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Applied and Environmental Microbiology, March 2009, p. 1635-1641, Vol. 75, No. 6
0099-2240/09/$08.00+0     doi:10.1128/AEM.02027-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Reengineering of a Corynebacterium glutamicum L-Arginine and L-Citrulline Producer

Masato Ikeda,^1,2* Satoshi Mitsuhashi,¹ Kenji Tanaka,¹ and Mikiro Hayashi¹

Bioprocess Development Center, Kyowa Hakko Bio Co., Ltd., Tsukuba, Ibaraki 305-0841, Japan,¹ Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, Nagano 399-4598, Japan²

Received 2 September 2008/ Accepted 6 January 2009

Toward the creation of a robust and efficient producer of L-arginine and L-citrulline (arginine/citrulline), we have performed reengineering of a Corynebacterium glutamicum strain by using genetic information of three classical producers. Sequence analysis of their arg operons identified three point mutations (argR123, argG92^up, and argG45) in one producer and one point mutation (argB26 or argB31) in each of the other two producers. Reconstitution of the former three mutations or of each argB mutation on a wild-type genome led to no production. Combined introduction of argB26 or argB31 with argR123 into a wild type gave rise to arginine/citrulline production. When argR123 was replaced by an argR-deleted mutation (argR), the production was further increased. The best mutation set, argR and argB26, was used to screen for the highest productivity in the backgrounds of different wild-type strains of C. glutamicum. This yielded a robust producer, RB, but the production was still one-third of that of the best classical producer. Transcriptome analysis revealed that the arg operon of the classical producer was much more highly upregulated than that of strain RB. Introduction of leuC456, a mutation derived from a classical L-lysine producer and provoking global induction of the amino acid biosynthesis genes, including the arg operon, into strain RB led to increased production but incurred retarded fermentation. On the other hand, replacement of the chromosomal argB by heterologous Escherichia coli argB, natively insensitive to arginine, caused a threefold-increased production without retardation, revealing that the limitation in strain RB was the activity of the argB product. To overcome this, in addition to argB26, the argB31 mutation was introduced into strain RB, which caused higher deregulation of the enzyme and resulted in dramatically increased production, like the strain with E. coli argB. This reconstructed strain displayed an enhanced performance, thus allowing significantly higher productivity of arginine/citrulline even at the suboptimal 38°C.

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* Corresponding author. Mailing address: Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, Nagano 399-4598, Japan. Phone: 81-265-77-1614. Fax: 81-265-77-1629. E-mail: mikeda{at}shinshu-u.ac.jp

Published ahead of print on 9 January 2009.

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Applied and Environmental Microbiology, March 2009, p. 1635-1641, Vol. 75, No. 6
0099-2240/09/$08.00+0     doi:10.1128/AEM.02027-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.