Type II thioesterase ScoT is required for coelimycin production by the modular polyketide synthase Cpk of Streptomyces coelicolor A3(2).

Type II thioesterases were shown to maintain efficiency of modular type I polyketide synthases and nonribosomal peptide synthetases by removing acyl residues blocking extension modules. We found that thioesterase ScoT from Streptomyces coelicolor A3(2) is required for the production of the yellow-pigmented coelimycin by the modular polyketide synthase Cpk. No production of coelimycin was observed in cultures of scoT disruption mutant. Polyketide production was restored upon complementation with an intact copy of the scoT gene. An enzymatic assay showed that ScoT thioesterase can hydrolyse a 12-carbon acyl chain but the activity is too low to play a role in product release from the polyketide synthase. We conclude that ScoT is an editing enzyme necessary to maintain the activity of polyketide synthase Cpk. We provide a HPLC based method to measure the amount of coelimycin P2 in a culture medium.

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Type II Thioesterase ScoT, Associated with Streptomyces coelicolor A3(2) Modular Polyketide Synthase Cpk, Hydrolyzes Acyl Residues and Has a Preference for Propionate

Applied and Environmental Microbiology ◽

10.1128/aem.01371-08 ◽

2008 ◽

Vol 75 (4) ◽

pp. 887-896 ◽

Cited By ~ 17

Author(s):

Magdalena Kotowska ◽

Krzysztof Pawlik ◽

Aleksandra Smulczyk-Krawczyszyn ◽

Hubert Bartosz-Bechowski ◽

Katarzyna Kuczek

Keyword(s):

Substrate Specificity ◽

Kinetic Parameters ◽

Hybrid Systems ◽

Active Site ◽

Polyketide Synthase ◽

Streptomyces Coelicolor ◽

Polyketide Synthases ◽

Type Ii ◽

Starter Unit ◽

Modular Polyketide Synthase

ABSTRACT Type II thioesterases (TE IIs) were shown to maintain the efficiency of polyketide synthases (PKSs) by removing acyl residues blocking extension modules. However, the substrate specificity and kinetic parameters of these enzymes differ, which may have significant consequences when they are included in engineered hybrid systems for the production of novel compounds. Here we show that thioesterase ScoT associated with polyketide synthase Cpk from Streptomyces coelicolor A3(2) is able to hydrolyze acetyl, propionyl, and butyryl residues, which is consistent with its editing function. This enzyme clearly prefers propionate, in contrast to the TE IIs tested previously, and this indicates that it may have a role in control of the starter unit. We also determined activities of ScoT mutants and concluded that this enzyme is an α/β hydrolase with Ser90 and His224 in its active site.

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LeuRS can leucylate type I and type II tRNALeus in Streptomyces coelicolor

Nucleic Acids Research ◽

10.1093/nar/gkz443 ◽

2019 ◽

Vol 47 (12) ◽

pp. 6369-6385

Author(s):

Jia-Yi Fan ◽

Qian Huang ◽

Quan-Quan Ji ◽

En-Duo Wang

Keyword(s):

Tertiary Structure ◽

Streptomyces Coelicolor ◽

Catalytic Efficiency ◽

Type I ◽

Type Ii ◽

Specific Domain ◽

Recognition Mechanism ◽

Variable Loop

Abstract Transfer RNAs (tRNAs) are divided into two types, type I with a short variable loop and type II with a long variable loop. Aminoacylation of type I or type II tRNALeu is catalyzed by their cognate leucyl-tRNA synthetases (LeuRSs). However, in Streptomyces coelicolor, there are two types of tRNALeu and only one LeuRS (ScoLeuRS). We found that the enzyme could leucylate both types of ScotRNALeu, and had a higher catalytic efficiency for type II ScotRNALeu(UAA) than for type I ScotRNALeu(CAA). The results from tRNA and enzyme mutagenesis showed that ScoLeuRS did not interact with the canonical discriminator A73. The number of nucleotides, rather than the type of base of the variable loop in the two types of ScotRNALeus, was determined as important for aminoacylation. In vitro and in vivo assays showed that the tertiary structure formed by the D-loop and TψC-loop is more important for ScotRNALeu(UAA). We showed that the leucine-specific domain (LSD) of ScoLeuRS could help LeuRS, which originally only leucylates type II tRNALeu, to aminoacylate type I ScotRNALeu(CAA) and identified the crucial amino acid residues at the C-terminus of the LSD to recognize type I ScotRNALeu(CAA). Overall, our findings identified a rare recognition mechanism of LeuRS to tRNALeu.

