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Multiplexed integrating plasmids for engineering of the erythromycin gene cluster for expression in Streptomyces spp. and combinatorial biosynthesis.

Fayed B, Ashford DA, Hashem AM, Amin MA, El Gazayerly ON, Gregory MA, Smith MC - Appl. Environ. Microbiol. (2015)

Bottom Line: A further pair of integrating plasmids, both derived from the ϕC31 int/attP locus, were constructed carrying a gene cassette for glycosylation of the aglycone intermediates, with or without the tailoring gene, eryF, required for the synthesis of erythronolide B (EB).Liquid chromatography-mass spectrometry of the metabolites indicated the production of angolosaminyl-6-dEB and angolosaminyl-EB.The advantages of using multiplexed integrating plasmids for engineering expression and for combinatorial biosynthesis were demonstrated.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of York, York, United Kingdom Chemistry of Natural and Microbial Products Department, National Research Centre, Cairo, Egypt.

No MeSH data available.


Overview of the biosynthesis of erythromycin. (A) Map of the erythromycin gene cluster from S. erythraea NRRL 2338. (B) Synthesis of erythromycin A showing the intermediates 6-deoxyerythronolide B (6-dEB) and erythronolide B (EB). (C) Four activated sugars relevant to the present study.
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Figure 1: Overview of the biosynthesis of erythromycin. (A) Map of the erythromycin gene cluster from S. erythraea NRRL 2338. (B) Synthesis of erythromycin A showing the intermediates 6-deoxyerythronolide B (6-dEB) and erythronolide B (EB). (C) Four activated sugars relevant to the present study.

Mentions: To exemplify the use of multiplexed integrating vectors to facilitate genetic manipulation and combinatorial biosynthesis of antibiotic pathways, we chose the erythromycin biosynthesis cluster (Fig. 1A). Erythromycin A is a bacteriostatic macrolide antibiotic produced from Saccharopolyspora erythraea (formerly Streptomyces erythreus) (13). The biosynthesis of erythromycin can be divided into two stages (14). First, the modular polyketide synthase (PKS) complex, 6-deoxyerythronolide B (6-dEB) synthase, catalyzes the sequential condensation of proprionyl-coenzyme A (CoA) and six methylmalonyl-CoA precursors to generate 6-dEB, the first isolatable intermediate in the pathway (Fig. 1). The second stage is the conversion of 6-dEB to erythromycin A, starting with the conversion of 6-dEB to erythronolide B (EB) by EryF hydroxylase (15). Two deoxysugars are then transferred to the aglycone ring to generate the first bioactive intermediate, erythromycin D (15); EryBV glycosyltransferase transfers l-mycarose to yield 3-O-mycarosylerythronolide B and then EryCIII, activated by EryCII, transfers d-deoxydesosamine to the C-5 hydroxyl (14, 16). The genes required for the biosynthesis of the activated sugars, TDP-deoxymycarose and TDP-deoxydesosamine, are all encoded within the erythromycin gene cluster (16). The final two tailoring steps, hydroxylation of C-12 by EryK hydroxylase (17), and methylation the 3′-OH of mycarose by EryG methyltransferase lead to the final product erythromycin A (18) (Fig. 1).


Multiplexed integrating plasmids for engineering of the erythromycin gene cluster for expression in Streptomyces spp. and combinatorial biosynthesis.

Fayed B, Ashford DA, Hashem AM, Amin MA, El Gazayerly ON, Gregory MA, Smith MC - Appl. Environ. Microbiol. (2015)

Overview of the biosynthesis of erythromycin. (A) Map of the erythromycin gene cluster from S. erythraea NRRL 2338. (B) Synthesis of erythromycin A showing the intermediates 6-deoxyerythronolide B (6-dEB) and erythronolide B (EB). (C) Four activated sugars relevant to the present study.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4644662&req=5

Figure 1: Overview of the biosynthesis of erythromycin. (A) Map of the erythromycin gene cluster from S. erythraea NRRL 2338. (B) Synthesis of erythromycin A showing the intermediates 6-deoxyerythronolide B (6-dEB) and erythronolide B (EB). (C) Four activated sugars relevant to the present study.
Mentions: To exemplify the use of multiplexed integrating vectors to facilitate genetic manipulation and combinatorial biosynthesis of antibiotic pathways, we chose the erythromycin biosynthesis cluster (Fig. 1A). Erythromycin A is a bacteriostatic macrolide antibiotic produced from Saccharopolyspora erythraea (formerly Streptomyces erythreus) (13). The biosynthesis of erythromycin can be divided into two stages (14). First, the modular polyketide synthase (PKS) complex, 6-deoxyerythronolide B (6-dEB) synthase, catalyzes the sequential condensation of proprionyl-coenzyme A (CoA) and six methylmalonyl-CoA precursors to generate 6-dEB, the first isolatable intermediate in the pathway (Fig. 1). The second stage is the conversion of 6-dEB to erythromycin A, starting with the conversion of 6-dEB to erythronolide B (EB) by EryF hydroxylase (15). Two deoxysugars are then transferred to the aglycone ring to generate the first bioactive intermediate, erythromycin D (15); EryBV glycosyltransferase transfers l-mycarose to yield 3-O-mycarosylerythronolide B and then EryCIII, activated by EryCII, transfers d-deoxydesosamine to the C-5 hydroxyl (14, 16). The genes required for the biosynthesis of the activated sugars, TDP-deoxymycarose and TDP-deoxydesosamine, are all encoded within the erythromycin gene cluster (16). The final two tailoring steps, hydroxylation of C-12 by EryK hydroxylase (17), and methylation the 3′-OH of mycarose by EryG methyltransferase lead to the final product erythromycin A (18) (Fig. 1).

Bottom Line: A further pair of integrating plasmids, both derived from the ϕC31 int/attP locus, were constructed carrying a gene cassette for glycosylation of the aglycone intermediates, with or without the tailoring gene, eryF, required for the synthesis of erythronolide B (EB).Liquid chromatography-mass spectrometry of the metabolites indicated the production of angolosaminyl-6-dEB and angolosaminyl-EB.The advantages of using multiplexed integrating plasmids for engineering expression and for combinatorial biosynthesis were demonstrated.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of York, York, United Kingdom Chemistry of Natural and Microbial Products Department, National Research Centre, Cairo, Egypt.

No MeSH data available.