Fermentative Production of Chloramphenicol-analogs (Corynecins) from Sucrose

Corynecins (N-acyl derivatives of d-(?)-threo-p-nitrophenyl serinol), which were firstly discovered in the culture broth of Corynebacterium hydrocarboclastus KY 4339 grown on n-alkane were also produced when n-alkane was replaced by sucrose. The corynecins production in the sucrose-medium was significantly stimulated by the supplement of molasses. On the basis of the composition of ingredients in molasses, the preferable culture medium was designed for the production of corynecins from sucrose. This semi-synthetic medium is consisted of low concentration of phosphate, high concentration of potassium chloride, inositol, fructose and yeast extract in addition to ordinary mineral salts. By controlling the pH of the medium at the neutral range and keeping the aeration at a relatively high level, approximately 4 g of corynecins (as l-base) per liter of the medium were produced using a 5-liter jar fermentor.     

Immunochemical assay applied to mycotoxin biosynthesis: ELISA comparison of sterigmatocystin production byAspergillus versicolor andAspergillus nidulans

Conventional thin layer and instrumental methods for analyzing mycotoxins and their precursors are time-consuming and make the investigation of mycotoxin biosynthesis particularly difficult. As an alternative, sensitive enzyme-liked immunosorbent assays (ELISAs) can be utilized to analyze for these compounds. In this report, sterigmatocystin production in test tube cultures ofAspergillus versicolor ATCC 18643 andAspergillus nidulans ATCC 32610 were compared using competitive ELISA. Polyclonal antiserum that was prepared against a sterigmatocystin hemiacetal-bovine serum albumin conjugate exhibited greatest specificity for sterigmatocystin hemiacetal and sterigmatocystin with less reactivity for O-methylsterigmatocystin. The antiserum could be used to detect as little as 50 ng/ml sterigmatocystin in ELISA. Direct ELISA could be performed on diluted culture broth and on mycelial extracts solubilized with N,N-dimethylformamide.Apergillus versicolor ATCC 18643 produced more sterigmatocystin in SLS medium than in YES medium, and showed maximal levels at between 9 to 12 days incubation. Approximately 75% of sterigmatocystin was detectable in mycelium (254g/ml culture) compared to the extracellular fraction (87g/ml culture).Aspergillus nidulans exhibited qualitatively similar patterns of growth and toxigenesis in SLS medium but accumulated maximal levels of only 15g mycelial sterigmatocystin/ml culture and 5g extracellular sterigmatocystin/ml broth, respectively.     

L-Tyrosine production by Polyauxotrophic Mutants of Corynebacterium glutamicum

Polyauxotrophic mutants of Corynebacterium glutamicum which have additional requirements to L-phenylalanine were derived from L-tyrosine producing strains of phenylalanine auxotrophs, C. glutamicum KY 9189 and C. glutamicum KY 10233, and screened for L-tyrosine production. The increase of L-tyrosine production was noted in many auxotrophic mutants derived from both strains. Especially some double auxotrophs which require phenylalanine and purine, phenylalanine and histidine, or phenylalanine and cysteine produced significantly higher amounts of L-tyrosine compared to the parents, A phenylalanine and purine double auxotrophic strain LM–96 produced L-tyrosine at a concentration of 15.1 mg per ml in the medium containing 20% sucrose. L-Tyrosine production by the strain decreased at high concentrations of L-phenylalanine.     

Production of Nucleic Acid-Related Substances by Fermentative Processes

A seed medium and a fermentation medium for nucleotide fermentations such as 5′ IMP, 5′GMP (plus GDP and GTP) and 5′AMP (plus ADP and ATP) with Brevibacterirm ammoniagenes ATCC 6872 were entirely chemically defined, with the use of a mixture of five amino acids.

As a result, the presence of Zn²⁺, Fe²⁺ and Ca²+ in addition to Mn²⁺ was found to be essential for the nucleotide fermentations. In particular, Zn²⁺ levels as well as Mn²⁺ affected nucleotide productions remarkably. Various fermentations proceeded favorably only when suboptimum levels of manganese (20~30 μg/liter) and zinc (100~200 μg/liter) were simultaneously present. This effect of trace metals was attributed to the fact that the excretion of R5P, a precursor of nucleotides, and those enzymes catalyzing reactions synthesizing nucleotides from R5P, ATP and purine bases were greatly stimulated by trace metals in cooperation with two vitamins, Ca-pantothenate and thiamine, and presumably high concentrations of phosphate and magnesium.

