Towards Methionine Overproduction in Corynebacterium glutamicum - Methanethiol and Dimethyldisulfide as Reduced Sulfur Sources
Towards Methionine Overproduction in Corynebacterium glutamicum - Methanethiol and Dimethyldisulfide as Reduced Sulfur Sources
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DOI：
10.4014/jmb.1002.02018
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发表时间：
2010-08-01
影响因子：
2.8
通讯作者：
Wittmann, Christoph
中科院分区：
文献类型：
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作者：
Bolten, Christoph J.;Schroeder, Hartwig;Wittmann, Christoph
本研究以甲硫醇和二甲基二硫醚为硫源，对谷氨酸棒杆菌合成蛋氨酸进行了研究。计算机模拟途径分析预测这些还原化合物的甲硫氨酸产率较高，前提是它们可被利用。野生型细胞能够在甲硫醇和二甲基二硫醚作为唯一硫源上生长。同位素标记的突变株，表现出有针对性的修改蛋氨酸生物合成的研究，提供了详细的洞察到潜在的途径参与的同化甲烷和二甲基二硫醚。这两种硫化合物都作为一个完整的分子结合，将末端S-CH（3）基团添加到O-乙酰基高丝氨酸上。在该反应中，甲硫氨酸直接形成。MetY（O-乙酰高丝氨酸巯基解酶）被鉴定为催化该反应的酶。metY的缺失导致甲硫氨酸营养缺陷型菌株生长在以甲硫醇或二甲基二硫醚作为唯一硫源上。在Delta metY背景中基于质粒的metY过表达恢复了以甲硫醇或二甲基二硫醚作为唯一硫源生长的能力。体外研究表明，C.谷氨酸野生型的MetY对甲硫醇（63 mU/mg）和二甲基二硫化物（61 mU/mg）的活性相对较低。metY的过表达将体外活性增加至1，780 mU/mg，并且有益于甲硫氨酸生产，因为在工程菌株中细胞内甲硫氨酸库增加了2倍。这种积极作用受到metY底物0-乙酰基高丝氨酸耗尽的限制，表明需要针对竞争性生产菌株的进一步代谢工程靶标。
In the present work, methanethiol and dimethyldisulfide were investigated as sulfur sources for methionine synthesis in Corynebacterium glutamicum. In silico pathway analysis predicted a high methionine yield for these reduced compounds, provided that they could be utilized. Wild-type cells were able to grow on both methanethiol and dimethyldisulfide as sole sulfur sources. Isotope labeling studies with mutant strains, exhibiting targeted modification of methionine biosynthesis, gave detailed insight into the underlying pathways involved in the assimilation of methanethiol and dimethyldisulfide. Both sulfur compounds are incorporated as an entire molecule, adding the terminal S-CH(3) group to O-acetylhomoserine. In this reaction, methionine is directly formed. MetY (O-acetylhomoserine sulfhydrylase) was identified as the enzyme catalyzing the reaction. The deletion of metY resulted in methionine auxotrophic strains grown on methanethiol or dimethyldisulfide as sole sulfur sources. Plasmid-based overexpression of metY in the Delta metY background restored the capacity to grow on methanethiol or dimethyldisulfide as sole sulfur sources. In vitro studies with the C. glutamicum wild type revealed a relatively low activity of MetY for methanethiol (63 mU/mg) and dimethyldisulfide (61 mU/mg). Overexpression of metY increased the in vitro activity to 1,780 mU/mg and was beneficial for methionine production, since the intracellular methionine pool was increased 2-fold in the engineered strain. This positive effect was limited by a depletion of the metY substrate O-acetylhomoserine, suggesting a need for further metabolic engineering targets towards competitive production strains.