A dinucleotide mutation in dihydrodipicolinate synthase of Nicotiana sylvestris leads to lysine overproduction

A dinucleotide mutation in dihydrodipicolinate synthase of Nicotiana sylvestris leads to lysine... By applying a mutagenesis/selection procedure to obtain resistance to a lysine analog, S‐(2‐aminoethyl)l‐cysteine (AEC), a lysine overproducing mutant in Nicotiana sylvestris was isolated. Amino acid analyses performed throughout plant development and of different organs of the N. sylvestris RAEC‐1 mutant, revealed a developmental‐dependent accumulation of free lysine. Lysine biosynthesis in the RAEC‐1 mutant was enhanced due to a lysine feedback‐desensitized dihydrodipicolinate synthase (DHDPS). Several molecular approaches were undertaken to identify the nucleotide change in the dhdps‐r1 gene, the mutated gene coding for the lysine‐desensitized enzyme. The enzyme was purified from wild‐type plants for amino end microsequencing and 10 amino acids were identified. Using dicotyledon dhdps probes, a genomic fragment was cloned from an enriched library of DNA from the homozygote RAEC‐1 mutant plant. A dhdps cDNA, putatively full‐length, was isolated from a tobacco cDNA library. Nucleotide sequence analyses confirmed the presence of the previously identified amino end preceded by a chloroplast transit peptide sequence. Nucleotide sequence comparisons, enzymatic and immunological analyses revealed that the tobacco cDNA corresponds to a normal type of DHDPS, lysine feedback‐regulated, and the genomic fragment to the mutated DHDPS, insensitive to lysine inhibition. Functional complementation of a DHDPS‐deficient Escherichia coli strain was used as an expression system. Reconstruction between the cDNA and genomic fragment led to the production of a cDNA producing an insensitive form of DHDPS. Amino acid sequence comparisons pointed out, at position 104 from the first amino acid of the mature protein, the substitution of Asn to lleu which corresponds to a dinucleotide mutation. This change is unique to the dhdps‐r1 gene when compared with the wild‐type sequence. The identification of the nucleotide and amino acid change of the lysine‐desensitized DHDPS from RAEC‐1 plant opens new perspectives for the improvement of the nutritional value of crops and possibly to develop a new plant selectable marker. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Plant Journal Wiley

A dinucleotide mutation in dihydrodipicolinate synthase of Nicotiana sylvestris leads to lysine overproduction

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Publisher
Wiley
Copyright
Copyright © 1995 Wiley Subscription Services, Inc., A Wiley Company
ISSN
0960-7412
eISSN
1365-313X
D.O.I.
10.1046/j.1365-313X.1995.08050733.x
Publisher site
See Article on Publisher Site

Abstract

By applying a mutagenesis/selection procedure to obtain resistance to a lysine analog, S‐(2‐aminoethyl)l‐cysteine (AEC), a lysine overproducing mutant in Nicotiana sylvestris was isolated. Amino acid analyses performed throughout plant development and of different organs of the N. sylvestris RAEC‐1 mutant, revealed a developmental‐dependent accumulation of free lysine. Lysine biosynthesis in the RAEC‐1 mutant was enhanced due to a lysine feedback‐desensitized dihydrodipicolinate synthase (DHDPS). Several molecular approaches were undertaken to identify the nucleotide change in the dhdps‐r1 gene, the mutated gene coding for the lysine‐desensitized enzyme. The enzyme was purified from wild‐type plants for amino end microsequencing and 10 amino acids were identified. Using dicotyledon dhdps probes, a genomic fragment was cloned from an enriched library of DNA from the homozygote RAEC‐1 mutant plant. A dhdps cDNA, putatively full‐length, was isolated from a tobacco cDNA library. Nucleotide sequence analyses confirmed the presence of the previously identified amino end preceded by a chloroplast transit peptide sequence. Nucleotide sequence comparisons, enzymatic and immunological analyses revealed that the tobacco cDNA corresponds to a normal type of DHDPS, lysine feedback‐regulated, and the genomic fragment to the mutated DHDPS, insensitive to lysine inhibition. Functional complementation of a DHDPS‐deficient Escherichia coli strain was used as an expression system. Reconstruction between the cDNA and genomic fragment led to the production of a cDNA producing an insensitive form of DHDPS. Amino acid sequence comparisons pointed out, at position 104 from the first amino acid of the mature protein, the substitution of Asn to lleu which corresponds to a dinucleotide mutation. This change is unique to the dhdps‐r1 gene when compared with the wild‐type sequence. The identification of the nucleotide and amino acid change of the lysine‐desensitized DHDPS from RAEC‐1 plant opens new perspectives for the improvement of the nutritional value of crops and possibly to develop a new plant selectable marker.

Journal

The Plant JournalWiley

Published: Nov 1, 1995

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