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

Abstract

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.

Application of Acetate as a Substrate for the Production of Value-Added Chemicals in Escherichia coli

“…In nature, mevalonate can be synthesized via the mevalonate pathway in most eukaryotes and higher plants. A complete biosynthetic pathway for mevalonate using sugars as substrates was constructed in recombinant E. coli [ 42 , 43 ]. In 2018, Xu et al constructed a recombinant E. coli strain with acetate as the sole carbon source for the production of mevalonate [ 44 ].…”

Advances in the optimization of central carbon metabolism in metabolic engineering

“…Metabolic engineering employs genetic engineering techniques to increase the yield of target products by modifying metabolic pathways within cells. The most common methods in metabolic engineering include manipulation of promoter and copy number of target enzymes [ 11 , 12 ], transcription factor regulation [ 13 ], fusion protein construction [ 14 ], protein scaffold assembly [ 15 ], organelle compartmentalization [ 16 ], and dynamic regulatory engineering [ 17 ]. The integration of these approaches enables complex and sophisticated metabolic pathway optimization of chassis cells to develop desired optimization strategies such as increasing the metabolic flux of target product-related pathways [ 18 ], blocking or attenuating other target product-consuming pathways [ 19 ], increasing the catalytic rate of rate-limiting steps [ 20 ], and introducing heterologous metabolic pathways [ 21 ].…”