Genome shuffling of Saccharomyces cerevisiae for enhanced glutathione yield and relative gene expression analysis using fluorescent quantitation reverse transcription polymerase chain reaction.
Genome shuffling is an efficient and promising approach for the rapid improvement of microbial phenotypes. In this study, genome shuffling was applied to enhance the yield of glutathione produced by Saccharomyces cerevisiae YS86. Six isolates with subtle improvements in glutathione yield were obtained from populations generated by ultraviolet (UV) irradiation and nitrosoguanidine (NTG) mutagenesis. These yeast strains were then subjected to recursive pool-wise protoplast fusion. A strain library that was likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both UV irradiation and heat treatments. After two rounds of genome shuffling, a high-yield recombinant YSF2-19 strain that exhibited 3.2- and 3.3-fold increases in glutathione production in shake flask and fermenter respectively was obtained. Comparative analysis of synthetase gene expression was conducted between the initial and shuffled strains using FQ (fluorescent quantitation) RT-PCR (reverse transcription polymerase chain reaction). Delta CT (threshold cycle) relative quantitation analysis revealed that glutathione synthetase gene (GSH-I) expression at the transcriptional level in the YSF2-19 strain was 9.9-fold greater than in the initial YS86. The shuffled yeast strain has a potential application in brewing, other food, and pharmaceutical industries. Simultaneously, the analysis of improved phenotypes will provide more valuable data for inverse metabolic engineering.
State Key Laboratory of Biological Fermentation Engineering of Beer, Qingdao 266061, PR China; Tsingtao Brewery Co. Ltd., Qingdao 266061, PR China; The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi 214122, PR China.
Hua
H
Yin
State Key Laboratory of Biological Fermentation Engineering of Beer, Qingdao 266061, PR China; Tsingtao Brewery Co. Ltd., Qingdao 266061, PR China.
Yanlin
Y
Ma
State Key Laboratory of Biological Fermentation Engineering of Beer, Qingdao 266061, PR China; Tsingtao Brewery Co. Ltd., Qingdao 266061, PR China. Electronic address: dengyang719@hotmail.com.
Yang
Y
Deng
College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, PR China.
Zhenbo
Z
Xu
College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, PR China.
Junyan
J
Liu
College of Food Science and Engineering, Henan University of Science and Technology, Tianjing Road, Luoyang 471003, PR China.
Junfeng
J
Zhao
State Key Laboratory of Biological Fermentation Engineering of Beer, Qingdao 266061, PR China; Tsingtao Brewery Co. Ltd., Qingdao 266061, PR China; The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi 214122, PR China.
Jianjun
J
Dong
State Key Laboratory of Biological Fermentation Engineering of Beer, Qingdao 266061, PR China; Tsingtao Brewery Co. Ltd., Qingdao 266061, PR China.
Junhong
J
Yu
State Key Laboratory of Biological Fermentation Engineering of Beer, Qingdao 266061, PR China; Tsingtao Brewery Co. Ltd., Qingdao 266061, PR China.
Zongming
Z
Chang
D019149:Bioreactors; D032621:DNA Shuffling / Q000379:methods*; D015870:Gene Expression; D016681:Genome, Fungal; D005978:Glutathione / Q000235:genetics / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D016296:Mutagenesis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
D005978:Glutathione
FQ RT-PCR; Genome shuffling; Glutathione; Saccharomyces cerevisiae