Characterization and utilization of methyltransferase for apramycin production in Streptoalloteichus tenebrarius
Junyang Sun1, Hongjing Gao1, Danyang Yan1
1School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, People's Republic of China.
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%.
Insights
Researchers identified AprI as a key enzyme in apramycin biosynthesis, reducing impurities like demethyl-apramycin and carbamyltobramycin. This work enhances apramycin production for veterinary use.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Apramycin, a unique aminoglycoside antibiotic, is vital in veterinary medicine for treating bacterial infections.
- Despite its long history, the complete biosynthetic pathway of apramycin remains largely unelucidated.
- Understanding apramycin biosynthesis is crucial for improving its production and purity.
Purpose of the Study:
- To identify the 7'-N-methyltransferase responsible for apramycin biosynthesis.
- To characterize the role of the identified enzyme in the pathway.
- To engineer the apramycin biosynthetic pathway for enhanced production and reduced impurities.
Main Methods:
- Identification and characterization of a HemK family methyltransferase, AprI.
- Biochemical assays to confirm AprI's enzymatic activity (demethyl-aprosamine to aprosamine conversion).
- Gene disruption of aprI to analyze its impact on apramycin production.
- Genetic engineering strategies involving overexpression of aprI and disruption of tobramycin biosynthesis genes (e.g., tobM2).
Main Results:
- AprI was confirmed as the 7'-N-methyltransferase in apramycin biosynthesis.
- Disruption of aprI led to the accumulation of demethyl-apramycin, a previously unrecognized impurity.
- Overexpression of aprI and disruption of tobM2 significantly increased apramycin titer (2227 to 2331 mg/L) and purity.
- Impurity levels of demethyl-apramycin decreased from 196 to 51 mg/L, and carbamyltobramycin was eliminated.
Conclusions:
- AprI is a critical enzyme for apramycin production, catalyzing a key N-methylation step.
- Genetic manipulation of apramycin and tobramycin biosynthetic pathways can enhance apramycin yield and purity.
- This study provides a foundation for further metabolic engineering of apramycin production in Streptoalloteichus tenebrarius.
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