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

Abstract

The prephenate dehydratase gene was cloned from a mutant of Brevibacterium lactofermenturn, AJl1957 that produced enzyme free from feedback inhibition. The recombinant plasmids pPHI 1 and pPH14 complemented a phenylalanine auxotroph of B. lactofermenturn, 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 Bo 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 5.5g/1 L-phenylalanine, 6.8 g/1 L-tyrosine and 0.3 g/1 anthranilate turned into a potent L-phenylalanine producer producing 18.2 g/1 Lphenylalanine and 1.0 g/1 L-tyrosine by-product.

Metabolic regulation and overproduction of primary metabolites

“…DHAPS in the wild-type was inhibited cumulatively by phenylalanine and tyrosine whereas PD was inhibited by phenylalanine. Cloning of the gene encoding PD from a desensitized mutant and the gene encoding desensitized DAHPS increased the enzyme activities and yielded a strain producing 18 g l -1 phenylalanine, 1 g l -1 tyrosine and no anthranilate (Ito et al, 1990a). Further cloning of a recombinant plasmid expressing desensitized 3-deoxy-Darabinoheptulosonate-7-phosphate synthase increased production to 26 g l -1 phenylalanine (Ito et al, 1991).…”

“…DHAPS in the wild‐type was inhibited cumulatively by phenylalanine and tyrosine whereas PD was inhibited by phenylalanine. Cloning of the gene encoding PD from a desensitized mutant and the gene encoding desensitized DAHPS increased the enzyme activities and yielded a strain producing 18 g l −1 phenylalanine, 1 g l −1 tyrosine and no anthranilate ( Ito et al. , 1990a ).…”

Optimization of L‐phenylalanine production of Corynebacterium glutamicum under product feedback inhibition by elevated oxygen transfer rate

“…By genetic engineering techniques, the bottleneck in the biosynthetic pathway was eliminated by amplifying the deregulated gene coding for the ratelimiting enzyme, resulting in signi®cant improvement in yields. The prephenate dehydratase is determined to be the major control point of phenylalanine biosynthesis in some coryneform bacteria (Ikeda and Katsumata, 1992;Ito et al, 1990). However, the target of action of phe-nylalanine analogue and the structure elements involved in the feedback regulation of this enzyme have not been fully elucidated.…”

Phenylalanine production by metabolically engineered Corynebacterium glutamicum with the pheA gene of Escherichia coli

“…Besides the classical genetic Correspondence to: R. Katsumata approaches, the recent development of host-vector systems for these bacteria Santamaria et al 1984;Yoshihama et al 1985) enabled further strain improvement with the use of recombinant DNA technology. Large increases in phenylalanine production have been achieved by amplifying the rate-limiting enzymes in existing phenylalanine producers (Ozaki et al 1985;Ito et al 1990). Metabolic conversion from tryptophan production to phenylalanine production by amplifying the branch-point enzymes was also reported (Ikeda and Katsumata 1992).…”