Combinatorial biosynthesis of lipopeptide antibiotics in Streptomyces roseosporus

Daptomycin is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus. Cubicin (daptomycin-for-injection) was approved in 2003 by the FDA to treat skin and skin structure infections caused by Gram-positive pathogens. Daptomycin is particularly significant in that it represents the first new natural product antibacterial structural class approved for clinical use in three decades. The daptomycin gene cluster contains three very large genes (dptA, dptBC, and dptD) that encode the nonribosomal peptide synthetase (NRPS). The related cyclic lipopeptide A54145 has four NRPS genes (lptA, lptB, lptC, and lptD), and calcium dependent antibiotic (CDA) has three (cdaPS1, cdaPS2, and cdaPS3). Mutants of S. roseosporus containing deletions of one or more of the NRPS genes have been trans-complemented with dptA, dptBC, and dptD by inserting these genes under the control of the ermEp* promoter into separate conjugal cloning vectors containing phiC31 or IS117 attachment (attP int) sites; delivering the plasmids into S. roseosporus by conjugation from Escherichia coli; and inserting the plasmids site-specifically into the chromosome at the corresponding attB sites. This trans-complementation system was used to generate subunit exchanges with lptD and cdaPS3 and the recombinants produced novel hybrid molecules. Module exchanges at positions D: -Ala(8) and D: -Ser(11) in the peptide have produced additional novel derivatives of daptomycin. The approaches of subunit exchanges and module exchanges were combined with amino acid modifications of Glu at position 12 and natural variations in lipid side chain starter units to generate a combinatorial library of antibiotics related to daptomycin. Many of the engineered strains produced levels of novel molecules amenable to isolation and antimicrobial testing, and most of the compounds displayed antibacterial activities.     

Experimental analysis of the oil addition effect on mycelia and polysaccharide productions in Ganoderma lucidum submerged culture

The effect of corn oil addition on mycelium growth and polysaccharide productions in the medicinal mushroom Ganoderma lucidum was studied. The results showed that when a level of 2% corn oil was added at the beginning of culture, the biomass and polysaccharide productions reached a maximum of 12.9 and 1.038 g/L, respectively, during 13-day cultivation. The pH variation along with morphology observation in culture provided an indirect inference to the promotional effect of oil addition. Moreover, a curve fitting analysis was carried out to assay the elevated effect on biomass and exopolysaccharide productions in oil added culture. The experimental data of substrates consumption and products formation in culture with oil addition were predicted through the fitting equations obtained in single carbon source culture. The numerical results further clarified the stimulatory effects of oil addition in G. lucidum culture.     

Effect of the Medium Composition and Cultivation Conditions on Sporulation in Chemolithotrophic Bacteria

The possibility of regulating endospore formation by changing cultivation conditions was for the first time shown in acidophilic chemolithotrophic bacteria Sulfobacillus thermosulfidooxidans type strain 1269 and the thermotolerant strain K1 formerly described as S. thermosulfidooxidans subsp. thermotolerans. Suppression of sporulation occurred when these strains were cultured in Manning's liquid medium with yeast extract. This medium was optimized by gradually reducing the concentrations of ferrous iron salts (the source of energy), phosphorous, nitrogen, and yeast extract and simultaneously increasing the concentrations of calcium, magnesium, and manganese (the elements important for sporogenesis) to attain higher yields of endospores by strains 1269 and K1. As a result, a new medium A was proposed, in which, under aeration, the life cycle of the strains studied culminated in sporulation at a level of 45 and 60%, respectively, of the total cell number. In a series of additional tests, the growth temperature and medium pH were adjusted to obtain the maximum yield of endospores. The optimal ranges found were 40–50°C and pH 1.8–2.2 for strain 1269 and 35–40°C and pH 2.5–2.7 for strain K1. An even higher yield of endospores, amounting to 55 and 75% for strains 1269 and K1, respectively, was obtained when the above growth conditions were combined (growth on medium A at optimal temperatures and pH under static conditions). Our results suggest a new approach to optimizing sporulation by acidophilic chemolithotrophs, which consists in limiting the energy and nutrient sources and using temperature and pH values within the tolerance bounds of these cultures but outside their growth optimum ranges.     

