Production of l-Tryptophan by Azaserine-resistant Mutants of Brevibacterium flavum

Azaserine-resistant mutants derived from a 5-fluorotryptophan-resistant, l-tryptophan-producing mutant of Brevibacterium flavum, accumulated 10.3 g/liter of l-tryptophan at maximum. The production increased to 11.4 g/liter when l-serine was added. In the mutant, only anthranilate synthase among enzymes of the tryptophan-specific bio synthetic pathway increased in activity to a 2-fold higher level than that in the parent strain, No. 187. Sensitivity of anthranilate synthase to the feedback inhibition was not altered by the mutation. Activity of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, the first common enzyme for aromatic amino acid biosynthesis, also increased 2.7-fold and was less sensitive to the feedback inhibition by phenylalanine and tyrosine. Tryptophan transport activity in strain A-100 was similar as that in the parent. Azaserine inhibited anthranilate synthase activity by 50% at 0.075 mm. The inhibition was of a mixed type with respect to both the two substrates. Anthranilate synthase of strain A-100 was inhibited in a similar manner to that of the parent.     

l-Serine Production Improved by Analogue-resistant Mutants of a Methanol-utilizing Bacterium

In the higher plant, Arabidopsis thaliana, histidine-to-aspartate (His-to-Asp) phosphorelay signal transduction systems play crucial roles in propagation of environmental stimuli, including plant hormones. This plant has 11 sensor His-kinases, 5 histidine-containing phosphotransfer (HPt) factors (AHPs), and 20 response regulators (ARRs). To gain new insight into the functions of these phosphorelay components, their intracellular localization was examined with use of GFP-fusion proteins, constructed for certain representatives of HPt factors (AHP2) and type-A and type-B ARRs (ARR6/ARR7 and ARR10, respectively). The results showed that AHP2 is mainly located in the cytoplasmic space, while both the types of ARRs have an ability to enter preferentially into the nuclei, if not exclusively. Together with the results from an in vitro phosphorelay assay with AHP2 and ARRs, these results are discussed, in terms of a geneal framework of the Arabidopsis His-to-Asp phosphorelay network.     

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.     

Tryptophan Synthase and Production of l-Tryptophan in Regulatory Mutants

A 5-fluorotryptophan-resistant mutant of Brevibacterium flavum, No. 187, accumulated 2.6 g of indole 3-glycerol (InG) in addition to 8.0 g of l-tryptophan per liter in the culture medium. The addition of l-serine to the medium decreased the accumulation of InG and increased that of l-tryptophan up to a concentration of 10.3 g/liter, while the addition of l-tryptophan increased the InG accumulation, suggesting that InG was formed by hydrolysis of indole 3-glycerol phosphate (InGP), the substrate of tryptophan synthase (TS) which catalyzed the final step reaction of tryptophan biosynthesis. Then, in order to examine the mechanism of the InG accumulation, TS was purified from tryptophan auxotroph, TA-60. The reaction mechanism of TS was Ordered Bi Bi with Km’s of 0.63 and 0.038 mm for serine and InGP, respectively. Tryptophan, a product of the TS reaction, inhibited TS competitively with respect to serine and the Ki for tryptophan was estimated to be 2.0 mm. On the other hand, anthranilate synthase (AS), the first enzyme in the tryptophan biosynthetic pathway, was much less sensitive to the feedback inhibition by tryptophan in strain No. 187 than in the wild strain. The tryptophan concentration giving 50% inhibition of AS in strain No. 187 was estimated to be 2.4 mm, almost comparable to that of TS, 7.7 mm. From these results, it was concluded that the accumulation of InG in strain No. 187 would result from the product inhibition of TS by the tryptophan accumulated.     

Thermostability of Pseudomonas hydrogenothermophila Cytochrome c-552

The alcohol-fermenting yeast Torulaspora delbrueckii No. 3110 was less tolerant to high temperature than Saccharomyces cerevisiae IFO 0224 as measured by alcohol fermentation during mild agitation: at 40°C, ethanol production of the two yeasts was 0.8 and 5.2 wt% respectively. The No. 3110 cells had much unsaturated fatty acid (C18:2) and little ergosterol, which suggests that the low tolerance might be caused by high membrane fluidity. Two types of miconazole-resistant mutants were isolated and characterized. Strain M47 had less unsaturated fatty acid and was found to be more temperature tolerant than No. 3110. Strain M59 was defective in ergosterol synthesis and was less temperature tolerant than No. 3110. These results indicate the importance of membrane rigidity in temperature tolerance.

