CIESC Journal

• BIOTECHNOLOGY & BIOENGINEERING • 上一篇    下一篇

大肠杆菌中表达来源于Agrobacterium radibacter的新hamA基因以生产5-氨基乙酰丙醇

刘晓侠; 林建平; 秦钢; 岑沛霖   

  1. Institute of Bioengineering, College of Materials Science and Chemical Engineering, Zhejiang University, Hangzhou 310027, China
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2005-08-28 发布日期:2005-08-28
  • 通讯作者: 刘晓侠

Expression of a New hemA Gene from Agrobacterium radiobacter in Escherichia coli for 5-Aminolevulinate Production

LIU Xiaoxia; LIN Jianping; QIN Gang; CEN Peilin   

  1. Institute of Bioengineering, College of Materials Science and Chemical Engineering, Zhejiang University, Hangzhou 310027, China
  • Received:1900-01-01 Revised:1900-01-01 Online:2005-08-28 Published:2005-08-28
  • Contact: LIU Xiaoxia

摘要: A new hemA gene encoding 5-aminolevulinate (ALA) synthase was cloned from Agrobacterium radiobacter zju-0121. The ALA synthase catalyzes the pyridoxal phosphate-dependent condensation of succinyl coenzyme A (succinyl-CoA) and glycine to produce ALA. Four plasmids carrying the A. radiobacter hemA gene were transformed into different E. coli strains. The effects of both genetic and physiological factors on the expression of ALA synthase and ALA production were studied. The results indicated that the final intracellular activity of ALA synthase and the production of ALA in different expression systems varied largely. Among them, the recombinant E. coli BL21 (DE3) harboring the expression plasmid pET28-A. R-hemA was the most suitable one. The effects of isopropyl-β-D-thiogalactopyranoside (IPTG) addition time, IPTG concentration, culture temperature and the initial concentration of precursors and glucose on the ALA production were also evaluated. The expressed ALA synthase accounted for about 23.7% of the intracellular soluble protein. The highest specific activity of ALA syn- thase was 13.8 nmol.min^-1.mg^-1 of intracellular soluble protein. In the batch culture of the recombinant E. coli, the extracellular ALA concentration reached 0.9g.L^-1.

关键词: hemA基因;5-氨基乙醛丙醇;大肠杆菌;基因表达

Abstract: A new hemA gene encoding 5-aminolevulinate (ALA) synthase was cloned from Agrobacterium radiobacter zju-0121. The ALA synthase catalyzes the pyridoxal phosphate-dependent condensation of succinyl coenzyme A (succinyl-CoA) and glycine to produce ALA. Four plasmids carrying the A. radiobacter hemA gene were transformed into different E. coli strains. The effects of both genetic and physiological factors on the expression of ALA synthase and ALA production were studied. The results indicated that the final intracellular activity of ALA synthase and the production of ALA in different expression systems varied largely. Among them, the recombinant E. coli BL21 (DE3) harboring the expression plasmid pET28-A. R-hemA was the most suitable one. The effects of isopropyl-β-D-thiogalactopyranoside (IPTG) addition time, IPTG concentration, culture temperature and the initial concentration of precursors and glucose on the ALA production were also evaluated. The expressed ALA synthase accounted for about 23.7% of the intracellular soluble protein. The highest specific activity of ALA syn- thase was 13.8 nmol.min^-1.mg^-1 of intracellular soluble protein. In the batch culture of the recombinant E. coli, the extracellular ALA concentration reached 0.9g.L^-1.

Key words: 5-aminolevulinate (ALA), ALA synthase, metabolic engineering