High succinic acid tolerance E.coli (DST160)

To understand cell responses against high succinate stress, a continuous culture was performed for 9 months using a wild Escherichia coli under gradually increasing succinate stress. The final adapted strain against high succinate stress, named DST160, was able to grow with no lag phase in medium containing 1.35 M of succinate stress while wild W3110 showed more than 38 h lag phase. The maximum growth rate and growth yield of DST160 under the stress condition were 0.20 h-1 and 0.192 g-cell/g-glucose, which were 53.8 % and 12.3 % higher than those of W3110 (0.13 h-1 and 0.171 g-cell/g-glucose, respectively). A single colony of E. coli W3110 was inoculated into a 15-mL tube containing 4 mL of LB medium. After 12 h cultivation in a shaking incubator at 37℃ and 220 rpm, pre-culture (1 mL) was transferred to a 250 mL-fermentor (Mini-chemostat fermentor; Biotron Inc., Bucheon, Korea) containing 100 mL medium consisted of M9-glucose minimal medium (3 g glucose , 0.8 g NH4Cl, 0.5 g NaCl, 7.5 g Na2HPO4•2H2O, 3 g KH2PO4 with separately added trace elements of 0.2 g MgSO4•7H2O, 0.1 g CaCl2, 1 mg thiamine) per liter supplemented with 254 mmole of succinate (30 g succinic acid) per liter. The initial pH of the medium was set to be pH 7.5 by adding 6N NaOH. The fermentor was operated at 37℃, 350 rpm with air flushing (100 mL/min.). After 12 h batch mode growth, feeding and outlet pumps were turned on at flow rate of 0.167 mL/min (Dilution rate=0.1 h-1). The fresh medium reservoir (10 L) contained the same contents and pH with the initial medium. Every 10 turnover time (100 h), the reservoir was changed with the same medium composition except succinate concentration. The succinate concentration of the reservoir was gradually increased by 25.4 ~ 84.7 mM (3 ~ 10 g/L of succinic acid) at a time than previous states depending on the cell concentrations at the outlet. When the cell concentration was reduced below O.D.=0.2, succinate feeding pump was temporarily stopped to go back to the slightly lower succinate concentration. The continuous culture was performed until the steady state when succinate in the reservoir reached 1.35 M (160 g/L in succinic acid form). The final adapted cells in 100 mL culture were harvested by centrifuge and frozen rapidly in a liquid nitrogen tank for further analyses including DNA microarray. To understand the change of up/down regulations in the high-succinate adapted E. coli, total mRNAs of control cells and the adapted E. coli cells were extracted using a RNA extraction kit (RNeasy mini kit; Quiagen, Hilden, Germany), and the transcriptomes were compared using DNA microarray. Hybridization and washing were accomplished by a facility of Digital Genomics Co (Seoul, Korea). DNA microarray were scanned using an Axon GenePix 4000B Scanner (Axon Instruments, Union City, CA, USA), and the scanned images were analyzed with GenePix Pro 3.0 software to obtain gene expression ratios.

为解析大肠杆菌（Escherichia coli）对高琥珀酸胁迫的细胞应答机制，本研究采用野生型大肠杆菌W3110，在逐步升高的琥珀酸胁迫条件下开展了为期9个月的连续培养实验。最终获得的高琥珀酸胁迫适应性菌株命名为DST160，该菌株可在含1.35 M琥珀酸的培养基中无延滞期生长；而野生型菌株W3110的延滞期则超过38小时。在该胁迫条件下，DST160的最大比生长速率为0.20 h⁻¹，细胞生长得率为0.192 g-细胞/g-葡萄糖，分别较W3110（0.13 h⁻¹、0.171 g-细胞/g-葡萄糖）提升53.8%与12.3%。

实验初始操作如下：挑取单菌落大肠杆菌W3110接种至含4 mL LB培养基的15 mL试管中，于37℃、220 rpm的摇床中培养12小时完成预培养；随后取1 mL预培养液转接至装有100 mL培养基的250 mL发酵罐（Mini-chemostat恒化器发酵罐，韩国Biotron公司，富川市），该培养基为M9葡萄糖基本培养基（每升含3 g葡萄糖、0.8 g氯化铵、0.5 g氯化钠、7.5 g磷酸氢二钠二水合物、3 g磷酸二氢钾，额外添加微量元素：每升含0.2 g七水合硫酸镁、0.1 g氯化钙、1 mg硫胺素），并补充254 mmol琥珀酸（即30 g琥珀酸）每升。培养基初始pH通过6 mol/L氢氧化钠溶液调节至7.5。发酵罐运行参数设置为：温度37℃、搅拌转速350 rpm、空气通气量100 mL/min。

待菌体完成12小时分批培养后，开启进料与出料泵，流速设为0.167 mL/min（稀释率=0.1 h⁻¹）。10 L容量的新鲜培养基储液罐内配制与初始发酵罐内相同组分及pH的培养基。每经过10个周转时间（即100小时），更换储液罐内的培养基，仅调整琥珀酸浓度。根据出料端的菌体浓度，每次将储液罐内琥珀酸浓度较上一阶段提升25.4~84.7 mM（即3~10 g/L琥珀酸）。当出料端菌体浓度降至OD值0.2以下时，暂时关闭琥珀酸进料泵，将琥珀酸浓度回落至略低水平。连续培养持续至储液罐内琥珀酸浓度达到1.35 M（即160 g/L琥珀酸形式）时达到稳态。将100 mL培养体系内的最终适应性菌体通过离心收集，随后置于液氮中快速冻存，用于后续包括DNA微阵列（DNA microarray）在内的各类分析。

为解析高琥珀酸适应性大肠杆菌的基因上调/下调调控变化，分别提取对照菌株与适应性菌株的总mRNA，采用RNA提取试剂盒（RNeasy mini试剂盒，德国Qiagen公司，希尔德市）完成提取，随后通过DNA微阵列技术比较二者的转录组（transcriptome）。杂交与洗涤步骤由韩国Digital Genomics公司（首尔市）的实验平台完成。使用Axon GenePix 4000B扫描仪（美国Axon Instruments公司，联合城，加利福尼亚州）扫描DNA微阵列芯片，通过GenePix Pro 3.0软件分析扫描图像，以获取基因表达比值数据。