Synthesis of the C50 Diastereomers of the C33-C51 Fragment of Stambomycin D

2022 ◽  
Author(s):  
Yongchen Wang ◽  
Venkaiah Chintalapudi ◽  
Haraldur Gudmundsson ◽  
Gregory L Challis ◽  
Edward Alexander Anderson
Keyword(s):  
Sequence Analysis ◽  
Polyketide Synthase ◽  
Genome Mining ◽  
Macrolide Antibiotics ◽  
Stereochemical Assignment

As products of genome mining, the stereochemical assignment of the macrolide antibiotics stambomycins A–D has been made on the basis of sequence analysis of the associated polyketide synthase, aside from...

scholarly journals Engineering the Erythromycin-Producing Strain Saccharopolyspora erythraea HOE107 for the Heterologous Production of Polyketide Antibiotics

2020 ◽  
Vol 11 ◽  
Author(s):  
Jin Lü ◽  
Qingshan Long ◽  
Zhilong Zhao ◽  
Lu Chen ◽  
Weijun He ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Polyketide Synthase ◽  
Streptomyces Coelicolor ◽  
Genome Mining ◽  
Bac Library ◽  
Gene Clusters ◽  
Artificial Chromosome ◽  
Saccharopolyspora Erythraea ◽  
Heterologous Production ◽  
Integration Sites

Bacteria of the genus Saccharopolyspora produce important polyketide antibiotics, including erythromycin A (Sac. erythraea) and spinosad (Sac. spinosa). We herein report the development of an industrial erythromycin-producing strain, Sac. erythraea HOE107, into a host for the heterologous expression of polyketide biosynthetic gene clusters (BGCs) from other Saccharopolyspora species and related actinomycetes. To facilitate the integration of natural product BGCs and auxiliary genes beneficial for the production of natural products, the erythromycin polyketide synthase (ery) genes were replaced with two bacterial attB genomic integration sites associated with bacteriophages ϕC31 and ϕBT1. We also established a highly efficient conjugation protocol for the introduction of large bacterial artificial chromosome (BAC) clones into Sac. erythraea strains. Based on this optimized protocol, an arrayed BAC library was effectively transferred into Sac. erythraea. The large spinosad gene cluster from Sac. spinosa and the actinorhodin gene cluster from Streptomyces coelicolor were successfully expressed in the ery deletion mutant. Deletion of the endogenous giant polyketide synthase genes pkeA1-pkeA4, the product of which is not known, and the flaviolin gene cluster (rpp) from the bacterium increased the heterologous production of spinosad and actinorhodin. Furthermore, integration of pJTU6728 carrying additional beneficial genes dramatically improved the yield of actinorhodin in the engineered Sac. erythraea strains. Our study demonstrated that the engineered Sac. erythraea strains SLQ185, LJ161, and LJ162 are good hosts for the expression of heterologous antibiotics and should aid in expression-based genome-mining approaches for the discovery of new and cryptic antibiotics from Streptomyces and rare actinomycetes.

scholarly journals Pyridoxal-5′-phosphate–dependent bifunctional enzyme catalyzed biosynthesis of indolizidine alkaloids in fungi

2019 ◽  
Vol 117 (2) ◽  
pp. 1174-1180 ◽  
Author(s):  
Guang Zhi Dai ◽  
Wen Bo Han ◽  
Ya Ning Mei ◽  
Kuang Xu ◽  
Rui Hua Jiao ◽  
...  
Keyword(s):  
Polyketide Synthase ◽  
Complex Structure ◽  
Genome Mining ◽  
Biosynthetic Gene Cluster ◽  
Major Facilitator Superfamily ◽  
Molecular Architecture ◽  
Widespread Occurrence ◽  
Indolizidine Alkaloids ◽  
Major Facilitator ◽  
Insight Into

