Rewiring the Glucose Transportation and Central Metabolic Pathways for Overproduction of N-Acetylglucosamine in Bacillus subtilis
Rewiring the Glucose Transportation and Central Metabolic Pathways for Overproduction of N-Acetylglucosamine in Bacillus subtilis
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DOI：
10.1002/biot.201700020
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发表时间：
2017-10-01
影响因子：
4.7
通讯作者：
Liu, Long
中科院分区：
文献类型：
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作者：
Gu, Yang;Deng, Jieying;Liu, Long
N-乙酰氨基葡萄糖（GlcNAc）是一种重要的氨基糖，广泛应用于医疗保健领域。在先前的研究中，已经通过途径设计和模块优化构建了用于微生物生产GlcNAc的重组枯草芽孢杆菌菌株BSGN 6-P-xylA-glmS-pP 43 NMK-GNA 1（BN 0-GNA 1）。在这里，通过重新连接葡萄糖转运和中枢代谢途径，进一步改善GlcNAc的产生。首先，磷酸转移酶系统（PTS）被三个基因的缺失阻断，（编码PTS系统转运蛋白亚基IIA YyzE），ypqE（编码PTS系统转运蛋白亚基IIA YpqE），和ptsG（编码PTS系统葡萄糖特异性EIICBA组分），导致摇瓶中GlcNAc滴度增加47.6%（从6.5 +/- 0.25至9.6 +/- 0.16 gL（-1））。然后，进行glcP和glcK基因的表达的强化和葡萄糖促进蛋白的优化以促进葡萄糖输入和磷酸化。接下来，通过起始密码子优化策略抑制GlcNAc合成的竞争性途径，即糖酵解、肽聚糖合成途径、戊糖磷酸途径和三羧酸循环，并且摇瓶中的GlcNAc滴度从10.8 +/- 0.25提高到13.2 +/- 0.31 gL（-1）。最终，在3 L补料分批生物反应器中，GlcNAc滴度进一步提高至42.1 +/- 1.1gL（-1），是原始菌株BN 0-GNA 1的1.72倍。该研究显示显著增强的GlcNAc生产，并且本文描述的代谢工程策略将可用于工程化其他原核微生物以用于生产GlcNAc和相关分子。
N-acetylglucosamine (GlcNAc) is an important amino sugar extensively used in the healthcare field. In a previous study, the recombinant Bacillus subtilis strain BSGN6-P-xylA-glmS-pP43NMK-GNA1 (BN0-GNA1) had been constructed for microbial production of GlcNAc by pathway design and modular optimization. Here, the production of GlcNAc is further improved by rewiring both the glucose transportation and central metabolic pathways. First, the phosphotransferase system (PTS) is blocked by deletion of three genes, yyzE (encoding the PTS system transporter subunit IIA YyzE), ypqE (encoding the PTS system transporter subunit IIA YpqE), and ptsG (encoding the PTS system glucose-specific EIICBA component), resulting in 47.6% increase in the GlcNAc titer (from 6.5 +/- 0.25 to 9.6 +/- 0.16 gL(-1)) in shake flasks. Then, reinforcement of the expression of the glcP and glcK genes and optimization of glucose facilitator proteins are performed to promote glucose import and phosphorylation. Next, the competitive pathways for GlcNAc synthesis, namely glycolysis, peptidoglycan synthesis pathway, pentose phosphate pathway, and tricarboxylic acid cycle, are repressed by initiation codon-optimization strategies, and the GlcNAc titer in shake flasks is improved from 10.8 +/- 0.25 to 13.2 +/- 0.31 gL(-1). Finally, the GlcNAc titer is further increased to 42.1 +/- 1.1gL(-1) in a 3-L fed-batch bioreactor, which is 1.72-fold that of the original strain, BN0-GNA1. This study shows considerably enhanced GlcNAc production, and the metabolic engineering strategy described here will be useful for engineering other prokaryotic microorganisms for the production of GlcNAc and related molecules.