Net flux (mmol/g DCW/hr) of reference state for various glycolysis vs pentose pathway split ratios.

<p>Net flux (mmol/g DCW/hr) of reference state for various glycolysis vs pentose pathway split ratios.</p

Algorithm for Ensemble Modeling.

<p>The algorithm is illustrated on the left, while the steps are described in detail on the right.</p

The metabolic network for the production of aromatic precursor 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP).

<p>Metabolites are denoted by capital letters, while enzyme abbreviations are in italics. The metabolite PEP feedback inhibits the enzyme phosphofructokinase (<i>pfk</i>). In the studied system, 2-dehydro-3-deoxyphosphoheptonate aldolase (<i>aroG</i>) had already been made feedback resistant, denoted by superscript “fbr”.</p

Summary of literature used for screening phenotypes.

<p>Summary of literature used for screening phenotypes.</p

Illustration of screening the ensemble of models using different literature phenotypes.

<p>Because the ensemble is being screened using the effect of different enzyme overexpressions on the same reference state, the final screened ensemble is independent of the path chosen, and can be represented by the intersection of the subsets screened by each phenotype. For demonstration, the results of screening for the reference flux with a glycolysis∶pentose phosphate pathway split ratio of 75∶25 are shown. The values indicate how many of the original ensemble of <i>n</i> = 1500 models match the given phenotype.</p

Dual overexpression kinetic phenomena for DAHP production.

<p>A) Illustration of the prediction for the dual overexpression of Tkt and Pps for the split ratio of 95∶5. 100% of the selected models exhibit the phenotype where when Tkt and Pps are simultaneously overexpressed, the combined effect is greater than the sum of the two individual overexpressions. B) The kinetic phenomenon in DAHP production illustrated. Pps overexpression does not increase DAHP production until Tkt is overexpressed. After removing this limitation, Pps overexpression has a dramatic effect on DAHP production, pushing yields near the theoretical limit of 86% mol DAHP/mol glucose.</p

Repeatability of Ensemble Modeling.

<p>Three repeats of 1500 models in each ensemble for a 95∶5 split ratio show very similar models retained after each screening step. Less than 10% variance in the number of models retained is observed at each screening step, with each ensemble selecting out 195, 212, and 207 models, respectively, after all screening.</p

The elementary reaction mechanisms.

<p>Mechanisms used for the variety of different metabolic reactions. Reactions modeled as 1 substrate to 1 product: HPr, EIIA, Pgi, Tpi, Gpm, Eno, Pgl, Rpe, Rpi, Pta, Ppc, Fum, Sdh, recycle reactions of ATP, NADH and NADPH. 2 substrate to 1 product reactions: EIIBC, AroG^fbr. 1 substrate to 2 products: EI, Fba, Pfl. 2 substrates to 2 products: Pfk, Gap, Pgk, Zwf, Gnd, Tkt, Tal, Pyk, Pps, Ack, Mdh.</p

Analysis of parameters in screened models relative to original ensemble.

<p>Parameter analysis for the glycolysis∶pentose phosphate pathway split ratio of 95∶5. A) The distribution of the enzyme fraction representing the free enzyme EI for the original ensemble (solid line) compared to the final screened ensemble of <i>n</i> = 195 models (dashed line). B) Hierarchical clustering by the sampled enzyme fractions indicates that the screened models (denoted by black bars) exist primarily in two distinct clusters. C) The distribution of enzyme fractions representing the free enzymes EI, Pyk & Ppc for each of the two clusters (dashed lines) relative to the original ensemble (solid line). Both clusters have distributions in EI that deviate significantly from the original ensemble. Only cluster 1 has a distribution for Pyk significantly different from the original ensemble, and only cluster 2 has a Ppc distribution that deviates from the original ensemble.</p

Abbreviations for metabolites and enzymes.

<p>Abbreviations for metabolites and enzymes.</p