Biofuels derived from microalgal lipids have demonstrated a promising potential as future renewable bioenergy. However, the production costs for microalgae-based biofuels are not economically competitive, and one strategy to overcome this limitation is to develop better-performing microalgal strains that have faster growth and higher lipid content through genetic screening and metabolic engineering. In this work, we present

Protein phosphorylation is a post-translational modification with widespread regulatory roles in both eukaryotes and prokaryotes. Using mass spectrometry, we performed a genome wide investigation of protein phosphorylation in the non-model organism and biofuel producer Zymomonas mobilis under anaerobic, aerobic, and N2-fixing conditions. Our phosphoproteome analysis revealed 125 unique phosphorylated proteins, belonging to major pathways such as glycolysis, TCA cycle, electron transport, nitrogen metabolism, and protein synthesis. Quantitative analysis revealed significant and widespread changes in protein phosphorylation across growth conditions. For example, we observed increased phosphorylation of nearly all glycolytic enzymes and a large fraction of ribosomal proteins during aerobic and N2-fixing conditions. We also observed substantial changes in the phosphorylation status of enzymes and regulatory proteins involved in nitrogen fixation and ammonia assimilation during N2-fixing conditions, including nitrogenase, the Rnf electron transport complex, the transcription factor NifA, GS-GOGAT cycle enzymes, and the PII regulatory protein. This suggested that protein phosphorylation may play an important role at regulating all aspects of nitrogen metabolism in Z. mobilis. This study provides new knowledge regarding the specific pathways and cellular processes that may be regulated by protein phosphorylation in this important industrial organism and provides a useful road map for future experiments that investigate the physiological role of specific phosphorylation events in Z. mobilis.

While much effort has been placed on comprehensive quantitative proteome analysis, certain applications demand the measurement of only a few target proteins from complex systems. Traditional approaches to targeted proteomics rely on nanoliquid chromatography (nLC) and targeted mass spectrometry (MS) methods, e.g., parallel reaction monitoring (PRM). However, the time requirement for nLC can limit the throughput of targeted proteomics. To achieve rapid and high-throughput targeted methods, here we show that nLC separations can be eliminated and replaced with direct infusion shotgun proteome analysis (DISPA) using high-field asymmetric waveform ion mobility spectrometry (FAIMS) with PRM. We demonstrate the application of DISPA-PRM for rapid targeted quantification of bacterial enzymes utilized in the production of biofuels by monitoring temporal expression in 72 metabolically engineered bacterial cultures in less than 2.5 h, with a measured dynamic range >1200-fold. We conclude that DISPA-PRM presents a valuable innovative tool with results comparable to nLC-MS/MS, enabling fast and rapid detection of targeted proteins in complex mixtures.