Concepts and criteria defining emerging microbiome applications.

In recent years, microbiomes and their potential applications for human, animal or plant health, food production and environmental management came into the spotlight of major national and international policies and strategies. This has been accompanied by substantial R&D investments in both public and private sectors, with an increasing number of products entering the market. Despite widespread agreement on the potential of microbiomes and their uses across disciplines, stakeholders and countries, there is no consensus on what defines a microbiome application. This often results in non-comprehensive communication or insufficient documentation making commercialisation and acceptance of the novel products challenging. To showcase the complexity of this issue we discuss two selected, well-established applications and propose criteria defining a microbiome application and their conditions of use for clear communication, facilitating suitable regulatory frameworks and building trust among stakeholders.

Microbiome Interconnectedness throughout Environments with Major Consequences for Healthy People and a Healthy Planet.

Microbiomes have highly important roles for ecosystem functioning and carry out key functions that support planetary health, including nutrient cycling, climate regulation, and water filtration. Microbiomes are also intimately associated with complex multicellular organisms such as humans, other animals, plants, and insects and perform crucial roles for the health of their hosts. Although we are starting to understand that microbiomes in different systems are interconnected, there is still a poor understanding of microbiome transfer and connectivity. In this review we show how microbiomes are connected within and transferred between different habitats and discuss the functional consequences of these connections. Microbiome transfer occurs between and within abiotic (e.g., air, soil, and water) and biotic environments, and can either be mediated through different vectors (e.g., insects or food) or direct interactions. Such transfer processes may also include the transmission of pathogens or antibiotic resistance genes. However, here, we highlight the fact that microbiome transmission can have positive effects on planetary and human health, where transmitted microorganisms potentially providing novel functions may be important for the adaptation of ecosystems.

Conserved unique peptide patterns (CUPP) online platform 2.0: implementation of +1000 JGI fungal genomes.

Carbohydrate-processing enzymes, CAZymes, are classified into families based on sequence and three-dimensional fold. Because many CAZyme families contain members of diverse molecular function (different EC-numbers), sophisticated tools are required to further delineate these enzymes. Such delineation is provided by the peptide-based clustering method CUPP, Conserved Unique Peptide Patterns. CUPP operates synergistically with the CAZy family/subfamily categorizations to allow systematic exploration of CAZymes by defining small protein groups with shared sequence motifs. The updated CUPP library contains 21,930 of such motif groups including 3,842,628 proteins. The new implementation of the CUPP-webserver, https://cupp.info/, now includes all published fungal and algal genomes from the Joint Genome Institute (JGI), genome resources MycoCosm and PhycoCosm, dynamically subdivided into motif groups of CAZymes. This allows users to browse the JGI portals for specific predicted functions or specific protein families from genome sequences. Thus, a genome can be searched for proteins having specific characteristics. All JGI proteins have a hyperlink to a summary page which links to the predicted gene splicing including which regions have RNA support. The new CUPP implementation also includes an update of the annotation algorithm that uses only a fourth of the RAM while enabling multi-threading, providing an annotation speed below 1 ms/protein.

The need for an integrated multi-OMICs approach in microbiome science in the food system.

Microbiome science as an interdisciplinary research field has evolved rapidly over the past two decades, becoming a popular topic not only in the scientific community and among the general public, but also in the food industry due to the growing demand for microbiome-based technologies that provide added-value solutions. Microbiome research has expanded in the context of food systems, strongly driven by methodological advances in different -omics fields that leverage our understanding of microbial diversity and function. However, managing and integrating different complex -omics layers are still challenging. Within the Coordinated Support Action MicrobiomeSupport (https://www.microbiomesupport.eu/), a project supported by the European Commission, the workshop "Metagenomics, Metaproteomics and Metabolomics: the need for data integration in microbiome research" gathered 70 participants from different microbiome research fields relevant to food systems, to discuss challenges in microbiome research and to promote a switch from microbiome-based descriptive studies to functional studies, elucidating the biology and interactive roles of microbiomes in food systems. A combination of technologies is proposed. This will reduce the biases resulting from each individual technology and result in a more comprehensive view of the biological system as a whole. Although combinations of different datasets are still rare, advanced bioinformatics tools and artificial intelligence approaches can contribute to understanding, prediction, and management of the microbiome, thereby providing the basis for the improvement of food quality and safety.

