Rewriting yeast central carbon metabolism for industrial isoprenoid production.

A bio-based economy has the potential to provide sustainable substitutes for petroleum-based products and new chemical building blocks for advanced materials. We previously engineered Saccharomyces cerevisiae for industrial production of the isoprenoid artemisinic acid for use in antimalarial treatments. Adapting these strains for biosynthesis of other isoprenoids such as ß-farnesene (C15H24), a plant sesquiterpene with versatile industrial applications, is straightforward. However, S. cerevisiae uses a chemically inefficient pathway for isoprenoid biosynthesis, resulting in yield and productivity limitations incompatible with commodity-scale production. Here we use four non-native metabolic reactions to rewire central carbon metabolism in S. cerevisiae, enabling biosynthesis of cytosolic acetyl coenzyme A (acetyl-CoA, the two-carbon isoprenoid precursor) with a reduced ATP requirement, reduced loss of carbon to CO2-emitting reactions, and improved pathway redox balance. We show that strains with rewired central metabolism can devote an identical quantity of sugar to farnesene production as control strains, yet produce 25% more farnesene with that sugar while requiring 75% less oxygen. These changes lower feedstock costs and dramatically increase productivity in industrial fermentations which are by necessity oxygen-constrained. Despite altering key regulatory nodes, engineered strains grow robustly under taxing industrial conditions, maintaining stable yield for two weeks in broth that reaches >15% farnesene by volume. This illustrates that rewiring yeast central metabolism is a viable strategy for cost-effective, large-scale production of acetyl-CoA-derived molecules.

Use of pantothenate as a metabolic switch increases the genetic stability of farnesene producing Saccharomyces cerevisiae.

We observed that removing pantothenate (vitamin B5), a precursor to co-enzyme A, from the growth medium of Saccharomyces cerevisiae engineered to produce ß-farnesene reduced the strain׳s farnesene flux by 70%, but increased its viability, growth rate and biomass yield. Conversely, the growth rate and biomass yield of wild-type yeast were reduced. Cultivation in media lacking pantothenate eliminates the growth advantage of low-producing mutants, leading to improved production upon scale-up to lab-scale bioreactor testing. An omics investigation revealed that when exogenous pantothenate levels are limited, acyl-CoA metabolites decrease, ß-oxidation decreases from unexpectedly high levels in the farnesene producer, and sterol and fatty acid synthesis likely limits the growth rate of the wild-type strain. Thus pantothenate supplementation can be utilized as a "metabolic switch" for tuning the synthesis rates of molecules relying on CoA intermediates and aid the economic scale-up of strains producing acyl-CoA derived molecules to manufacturing facilities.

Application of dynamic flux balance analysis to an industrial Escherichia coli fermentation.

We have developed a reactor-scale model of Escherichia coli metabolism and growth in a 1000 L process for the production of a recombinant therapeutic protein. The model consists of two distinct parts: (1) a dynamic, process specific portion that describes the time evolution of 37 process variables of relevance and (2) a flux balance based, 123-reaction metabolic model of E. coli metabolism. This model combines several previously reported modeling approaches including a growth rate-dependent biomass composition, maximum growth rate objective function, and dynamic flux balancing. In addition, we introduce concentration-dependent boundary conditions of transport fluxes, dynamic maintenance demands, and a state-dependent cellular objective. This formulation was able to describe specific runs with high-fidelity over process conditions including rich media, simultaneous acetate and glucose consumption, glucose minimal media, and phosphate depleted media. Furthermore, the model accurately describes the effect of process perturbations--such as glucose overbatching and insufficient aeration--on growth, metabolism, and titer.

Metabolic and morphological differences between rapidly proliferating cancerous and normal breast epithelial cells.

The metabolic and morphological characteristics of two human epithelial breast cell populations--MCF7 cells, a cancerous cell line, and 48R human mammary epithelial cells (48R HMECs), a noncancerous, finite lifespan cell strain--were compared at identical growth rates. Both cell types were induced to grow rapidly in nutrient-rich media containing 13C-labeled glucose, and the isotopic enrichment of cellular metabolites was quantified to calculate metabolic fluxes in key pathways. Despite their similar growth rates, the cells exhibited distinctly different metabolic and morphological profiles. MCF7 cells have an 80% smaller exposed surface area and contain 26% less protein per cell than the 48R cells. Surprisingly, rapidly proliferating 48R cells exhibited a 225% higher per-cell glucose consumption rate, a 250% higher per-cell lactate production rate, and a nearly identical per-cell glutamine consumption rate relative to the cancer cell line. However, when fluxes were considered on the basis of exposed area, the cancer cells were observed to have higher glucose, lactate, and glutamine fluxes, demonstrating superior transport capabilities per unit area of cell membrane. MCF7 cells also consumed amino acids at rates much higher than are generally required for protein synthesis, whereas 48R cells generally did not. Pentose phosphate pathway activity was higher in MCF7 cells, and the flux of glutamine to glutamate was less reversible. Energy efficiency was significantly higher in MCF7 cells, as a result of a combination of their smaller size and greater reliance on the TCA cycle than the 48R cells. These observations support evolutionary models of cancer cell metabolism and suggest targets for metabolic drugs in metastatic breast cancers.

Optimal design of metabolic flux analysis experiments for anchorage-dependent mammalian cells using a cellular automaton model.

