We present an experimental and computational pipeline for the generation of kinetic models of metabolism, and demonstrate its application to glycolysis in Saccharomyces cerevisiae. Starting from an approximate mathematical model, we employ a “cycle of knowledge” strategy, identifying the steps with most control over flux. Kinetic parameters of the individual isoenzymes within these steps are measured experimentally under a standardised set of conditions. Experimental strategies are applied to establish a set of in vivo concentrations for isoenzymes and metabolites. The data are integrated into a mathematical model that is used to predict a new set of metabolite concentrations and reevaluate the control properties of the system. This bottom-up modelling study reveals that control over the metabolic network most directly involved in yeast glycolysis is more widely distributed than previously thought.

The coenzyme B12-dependent photoreceptor protein, CarH, is a bacterial transcriptional regulator that controls the biosynthesis of carotenoids in response to light. On binding of coenzyme B12 the monomeric apoprotein forms tetramers in the dark, which bind operator DNA thus blocking transcription. Under illumination the CarH tetramer dissociates, weakening its affinity for DNA and allowing transcription. The mechanism by which this occurs is unknown. Here we describe the photochemistry in CarH that ultimately triggers tetramer dissociation; it proceeds via a cob(III)alamin intermediate, which then forms a stable adduct with the protein. This pathway is without precedent and our data suggest it is independent of the radical chemistry common to both coenzyme B12 enzymology and its known photochemistry. It provides a mechanistic foundation for the emerging field of B12 photobiology and will serve to inform the development of a new class of optogenetic tool for the control of gene expression.

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a b s t r a c tSystems Biology has a mission that puts it at odds with traditional paradigms of physics and molecular biology, such as the simplicity requested by Occam's razor and minimum energy/maximal efficiency. By referring to biochemical experiments on control and regulation, and on flux balancing in yeast, we show that these paradigms are inapt. Systems Biology does not quite converge with biology either: Although it certainly requires accurate 'stamp collecting', it discovers quantitative laws. Systems Biology is a science of its own, discovering own fundamental principles, some of which we identify here. Crown

Monoterpenoids offer potential as biocatalytically derived monomer feedstocks for high-performance renewable polymers. We describe a biocatalytic route to lactone monomers menthide and dihydrocarvide employing Baeyer-Villiger monooxygenases (BVMOs) from Pseudomonas sp. HI-70 (CPDMO) and Rhodococcus sp. Phi1 (CHMO) as an alternative to organic synthesis. The regioselectivity of dihydrocarvide isomer formation was controlled by site-directed mutagenesis of three key active site residues in CHMO. A combination of crystal structure determination, molecular dynamics simulations, and mechanistic modeling using density functional theory on a range of models provides insight into the origins of the discrimination of the wild type and a variant CHMO for producing different regioisomers of the lactone product. Ring-opening polymerizations of the resultant lactones using mild metal-organic catalysts demonstrate their utility in polymer production. This semisynthetic approach utilizing a biocatalytic step, non-petroleum feedstocks, and mild polymerization catalysts allows access to known and also to previously unreported and potentially novel lactone monomers and polymers.