Substrate‐activated expression of a biosynthetic pathway in Escherichia coli
Name
10.1002-biot.202000433.pdf
Size
1.78 MB
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Adobe PDF
Checksum (MD5)
657069f83b11b19efb2f13380e6b7c76
Author(s)
Ni, Cynthia
Fox, Kevin J.
Prather, Kristala L. J.
Date Issued
May 29, 2021
Journal
Biotechnology Journal
Publisher
Wiley
Citation
Ni, C., Fox, K. J., & Prather, K. L. J. (2021). Substrate-activated expression of a biosynthetic pathway in Escherichia coli. Biotechnol. J, 00, e2000433
Version
Author's final manuscript
Abstract
Microbes can facilitate production of valuable chemicals more sustainably than traditional chemical processes in many cases: they utilize renewable feedstocks, require less energy intensive process conditions, and perform a variety of chemical reactions using endogenous or heterologous enzymes. In response to the metabolic burden imposed by production pathways, chemical inducers are frequently used to initiate gene expression after the cells have reached sufficient density. While chemically inducible promoters are a common research tool used for pathway expression, they introduce a compound extrinsic to the process along with the associated costs. We developed an expression control system for a biosynthetic pathway for the production of d-glyceric acid that utilizes galacturonate as both the inducer and the substrate, thereby eliminating the need for an extrinsic chemical inducer.
Activation of expression in response to the feed is actuated by a galacturonate-responsive transcription factor biosensor. We constructed variants of the galacturonate biosensor with a heterologous transcription factor and cognate hybrid promoter, and selected for the best performer through fluorescence characterization. We showed that native E. coli regulatory systems do not interact with our biosensor and favorable biosensor response exists in the presence and absence of galacturonate consumption. We then employed the control circuit to regulate the expression of the heterologous genes of a biosynthetic pathway for the production d-glyceric acid that was previously developed in our lab. Productivity via substrate-induction with our control circuit was comparable to IPTG-controlled induction and significantly outperformed a constitutive expression control, producing 2.13 ± 0.03 g L−1 d-glyceric acid within 6 h of galacturonate substrate addition.
This work demonstrated feed-activated pathway expression to be an attractive control strategy for more readily scalable microbial biosynthesis.
MIT Department
Massachusetts Institute of Technology. Department of Chemical Engineering
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DOI of Published Version
https://doi.org/10.1002/biot.202000433
