Inside the microbial black box: a redox-centric framework for deciphering microbial metabolism.

Microbial metabolism influences the global climate and human health and is governed by the balance between NADH and NAD+ through redox reactions. Historically, oxidative (i.e., catabolism) and reductive (i.e., fermentation) pathways have been studied in isolation, obscuring the complete metabolic picture. However, new omics technologies and biotechnological tools now allow an integrated system-level understanding of the drivers of microbial metabolism through observation and manipulation of redox reactions. Here we present perspectives on the importance of viewing microbial metabolism as the dynamic interplay between oxidative and reductive processes and apply this framework to diverse microbial systems. Additionally, we highlight novel biotechnologies to monitor and manipulate microbial redox status to control metabolism in unprecedented ways. This redox-focused systems biology framework enables a more mechanistic understanding of microbial metabolism.

Identification of biological signatures of cruciferous vegetable consumption utilizing machine learning-based global untargeted stable isotope traced metabolomics.

In recent years there has been increased interest in identifying biological signatures of food consumption for use as biomarkers. Traditional metabolomics-based biomarker discovery approaches rely on multivariate statistics which cannot differentiate between host- and food-derived compounds, thus novel approaches to biomarker discovery are required to advance the field. To this aim, we have developed a new method that combines global untargeted stable isotope traced metabolomics and a machine learning approach to identify biological signatures of cruciferous vegetable consumption. Participants consumed a single serving of broccoli (n = 16), alfalfa sprouts (n = 16) or collard greens (n = 26) which contained either control unlabeled metabolites, or that were grown in the presence of deuterium-labeled water to intrinsically label metabolites. Mass spectrometry analysis indicated 133 metabolites in broccoli sprouts and 139 metabolites in the alfalfa sprouts were labeled with deuterium isotopes. Urine and plasma were collected and analyzed using untargeted metabolomics on an AB SCIEX TripleTOF 5,600 mass spectrometer. Global untargeted stable isotope tracing was completed using openly available software and a novel random forest machine learning based classifier. Among participants who consumed labeled broccoli sprouts or collard greens, 13 deuterium-incorporated metabolomic features were detected in urine representing 8 urine metabolites. Plasma was analyzed among collard green consumers and 11 labeled features were detected representing 5 plasma metabolites. These deuterium-labeled metabolites represent potential biological signatures of cruciferous vegetables consumption. Isoleucine, indole-3-acetic acid-N-O-glucuronide, dihydrosinapic acid were annotated as labeled compounds but other labeled metabolites could not be annotated. This work presents a novel framework for identifying biological signatures of food consumption for biomarker discovery. Additionally, this work presents novel applications of metabolomics and machine learning in the life sciences.

Sulforaphane and Sulforaphane-Nitrile Metabolism in Humans Following Broccoli Sprout Consumption: Inter-individual Variation, Association with Gut Microbiome Composition, and Differential Bioactivity.

SCOPE: The glucosinolate glucoraphanin from broccoli is converted to sulforaphane (SFN) or sulforaphane-nitrile (SFN-NIT) by plant enzymes or the gut microbiome. Human feeding studies typically observe high inter-individual variation in absorption and excretion of SFN, however, the source of this variation is not fully known. To address this, a human feeding trial to comprehensively evaluate inter-individual variation in the absorption and excretion of all known SFN metabolites in urine, plasma, and stool, and tested the hypothesis that gut microbiome composition influences inter-individual variation in total SFN excretion has been conducted. METHODS AND RESULTS: Participants (n = 55) consumed a single serving of broccoli or alfalfa sprouts and plasma, stool, and total urine are collected over 72 h for quantification of SFN metabolites and gut microbiome profiling using 16S gene sequencing. SFN-NIT excretion is markedly slower than SFN excretion (72 h vs 24 h). Members of genus Bifidobacterium, Dorea, and Ruminococcus torques are positively associated with SFN metabolite excretion while members of genus Alistipes and Blautia has a negative association. CONCLUSION: This is the first report of SFN-NIT metabolite levels in human plasma, urine, and stool following consumption of broccoli sprouts. The results help explain factors driving inter-individual variation in SFN metabolism and are relevant for precision nutrition.

