Metabolomics data from A universal metabolite repair enzyme removes a strong inhibitor of the TCA cycle

Metabolomics data was prepared as following. Freezer stocks of wt (BW25113) and ΔycgM E. coli were streaked on LB plates and 4 single colonies from each strain were grown for 24 h in M9 minimal medium (0.2% glucose). Cells were washed with 1x M9 salts and used to inoculate 5 mL culture tubes containing M9 medium with either 0.2% glucose (natural isotopic abundance) or 0.2% 13C6-glucose (fully labeled) to an initial OD600 = 0.05. Four replicate cultures of each genotype were grown in each media formulation by shaking at 37°C for ~4 h until cells reached mid-log growth phase (OD600 ~0.6; determined with a Thermo Scientific Genesys 30 spectrophotometer that can measure OD inside culture tubes). To minimize metabolic disturbances, a rapid harvesting protocol was used. Briefly, an equivalent of 1 mL at OD600 = 1.0 was quickly transferred to 1.5 mL polypropylene tubes, cells were pelleted in a microcentrifuge at full speed for 30 s, the supernatant was quickly aspirated and collection tubes were immediately snap frozen in liquid nitrogen. Samples were stored at -80°C prior to extraction. After collecting the mid-log samples, cultures were shaken for an additional 2 h until early stationary growth phase (OD600 ~1.2) and samples were harvested as above. Collection tubes were placed on dry ice, 0.2 mL of cold 90% methanol was added, and tubes were incubated at -80°C for 72 h. Samples were removed from the freezer, vortexed for 15 s, and incubated on ice for 3 h with vortexing every ~30 min. Afterwards samples were spun for 15 min in a microcentrifuge (16,000 g) at 4°C and supernatants were collected for analysis.
Metabolomic data were obtained using an ultra-performance liquid chromatography-electrospray ionization-hybrid quadrupole-orbitrap mass spectrometer (Ultimate® 3000 HPLC, Q Exactive™, Thermo Scientific) with an autosampler and with a sample vial block maintained at 4°C. Chromatographic separations were carried out on an SeQuant® ZIC®-cHILIC 3µm, 100Å, 100 x 2.1 mm column (Merck, Darmstadt, Germany) with column temperature 40°C, flow rate 0.40 mL/min, and a 2 μL injection volume. Mobile phases A: 0.1% formic acid in water and B: 0.1% formic acid in acetonitrile were delivered over a 23 min. gradient according to the following profile: initial 98% B, 2 min 98% B, 20 min 40% B, 22 min 98% B, 23 min 98% B. The MS conditions used were full scan (mass range 50-750 m/z, and 115-1000 m/z in separate analyses), resolution 70,000, desolvation temperature 350°C, spray voltage 3800 V, auxiliary gas flow rate 20, sheath gas flow rate 50, sweep gas flow rate 1, S-Lens RF level 50, and auxiliary gas heater temperature 300°C. Xcalibur™ software version 2.1 (Thermo Scientific) was used for data collection.
Tandem MS data were obtained using data dependent Top N acquisition (Full MS dd-MS/MS). Precursor ions (top 5 most abundant ions per scan) were sequentially fragmented in the HCD collision cell with normalized collision energies (NCE) of 10, 20, 30, 40, 50, and 60 for six independent injections of each sample. MS/MS scans were acquired with 17,500 resolution, target value 1.0 × 105, 50 ms maximum injection time, and isolation window of 4.0 m/z. Data files were converted from .RAW to .mzML and .mgf formats using the ProteoWizard tool MSConvertGUI. MZmine 2.53 was utilized for extracting exact-mass chromatographic data for isotope ratio calculations, for generating untargeted metabolite feature tables, and for tabulating peak heights from targeted exact masses for calculating relative signal intensity ratios.

