chemical skeleton. To our knowledge, this is the first comprehensive study detailing the isolation of actinobacteria from a marine sponge and novel secondary metabolites from these strains.

MIDDAS-M: Motif-Independent De Novo Detection of Secondary Metabolite Gene Clusters through the Integration of Genome Sequencing and Transcriptome Data

Many bioactive natural products are produced as “secondary metabolites” by plants, bacteria, and fungi. During the middle of the 20th century, several secondary metabolites from fungi revolutionized the pharmaceutical industry, for example, penicillin, lovastatin, and cyclosporine. They are generally biosynthesized by enzymes encoded by clusters of coordinately regulated genes, and several motif-based methods have been developed to detect secondary metabolite biosynthetic (SMB) gene clusters using the sequence information of typical SMB core genes such as polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS). However, no detection method exists for SMB gene clusters that are functional and do not include core SMB genes at present. To advance the exploration of SMB gene clusters, especially those without known core genes, we developed MIDDAS-M, a motif-independent de novo detection algorithm for SMB gene clusters. We integrated virtual gene cluster generation in an annotated genome sequence with highly sensitive scoring of the cooperative transcriptional regulation of cluster member genes. MIDDAS-M accurately predicted 38 SMB gene clusters that have been experimentally confirmed and/or predicted by other motif-based methods in 3 fungal strains. MIDDAS-M further identified a new SMB gene cluster for ustiloxin B, which was experimentally validated. Sequence analysis of the cluster genes indicated a novel mechanism for peptide biosynthesis independent of NRPS. Because it is fully computational and independent of empirical knowledge about SMB core genes, MIDDAS-M allows a large-scale, comprehensive analysis of SMB gene clusters, including those with novel biosynthetic mechanisms that do not contain any functionally characterized genes.

Novel β‐1,3‐, 1,6‐oligoglucan elicitor from Alternaria alternata 102 for defense responses in tobacco

A novel elicitor that induces chitinases in tobacco BY‐2 cells was isolated from Alternaria alternata 102. Six other fungi, including A. alternata IFO 6587, could not induce, or weakly induce chitinase activity. The purified elicitor was soluble in 75% methanol and showed the chitinase‐inducing activity when applied at concentrations of as low as 25 ng·mL−1. Structural determination by methylation analysis, reducing‐end analysis, MALDI‐TOF/MS, and NMR spectroscopy indicated that the elicitor was a mixture of β‐1,3‐, 1,6‐oligoglucans mostly with a degree of polymerization of between 8 and 17. Periodate oxidation of the elicitor suggested that the 1,6‐linked and nonreducing terminal residues are essential for the elicitor activity. Further analysis of the elicitor responses in BY‐2 cells indicated that the activity of this β‐1,3‐, 1,6‐glucan elicitor was about 1000 times more potent than that of laminarin, which is a known elicitor of defense responses in tobacco. Analyzing the expression of defense‐related genes indicated that a phenylalanine ammonia‐lyase gene and a coumaroyl‐CoA O‐methyltransferase gene were transiently expressed by this β‐1,3‐, 1,6‐glucan elicitor. The elicitor induced a weak oxidative burst but did not induce cell death in the BY‐2 cells. In the tissue of tobacco plants, this β‐1,3‐, 1,6‐glucan elicitor induced the expression of basic PR‐3 genes, the phenylpropanoid pathway genes, and the sesquiterpenoid pathway genes. In comparison with laminarin and laminarin sulfate, which are reported to be potent elicitors of defense responses in tobacco, the expression pattern of genes induced by the purified β‐1,3‐, 1,6‐glucan elicitor was more similar to that induced by laminarin than to that induced by laminarin sulfate.

Solophenols B–D and Solomonin: New Prenylated Polyphenols Isolated from Propolis Collected from The Solomon Islands and Their Antibacterial Activity.

