Wiriyathanawudhiwong Natthawut, Ohtsu Iwao, Li Zhao-Di, Mori Hirotada, Takagi Hiroshi
Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
Appl Microbiol Biotechnol. 2009 Jan;81(5):903-13. doi: 10.1007/s00253-008-1686-9. Epub 2008 Sep 17.
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.
L-半胱氨酸就其工业应用而言是一种重要的氨基酸。我们之前发现，通过增强生物合成活性和削弱降解途径相结合，重组大肠杆菌细胞可直接从葡萄糖中大量生产L-半胱氨酸。预计使用氨基酸外排系统可进一步提高L-半胱氨酸的产量。在此，我们利用系统全面的大肠杆菌K-12基因敲除突变体文库（Keio文库），鉴定出一个参与L-半胱氨酸输出的新基因。在3985个非必需基因突变体中，与野生型细胞相比，tolC基因敲除的细胞对L-半胱氨酸表现出超敏性。基因表达分析表明，编码外膜通道的tolC基因对大肠杆菌细胞耐受L-半胱氨酸至关重要。然而，L-半胱氨酸耐受性并非由AcrA和AcrB等依赖TolC的药物外排系统介导。似乎包括OmpA和OmpF在内的其他外膜孔蛋白也不参与依赖TolC的L-半胱氨酸耐受性。当将携带tolC基因的低拷贝数质粒导入具有增强的L-半胱氨酸生物合成、削弱的降解和改善的输出能力的大肠杆菌细胞时，转化体比仅携带载体的细胞表现出更高的L-半胱氨酸耐受性和产量。我们得出结论，TolC可能因其输出能力而在L-半胱氨酸耐受性中发挥重要作用，并且TolC过表达对大肠杆菌中L-半胱氨酸的生产有效。
