epsilon-Poly-L: -lysine producer, Streptomyces albulus, has feedback-inhibition resistant aspartokinase.
Streptomyces albulus NBRC14147 produces epsilon-poly-L: -lysine (epsilon-PL), which is an amino acid homopolymer antibiotic. Despite the commercial importance of epsilon-PL, limited information is available regarding its biosynthesis; the L: -lysine molecule is directly utilized for epsilon-PL biosynthesis. In most bacteria, L: -lysine is biosynthesized by an aspartate pathway. Aspartokinase (Ask), which is the first enzyme in this pathway, is subject to complex regulation such as through feedback inhibition by the end-product amino acids such as L: -lysine and/or L: -threonine. S. albulus NBRC14147 can produce a large amount of epsilon-PL (1-3 g/l). We therefore suspected that Ask(s) of S. albulus could be resistant to feedback inhibition to provide sufficient L: -lysine for epsilon-PL biosynthesis. To address this hypothesis, in this study, we cloned the ask gene from S. albulus and investigated the feedback inhibition of its gene product. As predicted, we revealed the feedback resistance of the Ask; more than 20% relative activity of Ask was detected in the assay mixture even with extremely high concentrations of L: -lysine and L: -threonine (100 mM each). We further constructed a mutated ask gene for which the gene product Ask (M68V) is almost fully resistant to feedback inhibition. The homologous expression of Ask (M68V) further demonstrated the increase in epsilon-PL productivity.
Hamano
Y
Y
Department of Bioscience, Fukui Prefectural University, 4-1-1 Matsuoka-Kenjojima, Eiheiji-cho, Fukui, 910-1195, Japan. hamano@fpu.ac.jp
Nicchu
I
I
Shimizu
T
T
Onji
Y
Y
Hiraki
J
J
Takagi
H
H
D001222:Aspartate Kinase / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D025461:Feedback, Physiological; D008239:Lysine / Q000096:biosynthesis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011107:Polylysine / Q000096:biosynthesis*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology / Q000378:metabolism*
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D011107:Polylysine; D001222:Aspartate Kinase; D008239:Lysine