Biosynthesis of Epimers C2 and C2a in the Gentamicin C Complex

Abstract

Gentamicin is a broad-spectrum aminoglycoside antibiotic widely used to treat life-threatening bacterial infections. The gentamicin C complex consists of gentamicin C1, gentamicin C1a, and epimers gentamicin C2 and gentamicin C2a. At present there is a generally accepted pathway of gentamicin biosynthesis, except for detailed understanding of the epimerization process involving gentamicins C2 and C2a. Here we have investigated the biosynthesis of these epimers. JI-20B-an intermediate in the gentamicin biosynthetic pathway-and its epimer JI-20Ba were generated by in-frame deletion within genP, which encodes a phosphotransferase that catalyzes the first step of 3',4'-bisdehydroxylation in gentamicin biosynthesis. GenB1 and GenB2 are aminotransferases with different substrate specificities and enantioselectivities. JI-20Ba, containing a 6'S chiral amine, a precursor of gentamicin C2a, was synthesized from G418 by GenQ/GenB1 through sequential oxidation/transamination at C-6'. GenQ/GenB2 catalyzed the synthesis of JI-20B, containing a 6'R chiral amine, a precursor of gentamicin C2, from G418. GenB2 catalyzed the epimerization of JI-20Ba/JI-20B and of gentamicins C2a/C2.

The bifunctional enzyme, GenB4, catalyzes the last step of gentamicin 3′,4′-di-deoxygenation via reduction and transamination activities

“…Importantly, when GenB4-GenB2 were combined to catalyze (3), the reaction efficiency was remarkably improved and produced the deamination product, 6′-deamino-6′-oxoverdamicin (1). Hence, we concluded that (2) was verdamicin C2a and that (3) was verdamicin C2 [21].…”

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

“…The production of gentamicin X2 from A2 involves four enzymes: oxidoreductase, transaminase, and two methyltransferases [ 6 ]. X2 can also be transformed into JI-20A by C-6ʹ dehydrogenase and transaminase [ 7 – 9 ]. Moreover, GenK is the methyltransferase of C-6ʹ in gentamicin, and gen K disruption generates a gentamicin C1a-overproducing strain [ 10 – 12 ].…”

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].…”