Cloning, heterologous expression and purification of an isocitrate lyase from Streptomyces clavuligerus NRRL 3585.
The glyoxylate cycle comprising isocitrate lyase (ICL) and malate synthase (MS) is an anaplerotic pathway essential for growth on acetate as the sole carbon source. The aceB gene, which encodes malate synthase has been previously cloned from Streptomyces clavuligerus NRRL 3585 and characterized. In this study, the aceA gene, encoding ICL from S. clavuligerus NRRL 3585, was obtained via genome walking experiments and PCR. The fully sequenced open reading frame encodes 436 amino acids with a deduced M(r) of 47.5 kDa, consistent with the observed M(r) (49-67.5 kDa) of most ICL enzymes reported so far. The cloned aceA gene was expressed in Escherichia coli BL21(lambdaDE3) cells, from which ICL was purified as a His-tagged product and its functionality demonstrated. Furthermore, the relationship between the carbon sources, growth and ICL activity in S. clavuligerus were investigated. Rapid growth was observed when the cells were cultured on 0.5% (w/v) glycerol, while delayed growth was observed when cells were grown on 0.5% (w/v) acetate. However, in both cases, high levels of ICL activity coincided with a cessation of growth, suggesting a late physiological role played by ICL in the natural host, S. clavuligerus.
Soh
B S
BS
Department of Microbiology, Faculty of Medicine, National University of Singapore, MD4A, 5 Science Drive 2, 117597, Singapore, Singapore.
Loke
P
P
Sim
T S
TS
D001426:Bacterial Proteins; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007522:Isocitrate Lyase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D009876:Operon; D010802:Phylogeny; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*
D001426:Bacterial Proteins; D004247:DNA; D007522:Isocitrate Lyase