Growth and ultrastructure of Streptomyces venezuelae during chloramphenicol production.

Streptomyces venezuelae (3022a) was grown in flask cultures and fermentors, using three media having differential effects on chloramphenicol production. Micromorphology, ultrastructure and chloramphenicol concentrations were studied during the growth cycle in each medium. Chloramphenicol production was greatest in the glycerol-serine-lactate (GSL) medium, less in the glycerol-nutrient broth-yeast extract (GNY) medium and very low in glucose-mineral salts (GA) medium. In GSL and GA, much growth was in the form of microcolonies, especially in flask cultures, while short hyphal fragments predominated in GNY. The major ultrastructural features were the high frequency of mesosomes in fragmenting hyphae in GNY, and electron-transparent zones which appeared during chloramphenicol synthesis in GSL. None of the structural abnormalities induced by chloramphenicol in sensitive organisms were observed in S. venezuelae despite high levels of the antibiotic in GSL medium.     

Effect of ammonium on chloramphenicol production by Streptomyces venezuelae in batch and continuous cultures

Cultures of Streptomyces venezuelae presented with a mixture of ammonium and an amino acid as nitrogen sources used both compounds together. Absence of ammonium repression of alternative nitrogen assimilation pathways was also observed when ammonium was added to cultures already growing on proline. The presence of ammonium in the medium ab initio depressed the yield of chloramphenicol. However, its addition to a culture growing on proline caused only a temporary inhibition of antibiotic synthesis, even when sufficient ammonium was added to create an excess. Continuous cultures supplied with ammonium as the growth-limiting nutrient showed no significant change in specific antibiotic production at different specific growth rates. The overall results indicate that in S. venezuelae neither nitrogen utilization pathways nor chloramphenicol biosynthesis is controlled by nitrogen repression.     

A plasmid involved in chloramphenicol production in Streptomyces venezuelae: evidence from genetic mapping.

To test the hypothesis that chloramphenicol production in Streptomyces venezuelae depends on the presence of a plasmid, mapping analysis was carried out by using eight markers in addition to chloramphenicol production and melanoid pigment formation. The sequence of the eight markers was determined on a circular linkage map as follows: -his-ade-str-leu-lys-met-ilv-pro-(his-). This sequence resulted in the frequency of quadruple crossover (q.c.o.) recombinants having the lowest value, 3-2 to 4-9%. However, the character of chloramphenicol non-production, which was obtained by incubating mycelia with acriflavin, was not required to explain the results. From these results and other tests, it is concluded that chloramphenicol production is controlled by a plasmid. This plasmid appeared to be non-transferable in conjugation.     

Induction of pristinamycins production inStreptomyces pristinaespiralis

Summary Pristinamycins production in a mutant ofStreptomyces pristinaespiralis (strain Cl6/4) blocked in pristinamycins biosynthesis was induced by different commercial lactones on solid media. The activity of the -lactone ring molecules was improved when the length of the substituted carbon chain was increased. Pristinamycins induction was also obtained with the endogenous A aactor ofS. griseus. The minimum effective concentration of the A factor was 250 times lower than that of exogenous lactones. Addition of exogenous -butyrolactone did not affect antibiotic production by a hyperproducing mutant ofStreptomyces pristinaespiralis (strain Pr11) even in the presence of surfactant like Tween 80 or Triton X 100. Three hours before the initiation of antibiotic production, the hyperproducing strain Pr11 produced an extracellular factor extractable with ethyl acetate at pH 7 and able to induce the antibiotic production of strain Cl6/4.     

Isolation and characterization of Streptomyces venezuelae mutants blocked in chloramphenicol biosynthesis

Twelve Streptomyces venezuelae mutants blocked in chloramphenicol biosynthesis were isolated. Two of these (Cm1-1 and Cm1-12) were apparently blocked in the conversion of chorismic acid to p-aminophenylalanine and three (Cm1-4, Cm1-5 and Cm1-8) accumulated p-aminophenylalanine and may have been blocked in the hydroxylation reaction that converted this intermediate to p-aminophenylserine. One mutant (Cm1-2) accumulated D-threo-1-p-nitrophenyl-2-propionamido-1,3-propanediol and D-threo-1-p-nitrophenyl-2-isobutyramido-1,3-propanediol, indicating that chlorination of the alpha-N-acyl group of chloramphenicol was blocked. The remaining six strains did not excrete any detectable chloramphenicol pathway intermediates.     

