NADH-driven polyhydroxybutyrate accumulation in E. coli dataset 2
Generation of polyhydroxybutyrate (PHB) as a fermentation product enables the coupling of growth and product generation. Moreover, the reduction of oxygen supply should reduce operative cost and increase product yield. Generation of PHB as a fermentation product depends on the in vivo activity of an NADH-preferring acetoacetyl-CoA reductase. Proof of this concept requires the characterization of an NADH-preferring acetoacetyl-CoA reductase and PHB accumulation in a species naturally incapable of doing so, for example, Escherichia coli. This dataset contains three folders. The Enzyme Kinetics data folder contains: (1) Enzyme stability assays (Selwyn tests). (2) Initial rates versus acetoacetyl-CoA concentration and progress curves from reactions catalyzed by the acetoacetyl-CoA reductase from Candidatus Accumulibacter phosphatis (AARCAp) and another enzyme (AARChimera), who is a modification of the acetoacetyl-CoA reductase from Cupriavidus necator where the original residues N37-S38-P39-R40-R41 were replaced by the residues E37-F38-D39-K40-P41 from AARCAp; (3) Scripts to analyze these kinetic data with the software DYNAFIT and files resulting from the analysis with DYNAFIT, (4) a MATLAB script to calculate the relative consumption of NADH and NADPH (5) a MATLAB script to calculate the metabolic flux capacity of the reaction catalyzed by an acetoacetyl-CoA reductase at different NADH/NAD ratios. The "Fermentations" folder contains: (1) An Excel file with the biomass composition of an engineered E. coli strain: ((F– λ– ilvG– rfb-50 rph-1 (DE3) ΔadhE ΔadhP ΔldhA Δpta ΔmhpF)) transformed with the plasmid pCOLA-phaCACnecatorphaBCAp-cscABK. phaCA genes encodes for the polyhydroxybutyrate synthase and the acetyl-coenzyme A acetyltransferase from C. necator respectively; phaBCAp gene encodes for the enzyme AARCAp; cscABK genes encodes for the sucrose hydrolase, the sucrose:proton symporter and fructose kinase from E. coli W, respectively. (2) The MATLAB file “ecolicore.mat” which contains an E. coli wild type in silico model. (3) A MATLAB script to generate an in silico model of the engineered strain, considering plasmid and protein burdens. (4) An in silico metabolic model of the engineered E. coli strain. (5) A MATLAB script to calculate the reconciled specific rates during the continuous growth of such strain. Two steady-states, characterized by different levels of oxygen limitation, were studied. Sucrose was employed as the sole carbon source and the dilution rate was D=0.1 1/h. (6) An Excel file with the observed unbalanced and reconciled specific rates of the engineered strain during the growth in the continuous culture. (7) A MATLAB script to calculate the reconciled rates and the metabolic fluxes distributions. (8) Metabolic fluxes distributions in an Excel table and over a Metabolic fluxes distribution map. The "Plasmids DNA sequence maps" folder contains the DNA sequence maps of different plasmids employed in this research.