M59 aaccumulated much less trehalose than No. 3110 did. Addition of trehalose to the permeabilized cell system of M59 restored the temperature sensitivity, but not when the trehalase inhibitor deoxynojirimycin was also added, which suggests that the accumulation and metabolism of trehalose is important for the expression of temperature tolerance.     

The Conversion of d-Tryptophan to l-Tryptophan in Cell Cultures of Tobacco

d-Tryptophan was converted to l-tryptophan in tissue cultures of tobacco, in whole cells treated with dimethylsulfoxide, and in cell-free extracts treated by Sephadex G-25 filtration. Evidence was obtained that tryptophanase, tryptophan pyrrolase, and transaminase activities were not involved. The data were best explained by the presence of a tryptophan racemase as the enzyme catalyzing the reaction. The possible role of d-tryptophan in the biosynthesis of indoleacetic acid is discussed.     

Excretion of metabolites by hydrogen bacteria I. Autotrophic and heterotrophic fermentations

Summary The excretion of metabolites by 48 wild-type and mutant strains belonging to various species and genera of aerobic hydrogen-oxidizing bacteria was studied. The cells were grown autotrophically and heterotrophically, and samples were analyzed by gas chromatrographic techniques. The following metabolites were identified and quantitatively determined: acetate, ethanol, malate, citrate, lactate, succinate, 2-propanol, 2-methylpropanoate, 3-methylbutanoate, cis-aconitate, acetone, 2-oxoglutarate, isocitrate, butanoate, and methanol. The excretion of the metabolites started when ammonia and oxygen became limiting. The concentrations reached a maximum, whereupon the excreted products were reconsumed.The total concentration of the metabolites identified reached 5 g/l. Maximum concentrations were measured when mutants of Alcaligenes eutrophus lacking the ability to accumulate poly-3-hydroxybutanoate were grown on fructose, gluconate, or lactate in the fermenter under conditions of ammonia limitation and when the carbon source was present in excess.     

Production of l-DOPA by Mutants of Pseudomonas melanogenum

Several kinds of mutants of Pseudomonas melanogenum were derived by mutational treatment with N-methyl-N’-nitro-N-nitrosoguanidine, and selected for 3,4-dihydroxyphenyl-l-alanine (l-DOPA) production by newly devised screening method which was carried out on agar plates based on violet-black colour formation by the reaction of l-DOPA with iron ion. Mutants tested were; glucose-insensitive mutant, cysteine-insensitive mutant, 3-amino-tyrosine-resistant mutant and p-fluorophenylalanine-resistant mutant. Some colonies isolated by monocolony procedure without mutagenic treatment were also tested. Among the 3-aminotyrosine-resistant mutants many good l-DOPA producers were found.

An 3-aminotyrosine-resistant mutant, strain ATN–36, produced 14 to 15 mg/ml of l-DOPA from 26 mg/ml of l-tyrosine (68 % in molar conversion ratio). When the cell concentration in reaction mixture was increased to 4-times the concentration of culture broth, l-DOPA production reached to 21 mg/ml from 52 mg/ml of tyrosine. An enzymatic basis of the high l-DOPA productivity of the improved mutants was found to be due to the increased tyrosinase activity (150 to 160% of the parental strain) of the mutants.     