Furthermore, it was revealed that some metals were able to control the amounts of nucleotides accumulated when they were added to the broth during fermentation. For example, Hg²⁺ and Ag⁺ could increase the amounts of 5′GMP or 5′AMP, and decrease those of GTP and ATP.

Growth responses of Brevibacterium ammoniagenes ATCC 6872, capable of accumulating purine nucleotides, were investigated by the use of completely defined media.

1. Casamino acids required for its growth could be replaced by a mixture of l-histidine, l-homoserine, glycine, d, l-alanine and l-lysine. A completely defined medium for nucleotide productions was thus established by the use of this mixture.

2. High levels of phosphate inhibited growth markedly, and this inhibition was overcome by the simultaneous addition 1) of hign levels of Mg²⁺ and 2) of Mn²⁺, 3) pantothenate and 4) thiamine. Ca²⁺ had also a stimulatory effect on the growth. Therefore, a clear growth response to Mn²⁺ levels and the requirement of the two vitamins for growth emerged only under the conditions of high phosphate and magnesium salts. These 4 factors were found entirely the same as factors essential for nucleotide accumulations by Br. ammoniagenes.

     

l-Tryptophan Production by 5-Methyltryptophan-resistant Mutants of Glutamate-producing Bacteria

1. Some of 5-methyltrypotophan (5MT)-resistant mutants derived from glutamate-producing bacteria such as Brevibacterium flavum, Corynebacterium acetoglutamicum and Micrococcus glutamicus produced a small amount of l-tryptophan, while tyrosine and phenylalanine auxotrophs of B. flavum did not.

2. 5-MT-resistant mutant derived from the auxotroph for tyrosine and phenylalanine produced 390 mg/liter of l-tryptophan at most. A mutant resistant to a higher concentration of 5MT, which was derived from a tyrosine and phenylalanine auxotrophic mutant which was resistant to a low concentration of 5MT, produced 660 mg/liter of l-tryptophan. Using this mutant, the effects of the concentrations of components of the culture medium on the l-tryptophan production were examined. The high concentration of l-tyrosine, but not l-phenylalanine, inhibited the l-tryptophan production. Using the improved culture medium, this strain produced 1.9 g/liter of l-tryptophan.     

Production ofl-phenylalanine by double auxotrophic mutants ofArthrobacter globiformis: Effect of temperature,trace salts and inoculum dose

Three tryptophan-plus-tyrosine double auxotrophic mutants were isolated from a biotin-requiring glutamate-producingArthrobacter globiformis. The mutants were found to producel-phenylalanine in a mineral salt medium. Further improvement ofl-phenylalanine production was achieved by isolation of mutants resistant to β-2-thienylalanine from these double auxotrophs. Temperature of 30 °C and a 4% inoculum dose were found to be optimum for phenylalanine production. Addition of some trace salts does not enhance phenylalanine yield. Under optimal cultural conditions one mutant yielded 6.8 g phenylalanine per L medium in flask culture.     

Influence of physical conditions of nutrient medium and sucrose on somatic embryogenesis of date palm

Shoot tips and leafy bud fragments removed from offshoots of adult date palms (Phoenix dactylifera L.) were cultured on a nutrient medium containing the Murashige and Skoog inorganic salts, 453 M 2,4-dichlorophenoxyacetic acid, 14.8 M N⁶-(2-isopentenyl)adenine and 3 g l^-1 activated charcoal to develop nodular callus after 8 months of culture. Callus was cultured in agar-solidified and stationary or shaken liquid media containing half-strength MS inorganic salts, 3 g l^-1 activated charcoal and different sucrose concentrations to study the influence of these factors on somatic embryogenesis. The best conditions for embryo development were culturing in liquid medium shaken at 100 rpm for a period of 2 weeks without sucrose, followed by culture on 3% sucrose.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2iP N⁶-(2-isopentenyl) adenine - MS Murashige & Skoog (1962) - rpm revolutions per minute     

Experimental cerebral lesions produced by inoculation with Basidiobolus strains

The aflatoxinogenesis of Aspergillus parasiticus is significantly enhanced by the presence, in the medium, of sterigmatocystin at a high level (35–50 g/ml); low concentrations, in the order of 175 g/ml, have no effect on the production of aflatoxins. During the period where the aflatoxinogenesis of the culture is high, no variation of the sterigmatocystin level is noted. Experiments with ¹⁴C-sterigmatocystin indicate that the mold does not utilize the metabolite itself as a precursor of aflatoxins.     

