Additional file 1 of Metabolic control analysis enables rational improvement of E. coli l-tryptophan producers but methylglyoxal formation limits glycerol-based production
Creators: name: Schoppel, Kristin
name: Trachtmann, Natalia
name: Korzin, Emil J.
name: Tzanavari, Angelina
name: Sprenger, Georg A.
name: Weuster-Botz, Dirk
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Space Science
subject: Microbiology
subject: FOS: Biological sciences
subject: Genetics
subject: Pharmacology
subject: Biotechnology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: Inorganic Chemistry

description: Additional file 1: Figure S1. Schematic representation of central carbon metabolism and L-tryptophan biosynthesis pathway. Depicted are the metabolites glucose-6-phosphate (G6P), fructose 1,6-bisphosphate (FBP), glyceraldehyde 3-phosphate (GAP), 3-phospho-D-glycerate (3PG), phosphoenolpyruvate (PEP), pyruvate (PYR), 3-phosphohydroxypyruvate (3PHP), O-phospho-L-serine (L-PSer) and L-serine (L-ser) from glycolysis and glycerol metabolism (GLYC &amp; GLYK), 6-phosphoglucono-1,5-lactone (6PG), ribulose 5-phosphate (Ru5P), xylulose 5-phosphate (X5P), ribose 5-phosphate (R5P), sedoheptulose 7-phosphate (S7P), erythrose 4-phosphate (E4P), fructose 6-phosphate (F6P) and 5-phospho-alpha-D-ribose 1-diphosphate (PRPP) from the pentose-phosphate-pathway (PPP), Acetyl-Coa (AcCoA) from the citric acid cycle (TCA), chorismate (CHOR), 3-dehydroquinate (3DHQ), 3-dehydroshikimate (3DHS), shikimate (SHIK) and chorismate (CHOR) from the chorismate biosynthesis pathway (CHOR) as well as anthranilate (ANTH), N-(5-phospho-D-ribosyl)anthranilate (PRAN), 1-(2-Carboxyphenylamino)-1-deoxy-D-ribulose 5-phosphate (CDRP), 3-Indolyl-glycerol 3-phosphate (IGP), indole (IND) and L-tryptophan (L-trp) from the L-tryptophan biosynthesis pathway (L-trp). Relevant genes are denoted in a white font with a coloured background. Genes, which were introduced into the new L-trpytophan producer strains (based on the initial producer strain E. coli NT1259) in additional genomic copies are highlighted in colors: serB in orange, aroB in green, trpC, coding for a monofunctional version of indole-glycerolphosphate synthase in yellow and trpB as well as trpA in blue. The modifications resulted in the new producer strains NT1259 trpCmt(NT1405), NT1259 trpBA (NT1438), NT1259 trpBA trpCmt (NT1439), NT1259 trpBA trpCmt aroB (NT1445), NT1259 trpBA trpCmt serB (NT1444), NT1259 trpBA trpCmt aroB serB (NT1446), which were used alongside the reference strain NT1259 in this study. Figure S2. Total amounts of L-tryptophan (L-trp) and cell dry weight (CDW) during fed-batch production of L-tryptophan with E. coli NT1259 pF112aroFBLKan on a 15 L scale (37 °C, pH 7.0, DO &gt; 30% air saturation) of three different processes (run 1-3). Vertical solid black lines indicate (i) the end of the batch phase (~10.4 h) and (ii) the beginning of the constant feeding phase/addition of IPTG (~45.1 h). The broken red line marks the process time for cell sampling (~48.7 h). Figure S3. Total amounts of L-tryptophan (L-trp) and cell dry weight (CDW) during fed-batch production of L-tryptophan with E. coli NT1405 pF112aroFBLKan on a 15 L scale (37 °C, pH 7.0, DO &gt; 30% air saturation). Vertical solid black lines indicate (i) the end of the batch phase (9.9 h) and (ii) the beginning of the constant feeding phase/addition of IPTG (45.1 h). The broken red line marks the process time for cell sampling (49.5 h). Figure S4. Total amounts of L-tryptophan (L-trp) and cell dry weight (CDW) during fed-batch production of L-tryptophan with E. coli NT1438 pF112aroFBLKan on a 15 L scale (37 °C, pH 7.0, DO &gt; 30% air saturation). Vertical solid black lines indicate (i) the end of the batch phase (10.4 h) and (ii) the beginning of the constant feeding phase/addition of IPTG (45.1 h). The broken red line marks the process time for cell sampling (48.8 h). Figure S5. Total amounts of L-tryptophan (L-trp) and cell dry weight (CDW) during fed-batch production of L-tryptophan with E. coli NT1439 pF112aroFBLKan on a 15 L scale (37 °C, pH 7.0, DO &gt; 30% air saturation). Vertical solid black lines indicate (i) the end of the batch phase (9.0 h) and (ii) the beginning of the constant feeding phase/addition of IPTG (44.7 h). The broken red line marks the process time for cell sampling (48.2 h). Figure S6. Total amounts of L-tryptophan (L-trp) and cell dry