Previously, the production of N-acetylglucosamine (GlcNAc) in Saccharomyces cerevisiae was improved by deletion of the genes encoding phosphofructokinase 2 (PFK-2) isoforms, which reduced the glycolytic flux by eliminating the pathway to produce fructose-2,6-bisphosphate, an allosteric activator of phosphofructokinase 1 (PFK-1). We further examined the effects of an additional reduction in glucose metabolic rate on N-acetylglucosamine production. Glucose uptake rate was lowered by expressing a gene encoding truncated glucose-sensing regulator (MTH1-ΔT). In addition, catalytically dead Cas9 (dCas9) was introduced in order to down-regulate the expression levels of PFK-1 and pyruvate kinase-1 (Pyk1). Finally, the three strategies were introduced into S. cerevisiae strains in a combinatorial way; the strain containing all three modules resulted in the highest N-acetylglucosamine production yield. The results showed that the three modules cooperatively reduced the glucose metabolism and improved N-acetylglucosamine production up to 3.0 g/L in shake flask cultivation.

中文翻译：

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三种降低葡萄糖代谢率的方法的组合，以提高酿酒酵母中N-乙酰氨基葡萄糖的生产

以前，酿酒酵母中N-乙酰氨基葡萄糖（GlcNAc）的产生是通过删除编码磷酸果糖激酶2（PFK-2）同工型的基因来改善的，该基因通过消除产生果糖2,6-二磷酸的途径来降低糖酵解通量，磷酸果糖激酶1（PFK-1）的变构活化剂。我们进一步检查了葡萄糖代谢速率的进一步降低对N-乙酰氨基葡萄糖生产的影响。通过表达编码截短型葡萄糖敏感调节剂（MTH1- ΔT的基因），降低了葡萄糖摄取率）。此外，为了下调PFK-1和丙酮酸激酶1（Pyk1）的表达水平，引入了催化死亡的Cas9（dCas9）。最后，将三种策略以组合方式引入酿酒酵母菌株中。包含所有三个模块的菌株导致最高的N-乙酰氨基葡萄糖产量。结果表明，在摇瓶培养中，这三个模块协同降低了葡萄糖的代谢并提高了N-乙酰氨基葡萄糖的产量，最高可达3.0 g / L。