Lechevalieria Aerocolonigenes Research Articles

Fermentative aminopyrrolnitrin production by metabolically engineered Corynebacterium glutamicum

Aminopyrrolnitrin (APRN), a natural halogenated phenylpyrrole derivative (HPD), has strong antifungal and antiparasitic activities. Additionally, it showed 2.8-fold increased photostability compared to pyrrolnitrin, a commercially available HPD with antimicrobial activity. For microbial production of APRN, we first engineered anthranilate phosphoribosyltransferase encoded by trpD from Corynebacterium glutamicum, resulting in a TrpDA162D mutation that exhibits feedback-resistant against l-tryptophan and higher substrate affinity compared to wild-type TrpD. Plasmid-borne expression of trpDA162D in C. glutamicum TP851 strain with two copies of trpDA162D in the genome led to the production of 3.1 g/L l-tryptophan in flask culture. Subsequent step for l-tryptophan chlorination into 7-chloro-l-tryptophan was achieved by introducing diverse sources of genes encoding tryptophan 7-halogenase (PrnA or RebH) and flavin reductase (Fre, PrnF, or RebF). The combined expression of prnA from Serratia grimesii or Serratia plymuthica with flavin reductase gene from Escherichia coli, Pseudomonas fluorescens, or Lechevalieria aerocolonigenes yielded higher production of 7-chloro-l-tryptophan in comparison to other sets of two-component systems. In the next step, production of putative monodechloroaminopyrrolnitrin (MDAP) from 7-chloro-l-tryptophan was achieved through the expression of prnB encoding MDAP synthase from S. plymuthica or P. fluorescens. Finally, an artificial APRN biosynthetic pathway was constructed by simultaneously expressing genes coding for tryptophan 7-halogenase, flavin reductase, MDAP synthase, and MDAP halogenase (PrnC) from different microbial sources within the l-tryptophan-producing TP851 strain. As prnC from S. grimesii or S. plymuthica was introduced into the host strain, which carried plasmids expressing prnA from S. plymuthica, fre from E. coli, and prnB from S. plymuthica, APN3639 and APN3638 accumulated 29.5 mg/L and 28.1 mg/L of APRN in the culture broth. This study represents the first report on the fermentative APRN production by metabolically engineered C. glutamicum.

Coupled CFD‐DEM simulation and experimental validation of morphology and productivity by particle‐induced mechanical stress on the filamentous system of Lechevalieria aerocolonigenes

Coupled CFD‐DEM simulation and experimental validation of morphology and productivity by particle‐induced mechanical stress on the filamentous system of <i>Lechevalieria aerocolonigenes</i>

Pyrizomicin A and B: structure and bioactivity of new thiazolyl pyridines from Lechevalieria aerocolonigenes K10-0216.

Two new antibiotics, designated pyrizomicin A and B, were isolated from the cultured broth of a rare actinomycete strain, Lechevalieria aerocolonigenes K10-0216, by silica gel and HPLC purification. The chemical structures of pyrizomicin A and B were elucidated as new thiazolyl pyridine compounds by nuclear magnetic resonance and mass spectrometry. Pyrizomicin A and B both showed antimicrobial activity.

Recombinant flavin-dependent halogenases are functional in tobacco chloroplasts without co-expression of flavin reductase genes.

Halogenation of natural compounds in planta is rare. Herein, a successful engineering of tryptophan 6-halogenation into the plant context by heterologous expression of the Streptomyces toxytricini Stth gene and localization of its enzymatic product in various tobacco cell compartments is described. When co-expressed with the flavin reductase rebF from Lechevalieria aerocolonigenes, Stth efficiently produced chlorinated tryptophan in the cytosol. Further, supplementation of KBr yielded the brominated metabolite. More strikingly, targeting of the protein to the chloroplasts enabled effective halogenation of tryptophan even in absence of the partner reductase, providing crucial evidence for sufficient, organelle-specific supply of the FADH2 cofactor to drive halogen integration. Incorporation of an alternative enzyme, the 7-halogenase RebH from L. aerocolonigenes, into the metabolic set-up resulted in the formation of 6,7-dichlorotryptophan. Finally, expression of tryptophan decarboxylase (tdc) in concert with stth led to the generation of 6-chlorotryptamine, a new-to-nature precursor of monoterpenoid indole alkaloids. In sum, the report highlights the tremendous application potential of plants as a unique chassis for the engineering of rare and valuable halogenated natural products, with chloroplasts as the cache of reduction equivalents driving metabolic reactions.

