NADH-driven polyhydroxybutyrate accumulation in E. coli dataset 1

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.聚羟基丁酸酯（polyhydroxybutyrate, PHB）作为发酵产物的生成，可实现菌体生长与产物合成的偶联。此外，降低供氧水平能够降低操作成本并提升产物得率。PHB作为发酵产物的生成，依赖于偏好NADH的乙酰乙酰辅酶A还原酶（acetoacetyl-CoA reductase）的体内活性。验证该概念需完成两项工作：(i) 对偏好NADH的乙酰乙酰辅酶A还原酶进行表征；(ii) 在天然无法合成PHB的物种（例如大肠杆菌Escherichia coli）中实现PHB积累。本数据集包含三个文件夹：第一个文件夹收录了两种酶催化反应的相关数据：一种是从富集Candidatus Accumulibacter phosphatis的培养物中分离得到的基因编码的乙酰乙酰辅酶A还原酶（AARCAp），另一种为嵌合酶AARChimera——该酶是对真养产碱杆菌（Cupriavidus necator）来源的乙酰乙酰辅酶A还原酶的改造体，将其原序列中的N37-S38-P39-R40-R41残基替换为AARCAp来源的E37-F38-D39-K40-P41残基。由于真养产碱杆菌来源的乙酰乙酰辅酶A还原酶偏好NADPH，上述氨基酸替换可用于验证这些残基在辅酶偏好性区分中的作用。两种酶的编码基因均克隆至载体pCOLA-duet-1中，并在大肠杆菌BL21(DE3)细胞中实现过表达。第二个文件夹收录了连续培养工程菌株的相关数据：该菌株为大肠杆菌((F– λ– ilvG– rfb-50 rph-1 (DE3) ΔadhE ΔadhP ΔldhA Δpta ΔmhpF))，转化有质粒pCOLA-phaCACnecatorphaBCAp-cscABK。其中，phaCA分别编码真养产碱杆菌的聚羟基丁酸酯合酶与乙酰辅酶A乙酰转移酶；phaB编码AARCAp酶；cscABK分别编码大肠杆菌W来源的蔗糖水解酶、蔗糖-质子同向转运蛋白与果糖激酶。phaCACnecatorphaBCAp的表达受T7启动子调控，cscABK的表达受cscA与cscBK之间的天然双向启动子调控。实验以蔗糖作为唯一碳源，通过100 μM异丙基-β-D-硫代半乳糖苷（IPTG）诱导phaCACnecatorphaBCAp的表达，质粒筛选无需添加抗生素。培养稀释速率为D=0.1 h⁻¹。本数据集附带一张表格，收录了两种不同氧限制水平下稳态的代谢通量分布计算结果。第三个文件夹收录了本研究中使用的不同质粒的DNA序列图谱。