Identification and Engineering of n-Butanol Biosensors By Transcriptome Analysis in Yeast
Metabolic Engineering Conference
2016
Metabolic Engineering 11
Poster Session
Rapid Fire Poster Session 2
Monday, June 27, 2016 - 4:30pm to 5:30pm
Increasing concerns about depleting crude reserves have renewed interests in the biological production of n-butanol, due to its desirable fuel properties and its role as a platform chemical. There are advantages to utilize Saccharomyces cerevisiae as an n-butanol producer, due to its high n-butanol tolerance, phage resistance, well-established genetic tools and compatibility to current industrial infrastructures. However, current production level of n-butanol in S. cerevisiae is far less promising.
In vivo metabolite biosensors would significantly accelerate the construction and optimization of yeast producer via dynamic control of cell metabolism and serve as important high-throughput screening tools for the selection of the individuals carrying the desired phenotype. Thus, we propose to develop a systematic approach for the identification and engineering of biosensors to detect and regulate n-butanol in S. cerevisiae using transcriptomics analysis. The genome-wide gene expression profiling of S. cerevisiae exposed to n-butanol, n-propanol, and ethanol allowed us to identify novel genes responsive to the treatment of a specific alcohol. The gene hit list from each alcohol was cross-referenced with each other to identify genes that were only differentially expressed for n-butanol. We identified 435 genes that were differentially expressed only under the treatment of n-butanol rather than ethanol and n-propanol. Many genes were hypothetical, associated with ncRNA processing, DNA replication, response to DNA damage, steroid metabolism, and tRNA modification. Forty-eight promoters were selected and can be divided into two groups. One group of 24 promoters were selected because their genes had showed highest differential expression and stringent P value; the other group of 24 promoters were selected because they had showed conserved sequences in their promoter areas. For each group, promoters were selected within the region from 1 to 1000 bp upstream of translation start, and fused with the gene of mCherry fluorescent protein. The resultant reporter cassettes were introduced into S. cerevisiae to evaluate promoter strength and specificity for n-butanol pressure. Three promoters were shown to significantly drive the expression of mCherry upon exposure to n-butanol in a dose-dependent manner, with n-butanol concentration ranging from 0 to 1% (v/v). All these three promoters were from the group contained conserved sequences, highlighting the importance of data mining and processing. Upon further testing by truncating promoter areas, we have found sequences that contributed to the n-butanol response, indicating the possibility of re-design and re-construction of more efficient n-butanol sensors.
Our work demonstrates a general method for the discovery of endogenous promoters as biosensors to monitor alcohols, particularly n-butanol. This method should be generally applicable and highly useful for the development of biosensors in both high-throughput screening of large diversified libraries and implementation of synthetic circuits for dynamic control of cell metabolism.