scholarly journals Engineered Biosynthesis of a Novel Amidated Polyketide, Using the Malonamyl-Specific Initiation Module from the Oxytetracycline Polyketide Synthase

2006 ◽  
Vol 72 (4) ◽  
pp. 2573-2580 ◽  
Author(s):  
Wenjun Zhang ◽  
Brian D. Ames ◽  
Shiou-Chuan Tsai ◽  
Yi Tang
Keyword(s):  
Polyketide Synthase ◽  
Major Product ◽  
Polyketide Synthases ◽  
Type Ii ◽  
Streptomyces Rimosus ◽  
Aromatic Polyketides ◽  
Starter Unit ◽  
Necessary And Sufficient ◽  
Bacterial Type

ABSTRACT Tetracyclines are aromatic polyketides biosynthesized by bacterial type II polyketide synthases (PKSs). Understanding the biochemistry of tetracycline PKSs is an important step toward the rational and combinatorial manipulation of tetracycline biosynthesis. To this end, we have sequenced the gene cluster of oxytetracycline (oxy and otc genes) PKS genes from Streptomyces rimosus. Sequence analysis revealed a total of 21 genes between the otrA and otrB resistance genes. We hypothesized that an amidotransferase, OxyD, synthesizes the malonamate starter unit that is a universal building block for tetracycline compounds. In vivo reconstitution using strain CH999 revealed that the minimal PKS and OxyD are necessary and sufficient for the biosynthesis of amidated polyketides. A novel alkaloid (WJ35, or compound 2) was synthesized as the major product when the oxy-encoded minimal PKS, the C-9 ketoreductase (OxyJ), and OxyD were coexpressed in CH999. WJ35 is an isoquinolone compound derived from an amidated decaketide backbone and cyclized with novel regioselectivity. The expression of OxyD with a heterologous minimal PKS did not afford similarly amidated polyketides, suggesting that the oxy-encoded minimal PKS possesses novel starter unit specificity.

scholarly journals Structural and functional analysis of two di-domain aromatase/cyclases from type II polyketide synthases

2015 ◽  
Vol 112 (50) ◽  
pp. E6844-E6851 ◽  
Author(s):  
Grace Caldara-Festin ◽  
David R. Jackson ◽  
Jesus F. Barajas ◽  
Timothy R. Valentic ◽  
Avinash B. Patel ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Functional Role ◽  
Functional Characterization ◽  
Polyketide Synthases ◽  
Type Ii ◽  
Aromatic Polyketides ◽  
Linear Poly ◽  
Bacterial Type

Aromatic polyketides make up a large class of natural products with diverse bioactivity. During biosynthesis, linear poly-β-ketone intermediates are regiospecifically cyclized, yielding molecules with defined cyclization patterns that are crucial for polyketide bioactivity. The aromatase/cyclases (ARO/CYCs) are responsible for regiospecific cyclization of bacterial polyketides. The two most common cyclization patterns are C7–C12 and C9–C14 cyclizations. We have previously characterized three monodomain ARO/CYCs: ZhuI, TcmN, and WhiE. The last remaining uncharacterized class of ARO/CYCs is the di-domain ARO/CYCs, which catalyze C7–C12 cyclization and/or aromatization. Di-domain ARO/CYCs can further be separated into two subclasses: “nonreducing” ARO/CYCs, which act on nonreduced poly-β-ketones, and “reducing” ARO/CYCs, which act on cyclized C9 reduced poly-β-ketones. For years, the functional role of each domain in cyclization and aromatization for di-domain ARO/CYCs has remained a mystery. Here we present what is to our knowledge the first structural and functional analysis, along with an in-depth comparison, of the nonreducing (StfQ) and reducing (BexL) di-domain ARO/CYCs. This work completes the structural and functional characterization of mono- and di-domain ARO/CYCs in bacterial type II polyketide synthases and lays the groundwork for engineered biosynthesis of new bioactive polyketides.

