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A series of systematic single-amino acid omissions have modulated the production of vitamin [B.sub.12] from nearly undetectable levels to levels that are 20-fold higher. cDNA microarray experiments are used for analyzing the transcriptional response of Lactobacillus reuteri to medium lacking cysteine.

Lactobacillus reuteri JCM1112 has produced [B.sub.12] and folate, but the folate/[B.sub.12] mass ratio is far below that it is desired for human consumption. Metabolic engineering is used by applying genetic and physiological approaches to improve this ratio and a generic and natural process is developed that has increased the folate production.

The detection of luciferase activity in stationary-phase cells of Lactococcus lactis is discussed. Findings reveal a significant improvement in the detection of luciferase in stationary-phase cells of Lactococcus lactis due to the addition of riboflavin or flavin mononucleotide to whole-cell assay systems.

Whooping cough is a highly contagious respiratory disease caused by Bordetella pertussis. Despite widespread vaccination, its incidence has been rising alarmingly, and yet, the physiology of B. pertussis remains poorly understood. We combined genome-scale metabolic reconstruction, a novel optimization algorithm, and experimental data to probe the full metabolic potential of this pathogen, using B. pertussis strain Tohama I as a reference. Experimental validation showed that B. pertussis secretes a significant proportion of nitrogen as arginine and purine nucleosides, which may contribute to modulation of the host response. We also found that B. pertussis can be unexpectedly versatile, being able to metabolize many compounds while displaying minimal nutrient requirements. It can grow without cysteine, using inorganic sulfur sources, such as thiosulfate, and it can grow on organic acids, such as citrate or lactate, as sole carbon sources, providing in vivo demonstration that its tricarboxylic acid (TCA) cycle is functional. Although the metabolic reconstruction of eight additional strains indicates that the structural genes underlying this metabolic flexibility are widespread, experimental validation suggests a role of strain-specific regulatory mechanisms in shaping metabolic capabilities. Among five alternative strains tested, three strains were shown to grow on substrate combinations requiring a functional TCA cycle, but only one strain could use thiosulfate. Finally, the metabolic model was used to rationally design growth media with >2-fold improvements in pertussis toxin production. This study thus provides novel insights into B. pertussis physiology and highlights the potential, but also the limitations, of models based solely on metabolic gene content. IMPORTANCE The metabolic capabilities of Bordetella pertussis, the causative agent of whooping cough, were investigated from a systems-level perspective. We constructed a comprehensive genome-scale metabolic model for B. pertussis and challenged its predictions experimentally. This systems approach shed light on new potential host-microbe interactions and allowed us to rationally design novel growth media with >2-fold improvements in pertussis toxin production. Most importantly, we also uncovered the potential for metabolic flexibility of B. pertussis (significantly larger range of substrates than previously alleged; novel active pathways allowing growth in minimal, nearly mineral nutrient combinations where only the carbon source must be organic), although our results also highlight the importance of strain-specific regulatory determinants in shaping metabolic capabilities. Deciphering the underlying regulatory mechanisms appears to be crucial for a comprehensive understanding of B. pertussis's lifestyle and the epidemiology of whooping cough. The contribution of metabolic models in this context will require the extension of the genome-scale metabolic model to integrate this regulatory dimension. [ABSTRACT FROM AUTHOR]

Recent functional genomics and genome-scale modeling approaches indicated that B12 production in Lactobacillus reuteri could be improved by optimization of the medium. Here we show that a series of systematic single-amino-acid omissions could significantly modulate the production of B12 from nearly undetectable levels (with omission of isoleucine) to levels 20-fold higher than the levels previously reported (with omission of cysteine). Using cDNA microarray experiments, we analyzed the transcriptional response of L. reuteri to medium lacking cysteine. The results supported the observed high level of B12 production and provided new avenues for future improvement of production of vitamin B12. [ABSTRACT FROM AUTHOR]

We observed that Ladobacillus 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. [ABSTRACT FROM AUTHOR]