Effect of Drug Transporter Genes on Cysteine Export and Overproduction in Escherichia coli
Effect of Drug Transporter Genes on Cysteine Export and Overproduction in Escherichia coli
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DOI：
10.1128/aem.02507-05
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发表时间：
2006-07
影响因子：
4.4
通讯作者：
S. Yamada;N. Awano;K. Inubushi;E. Maeda;S. Nakamori;K. Nishino;A. Yamaguchi;H. Takagi
中科院分区：
文献类型：
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作者：
S. Yamada;N. Awano;K. Inubushi;E. Maeda;S. Nakamori;K. Nishino;A. Yamaguchi;H. Takagi
摘要l-半胱氨酸是一种重要的工业氨基酸。我们以前发现了一个显着的生产L-半胱氨酸从葡萄糖重组大肠杆菌细胞表达改变cysE基因编码反馈抑制不敏感的丝氨酸乙酰转移酶。此外，较低水平的半胱氨酸脱氢酶（CD）活性，这是参与L-半胱氨酸降解，增加L-半胱氨酸的生产力在E。杆菌L-半胱氨酸外排系统的使用可能是有前途的L-半胱氨酸过度生产者的育种。除了YdeD和YfiK，这两种蛋白以前已被报道为大肠杆菌中的L-半胱氨酸输出蛋白外。大肠杆菌中，我们分析了33个推定的药物转运蛋白基因在大肠杆菌中的作用。大肠杆菌对L-半胱氨酸输出和过量生产的影响。acrD、acrEF、bcr、cusA、emrAB、emrKY、ybjYZ和yojIH基因的过表达逆转了tnaA（主要CD基因）破坏的E.大肠杆菌细胞。我们还发现，这八个基因的过表达降低了细胞内L-半胱氨酸水平培养后，在L-半胱氨酸的存在。氨基酸转运试验表明，Bcr过表达赋予双环霉素和四环素耐药性，特别是促进L-半胱氨酸输出驱动的能量来自质子梯度。当tnaA被破坏后，E.用携带bcr基因的质粒转化表达改变的cysE基因的大肠杆菌菌株，转化子比仅携带载体的细胞表现出更高的L-半胱氨酸产量。报告基因分析表明，bcr基因是组成型表达在一个实质性的水平。这些结果表明，多药物转运蛋白Bcr在主要的易化剂家族参与l-半胱氨酸输出和过量生产的基因工程大肠杆菌。coli细胞。
ABSTRACT l-Cysteine is an important amino acid in terms of its industrial applications. We previously found a marked production of l-cysteine from glucose in recombinant Escherichia coli cells expressing an altered cysE gene encoding feedback inhibition-insensitive serine acetyltransferase. Also, a lower level of cysteine desulfhydrase (CD) activity, which is involved in l-cysteine degradation, increased l-cysteine productivity in E. coli. The use of an l-cysteine efflux system could be promising for breeding l-cysteine overproducers. In addition to YdeD and YfiK, which have been reported previously as l-cysteine exporter proteins in E. coli, we analyzed the effects of 33 putative drug transporter genes in E. coli on l-cysteine export and overproduction. Overexpression of the acrD, acrEF, bcr, cusA, emrAB, emrKY, ybjYZ, and yojIH genes reversed the growth inhibition of tnaA (the major CD gene)-disrupted E. coli cells by l-cysteine. We also found that overexpression of these eight genes reduces intracellular l-cysteine levels after cultivation in the presence of l-cysteine. Amino acid transport assays showed that Bcr overexpression conferring bicyclomycin and tetracycline resistance specifically promotes l-cysteine export driven by energy derived from the proton gradient. When a tnaA-disrupted E. coli strain expressing the altered cysE gene was transformed with a plasmid carrying the bcr gene, the transformant exhibited more l-cysteine production than cells carrying the vector only. A reporter gene assay suggested that the bcr gene is constitutively expressed at a substantial level. These results indicate that the multidrug transporter Bcr in the major facilitator family is involved in l-cysteine export and overproduction in genetically engineered E. coli cells.