Abstract
BackgroundWith the development of engineering the microbial cell factories, biosensors have been used widely for regulation of cellular metabolism and high-throughput screening. However, most of the biosensors constructed in Saccharomyces cerevisiae are designed for transcriptional activation. Very few studies have dedicated to the development of genetic circuit for repressive regulation, which is also indispensable for the dynamic control of metabolism.ResultsIn this study, through transcriptional deactivation design, we developed transcription-factor-based biosensors to allow repressive regulation in response to ligand. Using a malonyl-CoA sensing system as an example, the biosensor was constructed and systematically engineered to optimize the dynamic range by comparing transcriptional activity of the activators, evaluating the positions and numbers of the operators in the promoter and comparing the effects of different promoters. A biosensor with 82% repression ratio was obtained. Based on this design principle, another two biosensors, which sense acyl-CoA or xylose and downregulate gene expression, were also successfully constructed.ConclusionsOur work systematically optimized the biosensors for repressive regulation in yeast for the first time. It provided useful framework to construct similar biosensors. Combining the widely reported biosensors for transcriptional activation with the biosensors developed here, it is now possible to construct biosensors with opposing transcriptional activities in yeast.
Highlights
With the development of engineering the microbial cell factories, biosensors have been used widely for regulation of cellular metabolism and high-throughput screening
Construction of the malonyl‐CoA repressive biosensor in S. cerevisiae and evaluation of the activation efficiency of different activation domain (AD) in biosensor In transcription factors (TFs)-based biosensor for transcriptional activation, TF binds to its operator in the promoter to inhibit transcription
Contrary to the biosensor for transcriptional activation which operator was inserted into the core promoter close to the TATA box, the operators of the TF were inserted into the upstream region of the promoter to design the biosensor for transcriptional repression
Summary
With the development of engineering the microbial cell factories, biosensors have been used widely for regulation of cellular metabolism and high-throughput screening. Very few studies have dedicated to the development of genetic circuit for repressive regulation, which is indispensable for the dynamic control of metabolism. Similar to biosensor for transcriptional activation, the genetic circuit for repressive regulation is indispensable to dynamically control a metabolic pathway or screen for high-producer cells. It was reported that two malonyl-CoA biosensors that had either activating or repressive transcriptional activities were constructed to dynamically regulate fatty acid biosynthesis synergistically in Escherichia coli [13]. To screen for high-producer cells, a biosensor to switch off the expression of toxic protein with the increase of the metabolites level was often required to generate a growth-based selection system [15]
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