RESUMO
Introduction: 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. Methods: Here, we use both CRISPR-guided DNA methylation and chemical inhibitors to control flux through the phenylpropanoid pathway in Taxus chinensis, 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 Taxus chinensis plant cell cultures using a CRISPR-guided plant DNA methyltransferase (NtDRM). For chemical inhibition of downstream steps in the pathway, we treated Taxus chinensis 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. Results: 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. Discussion: 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.
RESUMO
Centralized facilities for genetic engineering, or "biofoundries", offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technology, but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assessed via a timed "pressure test", in which 3 months were given to build organisms to produce 10 molecules unknown to us in advance. By applying a diversity of new approaches, we produced the desired molecule or a closely related one for six out of 10 targets during the performance period and made advances toward production of the others as well. Specifically, we increased the titers of 1-hexadecanol, pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne warhead underlying powerful antimicrobials, established a cell-free system for monoterpene production, produced an intermediate toward vincristine biosynthesis, and encoded 7802 individually retrievable pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran and barbamide were designed and constructed, but toxicity or analytical tools inhibited further progress. In sum, we constructed 1.2 Mb DNA, built 215 strains spanning five species ( Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces albovinaceus), established two cell-free systems, and performed 690 assays developed in-house for the molecules.