Niu Tengfei, Lv Xueqin, Liu Yanfeng, Li Jianghua, Du Guocheng, Ledesma-Amaro Rodrigo, Liu Long
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China.
Science Center for Future Foods, Jiangnan University, Wuxi, China.
Biotechnol Bioeng. 2021 Jan;118(1):383-396. doi: 10.1002/bit.27577. Epub 2020 Oct 8.
Bacillus subtilis is a preferred microbial host for the industrial production of nutraceuticals and a promising candidate for the synthesis of functional sugars, such as N-acetylglucosamine (GlcNAc). Previously, a GlcNAc-overproducer B. subtilis SFMI was constructed using glmS ribozyme 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 in B. 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 of nagBB and murQ, responsible for GlcNAc precursor degradation, further improved GlcNAc synthesis. The best engineered strain, B. subtilis FMIP34, 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 in B. 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.
枯草芽孢杆菌是工业生产营养保健品的首选微生物宿主，也是合成功能性糖类（如N - 乙酰葡萄糖胺（GlcNAc））的有潜力的候选菌株。此前，利用glmS核酶双调控工具构建了GlcNAc高产菌株枯草芽孢杆菌SFMI。在此，我们进一步进行工程改造，以增强从葡萄糖到GlcNAc合成的碳通量。结果，流向GlcNAc合成的通量增加引发了磷酸糖应激反应，导致细胞生长异常。遗憾的是，枯草芽孢杆菌中磷酸糖应激反应的机制尚未阐明。为了揭示应激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摄取来减缓葡萄糖利用，并加速磷酸糖的分解代谢反应。为了验证这些结果，我们过表达了磷酸酶YwpJ，其缓解了磷酸糖应激，并使我们能够鉴定出从6 - 磷酸葡萄糖胺合成GlcNAc的酶。此外，删除负责GlcNAc前体降解的nagBB和murQ，进一步提高了GlcNAc的合成。最佳工程菌株枯草芽孢杆菌FMIP34在摇瓶中使GlcNAc产量从11.5克/升提高到26.1克/升，并在30升补料分批生物反应器中产生了87.5克/升的GlcNAc。我们的结果不仅首次阐明了枯草芽孢杆菌中的磷酸糖应激反应机制，还展示了合理的代谢工程与对生理学和代谢的新见解相结合如何能够构建用于生产高价值化学品的高效微生物细胞工厂。
