(174bh) Orthogonal Ahl-Based Quorum Sensing Provides Wider Design Space for Optimal Regulation

Quorum sensing (QS) is a form of population-dependent, cell-to-cell communication found natively in bacteria that allows populations to regulate key functions as a collective. Engineers have employed this communication system to dynamically regulate gene expression in bioproduction. Amongst the wide range of QS systems, a large focus is on a class of well-studied systems that utilize signals containing an acyl homoserine lactone (AHL) group. These regulators have been effectively employed in a variety of applications. However, since these systems respond to ligands that share similar structures, they cannot be easily implemented in multi-layered regulation due to crosstalk. Interactions between the systems reduces the possible design space for optimal regulation as presence of the other signal can cause off-target activation. Several studies have searched for potential orthogonality between AHL systems and have identified the tra and rpa systems as the best candidates. Despite being identified in fluorescent co-culture studies, to our knowledge, they have not been utilized in any multi-layered regulation within a production strain. Here, these regulators were used to optimize the production of naringenin by using the tra system to activate expression of the naringenin pathway, while the rpa system controlled the downregulation of the competing pathways. This orthogonal pairing was compared to a strain previously optimized with the lux and esa systems, which respond to the same AHL signal and produced a naringenin titer of 463 μM in shake flasks. Even prior to optimization, the initial testing of the orthogonal pairing led to a >80% improvement in naringenin titer in a 48 well plate. This suggests that the orthogonality between multi-layered regulations provides a wider potential design space for optimization than single-input circuits, making the autoinducible QS systems of tra and rpa ideal candidates to various bioproduction strains.