Participation in a multicomponent lifestyle intervention for people with obesity improves glycated hemoglobin (HbA1c).

Introduction: Obesity is associated with compromised glucose metabolism. Hence, it is of interest to investigate if the lifestyle interventions used in the LIBRA-cohort, which aimed at not only weight loss, but also patient well-being, could also help obese patients improve glucose metabolism by evidence of reduced HbA1c. The aim of the study was to retrospectively investigate if patients who were referred to a lifestyle intervention for obesity, were able to alter HbA1c. Research design and methods: Patients with a BMI≥30 undergoing a 6-month lifestyle intervention, who also completed physical and mental health surveys and whose baseline and 6-month blood samples were available, were included in the analysis. For changes in HbA1c and body weight a clinically relevant change of 5≥mmom/mol and 5%≥, respectively, was chosen. Participants were divided into groups according to their baseline HbA1c level: "Diabetes": HbA1c of ≥6.5% (≥48 mmol/mol), "Prediabetes": HbA1c of 5.7% to 6.4% (39-47.99 mmol/mol) or "Normal" HbA1c <5.7% (<39 mmol/mol). Results: 180 patients met the stated inclusion criteria and these patients were divided into groups (median age (25th;75th quartile): Diabetes: n=47, age 54 (43;60), 51% women, Prediabetes: n=68, age 60 (50;66), 71% women and Normal: n=65, median age 61 (50;66), 85% women. Significant reductions were found in all three groups and specifically in the diabetes group HbA1c was reduced (mean [95%CI]) -5[-8;-2] mmol/mol from baseline to the end of the intervention. Furthermore, 35% of patients with prediabetes normalized their HbA1c (<39) and 30% patients with diabetes reduced their HbA1c <48. All groups had clinically relevant (≥5%) reductions in body weight (p<0.01). There was an association between body weight reduction and HbA1c reduction in the diabetes group (p<0.01). All groups reported improvements in physical health (p<0.01). Conclusion: In this retrospective cohort study, all patients achieved clinically relevant weight loss after participation in the lifestyle intervention and obese patients with diabetes achieved clinically relevant reductions in HbA1c after 6-months. More than 1/3 of patients with prediabetes normalized their HbA1c.

Discovery of novel secretome CAZymes from Penicillium sclerotigenum by bioinformatics and explorative proteomics analyses during sweet potato pectin digestion.

Novel selective enzymatic refining of sweet potato processing residues requires judicious enzyme selection and enzyme discovery. We prepared a pectinaceous cell wall polysaccharide fraction from sweet potato using an enzymatic a treatment to preserve the natural linkages and substitutions. Polysaccharide composition and linkage analysis data confirmed the pectinaceous polysaccharide fraction to be a rhamnogalacturonan I-rich fraction with a high content of arabinogalactan Type I. We hypothesized that the post-harvest tuber pathogenic fungus Penicillium sclerotigenum would harbor novel enzymes targeting selective sweet potato pectin modification. As part of the study, we also report the first genome sequence of P. sclerotigenum. We incubated the sweet potato pectinaceous fraction with P. sclerotigenum. Using proteomics accompanied by CUPP-bioinformatics analysis, we observed induced expression of 23 pectin-associated degradative enzymes. We also identified six abundantly secreted, induced proteins that do not correspond to known CAZymes, but which we suggest as novel enzymes involved in pectin degradation. For validation, the predicted CUPP grouping of putative CAZymes and the exo-proteome data obtained for P. sclerotigenum during growth on sweet potato pectin were compared with proteomics and transcriptomics data reported previously for pectin-associated CAZymes from Aspergillus niger strain NRRL3. The data infer that P. sclerotigenum has the capacity to express several novel enzymes that may provide novel opportunities for sweet potato pectin modification and valorization of sweet potato starch processing residues. In addition, the methodological approach employed represents an integrative systematic strategy for enzyme discovery.

Microbiome ethics, guiding principles for microbiome research, use and knowledge management.

