Chu Luan L, Dhakal Dipesh, Shin Hee J, Jung Hye J, Yamaguchi Tokutaro, Sohng Jae K
Department of Life Science and Biochemical Engineering, Sun Moon University, Asan, South Korea.
Department of BT Convergence Pharmaceutical Engineering, Sun Moon University, Asan, South Korea.
Front Microbiol. 2018 Jul 27;9:1671. doi: 10.3389/fmicb.2018.01671. eCollection 2018.
Flavonoids are one of the predominant groups of plant polyphenols, and these compounds have significant effects on human health and nutrition. Sulfated flavonoids have more favorable attributes compared to their parent compounds such as increased solubility, stability, and bioavailability. In this research, we developed a microbial system to produce sulfated naringenin using expressing a sulfotransferase (ST) from (At2g03770). This wild-type strain was used as a model system for testing clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) metabolic engineering strategies. Using synthetic sgRNA to mediate transcriptional repression of , a gene encoding 3'-phosphoadenosine-5'-phosphosulfate (PAPS) ST, which is involved in sulfur metabolism, resulted in an increase in intracellular PAPS accumulation by over 3.28-fold without impairing cell growth. Moreover, naringenin 7-sulfate production by engineering with its gene repressed in the open reading frame through CRISPRi was enhanced by 2.83-fold in compared with the wild-type control. To improve the efficiency of biotransformation, the concentration of , glucose, and substrate were optimized. The bioproductivity of naringenin 7-sulfate was 135.49 μM [∼143.1 mg (47.7 mg L)] in a 3-L fermenter at 36 h. These results demonstrated that the CRISPRi system was successfully applied for the first time in to develop an efficient microbial strain for production of a sulfated flavonoid. In addition, antibacterial and anticancer activities of naringenin 7-sulfate were investigated and found to be higher than the parent compound.
黄酮类化合物是植物多酚的主要类别之一，这些化合物对人类健康和营养具有重要影响。硫酸化黄酮类化合物与其母体化合物相比具有更有利的特性，如增加的溶解度、稳定性和生物利用度。在本研究中，我们开发了一种微生物系统，通过表达来自拟南芥（At2g03770）的磺基转移酶（ST）来生产硫酸化柚皮素。该野生型菌株被用作测试成簇规律间隔短回文重复序列（CRISPR）干扰（CRISPRi）代谢工程策略的模型系统。使用合成的sgRNA介导参与硫代谢的3'-磷酸腺苷-5'-磷酸硫酸（PAPS）ST编码基因的转录抑制，导致细胞内PAPS积累增加超过3.28倍，而不损害细胞生长。此外，通过CRISPRi在开放阅读框中抑制其基因对工程菌株进行改造后，柚皮素7-硫酸酯的产量与野生型对照相比提高了2.83倍。为了提高生物转化效率，对PAPS、葡萄糖和底物的浓度进行了优化。在3-L发酵罐中36小时时，柚皮素7-硫酸酯的生物生产力为135.49 μM [约143.1 mg（47.7 mg/L）]。这些结果表明，CRISPRi系统首次成功应用于酿酒酵母，以开发一种高效生产硫酸化黄酮类化合物的微生物菌株。此外，还研究了柚皮素7-硫酸酯的抗菌和抗癌活性，发现其活性高于母体化合物。