Transductional analysis of chloramphenicol biosynthesis genes in Streptomyces venezuelae.

Auxotrophs isolated from two chloramphenicol-nonproducing mutants of Streptomyces venezuelae included three requiring pyridoxal (Pxl-), VS248 (cml-11 pdx-2), VS253 (cml-11 pdx-3), and VS258 (cml-12 pdx-4), and one requiring thiosulfate, VS263 (cml-12 cys-28). Results of SV1-mediated transductions were consistent with the relative marker order cys-28-cml-12-cml-11-pdx-2,3,4,5, all of which were cotransducible and must therefore span less than 45 kilobases of DNA, the approximate length of DNA packaged by SV1. cys-28 was also cotransducible with arg-4 and arg-6, but arg and pdx were not cotransducible. Results of crosses with donors carrying any one of 11 cml mutations were consistent with the location of all cml mutations between cys-28 and pdx markers. Also, a new Pxl- auxotroph (pdx-6) and two new Cml- mutants were recovered after localized hydroxylamine mutagenesis of a cys-28 cml+ strain derived from VS263 by transduction.     

Role of the carbon source in regulating chloramphenicol production by Streptomyces venezuelae: studies in batch and continuous cultures

Both carbon- and nitrogen-limited media that supported a biphasic pattern of growth and chloramphenicol biosynthesis were devised for batch cultures of Streptomyces venezuelae. Where onset of the idiophase was associated with nitrogen depletion, a sharp peak of arylamine synthetase activity coincided with the onset of antibiotic production. The specific activity of the enzyme was highest when the carbon source in the medium was also near depletion at the trophophase-idiophase boundary. In media providing a substantial excess of carbon source through the idiophase, the peak specific activity was reduced by 75%, although the timing of enzyme synthesis was unaltered. Moreover, chemostat cultures in which the growth rate was limited by the glucose concentration in the input medium failed to show a decrease in specific production of chloramphenicol as the steady-state intracellular glucose concentration was increased. The results suggest that a form of "carbon catabolite repression" regulates synthesis of chloramphenicol biosynthetic enzymes during a trophophase-idiophase transition induced by nitrogen starvation. However, this regulatory mechanism does not establish the timing of antibiotic biosynthesis and does not function during nitrogen-sufficient growth in the presence of excess glucose.     

Effects of addition of chloramphenicol on the growth and ultrastructure of Streptomyces venezuelae

The effects of adding chloramphenicol before inoculation and during exponential growth of Streptomyces venezuelae (3022a) in fermentors were studied. The responses of the organism during synthesis of chloramphenicol (in a glycerol-serine-lactate medium) were compared with those in media supporting less (glycerol-nutrient broth-yeast extract) or no synthesis (glucosemineral salts). In systems where little or no synthesis of the chloramphenicol occurred, addition of the antibiotic induced micromorphological and ultrastructural abnormalities similar to those reported for sensitive bacteria. There was also an increase in the frequency of mesosomes and electron-light areas. It was suggested that the former may be associated with activity of chloramphenicol hydrolase and the latter with storage and/or excretion of the breakdown product; N-acetyl p-nitro-phenylserinol. When chloramphenicol synthesis occurred, addition of the antibiotic had less effect on the micromorphology or ultrastructure of S. venezuelae as permeability barriers to external chloramphenicol had been established. Electron-light areas were frequent, possibly being associated with storage and excretion of precursors of chloramphenicol.     

Propionate and the production of monensins inStreptomyces cinnamonensis

Variants resistant to propionate were prepared from a mutant strain of Streptomyces cinnamonensis producing predominantly monensin A. Using selected resistants the production of monensins (in media with higher concentrations of propionate) was examined. Stimulation of monensin synthesis by propionate was observed with 70% of the resistants studied. Propionate did not influence the ratio between monensin A and B production.     