Indolizidine alkaloids such as anticancer drugs vinblastine and vincristine are exceptionally attractive due to their widespread occurrence, prominent bioactivity, complex structure, and sophisticated involvement in the chemical defense for the producing organisms. However, the versatility of the indolizidine alkaloid biosynthesis remains incompletely addressed since the knowledge about such biosynthetic machineries is only limited to several representatives. Herein, we describe the biosynthetic gene cluster (BGC) for the biosynthesis of curvulamine, a skeletally unprecedented antibacterial indolizidine alkaloid from Curvularia sp. IFB-Z10. The molecular architecture of curvulamine results from the functional collaboration of a highly reducing polyketide synthase (CuaA), a pyridoxal-5′-phosphate (PLP)-dependent aminotransferase (CuaB), an NADPH-dependent dehydrogenase (CuaC), and a FAD-dependent monooxygenase (CuaD), with its transportation and abundance regulated by a major facilitator superfamily permease (CuaE) and a Zn(II)Cys6 transcription factor (CuaF), respectively. In contrast to expectations, CuaB is bifunctional and capable of catalyzing the Claisen condensation to form a new C–C bond and the α-hydroxylation of the alanine moiety in exposure to dioxygen. Inspired and guided by the distinct function of CuaB, our genome mining effort discovers bipolamines A−I (bipolamine G is more antibacterial than curvulamine), which represent a collection of previously undescribed polyketide alkaloids from a silent BGC in Bipolaris maydis ATCC48331. The work provides insight into nature’s arsenal for the indolizidine-coined skeletal formation and adds evidence in support of the functional versatility of PLP-dependent enzymes in fungi.

scholarly journals Biosynthesis of a Complex Yersiniabactin-Like Natural Product via themicLocus in Phytopathogen Ralstonia solanacearum

2011 ◽  
Vol 77 (17) ◽  
pp. 6117-6124 ◽  
Author(s):  
Martin F. Kreutzer ◽  
Hirokazu Kage ◽  
Peter Gebhardt ◽  
Barbara Wackler ◽  
Hans P. Saluz ◽  
...  
Keyword(s):  
Natural Product ◽  
Gene Cluster ◽  
Ralstonia Solanacearum ◽  
Insertional Mutagenesis ◽  
Polyketide Synthase ◽  
Genome Mining ◽  
Content Type ◽  
A Genome ◽  
Iron Deficient ◽  
Peptide Synthetase

ABSTRACTA genome mining study in the plant pathogenic bacteriumRalstonia solanacearumGMI1000 unveiled a polyketide synthase/nonribosomal peptide synthetase gene cluster putatively involved in siderophore biosynthesis. Insertional mutagenesis confirmed the respective locus to be operational under iron-deficient conditions and spurred the isolation of the associated natural product. Bioinformatic analyses of the gene cluster facilitated the structural characterization of this compound, which was subsequently identified as the antimycoplasma agent micacocidin. The metal-chelating properties of micacocidin were evaluated in competition experiments, and the cellular uptake of gallium-micacocidin complexes was demonstrated inR. solanacearumGMI1000, indicating a possible siderophore role. Comparative genomics revealed a conservation of the micacocidin gene cluster in defined, but globally dispersed phylotypes ofR. solanacearum.

scholarly journals Synthaser: A CD-search Enabled Python Toolkit for Analysing Domain Architecture of Fungal Secondary Metabolite Megasynth(Et)ases and Beyond

2021 ◽  
Author(s):  
Cameron LM Gilchrist ◽  
Yit Heng Chooi
Keyword(s):  
Web Application ◽  
Large Scale ◽  
Polyketide Synthase ◽  
Genome Mining ◽  
Domain Architecture ◽  
Fungal Genome ◽  
Nonribosomal Peptide ◽  
Conserved Domain ◽  
Search Tool

