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

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.

Highly efficient fermentation of 5-keto-D-fructose with Gluconobacter oxydans at different scales.

BACKGROUND: The global market for sweeteners is increasing, and the food industry is constantly looking for new low-caloric sweeteners. The natural sweetener 5-keto-D-fructose is one such candidate. 5-Keto-D-fructose has a similar sweet taste quality as fructose. Developing a highly efficient 5-keto-D-fructose production process is key to being competitive with established sweeteners. Hence, the 5-keto-D-fructose production process was optimised regarding titre, yield, and productivity. RESULTS: For production of 5-keto-D-fructose with G. oxydans 621H ΔhsdR pBBR1-p264-fdhSCL-ST an extended-batch fermentation was conducted. During fructose feeding, a decreasing respiratory activity occurred, despite sufficient carbon supply. Oxygen and second substrate limitation could be excluded as reasons for the decreasing respiration. It was demonstrated that a short period of oxygen limitation has no significant influence on 5-keto-D-fructose production, showing the robustness of this process. Increasing the medium concentration increased initial biomass formation. Applying a fructose feeding solution with a concentration of approx. 1200 g/L, a titre of 545 g/L 5-keto-D-fructose was reached. The yield was with 0.98 g5-keto-d-fructose/gfructose close to the theoretical maximum. A 1200 g/L fructose solution has a viscosity of 450 mPa∙s at a temperature of 55 °C. Hence, the solution itself and the whole peripheral feeding system need to be heated, to apply such a highly concentrated feeding solution. Thermal treatment of highly concentrated fructose solutions led to the formation of 5-hydroxymethylfurfural, which inhibited the 5-keto-D-fructose production. Therefore, fructose solutions were only heated to about 100 °C for approx. 10 min. An alternative feeding strategy was investigated using solid fructose cubes, reaching the highest productivities above 10 g5-keto-d-fructose/L/h during feeding. Moreover, the scale-up of the 5-keto-D-fructose production to a 150 L pressurised fermenter was successfully demonstrated using liquid fructose solutions (745 g/L). CONCLUSION: We optimised the 5-keto-D-fructose production process and successfully increased titre, yield and productivity. By using solid fructose, we presented a second feeding strategy, which can be of great interest for further scale-up experiments. A first scale-up of this process was performed, showing the possibility for an industrial production of 5-keto-D-fructose.

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.

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.

Rational and evolutionary engineering of Saccharomyces cerevisiae for production of dicarboxylic acids from lignocellulosic biomass and exploring genetic mechanisms of the yeast tolerance to the biomass hydrolysate.

BACKGROUND: Lignosulfonates are significant wood chemicals with a $700 million market, produced by sulfite pulping of wood. During the pulping process, spent sulfite liquor (SSL) is generated, which in addition to lignosulfonates contains hemicellulose-derived sugars-in case of hardwoods primarily the pentose sugar xylose. The pentoses are currently underutilized. If they could be converted into value-added chemicals, overall economic profitability of the process would increase. SSLs are typically very inhibitory to microorganisms, which presents a challenge for a biotechnological process. The aim of the present work was to develop a robust yeast strain able to convert xylose in SSL to carboxylic acids. RESULTS: The industrial strain Ethanol Red of the yeast Saccharomyces cerevisiae was engineered for efficient utilization of xylose in a Eucalyptus globulus lignosulfonate stream at low pH using CRISPR/Cas genome editing and adaptive laboratory evolution. The engineered strain grew in synthetic medium with xylose as sole carbon source with maximum specific growth rate (µmax) of 0.28 1/h. Selected evolved strains utilized all carbon sources in the SSL at pH 3.5 and grew with µmax between 0.05 and 0.1 1/h depending on a nitrogen source supplement. Putative genetic determinants of the increased tolerance to the SSL were revealed by whole genome sequencing of the evolved strains. In particular, four top-candidate genes (SNG1, FIT3, FZF1 and CBP3) were identified along with other gene candidates with predicted important roles, based on the type and distribution of the mutations across different strains and especially the best performing ones. The developed strains were further engineered for production of dicarboxylic acids (succinic and malic acid) via overexpression of the reductive branch of the tricarboxylic acid cycle (TCA). The production strain produced 0.2 mol and 0.12 mol of malic acid and succinic acid, respectively, per mol of xylose present in the SSL. CONCLUSIONS: The combined metabolic engineering and adaptive evolution approach provided a robust SSL-tolerant industrial strain that converts fermentable carbon content of the SSL feedstock into malic and succinic acids at low pH.in production yields reaching 0.1 mol and 0.065 mol per mol of total consumed carbon sources.. Moreover, our work suggests potential genetic background of the tolerance to the SSL stream pointing out potential gene targets for improving the tolerance to inhibitory industrial feedstocks.

