Metabolic Engineering Mevalonate Pathway Mediated by RNA Scaffolds for Mevalonate and Isoprene Production in Escherichia coli .

Co-localizing biochemical processes is a great strategy when expressing the heterologous metabolic pathway for product biosynthesis. The RNA scaffold is a flexible and efficient synthetic compartmentalization method to co-localize the enzymes involved in the metabolic pathway by binding to the specific RNA, binding domains fused with the engineered enzymes. Herein, we designed two artificial RNA scaffold structures─0D RNA scaffolds and 2D RNA scaffolds─using the reported aptamers PP7 and BIV-Tat and the corresponding RNA-binding domains (RBDs). We verified the interaction of the RBD and RNA aptamer in vitro and in vivo. Then, we determined the efficiencies of these RNA scaffolds by co-localizing fluorescent proteins. We employed the RNA scaffolds combined with the enzyme fusion strategies to increase the metabolic flux involved in the enzymes of the mevalonate pathway for mevalonate and isoprene production. Compared with the no RNA scaffold strain, the mevalonate levels of the 0D RNA scaffolds and 2D RNA scaffolds increased by 84.1% (3.13 ± 0.03 g/L) and 76.5% (3.00 ± 0.09 g/L), respectively. We applied the 0D RNA scaffolds for increasing the isoprene production by localizing the enzymes involved in a heterologous multi-enzyme pathway. When applying the RNA scaffolds for co-localizing the enzymes mvaE and mvaS , the isoprene production reached to 609.3 ± 57.9 mg/L, increasing by 142% compared with the no RNA scaffold strain. Our results indicate that the RNA scaffold is a powerful tool for improving the efficiencies of the reaction process in the metabolic pathway.