Selection of Streptomyces ambofaciens mutants that produce large quantities of spiramycin and determination of optimal conditions for spiramycin production.

The aim of this work was to develop a strategy to isolate a morphologically stable mutant of Streptomyces ambofaciens ATCC 15154 which produced high titers of spiramycin. The rationale was to grow a nitrosoguanidine-mutated population for many generations under nonselective conditions followed by two cycles of protoplast formation and regeneration. A total of 2,400 surviving colonies were then screened for spiramycin production and subsequently checked for stability. From this experiment, strain 6-37 was isolated that produced 181 mg of spiramycin per liter and only one morphological type. The parent strain (ATCC 15154) produced 107 mg of spiramycin per liter and four morphological types. Strain 6-37 was then mutated with nitrosoguanidine, and 14,000 colonies were screened for spiramycin production. From this experiment, five strains were isolated that produced titers ranging from 187 to 373 mg of spiramycin per liter. Subsequent media and time studies with these strains resulted in a fermentation that produced 1,728 mg of spiramycin per liter.     

Semicontinuous and Continuous Production of Chloroperoxidase by Caldariomyces fumago Immobilized in k-Carrageenan

Three strains of Caldariomyces fumago were immobilized in 4% k-carrageenan and tested for semicontinuous production of chloroperoxidase (CPO). Over an 80-day period, growing in defined medium, C. fumago strains CMI 89362 and ATCC 11925 produced enzyme concentrations of 99 and 71 mg/liter, respectively, during six production periods of 12 to 14 days, while C. fumago DAOM 137632 produced only 24 mg of CPO per liter during six growth periods of 10 days. CPO production was unaffected by various regimens of washing between transfers. Mycelial growth was primarily restricted to the head surface, and bead size increased linearly with time. Attempts to restrict growth but maintain CPO production were unsuccessful. Pigment production, fructose utilization, and pH change in the immobilized cell cultures compared closely with the growth characteristics of free cell cultures. By using an airlift tower fermentor with an external loop run with continuous medium replacement of 20 ml/h (D = 0.016), strain CMI 89362 in bead form produced CPO at 40 mg/liter for 11 days.     

Effects of glucose limitation on biomass and spiramycin production by Streptomyces ambofaciens

Spiramycin production by Streptomyces ambofaciens Sp181110 with glucose as the carbon source was studied under a controlled nutritional environment. In a batch culture, the glucose excess after ammonium depletion led to pyruvate and α-ketoglutarate accumulation. 85 mg/l of spiramycin were produced in less than 70 h during the stationary and maintenance phase on these acids after glucose exhaustion. Fed-batch strategy was designed to study spiramycin production without by-product formation and glucose accumulation. In these conditions, up to 150 mg/l were produced in less than 80 h during the stationary phase on glucose. The antibiotic titre was found independent of the glucose feeding under carbon limitation and the importance of putative intracellular reserves formed after nutrient exhaustion was suggested. Besides, spiramycin production was not inhibited by the limiting flux of glucose.     

Involvement of acyl coenzyme A oxidase isozymes in biotransformation of methyl ricinoleate into gamma-decalactone by Yarrowia lipolytica

We reported previously on the function of acyl coenzyme A (acyl-CoA) oxidase isozymes in the yeast Yarrowia lipolytica by investigating strains disrupted in one or several acyl-CoA oxidase-encoding genes (POX1 through POX5) (H. Wang et al., J. Bacteriol. 181:5140-5148, 1999). Here, these mutants were studied for lactone production. Monodisrupted strains produced similar levels of lactone as the wild-type strain (50 mg/liter) except for Deltapox3, which produced 220 mg of gamma-decalactone per liter after 24 h. The Deltapox2 Deltapox3 double-disrupted strain, although slightly affected in growth, produced about 150 mg of lactone per liter, indicating that Aox2p was not essential for the biotransformation. The Deltapox2 Deltapox3 Deltapox5 triple-disrupted strain produced and consumed lactone very slowly. On the contrary, the Deltapox2 Deltapox3 Deltapox4 Deltapox5 multidisrupted strain did not grow or biotransform methyl ricinoleate into gamma-decalactone, demonstrating that Aox4p is essential for the biotransformation.     

