Search

Abstract Hydroxycinnamic acids such as p-coumaric acid (CA) are chemically linked to lignin in grassy biomass with fairly labile ester bonds and therefore represent a straightforward opportunity to extract and valorize lignin components. In this work, we investigated the enzymatic conversion of CA extracted from lignocellulose to 4-vinylphenol (4VP) by expressing a microbial phenolic acid decarboxylase in Corynebacterium glutamicum, Escherichia coli, and Bacillus subtilis. The performance of the recombinant strains was evaluated in response to the substrate concentration in rich medium or a lignin liquor and the addition of an organic overlay to perform a continuous product extraction in batch cultures. We found that using undecanol as an overlay enhanced the 4VP titers under high substrate concentrations, while extracting > 97% of the product from the aqueous phase. C. glutamicum showed the highest tolerance to CA and resulted in the accumulation of up to 187 g/L of 4VP from pure CA in the overlay with a 90% yield when using rich media, or 17 g/L of 4VP with a 73% yield from CA extracted from lignin. These results indicate that C. glutamicum is a suitable host for the high-level production of 4VP and that further bioprocess engineering strategies should be explored to optimize the production, extraction, and purification of 4VP from lignin with this organism.

Targeted proteomics is a mass spectrometry-based protein quantification technique with high sensitivity, accuracy, and reproducibility. As a key component in the multi-omics toolbox of systems biology, targeted liquid chromatography-selected reaction monitoring (LC-SRM) measurements are critical for enzyme and pathway identification and design in metabolic engineering. To fulfill the increasing need for analyzing large sample sets with faster turnaround time in systems biology, high-throughput LC-SRM is greatly needed. Even though nanoflow LC-SRM has better sensitivity, it lacks the speed offered by microflow LC-SRM. Recent advancements in mass spectrometry instrumentation significantly enhance the scan speed and sensitivity of LC-SRM, thereby creating opportunities for applying the high speed of microflow LC-SRM without losing peptide multiplexing power or sacrificing sensitivity. Here, we studied the performance of microflow LC-SRM relative to nanoflow LC-SRM by monitoring 339 peptides representing 132 enzymes in Pseudomonas putida KT2440 grown on various carbon sources. The results from the two LC-SRM platforms are highly correlated. In addition, the response curve study of 248 peptides demonstrates that microflow LC-SRM has comparable sensitivity for the majority of detected peptides and better mass spectrometry signal and chromatography stability than nanoflow LC-SRM.

ABSTRACT The common environmental bacterium and opportunistic pathogen Pseudomonas aeruginosa encodes diverse metabolic pathways and associated regulatory networks allowing it to thrive in these different environments. In an effort to understand P. aeruginosa metabolism and detection of host-derived compounds, we previously identified CdhR and GbdR as members of the AraC transcription factor family that regulate catabolism of the quaternary amine compounds carnitine and glycine betaine, respectively. In this study, our goal was to further characterize regulation of carnitine catabolism by the transcription factor CdhR. CdhR binds in a concentration-dependent manner upstream of the carnitine catabolism operon promoter (PcaiXcdhCABhocS). We identified the CdhR binding site and determined that it overlaps with the GbdR binding site in the caiX-cdhR intergenic region. Carnitine catabolism is repressed by glucose and glycine betaine, and here we show this happens at the transcriptional level. Furthermore, we show that CdhR enhances its own expression and that GbdR contributes to cdhR expression by enhancing the level of basal expression. The intertwined regulation of caiX and cdhR transcription by GbdR and CdhR suggests that carnitine catabolism is under tight but tuneable control. IMPORTANCE Pathogens must metabolize host-derived compounds during infection and properly regulate the responsible pathways. Carnitine is a common eukaryotic-associated quaternary amine compound that can be catabolized by Pseudomonas aeruginosa. Here we expand on our understanding of how this metabolic pathway is regulated and provide details on how carnitine catabolism is intertwined with glycine betaine catabolism at the level of transcriptional control.