DNA amplification affects protease production and sporulation inStreptomyces fradiae

Chloramphenicol resistance is an unstable character inStreptomyces fradiae, since spontaneous chloramphenicol-sensitive (Cml^s) mutants arose at very high frequencies. One such Cml^s mutant, DM14, showed DNA amplification as well. Extracellular protease activity was tenfold higher in DM14 when compared with its wild-type parent. Protease activity decreased considerably in DM14 when treated with spectinomycin, a treatment that reduces the copy number of amplified units of DNA. Sporulation in DM14 was delayed in the presence of spectinomycin at a concentration of 5 g/ml, whereas the wild type was unaffected at that concentration. The results strongly indicated that the amplified DNA affected the two secondary metabolic functions, viz., protease production and the onset of sporulation in the mutant.     

Relation between growth rate and erythromycin production inStreptomyces erythraeus

Summary The relation between specific growth rate and specific rate of product formation was studied in phosphate-limited chemostat cultures ofStreptomyces erythraeus. Specific rates of formation were measured for both the final product, erythromycin A, and several of its biosynthetic precursors. In all cases rates of formation increased with inereasing growth rate, indicating that antibiotic production was strongly growth-linked.     

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.     

Catabolite repression in Streptomyces venezuelae. Induction of beta-galactosidase, chloramphenicol production, and intracellular cyclic adenosine 3',5'-monophosphate concentrations

Chloramphenicol production was studied in cultures of Streptomyces venezuelae growing in a simple buffered medium with ammonia as the nitrogen source and glucose, lactose, or a glucose-lactose mixture as the sole source of carbon. With each carbon source the antibiotic was formed during growth. In the glucose-lactose medium, the production pattern was biphasic; a marked decrease in the rate of synthesis was associated with depletion of glucose from the medium and a corresponding diauxie pause in growth. Cells of S. venezuelae contained an inducible beta-galactosidase. Induction by lactose was suppressed by glucose. Measurement of the concentration of intracellular adenosine 3',5'-cyclic monophosphate during growth of cultures with glucose or a glucose-lactose mixture as the source of carbon showed no appreciable changes coinciding with depletion of glucose or the onset of chloramphenicol biosynthesis. It is concluded that the cyclic nucleotide does not mediate selective nutrient utilization or control antibiotic biosynthesis in this organism.     

Heterologous production of epothilones B and D in Streptomyces venezuelae

Epothilones, produced from the myxobacterium Sorangium cellulosum, are potential anticancer agents that stabilize microtubules in a similar manner to paclitaxel. The entire epothilone biosynthetic gene cluster was heterologously expressed in an engineered strain of Streptomyces venezuelae bearing a deletion of pikromycin polyketide synthase gene cluster. The resulting strains produced approximately 0.1 μg/l of epothilone B as a sole product after 4 days cultivation. Deletion of an epoF encoding the cytochrome P450 epoxidase gave rise to a mutant that selectively produces 0.4 μg/l of epothilone D. To increase the production level of epothilones B and D, an additional copy of the positive regulatory gene pikD was introduced into the chromosome of both S. venezuleae mutant strains. The resulting strains showed enhanced production of corresponding compounds (approximately 2-fold). However, deletion of putative transport genes, orf3 and orf14 in the epothilone D producing S. venezuelae mutant strain, led to an approximately 3-fold reduction in epothilone D production. These results introduce S. venezuelae as an alternative heterologous host for the production of these valuable anticancer agents and demonstrate the possibility of engineering this strain as a generic heterologous host for the production of polyketides and hybrid polyketide-nonribosomal peptides.     

