novel methodology employing Corynebacterium glutamicum genome information to generate a new L-lysine-producing mutant

• Published in: Applied microbiology and biotechnology 2002, Vol.58 (2), p.217-223
• Main Authors: Ohnishi, J, Mitsuhashi, S, Hayashi, M, Ando, S, Yokoi, H, Ochiai, K, Ikeda, M
• Format: Article
• Language: English
• Subjects: Accumulation; aspartate kinase; Aspartate Kinase - genetics; Bacteria; batch fermentation; Biological and medical sciences; Biotechnology; Corynebacterium - genetics; Corynebacterium - growth & development; Corynebacterium - metabolism; Corynebacterium glutamicum; Culture Media; Fermentation; Fundamental and applied biological sciences. Psychology; genes; Genetic technics; Genome, Bacterial; Genomes; Genomics; hom gene; homoserine; Homoserine Dehydrogenase - genetics; Industrial Microbiology - methods; lysC gene; Lysine; Lysine - biosynthesis; Methods. Procedures. Technologies; microorganisms; Molecular Sequence Data; mutagenesis; Mutant screening; mutants; Mutation; Prokaryotes; pyc gene; pyruvate carboxylase; Pyruvate Carboxylase - genetics; Sequence Analysis, DNA; synergism; Synergistic effect
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-001-0883-6

Classical whole-cell mutagenesis has achieved great success in development of many industrial fermentation strains, but has the serious disadvantage of accumulation of uncharacterized secondary mutations that are detrimental to their performance. In the post-genomic era, a novel methodology which avoids this drawback presents itself. This "genome-based strain reconstruction" involves identifying mutations by comparative genomic analysis, defining mutations beneficial for production, and assembling them in a single wild-type background. Described herein is an initial challenge involving reconstruction of classically derived L-lysine-producing Corynebacterium glutamicum. Comparative genomic analysis for the relevant terminal pathways, the efflux step, and the anaplerotic reactions between the wildtype and production strains identified a Val-59 leads to Ala mutation in the homoserine dehydrogenase gene (hom), a Thr-311 leads to Ile mutation in the aspartokinase gene (lysC), and a Pro-458 leads to Ser mutation in the pyruvate carboxylase gene (pyc). Introduction of the hom and lysC mutations into the wild-type strain by allelic replacement resulted in accumulation of 8 g and 55 g of L-lysine/l, respectively, indicating that both these specific mutations are relevant to production. The two mutations were then reconstituted in the wild-type genome, which led to a synergistic effect on production (75 g/l). Further introduction of the pyc mutation resulted in an additional contribution and accumulation of 80 g/l after only 27 h. This high-speed fermentation achieved the highest productivity (3.0 g l-1 h-1) so far reported for microbes producing L-lysine in fed-batch fermentation.