NADH-driven polyhydroxybutyrate accumulation in E. coli dataset 1

Published: 9 October 2020| Version 1 | DOI: 10.17632/5cyg64cbrt.1
Karel Olavarria


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 (i) the characterization of an NADH-preferring acetoacetyl-CoA reductase and (ii) PHB accumulation in a species naturally incapable of doing so, for example, Escherichia coli. This dataset contains three folders. First folder contains data from reactions catalyzed by two enzymes: an acetoacetyl-CoA reductase encoded by a gene isolated from a Candidatus Accumulibacter phosphatis-enriched culture (AARCAp) 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. Because the acetoacetyl-CoA reductase from C. necator prefers NADPH, the amino acid replacement enabled to verified the role of those residues in cofactor discrimination. The genes encoding for both enzymes were cloned in the vector pCOLA-duet-1 and were over-expressed in E. coli BL21(DE3) cells. The second folder contains data from a continuous culture of the strain E. coli ((F– λ– ilvG– rfb-50 rph-1 (DE3) ΔadhE ΔadhP ΔldhA Δpta ΔmhpF)) transformed with the plasmid pCOLA-phaCACnecatorphaBCAp-cscABK. phaCA encodes for the polyhydroxybutyrate synthase and the acetyl-coenzyme A acetyltransferase from C. necator respectively; phaB encodes for the enzyme AARCAp; cscABK encodes for the respective sucrose hydrolase, the sucrose:proton symporter and fructose kinase from E. coli W. Expression of phaCACnecatorphaBCAp was under the control of a T7 promoter. Expression of cscABK was under the control of a native bi-directional promoter between cscA and cscBK. Sucrose was employed as the sole carbon source. Expression of phaCACnecatorphaBCap was induced with IPTG 100 microM. No antibiotic was necessary for plasmid selection. The dilution rate was D=0.1 1/h. We deposited here a table with the metabolic fluxes distributions calculated for two steady-states characterized by different levels of oxygen limitation. The third folder contains the DNA sequence maps of different plasmids employed in this research.


Steps to reproduce

For the continuous cultures, a defined medium with sucrose (15 g/L) as the sole carbon source was prepared as follows (per liter): 5 g of (NH4)2SO4, 2 g KH2PO4, 0.5 g MgSO4*7H2O, 5 g NaCl, 2 g NH4Cl, 0.001 g of thiamine, 100 μM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and 2 mL of a trace elements solution. The composition of the trace elements solution was (per liter): 15 g Na2EDTA*2H2O, 4.5 g ZnSO4*7H2O, 1 g MnCl2*4H2O, 0.3 g CoCl2*6H2O, 0.3 g CuSO4*5H2O, 0.4 g Na2MoO4*2H2O, 5 g CaCl2*2H2O, 3 g FeSO4*7H2O, 1 g H3BO3, 0.1 g KI. All the components of the medium were mix to a final volume of 49.5 L. The medium was neutralized inside the reactor. The medium was sterilized at 25 °C by filtration using a nitrocellulose filter with a pore size of 0.22 μm (Millipore, Germany). Four milliliters of BC Antifoam 86/013 (Basildon Chemicals, United Kingdom) dissolved in 0.5 L distilled water were sterilized in an autoclave and added to the medium.


Molecular Biology, Enzyme Kinetics, Continuous Cell Culture