Genetic engineering combined with random mutagenesis to enhance G418 production in Micromonospora echinospora View Full Text


Ontology type: schema:ScholarlyArticle     


Article Info

DATE

2014-09

AUTHORS

Xianpu Ni, Zhenpeng Sun, Hongyu Zhang, Han He, Zhouxiang Ji, Huanzhang Xia

ABSTRACT

G418, produced by fermentation of Micromonospora echinospora, is an aminoglycoside antibiotic commonly used in genetic selection and maintenance of eukaryotic cells. Besides G418, M. echinospora produces many G418 analogs. As a result, the G418 product always contains impurities such as gentamicin C1, C1a, C2, C2a, gentamicin A and gentamicin X2. These impurities are less potent but more toxic than G418, but the purification of G418 is difficult because it has similar properties to its impurities. G418 is an intermediate in the gentamicin biosynthesis pathway. From G418 the pathway proceeds via successive dehydrogenation and aminotransferation at the C-6' position to generate the gentamicin C complex, but genes responsible for these steps are still obscure. Through disruption of gacJ, which is deduced to encode a C-6' dehydrogenase, the biosynthetic impurities gentamicin C1, C1a, C2 and C2a were all removed, and G418 became the main product of the gacJ disruption strain. These results demonstrated that gacJ is in charge of conversion of the 6'-OH of G418 into 6'-NH2. Disruption of gacJ not only eliminates the impurities seen in the original strain but also improves G418 titers by 15-fold. G418 production was further improved by 26.6 % through traditional random mutagenesis. Through the use of combined traditional and recombinant genetic techniques, we produced a strain from which most impurities were removed and G418 production was improved by 19 fold. More... »

PAGES

1383-1390

Identifiers

URI

http://scigraph.springernature.com/pub.10.1007/s10295-014-1479-3

DOI

http://dx.doi.org/10.1007/s10295-014-1479-3

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1030088026

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/25031027


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