(63e) High-Throughput, Mass Spectrometry-Based Screening of Microbial Libraries to Produce Designer Free Fatty Acids with Custom Compositions

Free fatty acids (FFAs) produced from microbial cell factories provide a sustainable alternative to petroleum for producing value-added oleochemicals including biodiesels, fatty alcohols, olefins, and waxes. The physiochemical properties of these molecules depend on the chemical structures of fatty acyl chains, such as chain length, branching pattern, and degree of saturation. For microbially produced FFAs, however, it remains a major challenge to control acyl chain compositions via enzymatic and metabolic engineering. Directed evolution has demonstrated wide successes in engineering biosynthetic selectivity via iterative rounds of large-scale genetic mutagenesis and phenotypic screening, but no general platform is currently available to analyze FFA-producing strain libraries in high throughput.

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 platform—the 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.