The outer membrane TolC is involved in cysteine tolerance and overproduction in Escherichia coli
The outer membrane TolC is involved in cysteine tolerance and overproduction in Escherichia coli
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DOI：
10.1007/s00253-008-1686-9
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发表时间：
2009-01-01
影响因子：
5
通讯作者：
Takagi, Hiroshi
中科院分区：
文献类型：
--
作者：
Wiriyathanawudhiwong, Natthawut;Ohtsu, Iwao;Takagi, Hiroshi
L-半胱氨酸是一种具有重要工业应用价值的氨基酸。我们先前在重组大肠杆菌中发现，通过增强生物合成活性和减弱降解途径相结合，可以直接从葡萄糖中产生L半胱氨酸。L半胱氨酸生产的进一步改进有望使用氨基酸外排系统。在这里，我们使用一个系统和全面的基因中断的大肠杆菌K-12突变株(Keio突变株)来鉴定与L有关的新基因-半胱氨酸输出。在3,985个非必需基因突变体中，与野生型细胞相比，TolC突变细胞对L半胱氨酸表现出超敏反应。基因表达分析表明，编码外膜通道的TolC基因是大肠杆菌细胞对L半胱氨酸耐受所必需的。然而，L-半胱氨酸耐受不是由TolC依赖的药物外排系统如AcrA和AcrB介导的。此外，包括OmpA和OmpF在内的其他外膜蛋白似乎不参与TolC依赖的L半胱氨酸耐受。将携带tolC基因的低拷贝数重组载体导入大肠杆菌细胞，可增强细胞对L半胱氨酸的耐受性和产量，降低细胞的降解能力，促进半胱氨酸的输出。我们认为TolC在L-半胱氨酸耐受性中起重要作用可能是由于其输出能力，TolC的过表达对于在大肠杆菌中生产L-半胱氨酸是有效的。
l-Cysteine is an important amino acid in terms of its industrial applications. We previously found marked production of l-cysteine directly from glucose in recombinant Escherichia coli cells by the combination of enhancing biosynthetic activity and weakening the degradation pathway. Further improvements in l-cysteine production are expected to use the amino acid efflux system. Here, we identified a novel gene involved in l-cysteine export using a systematic and comprehensive collection of gene-disrupted E. coli K-12 mutants (the Keio collection). Among the 3,985 nonessential gene mutants, tolC-disrupted cells showed hypersensitivity to l-cysteine relative to wild-type cells. Gene expression analysis revealed that the tolC gene encoding the outer membrane channel is essential for l-cysteine tolerance in E. coli cells. However, l-cysteine tolerance is not mediated by TolC-dependent drug efflux systems such as AcrA and AcrB. It also appears that other outer membrane porins including OmpA and OmpF do not participate in TolC-dependent l-cysteine tolerance. When a low-copy-number plasmid carrying the tolC gene was introduced into E. coli cells with enhanced biosynthesis, weakened degradation, and improved export of l-cysteine, the transformants exhibited more l-cysteine tolerance and production than cells carrying the vector only. We concluded that TolC plays an important role in l-cysteine tolerance probably due to its export ability and that TolC overexpression is effective for l-cysteine production in E. coli.