From Discovery Proteomics to Process-Informed Monitoring in Biomanufacturing Chassis Development

Gaining control of existing biomanufacturing chassis organisms, such as Escherichia coli K12, and novel isolates, such as the salt-tolerant Halomonas bluephagenesis sp TD01 studied here, may be facilitated by the investigation and monitoring of their metabolic and regulatory processes, particularly through proteomics. Here, we consider the performance of a range of typically available proteomics platforms across a range of price points to map chassis organisms' metabolic pathways. A set of model bacterial samples was prepared from E. coli and H. bluephagenesis sp. TD01 in 1:1, 1:2, and 2:1 ratios and analyzed using five LC-MS systems. Of the 8222 proteins identified across all samples analyzed (4401 proteins from E. coli; 3821 from Halomonas sp. TD01), the TimsTOF and Exploris were able to achieve extensive proteome coverage, quantifying 5.5k and 5k proteins, respectively, with the ZenoTOF, Waters MRT, and the legacy Waters Vion, respectively, quantifying 3.5k, 1.3k, and ∼850 proteins at 1% FDR. Proteins comprising core metabolic pathways critical to biomanufacturing in these chassis' organisms can be quantified with all instruments. We characterize metabolic adaptation in H. bluephagenesis by showing that replacement of glucose with a carboxylic acid feedstock directs carbon flux toward potential butane precursors, as well as how the acquired data permits monitoring of the cobalamin (vitamin B₁₂) production pathway.