Metabolic engineering of E. coli for producing phloroglucinol from acetate
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BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Metabolic engineering of E. coli for producing phloroglucinol from acetate Shengzhu Yu 1 & Liwei Guo 1 & Luyao Zhao 1 & Zhenya Chen 1 & Yixin Huo 1,2 Received: 5 December 2019 / Revised: 22 March 2020 / Accepted: 26 March 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract 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. Keywords Phloroglucinol . PhlD . Acetate . CRISPRi . Optimization . Low-cost production
Introduction Phenolic compounds exist widely in various plants and have diverse physiological and pharmacological activities, such as antioxidant (Fang et al. 2009), antimicrobial (Lima et al. 2016), and antineoplastic activities (Alves et al. 2016). Phloroglucinol is a three-hydroxyl phenolic compound and possesses antivirus (Singh et al. 2010) and anti-inflammatory activities (Kim and Kim 2010). In addition, phloroglucinol is a crucial intermediate in the fields of pharmacy and organic synthesis. Many phloroglucinol derivatives have significant pharmacological activities and wide applications (Lee et al. 2003; Liliana et al. 2007;
* Zhenya Chen [email protected] * Yixin Huo [email protected] 1
Key Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, No. 5 South Zhongguancun Street, Beijing 100081, China
2
Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao 266101, China
Straessler 2010; Toghiani et al. 2008). Chemical synthesis is the main method for phloroglucinol production, and various compounds such as 2,4,6-trinitrotoluene (TNT) (Ahmad and Roberts 1995; Kastens and Kaplan 1950), phenylamine (Palmisano et al. 2010), 1,3,5-triisopropylbenzene (Ito et al
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