title: Three-pathway combination for glutathione biosynthesis in Saccharomyces cerevisiae.
abstract: BACKGROUND
  Glutathione (GSH), a pivotal non-protein thiol, can be biosynthesized through three pathways in different organisms: (1) two consecutive enzymatic reactions catalyzed by γ-glutamylcysteine synthetase (Gsh1 or GshA) and glutathione synthetase (Gsh2 or GshB); (2) a bifunctional γ-glutamylcysteine synthetase/glutathione synthetase (GshF); (3) an alternative condensation of γ-glutamyl phosphate synthesized by γ-glutamyl kinase (Pro1 or ProB) with cysteine to form γ-glutamylcysteine which was further conjugated to glycine by glutathione synthetase. The Gsh1 and Gsh2 of conventional GSH biosynthetic pathway or the bifunctional GshF reported previously have been independently modulated for GSH production. This study developed a novel three-pathway combination method to improve GSH production in Saccharomyces cerevisiae.
  
  
  RESULTS
  A bifunctional enzyme GshF of Actinobacillus pleuropneumoniae was functionally expressed in S. cerevisiae and Pro1 in proline biosynthetic pathway was exploited for improving GSH yield. Moreover, two fusion proteins Gsh2-Gsh1 and Pro1-GshB were constructed to increase the two-step coupling efficiency of GSH synthesis by mimicking the native domain fusion of GshF. The engineered strain W303-1b/FGP with three biosynthetic pathways presented the highest GSH concentration (216.50 mg/L) and GSH production of W303-1b/FGP was further improved by 61.37 % when amino acid precursors (5 mM glutamic acid, 5 mM cysteine and 5 mM glycine) were fed in shake flask cultures. In batch culture process, the recombinant strain W303-1b/FGP also kept high efficiency in GSH production and reached an intracellular GSH content of 2.27 % after 24-h fermentation.
  
  
  CONCLUSIONS
  The engineered strains harbouring three GSH pathways displayed higher GSH producing capacity than those with individually modulated pathways. Three-pathway combinatorial biosynthesis of GSH promises more effective industrial production of GSH using S. cerevisiae.
authors.affiliation: State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, 1 Xian Nong Tan St., 100050, Beijing, China. tangl@imm.ac.cn.
authors.forename: Liang
authors.initials: L
authors.lastname: Tang
authors.affiliation: College of Life Science, Qufu Normal University, 273165, Qufu, Shandong, China. wangweiwei_fly@126.com.
authors.forename: Weiwei
authors.initials: W
authors.lastname: Wang
authors.affiliation: Key Laboratory of Biosynthesis of Natural Products of National Health and Family Planning Commission, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, 1 Xian Nong Tan St., 100050, Beijing, China. zhouwl@imm.ac.cn.
authors.forename: Wenlong
authors.initials: W
authors.lastname: Zhou
authors.affiliation: College of Life Science, Qufu Normal University, 273165, Qufu, Shandong, China. 492793582@qq.com.
authors.forename: Kai
authors.initials: K
authors.lastname: Cheng
authors.affiliation: State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, 1 Xian Nong Tan St., 100050, Beijing, China. yangyan@imm.ac.cn.
authors.forename: Yan
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Key Laboratory of Biosynthesis of Natural Products of National Health and Family Planning Commission, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, 1 Xian Nong Tan St., 100050, Beijing, China. lmzluoxue@imm.ac.cn.
authors.forename: Minzhi
authors.initials: M
authors.lastname: Liu
authors.affiliation: Key Laboratory of Biosynthesis of Natural Products of National Health and Family Planning Commission, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, 1 Xian Nong Tan St., 100050, Beijing, China. chengkd@imm.ac.cn.
authors.forename: Kedi
authors.initials: K
authors.lastname: Cheng
authors.affiliation: State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, 1 Xian Nong Tan St., 100050, Beijing, China. wwang@imm.ac.cn.|Key Laboratory of Biosynthesis of Natural Products of National Health and Family Planning Commission, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, 1 Xian Nong Tan St., 100050, Beijing, China. wwang@imm.ac.cn.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
mesh_terms: D016977:Actinobacillus pleuropneumoniae / Q000235:genetics; D019149:Bioreactors; D005978:Glutathione / Q000096:biosynthesis*; D060847:Metabolic Engineering; D053858:Metabolic Networks and Pathways; D030781:Organisms, Genetically Modified / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D005978:Glutathione