L-Methionine production by double auxotrophic mutants of a ethionine resistant strain of Brevibacterium heali

A number of lysine plus threonine double auxotrophs have been isolated from a ethionine resistant methionine producing strain of Brevibacterium heali previously isolated from soil by mutagenesis with N-methyl N′-nitro-N-nitrosoguanidine in two steps. This strain excreted L-methionine in sufficient amounts. For the three potent mutants tested, the medium of ALFOLDI was judged to be the best. Biotin and ammonium nitrate were found to be optimal at 5 μg/l and at a 40 mM level, respectively. With such an optimal dose, the strain BhLT 27 yielded 25.5 g/l methionine in a flask culture containing methionine-analogue ethionine at a minimal inhibitory concentration.     

L-Phenylalanine production by double auxotrophic analogue resistant mutants of Arthrobacter globiformis

A number of tryptophan plus tyrosine double auxotrophic mutants isolated by the NTG treatment of a glutamate producing strain of Arthrobacter globiformis were found to excrete phenylalanine in a mineral salt medium. By controlling the pH of the medium to near neutrality, the active growth period could be extended up to 72 h and more phenylalanine was accumulated compared to the unregulated culture where the growth period took up to 48 h. Under optimum culture conditions, the best double auxotroph (TT-39) produced 3 g phenylalanine/l. Further improvement of phenylalanine production has been achieved by the step-by-step isolation of a mutant resistant to the phenylalanine analogues p-fluorophenylalanine (PFP) and β-2-thienylalanine (TA) from the TT-39 strain. Under optimum culture conditions, the best double auxotrophic analogue resistant mutant TT-39 PT^r-21 yielded 8.7 g/l phenylalanine.     

Threonine production by dihydrodipicolinate synthase-defective mutants of Brevibacterium flavum

A novel type of threonine-producing strains, dihydrodipicolinate synthase (DPS)-defective mutants of Brevibacterium flavum, was isolated as alpha-amino-beta-hydroxyvaleric acid (AHV)-resistant producers. The third selection markers used were a strong lysine inhibition of threonine production and a lower production of lysine than that of threonine in those derived from strains with feedback-sensitive and-resistant aspartokinase (AK), respectively. The maximum threonine production by these DPS-defective mutants was 13.7 g/l at the optimum concentration of DL-diaminopimelic acid (DAP) in a medium containing 100 g/l of glucose, comparable to that by the previously reported conventional producers with feedback-resistant homoserine dehydrogenase (HD(R)). The DPS-defective mutants with feedback-sensitive AK showed a slow but substantial growth in the absence of DAP and their growth was markedly stimulated by DAP, while those with feedback-resistant AK grew well in the absence of DAP and their growth was not promoted by DAP more than that of the parent strain. DPS-defective mutants with HD(R) were derived from an HD(R) mutant producing 10 g/l of L-threonine and selected as AHV-resistant mutants with a higher productivity. The maximum production was 16 g/l.     

Characterization of cultured tobacco cell lines resistant to ethionine, a methionine analog

Two cultured tobacco cell lines (Nicotiana tabacum L. cv Xanthi) were selected for resistance to growth inhibition by the methionine analog ethionine. Comparison of the free amino acid pool levels in these lines with those of the ethionine-sensitive parental line showed substantial accumulation of methionine (110×), threonine (18×), and lysine (5×). In vitro enzymic analysis of lysine-sensitive aspartate kinase activity showed the resistant lines to contain 16 times that found in the sensitive line. The lysine-sensitive enzymes from both resistant and sensitive lines coeluted from DEAE-cellulose and exhibited similar K_m values. Both showed identical lysine plus S-adenosylmethionine inhibition profiles suggesting that the elevated activity in the resistant lines is not due to a structural change in the lysine-sensitive enzyme but possibly to the level of its expression.     