Mangromicin C, a new analog of mangromicin.

Mangromicin analogs were discovered in a cultured broth of a rare actinomycete, Lechevalieria aerocolonigenes K10-0216.1,2 The mangromicin analogs showed antitrypanosomal and reactive oxygen species (ROS) scavenging bioactivities. Mangromicin analogs have unique structures, including common partial structures, notably a cyclopentadecane skeleton with a tetrahydrofuran unit and a 5,6-dihydro-4-hydroxy-2-pyrone moiety. In a previous report, we described eight new mangromicin analogs, which possessed anti-oxidative properties. At that time, we discovered mangromicin C (1) but were unable to determine its structure, which was clearly different from all the other analogs. We have now found that 1 has a structure which includes a tetrahydropyran ring (Figure 1). In this paper, we report the fermentation, isolation, structural elucidation and biological activity of 1.

Mangromicins, six new anti-oxidative agents isolated from a culture broth of the actinomycete, Lechevalieria aerocolonigenes K10-0216

We have been continually searching for novel chemical compounds from culture broths of various actinomycetes using a physicochemical screening system. During the course of this program, we have previously reported the discovery of two new natural products, designated mangromicins A and B, discovered in a broth of a rare actinomycete strain, Lechevalieria aerocolonigenes K10-0216. Mangromicins have a unique and rare structure, a cyclopentadecane skeleton with a tetrahydrofuran unit and a 5,6-dihydro-4-hydroxy-2-pyrone moiety. New mangromicin analogs were isolated by using an improved production medium. As a consequence, six analogs, together with mangromicins A and B, were isolated from a cultured broth of L. aerocolonigenes K10-0216. We named them mangromicins D, E, F, G, H and I. All mangromicins showed radical scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals and nitric oxide generated from LPS-stimulated RAW264.7 cells, a murine macrophage cell line. Among the analogs, mangromicins A and I showed the most potent DPPH radical scavenging activity and nitric oxide scavenging activity, respectively.

First total synthesis of the antifungal antibiotic thiobutacin

The first total synthesis of thiobutacin, a butanoic acid with antifungal activity recently isolated from the culture broth of a soil actinomycete, Lechevalieria aerocolonigenes strain VK-A9, is described. The five-step procedure involves readily available and cheap starting materials and can easily be transposed to the large scale. Fungal growth inhibition of thiobutacin is mediated by the pH of the growth medium. Maximum inhibitory activity was obtained between pH 6 and 7.

Novel oxidative strategies en route to rebeccamycin &amp; staurosporine

The indolocarbazoles rebeccamycin and staurosporine, originally isolated from the actinomycetes, Lechevalieria aerocolonigenes and Streptomyces longisporoflavus, have received much attention due to their strong anticancer activities. These natural products are biosynthesized by parallel pathways, involving complex oxidative transformations and couplings to fashion the fused six-ring aglycones from a starting pair of L-tryptophan molecules. This overall 10–14 electron oxidation of two L-tryptophans (or 7-chloro-L-tryptophans) is catalyzed by only four enzymes: RebO, RebD, RebP and RebC (or their Sta homologues). RebP and StaP, in particular, are of interest due to their ability to catalyze both aryl-aryl coupling and oxidative decarboxylation, effecting a net 4–8 electron oxidation to three distinct aglycone products. Their flavin partner enzymes, RebC and StaC, direct the oxidative flux of the reaction to yield only one of these aglycone forms. We have reconstituted the complete biosynthetic pathway to the rebeccamycin and staurosporine aglycones. This work reveals some of Nature's unique mechanistic strategies in constructing natural products by oxidative pathways.