weight (CDW) during fed-batch production of L-tryptophan with E. coli NT1445 pF112aroFBLKan on a 15 L scale (37 °C, pH 7.0, DO &gt; 30% air saturation). Vertical solid black lines indicate (i) the end of the batch phase (10.6 h) and (ii) the beginning of the constant feeding phase/addition of IPTG (45.7 h). The broken red line marks the process time for cell sampling (50.0 h). Figure S7. Total amounts of L-tryptophan (L-trp) and cell dry weight (CDW) during fed-batch production of L-tryptophan with E. coli NT1444 pF112aroFBLKan on a 15 L scale (37 °C, pH 7.0, DO &gt; 30% air saturation). Vertical solid black lines indicate (i) the end of the batch phase (18.8 h) and (ii) the beginning of the constant feeding phase/addition of IPTG (49.7 h). The broken red line marks the process time for cell sampling (52.8 h). Figure S8. Total amounts of L-tryptophan (L-trp) and cell dry weight (CDW) during fed-batch production of L-tryptophan with E. coli NT1446 pF112aroFBLKan on a 15 L scale (37 °C, pH 7.0, DO &gt; 30% air saturation) of three different processes (run 1-3). Vertical solid black lines indicate (i) the end of the batch phase (~10.1 h) and (ii) the beginning of the constant feeding phase/addition of IPTG (~45.6 h). The broken red line marks the process time for cell sampling (~48.3 h). Figure S9. Fed-batch production of L-tryptophan with E. coli NT1446 pF112aroFBLKan on a 15 L scale (37 °C, pH 7.0, DO &gt; 30% air saturation). A: Concentrations (unit: g L−1) of glycerol and ammonium. B: Concentrations (unit: g L−1) of L-tyrosine, L-phenylalanine, D-lactate, malate and pyruvate. Vertical solid black lines indicate (i) the end of the batch phase (9.83 h) and (ii) the beginning of the constant feeding phase/addition of IPTG (43.9 h). The broken red line marks the process time for cell sampling for the parallel metabolic perturbation studies (47.9 h). Figure S10. Heat map illustrating flux distributions (unit: mmol gCDW−1 h−1) in glycolysis and glycerol metabolism (GLYC &amp; GLYK), methylglyoxal pathway (MGO), TCA cycle (TCA), pentose-phosphate-pathway (PPP), L-serine biosynthesis (L-ser), chorismate biosynthesis (CHOR), L-phenylalanine and L-tyrosine biosynthesis (L-phe/ L-tyr) and L-tryptophan production (L-trp) derived by thermodynamics-based flux analysis (pyTFA), restricted by measured extracellular rates during the parallel short-term perturbation experiments in stirred-tank bioreactors for metabolic analysis of L-tryptophan producing E. coli cells. Fluxes are depicted for the reference L-tryptophan production process with glycerol as sole carbon source (Ref) and the analysis reactors with glycerol (Glyc), glucose (Gluc), pyruvate (Pyr) as well as succinate (Suc). Flux directions are defined as in the model iJO1366. Figure S11. Estimated Gibbs‘ energies of reactions (unit: kJ mol-1) for reactions of glycolysis and glycerol metabolism (GLYC &amp; GLYK), methylglyoxal pathway (MGO), TCA cycle (TCA), pentose-phosphate-pathway (PPP), L-serine biosynthesis (L-ser), chorismate biosynthesis (CHOR), L-phenylalanine and L-tyrosine biosynthesis (L-phe/ L-tyr) and L-tryptophan production (L-trp) of cells in the L-tryptophan fed-batch production process at the process time chosen for metabolic analysis, derived by thermodynamic flux analysis (pyTFA) in consideration of measured intracellular metabolite concentrations and Gibbs’ free energies of formation for intracellular pH of pH 7.5 and ionic strength of 0.15 M. Reaction energies are depicted for positive flux directions, estimated with thermodynamics-based flux analysis for the reference state. Figure S12. RT-qPCR analyzed relative gene expression (without unit) of mgsA gene relative to ftsZ gene of samples from the four analysis reactors with the supplied carbon sources glycerol (MA Glyc), glucose (MA Gluc), pyruvate (MA Pyr) and succinate (MA Suc). Figure S13. RT-qPCR analyzed relative gene expression (without unit) of the genes gloA, gloB, gloC, yeiG and dld relative to ftsZ gene of samples from the four analysis reactors with the supplied carbon sources glycerol (MA Glyc), glucose (MA Gluc), pyruvate (MA Pyr) and succinate (MA Suc). Figure S14. RT-qPCR analyzed relative gene expression (without unit) of the genes yaijL and yhbO relative to ftsZ gene of samples from the four analysis reactors with the supplied carbon sources glycerol (MA Glyc), glucose (MA Gluc), pyruvate (MA Pyr) and succinate (MA Suc).