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Design of a System of Biotransformations by means of stoichiometric network analysis Anne Kümmel, ETH Zürich © ETH Zürich | Taskforce Kommunikation Datum Introduction SBT-production mode SBT-operation mode cheap substrate Production of all required enzymes in a genetically engineered microorganism Production of the product molecule using the washed crude extract high-value product 25.08.05 SBT design 2 DHAP production path net reaction: ATP glucose + P 2.7.1.2 Glucokinase glk DHAP + lactate Glucose-6-phosphate 5.3.1.9 Phosphoglucose isomerase pgi NADH + H+ 1,3-Diphosphoglycerate 2.7.2.3 Phosphoglycerate kinase pgk ADP Fructose-6-Phosphate Fructose-1,6-bisphosphate 2.7.1.11 Phosphofructokinase pfkB Glyceraldehyde-3-phosphate 1.2.1.12 Glyceraldehyde-3-phosphate dehydrogenase gapA lower part: co-subtrate balancing 5.4.2.1 Phosphoglycerate mutase gpmA 2-Phosphoglycerate 4.2.1.11 Enolase eno Legend: NADH balancing Lactate ATP balancing NAD + SBT design upper part: DHAP synthesis Phosphoenolpyruvate ADP 2.7.1.40 Pyruvate kinase pyk 1.1.1.28 D-Lactate dehydrogenase ldhA SBT path 25.08.05 4.1.2.13 Fructosebisphosphate aldolase fba P + NAD+ ATP 3-Phosphoglycerate ADP ATP ADP ATP Pyruvate NADH + H + 3 Dilemma For SBT-operation, reactions that divert material into side products reduce the yield: Isolation of the production pathway is necessary! Elimination of side reactions can be achieved by gene deletions. For SBT-production, a viable organism is required: Side reactions may be essential for growth! Genes cannot be deleted as required since SBT path is part of the central carbon metabolism. Key question: To which extent can the production pathway be isolated from the rest of the metabolic network for the operation mode while maintaining viability in production mode? 25.08.05 SBT design 4 Identification of elimination targets Genome-scale metabolic reconstruction of E.coli as a compendium (iJR904): The 11 metabolites belonging to the SBT path participate in 90 other enzymatic reactions of E.coli. Of the 90 undesired reactions, 73 are probably not problematic since they rely on substrates or cofactors washed out of the SBT or proceed irreversibly in the “wrong“ direction. 17 side reactions potentially interfere with the SBT-operation Which of these reactions can be eliminated by gene deletion? 25.08.05 SBT design 5 Methodology Assumptions for systematic, model-driven mutant design: Gene essentiality is predicted by stoichiometric network analysis. Enzymes for reactions not active in the wild-type strain are not expressed, and gene deletion is thus not required. Mixtures of different carbohydrates are not tested in silico as they may be not consumed simultaneously in vivo. 1. Flux balance analysis (FBA) max (growth rate) s.t. metabolite balances available substrates Enzyme expression in wild type strain 2. Mixed-integer optimization max (number of gene knockouts) s.t. viability available substrates 25.08.05 Possible knockouts and rerouting of fluxes within the mutant strains SBT design 6 Calculation results Three different in silico media have been tested for production of a SBT path that is as far as possible isolated. The expression of the genes (blue) in the wild type among the corresponding 17 undesired reactions are shown in the table. The expressed genes have been classified to be either essential (red) or possible deletion targets (green). 25.08.05 M9 glucose gene KO possible? no gpsA no pgm no serA no tpiA no tktA, tktB yes zwf no ilvI not active fbp not active pps not active fsaA, fsaB not active mgsA gatY, gatZ, not active kbaY, kbaZ not active dxs not active dgoA not active mtlD not active manA not active srlD SBT design amino acids + M9 glucose amino acids + glycerol gene gene KO possible? KO possible? gpsA pgm serA tpiA tktA, tktB zwf ilvI fbp pps fsaA, fsaB mgsA gatY, gatZ, kbaY, kbaZ dxs dgoA mtlD manA srlD no no not active yes no not active not active not active not active not active not active not active not active not active not active not active not active gpsA pgm serA tpiA tktA, tktB zwf ilvI fbp pps fsaA, fsaB mgsA gatY, gatZ, kbaY, kbaZ dxs dgoA mtlD manA srlD yes no not active yes no not active not active no not active not active not active not active not active not active not active not active not active 7 Isolation procedure •Glucose: deletion of zwf gene and six remaining side reactions •Glucose plus amino acids: reduction of used enzymes •Glycerol plus amino acids: deletion of two genes but flux through an undesired enzyme as well Glucose ATP ADP ATP ADP Glucose-6-phosphate Fructose-6-Phosphate NADH + H + 1,3-Diphosphoglycerate Fructose-1,6-bisphosphate P + NAD + gapA Dihydroxyacetone phosphate Glyceraldehyde-3-phosphate ADP ATP 3-Phosphoglycerate Phosphoenolpyruvate 2-Phosphoglycerate ADP (a) tpiA mutant grown on glucose/amino acids or (b) tpiA/gpsA mutant grown on glycerol/amino acids. 25.08.05 Legend: SBT path Reactions eliminated by gene deletion Remaining side reactions which interfere with SBT operation Reactions for which the corresponding enzymes are not expressed SBT design ATP Pyruvate Lactate NAD + NADH + H 8 Conclusion and outlook One tkt gene is first target for proteomic switch. Thermodynamic analysis can be used to rank the proteomic switch targets. Experimental evaluation should determine the yield of DHAP for the SBTs produced by the two suggested strategies. The SBT path - although mainly consisting of the core metabolism can be isolated to large extent. Only three side reactions are left. 25.08.05 SBT design 9