Isolation,identification and characterisation of a soil bacterium producing an enzyme with l-phenylalanine oxidase activity

A gram-positive, mesophilic bacterium which assimilated l-phenylalanine but which failed to utilise l-tyrosine was isolated from soil. The isolate, identified as a strain of Bacillus carotarum, converted l-phenylalanine to phenylpyruvate with the initial step catalysed by an inducible, intracellular enzyme which possessed l-phenylalanine oxidase activity. Phenylalanine oxidase has not been previously reported in Gram-positive bacteria, although there are a few examples of non-specific l-amino acid oxidases with activity towards l-phenylalanine. The isolate grew abundantly on complex media but failed to synthesise significant amounts of the enzyme in the absence of l-phenylalanine. The highest enzyme levels were achieved in a chemically defined minimal salts medium containing the amino acid at 10 g/l as the primary carbon and energy source.     

Utilization of Hydrocarbons by Microorganisms

As already reported, Corynebacterium hydrocarboclastus S10B1 was able to accumulate a good deal of l-glutamate in a thiamine-deficient medium at the sole expense of n-alkanes, but unable to form l-glutamate in a thiamine-sufficient medium though an abundant cell growth was observed.

α-Ketoglutaric acid and dl-alanine were found to be produced in the same thiamine-deficient medium in which l-glutamate was accumulated. Both products formed from n-tetradecane by this organism were isolated from culture broth, purified and identified. The optimum concentration of thiamine in the culture medium was 3 to 5 µg per liter for their production. The maximum yields of α-ketoglutaric acid and dl-alanine reached 16 g and 1.5 g per liter in the calcium carbonate-added medium, respectively. However, the addition of more than 30 μg per liter of thiamine extremely repressed their accumulation.     

Determination of L-Cysteine with L-Methionine γ-Lyase

A chemically defined medium was devised to examine the growth, production and biochemical pathway of tetrocarcin A. The production of tetrocarcin A was greatly stimulated by l-feucine and its corresponding keto acid, α-ketoisocaproate, suggesting that l-leucine is involved in the biosynthesis of tetrocarcin A. About 10–12 μg/ml of tetrocarcin A was produced in a chemically defined medium consisting of 20 g sucrose, 2.5 g KNO₃, 5 g MgSO₄·7H₂O, 5 g KH₂PO₄ and 1 g l-leucine per liter of water (pH 7.0).     

Intracellular concentrations and metabolism of carbon compounds in tobacco callus cultures: effects of light and auxin

Callus cultures derived from pith tissue of Nicotiana tabacum were grown on two media either under continuous illumination or in complete darkness. The first medium limited greening ability of callus grown in the light (3 milligrams per liter naphthalene acetic acid, 0.3 milligram per liter 2-isopentenylaminopurine, Murashige and Skoog salts, and 2% sucrose). The second medium encouraged chlorophyll synthesis (greening) though not shoot formation (0.3 milligram per liter naphthalene acetic acid; 0.3 milligrans per liter 2-isopentylaminopurine). To measure intracellular concentrations, calli were grown for 15 days on these standard media containing [U-¹⁴C]sucrose. The dry weight proportions of the calli (as a fraction of fresh weight) and many metabolite concentrations nearly doubled in light-grown cells compared to dark-grown cells and increased 30 to 40% on low-auxin media relative to high-auxin media. Glutamine concentrations (from 4 to 26 millimolar) were very high, probably due to the NH₃ content of the media. Proline concentrations were 20-fold higher in calli grown on low-auxin media in the light (green cells), possibly a stress response to high osmotic potentials in these cells. To analyze sucrose metabolism, callus cells were allowed to take up 0.2% (weight per volume) [U-¹⁴C]sucrose for up to 90 minutes. In callus tissues and in pith sections from stems of tobacco plants, sucrose was primarily metabolized through invertase activity, producing equal amounts of labeled glucose and fructose. Respiration of ¹⁴CO₂ followed the labeling patterns of tricarboxylic acid cycle intermediates. Photorespiration activity was low.     