Key for sample names:
1st position letter: A = wild-type, B = OAT1 KO (ΔycgM)
2nd position number: 2 = natural glucose, 3 = 13C-glucose
3rd position letter: M = mid-log phase, S = early stationary phase
4th position number: 1-4 = biological replicates (mid-log and early stationary samples are paired; i.e., A2M1 and A2S1 were collected from the same culture tube)

example: A2M1 = wild type E. coli grown with natural glucose and harvested at mid-log, replicate 1

本数据集的代谢组学（metabolomics）样本制备流程如下。野生型（wild-type，菌株BW25113）与ΔycgM大肠杆菌的冷冻保存菌种划线接种于LB平板，每株菌株挑取4个单菌落，于含0.2%葡萄糖的M9基础培养基中培养24小时。用1×M9盐溶液洗涤菌体后，接种至含5 mL M9培养基的培养管中，培养基分别添加0.2%天然同位素丰度葡萄糖或0.2%全标记13C6-葡萄糖，调整初始光密度（OD600）至0.05。每种基因型对应每种培养基配方均设置4个重复培养物，于37℃摇床培养约4小时，至菌体处于对数中期（mid-log phase，OD600约为0.6；使用赛默飞世尔科技（Thermo Scientific）Genesys 30分光光度计直接测量培养管内的光密度）。为最大限度减少代谢扰动，本研究采用快速收集流程：简要流程为，取OD600为1.0的菌液1 mL当量，快速转移至1.5 mL聚丙烯离心管中，以高速离心30秒沉淀菌体，快速吸除上清液后，立即将收集管置于液氮中快速冷冻。样本在萃取前均保存于-80℃环境中。收集对数中期样本后，继续摇床培养2小时，至菌体处于早期稳定生长期（OD600约为1.2），随后按照上述流程收集样本。将收集管置于干冰上，加入0.2 mL预冷的90%甲醇溶液，于-80℃孵育72小时。从冰柜中取出样本，涡旋振荡15秒，置于冰上孵育3小时，每约30分钟涡旋一次。随后将样本于4℃、16000×g条件下离心15分钟，收集上清液用于后续分析。

代谢组学数据采用超高效液相色谱-电喷雾电离-混合四极杆-轨道阱质谱仪（Ultimate® 3000高效液相色谱仪、Q Exactive™质谱仪，赛默飞世尔科技（Thermo Scientific））获取，配备自动进样器，样本瓶托盘维持于4℃。色谱分离采用SeQuant® ZIC®-cHILIC色谱柱（3 μm粒径、100 Å孔径、100 × 2.1 mm，默克（Merck），德国达姆施塔特），柱温设置为40℃，流速0.40 mL/min，进样体积2 μL。流动相A为含0.1%甲酸的水溶液，流动相B为含0.1%甲酸的乙腈溶液，采用23分钟梯度洗脱，洗脱程序如下：初始阶段为98% B，2分钟时维持98% B，20分钟时降至40% B，22分钟时回升至98% B，23分钟时维持98% B。质谱参数设置如下：全扫描模式（分别采用50-750 m/z和115-1000 m/z两个质量范围），分辨率70000，脱溶剂温度350℃，喷雾电压3800 V，辅助气流量20，鞘气流量50，吹扫气流量1，S-Lens射频电平50，辅助气加热温度300℃。采用Xcalibur™ 2.1版软件（赛默飞世尔科技（Thermo Scientific））进行数据采集。

串联质谱（tandem MS）数据采用数据依赖型Top N采集模式（全扫描-数据依赖型二级质谱（Full MS dd-MS/MS））获取。对每个样本进行6次独立进样，每轮扫描中丰度排名前5的前体离子将在高能碰撞解离（HCD，high-energy collision dissociation）碰撞池中依次碎裂，归一化碰撞能量（NCE，normalized collision energy）分别设置为10、20、30、40、50和60。二级质谱扫描分辨率设置为17500，目标离子数1.0×10^5，最大注入时间50 ms，隔离窗口为4.0 m/z。使用ProteoWizard工具MSConvertGUI将原始数据文件（.RAW格式）转换为.mzML和.mgf格式。采用MZmine 2.53软件提取精确质量色谱数据以计算同位素比值，生成非靶向代谢物特征表，并针对靶向精确质量的峰高进行制表，以计算相对信号强度比。

样本命名规则如下：
1. 第一位字母：A代表野生型（wild-type）大肠杆菌，B代表OAT1敲除型（ΔycgM）大肠杆菌
2. 第二位数字：2代表天然葡萄糖培养基，3代表13C-葡萄糖培养基
3. 第三位字母：M代表对数中期，S代表早期稳定生长期
4. 第四位数字：1-4代表生物学重复（对数中期与早期稳定期样本为配对样本，例如A2M1与A2S1取自同一培养管）

示例：A2M1代表采用天然葡萄糖培养基培养、于对数中期收集的野生型大肠杆菌生物学重复1