Multiplexed Integrating Plasmids for Engineering of the Erythromycin Gene Cluster for Expression in Streptomyces spp. and Combinatorial Biosynthesis

Abstract

Bacteria in the genus Streptomyces and its close relatives are prolific producers of secondary metabolites with antibiotic activity. Genome sequencing of these bacteria has revealed a rich source of potentially new antibiotic pathways, whose products have never been observed. Moreover, these new pathways can provide novel genes that could be used in combinatorial biosynthesis approaches to generate unnatural analogues of existing antibiotics. We explore here the use of multiple orthologous integrating plasmid systems, based on the int/attP loci from phages TG1, SV1, and ϕBT1, to express the polyketide synthase (PKS) for erythromycin in a heterologous Streptomyces host. Streptomyces strains containing the three polyketide synthase genes eryAI, eryAII, and eryAIII expressed from three different integrated plasmids produced the aglycone intermediate, 6-deoxyerythronolide B (6-dEB). 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.

Integrating Vectors for Genetic Studies in the Rare ActinomyceteAmycolatopsis marina

“…The eryF gene was amplified from Saccharopolyspora erythraea BIOT-0666 genomic DNA using the primer pair pHG1A-for/pHG1A-rev, and inserted by In-Fusion cloning into pBF20(27) cut with Nhel and Pacl to form the plasmid pHG1A. The 3785 bp fragment containing the ϕC31 int/attP and hygromycin resistance gene was amplified from plasmid pBF27C(27), using the primer pair pHG1-for and pHG1-rev. Plasmid pHG1A was digested with Xbal and Nhel, and the 5668 bp fragment was ligated with the 3785 bp PCR fragment from pBF27C by In-Fusion to give the plasmid pHG1.…”

“…The integrating plasmid pHG4 contains the TG1 int / attP locus and the apramycin-resistance gene ( aac3 ( IV )) for selection (Figure 1A). The fragment containing oriT, aac3 ( IV ) and TG1 int/attP was amplified from plasmid pBF20(27) using the primer pair pHG4-for/pHG4-rev. The fragment was joined via In-Fusion cloning to the 3344 bp HindIII-Pacl fragment from pBF22(27) (containing the E. coli plasmid replication origin, the bla gene encoding resistance to ampicillin and the actll-orf4 / act1p expression cassette) to form the plasmid pHG4.…”

“…This plasmid was originally constructed for the expression of EryF . The eryF gene was amplified from Saccharopolyspora erythraea BIOT-0666 genomic DNA using the primer pair pHG1A-for/pHG1A-rev, and inserted by In-Fusion cloning into pBF20(27) cut with Nhel and Pacl to form the plasmid pHG1A. The 3785 bp fragment containing the ϕC31 int/attP and hygromycin resistance gene was amplified from plasmid pBF27C(27), using the primer pair pHG1-for and pHG1-rev.…”

Synthetic Biology of Natural Products

“…Although the proof-of-concept target was a complex pathway of primary metabolism, the applicability of the same tools for natural product pathway engineering is obvious. The availability of integrating plasmids in actinobacteria, which are particularly talented producers of natural products, enables powerful multiplexed approaches for the combinatorial assembly of biosynthetic pathway variants (Fayed et al 2015), which, in the future, could be applied not only for combinatorial biosynthesis, but also for more general refactoring strategies in these species.…”

Current Trends in Natural Products Research from the CBNP10 Symposium at Warwick

“…In vivo strategies are those where the enzymes are studied that are known to give the natural product of interest, which are then exploited either within the producing host or within another suitable and easy to culture microorganism, such as E. coli or the baker's yeast Saccharomyces cerevisiae. To this purpose, assembly of large and complex enzymes can be achieved using different technologies, such as that of the newly developed multiplexed integrating plasmids (Fayed et al, 2015), which was presented during the CBNP10 by Hong Gao, from Maggie Smith's group at the University of York. This approach uses phage-encoded serine integrases (enzymes that can cut precisely DNA and recombine it in a predictable way), and its efficacy was demonstrated by cloning and assembling the cluster for biosynthesis of the antibiotic erythromycin.…”