Enhanced lincomycin production by co-overexpression of metK1 and metK2 in Streptomyces lincolnensis
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Streptomyces lincolnensis is generally utilized for the production of lincomycin A (Lin-A), a clinically useful antibiotic to treat Gram-positive bacterial infections. Three methylation steps, catalyzed by three different S-adenosylmethionine (SAM)-dependent methyltransferases, are required in the biosynthesis of Lin-A, and thus highlight the significance of methyl group supply in lincomycin production. In this study, we demonstrate that externally supplemented SAM cannot be taken in by cells and therefore does not enhance Lin-A production. Furthermore, bioinformatics and in vitro enzymatic assays revealed there exist two SAM synthetase homologs, MetK1 (SLCG_1651) and MetK2 (SLCG_3830) in S. lincolnensis that could convert l-methionine into SAM in the presence of ATP. Even though we attempted to inactivate metK1 and metK2, only metK2 was deleted in S. lincolnensis LCGL, named as ΔmetK2. Following a reduction of the intracellular SAM concentration, ΔmetK2 mutant exhibited a significant decrease of Lin-A in comparison to its parental strain. Individual overexpression of metK1 or metK2 in S. lincolnensis LCGL either elevated the amount of intracellular SAM, concomitant with 15% and 22% increase in Lin-A production, respectively. qRT-PCR assays showed that overexpression of either metK1 or metK2 increased the transcription of lincomycin biosynthetic genes lmbA and lmbR, and regulatory gene lmbU, indicating SAM may also function as a transcriptional activator. When metK1 and metK2 were co-expressed, Lin-A production was increased by 27% in LCGL, while by 17% in a high-yield strain LA219X.
KeywordsStreptomyces lincolnensis Lincomycin S-adenosylmethionine (SAM) SAM synthetase
We are grateful to Dr. Sabrina Huber from Department of Biological Engineering at Massachusetts Institute of Technology for critical editing of the manuscript. This work was supported by the National Natural Science Foundation of China (31300081, 31570074, 31600064), the National Program on Key Basic Research Project (973 programs, 2013CB734000), Open Project of State Key Laboratory of Microbial Metabolism from Shanghai Jiao Tong University (MMLKF13-05), the Initial Foundation of Doctoral Scientific Research in Anhui University (01001904, J01001935), and the National Innovation Experiment Program for University Students (J10118516053).
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conflict of interest
The authors declare that they have no competing interests.
Research involving human and animal participants
This article does not contain any studies with human participants or animals performed by any of the authors.
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