scholarly journals Evidence for an iterative module in chain elongation on the azalomycin polyketide synthase

2016 ◽  
Vol 12 ◽  
pp. 2164-2172 ◽  
Author(s):  
Hui Hong ◽  
Yuhui Sun ◽  
Yongjun Zhou ◽  
Emily Stephens ◽  
Markiyan Samborskyy ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Polyketide Synthase ◽  
Strong Support ◽  
Artificial Chromosome ◽  
Chain Extension ◽  
Chain Elongation ◽  
Starter Unit ◽  
Polyketide Chain ◽  
Streptomyces Olivaceus ◽  
Streptomyces Malaysiensis

The assembly-line synthases that produce bacterial polyketide natural products follow a modular paradigm in which each round of chain extension is catalysed by a different set or module of enzymes. Examples of deviation from this paradigm, in which a module catalyses either multiple extensions or none are of interest from both a mechanistic and an evolutionary viewpoint. We present evidence that in the biosynthesis of the 36-membered macrocyclic aminopolyol lactones (marginolactones) azalomycin and kanchanamycin, isolated respectively from Streptomyces malaysiensis DSM4137 and Streptomyces olivaceus Tü4018, the first extension module catalyses both the first and second cycles of polyketide chain extension. To confirm the integrity of the azl gene cluster, it was cloned intact on a bacterial artificial chromosome and transplanted into the heterologous host strain Streptomyces lividans, which does not possess the genes for marginolactone production. When furnished with 4-guanidinobutyramide, a specific precursor of the azalomycin starter unit, the recombinant S. lividans produced azalomycin, showing that the polyketide synthase genes in the sequenced cluster are sufficient to accomplish formation of the full-length polyketide chain. This provides strong support for module iteration in the azalomycin and kanchanamycin biosynthetic pathways. In contrast, re-sequencing of the gene cluster for biosynthesis of the polyketide β-lactone ebelactone in Streptomyces aburaviensis has shown that, contrary to a recently-published proposal, the ebelactone polyketide synthase faithfully follows the colinear modular paradigm.

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 Engineering Anthracycline Biosynthesis toward Angucyclines

2003 ◽  
Vol 47 (4) ◽  
pp. 1291-1296 ◽  
Author(s):  
Mikko Metsä-Ketelä ◽  
Kaisa Palmu ◽  
Tero Kunnari ◽  
Kristiina Ylihonko ◽  
Pekka Mäntsälä
Keyword(s):  
Gene Cluster ◽  
Polyketide Synthase ◽  
Gene Cassette ◽  
Side Chain ◽  
Antibiotic Biosynthesis ◽  
Biosynthesis Gene Cluster ◽  
Biosynthesis Gene ◽  
Starter Unit

ABSTRACT The biosynthesis pathways of two anthracyclines, nogalamycin and aclacinomycin, were directed toward angucyclines by using an angucycline-specific cyclase, pgaF, isolated from a silent antibiotic biosynthesis gene cluster. Addition of pgaF to a gene cassette that harbored the early biosynthesis genes of nogalamycin resulted in the production of two known angucyclinone metabolites, rabelomycin and its precursor, UWM6. Substrate flexibility of pgaF was demonstrated by replacement of the nogalamycin minimal polyketide synthase genes in the gene cassette with the equivalent aclacinomycin genes together with aknE2 and aknF, which specify the unusual propionate starter unit in aclacinomycin biosynthesis. This modification led to the production of a novel angucyclinone, MM2002, in which the expected ethyl side chain was incorporated into the fourth ring.

scholarly journals 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”

2004 ◽  
Vol 48 (9) ◽  
pp. 3468-3476 ◽  
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.

scholarly journals Identification of polyketide synthase gene clusters in a phage P1-derived artificial chromosome library of a Philippine strain of Streptomyces sp. PCS3-D2

2019 ◽  
pp. 56-63
Author(s):  
Aileen Bayot Custodio ◽  
Edwin Plata Alcantara
Keyword(s):  
Gene Cluster ◽  
Polyketide Synthase ◽  
Genetic Manipulation ◽  
Gene Clusters ◽  
Artificial Chromosome ◽  
Restriction Enzyme Digestion ◽  
Type I ◽  
Putative Gene ◽  
Streptomyces Sp ◽  
Type Iii

