Optimization of glutathione production in batch and fed-batch cultures by the wild-type and recombinant strains of the methylotrophic yeast Hansenula polymorpha DL-1.
BACKGROUND
Tripeptide glutathione (gamma-glutamyl-L-cysteinyl-glycine) is the most abundant non-protein thiol that protects cells from metabolic and oxidative stresses and is widely used as medicine, food additives and in cosmetic industry. The methylotrophic yeast Hansenula polymorpha is regarded as a rich source of glutathione due to the role of this thiol in detoxifications of key intermediates of methanol metabolism. Cellular and extracellular glutathione production of H. polymorpha DL-1 in the wild type and recombinant strains which overexpress genes of glutathione biosynthesis (GSH2) and its precursor cysteine (MET4) was studied.


RESULTS
Glutathione producing capacity of H. polymorpha DL-1 depending on parameters of cultivation (dissolved oxygen tension, pH, stirrer speed), carbon substrate (glucose, methanol) and type of overexpressed genes of glutathione and its precursor biosynthesis during batch and fed-batch fermentations were studied. Under optimized conditions of glucose fed-batch cultivation, the glutathione productivity of the engineered strains was increased from ~900 up to ~ 2300 mg of Total Intracellular Glutathione (TIG) or GSH+GSSGin, per liter of culture medium. Meantime, methanol fed-batch cultivation of one of the recombinant strains allowed achieving the extracellular glutathione productivity up to 250 mg of Total Extracellular Glutathione (TEG) or GSH+GSSGex, per liter of the culture medium.


CONCLUSIONS
H. polymorpha is an competitive glutathione producer as compared to other known yeast and bacteria strains (Saccharomyces cerevisiae, Candida utilis, Escherichia coli, Lactococcus lactis etc.) with good perspectives for further improvement especially for production of extracellular form of glutathione.
Institute of Cell Biology NAS of Ukraine, Drahomanov Street, 14/16, Lviv, 79005 Ukraine.
Vira M
VM
Ubiyvovk
Vladimir M
VM
Ananin
Alexander Y
AY
Malyshev
Hyun Ah
HA
Kang
Andriy A
AA
Sibirny
D000332:Aerobiosis; D018533:Biomass; D015139:Blotting, Southern; D003470:Culture Media; D005285:Fermentation; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D005947:Glucose / Q000378:metabolism; D005721:Glutamate-Cysteine Ligase / Q000235:genetics / Q000378:metabolism; D005978:Glutathione / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D000432:Methanol; D010843:Pichia / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D003470:Culture Media; D005656:Fungal Proteins; D012333:RNA, Messenger; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D005947:Glucose; D000432:Methanol
Mol Cell Biol. 1992 Apr;12(4):1719-27
1549123
Microb Cell Fact. 2008 Jul 23;7:21
18651968
Cell Biol Int. 2006 Aug;30(8):665-71
16857395
Biotechnol Bioeng. 2001 Sep;76(2):175-85
11505387
Appl Microbiol Biotechnol. 2004 Dec;66(3):233-42
15480625
J Biol Chem. 2001 Apr 27;276(17):14279-88
11278957
Proteomics. 2004 Jul;4(7):2005-13
15221762
Biometals. 2006 Dec;19(6):593-9
16946986
Biotechnol Bioeng. 2009 Dec 1;104(5):911-9
19575437
Cell Biol Int. 2003;27(9):785-9
12972285
Appl Microbiol Biotechnol. 1999 Jun;51(6):800-7
10422227
Funct Integr Genomics. 2002 Sep;2(4-5):181-92
12192591
FEMS Yeast Res. 2003 Nov;4(2):207-15
14613885
Arch Microbiol. 2002 Feb;177(2):150-8
11807564
Metab Eng. 2009 Jul-Sep;11(4-5):234-42
19379821
Mol Biol Cell. 2005 Jan;16(1):218-30
15509654
FEMS Yeast Res. 2008 Nov;8(7):1164-74
18752627
Adv Microb Physiol. 2004;49:1-76
15518828
Enzyme Microb Technol. 2000 Jun 1;26(9-10):737-742
10862879
Eur J Biochem. 2000 Aug;267(16):4928-44
10931175
Microb Cell Fact. 2006 Dec 14;5:39
17169150
Free Radic Biol Med. 1991;11(1):81-128
1937131
Mol Reprod Dev. 2001 Dec;60(4):498-506
11746961
Anal Biochem. 1976 Jul;74(1):189-97
962073
Biotechnology (N Y). 1992 Dec;10(12):1550-6
1369204
FEMS Yeast Res. 2002 Aug;2(3):327-32
12702282