Effect of pyk gene location on constructing dATP conversion E. coli strain.docVIP

Advances in Bioscience and Biotechnology, 2013, 4, 75-80 ABB /10.4236/abb.2013.41011 Published Online January 2013 (/journal/abb/) Effect of pyk gene location on constructing dATP conversion E. coli strain Xiujuan Xin, Jie Bao * State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China * Email: jbao@ Received 18 November 2012; revised 30 December 2012; accepted 10 January 2013 ABSTRACT cleoside triphosphates (dATP) [1]. People should express and purify two enzymes individually, this complicated the procedure and increased the cost. A dATP conversion E. coli strain could be con- structed when both pyk and adk1 gene were expressed successfully. pyk gene encodes pyruvate kinase (PK) could be expressed, when inserted it before adk1 gene which encodes adenylate kinase (AK) in plasmid pET-pyk-adk1 after transform into E. coli and the recombinant could be used to convert dATP from dAMP. Another plasmid pET-adk1-pyk, which in- serted pyk gene behind of adk1, the recombinant E. coli transformed with this plasmid could not convert dAMP into dATP, pyk gene cannot be translated in this recombinant. The different translation levels of pyk with gene location switching caused mainly by the different secondary structures formed by the 5’-un- translation regions and the gene sequence of its 5’-terminal. The dATP product E. coli strain could be constructed when cloned pyk gene at an optimum lo- cation. To clone two or more genes into one host was feasible in recently. In order to simplify the biosynthesis process, two kinases were cloned onto a high copy plasmid pET- 17b and performed overexpression in E. coli BL21. The recombinants are used to catalyze dATP from dAMP after inducting protein expression of plasmid pET-pyk- adk1. However, the poor expression spectrum of pyk gene on the pET-adk1-pyk plasmid pr