Organic Carbon Utilization by Obligately and Facultatively Autotrophic Beggiatoa Strains in Homogeneous and Gradient Cultures

Marine Beggiatoa strains MS-81-6 and MS-81-1c are filamentous gliding bacteria that use hydrogen sulfide and thiosulfate as electron donors for chemolithotrophic energy generation. They are known to be capable of chemolithoautotrophic growth in sulfide gradient media; here we report the first successful bulk cultivation of these strains in a defined liquid medium. To investigate their nutritional versatilities, strains MS-81-6 and MS-81-1c were grown in sulfide-oxygen gradient media supplemented with single organic compounds. Respiration rates and biomass production relative to those of controls grown in unsupplemented sulfide-limited media were monitored to determine whether organic compounds were utilized as sources of energy and/or cell carbon. With cells grown in sulfide gradient and liquid media, we showed that strain MS-81-6 strongly regulates two enzymes, the tricarboxylic acid cycle enzyme 2-oxoglutarate dehydrogenase and the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase, in response to the presence of organic carbon (acetate) in the growth medium. In contrast, strain MS-81-1c lacked 2-oxoglutarate dehydrogenase activity and regulated ribulose-1,5-bisphosphate carboxylase/oxygenase activity only slightly in response to organic substrates. Tracer experiments with radiolabeled acetate showed that strain MS-81-1c did not oxidize acetate to CO(inf2) but could synthesize approximately 20% of its cell carbon from acetate. On the basis of these results, we conclude that Beggiatoa strain MS-81-1c is an obligate chemolithoautotroph, while strain MS-81-6 is a versatile facultative chemolithoautotroph.     

Extracellular Polysaccharide Formed by Pseudomonas hydrogenovora in Autotrophic Culture and Its Physiological Activities

Gram negative hydrogen bacterium Pseudomonas hydrogenovora was found to excrete an anthrone-H₂SO₄ positive viscous polysaccharide. About 12 g/liter of the polysaccharide was produced autotrophically on gaseous hydrogen at the stationary phase of growth. Biosynthesis of the polysaccharide occurred under nitrogen-deficient condition. Its elementary composition was C: 39.29%, H: 6.23%, O: 49.67%, N: 0.21% and ash 4.6%. The polysaccharide contained galactose, glucose, mannose and rhamnose as its main components. The polysaccharide had anti-tobacco mosaic virus and anti-tumor activities.     

Metabolism of Phenol and Cresols by Mutants of Pseudomonas putida

Mutant strains of Pseudomonas putida strain U have been obtained which are deficient in enzymes of the degradative pathways of phenol and cresols. Mutant strains deficient in catechol 2, 3-oxygenase accumulated the appropriate catechol derivative from cresols. A mutant strain which would not grow on either phenol or a cresol was shown to be deficient in both 2-hydroxymuconic semialdehyde hydrolase and a nicotinamide adenine dinucleotide, oxidized form, (NAD(+))-dependent aldehyde dehydrogenase. When this strain was grown in the presence of phenol or a cresol, the appropriate product of meta fission of these compounds accumulated in the growth medium. A partial revertant of this mutant strain, which was able to grow on ortho- and meta-cresol but not para-cresol, was shown to have regained only the hydrolase activity. This strain was used to show that the products of meta ring fission of the cresols and phenol are metabolized as follows: (i) ortho- and meta-cresol exclusively by a hydrolase; (ii) para-cresol exclusively by a NAD(+)-dependent aldehyde dehydrogenase; (iii) phenol by both a NAD(+)-dependent dehydrogenase and a hydrolase in the approximate ratio of 5 to 1. This conclusion is supported by the substrate specificity and enzymatic activity of the hydrolase and NAD(+)-dependent aldehyde dehydrogenase enzymes of the wild-type strain. The results are discussed in terms of the physiological significance of the pathway. Properties of some of the mutant strains isolated are discussed.     

Characteristics of Autotrophic and Heterotrophic Denitrification by the Strain Pseudomonas sp. H117

Strain H117 was isolated from the Tang Yu reservoir. Based on the phylogenetic characteristics, strain H117, which was identified as Pseudomonas sp. strain H117, had the capability to utilize bicarbonate and sodium acetate as a carbon source under anaerobic conditions. Furthermore, the strain could grow on both autotrophic and heterotrophic media, and could perform both autotrophic and heterotrophic denitrification in the medium. Response surface methodology analysis demonstrated that the maximum degradation ratio of nitrate-occurred under the following conditions in the autotrophic medium: initial pH of 6.00, C/N ratio of 4.68 and temperature of 31.33°C. The maximum degradation ratio of nitrate occurred under the following conditions in the heterotrophic medium: initial pH of 6.16, C/N ratio of 8.23 and temperature of 28.48°C. Finally, the denitrification performance of strain H117 was evaluated under the optimum conditions. These results suggest that strain H117 has potential applications for the bioremediation of polluted groundwater.     