A phage P1-derived artificial chromosome (PAC) library was constructed from genomic DNA of Streptomyces sp. PCS3-D2. Polymerase chain reaction (PCR) screening of the PAC library revealed two clones, PAC16D and P222O, which were positively identified to harbor polyketide synthase (PKS) Type I and PKS Type III gene clusters, respectively. Restriction enzyme digestion showed that PAC16D and PAC222O contained a 130 kb and a 140 kb insert, respectively. Results of sequencing and bioinformatics analyses revealed that PAC16D comprised of a full-length PKS type I bafilomycin gene cluster while PAC222O harbored truncated siderophore and putative gene clusters as well as a complete PKS III biosynthetic gene cluster. The PKS III gene cluster had three genes similar to alkyl resorcinol biosynthetic genes, however majority of the novel gene cluster had little similarity to known PKS Type III gene clusters. The successful cloning and identification of these gene clusters from Streptomyces sp. PCS3-D2 serve as the jump off point to further genetic manipulation in order to produce the insecticidal natural product in a heterologous host.

scholarly journals Engineering of a minimal modular polyketide synthase, and targeted alteration of the stereospecificity of polyketide chain extension

1998 ◽  
Vol 5 (8) ◽  
pp. 407-412 ◽  
Author(s):  
Ines Böhm ◽  
Inès E. Holzbaur ◽  
Ulf Hanefeld ◽  
Jesus Cortési ◽  
Jim Staunton ◽  
...  
Keyword(s):  
Polyketide Synthase ◽  
Chain Extension ◽  
Polyketide Chain ◽  
Modular Polyketide Synthase

scholarly journals Sulfation and amidinohydrolysis in the biosynthesis of giant linear polyenes

2017 ◽  
Vol 13 ◽  
pp. 2408-2415 ◽  
Author(s):  
Hui Hong ◽  
Markiyan Samborskyy ◽  
Katsiaryna Usachova ◽  
Katharina Schnatz ◽  
Peter F Leadlay
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Late Stage ◽  
Rare Case ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Starter Unit ◽  
Linear Polyenes ◽  
Streptomyces Malaysiensis

Clethramycin from Streptomyces malaysiensis DSM4137, and mediomycins (produced together with clethramycin from Streptomyces mediocidicus), are near-identical giant linear polyenes apparently constructed from, respectively, a 4-guanidinobutanoate or 4-aminobutanoate starter unit and 27 polyketide extender units, and bearing a specific O-sulfonate modification at the C-29 hydroxy group. We show here that mediomycins are actually biosynthesised not by use of a different starter unit but by direct late-stage deamidination of (desulfo)clethramycin. A gene (slf) encoding a candidate sulfotransferase has been located in both gene clusters. Deletion of this gene in DSM4137 led to accumulation of desulfoclethramycin only, instead of a mixture of desulfoclethramycin and clethramycin. The mediomycin gene cluster does not encode an amidinohydrolase, but when three candidate amidinohydrolase genes from elsewhere in the S. mediocidicus genome were individually expressed in Escherichia coli and assayed, only one of them (medi4948), located 670 kbp away from the mediomycin gene cluster on the chromosome, catalysed the removal of the amidino group from desulfoclethramycin. Subsequent cloning of medi4948 into DSM4137 caused mediomycins A and B to accumulate at the expense of clethramycin and desulfoclethramycin, respectively, a rare case where an essential biosynthetic gene is not co-located with other pathway genes. Clearly, both desulfoclethramycin and clethramycin are substrates for this amidinohydrolase. Also, purified recombinant sulfotransferase from DSM4137, in the presence of 3'-phosphoadenosine-5'-phosphosulfate as donor, efficiently converted mediomycin B to mediomycin A in vitro. Thus, in the final steps of mediomycin A biosynthesis deamidination and sulfotransfer can take place in either order.

scholarly journals Elucidating the mechanism of fluorinated extender unit loading for improved production of fluorine-containing polyketides