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® 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®. 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.

Derivation and identification of a mechanistic model for a branched enzyme-catalyzed carboligation.

The kinetic description of enzyme-catalyzed reactions is a core task in biotechnology and biochemical engineering. In particular, mechanistic kinetic models help from the discovery of the biocatalyst throughout its application. Chemo- or enantioselective enzyme reactions often undergo two alternative pathways for the release of two different products from a central intermediate. For these types of reaction, no explicit reaction equations have been derived so far. To this end, we extend the commonly used Cleland's notation for branched reaction pathways and explicitly derive the rate expressions for two-coupled ordered bi-uni reactions. This mechanism also leads to a ping-pong bi-bi mechanism for a transfer reaction between the two products via the same central intermediate of the reaction system. Using the cross-ligation of benzaldehyde and propanal catalyzed by the thiamine diphosphate-dependent enzyme benzaldehyde lyase from Pseudomonas fluorescens yielding (R)-2-hydroxy-1-phenylbutan-1-one as a case study, we performed model-based experimental analysis to show that such a reaction mechanism can be modeled mechanistically and leads to reasonable kinetic parameters.

Simultaneous identification of reaction and inactivation kinetics of an enzyme-catalyzed carboligation.

Thiamine diphosphate (ThDP)-dependent enzymes catalyze a broad range of reactions with excellent enantioselectivity. Among these reactions, carboligations of aldehydes are of particular interest since the products, chiral hydroxy ketones, are valuable building blocks in the pharmaceutical industry. However, the substrates, for example, benzaldehyde, inactivate the biocatalysts, for example the ThDP-dependent benzaldehyde lyase from Pseudomonas fluorescens (PfBAL). Because only few mechanistic kinetic models for carboligation and simultaneous inactivation are available today, we quantitatively determined the reaction kinetics and inactivation of the self-carboligation of benzaldehyde yielding the product (R)-benzoin catalyzed by PfBAL directly from progress curves using model-based experimental analysis. Discrimination of several inactivation models identified the substrate-dependent inactivation by benzaldehyde to be significant. Sensitivity analysis and optimal experimental design improved parameter precision significantly, to between 4 and 26% relative standard deviation while maintaining the necessary number of 13 experiments moderate. The developed mechanistic kinetic model will enable to perform a model-based process optimization to circumvent the substrate-dependent enzyme inactivation. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1081-1092, 2018.

Production of the potential sweetener 5-ketofructose from fructose in fed-batch cultivation with Gluconobacter oxydans.

Sweeteners improve the dietary properties of many foods. A candidate for a new natural sweetener is 5-ketofructose. In this study a fed-batch process for the production of 5-ketofructose was developed. A Gluconobacter oxydans strain overexpressing a fructose dehydrogenase from G. japonicus was used and the sensory properties of 5-ketofructose were analyzed. The compound showed an identical sweet taste quality as fructose and a similar intrinsic sweet threshold concentration of 16.4 mmol/L. The production of 5-ketofructose was characterized online by monitoring of the respiration activity in shake flasks. Pulsed and continuous fructose feeding was realized in 2 L stirred tank reactors and maximum fructose consumption rates were determined. 5-Ketofructose concentrations of up to 489 g/L, product yields up to 0.98 g5-KF/gfructose and space time yields up to 8.2 g/L/h were reached highlighting the potential of the presented process.