Brzycki Newton Cassandra, Young Eric M, Roberts Susan C
Department of Chemical Engineering, Worcester Polytechnic Institute, Worcester, MA, United States.
Front Bioeng Biotechnol. 2023 Oct 17;11:1272811. doi: 10.3389/fbioe.2023.1272811. eCollection 2023.
Plant cell culture biomanufacturing is rapidly becoming an effective strategy for production of high-value plant natural products, such as therapeutic proteins and small molecules, vaccine adjuvants, and nutraceuticals. Many of these plant natural products are synthesized from diverse molecular building blocks sourced from different metabolic pathways. Even so, engineering approaches for increasing plant natural product biosynthesis have typically focused on the core biosynthetic pathway rather than the supporting pathways. Here, we use both CRISPR-guided DNA methylation and chemical inhibitors to control flux through the phenylpropanoid pathway in , which contributes a phenylalanine derivative to the biosynthesis of paclitaxel (Taxol), a potent anticancer drug. To inhibit PAL, the first committed step in phenylpropanoid biosynthesis, we knocked down expression of PAL in plant cell cultures using a CRISPR-guided plant DNA methyltransferase (NtDRM). For chemical inhibition of downstream steps in the pathway, we treated plant cell cultures with piperonylic acid and caffeic acid, which inhibit the second and third committed steps in phenylpropanoid biosynthesis: cinnamate 4-hydroxylase (C4H) and 4-coumaroyl-CoA ligase (4CL), respectively. Knockdown of PAL through CRISPR-guided DNA methylation resulted in a profound 25-fold increase in paclitaxel accumulation. Further, through the synergistic action of both chemical inhibitors and precursor feeding of exogenous phenylalanine, we achieve a 3.5-fold increase in paclitaxel biosynthesis and a similar reduction in production of total flavonoids and phenolics. We also observed perturbations to both activity and expression of PAL, illustrating the complex transcriptional co-regulation of these first three pathway steps. These results highlight the importance of controlling the metabolic flux of supporting pathways in natural product biosynthesis and pioneers CRISPR-guided methylation as an effective method for metabolic engineering in plant cell cultures. Ultimately, this work demonstrates a powerful method for rewiring plant cell culture systems into next-generation chassis for production of societally valuable compounds.
植物细胞培养生物制造正迅速成为生产高价值植物天然产物的有效策略，这些天然产物包括治疗性蛋白质和小分子、疫苗佐剂以及营养保健品。许多此类植物天然产物是由源自不同代谢途径的多种分子构件合成的。即便如此，增加植物天然产物生物合成的工程方法通常集中在核心生物合成途径而非支持途径上。在此，我们使用CRISPR引导的DNA甲基化和化学抑制剂来控制通过苯丙烷途径的通量，该途径为强效抗癌药物紫杉醇（泰素）的生物合成贡献一种苯丙氨酸衍生物。为抑制苯丙烷生物合成的首个关键步骤苯丙氨酸解氨酶（PAL），我们使用CRISPR引导的植物DNA甲基转移酶（NtDRM）在植物细胞培养物中敲低PAL的表达。为对该途径的下游步骤进行化学抑制，我们用胡椒酸和咖啡酸处理植物细胞培养物，它们分别抑制苯丙烷生物合成的第二个和第三个关键步骤：肉桂酸4-羟化酶（C4H）和4-香豆酰辅酶A连接酶（4CL）。通过CRISPR引导的DNA甲基化敲低PAL导致紫杉醇积累显著增加25倍。此外，通过化学抑制剂和外源苯丙氨酸前体饲喂的协同作用，我们使紫杉醇生物合成增加了3.5倍，总黄酮和酚类的产量也有类似程度的降低。我们还观察到PAL的活性和表达均受到干扰，这说明了这前三个途径步骤复杂的转录共调控。这些结果突出了控制天然产物生物合成中支持途径代谢通量的重要性，并开创了CRISPR引导的甲基化作为植物细胞培养物中代谢工程的有效方法。最终，这项工作展示了一种将植物细胞培养系统重新设计成用于生产具有社会价值化合物的下一代底盘的强大方法。
