Kosalková K, Marcos A T, Martín J F
Area de Microbiología, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, León 24071, Spain.
J Ind Microbiol Biotechnol. 2001 Oct;27(4):252-8. doi: 10.1038/sj.jim.7000192.
L-cysteine is a precursor of the penicillin, cephalosporin and cephamycin families of beta-lactam antibiotics. Cystathionine-gamma-lyase (encoded by the mecB gene), an enzyme that splits cystathionine releasing cysteine, is required for high-level cephalosporin production in methionine-supplemented medium. By amplification of the mecB gene in Acremonium chrysogenum C10, several transformants were obtained that produced 10-40% higher levels of cephalosporin. All selected transformants contained at least two or three copies of the mecB gene as shown by Southern hybridization with a probe internal to mecB. Two of these transformants, A. chrysogenum T27 and A. chrysogenum T58, showed 4- to 10-fold higher cystathionine-gamma-lyase activity than the control strain. Northern hybridizations indicated that the levels of the two mecB transcripts of 1.7 and 1.5 kb were greatly increased in transformants T27 and T58. Fermentor studies using controlled conditions confirmed that transformant T27 was a cephalosporin overproducer, reaching titers of nearly 2000 microg/ml of cephalosporin in Shen-defined medium that correlated with two- to fourfold higher cystathionine-gamma-lyase levels than in the control strain. Transformant T58 containing five- to sixfold higher levels of cystathionine-gamma-lyase in fermentor cultures showed a reduced growth rate and a slow cephalosporin accumulation rate. In conclusion, moderately increased levels of cystathionine-gamma-lyase stimulated cephalosporin production but very high levels of this enzyme were deleterious for growth and cephalosporin biosynthesis.
L-半胱氨酸是β-内酰胺抗生素中青霉素、头孢菌素和头霉素家族的前体。胱硫醚-γ-裂解酶（由mecB基因编码）是一种能分解胱硫醚释放半胱氨酸的酶，在补充蛋氨酸的培养基中，高水平头孢菌素的产生需要该酶。通过在产黄顶头孢霉C10中扩增mecB基因，获得了几个转化体，它们产生的头孢菌素水平比原来高10%-40%。如用mecB内部探针进行Southern杂交所示，所有筛选出的转化体至少含有两到三个mecB基因拷贝。其中两个转化体，产黄顶头孢霉T27和产黄顶头孢霉T58，其胱硫醚-γ-裂解酶活性比对照菌株高4至10倍。Northern杂交表明，1.7 kb和1.5 kb的两种mecB转录本水平在转化体T27和T58中大幅增加。在可控条件下进行的发酵罐研究证实，转化体T27是头孢菌素高产菌株，在沈氏定义培养基中头孢菌素滴度接近2000μg/ml，这与胱硫醚-γ-裂解酶水平比对照菌株高两到四倍相关。在发酵罐培养中胱硫醚-γ-裂解酶水平高五到六倍的转化体T58，其生长速率降低，头孢菌素积累速率缓慢。总之，胱硫醚-γ-裂解酶水平适度增加刺激了头孢菌素的产生，但该酶水平过高对生长和头孢菌素生物合成有害。
