Rapid exometabolome footprinting combined with multivariate statistics: A powerful tool for bioprocess optimization

Corynebacterium glutamicum is used as an industrial platform organism for amino acid production. Previously, the organism was utilized to produce l-histidine with research focusing on metabolic engineering approaches to increase titer and yield. Only a few studies have been published that provide information on bioprocess development, with media optimization and fed-batch cultivation procedure being particularly promising areas. In this work, we show how experimental setups such as miniature cultivation technology, dynamic and time-optimized LC-MS/MS metabolic footprinting tools, and automated workflows for the detection of local and global metabolic patterns can significantly accelerate bioprocess development. Potential media bottlenecks in form of phosphate and magnesium availability were identified by sensitivity analysis in parallelized microscale cultivation assisted by lab automation. A rapid dilute-and-shoot flow-injection-analysis tandem mass spectrometry approach was used to cope with the resulting cultivation throughput and allowed to quantify amino acids with 1 min per sample. We were able to increase the l-histidine titer of a C. glutamicum random mutagenesis mutant by a factor of 5.8 through process optimization while also identifying both known and previously unknown targets for additional strain improvements. The presented methodology can be seen as a supplement to traditional approaches in the field of bioprocess development.