Construction of hybrid bacterial deacetoxycephalosporin C synthases (expandases) by in vivo homeologous recombination

Homeologous recombination (recombination between partially homeologous DNA sequences) was used to produce novel functional deacetoxycephalosporin C synthase (expandase) enzymes in vivo which are hybrids of the Streptomyces clavuligerus and Nocardia lactamdurans enzymes. DNA sequencing of hybrids obtained in E. coli showed that recombination had occurred at several locations within conserved sequences as short as 2 bp. Recombination events obtained in a Streptomyces background resulted in expandases with altered activity on penicillin G as determined by bioassay and HPLC.     

Performance of a recombinant strain of<Emphasis Type="Italic"> Streptomyces lividans</Emphasis> for bioconversion of penicillin G to deacetoxycephalosporin G

We examined the performance of Streptomyces lividans strain W25 containing a hybrid expandase (deacetoxycephalosporin C synthase; DAOCS) gene, obtained by in vivo recombination between the expandase genes of S. clavuligerus and Nocardia lactamdurans for resting-cell bioconversion of penicillin G to deacetoxycephalosporin G. Strain W25 carried out a much more effective level of bioconversion than the previously used strain, S. clavuligerus NP1. The two strains also differed in the concentrations of FeSO₄ and α-ketoglutarate giving maximal activity. Whereas NP1 preferred 1.8 mM FeSO₄ and 1.3 mM α-ketoglutarate, recombinant W25 performed best at 0.45 mM FeSO₄ and 1.9 mM α-ketoglutarate. Electronic Publication     

扩环酶的研究进展及其应用前景

扩环酶,也叫脱乙酰氧基头孢菌素Ｃ合成酶,催化青霉素Ｎ扩环生成脱乙酰氧基头孢菌素Ｃ,是头孢菌素生物合成中的关键酶。在真菌中它是一个双功能酶,同时具有扩环酶和羟化酶的活性;而在细菌中扩环和羟化却是由两个独立的酶来承担的。近年来,扩环酶被纯化成均一蛋白,有关它的性质、分子结构以及基因结构等方面的研究都取得了飞速发展,并不断地应用基因工程的技术探索其在抗生素生产上的应用。