Mannitol Production by Pyrenochaeta terrestris

Pyrenochaeta terrestris (Hansen) Gorenz, J. C. Walker and Larson produces D-mannitol in the mycelium but not in the cutture filtrates when grown in a sucrose salts liquid medium. In the present study, P. terrestris was grown in stilt culture on a synthetic salts medium containing 30 g of sucrose per liter. After inoculation with a myceliat suspension, the mycelial mats were harvested and the dry weight and the amount of mannitol were determined. Maximum mycelial mat production occurred at 15 days after inoculation while the amount of mannitol was greatest at about 7 days after inoculation. The percentage of mannitot on a dry weight basis was maximal (20–25 per cent) within a few days after inoculation and decreased rapidly to 3–4 per cent at the time mycelial mat production was greatest. The same percentage of mannitot was produced when the fungus was grown in shake culture or when the sucrose was replaced by equivalent amounts of D-fructose, D-glucose, D-mannose, maltose, trehalose, and raffinose. Increasing the amount of sucrose or decreasing the amount of sodium nitrate increased the amount of mycelium produced but the percentage of mannitol in the mycelium remained about the same. Mannitot was reutilized when mycelial mats were transferred to a mineral medium without a carbon source. It was concluded that mannitot probably serves as a reserve carbohydrate in P. terrestris.     

Serum-free modified trichosel medium for the isolation ofTrichomonas vaginalis

A serum-free medium was developed for the isolation and cultivation ofTrichomonas veginalis. The basal medium was Trichosel fluid medium to which was added 20 g per liter of yeast extract, 2.5 ml per liter of whole milk, and 10 g per ml of cholesterol; the entire medium was then heat sterilized. This serum-free medium supported the growth and proliferation of small numbers of trichomonads and was successful for maintenance, serial passage, and clinical isolation of the organism from vaginal specimens.     

Growth and productivity of Beta vulgaris cell culture in fluidized bed reactors

Beta vulgaris cells were cultured in specially constructed fluidized bed reactors on Murashige and Skoog medium with 3% sucrose as a carbon source. Biomass density levels of approximately 17–18 g dry cell weight liter^–1 for 5 liter fermenters and 9–10 g dry cell weight liter^–1 for 50 liter reactor were observed. Depending on particular cell line, cell content of betalains reached levels ranging from 12 to 25 mg g dry cell weight^–1, representing the productivities of 14 to 17 mg of pigments per liter and day.The authors would like to thank the Chemap Tetra Laval group and the Kommission zur Förderung der wissenschaftlichen Forschung for the financial support of this project (KWF No. 2057.1).     

Establishment of a system of high-frequency embryogenesis from long-term cell suspension cultures of rice (Oryza sativa L.)

Summary Suspension cultures which maintained embryogenic potency for more than 18 months were established from excised immature embryos of rice (Oryza sativa L. cv. Konansou). The cultures were subcultured every three days in N6 medium supplemented with proline (10 mM), casein hydrolysate (300 mg/l), sucrose (30 g/l) and 2,4-D (1 mg/l). The frequency of embryogenesis from the embryogenetic suspension cultures reached about 90% when cell clusters (about 1 mm in diameter) were transferred to a solid medium which consisted of N6 medium, NAA (1 mg/l), kinetin (5 mg/l), sucrose (30 g/l) and Gelrite (2 g/l). When smaller clusters of cells (approximately 200–400 m in diameter) were transferred to a liquid medium which consisted of salts of N6 medium diluted with an equal volume of water plus sucrose (45 g/l), NAA (0.01 mg/l) and 4-PU (0.1 mg/l) at a cell density of 13 clusters/ml in 2 ml of medium, somatic embryogenesis was initated at high frequency (about 50%). Morphological evidence is provided to demonstrate that the regeneration occurred via embryogenesis. This is the first report of high-frequency embryogenesis in suspension cultures of rice cells.     

