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假定的转录阻遏物McbR是TetR家族的成员,参与谷氨酸棒杆菌中指导含硫氨基酸合成的代谢网络的调控。

The putative transcriptional repressor McbR, member of the TetR-family, is involved in the regulation of the metabolic network directing the synthesis of sulfur containing amino acids in Corynebacterium glutamicum.

作者信息

Rey Daniel Alexander, Pühler Alfred, Kalinowski Jörn

机构信息

Lehrstuhl für Genetik, Universität Bielefeld, Universitätsstrasse 25, D-33501 Bielefeld, Germany.

出版信息

J Biotechnol. 2003 Jun 12;103(1):51-65. doi: 10.1016/s0168-1656(03)00073-7.

Abstract

In order to isolate transcriptional regulatory proteins involved in L-methionine-dependent repression in Corynebacterium glutamicum, proteins binding to the putative promoter region upstream of the metY gene were isolated by DNA affinity chromatography. One of the isolated proteins was identified as a putative transcriptional repressor of the TetR-family by a mass spectrometry fingerprint technique based on the complete C. glutamicum genome sequence. The respective gene, designated mcbR, was deleted in the mutant strain C. glutamicum DR1. Using 2D-PAGE, the protein contents of the C. glutamicum wild type and the mutant strain DR1 grown in media with or without L-methionine supplementation were compared and a set of six proteins was identified. Their abundance was drastically enhanced in the mutant strain and no longer influenced by L-methionine added to the growth medium. The corresponding genes were identified by mass spectrometry fingerprint analysis. They included metY encoding O-acetyl-L-homoserine sulfhydrylase, metK encoding S-adenosyl-methionine synthethase, hom encoding homoserine dehydrogenase, cysK encoding L-cysteine synthase, cysI encoding an NADPH dependant sulfite reductase, and ssuD encoding an alkanesulfonate monooxygenase. Evidently, the putative transcriptional repressor McbR is involved in the regulation of the metabolic network directing the synthesis of L-methionine in C. glutamicum. The C. glutamicum mcbR mutant can be considered to represent a first step in the construction of an L-methionine production strain.

摘要

为了分离参与谷氨酸棒杆菌中L-甲硫氨酸依赖性阻遏的转录调控蛋白,通过DNA亲和层析分离了与metY基因上游假定启动子区域结合的蛋白。通过基于谷氨酸棒杆菌完整基因组序列的质谱指纹技术,鉴定出其中一种分离蛋白为TetR家族的假定转录阻遏物。在突变株谷氨酸棒杆菌DR1中缺失了相应的基因,命名为mcbR。使用二维聚丙烯酰胺凝胶电泳(2D-PAGE),比较了在添加或不添加L-甲硫氨酸的培养基中生长的谷氨酸棒杆菌野生型和突变株DR1的蛋白质含量,并鉴定出一组六种蛋白质。它们在突变株中的丰度大幅提高,并且不再受添加到生长培养基中的L-甲硫氨酸的影响。通过质谱指纹分析鉴定了相应的基因。它们包括编码O-乙酰-L-高丝氨酸硫氢酶的metY、编码S-腺苷甲硫氨酸合成酶的metK、编码高丝氨酸脱氢酶的hom、编码L-半胱氨酸合成酶的cysK、编码NADPH依赖性亚硫酸盐还原酶的cysI以及编码链烷磺酸盐单加氧酶的ssuD。显然,假定的转录阻遏物McbR参与了谷氨酸棒杆菌中指导L-甲硫氨酸合成的代谢网络的调控。谷氨酸棒杆菌mcbR突变体可被视为构建L-甲硫氨酸生产菌株的第一步。

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