Gut enterotype-dependent modulation of gut microbiota and their metabolism in response to xanthohumol supplementation in healthy adults.

Xanthohumol (XN), a polyphenol found in the hop plant (Humulus lupulus), has antioxidant, anti-inflammatory, prebiotic, and anti-hyperlipidemic activity. Preclinical evidence suggests the gut microbiome is essential in mediating these bioactivities; however, relatively little is known about XN's impact on human gut microbiota in vivo. We conducted a randomized, triple-blinded, placebo-controlled clinical trial (ClinicalTrials.gov NCT03735420) to determine safety and tolerability of XN in healthy adults. Thirty healthy participants were randomized to 24 mg/day XN or placebo for 8 weeks. As secondary outcomes, quantification of bacterial metabolites and 16S rRNA gene sequencing were utilized to explore the relationships between XN supplementation, gut microbiota, and biomarkers of gut health. Although XN did not significantly change gut microbiota composition, it did re-shape individual taxa in an enterotype-dependent manner. High levels of inter-individual variation in metabolic profiles and bioavailability of XN metabolites were observed. Moreover, reductions in microbiota-derived bile acid metabolism were observed, which were specific to Prevotella and Ruminococcus enterotypes. These results suggest interactions between XN and gut microbiota in healthy adults are highly inter-individualized and potentially indicate that XN elicits effects on gut health in an enterotype-dependent manner.

Microbial polyphenol metabolism is part of the thawing permafrost carbon cycle.

With rising global temperatures, permafrost carbon stores are vulnerable to microbial degradation. The enzyme latch theory states that polyphenols should accumulate in saturated peatlands due to diminished phenol oxidase activity, inhibiting resident microbes and promoting carbon stabilization. Pairing microbiome and geochemical measurements along a permafrost thaw-induced saturation gradient in Stordalen Mire, a model Arctic peatland, we confirmed a negative relationship between phenol oxidase expression and saturation but failed to support other trends predicted by the enzyme latch. To inventory alternative polyphenol removal strategies, we built CAMPER, a gene annotation tool leveraging polyphenol enzyme knowledge gleaned across microbial ecosystems. Applying CAMPER to genome-resolved metatranscriptomes, we identified genes for diverse polyphenol-active enzymes expressed by various microbial lineages under a range of redox conditions. This shifts the paradigm that polyphenols stabilize carbon in saturated soils and highlights the need to consider both oxic and anoxic polyphenol metabolisms to understand carbon cycling in changing ecosystems.

Sulforaphane Bioavailability in Healthy Subjects Fed a Single Serving of Fresh Broccoli Microgreens.

Cruciferous vegetable consumption is associated with numerous health benefits attributed to the phytochemical sulforaphane (SFN) that exerts antioxidant and chemopreventive properties, among other bioactive compounds. Broccoli sprouts, rich in SFN precursor glucoraphanin (GRN), have been investigated in numerous clinical trials. Broccoli microgreens are similarly rich in GRN but have remained largely unexplored. The goal of this study was to examine SFN bioavailability and the microbiome profile in subjects fed a single serving of fresh broccoli microgreens. Eleven subjects participated in a broccoli microgreens feeding study. Broccoli microgreens GRN and SFN contents and stability were measured. Urine and stool SFN metabolite profiles and microbiome composition were examined. Broccoli microgreens had similar GRN content to values previously reported for broccoli sprouts, which was stable over time. Urine SFN metabolite profiles in broccoli microgreens-fed subjects were similar to those reported previously in broccoli sprouts-fed subjects, including the detection of SFN-nitriles. We also reported the detection of SFN metabolites in stool samples for the first time. A single serving of broccoli microgreens did not significantly alter microbiome composition. We showed in this study that broccoli microgreens are a significant source of SFN. Our work provides the foundation for future studies to establish the health benefits of broccoli microgreens consumption.

Interplay between Cruciferous Vegetables and the Gut Microbiome: A Multi-Omic Approach.