Synthesis of cephalosporin C by a methionine analogue resistant mutant of Cephalosporium acremonium

Summary DL-seleno-methionine resistant mutants of Cephalosporium acremonium were isolated which have an enhanced capacity to utilized sulfate for the synthesis of cephalosporin C. Of these mutants, one designated as SMR-I3 produced three-fold more cephalosporin C from sulfate than its parent CW19. Mutant SMR-I3 required less dl-methionine for maximal synthesis of cephalosporin C, but an excess of dl-methionine inhibited the synthesis of the antibiotic. Furthermore, the mutant accumulated excessive methionine in the amino acid pool and possessed superior activity for sulfate uptake. These observations indicate that in the mutant SMR-I3, the biosynthesis of methionine from sulfate is very active and excess methionine becomes available for the synthesis of cephalosporin C.     

Threonine analogue resistant mutants of Escherichia coli K-12

Summary Mutants of Escherichia coli K-12 resistant to a threonine analogue (-amino--hydroxy valeric acid) were predominantly resistant to ethionine and overproduced both threonine and methionine (2 mg/ml each). Novelty of the mutants is discussed.     

Production of methanethiol from methionine by Brevibacterium linens CNRZ 918

The conditions under which Brevibacterium linens CNRZ 918, a strain isolated from the surface smear flora of Gruyère de Comté cheese, produced methanethiol from methionine were studied. Demethiolation was estimated from the methanethiol production capacity of resting cells. Methionine was demethiolated mainly during the exponential growth phase of the organism during which time the cells were rod-shaped and had a generation time of 5 h, and the medium became alkaline. At the end of growth (pH 9) the cells were coccoid, and produced only very little methanethiol. The production of methanethiol required the presence of methionine in the culture medium, this reflecting the probable induction of the enzyme systems involved. Glucose favoured growth and inhibited production of methanethiol. Lactate favoured both growth and methanethiol production. Resting rod cells also produced methanethiol from structural analogues of methionine and from methionine-containing peptides. The apparent kinetic constants of the production of methanethiol for rod and coccoid cells were respectively Km = 14 mM and 46 mM, Vmax = 208 nkat g-1 and 25 nkat g-1. The optimum temperature and pH for production were 30 degrees C and pH 8. Azide or malonate favoured the production of methanethiol by resting cells, whereas chloramphenicol had no effect.     

Penicillin production by mutants ofPenicillium chrysogenum resistant to polyene macrolide antibiotics

Summary Polyene-resistant mutants ofPenicillium chrysogenum Wis. 54–1255 have been obtained by stepwise selection in increasing concentrations of polyene antibiotics. From the parent strain, sensitive to 10 g/ml of polyene antibiotics, mutants resistant to fungimycin (1.3 mg/ml), amphotericin B (0. 5 mg/ml), or nystatin (167 g//ml) were obtained. Their penicillin production is different from that of the parent strain and in particular some of the fungimycin-resistant mutants produce higher levels of penicillin.     

Increase of fungitoxicity of mercuric chloride by methionine,ethionine and S-methylcysteine

The fungitoxicity of mercuric chloride to Aspergillus niger was increased in the presence of d-, l-, dl-methionine, dl-ethionine, dl-S-methylcysteine or sodium methylmercaptide. The same effect was observed with methionine for two other fungi investigated: Cladosporium cucumerinum and Scopulariopsis brevicaulis. It is suggested that this effect can be ascribed to the formation of CH₃SHg⁺ or (CH₃S)₂Hg, or the corresponding ethyl compounds. CH₃SHgCl and (CH₃S)₂Hg were synthetically prepared and proved indeed far more fungitoxic than HgCl₂. The hypothesis was further substantiated by the observation that A. niger rapidly converts dl-methionine into CH₃SH, which undoubtedly reacts with Hg²⁺ to give the above mentioned methylthiomercury compounds.     

Ethanol production by thermotolerant yeast and its UV resistant mutants.

