title: Characterization of UGPase from Aureobasidium pullulans NRRL Y-12974 and Application in Enhanced Pullulan Production.
abstract: UDPG pyrophosphatase (UGPase) plays an important role in carbohydrate metabolism, catalyzing a reversible production of uridine diphosphate glucose (UDPG) and pyrophosphate (PPi) from Glc-1-P and UTP. UGPase gene from Aureobasidium pullulans NRRL Y-12974 was cloned, overexpressed in Escherichia coli. The recombinant UGPase possess molecular mass of 55 KDa and specific activity of 7.33 U/mg protein. The K m values of rUGPase were 5.045 μM against UTP and 3.333 μM against Glc-1-P. The V max values of rUGPase were 3.467 μM min(-1)against UTP and 2.817 μM min(-1) against Glc-1-P. And, it does not catalyze Glc-1-P and ATP, nor galactose-1-P and UTP. Homolgous expression of UGPase in native organism can improve the intracellular UDPG concentration by 4.7-fold time. The yield of pullulan in engineering strain A4 was improved to 18.2 g g(-1) cell dry weight which is 1.3-fold time of parent strain. No obvious change of growth was found between engineering strain and parent strain. To the best of our knowledge, this is the first report of improving pullulan yield in A. pullulans using metabolic engineering technique.
authors.affiliation: College of Medicine, Hangzhou Normal University, No.1378 Wenyi Xi Road, Hangzhou, 311121, Zhejiang, People's Republic of China. lihaifengwater@163.com.
authors.forename: Haifeng
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of Microbiology, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, 310051, Zhejiang, People's Republic of China.
authors.forename: Yunyi
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: College of Medicine, Hangzhou Normal University, No.1378 Wenyi Xi Road, Hangzhou, 311121, Zhejiang, People's Republic of China.
authors.forename: Yunyun
authors.initials: Y
authors.lastname: Gao
authors.affiliation: College of Medicine, Hangzhou Normal University, No.1378 Wenyi Xi Road, Hangzhou, 311121, Zhejiang, People's Republic of China.
authors.forename: Yuanyang
authors.initials: Y
authors.lastname: Lan
authors.affiliation: College of Chemistry and Materials Science, Hangzhou Normal University, Hangzhou, 311121, Zhejiang, People's Republic of China.
authors.forename: Xiaopu
authors.initials: X
authors.lastname: Yin
authors.affiliation: College of Medicine, Hangzhou Normal University, No.1378 Wenyi Xi Road, Hangzhou, 311121, Zhejiang, People's Republic of China.
authors.forename: Lifeng
authors.initials: L
authors.lastname: Huang
mesh_terms: D001203:Ascomycota / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005936:Glucans / Q000096:biosynthesis*; D014158:Transcription, Genetic; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000378:metabolism*
chemical_list: D005936:Glucans; C009109:pullulan; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase
keywords: Aureobasidium pullulans; Expression; Pullulan; UDPG pyrophosphatase