Brassica vegetables contain a multitude of bioactive compounds that prevent and suppress cancer and promote health. Evidence suggests that the gut microbiome may be essential in the production of these compounds; however, the relationship between specific microbes and the abundance of metabolites produced during cruciferous vegetable digestion are still unclear. We utilized an ex vivo human fecal incubation model with in vitro digested broccoli sprouts (Broc), Brussels sprouts (Brus), a combination of the two vegetables (Combo), or a negative control (NC) to investigate microbial metabolites of cruciferous vegetables. We conducted untargeted metabolomics on the fecal cultures by LC-MS/MS and completed 16S rRNA gene sequencing. We identified 72 microbial genera in our samples, 29 of which were significantly differentially abundant between treatment groups. A total of 4499 metabolomic features were found to be significantly different between treatment groups (q ≤ 0.05, fold change > 2). Chemical enrichment analysis revealed 45 classes of compounds to be significantly enriched by brassicas, including long-chain fatty acids, coumaric acids, and peptides. Multi-block PLS-DA and a filtering method were used to identify microbe−metabolite interactions. We identified 373 metabolites from brassica, which had strong relationships with microbes, such as members of the family Clostridiaceae and genus Intestinibacter, that may be microbially derived.

Metabolic Fate of Dietary Glucosinolates and Their Metabolites: A Role for the Microbiome.

Robust evidence shows that phytochemicals from cruciferous vegetables, like broccoli, are associated with numerous health benefits. The anti-cancer properties of these foods are attributed to bioactive isothiocyanates (ITCs) and indoles, phytochemicals generated from biological precursor compounds called glucosinolates. ITCs, and particularly sulforaphane (SFN), are of intense interest as they block the initiation, and suppress the progression of cancer, through genetic and epigenetic mechanisms. The efficacy of these compounds is well-demonstrated in cell culture and animal models, however, high levels of inter-individual variation in absorption and excretion of ITCs is a significant barrier to the use of dietary glucosinolates to prevent and treat disease. The source of inter-individual ITC variation has yet to be fully elucidated and the gut microbiome may play a key role. This review highlights evidence that the gut microbiome influences the metabolic fate and activity of ITCs. Human feeding trials have shown inter-individual variations in gut microbiome composition coincides with variations in ITC absorption and excretion, and some bacteria produce ITCs from glucosinolates. Additionally, consumption of cruciferous vegetables can alter the composition of the gut microbiome and shift the physiochemical environment of the gut lumen, influencing the production of phytochemicals. Microbiome and diet induced changes to ITC metabolism may lead to the decrease of cancer fighting phytochemicals such as SFN and increase the production of biologically inert ones like SFN-nitrile. We conclude by offering perspective on the use of novel "omics" technologies to elucidate the interplay of the gut microbiome and ITC formation.

Composition of the Gut Microbiome Influences Production of Sulforaphane-Nitrile and Iberin-Nitrile from Glucosinolates in Broccoli Sprouts.

Isothiocyanates, such as sulforaphane and iberin, derived from glucosinolates (GLS) in cruciferous vegetables, are known to prevent and suppress cancer development. GLS can also be converted by bacteria to biologically inert nitriles, such as sulforaphane-nitrile (SFN-NIT) and iberin-nitrile (IBN-NIT), but the role of the gut microbiome in this process is relatively undescribed and SFN-NIT excretion in humans is unknown. An ex vivo fecal incubation model with in vitro digested broccoli sprouts and 16S sequencing was utilized to explore the role of the gut microbiome in SFN- and IBN-NIT production. SFN-NIT excretion was measured among human subjects following broccoli sprout consumption. The fecal culture model showed high inter-individual variability in nitrile production and identified two sub-populations of microbial communities among the fecal cultures, which coincided with a differing abundance of nitriles. The Clostridiaceae family was associated with high levels, while individuals with a low abundance of nitriles were more enriched with taxa from the Enterobacteriaceae family. High levels of inter-individual variation in urine SFN-NIT levels were also observed, with peak excretion of SFN-NIT at 24 h post broccoli sprout consumption. These results suggest that nitrile production from broccoli, as opposed to isothiocyanates, could be influenced by gut microbiome composition, potentially lowering efficacy of cruciferous vegetable interventions.