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Publisher DOI: 10.1002/elsc.202100047
Title: Improvement of glycine biosynthesis from one-carbon compounds and ammonia catalyzed by the glycine cleavage system in vitro
Language: English
Authors: Xu, Yingying 
Ren, Jie 
Wang, Wei 
Zeng, An-Ping  
Keywords: C1 assimilation; glycine cleavage system; glycine synthesis; H protein; in vitro metabolic engineering
Issue Date: Jan-2022
Publisher: Wiley
Source: Engineering in Life Sciences 22 (1): 40-53 (2022-01)
Abstract (english): 
Glycine cleavage system (GCS) plays a central role in one-carbon (C1) metabolism and receives increasing interest as a core part of the recently proposed reductive glycine pathway (rGlyP) for assimilation of CO2 and formate. Despite decades of research, GCS has not yet been well understood and kinetic data are barely available. This is to a large degree because of the complexity of GCS, which is composed of four proteins (H, T, P, and L) and catalyzes reactions involving different substrates and cofactors. In vitro kinetics of reconstructed microbial multi-enzyme glycine cleavage/synthase system is desired to better implement rGlyP in microorganisms like Escherichia coli for the use of C1 resources. Here, we examined in vitro several factors that may affect the rate of glycine synthesis via the reverse GCS reaction. We found that the ratio of GCS component proteins has a direct influence on the rate of glycine synthesis, namely higher ratios of P protein and especially H protein to T and L proteins are favorable, and the carboxylation reaction catalyzed by P protein is a key step determining the glycine synthesis rate, whereas increasing the ratio of L protein to other GCS proteins does not have significant effect and the ratio of T protein to other GCS proteins should be kept low. The effect of substrate concentrations on glycine synthesis is quite complex, showing interdependence with the ratios of GCS component proteins. Furthermore, adding the reducing agent dithiothreitol to the reaction mixture not only results in great tolerance to high concentration of formaldehyde, but also increases the rate of glycine synthesis, probably due to its functions in activating P protein and taking up the role of L protein in the non-enzymatic reduction of Hox to Hred. Moreover, the presence of some monovalent and divalent metal ions can have either positive or negative effect on the rate of glycine synthesis, depending on their type and their concentration.
DOI: 10.15480/882.4127
ISSN: 1618-2863
Journal: Engineering in life sciences 
Institute: Bioprozess- und Biosystemtechnik V-1 
Document Type: Article
Project: Publikationsfonds 2021 
More Funding information: Open access funding enabled and organized by Projekt DEAL.
License: CC BY 4.0 (Attribution) CC BY 4.0 (Attribution)
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