Yu Shengzhu, Guo Liwei, Zhao Luyao, Chen Zhenya, Huo Yixin
Key Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, No. 5 South Zhongguancun Street, Beijing, 100081, China.
Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao, 266101, China.
Appl Microbiol Biotechnol. 2020 Sep;104(18):7787-7799. doi: 10.1007/s00253-020-10591-2. Epub 2020 Jul 31.
Phloroglucinol is a three-hydroxyl phenolic compound and has diverse physiological and pharmacological activities such as antivirus and anti-inflammatory activities. Chemical synthesis of phloroglucinol suffered from many drawbacks such as high cost and environmental pollution. To avoid the above issues, microbial phloroglucinol biosynthesis was successfully accomplished in this study, while the abundant and low-cost acetate was used as the main carbon source. Firstly, the toxicity of phloroglucinol was tested, and E. coli BL21(DE3) could tolerate 5 g/L phloroglucinol. The ability of phloroglucinol synthase (PhlD) for catalyzing malonyl-CoA to phloroglucinol was confirmed, and E. coli BL21(DE3) expressing PhlD and acetyl-CoA carboxylase (ACCase) could produce 1107 ± 12 mg/L phloroglucinol from glucose. Then, E. coli BL21(DE3) was engineered to utilize acetate to produce 228 ± 15 mg/L phloroglucinol. Then, the endogenous citrate synthase (GltA) which could catalyze oxaloacetate and acetyl-CoA generated from acetate to citrate was knocked down by CRISPRi system in order to enhance the carbon flux for phloroglucinol production, and the titer was improved to 284 ± 8 mg/L. This work demonstrated that acetate could be used as low-cost substrate to achieve the biosynthesis of phloroglucinol and provided an example of effective utilization of acetate.
间苯三酚是一种三羟基酚类化合物，具有多种生理和药理活性，如抗病毒和抗炎活性。间苯三酚的化学合成存在许多缺点，如成本高和环境污染。为避免上述问题，本研究成功实现了间苯三酚的微生物合成，同时使用丰富且低成本的乙酸盐作为主要碳源。首先，测试了间苯三酚的毒性，大肠杆菌BL21(DE3)能够耐受5 g/L的间苯三酚。证实了间苯三酚合酶（PhlD）催化丙二酰辅酶A生成间苯三酚的能力，表达PhlD和乙酰辅酶A羧化酶（ACCase）的大肠杆菌BL21(DE3)能够从葡萄糖中产生产出1107±12 mg/L的间苯三酚。然后，对大肠杆菌BL21(DE3)进行工程改造，使其利用乙酸盐产生产出228±15 mg/L的间苯三酚。接着，为了增强间苯三酚生产的碳通量，通过CRISPRi系统敲低了能够催化乙酸盐产生的草酰乙酸和乙酰辅酶A生成柠檬酸的内源性柠檬酸合酶（GltA），产量提高到了284±8 mg/L。这项工作表明乙酸盐可以作为低成本底物实现间苯三酚的生物合成，并提供了有效利用乙酸盐的实例。
