Dynamic control over feedback regulatory mechanisms improves NADPH fluxes and xylitol biosynthesis in engineeredE. coli

Abstract

We report improved NADPH flux and xylitol biosynthesis in engineered E. coli. Xylitol is produced from xylose via an NADPH dependent reductase. We utilize two-stage dynamic metabolic control to compare two approaches to optimize xylitol biosynthesis, a stoichiometric approach, wherein competitive fluxes are decreased, and a regulatory approach wherein the levels of key regulatory metabolites are reduced. The stoichiometric and regulatory approaches lead to a 16 fold and 100 fold improvement in xylitol production, respectively. Strains with reduced levels of enoyl-ACP reductase and glucose-6-phosphate dehydrogenase, led to altered metabolite pools resulting in the activation of the membrane bound transhydrogenase and a new NADPH generation pathway, namely pyruvate ferredoxin oxidoreductase coupled with NADPH dependent ferredoxin reductase, leading to increased NADPH fluxes, despite a reduction in NADPH pools. These strains produced titers of 200 g/L of xylitol from xylose at 86% of theoretical yield in instrumented bioreactors. We expect dynamic control over enoyl-ACP reductase and glucose-6-phosphate dehydrogenase to broadly enable improved NADPH dependent bioconversions.HighlightsDecreases in NADPH pools lead to increased NADPH fluxesPyruvate ferredoxin oxidoreductase coupled with NADPH-ferredoxin reductase improves NADPH production in vivo.Dynamic reduction in acyl-ACP/CoA pools alleviate inhibition of membrane bound transhydrogenase and improve NADPH fluxXylitol titers > 200g/L in fed batch fermentations with xylose as a sole feedstock.

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).…”

“…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). Firstly, we evaluated the impact of silencing both promoters driving gltA expression.…”

“…[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.…”

“…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.…”