Crispri-Guided Balancing of a Biosynthetic Pathway Increases Terpenoids Production

Simple and efficient regulation of gene expression is essential to the study of synthetic gene networks and to the biotechnological applications. In particular, well-modulated transcription of metabolic pathway genes plays a key role in maximizing product yield and minimizing metabolic burden caused by heterologous expression of multiple genes in operon context. CRISPR interference (CRISPRi) has shown tremendous promise as an efficient tool for transcriptional regulation, but there remain only a handful of applications described to date. Here, we report the systematic regulation of a biosynthetic mevalonate pathway that has been used for microbial production of the most numerous and diverse natural products, terpenoids. A tunable CRISPRi system was created for fine-tuning a metabolic pathway and implemented to efficiently channel carbon flux toward terpenoids synthesis in engineered Escherichia coli harboring exogenous mevalonate pathway and plant-derived terpene synthases through balancing the expression of multiple genes, resulting in enhanced production of (-)-α-bisabolol (C15), and lycopene (C40) along with alleviating the inhibition of cell growth. Coupling the CRISPRi to cell growth through regulation of endogenous essential gene further increased the production of isoprene (C5). Overall, CRISPRi is a platform for systematic modulation of synthetic gene expression to balance the synthetic metabolic pathways and subsequently for developing microbes as a smart cell factory to produce other industrially valuable terpenoids.