Production and statistical optimization of Paromomycin by Streptomyces rimosus NRRL 2455 in solid state fermentation.

BACKGROUND: Paromomycin is a 2-deoxystreptamine aminocyclitol aminoglycoside antibiotic with broad spectrum activity against Gram-negative, Gram-positive bacteria and many protozoa. This study introduces a strategy for paromomycin production through solid-state fermentation using Streptomyces rimosus subsp. paromomycinus NRRL 2455. Solid state fermentation has gained enormous attention in the development of several products because of their numerous advantages over submerged liquid fermentation. After selecting the best solid substrate, a time course study of paromomycin production was carried out followed by optimization of environmental conditions using response surface methodology. Paromomycin yields obtained using this technique were also compared to those obtained using submerged liquid fermentation. RESULTS: Upon screening of 6 different substrates, maximum paromomycin concentration (0.51 mg/g initial dry solids) was obtained with the cost-effective agro-industrial byproduct, corn bran, impregnated with aminoglycoside production media. Optimization of environmental conditions using D-optimal design yielded a 4.3-fold enhancement in paromomycin concentration reaching 2.21 mg/g initial dry solids at a pH of 8.5, inoculum size of 5% v/w and a temperature of 30 °C. CONCLUSION: Compared to submerged liquid fermentation, solid state fermentation resulted in comparable paromomycin concentrations, cost reduction of raw materials, less energy consumption and waste water discharge, which have major implications in industrial fermentation. Therefore, solid state fermentation is a promising alternative to submerged liquid fermentation for paromomycin production. To the best of our knowledge, this is the first report on the optimized paromomycin production through solid state fermentation process.

Paromomycin production from Streptomyces rimosus NRRL 2455: statistical optimization and new synergistic antibiotic combinations against multidrug resistant pathogens.

BACKGROUND: Response surface methodology (RSM) employing Box-Behnken design was used to optimize the environmental factors for the production of paromomycin, a 2 deoxystreptamine aminocyclitol aminoglycoside antibiotic, (2DOS-ACAGA) from Streptomyces (S.) rimosus NRRL 2455. Emergence of bacterial resistance caught our attention to consider the combination of antimicrobial agents. The effect of paromomycin combination with other antimicrobial agents was tested on some multiple drug resistant isolates. To the best of our knowledge, this is the first report on optimization of paromomycin production from S. rimosus NRRL 2455. A Quadratic model and response surface method were used by choosing three model factors; pH, incubation time and inoculum size. A total of 17 experiments were done and the response of each experiment was recorded. Concerning the effect of combining paromomycin with different antimicrobial agents, it was tested using the checkerboard assay against six multidrug resistant (MDR) pathogens including; Pseudomonas (P.) aeruginosa (2 isolates), Klebsiella (K.) pneumoniae, Escherichia (E.) coli, methicillin sensitive Staphylococcus aureus (MSSA) and methicillin resistant Staphylococcus aureus (MRSA). Paromomycin was tested in combination with ceftriaxone, ciprofloxacin, ampicillin/sulbactam, azithromycin, clindamycin and doxycycline. RESULTS: The optimum conditions for paromomycin production were a pH of 6, an incubation time of 8.5 days and an inoculum size of 5.5% v/v using the optimized media (soybean meal 30 g/L, NH4CL 4 g/L, CaCO3 5 g/L and glycerol 40 ml/L), 28 °C incubation temperature, and 200 rpm agitation rate that resulted in 14 fold increase in paromomycin production as compared to preliminary fermentation level using the basal medium. The tested antibiotic combinations showed either synergistic effect on paromomycin activity on most of the tested MDR pathogens (45.83%), additive effect in 41.67% or indifferent effect in 12.5%. CONCLUSION: RSM using multifactorial design was a helpful and a reliable method for paromomycin production. Paromomycin combination with ceftriaxone, ciprofloxacin, ampicillin/sulbactam, azithromycin, clindamycin or doxycycline showed mostly synergistic effect on certain selected clinically important MDR pathogens.

Development of antibiotic-overproducing strains by site-directed mutagenesis of the rpsL gene in Streptomyces lividans.

Certain rpsL (which encodes the ribosomal protein S12) mutations that confer resistance to streptomycin markedly activate the production of antibiotics in Streptomyces spp. These rpsL mutations are known to be located in the two conserved regions within the S12 protein. To understand the roles of these two regions in the activation of silent genes, we used site-directed mutagenesis to generate eight novel mutations in addition to an already known (K88E) mutation that is capable of activating antibiotic production in Streptomyces lividans. Of these mutants, two (L90K and R94G) activated antibiotic production much more than the K88E mutant. Neither the L90K nor the R94G mutation conferred an increase in the level of resistance to streptomycin and paromomycin. Our results demonstrate the efficacy of the site-directed mutagenesis technique for strain improvement.

Transcriptional mapping of the promoter of the aminoglycoside acetyltransferase gene (aacC9) of neomycin-producing Micromonospora chalcea.

