Corynebacterium glutamicum ATP-phosphoribosyl transferases suitable for L-histidine production - Strategies for the elimination of feedback inhibition
Corynebacterium glutamicum ATP-phosphoribosyl transferases suitable for L-histidine production - Strategies for the elimination of feedback inhibition
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DOI：
10.1016/j.jbiotec.2015.04.001
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发表时间：
2015-07-20
影响因子：
4.1
通讯作者：
Kalinowski, Joern
中科院分区：
文献类型：
--
作者：
Kulis-Horn, Robert K.;Persicke, Marcus;Kalinowski, Joern
谷氨酸棒杆菌中L-组氨酸的生物合成主要是通过L-组氨酸反馈抑制催化该途径第一步的磷酸核糖转移酶组氨酸来调节的。消除这种反馈抑制是发展L组氨酸生产菌株的第一步，也是最重要的一步。为此，采用了随机诱变和合理的酶重新设计相结合的方法。对有毒的L-组氨酸类似物β-(2-噻唑基)-DL-丙氨酸(2-TA)自发产生抗性的突变体在组氨酸的C端调节域发现了新的和不可预测的突变，导致反馈阻力增加。此外，整个C末端调控结构域的缺失以及催化区功能突变S143F的获得导致了一个组氨酸突变体，即使在L-组氨酸浓度接近溶解度极限的情况下，该突变体仍然具有高活性。值得注意的是，S143F突变本身就引发了反馈放松调控，首次揭示了HisG催化区域中参与反馈调控机制的氨基酸残基。此外，我们还研究了HisG突变对L-组氨酸产生的不同水平的影响。这包括对不同表达系统的分析，包括基于质粒和染色体的过度表达，以及选择密码子对HisG突变的重要性。结构域缺失、单一氨基酸交换、密码子选择和基于染色体的过度表达相结合，导致生产菌株积累了约0.5gl(-1)L组氨酸，展示了不同方法的附加值。(C)由爱思唯尔出版的2015年。
L-Histidine biosynthesis in Corynebacterium glutamicum is mainly regulated by L-histidine feedback inhibition of the ATP-phosphoribosyltransferase HisG that catalyzes the first step of the pathway. The elimination of this feedback inhibition is the first and most important step in the development of an L-histidine production strain. For this purpose, a combined approach of random mutagenesis and rational enzyme redesign was performed. Mutants spontaneously resistant to the toxic L-histidine analog beta-(2-thiazolyl)-DL-alanine (2-TA) revealed novel and unpredicted mutations in the C-terminal regulatory domain of HisG resulting in increased feedback resistance. Moreover, deletion of the entire C-terminal regulatory domain in combination with the gain of function mutation S143F in the catalytic domain resulted in a HisG variant that is still highly active even at L-histidine concentrations close to the solubility limit. Notably, the S143F mutation on its own provokes feedback deregulation, revealing for the first time an amino acid residue in the catalytic domain of HisG that is involved in the feedback regulatory mechanism. In addition, we investigated the effect of hisG mutations for L-histidine production on different levels. This comprised the analysis of different expression systems, including plasmid- and chromosome-based overexpression, as well as the importance of codon choice for HisG mutations. The combination of domain deletions, single amino acid exchanges, codon choice, and chromosome-based overexpression resulted in production strains accumulating around 0.5 gl(-1) L-histidine, demonstrating the added value of the different approaches. (C) 2015 Published by Elsevier B.V.