Biosynthetic pathways for selected glycopeptides will be analysed and optimized to mobilize the full potential of the actinomycete Cell Factory in the development and production of improved antibiotics. Glycopeptides are structurally complex molecules so their accessibility to chemistry is severely limited, but room for improvement has been demonstrated. In order to have an impact on the health of EU citizens, improved antibiotics need to be discovered and effectively developed. The objectives of this proposal are the development of effective tools for the engineering of bacterial producers of glycopeptides, the discovery of improved glycopeptide antibiotics and the rapid development of promising antibiotics through optimization of the production process. To achieve these objectives, this proposal gathers an inter-disciplinary team of academic and industrial partners, recognized leaders in the application of tools such as combinatorial biochemistry, biotransformation, protein engineering, artificial chromosomes, synthetic chemistry, molecular genetics, metabolic flux analysis, high throughput screening and medical microbiology.
Most of the expected objectives and deliverables were achieved. We have
a) completed the DNA sequences of three glycopeptide clusters;
b) produced glycopeptide intermediates by genetic manipulation of producer strains;
c) overexpressed active oxidases and glycosyltransferases;
d) developed a gene transfer system for Nonomuraea and A. mediterranei;
e) established a defined medium for Nonomuraea growth and mapped metabolic fluxes;
f) implemented a HTS program for hybrid glycosyltransferases;
g) generated novel glycopeptides by mutasynthesis;
h) generated detailed information on parameters affecting A40926 production.

Through these achievements, we can now effectively manipulate two glycopeptide producer strains; we have established the feasibility and limitations to produce novel glycopeptides through chemical, enzymatic, biotransformation and genetic approaches; and we have used the information achieved through the project to develop an efficient process for A40926 production.