Flux Balance Analysis of Dynamic Metabolism in Shewanella oneidensis MR-1 Using a Static Optimization Approach

Shewanella bacteria are facultative anaerobes isolated from aquatic and sedimentary environments (Hau and Gralnick 2007) with a broad capacity for reduction of multiple electron receptors (Pinchuk et al. 2009; Serres and Riley 2006), including Fe(III), Mn(IV), sulfur, nitrate, and fumarate. With the accomplishment of complete genome sequencing of several Shewanella bacteria, the general pictures of the carbon metabolism have been revealed (Serres and Riley 2006). metabolism. One of the most physiological methods to decipher the time-variant metabolic regulation is to determine the dynamic distribution of intracellular metabolic fluxes since it reveals the final response of cellular metabolism to genomic, transcriptional and post-transcriptional regulations (Sauer 2006; Tang et al. 2009). In order to track the dynamic intracellular metabolic regulation, dynamic flux balance analysis (DFBA) was developed (Mahadevan et al. 2002), in which cell growth phase was divided into numerous stages, assuming that at each stage a new metabolic steady state was maintained. All the metabolic fluxes were then searched to satisfy the objective functions set for each stage. By solving this nonlinear optimization model using a cutting-edge nonlinear optimization solver (IPOPT), we confirmed the changing of carbon sources for the growth of Shewanella oneidensis MR-1 and deciphered the dynamic regulation of intracellular metabolism

Carbohydrate Metabolism and Carbon Fixation in Roseobacter denitrificans OCh114

The Roseobacter clade of aerobic marine proteobacteria, which compose 10–25% of the total marine bacterial community, has been reported to fix CO2, although it has not been determined what pathway is involved. In this study, we report the first metabolic studies on carbohydrate utilization, CO2 assimilation, and amino acid biosynthesis in the phototrophic Roseobacter clade bacterium Roseobacter denitrificans OCh114. We develop a new minimal medium containing defined carbon source(s), in which the requirements of yeast extract reported previously for the growth of R. denitrificans can be replaced by vitamin B12 (cyanocobalamin). Tracer experiments were carried out in R. denitrificans grown in a newly developed minimal medium containing isotopically labeled pyruvate, glucose or bicarbonate as a single carbon source or in combination. Through measurements of 13C-isotopomer labeling patterns in protein-derived amino acids, gene expression profiles, and enzymatic activity assays, we report that: (1) R. denitrificans uses the anaplerotic pathways mainly via the malic enzyme to fix 10–15% of protein carbon from CO2; (2) R. denitrificans employs the Entner-Doudoroff (ED) pathway for carbohydrate metabolism and the non-oxidative pentose phosphate pathway for the biosynthesis of histidine, ATP, and coenzymes; (3) the Embden-Meyerhof-Parnas (EMP, glycolysis) pathway is not active and the enzymatic activity of 6-phosphofructokinase (PFK) cannot be detected in R. denitrificans; and (4) isoleucine can be synthesized from both threonine-dependent (20% total flux) and citramalate-dependent (80% total flux) pathways using pyruvate as the sole carbon source

Dynamic Metabolic Flux Analysis of Shewanella oneidensis MR-1 Central Metabolisms [abstract]

Only abstract of poster available.Track II: Transportation and BiofuelsShewanella oneidensis MR-1 have received significant attention because of their versatile carbon metabolisms and potential to engage in bioremediation of toxic metal compounds and microbial fuel cell applications. In active growth phase using lactate as the carbon source, MR-1 shows a dynamic metabolism. MR-1 produces a significant amount of pyruvate and acetate when the lactate is excess in the medium. When the energy favorable carbon source (lactate) is depleted, MR-1 will utilize the waste product (pyruvate and acetate) for their growth. In response to the switch of carbon sources during the growth, the central metabolism (TCA cycle, glyoxylate shunt and futile pathways) of MR-1 also changes. To describe this dynamic metabolism, we combine the enzyme kinetic modeling with the isotopomer analysis to quantitatively understand the regulation of metabolic network and profile the flux distribution as the function of the time. Using MATLAB based ODE tool box, we can solve the dynamic metabolism and predict the growth/extracellular metabolites production. Meanwhile, we may improve the model predictions using the constraints from the labeling information. The isotopomer information can also provide us the insight into the regulation of central metabolic pathways during MR-1 growth. Such isotopomer assisted dynamic flux model may be potentially used in other biological systems including the biofuel producers or microbial communities

Conditional protein degradation in Yarrowia lipolytica using the auxin-inducible degron

Conditional protein degradation is a powerful tool for controlled protein knockdown. The auxin-inducible degron (AID) technology uses a plant auxin to induce depletion of degron-tagged proteins, and it has been shown to be functional in several non-plant eukaryotes. In this study, we demonstrated AID-based protein knockdown in an industrially important oleaginous yeas

Incomplete Wood-Ljungdahl pathway facilitates one-carbon metabolism in organohalide-respiring Dehalococcoides mccartyi.

The acetyl-CoA "Wood-Ljungdahl" pathway couples the folate-mediated one-carbon (C1) metabolism to either CO2 reduction or acetate oxidation via acetyl-CoA. This pathway is distributed in diverse anaerobes and is used for both energy conservation and assimilation of C1 compounds. Genome annotations for all sequenced strains of Dehalococcoides mccartyi, an important bacterium involved in the bioremediation of chlorinated solvents, reveal homologous genes encoding an incomplete Wood-Ljungdahl pathway. Because this pathway lacks key enzymes for both C1 metabolism and CO2 reduction, its cellular functions remain elusive. Here we used D. mccartyi strain 195 as a model organism to investigate the metabolic function of this pathway and its impacts on the growth of strain 195. Surprisingly, this pathway cleaves acetyl-CoA to donate a methyl group for production of methyl-tetrahydrofolate (CH3-THF) for methionine biosynthesis, representing an unconventional strategy for generating CH3-THF in organisms without methylene-tetrahydrofolate reductase. Carbon monoxide (CO) was found to accumulate as an obligate by-product from the acetyl-CoA cleavage because of the lack of a CO dehydrogenase in strain 195. CO accumulation inhibits the sustainable growth and dechlorination of strain 195 maintained in pure cultures, but can be prevented by CO-metabolizing anaerobes that coexist with D. mccartyi, resulting in an unusual syntrophic association. We also found that this pathway incorporates exogenous formate to support serine biosynthesis. This study of the incomplete Wood-Ljungdahl pathway in D. mccartyi indicates a unique bacterial C1 metabolism that is critical for D. mccartyi growth and interactions in dechlorinating communities and may play a role in other anaerobic communities