We have studied the promoter of the gene encoding aminoglycoside acetyltransferase (aacC9) in neomycin-producing Micromonospora chalcea. S1 nuclease mapping showed that the transcription initiation point of this gene is at the translation start point, with no evidence of a conventional ribosome-binding site. The aac of paromycin-producing Streptomyces rimosus forma paromomycinus shows the same characteristic; there is no homology in the promoter regions of the two genes, whereas the coding sequences are very similar.

6"'-Deamino-6"'-hydroxy derivatives, as intermediates in the biosynthesis of neomycin and paromomycin.

From broths of a neomycin producing Streptomyces fradiae and of a mutant of Streptomyces rimosus forma paromomycinus respectively, 6"'-deamino-6"'-hydroxyneomycin and 6"'-deamino-6"'hydroxyparomomycin were obtained and their structures established by mass and 13C-NMR spectroscopy and by the study of hydrolytic fragments. These new compounds, which are both present as two epimers at C-5"', are suggested as intermediates in the biosynthesis of the parent antibiotics. The place and the mechanism of the 5"'-epimerisation and of the 6"'-amination are discussed.

[Physiology and biochemistry of streptomycetes. X. Biological degradation of paromomycin and alkaline phosphatase activity depending on antibiotic production by Streptomyces albus var. metamycinus nov. var]. / Physiologie und Biochemie der Streptomyceten. X. Biologischer Abbau des Paromomycins und Aktivität der alkalischen Phosphatase in Abhängigkeit von der Antibioticumbildung bei Streptomyces albus var. metamycinus nov. var.

After adding 14C-paromomycin to the fermentation broth we observed a varying course of decomposition of the antibiotic, which is dependent on the intensity of paromomycin biosynthesis running simultaneously. At a reduced rate of antibiotic biosynthesis, the activity of alkaline phosphatase is lower than with an increased rate of production. This applies for mycelium as well as for broth.

[Physiology and biochemistry of streptomycetes. XI. Different incorporation of D-glucose-u-14C into the paromomycin isomers and the precursors of paromomycin I]. / Physiologie und Biochemie der Streptomyceten. XI. Unterschiedlicher Einbau von D-glucose-u-14C in die Paromomycin-Isomere und in die Baustein von Paromomycin I.

During application of D-glucose-u-14C paromomycine II is higher labelled and shows a different dependence on the application time than paromomycine I, which is isomer at the paromose part. For the two paromose isomeres different rates of synthesis are supposed that change nonproportionally to each other. The distribution of radioactivity in paromomycine I shows that there is no fragmentation of the glucose chain during the biosynthesis of glucosamine, ribose, and paromose I. As to the 2-deoxystreptamine the result has not been ascertained.

[Physiology and biochemistry of streptomycetes. XIII. Biosynthesis of paromomycin by Streptomyces albus var. metamycinus nov. var. supplied with 14C-glucose, 14C-glucosamine, 14C-2-desoxystreptamine and 14C-ribose]. / Physiologie und Biochemie der Streptomyceten. XIII. Biosynthese von Paromomycin unter Einsatz von 14C-Glucose,-Glucosamin, -2-Desoxystreptamin und-Ribose durch Streptomyces albus var. metamycinus nov. var.

Distribution of radioactivity in paromomycin ascertained after application of 14C-D-glucose, 14C-D-glucosamine, 14C-2-deoxystreptamine, respectively, 14C-D-ribose is taken as basis for a biosynthesis scheme: While ribose bound in the antibiotic originates from glucose by oxidation and following decarboxylation, glucosamine is formed via fructose-6-phosphate. Paromose I arises from glucosamine, but not the cyclohexan derivative 2-deoxystreptamine, whose biosynthesis pathway is directly branching off glucose.

Aminoglycoside 3'-phosphotransferases I and II in Pseudomonas aeruginosa.

Aminoglycoside 3'-phosphotransferases I and II in three strains of Pseudomonas aeruginosa were studied in comparison with those in two strains of R factor-carrying Escherichia coli. The strain TI-13 of P. aeruginosa produced the former and strain H-9 the latter. Strain B-13 produced the both enzymes. The 3'-phosphotransferases of type I in P. aeruginosa TI-13, B-13 and E. coli K12 J5 R11-2 were different from each other in chromatographic behavior, molecular weight, pH optimum, and Ii. The 3'-phosphotransferase of type II in P. aeruginosa H-9 and E. coli JR66/W677 showed the same behavior.

[Physiology and biochemistry of streptomycetes. III. Incorporation of D-glucose-u-14C in paramomycin as indicator of antibiotic biosynthesis by Streptomyces albus var. metamycinus nov. var]. / Phsiologie und Biochemie der Streptomyceten. III. Einbau von D-Glucose-u-14C in Paromomucin als Indikator für die Antibioticabildung durch Streptomyces albus var. metamycinus nov. var.

Paromomycin was isolated from culture filtrates of Streptomyces albus var. metamycinus nov. var. after feeding the growing cultures with D-glucose-u-14C. From the different incorporation rates conclusions concerning different features of the paromomycin biosynthesis (utilization of the carbon source, proportional and disproportional changes of the rates of synthesis) could be drawn. Uptake and metabolism of glucose are discussed.