Abstract
Glycopeptide antibiotics (GPAs) are essential for the treatment of severe infectious diseases caused by Gram-positive bacteria. The emergence and spread of GPA resistance have propelled the search for more effective GPAs. Given their structural complexity, genetic intractability, and low titer, expansion of GPA chemical diversity using synthetic or medicinal chemistry remains challenging. Here we describe a synthetic biology platform, GPAHex (GPA Heterologous expression), which exploits the genes required for the specialized GPA building blocks, regulation, antibiotic transport, and resistance for the heterologous production of GPAs. Application of the GPAHex platform results in: (1) a 19-fold increase of corbomycin titer compared to the parental strain, (2) the discovery of a teicoplanin-class GPA from an Amycolatopsis isolate, and (3) the overproduction and characterization of a cryptic nonapeptide GPA. GPAHex provides a platform for GPA production and mining of uncharacterized GPAs and provides a blueprint for chassis design for other natural product classes.
Highlights
Glycopeptide antibiotics (GPAs) are essential for the treatment of severe infectious diseases caused by Gram-positive bacteria
We introduced genes encoding the gatekeepers of the shikimate pathway, 3deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase and chorismate mutase, from S. toyocaensis NRRL15009, downstream from the remaining A47934 biosynthetic genes resulting in plasmid pAMX4 (Fig. 1b and Supplementary Fig. 1). pAMX4 was delivered into S. coelicolor M115428 and integrated into the attBφC31 site to generate the GPAHex chassis, S. coelicolor M1154/ pAMX4
We developed a protocol for cloning of large natural products (NPs) biosynthetic gene clusters (BGCs) by constructing vector pCGW, which replaces the SuperCos I backbone of pCAP03-aac(3)IV with the copy number control replicon bearing ‘oriV-ori2-repE-sopABC’ cassette from pBAClacZ. pCGW can be maintained at 1–2 copies/cell when supplemented with 0.2 % D-glucose; it can be conditionally induced to ~100 copies/cell by adding 1 mM Larabinose in a trfA+ E. coil strains such as E. coli EPI300, which are critical for stable maintenance of large exogenous DNA32
Summary
Glycopeptide antibiotics (GPAs) are essential for the treatment of severe infectious diseases caused by Gram-positive bacteria. Vancomycin resistance occurs through reprogramming of cell wall biosynthesis, mediated by a vanRSHAX resistance cassette that replaces the D-Ala-D-Ala termini with D-Ala-D-Lactate This change of an amide linkage to an ester decreases the affinity of vancomycin to the D-Ala-D-Ala terminus by 1000 fold, resulting in a commensurate increase in the antibiotic minimal inhibitory concentration (MIC)[2,3,4]. The spread of vancomycin resistance across the globe and in most health care settings warrants the investigation of GPAs with improved efficacy To this end, three semi-synthetic derivatives of NP GPAs, telavancin, dalbavancin, and oritavancin, have been successfully introduced to the clinic[6,7,8,9,10,11]. Because they do not bind D-Ala-D-Ala, they are not susceptible to the canonical D-Ala-D-Lac-mediated GPA resistance and offer a class of promising antibiotics
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