Biosynthesis of Heterocyclic Antibiotics in Actinomycetes and an Approach to Synthesize the Natural Compounds

Staurosporine and rebeccamycin are natural antitumor compounds produced by actinomycetes. Indolocarbazole biosynthetic gene clusters have been cloned from both Streptomyces sp. TP-A0274 and Lechevalieria aerocolonigenes ATCC39243. Staurosporine biosynthetic gene cluster consists of 15 orfs spanning 22 kb, and rebeccamycin biosynthetic gene cluster consists of 10 orfs spanning 16 kb. Gene disruption and bioconversion experiments revealed that indole-3-pyruvic acid, which is derived from tryptophan, is coupled to yield chromopyrrolic acid, and then this dicarboxylic bisindole compound is transformed into the indolocarbazole skeleton via two oxidation steps. Goadsporin is another heterocyclic antibiotic produced by Streptomyces sp. TP-A0584. It is a polypeptide antibiotic containing the thiazole and oxazole rings. The biosynthetic goadsporin gene cluster consists of 10 genes: a structural gene godA and nine genes involved in post-translational modification, immunity, and transcriptional regulation. It is particularly noteworthy that godI, which shows high similarity to the subunit of the “signal recognition particle,” plays an important role in goadsporin immunity. Furthermore, combinatorial biosynthesis was applied to produce the indolocarbazole and goadsporin analogs, and as a result, 14 analogs of indolocarbazole and 4 of goadsporin were produced.

Thiobutacin, a Novel Antifungal and Antioomycete Antibiotic from Lechevalieria aerocolonigenes

A novel butanoic acid, thiobutacin (1), 4-(2-aminophenyl)-4-oxo-2-methylthiobutanoic acid (C11H13NO3S), was isolated from the culture broth of a soil actinomycete, Lechevalieria aerocolonigenes strain VK-A9. The structure of thiobutacin (1) was elucidated on the basis of the extensive 2D NMR spectral data including 1H-1H COSY, HMBC, HMQC, ROESY, and NOESY. Thiobutacin (1) showed antioomycete and antifungal activity against phytopathogenic Phytophthora capsici and Botrytis cinerea and the yeast Saccharomyces cerevisiae.

PTOYAMAcos, pTYM18, and pTYM19, actinomycete-Escherichia coli integrating vectors for heterologous gene expression.

A novel shuttle integration cosmid vector (pTOYAMAcos), based on pKU402, and shuttle integration vectors (pTYM18 and pTYM19) were constructed for the cloning of actinomycete DNA and its heterologous expression. These vectors contain oriT of an IncP transmissible plasmid in order to transfer genes by conjugation from Escherichia coli to actinomycetes, and they also contain int derived from actinophage phiC31 in order to integrate site-specifically into the chromosomal DNA. pTOYAMAcos contains the lambdacos site to promote packaging of vectors containing 35 to approximately 45-kb DNA fragments into lambda particles. pTYM18 and pTYM19 contain kanamaycin and thiostrepton resistance genes, respectively, and have multiple cloning sites including EcoRI and HindIII sites, which are available for blue/white screening in E. coli. To demonstrate the utility of these vectors, we expressed the entire gene cluster for rebeccamycin biosynthesis from Lechevalieria aerocolonigenes using pTOYAMAcos and detected rebeccamycin production in transformed S. lividans. In addition, we demonstrated the utility of pTYM 19 in a gene-disruption complementation test. L. aerocolonigenes deltarebC strain, which is defective in rebeccamycin production because of a rebC deletion, was restored to rebeccamycin production by complemention by rebC cloned in pTYM 19.