Gluconate Metabolism by a Thiamine-requiring Species of Corynebacterium

A thiamine-requiring strain of Corynebacterium was found to accumulate a ketopentose extracellularly from gluconate. The ketopentose was isolated from the culture medium and identified as d-ribulose. The accumulation of d-ribulose was significantly influenced by the concentration of thiamine in the medium. The maximum yield of d-ribulose was obtained at a thiamine concentration of 10 μg per liter, whereas good growth was favored at thiamine concentrations greater than 50 μg per liter. The accumulation of d-ribulose reached the concentration of 9.5 mg per ml after cessation of cell growth in shake culture at 30°C in a medium containing 6% potassium gluconate as a carbon source.     

Levan production byBacillus polymyxa

Summary A levan-producing bacterium was isolated from soils and its characteristics for polysaccharide synthesis were studied. A series of enrichment and plating techniques enabled the isolation of a levan-producing bacterium from closely related contaminants. Cultural and physiological characteristics of the isolate identified the organism an a strain ofBacillus polymyxa. The organism produced about 40 g extracellular polysaccharide per liter of sucrose medium, which was about three times more yield than levan obtained from known levan producers. The highest amount of polysaccharide was on a 8% sucrose medium. Hydrolysis of the product showed that the polysaccharide consisted entirely ofd-fructose, and¹³C.n.m.r. spectra confirmed that the product was levan, a fructose polymer linked by B-(26) fructofuranosyl linkage.     

l-Phenylalanine production by double auxotrophic mutants ofArthrobacter globiformis

A number of tryptophan-plus-tyrosine double auxotrophs have been isolated from a glutamate producingArthrobacter globiformis excretingl-phenylalanine by two-step mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine. For the three potent mutants tested the medium of Alföldi was found to be the best. The optimum tryptophan, tyrosine and biotin concentrations for phenylalanine production of these mutants were 0.5 mmol/L, 0.1 mmol/L and 5 μg/L, respectively. At these levels strain TT-39 yielded 2.6 g phenylalanine per L of medium in flask culture with glucose (350 mmol/L) and NH₄Cl (60 mmol/L).     

Transformation of five grape rootstocks with plant virus genes and a virE2 gene from Agrobacterium tumefaciens

Summary To facilitate the development of transgenic grapevines that are resistant to grapevine fanleaf virus (GFLV), grapevine leafroll-associated closterovirus (GLRaV-3) and crown gall diseases, we developed a rapid system for regenerating root-stocks: Couderc 3309, Vitis riparia ‘Gloire de Montpellier’, Teleki 5C, Millardet et De Grasset 101-14, and 110 Richter via somatic embryogenesis. Embryo culture and grape regeneration were accomplished with four media. Embryogenic calluses from anthers were induced in the initiation medium [MS basic medium containing 20 g sucrose per L, 1.1 mg 2,4-dichlorophenoxyacetic acid (2,4-D) per L, 0.2 mg N⁶-benzyladenine (BA) per L, and 0.8% Noble agar). The percentage of anthers that developed into embryogenic calli ranged from 2 to 16.3% depending on the rootstock. Calluses with early globular stage embryos were cocultivated with Agrobacterium tumefaciens strain C58Z707 containing the gene constructs of interest. The genes were sense-oriented translatable and antisense coat protein genes from GFLV and GLRaV-3, a truncated HSP90-related gene of GLRaV-3 (43K), and a virE2 del B gene from A. tumefaciens strain C58. Twenty independent transformation experiments were performed on five rootstocks. After 3–4 mo. under kanamycin selection, secondary embryos were recovered on differentiation medium (1/2 MS salts with 10 g sucrose per L, 4.6 g glycerol per L, and 0.8% Noble agar). Embryos that were transformed were regenerated on a medium containing MS salts with 20 g sucrose per L, 4.6 g glycerol per L, 1 g casein hydrolysate per L, and 0.8% Noble agar. Elongated embryos were then transferred to a rooting medium supplemented with 0.1 mg BA per L, 3 g activated charcoal per L, 1.5% sucrose, and 0.65% Bacto agar. A total of 928 independent putative transgenic plants were propagated in the greenhouse. All plants were tested for neomycin phosphotransferase II expression by enzyme-linked immunosorbent assay (ELISA). The presence of transgenes was assessed by polymerase chain reaction and Southern analysis. ELISA revealed various levels of expression of GFLV coat protein in transgenic plants of Couderc 3309. The transgenic rootstocks that have been generated are being screened to determine whether transgenes have conferred resistance to the virus and crown gall diseases.