Systems-level characterization of probiotic bifidobacteria - Towards rational optimization of industrial production

Probiotic Bifidobacterium strains contribute to a healthy gut microbiota of their hosts. Increasing public awareness of this positive effect has resulted in a growing demand for these microorganisms. During industrial production, probiotic microorganisms encounter environmental stressors, which can negatively impact their viability and health-promoting benefits. In this thesis, the current state of knowledge on robustness, stability, and stress physiology in bifidobacteria is reviewed, and the robust and stable Bifidobacterium animalis subsp. lactis BB-12\uae and the more sensitive Bifidobacterium longum subsp. longum BB-46 are investigated in detail. The aim of this thesis was to compare the metabolism and physiology of BB-12\uae and BB-46, and to identify key determinants of growth and viability. The applied approach relied on the integration of constraint-based modeling, classical physiological analyses, and omics analyses. Strain-specific, thoroughly curated, genome-scale metabolic models were built for BB-12\uae and BB-46, and were applied to identity their nutritional requirements. This allowed for the formulation of a chemically defined medium that supported growth of both strains. The models and medium are valuable tools for optimizing industrial production of these two strains. BB-12\uae and BB-46 were studied in lab-scale cultivations in the newly formulated medium to identify correlations between cellular characteristics, robustness, and stability of bifidobacteria. Transcriptomic analysis revealed consistently higher expression of several stress-associated genes (e.g., chaperones) in BB-12\uae as compared to BB-46, which may explain the higher stress tolerance of BB-12\uae. Upregulation of genes related to DNA repair in BB-46 coincided with increased robustness and stability in stationary compared to exponential phase. The composition of the cultivation medium had a considerable impact on growth and stability of BB-12\uae and BB-46. The cell membrane fatty acid profile was identified as a key determinant of robustness and stability, by omitting Tween\uae 80 from the medium. An unsaturated to saturated fatty acid ratio below or around one was found to be beneficial. Moreover, a complex nitrogen source was found to reduce the survival of BB-46, and an increased cell size of BB-12\uae in complex MRS medium was proposed to contribute to its poor survival under this condition. To assess for possible correlations between gene content and the strain physiology under stress conditions, the genomes of 171 Bifidobacterium strains, including BB-12\uae and BB-46, were screened for the presence of known stress-associated genes, resulting in the postulation of putative genotype-phenotype correlations. The long-term objective is to use the knowledge gained in this work to guide rational optimization of industrial production processes involving probiotic bifidobacteria

Genome-Wide Assessment of Stress-Associated Genes in Bifidobacteria

Over the last decade, the genomes of several Bifidobacterium strains have been sequenced, delivering valuable insights into their genetic makeup. However, bifidobacterial genomes have not yet been systematically mined for genes associated with stress response functions and their regulation. In this work, a list of 76 genes related to stress response in bifidobacteria was compiled from previous studies. The prevalence of the genes was evaluated among the genome sequences of 171 Bifidobacterium strains. Although genes of the protein quality control and DNA repair systems appeared to be highly conserved, genome-wide in silico screening for consensus sequences of putative regulators suggested that the regulation of these systems differs among phylogenetic groups. Homologs of multiple oxidative stress-associated genes are shared across species, albeit at low sequence similarity. Bee isolates were confirmed to harbor unique genetic features linked to oxygen tolerance. Moreover, most studied Bifidobacterium adolescentis and all Bifidobacterium angulatum strains lacked a set of reactive oxygen species-detoxifying enzymes, which might explain their high sensitivity to oxygen. Furthermore, the presence of some putative transcriptional regulators of stress responses was found to vary across species and strains, indicating that different regulation strategies of stress-associated gene transcription contribute to the diverse stress tolerance. The presented stress response gene profiles of Bifidobacterium strains provide a valuable knowledge base for guiding future studies by enabling hypothesis generation and the identification of key genes for further analyses. IMPORTANCE Bifidobacteria are Gram-positive bacteria that naturally inhabit diverse ecological niches, including the gastrointestinal tract of humans and animals. Strains of the genus Bifidobacterium are widely used as probiotics, since they have been associated with health benefits. In the course of their production and administration, probiotic bifidobacteria are exposed to several stressors that can challenge their survival. The stress tolerance of probiotic bifidobacteria is, therefore, an important selection criterion for their commercial application, since strains must maintain their viability to exert their beneficial health effects. As the ability to cope with stressors varies among Bifidobacterium strains, comprehensive understanding of the underlying stress physiology is required for enabling knowledge-driven strain selection and optimization of industrial-scale production processes

Identifying the essential nutritional requirements of the probiotic bacteria Bifidobacterium animalis and Bifidobacterium longum through genome-scale modeling

Although bifidobacteria are widely used as probiotics, their metabolism and physiology remain to be explored in depth. In this work, strain-specific genome-scale metabolic models were developed for two industrially and clinically relevant bifidobacteria, Bifidobacterium animalis subsp. lactis BB-12\uae and B. longum subsp. longum BB-46, and subjected to iterative cycles of manual curation and experimental validation. A constraint-based modeling framework was used to probe the metabolic landscape of the strains and identify their essential nutritional requirements. Both strains showed an absolute requirement for pantethine as a precursor for coenzyme A biosynthesis. Menaquinone-4 was found to be essential only for BB-46 growth, whereas nicotinic acid was only required by BB-12\uae. The model-generated insights were used to formulate a chemically defined medium that supports the growth of both strains to the same extent as a complex culture medium. Carbohydrate utilization profiles predicted by the models were experimentally validated. Furthermore, model predictions were quantitatively validated in the newly formulated medium in lab-scale batch fermentations. The models and the formulated medium represent valuable tools to further explore the metabolism and physiology of the two species, investigate the mechanisms underlying their health-promoting effects and guide the optimization of their industrial production processes