Abstract Background: Fungi are prolific producers of secondary metabolites (SMs), which are bioactive small molecules with important applications in medicine, agriculture and other industries. The backbones of a large proportion of fungal SMs are generated through the action of large, multi-domain megasynth(et)ases such as polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). The structure of these backbones is determined by the domain architecture of the corresponding megasynth(et)ase, and thus accurate annotation and classification of these architectures is an important step in linking SMs to their biosynthetic origins in the genome. Results: Here we report synthaser, a Python package leveraging the NCBI's conserved domain search tool for remote prediction and classification of fungal megasynth(et)ase domain architectures. synthaser is capable of batch sequence analysis, and produces rich textual output and interactive visualisations which allow for quick assessment of the megasynth(et)ase diversity of a fungal genome. synthaser uses a hierarchical rule-based classification system, which can be extensively customised by the user through a web application (http://gamcil.github.io/synthaser). We show that synthaser provides more accurate domain architecture predictions than comparable tools which rely on curated profile hidden Markov model (pHMM)-based approaches; the utilisation of the NCBI conserved domain database also allows for significantly greater flexibility compared to pHMM approaches. In addition, we demonstrate how synthaser can be applied to large scale genome mining pipelines through the construction of an Aspergillus PKS similarity network. Conclusions: synthaser is an easy to use tool that represents a significant upgrade to previous domain architecture analysis tools. synthaser is freely available under a MIT license from PyPI (https://pypi.org/project/synthaser) and GitHub (https://github.com/gamcil/synthaser). Keywords: secondary metabolism, domain architecture, polyketide synthase, nonribosomal peptide synthetase, bioinformatics, software

scholarly journals In Tuber Biocontrol of Potato Late Blight by a Collection of Phenazine-1-Carboxylic Acid-Producing Pseudomonas spp.

2021 ◽  
Vol 9 (12) ◽  
pp. 2525
Author(s):  
Geneviève Léger ◽  
Amy Novinscak ◽  
Adrien Biessy ◽  
Simon Lamarre ◽  
Martin Filion
Keyword(s):  
Carboxylic Acid ◽  
Late Blight ◽  
Polyketide Synthase ◽  
Genome Mining ◽  
Potato Late Blight ◽  
Genetic Determinants ◽  
Pseudomonas Spp ◽  
Disease Reduction ◽  
Biocontrol Activity ◽  
Different Levels

Phenazine-1-carboxylic acid (PCA) produced by plant-beneficial Pseudomonas spp. is an antibiotic with antagonistic activities against Phytophthora infestans, the causal agent of potato late blight. In this study, a collection of 23 different PCA-producing Pseudomonas spp. was confronted with P. infestans in potato tuber bioassays to further understand the interaction existing between biocontrol activity and PCA production. Overall, the 23 strains exhibited different levels of biocontrol activity. In general, P. orientalis and P. yamanorum strains showed strong disease reduction, while P. synxantha strains could not effectively inhibit the pathogen’s growth. No correlation was found between the quantities of PCA produced and biocontrol activity, suggesting that PCA cannot alone explain P. infestans’ growth inhibition by phenazine-producing pseudomonads. Other genetic determinants potentially involved in the biocontrol of P. infestans were identified through genome mining in strains displaying strong biocontrol activity, including siderophores, cyclic lipopeptides and non-ribosomal peptide synthase and polyketide synthase hybrid clusters. This study represents a step forward towards better understanding the biocontrol mechanisms of phenazine-producing Pseudomonas spp. against potato late blight.

scholarly journals Characterization of transglutaminase homologues found in andrimid biosynthesis

2014 ◽  
Vol 70 (a1) ◽  
pp. C476-C476
Author(s):  
Janice Reimer ◽  
T Schmeing
Keyword(s):  
Amino Acids ◽  
Polyketide Synthase ◽  
Genome Mining ◽  
Protein Domain ◽  
Amide Bond ◽  
Free Standing ◽  
Remarkable Feature ◽  
Nonribosomal Peptide ◽  
Peptide Carrier

