Improvement of nikkomycin production by enhanced copy of sanU and sanV in Streptomyces ansochromogenes and characterization of a novel glutamate mutase encoded by sanU and sanV.
Previous studies revealed that two genes-sanU and sanV were associated with nikkomycin biosynthesis in Streptomyces ansochromogenes. A plasmid used to increase an extra copy of sanU and sanV was constructed and introduced into wild-type strain. HPLC results showed that nikkomycin production of recombinant strain was about 1.8 fold than that of wild-type strain. RT-PCR analysis indicated that the transcriptional level of sanU and sanV in this recombinant strain was about two folds than that of wild-type strain. The sanU and sanV were expressed in E. coli BL21 (DE3). SanU and SanV were purified individually. SanU and SanV assembled with coenzyme B12 to form a complete enzyme in vitro, which showed glutamate mutase activity. The glutamate mutase converted L-glutamate toL-threo-beta-Methylaspartic acid, and then l-threo-beta-Methylaspartic acid was probably deaminated to form 2-oxo-3-methylsuccinic acid to join biosynthetic pathway of the peptidyl moiety HPHT in S. ansochromogenes. SanU is the coenzyme B12-binding component and more than two folds of SanU are required for maximal enzyme activity. The optimal pH and temperature for the formed enzyme are 7.5-8.5 and 35-42 degrees C, respectively. Sulfhydryl compounds are important for activity of the reassembled enzyme.
Li
Yirong
Y
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Science, Beijing 100080, PR China.
Ling
Hongbo
H
Li
Wenli
W
Tan
Huarong
H
D000617:Aminoglycosides / Q000096:biosynthesis* / Q000235:genetics; D004789:Enzyme Activation; D004795:Enzyme Stability; D018628:Gene Dosage; D024861:Genetic Enhancement / Q000379:methods*; D019751:Intramolecular Transferases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D013045:Species Specificity; D013302:Streptomyces / Q000145:classification / Q000235:genetics* / Q000378:metabolism*
D000617:Aminoglycosides; C011952:nikkomycin; D019751:Intramolecular Transferases; C021570:methylaspartate mutase