Dataset Information

Quantitative Proteogenomics and the Reconstruction of the Metabolic Pathway in Lactobacillus mucosae LM1.

ABSTRACT: Lactobacillus mucosae is a natural resident of the gastrointestinal tract of humans and animals and a potential probiotic bacterium. To understand the global protein expression profile and metabolic features of L. mucosae LM1 in the early stationary phase, the QExactive(TM) Hybrid Quadrupole-Orbitrap Mass Spectrometer was used. Characterization of the intracellular proteome identified 842 proteins, accounting for approximately 35% of the 2,404 protein-coding sequences in the complete genome of L. mucosae LM1. Proteome quantification using QExactive(TM) Orbitrap MS detected 19 highly abundant proteins (> 1.0% of the intracellular proteome), including CysK (cysteine synthase, 5.41%) and EF-Tu (elongation factor Tu, 4.91%), which are involved in cell survival against environmental stresses. Metabolic pathway annotation of LM1 proteome using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database showed that half of the proteins expressed are important for basic metabolic and biosynthetic processes, and the other half might be structurally important or involved in basic cellular processes. In addition, glycogen biosynthesis was activated in the early stationary phase, which is important for energy storage and maintenance. The proteogenomic data presented in this study provide a suitable reference to understand the protein expression pattern of lactobacilli in standard conditions.

SUBMITTER: Pajarillo EA

PROVIDER: S-EPMC4670900 | biostudies-other | 2015

REPOSITORIES: biostudies-other

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Quantitative Proteogenomics and the Reconstruction of the Metabolic Pathway in Lactobacillus mucosae LM1.

Pajarillo Edward Alain B EA Kim Sang Hoon SH Lee Ji-Yoon JY Valeriano Valerie Diane V VD Kang Dae-Kyung DK

Korean journal for food science of animal resources 20151031 5

Lactobacillus mucosae is a natural resident of the gastrointestinal tract of humans and animals and a potential probiotic bacterium. To understand the global protein expression profile and metabolic features of L. mucosae LM1 in the early stationary phase, the QExactive(TM) Hybrid Quadrupole-Orbitrap Mass Spectrometer was used. Characterization of the intracellular proteome identified 842 proteins, accounting for approximately 35% of the 2,404 protein-coding sequences in the complete genome of L ...[more]

PMID: 26761899

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Project description:BackgroundLactobacillus reuteri is a heterofermentative Lactic Acid Bacterium (LAB) that is commonly used for food fermentations and probiotic purposes. Due to its robust properties, it is also increasingly considered for use as a cell factory. It produces several industrially important compounds such as 1,3-propanediol and reuterin natively, but for cell factory purposes, developing improved strategies for engineering and fermentation optimization is crucial. Genome-scale metabolic models can be highly beneficial in guiding rational metabolic engineering. Reconstructing a reliable and a quantitatively accurate metabolic model requires extensive manual curation and incorporation of experimental data.ResultsA genome-scale metabolic model of L. reuteri JCM 1112T was reconstructed and the resulting model, Lreuteri_530, was validated and tested with experimental data. Several knowledge gaps in the metabolism were identified and resolved during this process, including presence/absence of glycolytic genes. Flux distribution between the two glycolytic pathways, the phosphoketolase and Embden-Meyerhof-Parnas pathways, varies considerably between LAB species and strains. As these pathways result in different energy yields, it is important to include strain-specific utilization of these pathways in the model. We determined experimentally that the Embden-Meyerhof-Parnas pathway carried at most 7% of the total glycolytic flux. Predicted growth rates from Lreuteri_530 were in good agreement with experimentally determined values. To further validate the prediction accuracy of Lreuteri_530, the predicted effects of glycerol addition and adhE gene knock-out, which results in impaired ethanol production, were compared to in vivo data. Examination of both growth rates and uptake- and secretion rates of the main metabolites in central metabolism demonstrated that the model was able to accurately predict the experimentally observed effects. Lastly, the potential of L. reuteri as a cell factory was investigated, resulting in a number of general metabolic engineering strategies.ConclusionWe have constructed a manually curated genome-scale metabolic model of L. reuteri JCM 1112T that has been experimentally parameterized and validated and can accurately predict metabolic behavior of this important platform cell factory.

Project description:Lactobacillus plantarum is the lactic acid bacterial species most frequently found in plant-food fermentations where hydroxycinnamic acids are abundant. L. plantarum efficiently decarboxylates these compounds and also reduces them, yielding substituted phenylpropionic acids. Although the reduction step is known to be induced by a hydroxycinnamic acid, the enzymatic machinery responsible for this reduction pathway has not been yet identified and characterized. A previous study on the transcriptomic response of L. plantarum to p-coumaric acid revealed a marked induction of two contiguous genes, lp_1424 and lp_1425, encoding putative reductases. In this work, the disruption of these genes abolished the hydroxycinnamate reductase activity of L. plantarum, supporting their involvement in such chemical activity. Functional in vitro studies revealed that Lp_1425 (HcrB) exhibits hydroxycinnamate reductase activity but was unstable in solution. In contrast, Lp_1424 (HcrA) was inactive but showed high stability. When the hcrAB genes were co-overexpressed, the formation of an active heterodimer (HcrAB) was observed. Since L. plantarum reductase activity was only observed on hydroxycinnamic acids (o-coumaric, m-coumaric, p-coumaric, caffeic, ferulic, and sinapic acids), the presence of a hydroxyl group substituent on the benzene ring appears to be required for activity. In addition, hydroxycinnamate reductase activity was not widely present among lactic acid bacteria, and it was associated with the presence of hcrAB genes. This study revealed that L. plantarum hydroxycinnamate reductase is a heterodimeric NADH-dependent coumarate reductase acting on a carbon-carbon double bond.IMPORTANCELactobacillus plantarum is a bacterial species frequently found in the fermentation of vegetables where hydroxycinnamic acids are present. The bacterial metabolism on these compounds during fermentation plays a fundamental role in the biological activity of hydroxycinnamates. L. plantarum strains exhibit an as yet unknown reducing activity, transforming hydroxycinnamates to substituted phenylpropionic acids, which possess higher antioxidant activity than their precursors. The protein machinery involved in hydroxycinnamate reduction, HcrAB, was genetically identified and characterized. The heterodimeric NADH-dependent coumarate reductase HcrAB described in this work provides new insights on the L. plantarum metabolic response to counteract the stressful conditions generated by food phenolics.

Dataset Information

Quantitative Proteogenomics and the Reconstruction of the Metabolic Pathway in Lactobacillus mucosae LM1.

Publications

Quantitative Proteogenomics and the Reconstruction of the Metabolic Pathway in Lactobacillus mucosae LM1.

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