Article
Version 1
Preserved in Portico This version is not peer-reviewed
A Synthetic Pathway for the Production of Benzylsuccinate in Escherichia coli
Version 1
: Received: 5 December 2023 / Approved: 6 December 2023 / Online: 6 December 2023 (07:16:11 CET)
A peer-reviewed article of this Preprint also exists.
Mock, J.; Schühle, K.; Linne, U.; Mock, M.; Heider, J. A Synthetic Pathway for the Production of Benzylsuccinate in Escherichia coli. Molecules 2024, 29, 415. Mock, J.; Schühle, K.; Linne, U.; Mock, M.; Heider, J. A Synthetic Pathway for the Production of Benzylsuccinate in Escherichia coli. Molecules 2024, 29, 415.
Abstract
Benzylsuccinate is generated in anaerobic toluene degradation by radical addition of toluene to fumarate and further degraded to benzoyl-CoA by a beta-oxidation pathway. Using metabolic modules for benzoate transport and activation to benzoyl-CoA and the enzymes of benzylsuccinate beta-oxidation, we established an artificial pathway for benzylsuccinate production in Escherichia coli, which is based of its degradation pathway running in reverse. Benzoate is supplied to the medium, but needs to be converted to benzoyl-CoA by an uptake transporter and a benzoate-CoA ligase or CoA-transferase. In contrast, the second substrate succinate is endogenously produced from glucose under anaerobic conditions, and the constructed pathway includes a succinyl-CoA:benzylsuccinate CoA-transferase that activates it to the CoA-thioester. We present first evidence for the feasibility of this pathway and explore product yields under different growth conditions. Compared to aerobic cultures, the product yield increased more than 1000-fold in anaerobic glucose-fermenting cultures and showed further improvement under fumarate-respiring conditions. An important bottleneck to overcome appears to be product excretion, based on much higher recorded intracellular concentrations of benzylsuccinate, compared to those excreted. While no export system is known for benzylsuccinate, we observed an increased product yield after adding an unspecific mechanosensitive channel to the constructed pathway.
Keywords
anaerobic toluene degradation; reverse -oxidation; synthetic pathway; transport; CoA ligase; CoA-transferase; benzylsuccinate
Subject
Biology and Life Sciences, Biochemistry and Molecular Biology
Copyright: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Comments (0)
We encourage comments and feedback from a broad range of readers. See criteria for comments and our Diversity statement.
Leave a public commentSend a private comment to the author(s)
* All users must log in before leaving a comment