School of Life Sciences, School of Chemistry and Chemical Engineering, Institute of Physical Science and Information Technology, Anhui University, Hefei, 230601, China.
State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai, 200240, China.
J Ind Microbiol Biotechnol. 2018 May;45(5):345-355. doi: 10.1007/s10295-018-2029-1. Epub 2018 Mar 24.
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.
林肯链霉菌通常用于生产林可霉素 A（Lin-A），这是一种用于治疗革兰氏阳性细菌感染的临床有效抗生素。在林可霉素 A 的生物合成中，需要经过三个不同的 S-腺苷甲硫氨酸（SAM）依赖性甲基转移酶催化的甲基化步骤，因此凸显了甲基供体在林可霉素生产中的重要性。在本研究中，我们证明了细胞不能摄取外部补充的 SAM，因此不会提高林可霉素 A 的产量。此外，生物信息学和体外酶促分析表明，林肯链霉菌中存在两种 SAM 合成酶同源物，MetK1（SLCG_1651）和 MetK2（SLCG_3830），它们可以在 ATP 存在的情况下将 L-蛋氨酸转化为 SAM。尽管我们试图使 metK1 和 metK2 失活，但只有 metK2 在林肯链霉菌 LCGL 中被删除，命名为 ΔmetK2。当细胞内 SAM 浓度降低时，与亲本菌株相比，ΔmetK2 突变体的林可霉素 A 产量显著下降。在林肯链霉菌 LCGL 中单独过表达 metK1 或 metK2 分别提高了细胞内 SAM 的含量，并使林可霉素 A 的产量分别增加了 15%和 22%。qRT-PCR 检测表明，metK1 或 metK2 的过表达均增加了林可霉素生物合成基因 lmbA 和 lmbR 以及调节基因 lmbU 的转录，表明 SAM 也可能作为转录激活剂发挥作用。当 metK1 和 metK2 同时过表达时，LCGL 中的林可霉素 A 产量增加了 27%，而在高产菌株 LA219X 中增加了 17%。
