(390c) Tailoring Pathways for Balanced Expression by Short Mufflers

Production of biosynthesized compounds requires the construction of complex metabolic pathways and optimization of these pathways is usually sought to enable high-yield production. Interesting approaches have been demonstrated in the last decade to fine-tune the expression level of individual genes to achieve a balanced pathway expression. Examples include engineering of regulatory elements, such as promoters, terminators, ribosome binding sites and enhancers.

In this work, we develop a novel strategy to rapidly modulate gene expression in individual cassettes by using short library-generated muffler sequences. A study using GFP as a reporter showed that significant variation in gene expression was easily obtained when randomizing 30 bp sequences flanking the GFP expression cassette. This approach was subsequently applied to carotenoid biosynthetic pathway and cellobiose utilizing pathway, leading to identification of variants with balanced expression in a very effective manner. This success supports that coupling of library creation using expression mufflers to high-throughput screening methods is a useful approach in the construction of tailored pathways.