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ClusterCAD: a computational platform for type I modular polyketide synthase design

Nucleic Acids Research ◽

10.1093/nar/gkx893 ◽

2017 ◽

Vol 46 (D1) ◽

pp. D509-D515 ◽

Cited By ~ 40

Author(s):

Clara H Eng ◽

Tyler W H Backman ◽

Constance B Bailey ◽

Christophe Magnan ◽

Héctor García Martín ◽

...

Keyword(s):

Polyketide Synthase ◽

Type I ◽

Modular Polyketide Synthase ◽

Computational Platform

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Cloning, Sequencing, and Functional Analysis of an Iterative Type I Polyketide Synthase Gene Cluster for Biosynthesis of the Antitumor Chlorinated Polyenone Neocarzilin in “Streptomyces carzinostaticus”

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.9.3468-3476.2004 ◽

2004 ◽

Vol 48 (9) ◽

pp. 3468-3476 ◽

Cited By ~ 43

Author(s):

Miyuki Otsuka ◽

Koji Ichinose ◽

Isao Fujii ◽

Yutaka Ebizuka

Keyword(s):

Gene Cluster ◽

Polyketide Synthase ◽

Streptomyces Coelicolor ◽

Open Reading Frames ◽

Modular Organization ◽

Type I ◽

Orf3 Protein ◽

Polyketide Synthase Gene ◽

Polyketide Chain ◽

Reading Frames

ABSTRACT Neocarzilins (NCZs) are antitumor chlorinated polyenones produced by “Streptomyces carzinostaticus” var. F-41. The gene cluster responsible for the biosynthesis of NCZs was cloned and characterized. DNA sequence analysis of a 33-kb region revealed a cluster of 14 open reading frames (ORFs), three of which (ORF4, ORF5, and ORF6) encode type I polyketide synthase (PKS), which consists of four modules. Unusual features of the modular organization is the lack of an obvious acyltransferase domain on modules 2 and 4 and the presence of longer interdomain regions more than 200 amino acids in length on each module. Involvement of the PKS genes in NCZ biosynthesis was demonstrated by heterologous expression of the cluster in Streptomyces coelicolor CH999, which produced the apparent NCZ biosynthetic intermediates dechloroneocarzillin A and dechloroneocarzilin B. Disruption of ORF5 resulted in a failure of NCZ production, providing further evidence that the cluster is essential for NCZ biosynthesis. Mechanistic consideration of NCZ formation indicates the iterative use of at least one module of the PKS, which subsequently releases its product by decarboxylation to generate an NCZ skeleton, possibly catalyzed by a type II thioesterase encoded by ORF7. This is a novel type I PKS system of bacterial origin for the biosynthesis of a reduced polyketide chain. Additionally, the protein encoded by ORF3, located upstream of the PKS genes, closely resembles the FADH2-dependent halogenases involved in the formation of halometabolites. The ORF3 protein could be responsible for the halogenation of NCZs, presenting a unique example of a halogenase involved in the biosynthesis of an aliphatic halometabolite.

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Deletion of a regulatory gene within the cpk gene cluster reveals novel antibacterial activity in Streptomyces coelicolor A3(2)

Microbiology ◽

10.1099/mic.0.038281-0 ◽

2010 ◽

Vol 156 (8) ◽

pp. 2343-2353 ◽

Cited By ~ 107

Author(s):

Marco Gottelt ◽

Stefan Kol ◽

Juan Pablo Gomez-Escribano ◽

Mervyn Bibb ◽

Eriko Takano

Keyword(s):

Antibacterial Activity ◽

Gene Cluster ◽

Polyketide Synthase ◽

Streptomyces Coelicolor ◽

Regulatory Gene ◽

Feedback Mechanism ◽

Biologically Active ◽

Transcriptional Analysis ◽

Type I ◽

Negative Feedback Mechanism

Genome sequencing of Streptomyces coelicolor A3(2) revealed an uncharacterized type I polyketide synthase gene cluster (cpk). Here we describe the discovery of a novel antibacterial activity (abCPK) and a yellow-pigmented secondary metabolite (yCPK) after deleting a presumed pathway-specific regulatory gene (scbR2) that encodes a member of the γ-butyrolactone receptor family of proteins and which lies in the cpk gene cluster. Overproduction of yCPK and abCPK in a scbR2 deletion mutant, and the absence of the newly described compounds from cpk deletion mutants, suggest that they are products of the previously orphan cpk biosynthetic pathway in which abCPK is converted into the yellow pigment. Transcriptional analysis suggests that scbR2 may act in a negative feedback mechanism to eventually limit yCPK biosynthesis. The results described here represent a novel approach for the discovery of new, biologically active compounds.

