Showing papers by "Arnold L. Demain published in 1994"
TL;DR: The new pigments derived from the hydrophobic leucine were more hydrophilic than the conventional red pigments (lacking an amino acid side-chain) and were extracellularly produced.
Abstract: The formation of soluble Monascus red pigments is strongly positively and negatively regulated by different amino acids. Leucine, valine, lysine, and methionine had strong negative effects on pigment formation. Leucine supported poor pigment formation when used as sole nitrogen source in fermentations, yet it neither repressed pigment synthase(s) nor inhibited its action. The new pigments derived from the hydrophobic leucine were more hydrophilic than the conventional red pigments (lacking an amino acid side-chain) and were extracellularly produced. Therefore, the low level of red pigments produced when leucine was the nitrogen source was not due to feed-back regulation by cell-bound leucine pigments. The negative effect of leucine was caused by enhanced decay of pigment synthase(s). The enhanced decay was not due simply to de novo synthesis of a leucine-induced protease.
45 citations
TL;DR: DNA sequencing showed that this region encodes the entire LAT gene (lat), required for the conversion of L-lysine to the beta-lactam precursor L-alpha-aminoadipic acid (alpha-AAA), as well as the upstream half of the ACVS gene (pcbAB).
Abstract: Streptomyces clavuligerus produces the β-lactam antibiotics penicillin N, O-carbamoyldeacetycelcephalosporin C and cephamycin C. We characterized a wild-type DNA region which restores antibiotic formation to a mutant strain named NP1, previously shown to exhibit depressed activities for two early enzymes of cephalosporin synthesis, δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and isopenicillin N synthase (IPNS). L-Lysine e-aminotransferase (LAT) assays and α-AAA feeding experiments suggested that strain NP1 is a lat mutant. NP1 recovered LAT, ACVS and IPNS activities when transformed with the cloned region. DNA sequencing showed that this region encodes the entire LAT gene (lat), required for the conversion of L-lysine to the β-lactam precursor L-α-aminoadipic acid (α-AAA), as well as the upstream half of the ACVS gene (pcbAB). The activities of ACVS and IPNS appear to depend upon LAT expression. Gene fusions constructed to investigate promoter activities in the cloned region support a model of interdependence in the expression of the genes for LAT, ACVS and IPNS (pcbC).
29 citations
TL;DR: It is suggested that such oxygenated heterolactic fermentation processes should be analyzed as fed-batch fermentations with oxygen as the limiting substrate, thus offering an alternative mechanism for controlling acetic acid formation.
Abstract: Controlled aeration ofLeuconostoc mesenteroides was studied as a possible mechanism for control of the formation of acetic acid a metabolite of major influence on the taste of lactic fermented foods. Fermentations were carried out in small scale in a medium in which growth was limited by the buffer capacity only. Ethanol and acetic acid formed during the fermentation were analyzed by rapid head space gas chromatography, and the ratio of the molar concentrations of these two volatiles quantitatively predicted the balance between the formation of acetic acid and lactic acid. The oxygen concentration during the fermentations decreased rapidly to zero, meaning that oxygen transfer was limited by the volumetric oxygen transfer rate,k1aC*. A linear correlation between k1aC* and the quantity of acetic acid produced was established, and it is suggested that such oxygenated heterolactic fermentation processes should be analyzed as fed-batch fermentations with oxygen as the limiting substrate. Addition of fructose in limited amounts leads to the formation of one half mole of acetic acid for each mole fructose, thus offering an alternative mechanism for controlling acetic acid formation.
8 citations
TL;DR: Interference in cephalosporin production by growth in ammonium salts involves synthase inhibition by intracellular alanine, in addition to the known role of ammonium in synthase repression.
Abstract: It is known that excess ammonium supply decreases cephalosporin production and represses cephalosporin synthases. We wondered whether an additional important effect could be inhibition of synthase action by alanine. We had previously shown that ammonium addition induced alanine dehydrogenase and increased intracellular alanine and that alanine could inhibit resting cell synthesis of cephalosporins. In the present work we confirm the alanine inhibition of antibiotic production by resting cells. We foundl-alanine inhibited three of the four synthases tested: ACV synthetase, cyclase and expandase; the epimerase was not inhibited. These data suggest that interference in cephalosporin production by growth in ammonium salts involves synthase inhibition by intracellular alanine, in addition to the known role of ammonium in synthase repression.
4 citations
TL;DR: Four cephamycin non- (or low-) producing mutants are pleiotropic in that they have undetectable or markedly diminished levels of ACVS and cyclase; two mutants almost completely lack LAT activity.
Abstract: The early steps of cephamycin synthesis by S. clavuligerus are catalyzed sequentially by lysine epsilon-aminotransferase (LAT), delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and isopenicillin N synthase (cyclase, IPNS). The genes (lat, pcbAB, and pcbC, respectively) are closely linked in the same order as the enzymes act in the biosynthetic pathway and are transcribed in the same direction. Four cephamycin non- (or low-) producing mutants are pleiotropic in that they have undetectable or markedly diminished levels of ACVS and cyclase; two mutants almost completely lack LAT activity. All four mutants are complemented in cephamycin formation by transformation with pNBR1, a plasmid containing a 7.2-kb genomic region of S. clavuligerus in vector pIJ702. The cloned DNA was found to possess no part of the cyclase gene, but instead it contained lat and the 5' upstream part of pcbAB. Doran et al. reported that the 31-bp region between pcbAB and pcbC contains no recognizable promoter or transcription termination sequences. We found that there are 153 bp between the lat ORF and the pcbAB start codon. A potential transcriptional terminator begins 4 to 6 bp downstream of the lat ORF. In the 111-bp segment between the end of the "terminator" and the pcbAB start codon, there are no Streptomyces-like or Escherichia coli-like promoter consensus sequences. However, upstream of the "terminator," that is, in the downstream portion of the lat ORF, are two regions resembling a Streptomyces consensus promoter. Promoter activity in gene fusion constructions was demonstrated in this region. A third potential promoter is upstream of the lat ORF, but only the--10 part is on the cloned DNA. The mechanism by which the cloned DNA (containing lat, the 5' part of pcbAB, and the intervening sequence) influences the expression of the downstream genes encoding ACVS and IPNS, even in strains that possess LAT activity, is an intriguing target of future investigation.
2 citations