The elucidation of phosphosugar stress response inBacillus subtilisguides strain engineering for highN-acetylglucosamine production
The elucidation of phosphosugar stress response inBacillus subtilisguides strain engineering for highN-acetylglucosamine production
复制标题
枯草芽孢杆菌磷糖应激反应的阐明指导高产 N-乙酰氨基葡萄糖的菌株工程
DOI：
10.1002/bit.27577
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发表时间：
2020-10-08
影响因子：
3.8
通讯作者：
Liu, Long
中科院分区：
文献类型：
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作者：
Niu, Tengfei;Lv, Xueqin;Liu, Long
枯草芽孢杆菌是一种用于营养素工业生产的首选微生物宿主，也是合成功能性糖的有前途的候选者，例如N-乙酰葡萄糖胺（GLCNAC）。以前是GlcNAC-Overroducerb。使用Glmsribozyme双调节工具构建了枯草菌。在此，我们进一步设计了从葡萄糖到GlcNAC合成的碳通量。结果，向GlcNAC合成的通量增加触发了磷糖应激反应，从而导致细胞异常的生长。不幸的是，未阐明INB的磷糖应激反应的机制。枯草脂。为了揭示应力机制并克服其在生物生产中的负面影响，我们进行了比较转录组分析。结果表明，细胞通过抑制葡萄糖进口和加速磷果糖的分解代谢反应来减缓葡萄糖的利用。为了验证这些结果，我们过度表达了磷酸酶YWPJ，从而缓解了磷酸盐应激，并使我们能够鉴定出负责从GlcNAC 6-磷酸盐的GlcNAC合成的酶。此外，负责GLCNAC前体降解的纳格班班班班尔克的缺失进一步改善了GlcNAC合成。最好的工程菌株b。在奶昔烧瓶中将GlcNAC滴度从11.5增加到26.1 g/L，并在30-l喂食生物反应器中产生87.5 g/L GlcNAC。我们的结果不仅首次阐明了磷光应力反应机制INB。枯草厂，但还展示了理性代谢工程与对生理学和代谢的新见解的结合，允许建造高效的微生物细胞工厂来生产高价值化学物质。
Bacillus subtilisis a preferred microbial host for the industrial production of nutraceuticals and a promising candidate for the synthesis of functional sugars, such asN-acetylglucosamine (GlcNAc). Previously, a GlcNAc-overproducerB. subtilisSFMI was constructed usingglmSribozyme dual-regulatory tool. Herein, we further engineered to enhance carbon flux from glucose towards GlcNAc synthesis. As a result, the increased flux towards GlcNAc synthesis triggered phosphosugar stress response, which caused abnormal cell growth. Unfortunately, the mechanism of phosphosugar stress response had not been elucidated inB. subtilis. To reveal the stress mechanism and overcome its negative effect in bioproduction, we performed comparative transcriptome analysis. The results indicate that cells slow glucose utilization by repression of glucose import and accelerate catabolic reactions of phosphosugar. To verify these results, we overexpressed the phosphatase YwpJ, which relieved phosphosugar stress and allowed us to identify the enzyme responsible for GlcNAc synthesis from GlcNAc 6-phosphate. In addition, the deletion ofnagBBandmurQ, responsible for GlcNAc precursor degradation, further improved GlcNAc synthesis. The best engineered strain,B. subtilisFMIP34, increased GlcNAc titer from 11.5 to 26.1 g/L in shake flasks and produced 87.5 g/L GlcNAc in 30-L fed-batch bioreactor. Our results not only elucidate, for the first time, the phosphosugar stress response mechanism inB. subtilis, but also demonstrate how the combination of rational metabolic engineering with novel insights into physiology and metabolism allows the construction of highly efficient microbial cell factories for the production of high-value chemicals.