Application of metabolic engineering to improve both the production and use of biotech indigo
Application of metabolic engineering to improve both the production and use of biotech indigo
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DOI：
10.1038/sj/jim/7000228
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发表时间：
2002-03-01
影响因子：
3.4
通讯作者：
Weyler, W
中科院分区：
文献类型：
--
作者：
Berry, A;Dodge, TC;Weyler, W
研究了重组大肠杆菌发酵葡萄糖生产靛蓝的工艺。这是通过修改色氨酸途径，导致高水平的吲哚生产，并添加编码萘双加氧酶（NDO）的恶臭假单胞菌基因。与一种过量生产靛蓝的菌株相比，第一种生产靛蓝的菌株生产的靛蓝不到预期数量的一半。在从靛蓝发酵收集的细胞中观察到芳香族blosynthesis的第一种酶，3-脱氧-D-阿拉伯庚酮糖酸7-磷酸（DAHP）合酶（aroG（fbr）基因产物）的严重失活。随后的体外实验表明，DAHP合酶被灭活暴露于吲哚酚到靛蓝的自发化学转化。此后，通过增加aroG（fbr）的基因剂量或通过扩增tktA（转酮醇酶）基因或灭活丙酮酸激酶的两种同工酶来增加体内DAHP合酶的底物可用性，从而提高靛蓝的产量。通过结合所有三种增强细胞中DAHP形成的策略，实现了靛蓝产量增加60%。然后进一步应用代谢工程来消除吲哚酚自发转化为靛蓝的副产物，从而解决了在最终牛仔布染色应用中使用生物靛蓝的严重问题。
A fermentation process was developed for production of indigo from glucose using recombinant Escherichia coli. This was achieved by modifying the tryptophan pathway to cause high-level indole production and adding the Pseudomonas putida genes encoding naphthalene dioxygenase (NDO). In comparison to a tryptophan-overproducing strain, the first indigo-producing strain made less than half of the expected amount of indigo. Severe inactivation of the first enzyme of aromatic blosynthesis, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (the aroG(fbr) gene product), was observed in cells collected from indigo fermentations. Subsequent in vitro experiments revealed that DAHP synthase was inactivated by exposure to the spontaneous chemical conversion of indoxyl to indigo. Indigo production was thereafter improved by increasing the gene dosage of aroG(fbr) or by increasing substrate availability to DAHP synthase in vivo by either amplifying the tktA (transketolase) gene or inactivating both isozymes of pyruvate kinase. By combining all three strategies for enhancing DAHP formation in the cell, a 60% increase in indigo production was achieved. Metabolic engineering was then further applied to eliminate a byproduct of the spontaneous conversion of indoxyl to indigo, thereby solving a serious problem with the use of bio-indigo in the final denim dyeing application.