Hydrolytic Enzymes and Sporulation in Bacillus intermedius

The investigation of the activity of extracellular hydrolytic enzymes and sporulation in the bacterium Bacillus intermedius 3-19 showed that the activity of ribonuclease is maximal in the glucose-containing growth medium, in which sporulation is suppressed. At the sporulation stages II–IV, the synthesis of phosphatase was not regulated by the factors that influence this synthesis in the phase of growth retardation. Caseinolytic activity exhibited two peaks. The first peak was observed when thiol-dependent proteinase began accumulating in the medium. The second peak corresponded to the late stages of sporulation, i.e., the stages of spore maturation and the autolysis of sporangium. The regulatory relationship between proteinase synthesis and sporulation and the possible role of extracellular phosphatases and proteinases in the sporulation are discussed.     

Bioconversion capacity ofStreptomyces sp GE44282, a producer of the antibiotics heneicomycin and aurodox

Streptomyces sp GE44282 was isolated in the course of a screening program for novel antibiotics. It co-produces heneicomycin and aurodox, two kirromycin-type antibiotics, which differ by the presence of an hydroxyl group at the C30 position of aurodox. Heneicomycin is converted into aurodox both by growing and resting cells ofStreptomyces sp GE44282 and by the producer of aurodox,Streptomyces goldiniensis ATCC 21386. This bioconversion of heneicomycin is substrate-specific and is not observed using the producer of heneicomycin,Streptomyces filippiniensis NRRL 11044. The three strains show very similar taxonomic characteristics. These results suggest that heneicomycin is a precursor of aurodox, the production of which depends on the bioconversion capability expressed by the strain.     

Synthesis and Secretion of Proteinases by Bacillus intermedius in the Late Stages of Sporulation

In the late stages of sporulation, cells of Bacillus intermedius 3-19 secreted into the medium two proteinases, glutamyl endopeptidase and subtilisin, whose maximum activities were recorded in the 40th and 44th hours of growth, respectively. By estimating -galactosidase activity as a marker of cytoplasmic membrane integrity, it was revealed that the accumulation of these proteinases in the medium was a result of their secretion and not of lysis of the cell envelope. Concentrations of peptone and inorganic phosphate ensuring the maximum production of the enzymes were established. Ammonium ions were shown to inhibit the production of proteinases by the mechanism of repression by nitrogen metabolites.     

Trends in the fertilization ofStreptomyces coelicolor A3(2)

SCP1 and SCP2 (in the SCP2^* state) fertility plasmids ofStreptomyces coelicolor A3(2) elicit recombination in SCP1⁺×SCP1^- or SCP2^*×SCP2^- crosses. The rate is essentially constant (c. 10^-4) if referred to the plasmid-less parent, irrespective of extreme variations in the parent balance. In interrupted matings the alleles of the plasmid-less parent gradually increase in frequency in successive samples. The mobilization of the chromosome of the plasmid-less strain appears to be the primary event in merozygote formation.     

Isolation and characterization of stable mutants ofStreptomyces peucetius defective in daunorubicin biosynthesis

Daunorubicin and its derivative doxorubicin are antitumour anthracycline antibiotics produced byStreptomyces peucetius. In this study we report isolation of stable mutants ofS. peucetius blocked in different steps of the daunorubicin biosynthesis pathway. Mutants were screened on the basis of colony colour since producer strains are distinctively coloured on agar plates. Different mutants showed accumulation of aklaviketone, ε-rhodomycinone, maggiemycin or 13-dihydrocarminomycin in their culture filtrates. These results indicate that the mutations in these isolates affect steps catalysed bydnrE (mutants SPAK and SPMAG),dnrS (SPFS and SPRHO) anddoxA (SPDHC) gene products.     

Feather degradation by Kocuria rosea in submerged culture

A strain of Kocuria rosea with keratinolytic activity was studied. In batch culture, the optimum temperature for feather degradation, bacterial growth and protease secretion was at 40 °C. A specific growth rate of 0.17 h⁻¹ was attained in basal medium with feathers as fermentation substrate. Under these conditions, after 36 h of incubation, biomass and caseinolytic activity reached 3.2 g/l and 0.15 U/ml, respectively. Extracellular protease secretion was associated with the exponential growth phase. In batch fermentation, feather degradation up to 51% in 72 h was obtained with a conversion yield in biomass of 0.32 g/g. No organic acids were detected in the fermentation broth in significant amount. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sporulation of Streptomyces griseus in submerged culture.