Metabolic flux analysis (MFA) is widely used to quantify metabolic pathway activity. Typical applications involve isotopically labeled substrates, which require both metabolic and isotopic steady states for simplified data analysis. For bacterial systems, these steady states are readily achieved in chemostat cultures. However, mammalian cells are often anchorage dependent and experiments are typically conducted in batch or fed-batch systems, such as tissue culture dishes or microcarrier-containing bioreactors. Surface adherence may cause deviations from exponential growth, resulting in metabolically heterogeneous populations and a varying number of cellular "nearest neighbors" that may affect the observed metabolism. Here, we discuss different growth models suitable for deconvoluting these effects and their application to the design and optimization of MFA experiments employing surface-adherent mammalian cells. We describe a stochastic two-dimensional (2D) cellular automaton model, with empirical descriptions of cell number and non-growing cell fraction, suitable for easy application to most anchorage-dependent mammalian cell cultures. Model utility was verified by studying the impact of contact inhibition on the growth rate, specific extracellular flux rates, and isotopic labeling in lactate for MCF7 cells, a commonly studied breast cancer cell line. The model successfully defined the time over which exponential growth and a metabolically homogeneous growing cell population could be assumed. The cellular automaton model developed is shown to be a useful tool in designing optimal MFA experiments.

A kinetic model describing Shewanella oneidensis MR-1 growth, substrate consumption, and product secretion.

Aerobic growth of Shewanella oneidensis MR-1 in minimal lactate medium was studied in batch cultivation. Acetate production was observed in the middle of the exponential growth phase and was enhanced when the dissolved oxygen (DO) concentration was low. Once the lactate was nearly exhausted, S. oneidensis MR-1 used the acetate produced during growth on lactate with a similar biomass yield as lactate. A two-substrate Monod model, with competitive and uncompetitive substrate inhibition, was devised to describe the dependence of biomass growth on lactate, acetate, and oxygen and the acetate growth inhibition across a broad range of concentrations. The parameters estimated for this model indicate interesting growth kinetics: lactate is converted to acetate stoichiometrically regardless of the DO concentration; cells grow well even at low DO levels, presumably due to a very low K(m) for oxygen; cells metabolize acetate (maximum specific growth rate, micro(max,A) of 0.28 h(-1)) as a single carbon source slower than they metabolize lactate (micro(max,L) of 0.47 h(-1)); and growth on acetate is self-inhibiting at a concentration greater than 10 mM. After estimating model parameters to describe growth and metabolism under six different nutrient conditions, the model was able to successfully estimate growth, oxygen and lactate consumption, and acetate production and consumption under entirely different growth conditions.

Anaerobic central metabolic pathways in Shewanella oneidensis MR-1 reinterpreted in the light of isotopic metabolite labeling.

It has been proposed that during growth under anaerobic or oxygen-limited conditions, Shewanella oneidensis MR-1 uses the serine-isocitrate lyase pathway common to many methylotrophic anaerobes, in which formaldehyde produced from pyruvate is condensed with glycine to form serine. The serine is then transformed through hydroxypyruvate and glycerate to enter central metabolism at phosphoglycerate. To examine its use of the serine-isocitrate lyase pathway under anaerobic conditions, we grew S. oneidensis MR-1 on [1-13C]lactate as the sole carbon source, with either trimethylamine N-oxide (TMAO) or fumarate as an electron acceptor. Analysis of cellular metabolites indicated that a large percentage (>70%) of lactate was partially oxidized to either acetate or pyruvate. The 13C isotope distributions in amino acids and other key metabolites indicate that under anaerobic conditions, although glyoxylate synthesized from the isocitrate lyase reaction can be converted to glycine, a complete serine-isocitrate pathway is not present and serine/glycine is, in fact, oxidized via a highly reversible degradation pathway. The labeling data also suggest significant activity in the anapleurotic (malic enzyme and phosphoenolpyruvate carboxylase) reactions. Although the tricarboxylic acid (TCA) cycle is often observed to be incomplete in many other anaerobes (absence of 2-oxoglutarate dehydrogenase activity), isotopic labeling supports the existence of a complete TCA cycle in S. oneidensis MR-1 under certain anaerobic conditions, e.g., TMAO-reducing conditions.

Estradiol stimulates the biosynthetic pathways of breast cancer cells: detection by metabolic flux analysis.

Selective estrogen receptor (ER) modulators are highly successful breast cancer therapies, but they are not effective in patients with ER negative and selective estrogen receptor modulator (SERM)-resistant tumors. Understanding the mechanisms of estrogen-stimulated proliferation may provide a route to design estrogen-independent therapies that would be effective in these patients. In this study, metabolic flux analysis was used to determine the intracellular fluxes that are significantly affected by estradiol stimulation in MCF-7 breast cancer cells. Intracellular fluxes were calculated from nuclear magnetic resonance (NMR)-generated isotope enrichment data and extracellular metabolite fluxes, using a specific flux analysis algorithm. The metabolic pathway model used by the algorithm includes glycolysis, the tricarboxylic acid cycle (TCA cycle), the pentose phosphate pathway, glutamine catabolism, pyruvate carboxylase, and malic enzyme. The pathway model also incorporates mitochondrial compartmentalization and reversible trans-mitochondrial membrane reactions to more accurately describe the role of mitochondria in cancer cell proliferation. Flux results indicate that estradiol significantly increases carbon flow through the pentose phosphate pathway and increases glutamine consumption. In addition, intra-mitochondrial malic enzyme was found to be inactive and the malate-aspartate shuttle (MAS) was only minimally active. The inactivity of these enzymes indicates that glutamine is not oxidized within mitochondria, but is consumed primarily to provide biosynthetic precursors. The excretion of glutamine carbons from the mitochondria has the secondary effect of limiting nicotinamide adenine dinucleotide (NADH) recycle, resulting in NADH buildup in the cytosol and the excretion of lactate. The observed dependence of breast cancer cells on pentose phosphate pathway activity and glutamine consumption for estradiol-stimulated biosynthesis suggests that these pathways may be targets for estrogen-independent breast cancer therapies.