Three new prenylated flavonoids, namely, solophenols B (1), C (2), and D (3), as well as a new prenylated stilbene, solomonin (4), were isolated from propolis collected from the Solomon Islands. In addition, 17 known compounds were identified. The structures of the new compounds were determined by a combination of methods, including mass spectrometry and NMR. These new compounds and several known compounds were tested for antibacterial activity against Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa. Most of them exhibited potent antibacterial activity. These findings may indicate that propolis from the Solomon Islands has potential applications as an ingredient in food additives or pharmaceuticals.

New glycosylated derivatives of versipelostatin, the GRP78/Bip molecular chaperone down-regulator, from Streptomyces versipellis 4083-SVS6

Four novel glycosylated derivatives of versipelostatin (1), versipelostatins B-E (2-5), were isolated from the culture broth of Streptomyces versipellis 4083-SVS6. The inhibitory activities of the isolated compounds against the expression of molecular chaperone GRP78 induced by 2-deoxyglucose were evaluated. Of the five versipelostatin family members, 1 and 4 were the more potent with IC(50) values of 3.5 and 4.3 microM. These results suggest that the alpha-L-oleandropyranosyl (1-->4)-beta-D-digitoxopyranosyl residue in the sugar moiety may play an important role in down-regulating GRP78 expression induced by 2-deoxyglucose.

Relationship between Response to and Production of the Aerial Mycelium-inducing Substances Pamamycin-607 and A-factor

Respectively, exogenous pamamycin-607 and A-factor restored or stimulated aerial mycelium formation in 30 (67%) and 6 (13%) of 45 Streptomyces strains, and both restored or stimulated it in 5 strains (11%). Pamamycin-607 production was detected in 3 of those strains that responded to pamamycin-607. These findings indicate that pamamycin-607 acts on the common regulatory system for aerial mycelium formation in Streptomyces spp. but is not a universal autoregulator. Increased or decreased antibacterial production occurred in 5 strains in association with aerial mycelium formation by pamamycin-607 or A-factor.

Analysis of the biological activity of a novel 24-membered macrolide JBIR-19 in Saccharomyces cerevisiae by the morphological imaging program CalMorph

To investigate the biological activity of a novel 24-membered macrolide compound, JBIR-19, isolated from the culture broth of the entomopathogenic fungus Metarhizium sp. fE61, morphological changes in yeast cells were examined using the automated image-processing program CalMorph. Principal components analysis was used to elucidate dynamic changes in the phenotypes, revealing two independent effects of JBIR-19 in yeast cells: bud elongation and increased size of the actin region. Using a fitness assay, we identified the genes required for robust growth in the presence of JBIR-19. Among these were CCW12, YLR111W, and DHH1, which are also involved in abnormal bud morphology. Based on these results and others, we predict intracellular targets of JBIR-19 and its functional interactions.

Characterization of Giant Modular PKSs Provides Insight into Genetic Mechanism for Structural Diversification of Aminopolyol Polyketides

Polyketides form many clinically valuable compounds. However, manipulation of their biosynthesis remains highly challenging. An understanding of gene cluster evolution provides a rationale for reprogramming of the biosynthetic machinery. Herein, we report characterization of giant modular polyketide synthases (PKSs) responsible for the production of aminopolyol polyketides. Heterologous expression of over 150 kbp polyketide gene clusters successfully afforded their products, whose stereochemistry was established by taking advantage of bioinformatic analysis. Furthermore, phylogenetic analysis of highly homologous but functionally diverse domains from the giant PKSs demonstrated the evolutionary mechanism for structural diversification of polyketides. The gene clusters characterized herein, together with their evolutionary insights, are promising genetic building blocks for de novo production of unnatural polyketides.

Viable Cell Detection by the Combined Use of Fluorescent Glucose and Fluorescent Glycine

The combined use of a fluorescent glucose (2NBDG) and a fluorescent glycine (NBD-Gly) was tried for the detection of viable cells of significant foodborne pathogenic strains in addition to several Escherichia coli strains and coliforms. Thirty-five out of 41 strains showed marked uptake of 2NBDG but 6 strains were not able to take in 2NBDG. Five out of these 6 strains showed NBD-Gly uptake.