A wild-type strain of Streptomyces griseus forms spores both on solid media (aerial spores) and in liquid culture (submerged spores). Both spore types are highly resistant to sonication, but only aerial spores are resistant to lysozyme digestion. Electron micrographs suggest that lysozyme sensitivity may result from the thinner walls of the submerged spores. Studies of the life cycle indicate that neither streptomycin excretion nor extracellular protease activity is required for sporulation: the analysis of mutants, however, suggests that antibiotic production may be correlated with the ability to sporulate. A method was devised to induce the rapid sporulation of S. griseus in a submerged culture. This method, which depends on nutrient deprivation, was used to determine that either ammonia or phosphate starvation can trigger sporulation and that the enzyme glutamine synthetase may be useful as a sporulation marker after phosphate deprivation.     

Endospore formation by <Emphasis Type="Italic">Streptomyces avermitilis</Emphasis> in submerged culture

The ability of streptomycetes to form endospores during their life cycle was studied in submerged cultures of Streptomyces avermitilis. Submerged S. avermitilis spores were most intensely formed (1) during the culture development cycle on synthetic medium CP1 with glucose under phosphate limitation and (2) in autolysing cell suspensions of high density obtained by tenfold concentration in phosphate buffer (pH 7.2) with 0.2% CaCl₂ of stationary-phase cells grown in synthetic medium. Endospores of S. avermitilis formed in submerged cultures shared the major characteristics of specialized microbial resting forms: heat resistance, resistance to lysozyme, ability to retain the main species-specific features, and ultrastructural organization characteristic of endospores. They can be considered a resting form of streptomycetes alternative to the spores formed exogenously on aerial mycelium in surface cultures.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 204–214.Original Russian Text Copyright © 2005 by Filippova, Gorbatyuk, Poglazova, Soina, Kuznetsov, El-Registan.     

Production of <Emphasis Type="Italic">Pleurotus sajor-caju</Emphasis> strain PS-2001 biomass in submerged culture

Mushrooms or fruiting bodies of many basidiomycetes are commonly produced in solid-state fermentation, generally after 20-60 days of growth. However, it is also possible to produce biomass from these fungi, in submerged fermentation in shorter time. This work was aimed at evaluating biomass production with the basidiomycete Pleurotus sajor-caju, in a submerged process and to determine the proportion of chemical components of this biomass. Initially, an optimization of the culture medium was done to produce a faster growth of microbial mass by changing the concentrations of ammonium sulfate, soy protein and yeast extract. Using the optimized culture medium, values of approximately 5.5 g L(-1) of biomass in a medium with 10 g L(-1) of glucose were attained. When the optimized culture medium was tested in a 5-L stirred tank bioreactor, using 10 g L(-1) of glucose or sucrose as carbon source, values of 8.18 and 5.94 g L(-1) of biomass concentration were obtained, respectively. In the medium with glucose, high yields (0.82 g g(-1)) and productivity of 0.085 g L(-1) h(-1) were obtained. The exopolysaccharide content (1.58 g dry matter L(-1)) in the culture was higher in the fermentation with sucrose. The nutritional composition of the biomass obtained in the submerged fermentation was similar to that of the fruiting body in terms of quantities of total carbohydrates, ash and calories, but total fat and protein were higher.     

Disruption of sabR affects nikkomycin biosynthesis and morphogenesis in Streptomyces ansochromogenes

The gene, sabR, encoding a receptor for -butyrolactone, was cloned from the genomic DNA of Streptomyces ansochromogenes 7100. Its deduced protein shows strong homology to several -butyrolactone-binding proteins in Streptomyces. Disruption of sabR retarded nikkomycin production in liquid media containing glucose or glycerol as carbon source. Sporulation of sabR disruption mutants was earlier than the parent strain on solid media with glucose or glycerol as carbon source. However, disruption of sabR had no effect on either nikkomycin production or sporulation on media containing mannitol as carbon source, suggesting that sabR is a pleiotropic regulatory gene that controls the onset of nikkomycin production and sporulation in S. ansochromogenes and is related to the utilization of carbon source.     

