(63e) High-Throughput, Mass Spectrometry-Based Screening of Microbial Libraries to Produce Designer Free Fatty Acids with Custom Compositions
To overcome this grand challenge, we developed mass spectrometry (MS)-based platforms for rapid profiling of FFA compositions from microbial variants in the formats of both colony and liquid cultures. Particularly, matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF) MS was applied to achieve rapid phenotyping at a rate of 1 s per sample. The relative ratios of FFA molecules quantified using MALDI-ToF MS correlated well with those obtained using GC-MS, which generally takes >10 min to analyze one sample. To further streamline the creation and screening of microbial libraries, machine vision and acoustic lipid handling were applied to interface off-line mass spectrometric analysis with a robotic platformâthe Illinois Biological Foundry for Advanced Biomanufacturing. Such integration and automation greatly accelerated the design-build-test-learn cycles. We applied our platform to engineer substrate specificities of both fatty acid synthetase (FAS) and acyl-ACP thioesterase (TE) in Saccharomyces cerevisiae. Co-expression of FAS and TE variants enables synergistic improvement towards the production of medium-chain fatty acids of select chain lengths. For future work, we aim to improve quantification capability of MALDI-ToF MS profiling by reducing sample preparation variations and including internal standards. Enhanced quantitative analysis coupled with pathway and genome engineering may enable high-throughput isolation of strain mutants with improved titers of FFA production.