Fluorene Cometabolism by Rhodococcus rhodochrous and Pseudomonas fluorescens Cultures

The transformation of fluorene by Rhodococcus rhodochrous strain 172 grown on sucrose and Pseudomonas fluorescens strain 26K grown on glycerol was studied as a function of the substrate concentration and the growth phase. Under certain cultivation conditions, fluorene was completely consumed from the medium. The specific transformation rate of fluorene was considerably higher when it was transformed in the presence of the cosubstrates than when it served as the sole carbon source. An approach to the evaluation of the specific transformation rate of fluorene during batch cultivations is proposed.     

L-Serine Production by Temperature-sensitive Mutants of Methanol-utilizing Bacterium Pseudomonas MS 31

Temperature-sensitive mutants producing L-serine efficiently from glycine were obtained from the facultative methylotroph Pseudomonas MS 31. Forty-five mutant strains showed adequate growth on methanol at 30°C but little or no growth at 37°C. Fourteen of these mutants produced L- serine more efficiently than the wild-type strain. The typical mutant strain ts 162 showed a high conversion rate in glycine-to-L-serine when the cultivation temperature was changed from a permissive (30°C) to non-permissive state (38?42°C) together with the addition of glycine and methanol after adequate growth. The mutant strain accumulated 6.8 mg L-serine from 12 mg glycine per ml culture under optimum conditions. The reduction of L-serine degrading activity in the mutant strain seemed to contribute to the high productivity of L-serine.     

Excretion of Glycolate, Mesotartrate and Isocitrate Lactone by Synchronized Cultures of Ankistrodesmus braunii

Fixation of ¹⁴CO₂ by synchronized cultures of Ankistrodesmus braunii was highest for young growing cells, low for mature cells, and lowest for dividing cells. The amount of ¹⁴C excreted during photosynthesis followed the same trend. Cells at the end of the growing phase, after 10 hours of a 16-hour light phase, excreted nearly 35% of the total ¹⁴C fixed as one product, glycolate. Dividing cells from the dark phase, when tested in the light, excreted only 4% as much glycolate-¹⁴C as the young growing cells. Dividing cells also excreted as much mesotartrate as glycolate and also some isocitrate lactone and an unidentified acid. None of these excreted acids were found inside the cells in significant amounts. Methods for isolation and identification of the excreted acids are present. With ¹⁴C-labeled algae, it was shown that the excretion of glycolate was light-dependent and inhibited by 1,1-dimethyl-3-(p-chlorophenyl) urea. The excretion of labeled mesotartrate, isocitrate lactone, and an unknown acid, but not glycolate, also occurred in the dark. The excreted mesotartrate was predominantly carboxyl-labeled even after long periods of ¹⁴CO₂ fixation. Since glycolate is known to be uniformly labeled, glycolate could not be the precursor of the carboxyl-labeled mesotartrate. The reason for the specific excretion of glycolate, mesotartrate, and isocitrate lactone is not known, but the metabolism of all three acids by the algae may be limited and each can form dilactides or lactones by dehydration. In this context isocitrate lactone was excreted rather than the free acid.     

Callus Cultures of Tomato Mutants II. Carotenoid Formation

Carotenoid synthesis in the fruit of tomato mutants is under gene control. Since undifferentiated plant cells are presumably totipoltent, the callus tissue should be able to produce the carotenoids typical of the genotype. The carotenoids of masses of callus derived from hypocotyls of the red ghost and tangerine tomatoes were extracted and characterized by means of column and thin layer chromatography, chemical and spectral properties. The red ghost callus contained predominantly phytoene and phytofluene; the tangerine callus had primarily ζ-carotene and prolycopene. Thus under our cultural conditions, carotenoids typical of the fruit rather than the leaf were produced, albeit in much reduced quantities.