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Streptomyces coelicolor DNA homologous with acyltransferase domains of type I polyketide synthase gene complex

FEMS Microbiology Letters ◽

10.1111/j.1574-6968.1997.tb12773.x ◽

2006 ◽

Vol 157 (1) ◽

pp. 195-200 ◽

Cited By ~ 8

Author(s):

Katarzyna Kuczek ◽

Krzysztof Pawlik ◽

Magdalena Kotowska ◽

Marian Mordarski

Keyword(s):

Polyketide Synthase ◽

Streptomyces Coelicolor ◽

Type I ◽

Gene Complex ◽

Polyketide Synthase Gene

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Biosynthesis of Cell Envelope-Associated Phenolic Glycolipids in Mycobacterium marinum

Journal of Bacteriology ◽

10.1128/jb.02546-14 ◽

2015 ◽

Vol 197 (6) ◽

pp. 1040-1050 ◽

Cited By ~ 13

Author(s):

Olivia Vergnolle ◽

Sivagami Sundaram Chavadi ◽

Uthamaphani R. Edupuganti ◽

Poornima Mohandas ◽

Catherine Chan ◽

...

Keyword(s):

Polyketide Synthase ◽

Cell Envelope ◽

Acyl Chain ◽

Deep Understanding ◽

Fatty Acyl ◽

Chain Extension ◽

Type I ◽

Content Type ◽

Iterative Polyketide Synthase ◽

Phenolic Glycolipids

Phenolic glycolipids (PGLs) are polyketide synthase-derived glycolipids unique to pathogenic mycobacteria. PGLs are found in several clinically relevant species, including variousMycobacterium tuberculosisstrains,Mycobacterium leprae, and several nontuberculous mycobacterial pathogens, such asM. marinum. Multiple lines of investigation implicate PGLs in virulence, thus underscoring the relevance of a deep understanding of PGL biosynthesis. We report mutational and biochemical studies that interrogate the mechanism by which PGL biosynthetic intermediates (p-hydroxyphenylalkanoates) synthesized by the iterative polyketide synthase Pks15/1 are transferred to the noniterative polyketide synthase PpsA for acyl chain extension inM. marinum. Our findings support a model in which the transfer of the intermediates is dependent on ap-hydroxyphenylalkanoyl-AMP ligase (FadD29) acting as an intermediary between the iterative and the noniterative synthase systems. Our results also establish thep-hydroxyphenylalkanoate extension ability of PpsA, the first-acting enzyme of a multisubunit noniterative polyketide synthase system. Notably, this noniterative system is also loaded with fatty acids by a specific fatty acyl-AMP ligase (FadD26) for biosynthesis of phthiocerol dimycocerosates (PDIMs), which are nonglycosylated lipids structurally related to PGLs. To our knowledge, the partially overlapping PGL and PDIM biosynthetic pathways provide the first example of two distinct, pathway-dedicated acyl-AMP ligases loading the same type I polyketide synthase system with two alternate starter units to produce two structurally different families of metabolites. The studies reported here advance our understanding of the biosynthesis of an important group of mycobacterial glycolipids.

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DDAP: docking domain affinity and biosynthetic pathway prediction tool for type I polyketide synthases

10.1101/637405 ◽

2019 ◽

Author(s):

Tingyang Li ◽

Ashootosh Tripathi ◽

Fengan Yu ◽

David H. Sherman ◽

Arvind Rao

Keyword(s):

Polyketide Synthase ◽

High Efficiency ◽

Prediction Algorithm ◽

Type I ◽

Biosynthetic Pathways ◽

Data Set ◽

Link Type ◽

Pathway Prediction ◽

Modular Polyketide Synthase ◽

Popular Rule

AbstractSummaryDDAP is a tool for predicting the biosynthetic pathways of the products of type I modular polyketide synthase (PKS) with the focus on providing a more accurate prediction of the ordering of proteins and substrates in the pathway. In this study, the module docking domain (DD) affinity prediction performance on a hold-out testing data set reached AUC = 0.88; the MRR of pathway prediction reached 0.67. DDAP has advantages compared to previous informatics tools in several aspects: (i) it does not rely on large databases, making it a high efficiency tool, (ii) the predicted DD affinity is represented by a probability (0 to 1), which is more intuitive than raw scores, (iii) its performance is competitive compared to the current popular rule-based algorithm. To the best of our knowledge, DDAP is so far the first machine learning based algorithm for type I PKS pathway prediction. We also established the first database of type I modular PKSs, featuring a comprehensive annotation of available docking domains information in bacterial biosynthetic pathways.Availability and implementationThe DDAP database is available at https://tylii.github.io/ddap. The prediction algorithm DDAP is freely available on GitHub (https://github.com/tylii/ddap) and released under the MIT [email protected]

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