Characterization and utilization of methyltransferase for apramycin production in Streptoalloteichus tenebrarius

Abstract

A structurally unique aminoglycoside produced in Streptoalloteichus tenebrarius, Apramycin is used in veterinary medicine or the treatment of Salmonella, Escherichia coli and Pasteurella multocida infections. Although apramycin was discovered nearly 50 years ago, many biosynthetic steps of apramycin remain unknown. In this study, we identified a HemK family methyltransferase, AprI, to be the 7’-N-methyltransferase in apramycin biosynthetic pathway. Biochemical experiments showed that AprI converted demethyl-aprosamine to aprosamine. Through gene disruption of aprI, we identified a new aminoglycoside antibiotic demethyl-apramycin as the main product in aprI disruption strain. The demethyl-apramycin is an impurity in apramycin product. In addition to demethyl-apramycin, carbamyltobramycin is another major impurity. However, unlike demethyl-apramycin, tobramycin is biosynthesized by an independent biosynthetic pathway in S. tenebrarius. The titer and rate of apramycin were improved by overexpression of the aprI and disruption of the tobM2, which is a crucial gene for tobramycin biosynthesis. The titer of apramycin increased from 2227±320 mg/L to 2331±210 mg/L, while the titer of product impurity demethyl-apramycin decreased from 196±36 mg/L to 51±9 mg/L. Moreover, the carbamyltobramycin titer of the wild-type strain was 607±111 mg/L and that of the engineering strain was null. The rate of apramycin increased from 68% to 87%, and that of demethyl-apramycin decreased from 1.17% to 0.34%.

Biosynthetic Origin of the Octose Core and Its Mechanism of Assembly during Apramycin Biosynthesis

“…If the retro-aldol bond cleavage between C2−C3 in 12/16 is the first step as proposed in mechanism A, then AprG may also catalyze degradation of 12/16 even in the absence of an acceptor. Indeed, upon incubation of AprG with 16 alone, Dthreose (21) and acetamidoacetaldehyde (25) were both detected by LC-MS and coelution with standards (Figure S11 and Section S9.2 in the SI). Furthermore, the incubation of lividamine (5) with BtrQ/AprG/AprU in the presence of 25, which is expected to be in equilibrium with 24 in free solution, also led to the production of phosphorylated octose 8 based on LC-MS analysis (Figure S16B(a)).…”

“…Biosynthetic pathway for apramycin (1). 17,21,24,25 Conversion of paromamine (4) to lividamine (5) is catalyzed by AprD4 and AprD3. 17−19 Subsequent AprQ mediated C6′ oxidation of 5 to oxolividamine (6) 17 sets the stage for octose assembly.…”

“…Biosynthetic pathway for apramycin ( 1 ). ,,, Conversion of paromamine ( 4 ) to lividamine ( 5 ) is catalyzed by AprD4 and AprD3. − Subsequent AprQ mediated C6′ oxidation of 5 to oxolividamine ( 6 ) sets the stage for octose assembly. The final steps include AprI catalyzed N -7′ methylation of 9 to give 10 , AprH mediated attachment of a 4-aminosugar, and AprZ catalyzed dephosphorylation of 11 to yield 1 .…”

“…How apramycin ( 1 ) is assembled has attracted much attention over the years. − The identification of its biosynthetic gene cluster ( apr cluster) − lead to significant progress in understanding the biological formation of apramycin (see Figure ). Apramycin ( 1 ) contains five amino groups at C1, C3, C2′, C7′ and C4″.…”