Molecular breeding of a Brevibacterium lactofermentum L-phenylalanine producer using a cloned prephenate dehydratase gene

• Published in: Applied microbiology and biotechnology 1990-05, Vol.33 (2), p.190-195
• Main Authors: Ito, H, Sato, K, Matsui, K, Sano, K, Enei, H, Hirose, Y
• Format: Article
• Language: English
• Subjects: amino acid metabolism; Bacteriology; Biological and medical sciences; Brevibacterium; Brevibacterium lactofermentum; cloning; enzyme activity; Fundamental and applied biological sciences. Psychology; gene expression; genetic transformation; hydro-lyases; Microbiology; Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains; phenylalanine; plasmid vectors; recombinant DNA; structural genes
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/bf00176523

The prephenate dehydratase gene was cloned from a mutant of Brevibacterium lactofermentum, AJ11957 that produced enzyme free from feedback inhibition. The recombinant plasmids pPH11 and pPH14 complemented a phenylalanine auxotroph of B. lactofermentum, A-15, provided the transformant with the desensitized enzyme and caused an increased level of the enzyme compared to that of a wild strain. Plasmid pPH14 was introduced into L-phenylalanine producers genetically induced from B. lactofermentum; MF358 and FP-1 excreting L-tyrosine and anthranilate, respectively, as by-products. Both transformants predominantly accumulated L-phenylalanine at the expense of by-product formation. Co-existence of pPH14 and pTAR16, a recombinant plasmid expressing desensitized 3-deoxy-D-arabino-hepturosonate-7-phosphate synthase had a marked effect on further improvement in L-phenylalanine productivity, accompanied by an increase in the corresponding enzyme activity. The parent, MF358, accumulating 5.5 g/l L-phenylalanine, 6.8 g/l L-tyrosine and 0.3 g/l anthranilate turned into a potent L-phenylalanine producer producing 18.2 g/l L-phenylalanine and 1.0 g/l L-tyrosine by-product.