(102e) Investigating Non-Coding Sequences with Regulator-like Function on Pathway Expression Balance and Plasmid Stability Improvement

Production of biosynthesized compounds requires the construction of complex metabolic pathways. A delicate balance is essential to meet production needs without compromising cell fitness. Considering that the identification of bottlenecks and sequential manipulations are often time-consuming and labor intensive, we focused on developing a combinatorial approach to fine-tune pathway expression.

In this talk, we will first report a novel strategy based on the use of a library of short perturbation sequences to modulate expression of gene cassettes. The versatility of the strategy was confirmed by implementation on a reporter gene, the cellobiose utilization pathway, and the beta-carotene pathway. Results indicated that the use of this approach effectively balanced gene expression in a pathway as evidenced by significant increased growth rate and production of the target product.

Based on the success of using sequences as short as 30 bp to improve pathway balanced expression, we sought to apply this concept in a different setting. Yarrowia lipolytica is a non-conventional yeast host whose heterologous expression often relies on genome integration due to the instability of plasmid systems. We investigated the role that a native non-coding spacer residing between the origin of replication and centromere plays on plasmid stability. Results indicated that the effect of modifying a native spacer is minimal, while modifications of a nonnative spacer caused drastic improvement of plasmid stability, even surpassing the native threshold. Collectively, these studies show the importance of neighboring sequences and how they can impact the function of coding elements nearby, providing a new space for design for the synthetic biology community.