The advent of genome-scale models of metabolism has laid the foundation for the development of computational procedures for suggesting genetic manipulations that lead to overproduction. In this work, the computational OptKnock framework is introduced for suggesting gene deletion strategies leading to the overproduction of chemicals or biochemicals in E. coli. This is accomplished by ensuring that a drain towards growth resources (i.e., carbon, redox potential, and energy) must be accompanied, due to stoichiometry, by the production of a desired product. Computational results for gene deletions for succinate, lactate, and 1,3-propanediol (PDO) production are in good agreement with mutant strains published in the literature. While some of the suggested deletion strategies are straightforward and involve eliminating competing reaction pathways, many others suggest complex and nonintuitive mechanisms of compensating for the removed functionalities. Finally, the OptKnock procedure, by coupling biomass formation with chemical production, hints at a growth selection/adaptation system for indirectly evolving overproducing mutants.

This paper introduces the hierarchical computational framework OptStrain aimed at guiding pathway modifications, through reaction additions and deletions, of microbial networks for the overproduction of targeted compounds. These compounds may range from electrons or hydrogen in biofuel cell and environmental applications to complex drug precursor molecules. A comprehensive database of biotransformations, referred to as the Universal database (with >5700 reactions), is compiled and regularly updated by downloading and curating reactions from multiple biopathway database sources. Combinatorial optimization is then used to elucidate the set(s) of non-native functionalities, extracted from this Universal database, to add to the examined production host for enabling the desired product formation. Subsequently, competing functionalities that divert flux away from the targeted product are identified and removed to ensure higher product yields coupled with growth. This work represents an advancement over earlier efforts by establishing an integrated computational framework capable of constructing stoichiometrically balanced pathways, imposing maximum product yield requirements, pinpointing the optimal substrate(s), and evaluating different microbial hosts. The range and utility of OptStrain are demonstrated by addressing two very different product molecules. The hydrogen case study pinpoints reaction elimination strategies for improving hydrogen yields using two different substrates for three separate production hosts. In contrast, the vanillin study primarily showcases which non-native pathways need to be added into Escherichia coli. In summary, OptStrain provides a useful tool to aid microbial strain design and, more importantly, it establishes an integrated framework to accommodate future modeling developments.[Supplemental material is available online at www.genome.org. The Universal database can be found at http://fenske.che.psu.edu/Faculty/CMaranas/pubs.html.]A fundamental goal in systems biology is to elucidate the complete "palette" of biotransformations accessible to nature in living systems. This goal parallels the continuing quest in biotechnology to construct microbial strains capable of accomplishing an ever-expanding array of desired biotransformations. These biotransformations are aimed at products that range from simple precursor chemicals (Nakamura and Whited 2003;Causey et al. 2004) or complex molecules such as carotenoids (Misawa et al. 1991), to electrons in biofuel cells (Liu et al. 2004) or batteries (Bond et al. 2002;Bond and Lovley 2003), to even microbes capable of precipitating heavy metal complexes in bioremediation applications (Finneran et al. 2002;Lovley 2003;Methe et al. 2003). Recent developments in molecular biology and recombinant DNA technology have ushered in a new era in the ability to shape the gene content and expression levels for microbial production strains in a direct and targeted fashion (Stephanopoulos 2002). The astounding range and diversity of these newly acquired capabili...

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A wide variety of commercial products can be potentially made from monomeric sugars produced by the dilute acid hydrolysis of lignocellulosic biomass. However, this process is accompanied by side products such as furfural that hinder microbial growth and fermentation. To investigate the mechanism of furfural inhibition, mRNA microarrays of an ethanologenic strain of Escherichia coli (LY180) were compared immediately prior to and 15 min after a moderate furfural challenge. Expression of genes and regulators associated with the biosynthesis of cysteine and methionine was increased by furfural, consistent with a limitation of these critical metabolites. This was in contrast to a general stringent response and decreased expression of many other biosynthetic genes. Of the 20 amino acids individually tested as supplements (100 M each), cysteine and methionine were the most effective in increasing furfural tolerance with serine (precursor of cysteine), histidine, and arginine of lesser benefit. Supplementation with other reduced sulfur sources such as D-cysteine and thiosulfate also increased furfural tolerance. In contrast, supplementation with taurine, a sulfur source that requires 3 molecules of NADPH for sulfur assimilation, was of no benefit. Furfural tolerance was also increased by inserting a plasmid encoding pntAB, a cytoplasmic NADH/NADPH transhydrogenase. Based on these results, a model is proposed for the inhibition of growth in which the reduction of furfural by YqhD, an enzyme with a low K m for NADPH, depletes NADPH sufficiently to limit the assimilation of sulfur into amino acids (cysteine and methionine) by CysIJ (sulfite reductase).Lignocellulose contains up to 70% carbohydrate by weight (35 to 45% cellulose and 20 to 35% hemicellulose) and represents an excellent potential source of sugars for microbial conversion into renewable fuels, plastics, and other chemicals (9,13,15,38). Prior to fermentation, these carbohydrate polymers must be converted to soluble sugars. Hemicellulose can be conveniently hydrolyzed to sugar monomers using dilute mineral acids. However, this process is accompanied by side products that inhibit microbial growth (20,21,29,30,(44)(45)(46). Furfural, the dehydration product of pentose sugars, is one of the most important such inhibitors (1). Previous studies have shown that furfural levels directly correlate with toxicity (20, 21). Overliming treatments that render hemicellulose hydrolysates fermentable also reduce the levels of furfural. Full toxicity in overlimed hydrolysates is restored by the addition of furfural. Of the many components of hydrolysates that have been tested for toxicity, only furfural was found to potentiate the toxicity of other agents in binary combinations (45).A number of approaches have been used to investigate the mechanism of furfural action. Furfural and 5-hydroxymethyl furfural (dehydration product from hexose sugars) have been previously proposed to inhibit growth by damaging DNA (3,16,36), inhibiting glycolysis and glycolytic enzymes (5,11,25), an...