Conjugational fertility and location of chloramphenicol biosynthesis genes on the chromosomal linkage map of Streptomyces venezuelae

In Streptomyces venezuelae fertility, defined as chromosomal gene recombination, was enhanced over 1000-fold when one parent in a biparental conjugational cross lacked the physically-undetected plasmid SVP1, as compared with crosses in which both parents carried SVP1. The existence of SVP1 and at least two other fertility plasmids, SVP2 and SVP3, was detected in S. venezuelae by 'lethal zygosis' elicited by a plasmid-plus mycelium in contact with a plasmid-minus mycelium. Conjugational crosses were used to construct a linkage map of S. venezuelae which was highly consistent with the map of analogous loci in S. coelicolor A3(2). A cluster of genes governing chloramphenicol biosynthesis was located near arg, cys and pdxB genes at a position roughly equivalent to the 1-2 o'clock region of the S. coelicolor A3(2) map.     

Enhanced flavonoid production in Streptomyces venezuelae via metabolic engineering

Metabolic engineering of plant-specific phenylpropanoid biosynthesis has attracted an increasing amount of attention recently, owing to the vast potential of flavonoids as nutraceuticals and pharmaceuticals. Recently, we have developed a recombinant Streptomyces venezuelae as a heterologous host for the production of flavonoids. In this study, we successfully improved flavonoid production by expressing two sets of genes predicted to be involved in malonate assimilation. The introduction of matB and matC encoding for malonyl-CoA synthetase and the putative dicarboxylate carrier protein, respectively, from Streptomyces coelicolor into the recombinant S. venezuelae strains expressing flavanone and flavone biosynthetic genes resulted in enhanced production of both flavonoids.     

Biochemistry and genetics of chloramphenicol production

In Streptomyces venezuelae, chloramphenicol is derived by an unusual diversion of chorismate, the branchpoint intermediate of the pathway involved in the biosynthesis of aromatic amino acids. In the chloramphenicol-producing organism, the DAHP synthetase was neither feedback inhibited nor repressed. Chorismate mutase was not repressed or inhibited by the intermediates or end-products of the shikimate-chorismate pathway. However, anthranilate synthetase and prephenate dehydratase are feedback inhibited by tryptophan and phenylalanine, respectively. During growth, when primary metabolism is not perfectly coordinated, decreasing demand for aromatic amino acids results in shunting of chorismate towards chloramphenicol biosynthesis.The endogenous synthesis of chloramphenicol produced by Streptomyces venezuelae is inhibited by the increasing concentration of chloramphenicol in the medium. Arylamine synthetase, the first enzyme involved in chloramphenicol biosynthesis, is repressed by the secreted chloramphenicol, by dl-p-aminophenylalanine and l-threo-p-aminophenylserinol. The excess intracellular chorismate pool is diverted to other aromatic shunt metabolites if biosynthesis of chloramphenicol is inhibited. There appears to be a glutamine binding protein subunit which is shared by several enzymes involved in amination of the aromatic ring of chorismate.Chloramphenicol producing organism also inactivated intracellular chloramphenicol. However, the resistance of the streptomycetes is due to inducible impermeability of the organism to chloramphenicol during antibiotic production. Streptomyces venezuelae is sensitive to chloramphenicol when it is not engaged in antibiotic production. The resistance to and production of chloramphenicol are induced simultaneously.A linkage map for 17 marker loci using Streptomyces venezuelae has been constructed. Restriction enzyme map of a plasmid from the chloramphenicol-producing streptomycetes has also been developed. The role of the plasmid in chloramphenicol biosynthesis and the life-cycle of the Streptomyces venezuelae is not yet understood.     

Effects of reduced oxygen on growth and antibiotic production inStreptomyces clavuligerus

Summary The effects of reduced oxygen on growth and antibiotic production were studied in batch cultures ofStreptomyces clavuligerus in defined media. Antibiotic levels were unaffected by reduced oxygen for the first 50 hours of fermentation. After growth ceased, antibiotic concentrations dropped by a factor of about three under reduced oxygen, while the antibiotic concentration was stable when air was used. This suggests that enzymes for hydrolyzing antibiotics may be regulated by the aeration conditions.     

Genetic improvement of glucose isomerase production inStreptomyces nigrificans by ultraviolet irradiation

Summary Glucose isomerase activity inStreptomyces nigrificans 3014, a producer of antibiotics of the chromomycin group., increased by 198 % as a result of a two-step improvement procedure including the selection of more active variants among antibiotic-producing isolates from population treated with ultraviolet light.