鲨肌醇(scyllo-inositol,SI)是治疗阿尔茨海默病的一种有前景的药物。谷氨酸棒杆菌具有合成SI的能力,葡萄糖-6-磷酸(glucose-6-phosphate,G6P)在肌醇磷酸合酶和肌醇单磷酸酶的作用下生成肌肌醇(myo-inositol,MI),再通过MI脱氢酶IolG和SI脱氢酶IolW催化得到SI,但并不积累SI。为了实现SI的从头合成,该研究敲除了谷氨酸棒杆菌SN01中除iolW和iolG以外的肌醇分解代谢基因,整合了外源肌醇磷酸合酶和肌醇单磷酸酶基因,并用强启动子PtacM替换iolW天然启动子,菌株SD-1成功从20 g/L MI转化得到18.65 g/L SI,但不能从葡萄糖从头合成SI。随后,分别采用生长依赖型启动子和Esa群体感应系统下调G6P竞争途径的通量,并过表达内源肌醇磷酸合酶编码基因ino1,菌株SDI-1可以以葡萄糖为底物从头合成2.26 g/L SI。此外,该研究还探索了提高前体G6P的富集以及增加辅因子NAD+供应对SI从头合成的影响,最佳菌株SDN-2最终可以合成2.29 g/L SI,为目前报道的微生物以葡萄糖为底物从头合成SI的最高产量。
Scyllo-inositol (SI) is a promising drug for the treatment of Alzheimer’s disease.Corynebacterium glutamicum has the inherent ability to synthesize SI, Myo-inositol 1-phosphate synthase and myo-inositol 1-phosphatase catalyze the conversion of glucose-6-phosphate (G6P) to myo-inositol (MI), and MI dehydrogenase IolG and SI dehydrogenase IolW thereafter convert MI into SI.But C.glutamicum cannot accumulate SI.To achieve de novo synthesis of SI, this study knocked out the inositol catabolic genes except iolW and iolG in C.glutamicum SN01, integrated the exogenous myo-inositol 1-phosphate synthase and myo-inositol 1-phosphatase genes, and replaced the iolW native promoter with the strong promoter PtacM.The strain SD-1 successfully converted 20 g/L MI to 18.65 g/L SI, but could not produce SI from glucose.Subsequently, the growth-dependant promoter or the Esa quorum-sensing system were used to down-regulate the flux of the G6P competition pathways, and the endogenous myo-inositol 1-phosphate synthase coding gene ino1 was overexpressed.Strain SDI-1 could synthesize 2.26 g/L SI from glucose.In addition, the effects of increasing the enrichment of precursor G6P and the supply of co-factor NAD+ on the SI de novo synthesis were explored in this study.The best strain SDN-2 could finally synthesize 2.29 g/L SI, which is the highest titer of de novo SI synthesis from glouse by microorganisms.
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