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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.

The opportunistic pathogen Pseudomonas aeruginosa can metabolize carnitine and O-acylcarnitines, which are abundant in host muscle and other tissues. Acylcarnitines are metabolized to carnitine and a fatty acid. The liberated carnitine and its catabolic product, glycine betaine, can be used as osmoprotectants, to induce the secreted phospholipase C PlcH, and as sole carbon, nitrogen and energy sources. P. aeruginosa is incapable of de novo synthesis of carnitine and acylcarnitines, therefore they must be imported from an exogenous source. In this study, we present the first characterization of bacterial acylcarnitine transport. Short-chain acylcarnitines are imported by the ABC transporter CaiX-CbcWV. Medium- and long-chain acylcarnitines (MCACs and LCACs) are hydrolysed extracytoplasmically and the free carnitine is transported primarily through CaiX-CbcWV. These findings suggest that the periplasmic protein CaiX has a binding pocket that permits short acyl chains on its carnitine ligand and that there are one or more secreted hydrolases that cleave MCACs and LCACs. To identify the secreted hydrolase(s), we used a saturating genetic screen and transcriptomics followed by phenotypic analyses, but neither led to identification of a contributing hydrolase, supporting but not conclusively demonstrating redundancy for this activity.

Carnitine is a quaternary amine compound found at high concentration in animal tissues, particularly muscle, and is most well studied for its contribution to fatty acid transport into mitochondria. In bacteria, carnitine is an important osmoprotectant, and can also enhance thermotolerance, cryotolerance and barotolerance. Carnitine can be transported into the cell or acquired from metabolic precursors, where it can serve directly as a compatible solute for stress protection or be metabolized through one of a few distinct pathways as a nutrient source. In this review, we summarize what is known about carnitine physiology and metabolism in bacteria. In particular, recent advances in the aerobic and anaerobic metabolic pathways as well as the use of carnitine as an electron acceptor have addressed some long-standing questions in the field.

Efficient catabolism of host-derived compounds is essential for bacterial survival and virulence. While these links in intracellular bacteria are well studied, such studies in extracellular bacteria lag behind, mostly for technical reasons. The field has identified important metabolic pathways, but the mechanisms by which they impact infection and in particular, establishing the importance of a compound's catabolism versus alternate metabolic roles has been difficult. In this review we will examine evidence for catabolism during extracellular bacterial infections in animals and known or potential roles in virulence. In the process, we point out key gaps in the field that will require new or newly adapted techniques. J. Cell. Biochem. 115: 217–223, 2014. © 2013 Wiley Periodicals, Inc.

Pseudomonas aeruginosa displays tremendous metabolic diversity, controlled in part by the abundance of transcription regulators in the genome. We have been investigating P. aeruginosa's response to the host, particularly changes regulated by the host-derived quaternary amines choline and glycine betaine (GB). We previously identified GbdR as an AraC family transcription factor that directly regulates choline acquisition from host phospholipids (via binding to plcH and pchP promoters), is required for catabolism of the choline metabolite GB, and is an activator that induces transcription in response to GB or dimethylglycine. Our goal was to characterize the GbdR regulon in P. aeruginosa by using genetics and chemical biology in combination with transcriptomics and in vitro DNA-binding assays. Here we show that GbdR activation regulates transcription of 26 genes from 12 promoters, 11 of which have measureable binding to GbdR in vitro. The GbdR regulon includes the genes encoding GB, dimethylglycine, sarcosine, glycine, and serine catabolic enzymes and the BetX and CbcXWV quaternary amine transport proteins. We characterized the GbdR consensus binding site and used it to identify that the recently characterized acetylcholine esterase gene, choE (PA4921), is also regulated by GbdR. The regulon member not directly controlled by GbdR is the secreted lipase gene lipA, which was also the only regulon member repressed under GbdR-activating conditions. Determination of the GbdR regulon provides deeper understanding of how GbdR links bacterial metabolism and virulence. Additionally, identification of two uncharacterized regulon members suggests roles for these proteins in response to choline metabolites.

To survive in various environments, from host tissue to soil, opportunistic bacterial pathogens must be metabolically flexible and able to use a variety of nutrient sources. We are interested in Pseudomonas aeruginosa 's catabolism of quaternary amine compounds that are prevalent in association with eukaryotes. Carnitine and acylcarnitines are abundant in animal tissues, particularly skeletal muscle, and are used to shuttle fatty acids in and out of the mitochondria, where they undergo β-oxidation. We previously identified the genes required for carnitine catabolism as the first four genes in the carnitine operon ( caiX-cdhCAB ; PA5388 to PA5385 ). However, the last gene in the operon, PA5384 , was not required for carnitine catabolism. We were interested in determining the function of PA5384. Bioinformatic analyses along with the genomic location of PA5384 led us to hypothesize a role for PA5384 in acylcarnitine catabolism. Here, we have characterized PA5384 as an l -enantiomer-specific short-chain acylcarnitine hydrolase that is required for growth and hydrolysis of acetyl- and butyrylcarnitine to carnitine and the respective short-chain fatty acid. The liberated carnitine and its downstream catabolic product, glycine betaine, are subsequently available to function as osmoprotectants in hyperosmotic environments and induce transcription of the virulence factor phospholipase C, plcH . Furthermore, we confirmed that acylcarnitines with 2- to 16-carbon chain lengths, except for octanoylcarnitine (8 carbons), can be utilized by P. aeruginosa as sole carbon and nitrogen sources. These findings expand our knowledge of short-chain acylcarnitine catabolism and also point to remaining questions related to acylcarnitine transport and hydrolysis of medium- and long-chain acylcarnitines.