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%.

Keywords: Streptoalloteichus tenebrarius; aprI gene; Apramycin biosynthesis pathway; Methyltransferase.

Fig. 1

Apramycin biosynthetic gene cluster, tobramycin biosynthetic gene cluster and proposed apramycin biosynthetic pathway. (A) Apramycin biosynthetic gene cluster and tobramycin biosynthetic gene cluster. (B) Proposed apramycin biosynthetic pathway.

Fig. 2

Identification of AprI as the methyltransferase for 7’-N methylation. (A) Schematic representation of the aprI deletion. (B) Verification of ΔaprI mutant by PCR. PCR amplification with wild-type strain chromosome generated a 4.8 kb fragment, and ΔaprI strain chromosome generated a 4.0 kb fragment. (C) The analysis of products by TLC from wild-type S. tenebrarius and the ∆aprI. (D) HPLC analysis was performed by using an evaporative light-scattering detector (ELSD); (i) apramycin standard, (ii) wild-type S. tenebrarius, (iii) ∆aprI, and (iv) ∆aprI containing an aprI-expressing plasmid.

Fig. 3

Characterization of AprI as a SAM-dependent methyltransferase. (A) SDS-PAGE analysis of AprI. (B) HPLC-ELSD analysis of the conversion of demethyl-aprosamine by AprI in vitro; (i) demethyl-aprosamine, (ii) control reaction in which AprI was omitted, and (iii) AprI reaction with demethyl-aprosamine and SAM. AprI converted demethyl-aprosamine into aprosamine. (C) HPLC-ELSD analysis of the conversion of demethyl-apramycin by AprI in vitro. (i) demethyl-apramycin, (ii) apramycin, and (iii) AprI reaction with demethyl-apramycin and SAM. AprI could not convert demethyl-apramycin into apramycin. (D) The 7’-N position methylation biosynthetic route of apramycin. AprI converts demethyl-aprosamine into aprosamine. However, AprI cannot convert demethyl-apramycin into apramycin.

Fig. 4

Construction and confirmation of the aprI overexpressing strain S. tenebrarius IB. (A) Schematic representation of the S. tenebrarius IB construction via homologous recombination. (B) Verification of S. tenebrarius IB homologous recombination events. Primers PhrdB-up and IB-P3 were used and PCR amplification showed that S. tenebrarius IB had a 1.2 kb fragment. (C) Analysis of secondary metabolite yields.