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First, we report the application of stable isotope dilution theory in metabolome characterization of aerobic glucose limited chemostat culture of S. cerevisiae CEN.PK 113-7D using liquid chromatography-electrospray ionization MS/MS (LC-ESI-MS/MS). A glucose-limited chemostat culture of S. cerevisiae was grown to steady state at a specific growth rate (mu)=0.05 h(-1) in a medium containing only naturally labeled (99% U-12C, 1% U-13C) carbon source. Upon reaching steady state, defined as 5 volume changes, the culture medium was switched to chemically identical medium except that the carbon source was replaced with 100% uniformly (U) 13C labeled stable carbon isotope, fed for 4 h, with sampling every hour. We observed that within a period of 1 h approximately 80% of the measured glycolytic metabolites were U-13C-labeled. Surprisingly, during the next 3 h no significant increase of the U-13C-labeled metabolites occurred. Second, we demonstrate for the first time the LC-ESI-MS/MS-based quantification of intracellular metabolite concentrations using U-13C-labeled metabolite extracts from chemostat cultivated S. cerevisiae cells, harvested after 4 h of feeding with 100% U-13C-labeled medium, as internal standard. This method is hereby termed "Mass Isotopomer Ratio Analysis of U-13C Labeled Extracts" (MIRACLE). With this method each metabolite concentration is quantified relative to the concentration of its U-13C-labeled equivalent, thereby eliminating drawbacks of LC-ESI-MS/MS analysis such as nonlinear response and matrix effects and thus leads to a significant reduction of experimental error and work load (i.e., no spiking and standard additions). By coextracting a known amount of U-13C labeled cells with the unlabeled samples, metabolite losses occurring during the sample extraction procedure are corrected for.

Plasmids are mobile genetic elements that play a key role in the evolution of bacteria by mediating genome plasticity and lateral transfer of useful genetic information. Although originally considered to be exclusively circular, linear plasmids have also been identified in certain bacterial phyla, notably the actinomycetes. In some cases, linear plasmids engage with chromosomes in an intricate evolutionary interplay, facilitating the emergence of new genome configurations by transfer and recombination or plasmid integration. Genome sequencing of Streptomyces clavuligerus ATCC 27064, a Gram-positive soil bacterium known for its production of a diverse array of biotechnologically important secondary metabolites, revealed a giant linear plasmid of 1.8 Mb in length. This megaplasmid (pSCL4) is one of the largest plasmids ever identified and the largest linear plasmid to be sequenced. It contains more than 20% of the putative protein-coding genes of the species, but none of these is predicted to be essential for primary metabolism. Instead, the plasmid is densely packed with an exceptionally large number of gene clusters for the potential production of secondary metabolites, including a large number of putative antibiotics, such as staurosporine, moenomycin, β-lactams, and enediynes. Interestingly, cross-regulation occurs between chromosomal and plasmid-encoded genes. Several factors suggest that the megaplasmid came into existence through recombination of a smaller plasmid with the arms of the main chromosome. Phylogenetic analysis indicates that heavy traffic of genetic information between Streptomyces plasmids and chromosomes may facilitate the rapid evolution of secondary metabolite repertoires in these bacteria.

This article presents the dynamic responses of several intra- and extracellular components of an aerobic, glucose-limited chemostat culture of Saccharomyces cerevisiae to glucose and ethanol pulses within a time window of 75 sec. Even though the ethanol pulse cannot perturb the glycolytic pathway directly, a distinct response of the metabolites at the lower part of glycolysis was found. We suggest that this response is an indirect effect, caused by perturbation of the NAD/NADH ratio, which is a direct consequence of the conversion of ethanol into acetaldehyde. This effect of the NAD/NADH ratio on glycolysis might serve as an additional explanation for the observed decrease of 3PG, 2PG, and PEP during a glucose pulse. The responses measured during the ethanol pulse were used to evaluate the allosteric regulation of glycolysis. Our results confirm that FBP stimulates pyruvate kinase and suggest that this effect is pronounced. Furthermore, it appears that PEP does not play an important role in the allosteric regulation of phosphofructo kinase.

Nylon-6 is a bulk polymer used for many applications. It consists of the non-natural building block 6-aminocaproic acid, the linear form of caprolactam. Via a retro-synthetic approach, two synthetic pathways were identified for the fermentative production of 6-aminocaproic acid. Both pathways require yet unreported novel biocatalytic steps. We demonstrated proof of these bioconversions by in vitro enzyme assays with a set of selected candidate proteins expressed in Escherichia coli. One of the biosynthetic pathways starts with 2-oxoglutarate and contains bioconversions of the ketoacid elongation pathway known from methanogenic archaea. This pathway was selected for implementation in E. coli and yielded 6-aminocaproic acid at levels up to 160 mg/L in lab-scale batch fermentations. The total amount of 6-aminocaproic acid and related intermediates generated by this pathway exceeded 2 g/L in lab-scale fed-batch fermentations, indicating its potential for further optimization toward large-scale sustainable production of nylon-6.