Search citation statements
Paper Sections
Citation Types
Year Published
Publication Types
Relationship
Authors
Journals
Xenobiology is an emerging field that focuses on the extension and redesign of biological systems through the use of laboratory-derived xenomolecules, which are molecules that are new to the metabolism of the cell. Despite the enormous potential of using xenomolecules in living organisms, most noncanonical building blocks still need to be supplied externally, and often poor uptake into cells limits wider applicability. To improve the cytosolic availability of noncanonical molecules, a synthetic transport system based on portage transport was developed, in which molecules of interest “cargo” are linked to a synthetic transport vector that enables piggyback transport through the alkylsulfonate transporter (SsuABC) of Escherichia coli. Upon cytosolic delivery, the vector-cargo conjugate is enzymatically cleaved by GGTxe, leading to the release of the cargo molecule. To deepen our understanding of the synthetic transport system, we focused on the characterization and further development of the enzymatic cargo release step. Hence, the substrate scope of GGTxe was characterized using a library of structurally diverse vector-cargo conjugates and MS/MS-based quantification of hydrolysis products in a kinetic manner. The resulting substrate tolerance characterization revealed that vector-amino acid conjugates were significantly unfavored. To overcome this shortcoming, a selection system based on metabolic auxotrophy complementation and directed evolution of GGTxe was established. In a directed evolution campaign, we improved the enzymatic activity of GGTxe for vector-amino acid conjugates and revealed the importance of residue D386 in the cargo unloading step.
Xenobiology is an emerging field that focuses on the extension and redesign of biological systems through the use of laboratory-derived xenomolecules, which are molecules that are new to the metabolism of the cell. Despite the enormous potential of using xenomolecules in living organisms, most noncanonical building blocks still need to be supplied externally, and often poor uptake into cells limits wider applicability. To improve the cytosolic availability of noncanonical molecules, a synthetic transport system based on portage transport was developed, in which molecules of interest “cargo” are linked to a synthetic transport vector that enables piggyback transport through the alkylsulfonate transporter (SsuABC) of Escherichia coli. Upon cytosolic delivery, the vector-cargo conjugate is enzymatically cleaved by GGTxe, leading to the release of the cargo molecule. To deepen our understanding of the synthetic transport system, we focused on the characterization and further development of the enzymatic cargo release step. Hence, the substrate scope of GGTxe was characterized using a library of structurally diverse vector-cargo conjugates and MS/MS-based quantification of hydrolysis products in a kinetic manner. The resulting substrate tolerance characterization revealed that vector-amino acid conjugates were significantly unfavored. To overcome this shortcoming, a selection system based on metabolic auxotrophy complementation and directed evolution of GGTxe was established. In a directed evolution campaign, we improved the enzymatic activity of GGTxe for vector-amino acid conjugates and revealed the importance of residue D386 in the cargo unloading step.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.