Nonribosomal peptide synthetases (NRPSs) are multimodular enzymes that synthesize products with diverse structures and activities ranging from antibiotics to industrial solvents. They are arranged into an assembly line of modules where each module is responsible for incorporating a specific monomer into the final nonribosomal peptide (NRP). Diversity in NRPs stems from the fact that NRPSs utilize not only the 20 proteinogenic amino acids, but also include nonproteinogenic amino acids, fatty acids, and alpha-hydroxy acids as building block substrates. Andrimid is a NRP antibiotic that inhibits membrane biosynthesis by blocking bacterial acetyl coenzyme A carboxylases. It is synthesized in a hybrid NRPS-polyketide synthase (NRPS-PKS) using a fatty acid, phenylalanine, valine, and glycine. A remarkable feature of this synthetic system is that instead of a normal condensation domain, it uses two atypical free-standing proteins with homology to transglutaminases to catalyze the formation of the first and second amide bonds. We are characterizing the action of transglutaminase homologues (TGH) in andrimid synthesis using biochemical assays and X-ray crystallography. Initial investigations of the andrimid biosynthetic cluster found in Panteao agglomerans focused on the TGH, AdmF, which catalyzes the formation of the first amide bond. Crystallization trials have been initiated on AdmF in its apo form and in complex with its interacting binding partner, the peptide carrier protein domain AdmI. To date, only a few andrimid producing bacteria have been discovered. Using genome mining, a biosynthetic cluster homologous to the andrimid biosynthetic cluster found in Panteao agglomerans was identified in Vibrio coralliilyticus. The two TGHs, CoraF and CoraS, were cloned, expressed and purified, and crystallization trials are underway. Our progress in biochemical and biophysical characterization of AdmF, CoraF, and CoraS will be presented.

scholarly journals Doxorubicin Overproduction in Streptomyces peucetius: Cloning and Characterization of the dnrU Ketoreductase anddnrV Genes and the doxA Cytochrome P-450 Hydroxylase Gene

1999 ◽  
Vol 181 (1) ◽  
pp. 305-318 ◽  
Author(s):  
Natalie Lomovskaya ◽  
Sharee L. Otten ◽  
Yukiko Doi-Katayama ◽  
Leonid Fonstein ◽  
Xiao-Chun Liu ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Double Mutant ◽  
Polyketide Synthase ◽  
Enzymatic Reactions ◽  
Triple Mutant ◽  
Streptomyces Peucetius ◽  
Polyketide Synthase Gene ◽  
Notable Increase ◽  
Cytochrome P 450

Doxorubicin-overproducing strains of Streptomyces peucetius ATCC 29050 can be obtained through manipulation of the genes in the region of the doxorubicin (DXR) gene cluster that containsdpsH, the dpsG polyketide synthase gene, the putative dnrU ketoreductase gene, dnrV, and thedoxA cytochrome P-450 gene. These five genes were characterized by sequence analysis, and the effects of replacingdnrU, dnrV, doxA, ordpsH with mutant alleles and of doxAoverexpression on the production of the principal anthracycline metabolites of S. peucetius were studied. The exact roles of dpsH and dnrV could not be established, although dnrV is implicated in the enzymatic reactions catalyzed by DoxA, but dnrU appears to encode a ketoreductase specific for the C-13 carbonyl of daunorubicin (DNR) and DXR or their biosynthetic precursors. The highest DXR titers were obtained in a dnrX dnrU (N. Lomovskaya, Y. Doi-Katayama, S. Filippini, C. Nastro, L. Fonstein, M. Gallo, A. L. Colombo, and C. R. Hutchinson, J. Bacteriol. 180:2379–2386, 1998) double mutant and a dnrX dnrU dnrH (C. Scotti and C. R. Hutchinson, J. Bacteriol. 178:7316–7321, 1996) triple mutant. Overexpression of doxA in adoxA::aphII mutant resulted in the accumulation of DXR precursors instead of in a notable increase in DXR production. In contrast, overexpression of dnrV and doxAjointly in the dnrX dnrU double mutant or the dnrX dnrU dnrH triple mutant increased the DXR titer 36 to 86%.

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