Dynamic control over feedback regulation identifies pyruvate-ferredoxin oxidoreductase as a central metabolic enzyme in stationary phaseE. coli

Abstract

We demonstrate the use of two-stage dynamic metabolic control to manipulate feedback regulation in central metabolism and improve biosynthesis in engineered E. coli. Specifically, we report the impact of dynamic control over two central metabolic enzymes: citrate synthase, and glucose-6-phosphate dehydrogenase, on stationary phase fluxes. Firstly, reduced citrate synthase levels lead to a reduction in α-ketoglutarate, which is an inhibitor of sugar transport, resulting in increased glucose uptake and glycolytic fluxes. Reduced glucose-6-phosphate dehydrogenase activity activates the SoxRS regulon and expression of pyruvate-ferredoxin oxidoreductase, which is in turn responsible for large increases in acetyl-CoA production. These two mechanisms lead to the improved stationary phase production of citramalic acid enabling titers of 126±7g/L. These results identify pyruvate oxidation via the pyruvate-ferredoxin oxidoreductase as a “central” metabolic pathway in stationary phase and highlight the potential of improving fluxes by manipulating essential central regulatory mechanisms using two-stage dynamic metabolic control.HighlightsDynamic reduction in α-keto-glutarate pools alleviate inhibition of PTS dependent transport improving stationary phase sugar uptake.Dynamic reduction in glucose-6-phosphate dehydrogenase activates pyruvate flavodoxin/ferredoxin oxidoreductase and improves stationary acetyl-CoA flux.Pyruvate flavodoxin/ferredoxin oxidoreductase is responsible for large stationary phase acetyl-CoA fluxes under aerobic conditions.Production of citramalate to titers 126 ± 7g/L at > 90 % of theoretical yield

Two-stage Dynamic Deregulation of Metabolism Improves Process Robustness & Scalability in EngineeredE. coli

“…As mentioned above we have previously reported the identification of valves which lead to deregulation of glucose uptake (GltA), acetyl-CoA flux (Zwf), and NADPH synthesis through the membrane bound transhydrogenase (FabI). 21,22 Additionally, we have reported that proteolytic degradation of the soluble transhydrogenase (UdhA) can improve NADPH pools. 21 As depicted in Figure 2a, alanine production requires pyruvate and NADPH, so we designed strains with combinations of valves reducing levels of GltA ("G" Valves), FabI ("F" Valves), and UdhA ("G" Valves).…”

“…Generally the best performing strains had "F", "G" and "U" valves, which is consistent with the previously reported effects of these valves. 21,22 Aditional studies are required to better understand how the variations in multiple enzyme activities contribute to production.…”

“…[18][19][20][21][22][23][24][25][26][27] Two-stage dynamic control, which decouples growth from production, offers additional potential benefits in large scale bioprocesses, enabling metabolic states to be pushed beyond the boundaries required for growth. 8,21,22,24,28 We have recently implemented two-stage dynamic control in the commonly used and well characterized microbe, E. coli , by utilizing synthetic metabolic valves, which rely on the combination of proteolysis and gene silencing to dynamically reduce levels of key enzymes in the context of a standardized two-stage phosphate depleted process (as illustrated in Figure 1) 17,21,22,29 Proteolysis is accomplished by appending C-terminal degron (DAS+4) tags to a given gene and silencing via expression of the native E. coli CRISPR CASCADE system as well as silencing gRNAs (from pCASCADE plasmids). 30,31 Importantly, in these studies where we produced the organic acid citramalate 22 and the sugar alcohol xylitol, 21 we identified that dynamic deregulation of metabolism was a fundamental mechanism to improve stationary phase metabolic fluxes.…”

“…8,21,22,24,28 We have recently implemented two-stage dynamic control in the commonly used and well characterized microbe, E. coli , by utilizing synthetic metabolic valves, which rely on the combination of proteolysis and gene silencing to dynamically reduce levels of key enzymes in the context of a standardized two-stage phosphate depleted process (as illustrated in Figure 1) 17,21,22,29 Proteolysis is accomplished by appending C-terminal degron (DAS+4) tags to a given gene and silencing via expression of the native E. coli CRISPR CASCADE system as well as silencing gRNAs (from pCASCADE plasmids). 30,31 Importantly, in these studies where we produced the organic acid citramalate 22 and the sugar alcohol xylitol, 21 we identified that dynamic deregulation of metabolism was a fundamental mechanism to improve stationary phase metabolic fluxes. Specifically, i) reducing citrate synthase (GltA) levels reduced -ketoglutarate pools alleviating -ketoglutarate mediated inhibition of glucose uptake, 22 ii) reducing glucose-6-phosphate dehydrogenase (Zwf) levels reduces NADPH pools activating the SoxRS regulon and increasing expression of pyruvate ferredoxin oxidoreductase and acetyl-CoA flux, 22 and iii) decreasing enoyl-ACP reductase (FabI) activity decreases fatty acid metabolite pools alleviating inhibition of the membrane bound transhydrogenase.…”

“…30,31 Importantly, in these studies where we produced the organic acid citramalate 22 and the sugar alcohol xylitol, 21 we identified that dynamic deregulation of metabolism was a fundamental mechanism to improve stationary phase metabolic fluxes. Specifically, i) reducing citrate synthase (GltA) levels reduced -ketoglutarate pools alleviating -ketoglutarate mediated inhibition of glucose uptake, 22 ii) reducing glucose-6-phosphate dehydrogenase (Zwf) levels reduces NADPH pools activating the SoxRS regulon and increasing expression of pyruvate ferredoxin oxidoreductase and acetyl-CoA flux, 22 and iii) decreasing enoyl-ACP reductase (FabI) activity decreases fatty acid metabolite pools alleviating inhibition of the membrane bound transhydrogenase. 21 The deregulation of central metabolic pathways enabled relatively high titers in instrumented reactors, reaching 125g/L in the case of citramalate and 200g/L in the case of xylitol.…”