Genetic characterization of enzymes involved in the priming steps of oxytetracycline biosynthesis in Streptomyces rimosus

Microbiology ◽  
2011 ◽  
Vol 157 (8) ◽  
pp. 2401-2409 ◽  
Author(s):  
Peng Wang ◽  
Xue Gao ◽  
Yit-Heng Chooi ◽  
Zixin Deng ◽  
Yi Tang
Keyword(s):  
Homology Modelling ◽  
Streptomyces Rimosus ◽  
Wild Type ◽  
Aromatic Polyketides ◽  
Starter Unit ◽  
Priming Process ◽  
Hydrolysis Of ◽  
Bacterial Type

Tetracyclines are clinically important aromatic polyketides whose biosynthesis is catalysed by bacterial type II polyketide synthases (PKSs). Tetracyclines are biosynthesized starting with an amide-containing malonamate starter unit and the resulting C-2 carboxyamide is critical for the antibiotic activities. In this work, we genetically verified that an amidotransferase, OxyD, and a thiolase, OxyP, are involved in the biosynthesis and incorporation of the starter unit. First, two mutations, R248T and D268N, were found to be present in OxyD* encoded in Streptomyces rimosus ATCC 13224, a strain that produces the acetate-primed 2-acetyl-2-decarboxyamido-oxytetracycline (ADOTC) instead of the malonamate-primed oxytetracycline (OTC). Homology modelling suggested that in particular D268N may inactivate OxyD. Complementation of S. rimosus ATCC 13224 with wild-type OxyD restored OTC biosynthesis, thereby confirming the essential role of OxyD in the synthesis of the amide starter unit. Second, using a series of knockout and complementation approaches, we demonstrated that OxyP is most likely involved in maintaining fidelity of the amide-priming process via hydrolysis of the competing acetate priming starter units. While the inactivation of OxyP does not eliminate OTC biosynthesis, the ratio of acetate-primed ADOTC to malonamate-primed OTC is significantly increased. This suggests that OxyP plays an ancillary role in OTC biosynthesis and is important for minimizing the levels of ADOTC, a shunt product that has much weaker antibiotic activities than OTC.

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

2008 ◽  
Vol 75 (4) ◽  
pp. 887-896 ◽  
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.

scholarly journals Policing starter unit selection of the enterocin type II polyketide synthase by the type II thioesterase EncL

2011 ◽  
Vol 19 (22) ◽  
pp. 6633-6638 ◽  
Author(s):  
John A. Kalaitzis ◽  
Qian Cheng ◽  
Dario Meluzzi ◽  
Longkuan Xiang ◽  
Miho Izumikawa ◽  
...  
Keyword(s):  
Polyketide Synthase ◽  
Type Ii ◽  
Unit Selection ◽  
Starter Unit ◽  
Selection Of

Effect of the DaunorubicindpsHGene on the Choice of Starter Unit and Cyclization Pattern Reveals That Type II Polyketide Synthases Can Be Unfaithful yet Intriguing

1997 ◽  
Vol 119 (31) ◽  
pp. 7392-7393 ◽  
Author(s):  
Martin Gerlitz ◽  
Guido Meurer ◽  
Evelyn Wendt-Pienkowski ◽  
Krishnamurthy Madduri ◽  
C. Richard Hutchinson
Keyword(s):  
Polyketide Synthases ◽  
Type Ii ◽  
Starter Unit

scholarly journals Type II fatty acid and polyketide synthases: deciphering protein–protein and protein–substrate interactions

2018 ◽  
Vol 35 (10) ◽  
pp. 1029-1045 ◽  
Author(s):  
Aochiu Chen ◽  
Rebecca N. Re ◽  
Michael D. Burkart
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Polyketide Synthase ◽  
Polyketide Synthases ◽  
Type Ii ◽  
Substrate Interactions ◽  
Protein Substrate ◽  
Functional Roles

Metabolites from type II fatty acid synthase (FAS) and polyketide synthase (PKS) pathways differ broadly in their identities and functional roles.

scholarly journals Functional Analyses of Oxygenases in Jadomycin Biosynthesis and Identification of JadH as a Bifunctional Oxygenase/Dehydrase

2005 ◽  
Vol 280 (23) ◽  
pp. 22508-22514 ◽  
Author(s):  
Yi-Hua Chen ◽  
Chen-Chen Wang ◽  
Lisa Greenwell ◽  
Uwe Rix ◽  
Dirk Hoffmeister ◽  
...  
Keyword(s):  
Polyketide Synthase ◽  
Streptomyces Lividans ◽  
Gene Products ◽  
General Phenomenon ◽  
Heterologous Host ◽  
Aromatic Polyketides ◽  
Biochemical Analyses ◽  
Functional Analyses

