Development of carbon plasma-coated multiwell plates for high-throughput mass spectrometric analysis of highly lipophilic fermentation products.
Classical approaches to strain improvement and metabolic engineering rely on rapid qualitative and quantitative analyses of the metabolites of interest. As an analytical tool, mass spectrometry (MS) has proven to be efficient and nearly universally applicable for timely screening of metabolites. Furthermore, gas chromatography (GC)/MS- and liquid chromatography (LC)/MS-based metabolite screens can often be adapted to high-throughput formats. We recently engineered a Saccharomyces cerevisiae strain to produce taxa-4(5),11(12)-diene, the first pathway-committing biosynthetic intermediate for the anticancer drug Taxol, through the heterologous and homologous expression of several genes related to isoprenoid biosynthesis. To date, GC/MS- and LC/MS-based high-throughput methods have been inherently difficult to adapt to the screening of isoprenoid-producing microbial strains due to the need for extensive sample preparation of these often highly lipophilic compounds. In the current work, we examined different approaches to the high-throughput analysis of taxa-4(5),11(12)-diene biosynthesizing yeast strains in a 96-deep-well format. Carbon plasma coating of standard 96-deep-well polypropylene plates allowed us to circumvent the inherent solvent instability of commonly used deep-well plates. In addition, efficient adsorption of the target isoprenoid product by the coated plates allowed rapid and simple qualitative and quantitative analyses of the individual cultures.
Heinig
Uwe
U
Fraunhofer Institut für Molekularbiologie und Angewandte Okologie, 52074 Aachen, Germany.
Scholz
Susanne
S
Dahm
Pia
P
Grabowy
Udo
U
Jennewein
Stefan
S
D000972:Antineoplastic Agents, Phytogenic / Q000032:analysis / Q000378:metabolism*; D002244:Carbon / Q000737:chemistry; D005285:Fermentation; D008401:Gas Chromatography-Mass Spectrometry / Q000295:instrumentation* / Q000379:methods; D057166:High-Throughput Screening Assays / Q000295:instrumentation* / Q000379:methods; D017239:Paclitaxel / Q000032:analysis / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013729:Terpenes / Q000032:analysis / Q000378:metabolism*
D000972:Antineoplastic Agents, Phytogenic; D013729:Terpenes; D002244:Carbon; D017239:Paclitaxel