State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, Jiangsu, China.
Microb Cell Fact. 2018 Sep 18;17(1):147. doi: 10.1186/s12934-018-0995-7.
Microbial biosynthesis of natural products holds promise for preclinical studies and treating diseases. For instance, pinocembrin is a natural flavonoid with important pharmacologic characteristics and is widely used in preclinical studies. However, high yield of natural products production is often limited by the intracellular cofactor level, including adenosine triphosphate (ATP). To address this challenge, tailored modification of ATP concentration in Escherichia coli was applied in efficient pinocembrin production.
In the present study, a clustered regularly interspaced short palindromic repeats (CRISPR) interference system was performed for screening several ATP-related candidate genes, where metK and proB showed its potential to improve ATP level and increased pinocembrin production. Subsequently, the repression efficiency of metK and proB were optimized to achieve the appropriate levels of ATP and enhancing the pinocembrin production, which allowed the pinocembrin titer increased to 102.02 mg/L. Coupled with the malonyl-CoA engineering and optimization of culture and induction condition, a final pinocembrin titer of 165.31 mg/L was achieved, which is 10.2-fold higher than control strains.
Our results introduce a strategy to approach the efficient biosynthesis of pinocembrin via ATP level strengthen using CRISPR interference. Furthermore coupled with the malonyl-CoA engineering and induction condition have been optimized for pinocembrin production. The results and engineering strategies demonstrated here would hold promise for the ATP level improvement of other flavonoids by CRISPRi system, thereby facilitating other flavonoids production.
微生物天然产物的生物合成有望用于临床前研究和治疗疾病。例如，乔松素是一种具有重要药理特性的天然类黄酮，广泛应用于临床前研究。然而，天然产物的高产量生产通常受到细胞内辅因子水平的限制，包括三磷酸腺苷 (ATP)。为了解决这一挑战，对大肠杆菌中 ATP 浓度进行了定制修饰，以实现乔松素的高效生产。
在本研究中，采用成簇规律间隔短回文重复 (CRISPR) 干扰系统筛选了几个与 ATP 相关的候选基因，其中 metK 和 proB 显示出提高 ATP 水平和增加乔松素产量的潜力。随后，优化了 metK 和 proB 的抑制效率，以达到适当的 ATP 水平，并增强乔松素的生产，使乔松素的产量增加到 102.02 mg/L。结合丙二酰辅酶 A 工程和培养及诱导条件的优化，最终乔松素的产量达到 165.31 mg/L，比对照菌株高 10.2 倍。
我们的研究结果介绍了一种通过 CRISPR 干扰强化 ATP 水平来提高乔松素高效生物合成的策略。此外，还优化了丙二酰辅酶 A 工程和诱导条件，以提高乔松素的产量。这里展示的结果和工程策略有望通过 CRISPRi 系统提高其他类黄酮的 ATP 水平，从而促进其他类黄酮的生产。
