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在枯草芽孢杆菌中优化甲基供体和减少前体降解途径以生产硒代甲基硒代半胱氨酸。

Optimized methyl donor and reduced precursor degradation pathway for seleno-methylselenocysteine production in Bacillus subtilis.

机构信息

Key Laboratory of Geriatric Nutrition and Health (Ministry of Education), Beijing Technology and Business University, Fucheng RD 11, Beijing, 100048, China.

China Food Flavor and Nutrition Health Innovation Center, Beijing Technology and Business University, Fucheng RD 11, Beijing, 100048, China.

出版信息

Microb Cell Fact. 2023 Oct 19;22(1):215. doi: 10.1186/s12934-023-02203-1.

Abstract

BACKGROUND

Seleno-methylselenocysteine (SeMCys) is an effective component of selenium supplementation with anti-carcinogenic potential that can ameliorate neuropathology and cognitive deficits. In a previous study, a SeMCys producing strain of Bacillus subtilis GBACB was generated by releasing feedback inhibition by overexpression of cysteine-insensitive serine O-acetyltransferase, enhancing the synthesis of S-adenosylmethionine as methyl donor by overexpression of S-adenosylmethionine synthetase, and expressing heterologous selenocysteine methyltransferase. In this study, we aimed to improve GBACB SeMCys production by synthesizing methylmethionine as a donor to methylate selenocysteine and by inhibiting the precursor degradation pathway.

RESULTS

First, the performance of three methionine S-methyltransferases that provide methylmethionine as a methyl donor for SeMCys production was determined. Integration of the NmMmt gene into GBACB improved SeMCys production from 20.7 to 687.4 μg/L. Next, the major routes for the degradation of selenocysteine, which is the precursor of SeMCys, were revealed by comparing selenocysteine hyper-accumulating and non-producing strains at the transcriptional level. The iscSB knockout strain doubled SeMCys production. Moreover, deleting sdaA, which is responsible for the degradation of serine as a precursor of selenocysteine, enhanced SeMCys production to 4120.3 μg/L. Finally, the culture conditions in the flasks were optimized. The strain was tolerant to higher selenite content in the liquid medium and the titer of SeMCys reached 7.5 mg/L.

CONCLUSIONS

The significance of methylmethionine as a methyl donor for SeMCys production in B. subtilis is reported, and enhanced precursor supply facilitates SeMCys synthesis. The results represent the highest SeMCys production to date and provide insight into Se metabolism.

摘要

背景

硒代甲基硒代半胱氨酸(SeMCys)是一种具有抗癌潜力的硒补充剂的有效成分,可改善神经病理学和认知缺陷。在之前的研究中,通过过表达半胱氨酸不敏感丝氨酸 O-乙酰转移酶来释放反馈抑制作用,通过过表达 S-腺苷甲硫氨酸合成酶来增强 S-腺苷甲硫氨酸作为甲基供体的合成,并表达异源硒代半胱氨酸甲基转移酶,生成了枯草芽孢杆菌 GBACB 的 SeMCys 产生菌株。在这项研究中,我们旨在通过合成甲硫氨酸作为甲基供体来甲基化硒代半胱氨酸,并抑制前体降解途径,来提高 GBACB 生产 SeMCys 的能力。

结果

首先,确定了三种甲硫氨酸 S-甲基转移酶的性能,这些酶为 SeMCys 生产提供甲基甲硫氨酸作为甲基供体。将 NmMmt 基因整合到 GBACB 中,将 SeMCys 的产量从 20.7 提高到 687.4μg/L。接下来,通过比较超积累和非生产型硒代半胱氨酸菌株的转录水平,揭示了硒代半胱氨酸(SeMCys 的前体)降解的主要途径。IscSB 敲除菌株使 SeMCys 产量增加了一倍。此外,删除负责降解丝氨酸(硒代半胱氨酸前体)的 sdaA,将 SeMCys 产量提高到 4120.3μg/L。最后,优化了摇瓶中的培养条件。该菌株能够耐受液体培养基中更高的亚硒酸盐含量,SeMCys 的产量达到 7.5mg/L。

结论

报告了甲基甲硫氨酸作为枯草芽孢杆菌中 SeMCys 生产的甲基供体的重要性,并且增强前体供应有助于 SeMCys 的合成。该结果代表了迄今为止最高的 SeMCys 产量,并为 Se 代谢提供了新的见解。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b313/10585787/8687e7c3b744/12934_2023_2203_Fig1_HTML.jpg

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