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Applied and Environmental Microbiology, October 2007, p. 6378-6385, Vol. 73, No. 20
0099-2240/07/$08.00+0     doi:10.1128/AEM.01249-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Fermentative Production of L-Alanyl-L-Glutamine by a Metabolically Engineered Escherichia coli Strain Expressing L-Amino Acid {alpha}-Ligase{triangledown} ,{dagger}

Kazuhiko Tabata1 and Shin-ichi Hashimoto2*

Tokyo BioFrontier Laboratories, Kyowa Hakko Kogyo Co. Ltd., 3-6-6 Ashahi-machi, Machida-shi, 194-8533 Tokyo, Japan,1 Technical Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., 1-1 Kyowa-cho, Hofu-shi, 747-8522 Yamaguchi, Japan2

Received 5 June 2007/ Accepted 13 August 2007

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.

* Corresponding author. Mailing address: Technical Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., 1-1 Kyowa-cho, Hofu-shi, 747-8522 Yamaguchi, Japan. Phone: 81-835-22-2518. Fax: 81-835-22-2466. E-mail: shashimoto{at}kyowa.co.jp

{triangledown} Published ahead of print on 24 August 2007.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, October 2007, p. 6378-6385, Vol. 73, No. 20
0099-2240/07/$08.00+0     doi:10.1128/AEM.01249-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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