Activation of cryptic milbemycin A4 production in Streptomyces sp. BB47 by the introduction of a functional bldA gene.

Streptomycetes are characterized by their ability to produce structurally diverse compounds as secondary metabolites and by their complex developmental life cycle, which includes aerial mycelium formation and sporulation. The production of secondary metabolites is growth-stage dependent, and generally coincides with morphological development on a solid culture. Streptomyces sp. BB47 produces several types of bioactive compounds and displays a bald phenotype that is devoid of an aerial mycelium and spores. Here, we demonstrated by genome analysis and gene complementation experiments that the bald phenotype arises from the bldA gene, which is predicted to encode the Leu-tRNAUUA molecule. Unlike the wild-type strain producing jomthonic acid A (1) and antarlide A (2), the strain complemented with a functional bldA gene newly produced milbemycin (3). The chemical structure of compound 3 was elucidated on the basis of various spectroscopic analyses, and was identified as milbemycin A4, which is an insecticidal/acaricidal antibiotic. These results indicate that genetic manipulation of genes involved in morphological development in streptomycetes is a valuable way to activate cryptic biosynthetic pathways.

Heterologous gene expression and characterization of two serine hydroxymethyltransferases from Thermoplasma acidophilum.

Serine hydroxymethyltransferase (SHMT) and threonine aldolase are classified as fold type I pyridoxal-5'-phosphate-dependent enzymes and engaged in glycine biosynthesis from serine and threonine, respectively. The acidothermophilic archaeon Thermoplasma acidophilum possesses two distinct SHMT genes, while there is no gene encoding threonine aldolase in its genome. In the present study, the two SHMT genes (Ta0811 and Ta1509) were heterologously expressed in Escherichia coli and Thermococcus kodakarensis, respectively, and biochemical properties of their products were investigated. Ta1509 protein exhibited dual activities to catalyze tetrahydrofolate (THF)-dependent serine cleavage and THF-independent threonine cleavage, similar to other SHMTs reported to date. In contrast, the Ta0811 protein lacks amino acid residues involved in the THF-binding motif and catalyzes only the THF-independent cleavage of threonine. Kinetic analysis revealed that the threonine-cleavage activity of the Ta0811 protein was 3.5 times higher than the serine-cleavage activity of Ta1509 protein. In addition, mRNA expression of Ta0811 gene in T. acidophilum was approximately 20 times more abundant than that of Ta1509. These observations suggest that retroaldol cleavage of threonine, mediated by the Ta0811 protein, has a major role in glycine biosynthesis in T. acidophilum.

Germination stimulatory activity of bacterial butenolide hormones from Streptomyces albus J1074 on seeds of the root parasitic weed Orobanche minor.

Damage caused by Orobanchaceae root parasitic weeds is a substantial agricultural problem for global food security. Many studies have been conducted to establish practical methods of control, but efforts are still required for successful management. Seed germination of root parasitic weeds requires host-derived germination stimulants including strigolactones (SLs). Studies on SLs have revealed that a butenolide ring is the essential moiety for SL activity as a germination stimulant. Interestingly, recent studies have revealed that butenolide hormones regulate the biosynthesis of secondary metabolites and mediate communication in actinomycete bacteria. Because of the structural similarity between SLs and the bacterial butenolides, we evaluated the germination stimulatory activity of butenolides isolated from Streptomyces albus J1074 on root parasitic weeds. These butenolides were found to specifically induce seed germination of Orobanche minor. Our findings contribute to understanding the molecular mechanisms of germination stimulant perception and to the development of a method for their biological control.

In Vitro Production of Coenzyme A Using Thermophilic Enzymes.

