title: Characterization of bifunctional L-glutathione synthetases from Actinobacillus pleuropneumoniae and Actinobacillus succinogenes for efficient glutathione biosynthesis.
abstract: Glutathione (GSH), an important bioactive substance, is widely applied in pharmaceutical and food industries. In this work, two bifunctional L-glutathione synthetases (GshF) from Actinobacillus pleuropneumoniae (GshFAp) and Actinobacillus succinogenes (GshFAs) were successfully expressed in Escherichia coli BL-21(DE3). Similar to the GshF from Streptococcus thermophilus (GshFSt), GshFAp and GshFAs can be applied for high titer GSH production because they are less sensitive to end-product inhibition (Ki values 33 and 43 mM, respectively). The active catalytic forms of GshFAs and GshFAp are dimers, consistent with those of GshFPm (GshF from Pasteurella multocida) and GshFSa (GshF from Streptococcus agalactiae), but are different from GshFSt (GshF from S. thermophilus) which is an active monomer. The analysis of the protein sequences and three dimensional structures of GshFs suggested that the binding sites of GshFs for substrates, L-cysteine, L-glutamate, γ-glutamylcysteine, adenosine-triphosphate, and glycine are highly conserved with only very few differences. With sufficient supply of the precursors, the recombinant strains BL-21(DE3)/pET28a-gshFas and BL-21(DE3)/pET28a-gshFap were able to produce 36.6 and 34.1 mM GSH, with the molar yield of 0.92 and 0.85 mol/mol, respectively, based on the added L-cysteine. The results showed that GshFAp and GshFAs are potentially good candidates for industrial GSH production.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.|Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, Aachen, 52074, Germany.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
authors.forename: Dezheng
authors.initials: D
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China. hwu@ecust.edu.cn.
authors.forename: Hui
authors.initials: H
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China. lizm@ecust.edu.cn.|Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 130 Meilong Road,Shanghai, 200237, Shanghai, China. lizm@ecust.edu.cn.
authors.forename: Zhimin
authors.initials: Z
authors.lastname: Li
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
authors.forename: Qin
authors.initials: Q
authors.lastname: Ye
mesh_terms: D000188:Actinobacillus / Q000201:enzymology*; D016977:Actinobacillus pleuropneumoniae / Q000201:enzymology*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D005978:Glutathione / Q000096:biosynthesis*; D005981:Glutathione Synthase / Q000235:genetics / Q000378:metabolism*; D005998:Glycine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D016979:Pasteurella multocida / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D013292:Streptococcus agalactiae / Q000235:genetics / Q000378:metabolism; D048249:Streptococcus thermophilus / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004151:Dipeptides; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D005981:Glutathione Synthase; D005978:Glutathione; D003545:Cysteine; C017341:gamma-glutamylcysteine; D005998:Glycine
keywords: Actinobacillus pleuropneumoniae; Actinobacillus succinogenes; Bifunctional glutathione synthetase; Escherichia coli; Glutathione; One-strain system