Version 1: Optimisation of aurodox production by Streptomyces goldiniensis.

Aurodox is a specialised metabolite of Streptomyces goldiniensis originally identified for its anti-streptococcal and anti-staphylococcal activity. More recently, there has been a renewed focus on the study of aurodox as the compound has been shown to inhibit virulence in both Enteropathogenic and Enterohemorrhagic Escherichia coli (EPEC/EHEC). Aurodox is able to inhibit host colonization in EPEC and EHEC infections via the downregulation of the Type III Secretion System – a major virulence factor in these pathogens. To further investigate activity, increased quantities of the compound are required for in-depth mechanism of action studies. To identify suitable aurodox production conditions, a number of relevant fermentation media were tested for enhanced aurodox yield, followed by bioassay and Liquid Chromatography Mass Spectrometry analysis. Glucose Yeast Extract medium (GYM) was identified as the leading production medium tested. Further analysis of S. goldiniensis growth in GYM medium established the optimum temporal range for aurodox production. A minimum growth period of 154 hours is required for detectable aurodox production in S. goldiniensis fermentations in liquid GYM medium. To improve overall aurodox titres, a heterologous expression (HE) approach was taken and four potential HE hosts were investigated. These data indicated that the aurodox biosynthetic gene cluster can be heterologously expressed in both S. collinus Tü365 and S. coelicolor M1152, with increased aurodox titres compared to those of S. goldiniensis. This work demonstrates how traditional physiological analysis and modern genetic techniques can be combined to improve natural product production for detailed mode-of-action studies.