(191h) Fine-Tuning of the Flavonoid Biosynthesis Pathway By Promoter Strategies

Promoter is one of the most important and basic tools to achieve diverse of synthetic biology goals. During the fine-tuning of metabolic or regulation networks, the limited number of well-characterized inducible promoters have made implementing complicated strategies difficult. In this study, 104 native promoter-5′-UTR complexes (PUTR) from E. coli were screened and characterized based on a series of RNA-seq data. The strength of the 104 PUTRs varied from 0.007% to 4630% of that of the P_BAD promoter in transcriptional level and from 0.1% to 137% in translational level. In order to further upregulate gene expression, a series of combinatorial PUTRs and cascade PUTRs were constructed by integrating strong transcriptional promoters with strong translational 5′-UTRs. Finally, two combinatorial PUTRs (P_ssrA-UTR_rpsT and P_dnaKJ-UTR_rpsT) and two cascade PUTRs (PUTR_ssrA-PUTR_infC-rplT and PUTR_alsRBACE-PUTR_infC-rplT) were identified as having the highest activity. These engineered PUTRs are stable for the expression different genes, such as the red fluorescence protein gene and the β-galactosidase gene. These PUTRs could be used for the efficient static regulation of the flavonoids biosynthesis pathway combined with ePathBrick and high-throughput screening process. Besides, combination of the strong PUTRs and a P_fdeR-dependent dynamic regulation system, fine-tuning of the naringenin biosynthesis pathway was achieved. The static and dynamic regulatory elements of screening and transformation obtained here will provide technical and theoretical references for metabolic engineering of microorganisms for the constitutive expression of complicated metabolic pathways.