Targeted disruption of homoserine dehydrogenase gene and its effect on cephamycin C production in Streptomyces clavuligerus

Abstract

The aspartate pathway of Streptomyces clavuligerus is an important primary metabolic pathway which provides substrates for beta-lactam synthesis. In this study, the hom gene which encodes homoserine dehydrogenase was cloned from the cephamycin C producer S. clavuligerus NRRL 3585 and characterized. The fully sequenced open reading frame encodes 433 amino acids with a deduced M (r) of 44.9 kDa. The gene was heterologously expressed in the auxotroph mutant Escherichia coli CGSC 5075 and the recombinant protein was purified. The cloned gene was used to construct a plasmid containing a hom disruption cassette which was then transformed into S. clavuligerus. A hom mutant of S. clavuligerus was obtained by insertional inactivation via double crossover, and the effect of hom gene disruption on cephamycin C yield was investigated by comparing antibiotic levels in culture broths of this mutant and in the parental strain. Disruption of hom gene resulted in up to 4.3-fold and twofold increases in intracellular free L-lysine concentration and specific cephamycin C production, respectively, during stationary phase in chemically defined medium.

Molecular Mechanisms of Persistence of Mutualistic Bacteria Photorhabdus in the Entomopathogenic Nematode Host

“…Induction of metL gene in P. temperata , which is known to be responsible for the synthesis of homoserine which serves as a precursor to threonine, lysine and methionine, reflects a growth-limiting concentration of these amino acids or an enhanced need for homoserine in the nematode as has been noted in Salmonella during infection of pigs [14]. Disruption of metL results in an increase in intracellular free lysine concentration during stationary phase in Streptomyces clavuligerus [15]. The fact that metL mutant supported nematode reproduction but had reduced persistence in the infective juveniles suggests that lysine may be helpful to H. bacteriophora growth and homoserine may be needed for P. temperata survival in the nematode.…”

Homologous expression of aspartokinase (ask) gene inStreptomyces clavuligerusand itshom-deleted mutant: Effects on cephamycin C production

“…In our previous study, the growth of the AK39 strain was rather poor in chemically-defined medium, CDM, in spite of a two-fold increased specific level of cephamycin C. 17 Some inhibitory effects of glycerol on cephamycin C production 18,19 as well as improvement of growth and cephamycin C yields by L-asparagine 20,21 have been previously reported. In the present study, the composition of CDM was submitted to several modifications to improve the growth of this strain and their transformants.…”

“…Cells were grown in mCDM, and samples were collected at intervals during cultivation. Intracellular free amino acids were extracted as described by Yılmaz et al 17 Following automated pre-column derivatization with o-phthaladehyde (OPA), amino acid analysis was performed by conventional reverse-phase Growth determination. Cultural growth was determined by DNA quantification with diphenylamine method adapted from Burton.…”

“…The disruption of hom resulted in a twofold increase in cephamycin C specific production in chemically defined medium. 17 The present study is aimed at the expression of multiple copies of the homologous ask gene in the wild strain of S. clavuligerus NRRL3585 and its hom-disrupted mutant, and to determine the effects of ask amplification on cephamycin C production.…”

Metabolomic analysis of biosynthesis mechanism of ε-polylysine produced by Streptomyces diastatochromogenes

“…By investigating the feedback of aspartate kinase, Hamano et al obtained a high-yield ε-PL producing strain (M68V) (Hamano et al 2007). By knocking out the homoserine dehydrogenase gene in Streptomyces clavuligerus, Yilmaz et al obtained a mutagenized strain with a 4.3-fold ε-PL yield compared with the original strain (Yılmaz et al 2008). Vanooyen et al found that reducing the activity of citrate synthase can buffer the increase of the substrate and thus obtained a high-yield lysine producing strain of Corynebacterium glutamicum (Vanooyen et al 2012).…”