Culture medium improvement for Isaria fumosorosea submerged conidia production

Submerged conidia and blastospores of the entomopathogenic fungus Isaria fumosorosea are produced in several liquid culture media. However, yields and the ecological fitness of these propagules vary according to culture media composition. In most culture media, hyphae, blastospores and submerged conidia are white but we found that in some media they develop a brown pigmentation. A dark pigment was extracted from brown-pigmented propagules and analyzed by IR spectroscopy. Adsorption bands coincided to those characteristics of melanins.Hadamard's matrices were employed in order to increase submerged conidia yields and brown pigmentation of fungal propagules. Media containing 20–30 mg/l of FeSO₄·7H₂O and 6–12 mg/l of CuSO₄·5H₂O allowed reaching the highest pigmentation (9 in a hedonic scale). A maximal concentration of submerged conidia of 1.0 (±1.2) × 10¹² cell/l was achieved after 120 h of liquid culture in a improved culture medium, containing 25 ml/l of Polyethylene glycol (MW 200), substance which enhanced submerged conidia production, reducing free mycelia or mycelial pellets formation. In the improved medium, it was estimated that more than 60% of produced biomass corresponded to submerged conidia and blastospores, while in other media, mycelia were the main product (80–97%).     

Production of an anti-complement exo-polymer produced by Auricularia auricula-judae in submerged culture

Exo-polymer (EP) was produced at 1.2 g l(-1) in submerged culture of Auricular auricula-judae. Crude EP (AJ-0) has 70% anti-complementary activity (inhibition of total complementary hemolysis 50%; ITCH50). The activating pathway of the complement system occurred through both the classical and alternative pathways, though the major pathway was the classical one. Fractionation of AJ-0 using Sepharose CL-6B gel chromatography gave three major fractions (AJ-Fr-I, II and III) of which the first was the most active. The mycelial growth and EP production of A. auricula-judae were optimal at pH 6, 25 degrees C and pH 5, 25 degrees C, respectively.     

Production of cyclodextrin glycosyltransferase by alkaliphilic <Emphasis Type="Italic">Bacillus circulans</Emphasis> in submerged and solid-state cultivation

Cyclodextrin glycosyltransferase (CGTase; E.C. 2.4.1.19) is an industrially important enzyme, which is used to produce cyclodextrins (CDs). In this research, we report the use of experimental factorial design to find the best conditions of pH and temperature for CGTase production by Bacillus circulans var. alkalophilus. The optimized calculated values for the tested variables were, respectively, pH 9.7 and temperature 36^oC, with a CGTase activity of 615 U mL⁻¹. The CGTase production was further studied with the optimized process parameters on submerged cultivations (SC) and solid-state cultivations (SSC) using soybean industrial fibrous residue (SIFR). The maximum CGTase activity obtained on SC was 1,155 U mL⁻¹ under aerobic conditions. Cell growth and CGTase synthesis in SSC using SIFR as substrate was excellent, with CGTase activity of 32,776 U g_(SIFR) ⁻¹. These results strongly support the use of SIFR for CGTase production since it is a non-expensive residue.     

Reduction of benthic macroinvertebrates due to waterfowl foraging on submerged vegetation during autumn migration

If present in large numbers, as during migration, herbivorous waterfowlmay reduce the amount of submerged vegetation. Because the vegetation is a keyfactor in shallow eutrophic lakes, removal of the green biomass can be expectedto affect also other biota that depend on the vegetation. We conducted anexperiment to determine how the abundance of chironomids andPisidium sp. were affected by intense foraging ofwaterfowlon the submerged plant Potamogeton pectinatus. This wasdone in Lake Ringsjön in southern Sweden, during the autumn migration ofthe birds. Three treatments, replicated six times, were used: (i) closed cagesthat excluded all waterfowl, (ii) semi-open cages that excluded only largewaterfowl (geese and swans), and (iii) open plots where all waterfowl couldfreely enter. Waterfowl densities were monitored during the experiment. Theresults suggest that the foraging of large waterfowl (swans) had a clearlynegative effect on macroinvertebrate abundance and aboveground biomass ofP. pectinatus. At the end of the experiment, the densityofchironomids was about 46% lower in the open than in the closed cages. Ingeneral, the density of Pisidium sp. tended to be lower inthe open plots. Small waterfowl alone did not seem to affect either thevegetation or macroinvertebrates. We suggest that thePisidium sp. was influenced at an early stage of grazing,when waterfowl foraged on aboveground biomass, whereas chironomids wereaffectedat a later stage, when swans were digging for below-ground tubers.     

Phosphate,ammonium, magnesium and iron nutrition ofStreptomyces hygroscopicus with respect to rapamycin biosynthesis

Summary Phosphate, ammonium and magnesium salts interfered with rapamycin production byStreptomyces hygroscopicus at concentrations optimal for growth. These observations point to the existence of phosphorus, magnesium and nitrogen-negative regulation mechanisms for rapamycin biosynthesis. On the other hand, Fe²⁺ stimulated rapamycin production at concentrations greater than that required for growth.