Coenzyme A (CoA) is an essential cofactor present in all domains of life and is involved in numerous metabolic pathways, including fatty acid metabolism, pyruvate oxidation through the tricarboxylic acid (TCA) cycle, and the production of secondary metabolites. This characteristic makes CoA a commercially valuable compound in the pharmaceutical, cosmetic, and clinical industries. However, CoA is difficult to accumulate in living cells at a high level, since it is consumed in multiple metabolic pathways, hampering its manufacturing by typical cell cultivation and extraction approaches. The feedback inhibition by CoA to a biosynthetic enzyme, pantothenate kinase (PanK), is also a serious obstacle for the high-titer production of CoA. To overcome this challenge, in vitro production of CoA, in which the CoA biosynthetic pathway was reconstructed outside cells using recombinant thermophilic enzymes, was performed. The in vitro pathway was designed to be insensitive to the feedback inhibition of CoA using CoA-insensitive type III PanK from the thermophilic bacterium Thermus thermophilus. Furthermore, a statistical approach using design of experiments (DOE) was employed to rationally determine the enzyme loading ratio to maximize the CoA production rate. Consequently, 0.94 mM CoA could be produced from 2 mM d-pantetheine through the designed pathway. We hypothesized that the insufficient conversion yield is attributed to the high Km value of T. thermophilus PanK toward ATP. Based on these observations, possible CoA regulation mechanisms in T. thermophilus and approaches to improve the feasibility of CoA production through the in vitro pathway have been investigated. IMPORTANCE The biosynthesis of coenzyme A (CoA) in bacteria and eukaryotes is regulated by feedback inhibition targeting type I and type II pantothenate kinase (PanK). Type III PanK is found only in bacteria and is generally insensitive to CoA. Previously, type III PanK from the hyperthermophilic bacterium Thermotoga maritima was shown to defy this typical characteristic and instead shows inhibition toward CoA. In the present study, phylogenetic analysis combined with functional analysis of type III PanK from thermophiles revealed that the CoA-sensitive behavior of type III PanK from T. maritima is uncommon. We cloned type III PanKs from Thermus thermophilus and Geobacillus sp. strain 30 and showed that neither enzyme's activities were inhibited by CoA. Furthermore, we utilized type III PanK for a one-pot cascade reaction to produce CoA.

Stable isotope and chemical inhibition analyses suggested the existence of a non-mevalonate-like pathway in the yeast Yarrowia lipolytica.

Methyl erythritol phosphate (MEP) is the metabolite found in the MEP pathway for isoprenoid biosynthesis, which is known to be utilized by plants, algae, and bacteria. In this study, an unprecedented observation was found in the oleaginous yeast Yarrowia lipolytica, in which one of the chromatographic peaks was annotated as MEP when cultivated in the nitrogen limiting condition. This finding raised an interesting hypothesis of whether Y. lipolytica utilizes the MEP pathway for isoprenoid biosynthesis or not, because there is no report of yeast harboring the MEP pathway. Three independent approaches were used to investigate the existence of the MEP pathway in Y. lipolytica; the spiking of the authentic standard, the MEP pathway inhibitor, and the 13C labeling incorporation analysis. The study suggested that the mevalonate and MEP pathways co-exist in Y. lipolytica and the nitrogen limiting condition triggers the utilization of the MEP pathway in Y. lipolytica.

Lavencidin, a polyene macrolide antibiotic from Streptomyces lavendulae FRI-5.

In our screening program for new biologically active compounds, a new polyene macrolide, lavencidin (1), along with known compound RKGS-A2215A (2), was isolated from the fermentation broth of Streptomyces lavendulae FRI-5 by changing the composition of liquid medium normally used for the strain. Their structures were elucidated by spectral methods (high-resolution fast-atom bombardment mass spectrometry (HRFABMS) and nuclear magnetic resonance (NMR)). Compound 1 includes a conjugated pentaene moiety together with six hydroxy groups and a carboxylic acid as a side chain. Lavencidin (1) showed moderate growth-inhibitory activity against yeast and was cytotoxic against human cancer cell lines with low-micromolar IC50 values.

Identification of biosynthetic genes for the ß-carboline alkaloid kitasetaline and production of the fluorinated derivatives by heterologous expression.

ß-Carboline alkaloids exhibit a broad spectrum of pharmacological and biological activities and are widely distributed in nature. Genetic information on the biosynthetic mechanism of ß-carboline alkaloids has not been accumulated in bacteria, because there are only a few reports on the microbial ß-carboline compounds. We previously isolated kitasetaline, a mercapturic acid derivative of a ß-carboline compound, from the genetically modified Kitasatospora setae strain and found a plausible biosynthetic gene cluster for kitasetaline. Here, we identified and characterized three kitasetaline (ksl) biosynthetic genes for the formation of the ß-carboline core structure and a gene encoding mycothiol-S-conjugate amidase for the modification of the N-acetylcysteine moiety by using heterologous expression. The proposed model of kitasetaline biosynthesis shows unique enzymatic systems for ß-carboline alkaloids. In addition, feeding fluorotryptophan to the heterologous Streptomyces hosts expressing the ksl genes led to the generation of unnatural ß-carboline alkaloids exerting novel/potentiated bioactivities.

Engineered production of kitasetalic acid, a new tetrahydro-ß-carboline with the ability to suppress glucose-regulated protein synthesis.

