SummaryWhereas intracellular carbon metabolism has emerged as an attractive drug target, the carbon sources of intracellularly replicating pathogens, such as the tuberculosis bacillus Mycobacterium tuberculosis, which causes long-term infections in one-third of the world’s population, remain mostly unknown. We used a systems-based approach—13C-flux spectral analysis (FSA) complemented with manual analysis—to measure the metabolic interaction between M. tuberculosis and its macrophage host cell. 13C-FSA analysis of experimental data showed that M. tuberculosis obtains a mixture of amino acids, C1 and C2 substrates from its host cell. We experimentally confirmed that the C1 substrate was derived from CO2. 13C labeling experiments performed on a phosphoenolpyruvate carboxykinase mutant revealed that intracellular M. tuberculosis has access to glycolytic C3 substrates. These findings provide constraints for developing novel chemotherapeutics.

Summary: 13C-based metabolic flux analysis (13C-MFA) is the state-of-the-art method to quantitatively determine in vivo metabolic reaction rates in microorganisms. 13CFLUX2 contains all tools for composing flexible computational 13C-MFA workflows to design and evaluate carbon labeling experiments. A specially developed XML language, FluxML, highly efficient data structures and simulation algorithms achieve a maximum of performance and effectiveness. Support of multicore CPUs, as well as compute clusters, enables scalable investigations. 13CFLUX2 outperforms existing tools in terms of universality, flexibility and built-in features. Therewith, 13CFLUX2 paves the way for next-generation high-resolution 13C-MFA applications on the large scale.Availability and implementation: 13CFLUX2 is implemented in C++ (ISO/IEC 14882 standard) with Java and Python add-ons to run under Linux/Unix. A demo version and binaries are available at www.13cflux.net.Contact: info@13cflux.net or k.noeh@fz-juelich.deSupplementary information: Supplementary data are available at Bioinformatics online.

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Methanol is already an important carbon feedstock in the chemical industry, but it has found only limited application in biotechnological production processes. This can be mostly attributed to the inability of most microbial platform organisms to utilize methanol as a carbon and energy source. With the aim to turn methanol into a suitable feedstock for microbial production processes, we engineered the industrially important but nonmethylotrophic bacterium Corynebacterium glutamicum toward the utilization of methanol as an auxiliary carbon source in a sugar-based medium. Initial oxidation of methanol to formaldehyde was achieved by heterologous expression of a methanol dehydrogenase from Bacillus methanolicus, whereas assimilation of formaldehyde was realized by implementing the two key enzymes of the ribulose monophosphate pathway of Bacillus subtilis: 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. The recombinant C. glutamicum strain showed an average methanol consumption rate of 1.7 ؎ 0.3 mM/h (mean ؎ standard deviation) in a glucose-methanol medium, and the culture grew to a higher cell density than in medium without methanol. In addition, [13 C]methanol-labeling experiments revealed labeling fractions of 3 to 10% in the m ؉ 1 mass isotopomers of various intracellular metabolites. In the background of a C. glutamicum ⌬ald ⌬adhE mutant being strongly impaired in its ability to oxidize formaldehyde to CO 2 , the m ؉ 1 labeling of these intermediates was increased (8 to 25%), pointing toward higher formaldehyde assimilation capabilities of this strain. The engineered C. glutamicum strains represent a promising starting point for the development of sugar-based biotechnological production processes using methanol as an auxiliary substrate.

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