CONSTRUCTION OF GLUTATHIONE OVERPRODUCERS IN THE YEAST HANSENULA POLYMORPHA Olena Kurylenko Department of Molecular Genetics and Biotechnology Institute of Cell Biology, NAS of Ukraine Glutathione and its physiological roles in cells Intracellular redox buffer Elimination of toxic endogenous metabolites Cell cycle regulation Cofactor of enzymes Detoxification of heavy metals and xenobiotics Sulfur and nitrogen source DNA and protein synthesis 2 Practical application of glutathione Preventing the oxidative spoilage of white wines, juices, fruits) Preservation of organs for surgery Pharmaceutical industry Supporting the immune system Bakery (bread texture, strength and extensibility of the dough) Food industry Flavour enhancer Anti-aging and anti-sun skin products GSH Cosmetic industry Component in creams and moisturizers, primarily to enhance the whitening effect on skin GSH producers Bacteria Escherichia coli Lactococcus lactis Yeasts Saccharomyces cerevisiae Candida utilis Hansenula polymorpha Belongs to the best studied yeasts with well developed tools for molecular research and completed genome sequence Grows on the methanol as a sole source of carbon and energy Glutathione synthesis γ-GCS GS The rate of GSH synthesis is controlled by The amount of γ-GCS present Availability of L-cysteine Feedback inhibition exerted by GSH on γ-GCS 5 Construction of glutathione overproducers in the yeast Hansenula polymorpha using metabolic engineering approaches GSH 2 H. polymorpha wild type strain WT mcGSH2 Intracellular glutathione nmol/mg proteim 350 300 250 WT 200 mc GSH2 150 100 50 0 24 h 96 h Ubiyvovk et al. BMC Biotechnology 2011, 11:8 MET4 Met4 is transcriptional activator that regulates the assimilation of extracellular sulfate into the sulfurcontaining amino acids methionine and cysteine. Met4, therefore, has an indirect effect on GSH biosynthesis by regulating the supply of cysteine. γ Glutamic acid α Cysteine Glycine Glutathione (GSH, γ-L-glutamyl-L-cysteinylglycine) Intracellular GSH production of H. polymorpha recombinant strains co-overexpressing MET4 and GSH2 genes Xb B prGAP Xh MET4 Sl GAP term Nd natNT2 lacZ Ad Nd bla pUC19/prGAP_MET4/NTC~ 5,9kb 1400 GSH, nmol/mg proteim 1200 1000 mcGSH2 (parental strain) 800 mcGSH2/MET4 (25 a) mcGSH2/MET4 (25 b) mcGSH2/MET4 (25 c) 600 400 200 0 24 h 48 h 72 h 96h Extracellular GSH production of H. polymorpha recombinant strains co-overexpressing MET4 and GSH2 genes Glucose 250 WT 150 mcGSH2 100 mcGSH2/MET4 mcGSH2/MET4 50 mcGSH2/MET4 0 0 1 2 3 4 5 6 Days Methanol 250 200 GSH, nmol/L GSH, nmol/L 200 WT 150 mcGSH2 100 mcGSH2/MET4 mcGSH2/MET4 50 mcGSH2/MET4 0 0 1 2 3 Days 4 5 6 Glutathione synthesis I Glu + Cys II ATP γ-GCS -Glu-Cys + Gly •Repression •Inhibition γ-GCS – gamma-glutamylcysteine synthetase GS – glutathione synthetase ATP GS GSH Mutagenesis of the GSH2 gene Construction of H. polymorpha strain with overexpressed native or modified forms of γ-glutamylcysteine synthase Xb B prGAP Xh Ad Sl GSH2mut GAP term LEU2_Hp bla pYT3/prGAP_GSH2mut ~ 10,3 kb H. polymorpha ∆gsh2 YNB Xb B prGAP Xh Ad Sl GSH2nat GAP term LEU2_Hp bla pYT3/prGAP_GSH2nat ~ 10,3 kb Resistance of selected transformants to different prooxidant agents as compared to strains carrying unmodified GSH2 gene YNB + 1,2,4-triazole 3 g/L 4 g/L 5 g/L GSH2mut GSH2nat YNB 1 g/L 2 g/L GSH2mut GSH2nat Conclusions and future prospects • Overexpression of MET4 gene coding for central regulator of sulfur metabolism lead to significant increase in intracellular and extracellular GSH level in the yeast H. polymorpha • The next step of this work is analysis of GSH level in obtained strains with modified γ - glutamylcysteine synthetase and identification of mutation in GSH2 gene, leading to enhanced GSH synthesis in the yeast H. polymorpha. Thank You for your attention! Acknowledgements Marianna Yurkiv Dr. Dmytruk K.V. Prof. Sybirny A.A.
© Copyright 2024 ExpyDoc
