Konosuke Sano

Phytase of the yeast Arxula adeninivorans

Of a number of yeasts screened for growth on phytic acid (inositol phosphates) as a sole source of carbon and phosphate, Arxula adeninivorans showed a particularly vigorous growth. This capacity was correlated with the presence of a high activity of secreted phytase. The crude enzyme showed an optimal temperature of at least 75 °C and an optimal pH of 4.5. The level of secreted enzyme far exceeded that of previously reported yeast phytases.

Expression of Escherichia coli promoters in Brevibacterium lactofermentum using the shuttle vector pEB003

Abstract A novel promoter probing vector for examining gene expression in a glutamic acid-producing bacterium Brevibacterium lactofermentum was constructed. This plasmid pEB003, a shuttle vector able to replicate either in B. lactofermentum or Escherichia coli , carries a promoter-less chloramphenicol acetyltransferase (CAT) gene as the index gene for probing promoter strength. By using pEB003, we demonstrated that the E. coli promoters, tac, trp and lac UV5 promoters, worked effectively in B. lactofermentum . As evaluated by the CAT activity, the relative strength of the promoters was the same in B. lactofermentum as in E. coli . Thus, B. lactofermentum seems to have a transcriptional machinery that is similar to that of E. coli .

In vitro proliferation of saffron (Crocus sativus L.) stigma

Excised young intact stigmas plus ovaries of Crocus sativus L. were cultured on Linsmaier-Skoog media supplemented with either a cytokinin or an auxin alone or in combinations. Benzyladenine and kinetin at concentrations of 0.1, 1, and 5 mgl-1 supported growth, and crocin was biosynthesized in the stigmas in vitro. Auxins had little effect. Young excised single stigmas or half ovaries were also cultured so as to form stigma-like structures in order to explore a possible new approach to industrial production of the spice, “saffron”. On Linsmaier-Skoog and Nitsch media supplemented with kinetin at concentration of 1 or 5 mgl-1 and alpha-naphthalene acetic acid or indole-butyric acid at concentration of 0.1 or 10 mgl-1 in combinations, stigma-like structures appeared directly and indirectly (through meristematic tissue), grew and matured. The maximum number of structures were 75 per half ovary. Three kinds of yellow pigments including crocin were tentatively identified by TLC in the stigma-like structures as was the case for the in vivo grown natural stigma, although the contents were lower.

Improvement of Escherichia coli strains overproducing lysine using recombinant DNA techniques

SummarySeveral genes of the lysine biosynthetic pathway were cloned separately on the high copy number plasmid pBR322 (Richaud et al. 1981). These hybrid plasmids were used to transform an Escherichia coli strain TOC R 21 that overproduces lysine due to mutations altering the aspartokinase reaction. The synthesis of lysine was studied in these different strains. It appears that only plasmids containing the dapA gene (encoding dihydrodipicolinate synthetase) lead to an increase in lysine production. This result allows us to identify this reaction as the limiting biosynthetic step in strain TOC R 21 and indicates that such a method of gene amplification can be used to improve strains overproducing metabolites.

Formation of alkaloids in suspension-cultured Colchicum autumnale

Abstract Undifferentiated callus tissues were induced from flowering shoots of Colchicum autumnale by treatment with 2 4-D. The callus tissues producing colchicine have been grown in modified Murashige & Skoog medium containing IBA and kinetin as growth factors. The type of nitrogen source in the medium influenced alkaloid formation. Although nitrate or ammonium as the sole nitrogen source inhibited the formation of colchicine as well as growth, the formation and growth were better with 20 mM ammonium plus 40 mM nitrate. Addition of SO 4 2− (20 mM markedly increased the formation of colchicine. At high concentrations, PO 4 3− , Ca 2+ , and Fe 2+ were inhibitory for formation. The identity of colchicine formed by callus was confirmed by UV and mass spectrometry analyses.

Colchicine precursors and the formation of alkaloids in suspension-cultured Colchicum autumnale

Abstract Various precursors of colchicine were added to cell suspension cultures of Colchicum autumnale and the formation of colchicine alkaloids was examined. Phenylalanine, tyrosine and methionine had no effect on the formation of colchicine, but p-coumaric acid, tyramine and demecolcine increased alkaloid formation. Production of colchicine occurred at maximum level when both p-coumaric acid and tyramine were provided together, suggesting a synergistic formation. However, concentrations of p-coumaric acid and tyramine were very low in the cultured cells in spite of high levels of phenylalanine and tyrosine. Activities of trans-cinnamic acid 4-hydroxylase and tyrosine decarboxylase, involved in the biosynthesis of both p-coumaric acid and tyramine, respectively, were very low in the callus tissues during growth, although light increased activity of phenylalanine ammonia-lyase. We conclude that p-coumaric acid and tyramine are triggers for the formation of colchicine alkaloids in Colchicum callus tissues.

Formation of alliin in the culture tissues of Allium sativum. Oxidation of S-allyl-l-cysteine

Abstract Addition of S -allyl- l -cysteine markedly increased the level of alliin in both shoot-forming and root-forming callus tissues of A. sativum .

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.

Novel host-vector system for selection and maintenance of plasmid-bearing, streptomycin-dependent Escherichia coli cells in antibiotic-free media

A novel system for selection and maintenance of cells carrying a recombinant plasmid has been developed, using the streptomycin-dependent (Smd) Escherichia coli 4D host and a plasmid vector carrying an rpsL gene from an Sm-resistant (Smr) mutant of E. coli which masks the Smd phenotype. Strain 4D carrying the Smr pBR322 plasmid can grow without Sm. Using this host-vector system, we can select for cells carrying an Smr recombinant plasmid and maintain them in antibiotic-free media.

Sequence analysis of the Brevibacterium lactofermentum trp operon.

SummaryBrevibacterium lactofermentum, a Gram-positive bacterium, is a commercially important amino acid producer. In this organism, the tryptophan biosynthetic enzymes are encoded within a 7725 bp HapII-BamHI fragment. Seven open reading frames were identified as trp genes by complementation tests with various B. lactofermentum and Escherichia coli tryptophan auxotrophs. Following the nomenclature established for E. coli and Serratia marcescens, the B. lactofermentum trp genes were designated trpL, trpE, trpG, trpD, trpC (including the trpF domain), trpB, and trpA. The organization of these genes is identical to that in S. marcescens. The nucleotide sequences of the putative ribosome-binding sites for the B. lactofermentum trp genes resemble those of E. coli and Bacillus subtilis. Computer analysis revealed that the trp enzymes of B. lactofermentum resemble the enzymes of the Gram-negative E. coli more closely than those of the Gram-positive B. subtilis.