A DRASTIC (Diffuse Reflectance Absorbance Spectroscopy Taking in Chemometrics) approach for the rapid analysis of microbial fermentation products: Quantification of aristeromycin and neplanocin A in Streptomyces citricolor broths

Abstract Microbial cultures can provide metabolites which are useful as structural templates for rational drug design. Increasing the titre of the metabolite is an important part of this process and is often achieved by random mutagenesis. As titre improved mutants derived by this method are extremely rare, many thousands need to be screened. screening mutants for increased metabolite production relies on methods such as assessing binding via the scintilation proximity assay or identifying an increase in concentration using chromatography. Such methods are typically restricted by the necessity to perform solvent extractions and, in the case of HPLC analysis, to optimise separation of the components of interest. Although the routine procedures can be automated, such multi-step screening processes are far from ideal. Diffuse reflectance absorbance infra-red spectroscopy provides an alternative rapid, automated, quantitative approach which yields more detailed information about chemical characteristics than, for example, the UV absorbance spectrum typically used in HPLC analysis. The method can also be employed non-invasively on unprocessed fermentation samples. We demonstrate the use of this spectroscopictechnique in combination with chemometrics for determining the concentrations of aristeromycin and neplanocin A in Streptomyces citricolor fermentations. The fermentation broths of a range of mutants previously obtained during a titre improvement programme were analysed by standard HPLC techniques and by automated diffuse reflectance absorbance infra-red spectroscopy. Chemometric processing of the infra-red spectra was performed using supervised and unsupervised multivariate calibration methods. DRASTIC proved to be a rapid and reliable method for the estimation of metabolite overproduction in cultures of biotechnological interest, and it was possible to discriminate cultures overproducing closely related molecules.