Feedback-resistant acetohydroxy acid synthase increases valine production in Corynebacterium glutamicum

被引:96
作者
Elisáková, V
Pátek, M
Holátko, J
Nesvera, JN
Leyval, D
Goergen, JL
Delaunay, S
机构
[1] Acad Sci Czech Republ, Inst Microbiol, CZ-14220 Prague 4, Czech Republic
[2] Inst Natl Polytech Lorraine, ENSAIA, Lab Bioprocedes Agroalimentaires, Vandoeuvre Les Nancy, France
[3] Inst Natl Polytech Lorraine, CNRS, Lab Sci Genie Chim, Vandoeuvre Les Nancy, France
关键词
D O I
10.1128/AEM.71.1.207-213.2005
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Acetohydroxy acid synthase (AHAS), which catalyzes the key reactions in the biosynthesis pathways of branched-chain amino acids (valine, isoleucine, and leucine), is regulated by the end products of these pathways. The whole Corynebacterium glutamicum ilvBNC operon, coding for acetohydroxy acid synthase (ilvBN) and acetohydroxy acid isomeroreductase (ilvC), was cloned in the newly constructed Escherichia coli-C. glutamicum shuttle vector pECKA (5.4 kb, Km(r)). By using site-directed mutagenesis, one to three amino acid alterations (mutations M8, M11, and M13) were introduced into the small (regulatory) AHAS subunit encoded by ilvN. The activity of AHAS and its inhibition by valine, isoleucine, and leucine were measured in strains carrying the ilvBNC operon with mutations on the plasmid or the ilvNM13 mutation within the chromosome. The enzyme containing the M13 mutation was feedback resistant to all three amino acids. Different combinations of branched-chain amino acids did not inhibit wild-type AHAS to a greater extent than was measured in the presence of 5 mM valine alone (about 57%). We infer from these results that there is a single binding (allosteric) site for all three amino acids in the enzyme molecule. The strains carrying the ilvNM13 mutation in the chromosome produced more valine than their wild-type counterparts. The plasmid-free C. glutamicum DeltailvA DeltapanB ilvNM13 strain formed 90 mM valine within 48 111 of cultivation in minimal medium. The same strain harboring the plasmid pECKAilvBNC produced as much as 130 mM valine under the same conditions.
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页码:207 / 213
页数:7
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