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.

聚羟基丁酸酯（polyhydroxybutyrate, PHB）作为发酵产物的合成过程，可实现菌体生长与产物生成的偶联。此外，降低供氧水平能够降低操作成本并提升产物得率。PHB的发酵合成依赖于偏好NADH的乙酰乙酰辅酶A还原酶的体内催化活性。验证该概念需完成两项核心工作：一是对偏好NADH的乙酰乙酰辅酶A还原酶进行表征，二是在天然不具备该合成能力的物种（例如大肠杆菌Escherichia coli）中实现PHB积累。

本数据集包含三个子文件夹：

1. 酶动力学数据（Enzyme Kinetics data）文件夹
(1) 酶稳定性测定实验（Selwyn试验）；
(2) 初始反应速率与乙酰乙酰辅酶A浓度的关系曲线，以及由候选累积假单胞菌（Candidatus Accumulibacter phosphatis, AARCAp）来源的乙酰乙酰辅酶A还原酶与另一嵌合酶（AARChimera）催化的反应进程曲线；该嵌合酶是对真养产碱菌（Cupriavidus necator）来源的乙酰乙酰辅酶A还原酶改造得到的，将其原有残基N37-S38-P39-R40-R41替换为AARCAp来源的E37-F38-D39-K40-P41残基；
(3) 用于使用DYNAFIT软件分析上述动力学数据的脚本文件，以及DYNAFIT分析得到的结果文件；
(4) 用于计算NADH与NADPH相对消耗量的MATLAB脚本；
(5) 用于计算不同NADH/NAD比例下乙酰乙酰辅酶A还原酶催化反应的代谢通量能力的MATLAB脚本。

2. 发酵实验（Fermentations）文件夹
(1) 包含工程化大肠杆菌菌株生物质组成信息的Excel文件：该菌株基因型为((F– λ– ilvG– rfb-50 rph-1 (DE3) ΔadhE ΔadhP ΔldhA Δpta ΔmhpF))，并导入了质粒pCOLA-phaCACnecatorphaBCAp-cscABK。其中，phaCA基因分别编码真养产碱菌（Cupriavidus necator）的聚羟基丁酸酯合酶与乙酰辅酶A乙酰转移酶；phaBCAp基因编码AARCAp酶；cscABK基因分别编码大肠杆菌W菌株来源的蔗糖水解酶、蔗糖-质子同向转运蛋白与果糖激酶；
(2) 包含大肠杆菌野生型计算机模拟代谢模型的MATLAB数据文件"ecolicore.mat"；
(3) 用于构建考虑质粒与蛋白表达负荷的工程菌株计算机模拟代谢模型的MATLAB脚本；
(4) 工程化大肠杆菌菌株的计算机模拟代谢模型；
(5) 用于计算该菌株连续培养过程中校正后比速率的MATLAB脚本；本研究针对两种不同氧限制水平的稳态进行了分析，以蔗糖作为唯一碳源，稀释率设置为D=0.1 h⁻¹；
(6) 包含该菌株在连续培养过程中实测的未校正与校正后比速率的Excel文件；
(7) 用于计算校正后速率与代谢通量分布的MATLAB脚本；
(8) 以Excel表格与代谢通量分布图形式呈现的代谢通量分布结果。

3. 质粒DNA序列图谱（Plasmids DNA sequence maps）文件夹
该文件夹包含本研究中使用的各类质粒的DNA序列图谱。