Genetic engineering combined with random mutagenesis to enhance G418 production inMicromonospora echinospora

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.

Assembly of a novel biosynthetic pathway for gentamicin B production in Micromonospora echinospora

“…Solid slanting medium was used for M. echinospora sporulation. The previously described media and culture conditions were used for gentamicin production [ 8 ].…”

Exclusive Production of Gentamicin C1a from Micromonospora purpurea by Metabolic Engineering

“…Most of the key steps in the biosynthesis of gentamicin C components have been elucidated in previous work ( Figure 2): genK is an S-Adenosyl methionine (SAM) dependent methylation gene and is responsible for C-6' methylation [11,12]; genP is thought to be responsible for 3', 4' double dehydroxylation [13,14]; genQ is related to Gentamicin C-6' dehydrogenation [15]; genB1 catalyzes the transformation of G418 to JI-20B and X2 to JI-20A [16]; genB2 involves in the epimerization of gentamicin C2 and C2a in vitro [16]; and genL is reported to be responsible for converting C1a to C2b and C2 to C1 of Micromonospora echinospora ATCC15835 [17]. Figure 2.…”

“…Previously, our team constructed an M. purpurea strain GK1101 to increase the titers of C1a [11], where C1 metabolic flux was blocked by inactivating the genK pathway ( Figure 2). However, this strain still produced a small amount of gentamicin C2b and an extra separation step is required to Most of the key steps in the biosynthesis of gentamicin C components have been elucidated in previous work ( Figure 2): genK is an S-Adenosyl methionine (SAM) dependent methylation gene and is responsible for C-6' methylation [11,12]; genP is thought to be responsible for 3', 4' double dehydroxylation [13,14]; genQ is related to Gentamicin C-6' dehydrogenation [15]; genB1 catalyzes the transformation of G418 to JI-20B and X2 to JI-20A [16]; genB2 involves in the epimerization of gentamicin C2 and C2a in vitro [16]; and genL is reported to be responsible for converting C1a to C2b and C2 to C1 of Micromonospora echinospora ATCC15835 [17]. Most of the key steps in the biosynthesis of gentamicin C components have been elucidated in previous work ( Figure 2): genK is an S-Adenosyl methionine (SAM) dependent methylation gene and is responsible for C-6' methylation [11,12]; genP is thought to be responsible for 3', 4' double dehydroxylation [13,14]; genQ is related to Gentamicin C-6' dehydrogenation [15]; genB1 catalyzes the transformation of G418 to JI-20B and X2 to JI-20A [16]; genB2 involves in the epimerization of gentamicin C2 and C2a in vitro [16]; and genL is reported to be responsible for converting C1a to C2b and C2 to C1 of Micromonospora echinospora ATCC15835 [17].…”

Recent progress on the development of antibiotics from the genus Micromonospora

“…The inactivation of gacD blocks the metabolic pathways from X2 to G418 and leads to the accumulation of gentamicin C1a [292]. Through the use of combined traditional and recombinant genetic techniques it was possible to obtain strain with improved G418 production by 19 fold with minimal by product formation [293]. gene cluster and biosyntheic pathway [300][301][302][303], and diazepinomicin biosynthetic pathway [304][305][306].…”