Heat maps showing global relative growth phenotype and comparison between measured and predicted values. (A) Growth rates of the multiple knockouts compared with wild type are shown in a heat map. Each row indicates the first gene deletion. Columns group indicates the second gene deletion, as well as the time point (24 or 48 h) and medium. The results for second mutations in talAB are enlarged in the lower panel. (B) RGIe and RGIs score are shown side by side for comparison. Tiles shown in black indicate conditions under which RGI scores were not calculated. The numbers at the bottom indicate the medium, as shown in panel A. The conditions mentioned in the text are highlighted by red boxes.

Pathways surrounding transaldolase. (A) Metabolic map of wild type during growth on xylose. (B) Currently accepted metabolism of transaldolase-deficient mutant (Tal deletion) on xylose and pgi-Tal deletion on glucose. Only the metabolites within the red circle could be produced in Tal deletion on xylose. Reactions indicated by blue arrows are additionally possible in the pgi-Tal knockout on glucose. (C) A map indicating a possible bypass pathway through deoxyribonucleosides (reactions shown in blue). The directions of the reactions shown in red are fixed to the reverse of the wild-type direction when the bypass is the sole source of carbon flux from xylose. (D) Possible metabolism of the Tal-deletion strain on gluconate, using currently known reactions. The reactions shown in red are required if ED pathway and/or the bypass shown in panel C is the sole source of carbon flux. (E) Relative amounts (talAB/wild type) of metabolite (box) and mRNA (diamond) are shown on the metabolic map. (F) Flux distribution of the wild type and talAB mutant growing on xylose. The novel reactions and the transaldolase reaction are shown in red and blue, respectively. The relative flux of each reaction (with incorporation of xylose set to 100) is shown at the respective position (upper, wild type; lower, talAB). See Supplementary Table IV B for detailed results.

Central carbon metabolism pathways examined in this study. Genes used for the first deletion are shown in red. Genes for the second deletion are boxed and abbreviations used in the text for the second deletion are indicated in boldface red if different from the deleted gene name. The reactions deleted by second deletion are shown with red arrows. For pckA and ppc, direction of the reaction catalyzing is shown by small arrows. The utilized carbon sources (blue) and entry points into central carbon metabolism are shown. See Supplementary Table I for gene and product names and Supplementary Table III for the abbreviations of metabolites not defined in the text.

Experimental and FBA-simulated conditions that exhibited the synthetic slow-growth phenotype. Within the 2177 cases for which both RGIe and RGIs scores were obtained, the number of cases exhibiting the synthetic slow-growth phenotype in either of experiment, FBA, or both of experiment and FBA are shown. Each class was subdivided into categories and the number of cases in each category is also shown. Most of the categories were defined by the first deletion, and the first deletion is indicated. Other categories were marked by ^*. ^*a: Either of aceE, aceF, or lpdA in the first deletion; ^*b: one each in the case of pfkA-Tal-xylose, tpiA-Pyk-glucose, and icdA-pgi-acetate; ^*c: zwf-pgi and pgi-zwf on glucose; ^*d: cases in which genes of the first deletion encoded the major isozyme; ^*e: cases having Tal as second deletion and explained by the novel reactions (each case was shown in Table IIB); and ^*f: icdA-aceA on acetate and casamino acid, gnd-pgi-glucose, and rpe-pgi-xylose. Cases classified into each category are shown in Supplementary Table II.

Mass isotopomer distribution of F6P. (A) Mass isotopomer distributions of F6P obtained from the wild type (WT) or the talAB mutant (talAB). m+n indicated the x-axis indicates the number of ¹³C incorporated, and on the y-axis indicates the ratio of each mass isotopomer. An average and s.d. value of results from two independent analyses are shown. (B) Math isotopomer distribution of F6P produced from the optimum fluxes result of MFA. The MFA results using the experimentally obtained isotopomer distributions from talAB were used to generate isotopomer distribution of F6P. The isotopomer distributions using the ‘current' pathway (current) or the ‘novel' pathway (novel), as well as the experimentally obtained one (exp.), are shown.