Pulsed-field gel electrophoresis analysis of the genome of Streptomyces ambofaciens strains

The genome of four Streptomyces ambofaciens strains from different geographical origins (ATCC15154, DSM40697, ETH9247 and ETH 11317) was analysed by pulsed-field gel electrophoresis (PFGE). The PFGE technique has allowed the study of the extrachromosomal content of these strains and the characterization of their genomic DNA by restriction analyses. Electrophoretic migration of undigested DNA allowed us to detect a 80 kb-length linear molecule with concatemeric forms in S. ambofaciens ATCC15154. These extrachromosomal molecules were shown to be homologous to the circular plasmid pSAM1 (80 kb) suggesting that pSAM1 could exist not only in circular form but also in linear form. In the same way a 45 kb-length linear molecule was detected in S. ambofaciens ETH9427 and ETH11317. In contrast, no extrachromosomal DNA could be detected in S. ambofaciens DSM40697. The analysis of the macrorestriction patterns using the rate-cutting enzymes AseI and DraI indicated a close relationship between the DSM- and ETH- strains. Indeed, three types of restriction patterns were distinguished: while S. ambofaciens ETH9427 and ETH11317 were characterized by the same pattern and share more than 75% of comigrating fragments with the strain DSM40697, S. ambofaciens ATCC15154 exhibited a restriction pattern different from the other three. The total genome sizes of S. ambofaciens ATCC15154, DSM40697, ETH9427 and ETH11317 were estimated to be about 6500, 8000, 8200 and 8200 kb, respectively.     

Effect of Aeration on Growth and Aflatoxin Production by Aspergillus flavus in Submerged Culture

Aspergillus flavus ATCC 15517 produced up to 212 mg per liter of total aflatoxin in submerged culture in aerated (3,000, 6,000, 9,000, and 12,000 ml/min) and agitated medium in 14-liter fermentors with 10 liters of medium consisting of 2% yeast extract and 10% sucrose. Aflatoxin production increased with time. A maximum of 212 mg/liter was produced at 9,000 ml/min aeration, whereas the yield decreased substantially at the lower aeration rates. Two other strains of A. flavus synthesized aflatoxin in smaller quantities.     

Nitrogen catabolite regulation of spiramycin production inStreptomyces ambofaciens

Spiramycin production byStreptomyces ambofaciens is controlled by the nitrogen source present in the culture medium. Thus, amino acids according to the mode of catabolism (transamination or deamination) influenced the spiramycin production differently. Arginine, whose catabolism led to an important excretion of ammonium, gave a slight spiramycin production of 5.3 mg. g^–1 dry cell weight; however, the introduction of an ammonium trapping agent [0.25% Mg₃(PO₄)₂] enhanced spiramycin production by 415%. The use of a neutral culture medium showed the existence of a critical phase during which the ammonium pulse had maximum negative effects on spiramycin production. Among these negative effects, the ammonium pulse provoked an increase in the growth rate, which was partially responsible for the decrease of the spiramycin production. The inhibitory effects of ammonium on spiramycin production were mitigated when the growth rate was controlled by the phosphate concentration. In addition, protease activities were limited on a culture medium in which ammonium was present and spiramycin production was null, whereas on lysine, where spiramycin production was favored, protease activities were higher.     

Microbial production of D-malate from maleate.

To produce D-malate from maleate by a microbial reaction, we screened a number of maleate-utilizing microorganisms for enzyme activity by an intact cell system. The strain which showed the best productivity among the 440 strains tested was identified taxonomically as Arthrobacter sp. strain MCI2612. The optical purity of the malate produced by this strain was 100% D type. The culture and reaction conditions for the production were studied for this strain. Addition of amino acids such as L-proline, L-histidine, and L-arginine to the culture medium promoted the formation of reaction activity as well as cell growth. Under optimum conditions, 87 g of D-malate per liter was produced in 20 h. The yield was 72 mol%.     

Cloning of spiramycin biosynthetic genes and their use in constructing Streptomyces ambofaciens mutants defective in spiramycin biosynthesis

Several cosmid clones from Streptomyces ambofaciens containing the spiramycin resistance gene srmB were introduced into S. fradiae PM73, a mutant defective in tylosin synthesis, resulting in tylosin synthesis. The DNA responsible for this complementation was localized to a 10.5-kilobase EcoRI fragment. A 32-kilobase DNA segment which included the srmB spiramycin resistance gene and DNA which complemented the defect in strain PM73 were mutagenized in vivo with Tn10 carrying the gene for Nmr (which is expressed in Streptomyces spp.) or in vitro by insertional mutagenesis with a drug resistance gene (Nmr) cassette. When these mutagenized DNA segments were crossed into the S. ambofaciens chromosome, three mutant classes blocked in spiramycin synthesis were obtained. One mutant accumulated two precursors of spiramycin, platenolide I and platenolide II. Two mutants, when cofermented with the platenolide-accumulating mutant, produced spiramycin. Tylactone supplementation of these two mutants resulted in the synthesis of a group of compounds exhibiting antibiotic activity. Two other mutants failed to coferment with any of the other mutants or to respond to tylactone supplementation.     

