Molecular mechanism of flagellar motor rotation arrest in bacterial zoospores of Actinoplanes missouriensis before germination.

Zoospores of the filamentous actinomycete Actinoplanes missouriensis swim vigorously using flagella and stop swimming to initiate germination in response to nutrient exposure. However, the molecular mechanisms underlying swimming cessation remain unknown. A protein (FtgA) of unknown function encoded by a chemotaxis gene cluster (che cluster-1) was found to be required for flagellar rotation arrest; the zoospores of ftgA-knockout mutants kept swimming awkwardly after germination.

Production of Phenyldiazene Derivatives Using the Biosynthetic Pathway of an Aromatic Diazo Group-Containing Natural Product from an Actinomycete.

The diazo group is an important functional group in organic synthesis because it confers high reactivity to the compounds and has been applied in various chemical reactions, such as the Sandmeyer reaction, Wolff rearrangement, cyclopropanation, and C-N bond formation with active methylene compounds. Previously, we revealed that 3-diazoavenalumic acid (3-DAA), which is potentially produced by several actinomycete species and contains an aromatic diazo group, is a biosynthetic intermediate of avenalumic acid. In this study, we aimed to construct a production system for phenyldiazene derivatives by adding several active methylene compounds to the culture of a 3-DAA-producing recombinant actinomycete.

Multimodal Molecular Imaging Reveals a Novel Membrane Component in Sporangia of the Rare Actinomycete .

The bacterium belongs to the genus , a prolific source of useful natural products. This microbe forms globular structures called sporangia, which contain many dormant spores. Recent studies using transmission electron microscopy have shown that the sporangium membrane has an unprecedented three-layer structure, but its molecular components remain unclear.

Involvement of a putative acyltransferase gene in sporangium formation in .

The actinomycete forms branched substrate mycelia during vegetative growth and produces terminal sporangia, each of which contains a few hundred spherical flagellated spores, from the substrate mycelia through short sporangiophores. Based on the observation that remodeling of membrane lipid composition is involved in the morphological development of A3(2), we hypothesized that remodeling of membrane lipid composition is also involved in sporangium formation in . Because some acyltransferases are presumably involved in the remodeling of membrane lipid composition, we disrupted each of the 22 genes annotated as encoding putative acyltransferases in the genome and evaluated their effects on sporangium formation.

Identification of the Cirratiomycin Biosynthesis Gene Cluster in Streptomyces Cirratus: Elucidation of the Biosynthetic Pathways for 2,3-Diaminobutyric Acid and Hydroxymethylserine.

Cirratiomycin, a heptapeptide with antibacterial activity, was isolated and characterized in 1981; however, its biosynthetic pathway has not been elucidated. It contains several interesting nonproteinogenic amino acids, such as (2S,3S)-2,3-diaminobutyric acid ((2S,3S)-DABA) and α-(hydroxymethyl)serine, as building blocks. Here, we report the identification of a cirratiomycin biosynthetic gene cluster in Streptomyces cirratus.

Identification of a putative cell wall-hydrolyzing amidase involved in sporangiospore maturation in .

is a filamentous bacterium that differentiates into terminal sporangia, each containing a few hundred spores. Previously, we reported that a cell wall-hydrolyzing -acetylglucosaminidase, GsmA, is required for the maturation process of sporangiospores in ; sporangia of the null mutant (Δ) strain released chains of 2-20 spores under sporangium dehiscence-inducing conditions. In this study, we identified and characterized a putative cell wall hydrolase (AsmA) that is also involved in sporangiospore maturation.

The gene is required for the early stages of sporangium formation in .

Unlabelled: In , multiple paralogs of SsgA-like proteins (SALPs) are involved in spore formation from aerial hyphae. However, the functions of SALPs have not yet been elucidated in other actinobacterial genera. Here, we report the primary function of an SsgB ortholog (AmSsgB) in , which develops terminal sporangia on the substrate mycelia via short sporangiophores.

Identification of the coumaric acid biosynthetic gene cluster in : insights into the diazotization-dependent deamination pathway.

Recently, we identified the biosynthetic gene cluster of avenalumic acid ( cluster) and revealed its entire biosynthetic pathway, resulting in the discovery of a diazotization-dependent deamination pathway. Genome database analysis revealed the presence of more than 100 cluster-related biosynthetic gene clusters (BGCs) in actinomycetes; however, their functions remained unclear. In this study, we focused on an cluster-related BGC in ( cluster), and revealed that it is responsible for coumaric acid biosynthesis by heterologous expression of the cluster and in vitro enzyme assays using recombinant Cma proteins.