The overarching biological impact of microbiomes on their hosts, and more generally their environment, reflects the co-evolution of a mutualistic symbiosis, generating fitness for both. Knowledge of microbiomes, their systemic role, interactions, and impact grows exponentially. When a research field of importance for planetary health evolves so rapidly, it is essential to consider it from an ethical holistic perspective. However, to date, the topic of microbiome ethics has received relatively little attention considering its importance. Here, ethical analysis of microbiome research, innovation, use, and potential impact is structured around the four cornerstone principles of ethics: Do Good; Don't Harm; Respect; Act Justly. This simple, but not simplistic approach allows ethical issues to be communicative and operational. The essence of the paper is captured in a set of eleven microbiome ethics recommendations, e.g., proposing gut microbiome status as common global heritage, similar to the internationally agreed status of major food crops.

Microbiome Research as an Effective Driver of Success Stories in Agrifood Systems - A Selection of Case Studies.

Increasing knowledge of the microbiome has led to significant advancements in the agrifood system. Case studies based on microbiome applications have been reported worldwide and, in this review, we have selected 14 success stories that showcase the importance of microbiome research in advancing the agrifood system. The selected case studies describe products, methodologies, applications, tools, and processes that created an economic and societal impact. Additionally, they cover a broad range of fields within the agrifood chain: the management of diseases and putative pathogens; the use of microorganism as soil fertilizers and plant strengtheners; the investigation of the microbial dynamics occurring during food fermentation; the presence of microorganisms and/or genes associated with hazards for animal and human health (e.g., mycotoxins, spoilage agents, or pathogens) in feeds, foods, and their processing environments; applications to improve HACCP systems; and the identification of novel probiotics and prebiotics to improve the animal gut microbiome or to prevent chronic non-communicable diseases in humans (e.g., obesity complications). The microbiomes of soil, plants, and animals are pivotal for ensuring human and environmental health and this review highlights the impact that microbiome applications have with this regard.

Changes in the Metagenome-Encoded CAZymes of the Rumen Microbiome Are Linked to Feed-Induced Reductions in Methane Emission From Holstein Cows.

Enteric methane (CH4) emission from cattle is strongly linked to the feeding regime and the rumen microbial community structure. Here, we report that feed-induced CH4-reducing effects correlate with specific alterations in the profile of the microbiome-encoded carbohydrate-active enzymes predicted from the rumen fluid metagenome. Rumen microbiome samples were obtained by mouth-tube sampling from 12 lactating Holstein cows after 3-4 weeks of feeding with three different concentrate-to-forage-ratio diets, i.e., standard, high, and extremely high levels of concentrate (4 cows per group; constant dry matter intake in the three groups). Increased inclusion of concentrate involved increased starch levels in the diet at the expense of fiber. The extreme diet resulted in 48% reduction of the CH4 emission per kg dry matter intake compared to the standard diet. From metagenome sequencing of the rumen fluid samples from each cow, 561 different microbial strains (bins) could be derived from analysis of 260 billion DNA base pairs. In the cows fed, the extreme diet, the relative abundance of the majority of the bins, was significantly altered compared to the other groups. Fibrobacterota and Verrucomicrobiota were less abundant in the Extreme group. Surprisingly, no significant abundance changes were observed among Archaea and Bacteroidota, although abundance changes of individual bins of these phyla were found. For each of the 561 bins, the functions of the metagenome-encoded carbohydrate-active enzymes were predicted by bioinformatics using conserved unique peptide pattern (CUPP) analysis. By linking each of the predicted molecular functions of the enzymes to their substrates, changes were found in the predicted abundance of the different enzyme types. Notably, the decreased CH4 emission of the extreme diet group was concurrent with a profound decrease in the xylan-active enzymes, targeting the xylan backbone ß-1,4-linkages, acetyl-, feruloyl-, and methyl-glucuronoyl substitutions in xylan. This work provides a first enzyme-conversion-based characterization of how extreme feeding, i.e., lowered forage, can drive rumen microbiome changes that support decreased CH4 emission via a changed carbohydrate-active enzyme profile. The data, furthermore, provide a metagenome-wide catalog of enzymes, underpinning the microbial conversion of different feed fibers (the enzymes attacking specific carbohydrate linkages) in the rumen of Holstein cows.