Microbiological Production of Gibberellic Acid in Glucose Media

Gibberellic acid production from various substrates was studied in 43 strains of Fusarium, among which F. moniliforme strain IOC-3326 was selected as the best producer. Experiments were carried out in shaker flasks and pilot plant fermentors. The results indicate that the best substrate for gibberellic acid production with this strain is composed of the following: glucose, 20 g; corn steep liquor, 25 g; ammonium nitrate, 2.6 g; monopotassium phosphate, 0.5 g; potassium sulfate, 0.2 g; and water, 1000 ml. Glucose, ammonium nitrate, and corn steep liquor were found to be critical. With this medium, maximal yields of 1196 mg per liter in shaker flasks and 997 mg per liter in fermentors were produced.     

Additional studies of histoplasmin formation

Summary Culture filtrates of 20 strains ofHistoplasma capsulatum were studied to determine the effect of certain growth conditions on histoplasmin formation. The presence of histoplasmin was denoted by an antigenic titer of 1:4 or higher with the complement fixation test.The data indicated that, in addition to verifying that the strain used affected histoplasmin formation, the morphological condition of the inoculum was extremely important. It was found that most strains which converted readily to the yeast phase at 37° C produced histoplasmin poorly. Tests with different volumes of media also showed that 500 ml volumes of culture media produced histoplasmin with higher titers than 3 liter volumes when cultured at 25° C for six months.Some additional histoplasmin could be liberated by sonification of the mycelial pad from culture filtrates which contained histoplasmin. A few strains produced high titer histoplasmin by the shake method if incubated for three months, but they had low titers after only six weeks.Complement fixation tests with sera from proven cases of histoplasmosis indicated that histoplasmin from a single strain ofH. capsulatum can give identical results with those obtained with histoplasmin from a pool ofH. capsulatum strains if H and M antigen components are present.     

Inactivation of Campylobacter jejuni by chlorine and monochloramine

Campylobacter jejuni and closely related organisms are important bacterial causes of acute diarrheal illness in the United States. Both endemic and epidemic infections have been associated with consuming untreated or improperly treated surface water. We compared susceptibility of three C. jejuni strains and Escherichia coli ATCC 11229 with standard procedures used to disinfect water. Inactivation of bacterial preparations with 0.1 mg of chlorine and 1.0 mg of monochloramine per liter was determined at pH 6 and 8 and at 4 and 25 degrees C. Under virtually every condition tested, each of the three C. jejuni strains was more susceptible than the E. coli control strain, with greater than 99% inactivation after 15 min of contact with 1.0 mg of monochloramine per liter or 5 min of contact with 0.1 mg of free chlorine per liter. Results of experiments in which an antibiotic-containing medium was used suggest that a high proportion of the remaining cells were injured. An animal-passaged C. jejuni strain was as susceptible to chlorine disinfection as were laboratory-passaged strains. These results suggest that disinfection procedures commonly used for treatment of drinking water to remove coliform bacteria are adequate to eliminate C. jejuni and further correlate with the absence of outbreaks associated with properly treated water.     

Inactivation of Campylobacter jejuni by chlorine and monochloramine.

Campylobacter jejuni and closely related organisms are important bacterial causes of acute diarrheal illness in the United States. Both endemic and epidemic infections have been associated with consuming untreated or improperly treated surface water. We compared susceptibility of three C. jejuni strains and Escherichia coli ATCC 11229 with standard procedures used to disinfect water. Inactivation of bacterial preparations with 0.1 mg of chlorine and 1.0 mg of monochloramine per liter was determined at pH 6 and 8 and at 4 and 25 degrees C. Under virtually every condition tested, each of the three C. jejuni strains was more susceptible than the E. coli control strain, with greater than 99% inactivation after 15 min of contact with 1.0 mg of monochloramine per liter or 5 min of contact with 0.1 mg of free chlorine per liter. Results of experiments in which an antibiotic-containing medium was used suggest that a high proportion of the remaining cells were injured. An animal-passaged C. jejuni strain was as susceptible to chlorine disinfection as were laboratory-passaged strains. These results suggest that disinfection procedures commonly used for treatment of drinking water to remove coliform bacteria are adequate to eliminate C. jejuni and further correlate with the absence of outbreaks associated with properly treated water.     

