Development of an optogenetic gene expression system inLactococcus lactisusing a split photoactivatable T7 RNA polymerase

Cellular processes can be modulated by physical means, such as light, which offers advantages over chemically inducible systems with respect to spatiotemporal control. Here we introduce an optogenetic gene expression system for Lactococcus lactis that utilizes a split T7 RNA polymerase linked to two variants of the Vivid regulators. Depending on the chosen photoreceptor variant, either ‘Magnets’ or ‘enhanced Magnets’, this system can achieve either high protein expression levels or low basal activity in the absence of light, exhibiting a fold induction close to 30, rapid expression kinetics, and heightened light sensitivity. This system functions effectively in liquid cultures and within cells embedded in hydrogel matrices, highlighting its potential in the development of novel engineered living materials capable of responding to physical stimuli such as light. The optogenetic component of this system is highly customizable, allowing for the adjustment of expression patterns through modifications to the promoters and/or engineered T7 RNA polymerase variants. We anticipate that this system can be broadly adapted to other Gram-positive hosts with minimal modifications required.