ß-Carboline alkaloids and related compounds show a broad spectrum of biological activities. We previously identified new members of the ß-carboline alkaloid family by using an engineered Kitasatospora setae strain and a heterologous Streptomyces host expressing the plausible biosynthetic genes, including the hypothetical gene kse_70640 (kslB). Here, we elucidated the chemical structure of a new tetrahydro-ß-carboline compound (named kitasetalic acid) that appeared in a heterologous Streptomyces host expressing the kslB gene alone. Kitasetalic acid suppressed the expression of glucose-regulated protein 78 (GRP78) without inducing cell death. This is the first report to show that a tetrahydro-ß-carboline compound regulates the expression of the GRP78 protein in cancer cell lines.

Cystargamide B, a cyclic lipodepsipeptide with protease inhibitory activity from Streptomyces sp.

We identified a new cyclic lipodepsipeptide, cystargamide B (1), from the mycelial extract of a Kaempferia galanga rhizome-derived actinomycete strain, Streptomyces sp. PB013. The planar structure was elucidated based on high resolution fast-atom bombardment mass spectrometry (HRFABMS) spectroscopy and one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopic data. The absolute configurations of the constituent amino acids were determined using advanced Marfey's method. Cystargamide B (1) includes rare structural units: a 5-hydroxytryptophan residue and a 2,3-epoxy fatty acid side chain. Notably, cystargamide B (1) inhibited the protease activity of the NS2B/NS3 complex from dengue virus.

Butenolides from Streptomyces albus J1074 Act as External Signals To Stimulate Avermectin Production in Streptomyces avermitilis.

In streptomycetes, autoregulators are important signaling compounds that trigger secondary metabolism, and they are regarded as Streptomyces hormones based on their extremely low effective concentrations (nM) and the involvement of specific receptor proteins. Our previous distribution study revealed that butenolide-type Streptomyces hormones, including avenolide, are a general class of signaling molecules in streptomycetes and that Streptomyces albus strain J1074 may produce butenolide-type Streptomyces hormones. Here, we describe metabolite profiling of a disruptant of the S. albusaco gene, which encodes a key biosynthetic enzyme for butenolide-type Streptomyces hormones, and identify four butenolide compounds from S. albus J1074 that show avenolide activity. The compounds structurally resemble avenolide and show different levels of avenolide activity. A dual-culture assay with imaging mass spectrometry (IMS) analysis for in vivo metabolic profiling demonstrated that the butenolide compounds of S. albus J1074 stimulate avermectin production in another Streptomyces species, Streptomyces avermitilis, illustrating the complex chemical interactions through interspecies signals in streptomycetes.IMPORTANCE Microorganisms produce external and internal signaling molecules to control their complex physiological traits. In actinomycetes, Streptomyces hormones are low-molecular-weight signals that are key to our understanding of the regulatory mechanisms of Streptomyces secondary metabolism. This study reveals that acyl coenzyme A (acyl-CoA) oxidase is a common and essential biosynthetic enzyme for butenolide-type Streptomyces hormones. Moreover, the diffusible butenolide compounds from a donor Streptomyces strain were recognized by the recipient Streptomyces strain of a different species, resulting in the initiation of secondary metabolism in the recipient. This is an interesting report on the chemical interaction between two different streptomycetes via Streptomyces hormones. Information on the metabolite network may provide useful hints not only to clarification of the regulatory mechanism of secondary metabolism, but also to understanding of the chemical communication among streptomycetes to control their physiological traits.

Characterization of the biosynthetic gene cluster for cryptic phthoxazolin A in Streptomyces avermitilis.

Phthoxazolin A, an oxazole-containing polyketide, has a broad spectrum of anti-oomycete activity and herbicidal activity. We recently identified phthoxazolin A as a cryptic metabolite of Streptomyces avermitilis that produces the important anthelmintic agent avermectin. Even though genome data of S. avermitilis is publicly available, no plausible biosynthetic gene cluster for phthoxazolin A is apparent in the sequence data. Here, we identified and characterized the phthoxazolin A (ptx) biosynthetic gene cluster through genome sequencing, comparative genomic analysis, and gene disruption. Sequence analysis uncovered that the putative ptx biosynthetic genes are laid on an extra genomic region that is not found in the public database, and 8 open reading frames in the extra genomic region could be assigned roles in the biosynthesis of the oxazole ring, triene polyketide and carbamoyl moieties. Disruption of the ptxA gene encoding a discrete acyltransferase resulted in a complete loss of phthoxazolin A production, confirming that the trans-AT type I PKS system is responsible for the phthoxazolin A biosynthesis. Based on the predicted functional domains in the ptx assembly line, we propose the biosynthetic pathway of phthoxazolin A.

Corrigendum: Total synthesis and absolute configuration of avenolide, extracellular factor in Streptomyces avermitilis.