Site-specific integration of plasmid pSAM2 in Streptomyces lividans and S. ambofaciens

Summary Streptomyces ambofaciens strain ATCC23877 contains the 11.1 kb plasmid pSAM2 stably integrated into its chromosome. This plasmidic sequence is able to loop out and to be transferred at high frequency to S. lividans where it is found simultaneously as both free and integrated plasmid. When a UV derivative of strain ATCC23877 (strain ATCC15154) is used, the resident copy of pSAM2 can be transferred to S. lividans, but only the integrated form is found in this strain. In both cases, the integration occurs at a unique chromosomal region through the same plasmidic integration site as that in strain ATCC23877. The resident copy of strain ATCC15154 can also be transferred at low frequency to S. ambofaciens DSM40697 (devoid of any pSAM2 sequence). In this case, as several copies of pSAM2 are integrated, the integration pattern is complicated. Integration of a complete pSAM2 sequence in this strain occurs in a region that hybridizes with the integration zones of S. lividans and of S. ambofaciens strain ATCC23877. Comparison of the cloned integration zone of S. lividans before and after the integration event showed that the restriction pattern of the resident pSAM2 in strain ATCC15154 is similar to that of the free form of pSAM2 found naturally in another UV derivative of strain ATCC23877 (strain JI3212).     

Production of ubiquinone-10 using bacteria

Among the bacterial strains known to contain ubiquinone-10, three strains, Agrobacterium tumefaciens KY-3085 (ATCC4452), Paracoccus denitrificans KY-3940 (ATCC19367) and Rhodobacter sphaeroides KY-4113 (FERM-P4675), were selected as excellent producers of this ubiquinone. The ubiquinone-10 production by the Agrobacterium and Rhodobacter strains was affected by aeration. An ethionine-resistant mutant (M-37) derived from A. tumefaciens KY-3085 promoted increased production of ubiquinone-10 (20% higher than the parent). Another Agrobacterium mutant (AU-55), which was induced by the successive addition of four genetic markers, showed a tolerance to the suppression of ubiquinone-10 production caused by aeration, and the fermentation time for production was remarkably shortened. The amount of ubiquinone-10 produced by this Agrobacterium mutant reached 180 mg/l in a 58 h culture. A green mutant (carotenoid-deficient mutant, Co-22-11) derived from R. sphaeroides KY-4113 produced 350 mg/l of ubiquinone-10 under culturing conditions with a limited supply of air, the ubiquinone-10 content being 8.7 mg/g-dry cell. In this case, the amount and content corresponded to 2.8 and 3.6 times larger than those given by the wild-type strain, respectively. A multiple-layer structure of cell membrane was observed in the highly ubiquinone-10 accumulating cell of the green mutant by electron microscopy. The amount of ubiquinone-10 produced by P. denitrificans was much lower than those of the other two strains.     

Siderophore production in relation to N2 fixation and iron uptake in pigeon pea-Rhizobium symbiosis

Thirty-one bradyrhizobial and rhizobial strains infecting pigeon pea were screened for siderophore production using Chrome Azurol S (CAS) agar plate as well as a CAS assay solution. Of a total of 31 strains only 23 showed siderophore production. Of the 23 siderophore-positive strains, 21 strains showed the production of hydroxamate while 6 strains showed the presence of catechol type of siderophore. A large variation in the quantity of hydroxamate and catechol produced by different rhizobial strains was observed (1.03–3.73 μg hydroxamate N per mg protein; 0.19–3.43 μmol/L of catechol per mg protein). Maximum nodule biomass was produced by strain PP-11 (CC-1020); strain G-14 formed minimum nodule biomass. Nitrogen contents of low, moderate and high siderophore-producing strains were 11.4, 15.4, 20.9 mg per plant, respectively, iron contents were 1445, 1768 and 2003 ppm, respectively. Siderophore production was related to N₂-fixing efficiency.     