A unique sigma/anti-sigma system in the actinomycete Actinoplanes missouriensis.

Bacteria of the genus Actinoplanes form sporangia that contain dormant sporangiospores which, upon contact with water, release motile spores (zoospores) through a process called sporangium dehiscence. Here, we set out to study the molecular mechanisms behind sporangium dehiscence in Actinoplanes missouriensis and discover a sigma/anti-sigma system with unique features. Protein σ contains a functional sigma factor domain and an anti-sigma factor antagonist domain, while protein SipA contains an anti-sigma factor domain and an anti-sigma factor antagonist domain.

Architecture of Actinoplanes missouriensis sporangia and zoospores visualized using quick-freeze deep-etch electron microscopy.

The architecture of sporangia and zoospores of Actinoplanes missouriensis was analyzed at a high resolution using quick-freeze deep-etch replica electron microscopy. This analysis revealed that (i) sporangia were surrounded by at least 2 membranous layers with smooth surfaces, (ii) zoospores were enclosed by a fibrillar layer, and (iii) flagella were generated in a restricted area on the zoospore surface.

characterization of nonribosomal peptide synthetase-dependent -(2-hydrazineylideneacetyl)serine synthesis indicates a stepwise oxidation strategy to generate the α-diazo ester moiety of azaserine.

Azaserine, a natural product containing a diazo group, exhibits anticancer activity. In this study, we investigated the biosynthetic pathway to azaserine. The putative azaserine biosynthetic gene () cluster, which contains 21 genes, including those responsible for hydrazinoacetic acid (HAA) synthesis, was discovered using bioinformatics analysis of the genome.

Identification and Analysis of the Biosynthetic Gene Cluster for the Hydrazide-Containing Aryl Polyene Spinamycin.

Natural products containing nitrogen-nitrogen (N-N) bonds have attracted much attention because of their bioactivities and chemical features. Several recent studies have revealed the nitrous acid-dependent N-N bond-forming machinery. However, the catalytic mechanisms of hydrazide synthesis using nitrous acid remain unknown.

The chromosomal evolutionary lineage of the genus Zygosaccharomyces.

Genome ploidy of Zygosaccharomyces rouxii is an intriguing topic in the field of industrial yeast research. However, the evolutionary relationship between the genome of Z. rouxii and other Zygosaccharomyces species is complex and not completely understood.

Engineering the Substrate Specificity of Toluene Degrading Enzyme XylM Using Biosensor XylS and Machine Learning.

Enzyme engineering using machine learning has been developed in recent years. However, to obtain a large amount of data on enzyme activities for training data, it is necessary to develop a high-throughput and accurate method for evaluating enzyme activities. Here, we examined whether a biosensor-based enzyme engineering method can be applied to machine learning.

Bacterial Avenalumic Acid Biosynthesis Includes Substitution of an Aromatic Amino Group for Hydride by Nitrous Acid Dependent Diazotization.

The diazo group is an important functional group that can confer biological activity to natural products owing to its high reactivity. Recent studies have revealed that diazo groups are synthesized from amino groups using nitrous acid in secondary metabolites of actinomycetes. However, genome database analysis indicated that there are still many diazo group-biosynthesizing enzymes for unknown biosynthetic pathways.

Mechanism-based cross-linking probes capture the Escherichia coli ketosynthase FabB in conformationally distinct catalytic states.

Ketosynthases (KSs) catalyse essential carbon-carbon bond-forming reactions in fatty-acid biosynthesis using a two-step, ping-pong reaction mechanism. In Escherichia coli, there are two homodimeric elongating KSs, FabB and FabF, which possess overlapping substrate selectivity. However, FabB is essential for the biosynthesis of the unsaturated fatty acids (UFAs) required for cell survival in the absence of exogenous UFAs.

Involvement of BldC in the Formation of Physiologically Mature Sporangium in Actinoplanes missouriensis.

AmBldD is a global transcriptional regulator that represses the transcription of several genes required for sporangium formation in Actinoplanes missouriensis. Here, we characterized one of the AmBldD regulons: , encoding an ortholog of BldC, which is a transcriptional regulator involved in the morphological development of Streptomyces. We determined the transcriptional start point of the ortholog by high-resolution S1 nuclease mapping and found an AmBldD box in its 5'-untranslated region.