Novel Insights into the Molecular Mechanisms Underlying Robustness and Stability in Probiotic Bifidobacteria

Probiotics are industrially and clinically important microorganisms. To exert their health-promoting effects, probiotic microorganisms must be administered at high counts, while maintaining their viability at the time of consumption. Some probiotic bifidobacteria are highly robust and shelf-stable, whereas others are difficult to produce, due to their sensitivity to stressors. This limits their potential use as probiotics. Here, we investigate the molecular mechanisms underlying the variability in stress physiologies of Bifidobacterium animalis subsp. lactis BB-12 and Bifidobacterium longum subsp. longum BB-46, by applying a combination of classical physiological characterization and transcriptome profiling. The growth behavior, metabolite production, and global gene expression profiles differed considerably between the strains. BB-12 consistently showed higher expression levels of multiple stress-associated genes, compared to BB-46. This difference, besides higher cell surface hydrophobicity and a lower ratio of unsaturated to saturated fatty acids in the cell membrane of BB-12, should contribute to its higher robustness and stability. In BB-46, the expression of genes related to DNA repair and fatty acid biosynthesis was higher in the stationary than in the exponential phase, which was associated with enhanced stability of BB-46 cells harvested in the stationary phase. The results presented herein highlight important genomic and physiological features contributing to the stability and robustness of the studied Bifidobacterium strains.IMPORTANCE Probiotics are industrially and clinically important microorganisms. To exert their health-promoting effects, probiotic microorganisms must be administered at high counts, while maintaining their viability at the time of consumption. In addition, intestinal survival and bioactivity are important criteria for probiotics. Although bifidobacteria are among the most well-documented probiotics, the industrial-scale production and commercialization of some Bifidobacterium strains is challenged by their high sensitivity to environmental stressors encountered during manufacturing and storage. Through a comprehensive comparison of the metabolic and physiological characteristics of 2 Bifidobacterium strains, we identify key biological markers that can serve as indicators for robustness and stability in bifidobacteria

Stress Response in Bifidobacteria

Bifidobacteria naturally inhabit diverse environments, including the gastrointestinal tracts of humans and animals. Members of the genus are of considerable scientific interest due to their beneficial effects on health and, hence, their potential to be used as probiotics. By definition, probiotic cells need to be viable despite being exposed to several stressors in the course of their production, storage, and administration. Examples of common stressors encountered by probiotic bifidobacteria include oxygen, acid, and bile salts. As bifidobacteria are highly heterogenous in terms of their tolerance to these stressors, poor stability and/or robustness can hamper the industrial-scale production and commercialization of many strains. Therefore, interest in the stress physiology of bifidobacteria has intensified in recent decades, and many studies have been established to obtain insights into the molecular mechanisms underlying their stability and robustness. By complementing traditional methodologies, omics technologies have opened new avenues for enhancing the understanding of the defense mechanisms of bifidobacteria against stress. In this review, we summarize and evaluate the current knowledge on the multilayered responses of bifidobacteria to stressors, including the most recent insights and hypotheses. We address the prevailing stressors that may affect the cell viability during production and use as probiotics. Besides phenotypic effects, molecular mechanisms that have been found to underlie the stress response are described. We further discuss strategies that can be applied to improve the stability of probiotic bifidobacteria and highlight knowledge gaps that should be addressed in future studies. Bifidobacteria naturally inhabit diverse environments, including the gastrointestinal tracts of humans and animals. Members of the genus are of considerable scientific interest due to their beneficial effects on health and, hence, their potential to be used as probiotics

Why are you stressed? A systems-level comparison of two Bifidobacterium strains with different stability characteristics

Bifidobacteria are widely used as probiotics, owing to their well-documented health-promoting properties. Throughout production, downstream processing and administration, probiotic bifidobacteria are exposed to various stressors that may affect the physiological state of the cells. The stability of bifidobacteria varies between strains and the sensitivity of some strains hampers their industrial production. Therefore, it is crucial to gain a more comprehensive, systems-level understanding of their metabolism and physiology, which may result in the identification of potential factors and metabolic bottlenecks influencing both growth and long-term viability. In this work, we compare the metabolic characteristics of two industrially relevant bifidobacterial strains that vary in their stability. Our approach relies on the integration of different phenotypic measurements using a constraint-based metabolic modelling framework. In order to identify the correlation between cellular characteristics and stability, the strains were studied in lab-scale pH-controlled fermentations in chemically-defined medium and their final stability was assessed. The cultivations were monitored by assessing growth and metabolite production. In addition, global transcriptome profiling and differential gene expression analysis were used to gain better insights into the metabolic differences between the strains. The genome-scale metabolic models of the strains were used as platforms for the integration of experimental data