title: Enzymatic synthesis of 2'-deoxyuridine by whole cell catalyst co-expressing uridine phosphorylase and thymidine phosphorylase through auto-induction system.
abstract: Genes encoding uridine phosphorylase (UP) and thymidine phosphorylase (TP) from Escherichia coli K12 were cloned and recombined respectively into plasmids pET-21a(+) and pET-28a(+). The recombinant plasmids BL21/pET21a-UP and BL21/pET28a-TP were co-transformed into E. coli BL21(DE3) to construct highly effective BTU strain (BL21/pET28a-TP, pET21a-UP) overexpressing both of UP and TP. BTU was cultivated in ZYM-Fe-5052 medium for 10 h and used as catalyst to synthesize 2'-deoxyuridine (dUR). It was found to increase the productivity of dUR by 8-9 fold when compared to wild E. coli K12 and E. coli BL21(DE3) strains. A series of experiments were carried out to find out the optimal conditions for synthesis of dUR. At 50°C, with 0.25‰ dry wt./v to catalyze the reaction of 2:1 β-thymidine: uracil (60 mM β-thymidine, 30 mM uracil), the conversion rate of dUR would reach 61.6% at 1 h, which was much higher than the rates obtained by BTU strain cultured in LB medium and induced by IPTG. This result proved co-expression and auto-induction were efficient methods in enhancing the expression quantity and activity of nucleoside phosphorylases, and they also had significant implications for large-scale industrial production of dUR and synthesis of other nucleoside derivatives.
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Shuli
authors.initials: S
authors.lastname: Xiong
authors.affiliation: School of Science, China University of Geosciences, Beijing 100083, China. Electronic address: wyb@cugb.edu.cn.
authors.forename: Yingbin
authors.initials: Y
authors.lastname: Wang
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Xi
authors.initials: X
authors.lastname: Wang
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Wang
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Li
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Guiyou
authors.initials: G
authors.lastname: Zhang
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Rongqing
authors.initials: R
authors.lastname: Zhang
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Liping
authors.initials: L
authors.lastname: Xie
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, China; Protein Science Laboratory of the Ministry of Education, Tsinghua University, Beijing 100084, China. Electronic address: hzwang@mail.tsinghua.edu.cn.
authors.forename: Hongzhong
authors.initials: H
authors.lastname: Wang
mesh_terms: D055162:Biocatalysis; D003857:Deoxyuridine / Q000378:metabolism*; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D013936:Thymidine / Q000378:metabolism; D013939:Thymidine Phosphorylase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D014543:Uridine Phosphorylase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*
chemical_list: D014543:Uridine Phosphorylase; D013939:Thymidine Phosphorylase; D013936:Thymidine; D003857:Deoxyuridine
keywords: 2′-Deoxyuridine; Auto-induction; Biocatalysis; Co-expression; Nucleoside phosphorylase