Fermentative production of L-alanyl-L-glutamine by a metabolically engineered Escherichia coli strain expressing L-amino acid alpha-ligase.
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.
Tabata
Kazuhiko
K
Technical Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., 1-1 Kyowa-cho, Hofu-shi, 747-8522 Yamaguchi, Japan.
Hashimoto
Shin-Ichi
S
D000596:Amino Acids / Q000378:metabolism*; D001709:Biotechnology; D003470:Culture Media; D004151:Dipeptides / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D005285:Fermentation; D005818:Genetic Engineering / Q000379:methods*; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D000596:Amino Acids; D003470:Culture Media; D004151:Dipeptides; D008025:Ligases; C054122:alanylglutamine