This corrects the article DOI: 10.1038/ja.2011.90.

Discovery of a new diol-containing polyketide by heterologous expression of a silent biosynthetic gene cluster from Streptomyces lavendulae FRI-5.

The genome of streptomycetes has the ability to produce many novel and potentially useful bioactive compounds, but most of which are not produced under standard laboratory cultivation conditions and are referred to as silent/cryptic secondary metabolites. Streptomyces lavendulae FRI-5 produces several types of bioactive compounds. However, this strain may also have the potential to biosynthesize more useful secondary metabolites. Here, we activated a silent biosynthetic gene cluster of an uncharacterized compound from S. lavendulae FRI-5 using heterologous expression. The engineered strain carrying the silent gene cluster produced compound 5, which was undetectable in the culture broth of S. lavendulae FRI-5. Using various spectroscopic analyses, we elucidated the chemical structure of compound 5 (named lavendiol) as a new diol-containing polyketide. The proposed assembly line of lavendiol shows a unique biosynthetic mechanism for polyketide compounds. The results of this study suggest the possibility of discovering more silent useful compounds from streptomycetes by genome mining and heterologous expression.

Discovering potential Streptomyces hormone producers by using disruptants of essential biosynthetic genes as indicator strains.

Autoregulators are low-molecular-weight signaling compounds that control the production of many secondary metabolites in actinomycetes and have been referred to as 'Streptomyces hormones'. Here, potential producers of Streptomyces hormones were investigated in 40 Streptomyces and 11 endophytic actinomycetes. Production of γ-butyrolactone-type (IM-2, VB) and butenolide-type (avenolide) Streptomyces hormones was screened using Streptomyces lavendulae FRI-5 (ΔfarX), Streptomyces virginiae (ΔbarX) and Streptomyces avermitilis (Δaco), respectively. In these strains, essential biosynthetic genes for Streptomyces hormones were disrupted, enabling them to respond solely to the externally added hormones. The results showed that 20% of each of the investigated strains produced IM-2 and VB, confirming that γ-butyrolactone-type Streptomyces hormones are the most common in actinomycetes. Unlike the γ-butyrolactone type, butenolide-type Streptomyces hormones have been discovered in recent years, but their distribution has been unclear. Our finding that 24% of actinomycetes (12 of 51 strains) showed avenolide activity revealed for the first time that the butenolide-type Streptomyces hormone is also common in actinomycetes.

Rakicidin F, a new antibacterial cyclic depsipeptide from a marine sponge-derived Streptomyces sp.

A new cyclic depsipeptide, rakicidin F (1), along with the known compound rakicidin C (2), was isolated from the fermentation broth of the marine sponge-derived actinomycete strain Streptomyces sp. GKU 220. Their structures were elucidated by interpreting the HRFABMS and NMR spectroscopic data. Rakicidin F (1) showed growth inhibitory activity against bacteria.The Journal of Antibiotics advance online publication, 2 August 2017; doi:10.1038/ja.2017.92.

Activation of cryptic phthoxazolin A production in Streptomyces avermitilis by the disruption of autoregulator-receptor homologue AvaR3.

The genomes of actinomycetes encode many cryptic novel/useful bioactive compounds, but access to these cryptic secondary metabolites remains limited. Streptomyces avermitilis predominantly produces three polyketide antibiotics (avermectin, filipin, and oligomycin) but has the potential to produce more secondary metabolites based on the number of cryptic biosynthetic gene clusters. Here, we extensively investigated the metabolite profiles of a gene disruptant of AvaR3 (an autoregulator receptor homologue), which is involved in the pleiotropic regulation of antibiotic production and cell morphology. Unlike the wild-type strain, the avaR3 mutant accumulated compound 3 in the culture. The chemical structure of compound 3 was elucidated on the basis of various spectroscopic analyses, and was identified as phthoxazolin A, a cellulose synthesis inhibitor. Bioassays demonstrated that compound 3 exerts growth inhibitory activity against a broad range of plant pathogenic oomycetes. Moreover, unlike avermectin production, phthoxazolin A (3) production was negatively controlled by avenolide, a new type of autoregulator in streptomycetes, through the function of AvaR3. These results suggest that the genetic manipulation of autoregulator receptor homologues would be a valuable tool for the discovery of cryptic bioactive compounds.

Identification and characterization of lbpA, an indigoidine biosynthetic gene in the γ-butyrolactone signaling system of Streptomyces lavendulae FRI-5.

