Design and Construction of a Synthetic Riboregulator-Based Platform for Metabolic Shunting of Pathways in Lactococcus lactis

Abstract

Microbial cell factories are subjected to rewiring of basic metabolism to enhance the carbon flux towards the desired product pathway. Conventionally, this metabolic engineering approach often involves over-expression of pathway genes and knocking out genes in the competing pathways. However, these approaches result in severe metabolic burden and eventual poor performance of the cells. Particularly, where biomass formation and product synthesis depend on common precursors, permanent changes like knocking out genes will only result in poor titer. Hence, temporary decoupling of biomass formation and product synthesis is considered to be a potential alternative. In this study, we designed synthetic riboregulators, which are RNA-based genetic switches, to shunt metabolic pathways in Lactococcus lactis bacteria, a GRAS organism employed as a cell factory for many biocompounds. The riboregulators are then subjected to evaluation by tagging the cis-repressive sequence (crRNA) to mCherry, a fluorescent reporter and regulated by the constitutive promoter, [Formula: see text]. The trans-activating RNA (taRNA) that interacts with the crRNA is placed under the control of another inducible promoter, [Formula: see text]. First, we observed that when there is no induction of taRNA, there is negligible fluorescence of mCherry indicating the successful repression of translation by the cis-sequence as expected. This has been further verified by comparing this expression level with the expression level of [Formula: see text]-mCherry without the cis-sequence, using fluorometer. Results from this analysis suggest that there is [Formula: see text]% repression by the designed crRNA sequence. Next, we induced the cells with 2[Formula: see text]ng/mL of nisin in the mid-log phase. Upon induction, there is a maximum of three fold increase in the fluorescence levels when compared to the uninduced cells, suggesting that the trans-activation takes place inside the live cells. However, further studies are necessary to optimize the cis-trans ratio to achieve better dynamic range for expression modulation and time window for operating metabolic shunting of competing pathways successfully in L. lactis.