(288f) Substrate-Activated Expression of a Biosynthetic Pathway in Escherichia coli | AIChE

(288f) Substrate-Activated Expression of a Biosynthetic Pathway in Escherichia coli


Ni, C. - Presenter, Massachusetts Institute of Technology
Fox, K. J., Massachusetts Institute of Technology
Prather, K., Massachusetts Institute of Technology
Microbial production leverages endogenous and heterologous enzymes to produce value-added chemicals. Overexpression of the genes encoding pathway enzymes can impose a metabolic burden to the host. There are many approaches to alleviating this burden, including using chemical inducers, such as IPTG, to delay expression, expressing genes from stationary phase promoters, or using feedback controllers that activate expression in response to a pathway intermediate. We developed a substrate-activated, feed-forward expression control strategy in which the necessary substrate of the pathway doubles as the inducer of heterologous pathway gene expression. We demonstrated this strategy on a D-glyceric acid pathway that utilizes galacturonate as a feed substrate. A galacturonate-responsive transcription factor was used to construct a galacturonate responsive biosensor. We constructed variants of the biosensor and selected the best performer through fluorescence characterization. The selected biosensor variant was used to control the heterologous gene expression of the D-glyceric acid biosynthetic pathway. We confirmed that expression was induced in presence of the substrate through qRT-PCR. Production via substrate-induction with our expression control circuit was comparable to IPTG-controlled induction and significantly outperformed constitutive expression. Our work demonstrates that substrate-activated pathway expression is an attractive control strategy for microbial production.