3-Amino-4-hydroxybenzoic acid production from glucose and/or xylose via recombinant Streptomyces lividans.

The aromatic compound 3-amino-4-hydroxybenzoic acid (3,4-AHBA) can be employed as a raw material for high-performance industrial plastics. The aim of this study is to produce 3,4-AHBA via a recombinant Streptomyces lividans strain containing griI and griH genes derived from Streptomyces griseus using culture medium with glucose and/or xylose, which are the main components in lignocellulosic biomass. Production of 3,4-AHBA by the recombinant S.

Enzymatic synthesis of non-natural flavonoids by combining geranyl pyrophosphate C6-methyltransferase and aromatic prenyltransferase.

Terpenoids are the largest class of natural products and are derived from C5 isoprene units. Recent discoveries of modification enzymes in native isoprene units before cyclization or transfer reactions have revealed that C5 units with additional carbon atoms are also used to produce terpenoids. These reports indicate that the utilization of these modification enzymes is useful for the enzymatic production of non-natural terpenoids.

Surface structure and nanomechanical properties of Actinoplanes missouriensis sporangia analyzed via atomic force microscopy.

The surface structures of the sporangia produced by Actinoplanes missouriensis were analyzed at high resolution in air and liquid via atomic force microscopy. Results revealed a dynamic change in sporangium surface structure in response to the amount of moisture. Furthermore, the Young's modulus of the sporangium surface (1.

The α/β Hydrolase AzpM Catalyzes Dipeptide Synthesis in Alazopeptin Biosynthesis Using Two Molecules of Carrier Protein-Tethered Amino Acid.

During the biosynthesis of alazopeptin, a tripeptide composed of two molecules of 6-diazo-5-oxo-L-norleucine (DON) and one of alanine, the α/β hydrolase AzpM synthesizes the DON-DON dipeptide using DON tethered to the carrier protein AzpF (DON-AzpF). However, whether AzpM catalyzes the condensation of DON-AzpF with DON or DON-AzpF remains unclear. Here, to distinguish between these two condensation possibilities, the reaction catalyzed by AzpM was examined in vitro using a DON analogue, azaserine (AZS).

Engineering of the Ligand Specificity of Transcriptional Regulator XylS by Deep Mutational Scanning.

Deep mutational scanning is a method for protein engineering. Here, we applied it to alter the ligand specificity of the transcriptional regulator XylS from to recognize toluic acid instead of the native ligand -toluic acid. For this purpose, we used an antibiotic resistance gene-based dual screening system, which was constructed for the directed evolution of XylS toward the above-mentioned ligand specificity.

Enhanced production of γ-amino acid 3-amino-4-hydroxybenzoic acid by recombinant Corynebacterium glutamicum under oxygen limitation.

Background: Bio-based aromatic compounds are of great interest to the industry, as commercial production of aromatic compounds depends exclusively on the unsustainable use of fossil resources or extraction from plant resources. γ-amino acid 3-amino-4-hydroxybenzoic acid (3,4-AHBA) serves as a precursor for thermostable bioplastics.

Results: Under aerobic conditions, a recombinant Corynebacterium glutamicum strain KT01 expressing griH and griI genes derived from Streptomyces griseus produced 3,4-AHBA with large amounts of amino acids as by-products.

Identification and Analysis of the Biosynthetic Gene Cluster for the Indolizidine Alkaloid Iminimycin in Streptomyces griseus.

Indolizidine alkaloids, which have versatile bioactivities, are produced by various organisms. Although the biosynthesis of some indolizidine alkaloids has been studied, the enzymatic machinery for their biosynthesis in Streptomyces remains elusive. Here, we report the identification and analysis of the biosynthetic gene cluster for iminimycin, an indolizidine alkaloid with a 6-5-3 tricyclic system containing an iminium cation from Streptomyces griseus.

Structural and Molecular Basis of the Catalytic Mechanism of Geranyl Pyrophosphate C6-Methyltransferase: Creation of an Unprecedented Farnesyl Pyrophosphate C6-Methyltransferase.

Prenyl pyrophosphate methyltransferases enhance the structural diversity of terpenoids. However, the molecular basis of their catalytic mechanisms is poorly understood. In this study, using multiple strategies, we characterized a geranyl pyrophosphate (GPP) C6-methyltransferase, BezA.