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

作　　者：LIU Xiaoxia（刘晓侠） LIN Jianping（林建平） QIN Gang（秦钢） CEN Peilin（岑沛霖） 

机构地区：[1]Institute of Bioengineering, College of Materials Science and Chemical Engineering, Zhejiang University, Hangzhou 310027, China

出　　处：《Chinese Journal of Chemical Engineering》2005年第4期522-528,共7页中国化学工程学报（英文版）

基　　金：Supported by the National Natural Science Foundation of China (No. 20306026)

摘　　要：A new hemA gene encoding 5-aminolevulinate (ALA) synthase was cloned from Agrobacterium ra- diobacter zju-0121. The ALA synthase catalyzes the pyridoxal phosphate-dependent condensation of succinyl coen- zyme 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 13.8nmol·min-1·mg-1 of intracellular soluble protein. In the batch culture of the recombinant E. coli, the extracellular ALA concentration reached 0.9 g·L-1.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：5-aminolevulinate （ALA） ALA synthase metabolic engineering 

分 类 号：Q55[生物学—生物化学] Q781