Streptomyces lavendulae FRI-5 produces the blue pigment indigoidine and other secondary metabolites (d-cycloserine and nucleoside antibiotics). The production of these useful compounds is controlled by a signaling cascade mediated by the γ-butyrolactone autoregulator IM-2. Previously we revealed that the far regulatory island includes the IM-2 receptor, the IM-2 biosynthetic enzyme, and several transcriptional regulators, and that it contributes to the regulation of indigoidine production in response to the signaling molecule. Here, we found that the vicinity of the far regulatory island includes the putative gene cluster for the biosynthesis of indigoidine and unidentified compounds, and demonstrated that the expression of the gene cluster is under the control of the IM-2 regulatory system. Heterologous expression of lbpA, encoding a plausible nonribosomal peptide synthetase, in the versatile model host Streptomyces avermitilis SUKA22 led to indigoidine production, which was enhanced dramatically by feeding of the indigoidine precursor l-glutamine. These results confirmed that LbpA is an indigoidine biosynthetic enzyme in the IM-2 signaling cascade.

Characterization of AvaR1, a butenolide-autoregulator receptor for biosynthesis of a Streptomyces hormone in Streptomyces avermitilis.

Streptomyces hormones, sometimes called as autoregulators, are important signaling molecules to trigger secondary metabolism across many Streptomyces species. We recently identified a butenolide-type autoregulator (termed avenolide) as a new class of Streptomyces hormone from Streptomyces avermitilis that produces important anthelmintic agent avermectin. Avenolide triggers the production of avermectin with minimum effective concentration of nanomolar. Here, we describe the characterization of avaR1 encoding an avenolide receptor in the regulation of avermectin production and avenolide biosynthesis. The disruption of avaR1 resulted in transcriptional derepression of avenolide biosynthetic gene with an increase in avenolide production, with no change in the avermectin production profile. Moreover, the avaR1 mutant showed increased transcription of avaR1. Together with clear DNA-binding capacity of AvaR1 toward avaR1 upstream region, it suggests that AvaR1 negatively controls the expression of avaR1 through the direct binding to the promoter region of avaR1. These findings revealed that the avenolide receptor AvaR1 functions as a transcriptional repressor for avenolide biosynthesis and its own synthesis.

Regulation of production of the blue pigment indigoidine by the pseudo γ-butyrolactone receptor FarR2 in Streptomyces lavendulae FRI-5.

The γ-butyrolactone autoregulator signaling cascade is widely distributed among Streptomyces species as an important regulatory system of secondary metabolism. In Streptomyces lavendulae FRI-5, a γ-butyrolactone autoregulator IM-2 and the IM-2 specific receptor FarA control production of the blue pigment indigoidine together with two types of antibiotics: d-cycloserine and the nucleoside antibiotics. Here, we demonstrated by in silico analysis that farR2 (a farA homologue), which is located in a cluster of regulatory genes including farA, belongs to the family of pseudoreceptor regulator genes, and that the expression of farR2 is controlled by the IM-2/FarA regulatory system. Disruption of farR2 resulted in delayed production of indigoidine and in transcriptional derepression of the clustered far regulatory genes. Moreover, FarR2 bound to the FarA-binding sequences in the promoter regions of the regulatory genes that were downregulated by FarR2.

Characterization of the biosynthetic gene cluster for maklamicin, a spirotetronate-class antibiotic of the endophytic Micromonospora sp. NBRC 110955.

Maklamicin, which is produced by the endophytic Micromonospora sp. NBRC 110955, is a spirotetronate-class antibiotic possessing anti-microbial activity against Gram-positive bacteria, and has several unique structural features different from other spirotetronates. Here we describe identification and characterization of the maklamicin biosynthetic (mak) gene cluster through draft genome sequencing, genomic library screening, and gene disruption. Sequence analysis revealed that a plausible maklamicin cluster resides in a 152 kb DNA region encoding 46 open reading frames, 24 of which can be assigned roles in the biosynthesis of polyketide backbone, spirotetronate or peripheral moieties, self-resistance and the regulation of maklamicin production. Disruption of the polyketide synthase (PKS) genes makA1 or makA4 resulted in a complete loss of maklamicin production, indicating that the type I modular PKS system is responsible for the biosynthesis of maklamicin. The mak gene cluster contained a set of biosynthetic genes for the formation of a tetronate moiety, which were found to be highly conserved in the gene clusters for spirotetronate antibiotics. Based on the estimated biosynthetic genes, we propose the biosynthetic pathway for maklamicin. Our findings provide not only insights on the biosynthetic mechanism of the unique structures in maklamicin, but also useful information to facilitate a comparative analysis of the spirotetronate biosynthetic pathways to expand the structural repertoire.