摘要
Metabolic engineering and synthetic biology endeavors benefit from promoters that perform consistently(or robustly)with respect to cellular growth phase(exponential and stationary)and fermentation scale(microtiter plates,tubes,flasks,and bioreactors).However,nearly all endogenous promoters(especially in Saccharomyces cerevisiae)do not perform in this manner.In this work,a hybrid promoter engineering strategy is leveraged to create novel synthetic promoters with robustness across these conditions.Using a multi-dimensional RNA-seq dataset,promoters with specific phase dependencies were identified.Fragments enriched with functional transcription factors were identified using MEME suite.These motif-containing fragments could impart activity dependence in the opposing condition.Specifically,we obtain two new promoters with high and consistent expression across both phases by increasing the exponential phase activity of the starting stationary-phase scaffold by 38 and 23-fold respectively.Further,we show that these promoters function consistently across various laboratory growth scales over time in a microtiter plate and in flasks.Overall,this work presents and validates a new strategy for engineering promoters in S.cerevisiae with high levels of expression that are robust to cellular growth phase and the scale of the culture.
基金
We acknowledge support from the Air Force Office of Scientific Research under Award No.FA9550-14-1-0089.Sequencing was conducted at the Genomic Sequencing and Analysis Facility(RRID no.SCR_021713)
flow cytometry as conducted at the Microscopy and Imaging Facility(RRID no.SCR_021756)at the UT Austin Center for Biomedical Research Support.
