Expression of salt-induced 2-Cys peroxiredoxin from Oryza sativa increases stress tolerance and fermentation capacity in genetically engineered yeast Saccharomyces cerevisiae.
Peroxiredoxins (Prxs), also termed thioredoxin peroxidases (TPXs), are a family of thiol-specific antioxidant enzymes that are critically involved in cell defense and protect cells from oxidative damage. In this study, a putative chloroplastic 2-Cys thioredoxin peroxidase (OsTPX) was identified by proteome analysis from leaf tissue samples of rice (Oryza sativa) seedlings exposed to 0.1 M NaCl for 3 days. To investigate the relationship between the OsTPX gene and the stress response, OsTPX was cloned into the yeast expression vector p426GPD under the control of the glyceraldehyde-3-phosphate dehydrogenase (GPD1) promoter, and the construct was transformed into Saccharomyces cerevisiae cells. OsTPX expression was confirmed by semi-quantitative reverse transcription-polymerase chain reaction and western blot analyses. OsTPX contained two highly conserved cysteine residues (Cys114 and Cys236) and an active site region (FTFVCPT), and it is structurally very similar to human 2-Cys Prx. Heterologous OsTPX expression increased the ability of the transgenic yeast cells to adapt and recover from reactive oxygen species (ROS)-induced oxidative stresses, such as a reduction of cellular hydroperoxide levels in the presence of hydrogen peroxide and menadione, by improving redox homeostasis. OsTPX expression also conferred enhanced tolerance to tert-butylhydroperoxide, heat shock, and high ethanol concentrations. Furthermore, high OsTPX expression improved the fermentation capacity of the yeast during glucose-based batch fermentation at a high temperature (40 °C) and at the general cultivation temperature (30 °C). The alcohol yield in OsTPX-expressing transgenic yeast increased by approximately 29 % (0.14 g g(-1)) and 21 % (0.12 g g(-1)) during fermentation at 40 and 30 °C, respectively, compared to the wild-type yeast. Accordingly, OsTPX-expressing transgenic yeast showed prolonged cell survival during the environmental stresses produced during fermentation. These results suggest that heterologous OsTPX expression increases acquired tolerance to ROS-induced oxidative stress by improving cellular redox homeostasis and improves fermentation capacity due to improved cell survival during fermentation, especially at a high temperature.
Advanced Bio-resource Research Center, Department of Biology, College of Natural Sciences, Kyungpook National University, #1370 Sankyuk-dong, Buk-gu, Daegu 702-701, Republic of Korea. 92kis@hanmail.net
Il-Sup
IS
Kim
Young-Saeng
YS
Kim
Ho-Sung
HS
Yoon
D003001:Cloning, Molecular; D000431:Ethanol / Q000378:metabolism; D005285:Fermentation; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D006801:Humans; D050296:Microbial Viability / Q000187:drug effects; D012275:Oryza / Q000201:enzymology* / Q000235:genetics; D018384:Oxidative Stress; D054464:Peroxiredoxins / Q000235:genetics / Q000378:metabolism*; D018515:Plant Leaves / Q000737:chemistry / Q000201:enzymology; D020543:Proteome / Q000032:analysis; D017382:Reactive Oxygen Species / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000502:physiology*; D013312:Stress, Physiological
D020543:Proteome; D017382:Reactive Oxygen Species; D000431:Ethanol; D054464:Peroxiredoxins; D005947:Glucose