Katsuaki Sato

Genetic breeding of L-tyrosine producer from Brevibacterium lactofermentum

A wild-type parent of Brevibacterium lactofermentum was converted into an L-Tyr producer by three steps of genetic breeding. First, acquirement of m-fluoro-D, L-phenylalanine resistance (1,000 microgram/ml) brought about MF1317 which produced 3.5 g/l of L-Tyr and a byproduct of 2.8 g/l of L-Phe. Second, increase in the drug resistance (5,000 microgram/ml) gave MF358 that produced 6.4 g/l of L-Tyr and a byproduct of 6.0 g/l of L-Phe. Third, an L-Phe auxotrophic mutant (FT-1) derived from MF358 accumulated 16 g/l of L-Tyr. In FT-1, L-Phe was not accumulated at all, but a small amount of anthranilate (0.4 g/l) was. A key enzyme in the biosynthesis of L-Tyr, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, was free from synergistic feedback inhibition by L-Tyr and L-Phe in the producers, and so L-Tyr accumulation occurred independently of L-Phe concentration in the production medium.

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

SummaryThe 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.

Application of a recombinant plasmid expressing desensitized 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase to the breeding of l-phenylalanine producers

Abstract A recombinant plasmid containing a desensitized 3-deoxy- d -arabinoheptulosonate-7-phosphate (DAHP) synthase gene was used for transformation of four strains of Brevibacterium lactofermentum : AJ12036 (a wild strain), no. 29 (a tyrosine auxotroph), MF358 (a phenylaianine analogue resistant mutant), and no. 123 (a phenylalanine analogue resistant and tyrosine auxotrophic mutant). The transformants exhibited improved productivities of aromatic amino acids as follows: (i) the introduction of the plasmid converted AJ12036 to a producer of both l -phenylalanine and l -tyrosine, (ii) transformants of no. 29 produced l -phenylalanine even in a medium with excess l -tyrosine, (iii) those of MF358 produced larger amounts of l -phenylalanine and l -tyrosine than the host strain, (iv) those of no. 123 produced larger amounts of l -phenylalanine than the host strain and the production did not decrease markedly even in a medium with excess l -tyrosine. The gene dosage effect of desensitized DAHP synthase on the production of l -phenylalanine and related compounds is discussed on the basis of these results.