Filipe Branco dos Santos

Exploring Metabolic Pathway Reconstruction and Genome-Wide Expression Profiling in Lactobacillus reuteri to Define Functional Probiotic Features

The genomes of four Lactobacillus reuteri strains isolated from human breast milk and the gastrointestinal tract have been recently sequenced as part of the Human Microbiome Project. Preliminary genome comparisons suggested that these strains belong to two different clades, previously shown to differ with respect to antimicrobial production, biofilm formation, and immunomodulation. To explain possible mechanisms of survival in the host and probiosis, we completed a detailed genomic comparison of two breast milk–derived isolates representative of each group: an established probiotic strain (L. reuteri ATCC 55730) and a strain with promising probiotic features (L. reuteri ATCC PTA 6475). Transcriptomes of L. reuteri strains in different growth phases were monitored using strain-specific microarrays, and compared using a pan-metabolic model representing all known metabolic reactions present in these strains. Both strains contained candidate genes involved in the survival and persistence in the gut such as mucus-binding proteins and enzymes scavenging reactive oxygen species. A large operon predicted to encode the synthesis of an exopolysaccharide was identified in strain 55730. Both strains were predicted to produce health-promoting factors, including antimicrobial agents and vitamins (folate, vitamin B12). Additionally, a complete pathway for thiamine biosynthesis was predicted in strain 55730 for the first time in this species. Candidate genes responsible for immunomodulatory properties of each strain were identified by transcriptomic comparisons. The production of bioactive metabolites by human-derived probiotics may be predicted using metabolic modeling and transcriptomics. Such strategies may facilitate selection and optimization of probiotics for health promotion, disease prevention and amelioration.

High-Level Folate Production in Fermented Foods by the B12 Producer Lactobacillus reuteri JCM1112

ABSTRACT We observed that Lactobacillus reuteri JCM1112 produces B12 and folate. However, the folate/B12 mass ratio found was far below that desired for human consumption (∼170:1). We used metabolic engineering applying genetic and physiological approaches to improve this ratio and developed a generic and natural process that significantly increases folate production.

The complete coenzyme B12 biosynthesis gene cluster of Lactobacillus reuteri CRL1098

The coenzyme B(12) production pathway in Lactobacillus reuteri has been deduced using a combination of genetic, biochemical and bioinformatics approaches. The coenzyme B(12) gene cluster of Lb. reuteri CRL1098 has the unique feature of clustering together the cbi, cob and hem genes. It consists of 29 ORFs encoding the complete enzymic machinery necessary for de novo biosynthesis. Transcriptional analysis showed it to be expressed as two tandem transcripts of approximately 22 and 4 kb, carrying cobD, cbiABCDETFGHJ, cobA/hemD, cbiKLMNQOP, sirA, hemACBL, and cobUSC, hemD, cobT, respectively. Both transcripts appear to be similarly regulated, and under the conditions assayed are induced in the late-exponential growth phase. Evidence for a regulatory mechanism of negative feedback inhibition by vitamin B(12) itself was observed. Comparative genomics analysis of the coding sequences showed them to be most similar to those coding for the anaerobic coenzyme B(12) pathways previously characterized in a few representatives of the genera Listeria and Salmonella. This contrasts with the trusted species phylogeny and suggests horizontal gene transfer of the B(12) biosynthesis genes. G+C content and codon adaptation index analysis is suggestive that the postulated transfer of these genes was not a recent event. Additional comparative genomics and transcriptional analysis of the sequences acquired during this study suggests a functional link between coenzyme B(12) biosynthesis and reuterin production, which might be implicated in Lb. reuteris success in colonizing the gastrointestinal tract. This information on gene organization, gene transcription and gene acquisition is relevant for the development of (fermented) foods and probiotics enriched in B(12).

Pseudovitamin B12 is the corrinoid produced by Lactobacillus reuteri CRL1098 under anaerobic conditions

We have reported previously on the ability of Lactobacillus reuteri to produce a compound with vitamin B 12 activity. Here we report on the chemical characterisation of this corrinoid‐like molecule. High performance liquid chromatography coupled to an ultraviolet diode array detector, mass spectrometry and nuclear magnetic resonance spectroscopy has enabled us to identify the compound as Co α ‐[ α ‐(7‐adenyl)]‐Co β ‐cyanocobamide or pseudovitamin B 12 . This molecule differs from cobalamin in the α ‐ligand, where it has adenine instead of 5,6‐dimethylbenzimidazole bound in a α ‐glycosidic linkage to C‐1 of ribose. L. reuteri is the first lactic acid bacterium in which the production of a cobalamin‐like molecule has been identified and the first microorganism reported to produce exclusively pseudo‐ B 12 .

Towards metagenome-scale models for industrial applications - the case of Lactic Acid Bacteria

We review the uses and limitations of modelling approaches that are in use in the field of Lactic Acid Bacteria (LAB). We describe recent developments in model construction and computational methods, starting from application of such models to monocultures. However, since most applications in food biotechnology involve complex nutrient environments and mixed cultures, we extend the scope to discuss developments in modelling such complex systems. With metagenomics and meta-functional genomics data becoming available, the developments in genome-scale community models are discussed. We conclude that exploratory tools are available and useful, but truly predictive mechanistic models will remain a major challenge in the field.