Production of eicosapentaenoic acid by marine bacteria

About 5,000 strains of marine microorganisms were screened for eicosapentaenoic acid (EPA)-producing ability, which was detected in 88 of them. All of the latter were found to be obligate aerobic, Gram-negative, motile, short rod-shaped bacteria. One strain, designated as SCRC-8132, showed a doubling time of 30 min at 25 degrees C and produced 20 mg/liter (4 mg/g dry cells) when cultured in a P-Y-M-Glucose medium for 18 h. The EPA to total fatty acids ratio was 24%. The strain produced 26 mg EPA/liter (15 mg/g dry cells) when cultured at 4 degrees C for 5 days, the EPA ratio being increased to 40%.     

Cholesterol assimilation by lactic acid bacteria and bifidobacteria isolated from the human gut

The objective of this study was to evaluate the effect of human gut-derived lactic acid bacteria and bifidobacteria on cholesterol levels in vitro. Continuous cultures inoculated with fecal material from healthy human volunteers with media supplemented with cholesterol and bile acids were used to enrich for potential cholesterol assimilators among the indigenous bacterial populations. Seven potential probiotics were found: Lactobacillus fermentum strains F53 and KC5b, Bifidobacterium infantis ATCC 15697, Streptococcus bovis ATCC 43143, Enterococcus durans DSM 20633, Enterococcus gallinarum, and Enterococcus faecalis. A comparative evaluation regarding the in vitro cholesterol reduction abilities of these strains along with commercial probiotics was undertaken. The degree of acid and bile tolerance of strains was also evaluated. The human isolate L. fermentum KC5b was able to maintain viability for 2 h at pH 2 and to grow in a medium with 4,000 mg of bile acids per liter. This strain was also able to remove a maximum of 14.8 mg of cholesterol per g (dry weight) of cells from the culture medium and therefore was regarded as a candidate probiotic.     

Citric acid production by Aspergillus niger on wet corn distillers grains

AIMS: To determine which citric acid-producing strain of Aspergillus niger utilized wet corn distillers grains most effectively to produce citric acid. METHODS AND RESULTS: Citric acid and biomass production by the fungal strains were analysed on the untreated grains or autoclaved grains using an enzyme assay and a gravimetric method respectively. Fungal citric acid production on the grains was found to occur on the untreated or autoclaved grains. The highest citric acid level on the grains was produced by A. niger ATCC 9142. The autoclaved grains supported less citric acid production by the majority of strains screened. Biomass production by the fungal strains on the untreated or autoclaved grains was quite similar. The highest citric acid yields for A. niger ATCC 9142, ATCC 10577, ATCC 11414, ATCC 12846 and ATCC 26550 were found on the untreated grains. Treatment of the grains had little effect on citric acid yields based on reducing sugars consumed by A. niger ATCC 9029 and ATCC 201122. CONCLUSIONS: It is feasible for citric acid-producing strains of A. niger to excrete citric acid on wet corn distillers grains whether the grains are treated or untreated. The most effective citric acid-producing strain of A. niger was ATCC 9142. SIGNIFICANCE AND IMPACT OF THE STUDY: The study shows that the ethanol processing co-product wet corn distillers grains could be utilized as a substrate for the commercial production of citric acid by A. niger without treatment of the grains.     

Involvement of Acyl Coenzyme A Oxidase Isozymes in Biotransformation of Methyl Ricinoleate into γ-Decalactone by Yarrowia lipolytica

We reported previously on the function of acyl coenzyme A (acyl-CoA) oxidase isozymes in the yeast Yarrowia lipolytica by investigating strains disrupted in one or several acyl-CoA oxidase-encoding genes (POX1 through POX5) (H. Wang et al., J. Bacteriol. 181:5140–5148, 1999). Here, these mutants were studied for lactone production. Monodisrupted strains produced similar levels of lactone as the wild-type strain (50 mg/liter) except for Δpox3, which produced 220 mg of γ-decalactone per liter after 24 h. The Δpox2 Δpox3 double-disrupted strain, although slightly affected in growth, produced about 150 mg of lactone per liter, indicating that Aox2p was not essential for the biotransformation. The Δpox2 Δpox3 Δpox5 triple-disrupted strain produced and consumed lactone very slowly. On the contrary, the Δpox2 Δpox3 Δpox4 Δpox5 multidisrupted strain did not grow or biotransform methyl ricinoleate into γ-decalactone, demonstrating that Aox4p is essential for the biotransformation.