Development and characterization of AND-gate dynamic controllers with a modular synthetic GAL1 core promoter in...

Expression of heterologous proteins in metabolic engineering endeavors can be detrimental to host cells due to increased usage of cellular resources. Dynamic controls, where protein expression can be triggered on-demand, are effective for the engineering and optimization of bio-catalysts towards robust cell growth and enhanced biochemical productivity. Here, we describe the development and characterization of AND-gate dynamic controllers in Saccharomyces cerevisiae which combine two dynamic control strategies, inducible promoters and sensing-regulation. These dynamic controllers were constructed based on synthetic hybrid promoters. Promoter enhancer sequences were fused to a synthetic GAL1 core promoter containing DNA binding sites for the binding of a repressor that reduced DNA affinity upon interaction with key intermediates in a biochemical pathway. As fatty acids are key intermediates for production of fatty alcohols, fatty acid esters, alkenes, and alkanes, which are advanced biofuels, we used the fatty acid responsive FadR repressor and its operator sequence to demonstrate the functionality of the dynamic controllers. We established that the synthetic GAL1 core promoter can be used as a modular promoter part for constructing synthetic hybrid promoters and conferring fatty acid inducibility. We further showed the performance of the AND-gate dynamic controllers, where two inputs (fatty acid and copper presence/phosphate starvation) were required to switch the AND-gate ON. This work provides a convenient platform for constructing AND-gate dynamic controllers, that is, promoters that combine inducible functionality with regulation of protein expression levels upon detection of key intermediates towards the engineering and optimization of bio-catalytic yeast cells.