Six thermotolerant yeasts were isolated at 37 degrees C from over-ripe grapes by serial dilution technique using glucose yeast extract medium. Purified yeast cultures were screened for ethanol production at 37 degrees C by batch fermentation, using cane molasses containing 20% sugars. Sugar conversion efficiency of these isolates varied from 66.0 to 88.5% and ethanol productivity from 1.11 to 1.73 ml/l/h. The highest ethanol producing isolate was exposed to UV radiations and 13 mutants were picked up from the UV treatment exhibiting 0.1 to 1.0%, survival. The UV mutants varied in cell size from parent as well as among themselves. Determination of ethanol produced by all the mutants revealed that only five mutants resulted in 4.5 to 6.2% increase in sugar conversion and 8.25 to 18.56% increase in ethanol concentration coupled with maximum ethanol productivity of 2.4 ml/l/h in 48 h of batch fermentation of cane molasses (20% sugars) at 37 degrees C temperature.     

A carbon source-dependent antimetabolite sensitivity test in Bacillus megaterium B71 for isolation of ethionine resistant mutants

Alteration of carbon sources significantly altered the analogue sensitivity of Bacillus megaterium B71. DL-Ethionine (ETN) was highly inhibitory with glucose, mannitol, sucrose, citrate, glycerol and arabinose. DL-Norleucine, L-homoserine and S(2'-aminoethyl)-L-cysteine were either highly inhibitory, slightly inhibitory or non-inhibitory depending on the carbon sources used. Maltose markedly overcame the inhibitory effect of ETN in liquid culture. Uninhibited growth was poor on citrate and arabinose when compared with other carbon sources. Six carbon sources showing comparable growth were used to determine minimum inhibitory concentrations (MIC) of the analogues. The MIC of ETN was highest (450 μg/ml) with maltose and lowest (4 μg/ml) with mannitol. ETN sensitivity was inversely related to the endogenous L-methionine pool size, and was relatively low with mannitol which was used to isolate ETN resistant mutants of B. megaterium B71. The best mutant BUE-118 produced 435 μg/ml of L-methionine.     

Single-gene knockout of a novel regulatory element confers ethionine resistance and elevates methionine production in Corynebacterium glutamicum

Despite the availability of genome data and recent advances in methionine regulation in Corynebacterium glutamicum, sulfur metabolism and its underlying molecular mechanisms are still poorly characterized in this organism. Here, we describe the identification of an ORF coding for a putative regulatory protein that controls the expression of genes involved in sulfur reduction dependent on extracellular methionine levels. C. glutamicum was randomly mutagenized by transposon mutagenesis and 7,000 mutants were screened for rapid growth on agar plates containing the methionine antimetabolite d,l-ethionine. In all obtained mutants, the site of insertion was located in the ORF NCgl2640 of unknown function that has several homologues in other bacteria. All mutants exhibited similar ethionine resistance and this phenotype could be transferred to another strain by the defined deletion of the NCgl2640 gene. Moreover, inactivation of NCgl2640 resulted in significantly increased methionine production. Using promoter lacZ-fusions of genes involved in sulfur metabolism, we demonstrated the relief of l-methionine repression in the NCgl2640 mutant for cysteine synthase, o-acetylhomoserine sulfhydrolase (metY) and sulfite reductase. Complementation of the mutant strain with plasmid-borne NCgl2640 restored the wild-type phenotype for metY and sulfite reductase.     

The biotransformation of t-butylacetonitrile and its boron-containing analogue trimethylamine-cyanoborane by Brevibacterium R312

The ability of the nitrile hydratase/amidase system from Brevibacterium R312 to biotransform tert-butylacetonitrile was studied with a view to their utilisation in the production of novel amino acids from isostructural compounds. Brevibacterium R312 was able to transform nitriles with this structure; however, the wide spectrum amidase from this organism was unable to biotransform the corresponding amide to the carboxylic acid. Received: 8 December 1996 / Accepted: 25 April 1997