Development and Characterization of Dynamic Controllers in Saccharomyces

Cerevisiae

Wei Suong Teo, Kai Sheng Hee, Matthew Wook Chang
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of
Singapore, 14 Medical Drive, 117599, Singapore
Synthetic Biology Research Consortium, National University of Singapore, 28
Medical Drive, 117456, Singapore
Expression of heterologous proteins in metabolic engineering endeavours 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. First, we developed and characterized 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 acid derived advanced biofuels, we used bacterial FadR repressor and operator 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 were required to switch the AND-gate ON. Second, we developed genetically encoded xylose sensor-regulators in yeast, where potential applications include dynamic regulation of xylose assimilating pathway enzymes and screening for efficient xylose transporters. Xylose sensor-regulators were created using bacterial XylR repressors and synthetic promoters with XylR operators. The operators are bound by XylR in the absence of xylose to repress transcription. Upon xylose detection, XylR DNA binding activity is antagonized, and the downstream yEGFP reporter expression increases. XylR was shown to repress gene expression from the synthetic promoters and exogenous xylose was able to regulate gene expression. We envision that these synthetic dynamic controllers will function as important tools for engineering and optimizing genetically engineered yeasts towards biochemical production.