A novel angucycline metabolite, 2,3-dehydro-UWM6, was identified in a jadH mutant of Streptomyces venezuelae ISP5230. Both UWM6 and 2,3-dehydro-UWM6 could be converted to jadomycin A or B by a ketosynthase α (jadA) mutant of S. venezuelae. These angucycline intermediates were also converted to jadomycin A by transformant of the heterologous host Streptomyces lividans expressing the jadFGH oxygenases in vivo and by its cell-free extracts in vitro; thus the three gene products JadFGH are implicated in catalysis of the post-polyketide synthase biosynthetic reactions converting UWM6 to jadomycin aglycone. Genetic and biochemical analyses indicate that JadH possesses dehydrase activity, not previously associated with polyketide-modifying oxygenase. Since the formation of aromatic polyketides often requires multiple dehydration steps, bifunctionality of oxygenases modifying aromatic polyketides may be a general phenomenon.

scholarly journals Characterization of the Biosynthetic Genes for 10,11-Dehydrocurvularin, a Heat Shock Response-Modulating Anticancer Fungal Polyketide from Aspergillus terreus

2013 ◽  
Vol 79 (6) ◽  
pp. 2038-2047 ◽  
Author(s):  
Yuquan Xu ◽  
Patricia Espinosa-Artiles ◽  
Vivien Schubert ◽  
Ya-ming Xu ◽  
Wei Zhang ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Response ◽  
Aspergillus Terreus ◽  
Polyketide Synthase ◽  
Polyketide Synthases ◽  
Content Type ◽  
Shock Response ◽  
Starter Unit ◽  
Acid Lactone ◽  
Resorcylic Acid Lactones

ABSTRACT10,11-Dehydrocurvularin is a prevalent fungal phytotoxin with heat shock response and immune-modulatory activities. It features a dihydroxyphenylacetic acid lactone polyketide framework with structural similarities to resorcylic acid lactones like radicicol or zearalenone. A genomic locus was identified from the dehydrocurvularin producer strainAspergillus terreusAH-02-30-F7 to reveal genes encoding a pair of iterative polyketide synthases (A. terreusCURS1 [AtCURS1] and AtCURS2) that are predicted to collaborate in the biosynthesis of 10,11-dehydrocurvularin. Additional genes in this locus encode putative proteins that may be involved in the export of the compound from the cell and in the transcriptional regulation of the cluster. 10,11-Dehydrocurvularin biosynthesis was reconstituted inSaccharomyces cerevisiaeby heterologous expression of the polyketide synthases. Bioinformatic analysis of the highly reducing polyketide synthase AtCURS1 and the nonreducing polyketide synthase AtCURS2 highlights crucial biosynthetic programming differences compared to similar synthases involved in resorcylic acid lactone biosynthesis. These differences lead to the synthesis of a predicted tetraketide starter unit that forms part of the 12-membered lactone ring of dehydrocurvularin, as opposed to the penta- or hexaketide starters in the 14-membered rings of resorcylic acid lactones. TetraketideN-acetylcysteamine thioester analogues of the starter unit were shown to support the biosynthesis of dehydrocurvularin and its analogues, with yeast expressing AtCURS2 alone. Differential programming of the product template domain of the nonreducing polyketide synthase AtCURS2 results in an aldol condensation with a different regiospecificity than that of resorcylic acid lactones, yielding the dihydroxyphenylacetic acid scaffold characterized by an S-type cyclization pattern atypical for fungal polyketides.

scholarly journals New insights into bacterial type II polyketide biosynthesis

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 172 ◽  
Author(s):  
Zhuan Zhang ◽  
Hai-Xue Pan ◽  
Gong-Li Tang
Keyword(s):  
Biological Activities ◽  
Genome Mining ◽  
Large Family ◽  
Combinatorial Biosynthesis ◽  
Type Ii ◽  
Polyketide Biosynthesis ◽  
Aromatic Polyketides ◽  
Starting Point ◽  
Biosynthetic Engineering ◽  
Bacterial Type

Bacterial aromatic polyketides, exemplified by anthracyclines, angucyclines, tetracyclines, and pentangular polyphenols, are a large family of natural products with diverse structures and biological activities and are usually biosynthesized by type II polyketide synthases (PKSs). Since the starting point of biosynthesis and combinatorial biosynthesis in 1984–1985, there has been a continuous effort to investigate the biosynthetic logic of aromatic polyketides owing to the urgent need of developing promising therapeutic candidates from these compounds. Recently, significant advances in the structural and mechanistic identification of enzymes involved in aromatic polyketide biosynthesis have been made on the basis of novel genetic, biochemical, and chemical technologies. This review highlights the progress in bacterial type II PKSs in the past three years (2013–2016). Moreover, novel compounds discovered or created by genome mining and biosynthetic engineering are also included.

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