Standardized assay medium to measure Lactococcus lactis enzyme activities while mimicking intracellular conditions.

ABSTRACT Knowledge of how the activity of enzymes is affected under in vivo conditions is essential for analyzing their regulation and constructing models that yield an integrated understanding of cell behavior. Current kinetic parameters for Lactococcus lactis are scattered through different studies and performed under different assay conditions. Furthermore, assay conditions often diverge from conditions prevailing in the intracellular environment. To establish uniform assay conditions that resemble intracellular conditions, we analyzed the intracellular composition of anaerobic glucose-limited chemostat cultures of L. lactis subsp. cremoris MG 1363. Based on this, we designed a new assay medium for enzyme activity measurements of growing cells of L. lactis, mimicking as closely as practically possible its intracellular environment. Procedures were optimized to be carried out in 96-well plates, and the reproducibility and dynamic range were checked for all enzyme activity measurements. The effects of freezing and the carryover of ammonium sulfate from the addition of coupling enzymes were also established. Activities of all 10 glycolytic and 4 fermentative enzymes were measured. Remarkably, most in vivo-like activities were lower than previously published data. Yet, the ratios of V max over measured in vivo fluxes were above 1. With this work, we have developed and extensively validated standard protocols for enzyme activity measurements for L. lactis.

A Practical Guide to Genome-Scale Metabolic Models and Their Analysis

Genome-scale metabolic reconstructions and their analysis with constraint-based modeling techniques have gained enormous momentum. It is a natural next step after sequencing of a genome, as a technique that links top-down systems biology analyses at genome scale with bottom-up systems biology modeling scrutiny. This chapter aims at (systems) biologists that have an interest in, but no extensive knowledge of, applying genome-scale metabolic reconstruction and modeling to their organism. Rather than being comprehensive--excellent and extensive reviews exist on every aspect of this field--we give a rather personal account on our experience with the process of reconstruction and modeling. First, we place genome-scale metabolic models in the spectrum of modeling approaches, and rather extensively discuss, for nonexperts, the central concept in constraint-based modeling: the solution space that is bounded through constraints on fluxes. We subsequently provide an overview of the different steps involved in metabolic reconstruction and modeling, pointing to aspects that we found difficult, important, not well enough addressed in the current reviews, or any combination thereof. In this way, we hope that this chapter serves as a practical guide through the field.

Functional identification in Lactobacillus reuteri of a PocR-like transcription factor regulating glycerol utilization and vitamin B12 synthesis

BackgroundLactobacillus reuteri harbors the genes responsible for glycerol utilization and vitamin B12 synthesis within a genetic island phylogenetically related to gamma-Proteobacteria. Within this island, resides a gene (lreu_1750) that based on its genomic context has been suggested to encode the regulatory protein PocR and presumably control the expression of the neighboring loci. However, this functional assignment is not fully supported by sequence homology, and hitherto, completely lacks experimental confirmation.ResultsIn this contribution, we have overexpressed and inactivated the gene encoding the putative PocR in L. reuteri. The comparison of these strains provided metabolic and transcriptional evidence that this regulatory protein controls the expression of the operons encoding glycerol utilization and vitamin B12 synthesis.ConclusionsWe provide clear experimental evidence for assigning Lreu_1750 as PocR in Lactobacillus reuteri. Our genome-wide transcriptional analysis further identifies the loci contained in the PocR regulon. The findings reported here could be used to improve the production-yield of vitamin B12, 1,3-propanediol and reuterin, all industrially relevant compounds.

Synechocystis: Not Just a Plug-Bug for CO2, but a Green E. coli

Following multiple reports warning for threats posed by raising levels of atmospheric CO2, it is of paramount importance that human society rapidly evolves to be sustainable. Processes relying on photosynthetic microorganisms, converting CO2 and water into compounds of interest, fueled by light, are very pertinent, particularly if not directly competing for arable land. Here, we identify specific research questions that remain to be targeted to exploit the full potential of cyanobacterial cell factories. We argue that this approach will be more likely to be successful if organisms such as Synechocystis are not perceived as mere chassis for CO2 fixation, but rather considered as the “green” E. coli.

Photosynthetic production of glycerol by a recombinant cyanobacterium

Cyanobacteria are prokaryotic organisms capable of oxygenic photosynthesis. Glycerol is an important commodity chemical. Introduction of phosphoglycerol phosphatase 2 from Saccharomyces cerevisiae into the model cyanobacterium Synechocystis sp. PCC6803 resulted in a mutant strain that produced a considerable amount of glycerol from light, water and COPhotosynthetic production . Mild salt stress (200 mM NaCl) on the cells led to an increase of the extracellular glycerol concentration of more than 20%. Under these conditions the mutant accumulated glycerol to an extracellular concentration of 14.3 mM after 17 days of culturing.