Characterization of 5-aminolevulinate synthase from Agrobacterium radiobacter, screening new inhibitors for 5-aminolevulinate dehydratase from Escherichia coli and their potential use for high 5-aminolevulinate production.
The hemA gene encoding 5-aminolevulinate synthase (ALAS) from Agrobacterium radiobacter zju-0121 showed 92.6% homology with that from A. radiobacter ATCC4718 and contained several rare codons. To enhance the expression of this gene, Escherichia coli Rosetta(DE3), which is a rare codon optimizer strain, was used as the host to construct an efficient recombinant strain. And the encoded protein was over-expressed as fusion protein and was purified by affinity purification on Ni-NTA agarose and by gel filtration chromatography on Sephadex G-25 Medium resin. The recombinant protein was partly characterized, and D-glucose, D-fructose, D-xylose, D-mannose, L-arabinose, D-galactose, lactose, sucrose and maltose were detected to have no distinct inhibition on this recombinant ALAS. Meanwhile, 20mM D-glucose or D-xylose inhibited about 20% activity of ALA dehydratase (ALAD) from Escherichia coli Rosetta(DE3). Combining D-xylose as a new inhibitor for ALAD with D-glucose in fed-batch culture and based on the optimal culture system using Rosetta(DE3)/pET28a-hemA, the yield of ALA achieved was 7.3g/l (56 mM) under the appropriate conditions in the fermenter.
Lin
Jianping
J
Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou, Zhejiang, China. linjp@zju.edu.cn
Fu
Weiqi
W
Cen
Peilin
P
D000624:5-Aminolevulinate Synthetase / Q000302:isolation & purification*; D016960:Agrobacterium tumefaciens / Q000187:drug effects / Q000201:enzymology*; D000622:Aminolevulinic Acid / Q000378:metabolism*; D002241:Carbohydrates / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D007477:Ions; D008670:Metals / Q000494:pharmacology; D000623:Porphobilinogen Synthase / Q000037:antagonists & inhibitors*; D011994:Recombinant Proteins / Q000302:isolation & purification; D013696:Temperature
D002241:Carbohydrates; D004791:Enzyme Inhibitors; D007477:Ions; D008670:Metals; D011994:Recombinant Proteins; D000622:Aminolevulinic Acid; D000624:5-Aminolevulinate Synthetase; D000623:Porphobilinogen Synthase