Tabata Kazuhiko, Hashimoto Shin-Ichi
Technical Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., 1-1 Kyowa-cho, Hofu-shi, 747-8522 Yamaguchi, Japan.
Appl Environ Microbiol. 2007 Oct;73(20):6378-85. doi: 10.1128/AEM.01249-07. Epub 2007 Aug 24.
In spite of its clinical and nutritional importance, l-alanyl-l-glutamine (Ala-Gln) has not been widely used due to the absence of an efficient manufacturing method. Here, we present a novel method for the fermentative production of Ala-Gln using an Escherichia coli strain expressing l-amino acid alpha-ligase (Lal), which catalyzes the formation of dipeptides by combining two amino acids in an ATP-dependent manner. Two metabolic manipulations were necessary for the production of Ala-Gln: reduction of dipeptide-degrading activity by combinatorial disruption of the dpp and pep genes and enhancement of the supply of substrate amino acids by deregulation of glutamine biosynthesis and overexpression of heterologous l-alanine dehydrogenase (Ald). Since expression of Lal was found to hamper cell growth, it was controlled using a stationary-phase-specific promoter. The final strain constructed was designated JKYPQ3 (pepA pepB pepD pepN dpp glnE glnB putA) containing pPE167 (lal and ald expressed under the control of the uspA promoter) or pPE177 (lal and ald expressed under the control of the rpoH promoter). Either strain produced more than 100 mM Ala-Gln extracellularly, in fed-batch cultivation on glucose-ammonium salt medium, without added alanine and glutamine. Because of the characteristics of Lal, no longer peptides (such as tripeptides) or dipeptides containing d-amino acids were formed.
尽管L-丙氨酰-L-谷氨酰胺(Ala-Gln)具有临床和营养重要性,但由于缺乏有效的生产方法,它尚未得到广泛应用。在此,我们提出了一种利用表达L-氨基酸α-连接酶(Lal)的大肠杆菌菌株发酵生产Ala-Gln的新方法,该酶以ATP依赖的方式通过结合两个氨基酸催化二肽的形成。生产Ala-Gln需要两种代谢操作:通过组合破坏dpp和pep基因降低二肽降解活性,以及通过解除谷氨酰胺生物合成的调控和异源L-丙氨酸脱氢酶(Ald)的过表达增强底物氨基酸的供应。由于发现Lal的表达会阻碍细胞生长,因此使用一个稳定期特异性启动子对其进行调控。构建的最终菌株命名为JKYPQ3(pepA pepB pepD pepN dpp glnE glnB putA),其含有pPE167(lal和ald在uspA启动子控制下表达)或pPE177(lal和ald在rpoH启动子控制下表达)。在葡萄糖-铵盐培养基上进行补料分批培养时,不添加丙氨酸和谷氨酰胺,这两种菌株中的任何一种都能在细胞外产生超过100 mM的Ala-Gln。由于Lal的特性,不会形成更长的肽(如三肽)或含有D-氨基酸的二肽。