2017 ◽  
Vol 114 (5) ◽  
pp. E660-E668 ◽  
Author(s):  
Omer Ad ◽  
Benjamin W. Thuronyi ◽  
Michelle C. Y. Chang
Keyword(s):  
Polyketide Synthase ◽  
Acyl Carrier Protein ◽  
Type System ◽  
Large Family ◽  
Product Yield ◽  
Building Blocks ◽  
Chain Extension ◽  
Chain Elongation ◽  
Single Chain ◽  
Extender Unit

Polyketides are a large family of bioactive natural products synthesized by polyketide synthase (PKS) enzyme complexes predominantly from acetate and propionate. Given the structural diversity of compounds produced using these two simple building blocks, there has been longstanding interest in engineering the incorporation of alternative extender units. We have been investigating the mechanism of fluorinated monomer insertion by three of the six different modules of the PKS involved in erythromycin biosynthesis (6-deoxyerythronolide B synthase, DEBS) to begin understanding the contribution of different steps, such as enzyme acylation, transacylation, C–C bond formation, and chain transfer, to the overall selectivity and efficiency of this process. In these studies, we observe that inactivation of acis-acyltransferase (AT) domain to circumvent its native extender unit preference leads concurrently to a change of mechanism in which chain extension with fluorine-substituted extender units switches largely to an acyl carrier protein (ACP)-independent mode. This result suggests that the covalent linkage between the growing polyketide chain and the enzyme is lost in these cases, which would limit efficient chain elongation after insertion of a fluorinated monomer. However, use of a standalonetrans-acting AT to complement modules with catalytically deficient AT domains leads to enzyme acylation with the fluoromalonyl-CoA extender unit. Formation of the canonical ACP-linked intermediate with fluoromalonyl-CoA allows insertion of fluorinated extender units at 43% of the yield of the wild-type system while also amplifying product yield in single chain-extension experiments and enabling multiple chain extensions to form multiply fluorinated products.

scholarly journals Reconstitution of the FK228 Biosynthetic Pathway Reveals Cross Talk between Modular Polyketide Synthases and Fatty Acid Synthase

2010 ◽  
Vol 77 (4) ◽  
pp. 1501-1507 ◽  
Author(s):  
Shane R. Wesener ◽  
Vishwakanth Y. Potharla ◽  
Yi-Qiang Cheng
Keyword(s):  
Fatty Acid ◽  
Cross Talk ◽  
Biosynthetic Pathway ◽  
Fatty Acid Synthase ◽  
Polyketide Synthase ◽  
Fatty Acid Biosynthesis ◽  
Chain Elongation ◽  
E Coli ◽  
Genetic Components ◽  
Polyketide Chain

ABSTRACTFunctional cross talk between fatty acid biosynthesis and secondary metabolism has been discovered in several cases in microorganisms; none of them, however, involves a modular biosynthetic enzyme. Previously, we reported a hybrid modular nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) pathway for the biosynthesis of FK228 anticancer depsipeptide inChromobacterium violaceumstrain 968. This pathway contains two PKS modules on the DepBC enzymes that lack a functional acyltransferase (AT) domain, and no apparent AT-encoding gene exists within the gene cluster or its vicinity. We report here that, through reconstitution of the FK228 biosynthetic pathway inEscherichia colicells, two essential genes,fabD1andfabD2, both encoding a putative malonyl coenzyme A (CoA) acyltransferase component of the fatty acid synthase complex, are positively identified to be involved in FK228 biosynthesis. Either gene product appears sufficient to complement the AT-less PKS modules on DepBC for polyketide chain elongation. Concurrently, a gene (sfp) encoding a putative Sfp-type phosphopantetheinyltransferase was identified to be necessary for FK228 biosynthesis as well. Most interestingly, engineeredE. colistrains carrying variable genetic components produced significant levels of FK228 under both aerobic and anaerobic cultivation conditions. Discovery of thetranscomplementation of modular PKSs by housekeeping ATs reveals natural product biosynthesis diversity. Moreover, demonstration of anaerobic production of FK228 by an engineered facultative bacterial strain validates our effort toward the engineering of novel tumor-targeting bioagents.

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