Effects of farnesol and the off-flavor derivative geosmin on Streptomyces tendae.

Effects of the sesquiterpene farnesol (3,7,11-trimethyl-2,6,10-dodecatrien-1-ol) and the sesquiterpene derivative geosmin (1,10-trans-dimethyl-trans-9-decalol) were investigated in a geosmin-producing actinomycete, Streptomyces tendae. Exposure to 300 microM farnesol reduced biomass (fresh matter) accumulation by 97% compared with biomass accumulation by controls, whereas an equal amount of geosmin did not affect biomass accumulation. Increasing exposure to farnesol corresponded with reduced optical density of the culture, reduced levels of geosmin, and reduced metabolic heat production compared with controls, while exogenous geosmin did not affect these parameters. Geosmin dissipated from unioculated medium more rapidly than farnesol, indicating that in addition to the lower toxicity of geosmin, the actual exposure to geosmin over time may be less than exposure to an equal amount of farnesol. Cultures grown on Actinomyces-B medium contained 99.5% less geosmin and were more sensitive to farnesol than those grown on Hickey-Tresner medium, indicating that geosmin synthesis was associated with reduced sensitivity to farnesol. Consumption of farnesyl moieties during geosmin synthesis may reduce the potential for farnesol-induced inhibition of growth and metabolism.     

Effects of several metals on spore,biomass, and geosmin production byStreptomyces tendae andPenicillium expansum

Cultures ofStreptomyces tendae andPenicillium expansum grown on Actinomyces and Czapek's media, respectively, were exposed to 5 mg L^–1 of manganese, magnesium, iron, cobalt, nickel, copper and zinc, supplied as sulfate salts. Only copper markedly increased geosmin (1, 10-dimethyl-9-decalol), biomass, and spore production. inductively coupled plasma-atomic emission spectrometric analysis ofS. tendae andP. expansum cells did not indicate an accumulation of copper. Both 1 and 5 mg L^–1 copper, as copper sulfate, increased total geosmin production in cultures ofS. tendae on several media, but decreased production on others, suggesting that substrate composition affects responses to copper.     

Nikkomycin production in pellets of Streptomyces tendae.

In previous submerged fermentation experiments mycelial suspensions of Streptomyces tendae were viscous and availability of oxygen limited the yield of nikkomycins (Nk), a complex of secondary metabolites which includes nucleoside-peptides with antibiotic activity. Increasing agitation improved oxygen transfer but consumed considerable power and shear-damaged cells. In this paper, cellular aggregates (pellets) were used to reduce viscosity and protect cells from shear. Under the conditions tested, specific productivity of S. tendae pellets increased with increasing size up to 1.4 mm diameter and then decreased. The maximal specific productivity of S. tendae pellets (44 mg Nk/g dry weight/h) occurred at a very low cell concentration. Pellet formation or high biomass concentration was required for the production of bioactive dipeptide and tripeptide Nks. It is speculated that accumulation of intermediates in large pellets activates the production of mature secondary metabolites.     

Nikkomycin production in pellets of Streptomyces tendae

S.E. VECHT-LIFSHITZ, Y. SASSON AND S. BRAUN. 1992. In previous submerged fermentation experiments mycelial suspensions of Streptomyces tendae were viscous and availability of oxygen limited the yield of nikkomycins (Nk), a complex of secondary metabolites which includes nucleoside-peptides with antibiotic activity. Increasing agitation improved oxygen transfer but consumed considerable power and shear-damaged cells. In this paper, cellular aggregates (pellets) were used to reduce viscosity and protect cells from shear. Under the conditions tested, specific productivity of S. tendae pellets increased with increasing size up to 1.4 mm diameter and then decreased. The maximal specific productivity of S. tendae pellets (44 mg Nk/g dry weight/h) occurred at a very low cell concentration. Pellet formation or high biomass concentration was required for the production of bioactive dipeptide and tripeptide Nks. It is speculated that accumulation of intermediates in large pellets activates the production of mature secondary metabolites.     

Effects of oxygen supply on the production of nikkomycin with immobilized cells of Streptomyces tendae

Summary For continuous production of the antibiotic nikkomycin immobilized cells have been used in a fluidized bed bioreactor. Cells of Streptomyces tendae were immobilized on sintered glass particles. Different biomass concentrations on the particles correspond to different thicknesses of mycelial layers because growth occurs only on the outer surface of the particles. The antibiotic productivity decreased with increasing layer thickness. In fermentations with higher concentrations of both biomass on the particles and dissolved oxygen levels of about 70% the productivity was also limited because of limited oxygen diffusion in the layers. Offprint requests to: H. U. Trück     

Continuous cultivation of Streptomyces tendae in different media

Summary The strain Streptomyces tendae is well suited for continuous cultivation because of its ability to grow and produce secondary metabolites simultaneously. Continuous culture experiments on defined medium show that growth is limited by nitrogen during steady state for the given medium composition. It is supposed that this also holds for complex medium. Production of antibiotics (several nikkomycins) occurs simultaneously with exponential growth. After switching from batch to continuous operation the fraction of biomass, consisting of pellets, decreases permanently.     

Plasmid transformation of Streptomyces tendae after heat attenuation of restriction.

Streptomyces tendae ATCC 31160 produces nikkomycin, a fungicide and insecticide that inhibits chitin synthases. Exposure of S. tendae protoplasts to 50 degrees C for 30 min is required for transformation (10(2) thiostrepton-resistant transformants micrograms of DNA-1) with plasmid pIJ702 or pIJ680 from Streptomyces lividans. pIJ702 and pIJ680 DNA isolated from the S. tendae transformants is efficient (10(6) to 10(7) transformants micrograms of DNA-1) in subsequent transformations of S. tendae protoplasts generated at 30 degrees C. PstI fails to cut the single PstI site in pIJ702 and cuts only one of the two PstI sites in pIJ680 DNA isolated from S. tendae transformants. Digests of plasmid DNA mixtures showed that plasmid DNA from S. tendae does not inhibit PstI activity. pIJ702 and pIJ680 DNA from S. tendae transformants was used to transform S. lividans to show that plasmid DNA remains unchanged, except for modification at some PstI sites in S. tendae, as a consequence of passage through S. tendae. The DNA modification is lost when S. lividans is transformed with plasmid DNA from S. tendae transformants. Since S. tendae modifies only some PstI sites, it appears the modification (presumably restriction activity also) activity in S. tendae recognizes a sequence that includes or overlaps the PstI hexanucleotide recognition sequence.     

Pellet formation and cellular aggregation in Streptomyces tendae

In submerged cultures, Streptomyces tendae tended to form fluffy spherical pellets of the noncoagulative type. An increase in the average pellet size could be attained by decreasing any of the following: shear rate, pH, temperature, or inoculum size. Conditions leading to oxygen limitation tended to reduce the average pellet size and induced pulpy growth, whereas oxygen sufficiency seemed to induce pellet formation. Factors inducing pellet formation simultaneously increased cell wall hydrophobicity. It is therefore proposed that the main forces inducing cellular aggregation in S. tendae are hydrophobic interactions of cell walls, and these interactions are controlled by availability of dissolved oxygen.     

Plasmid transformation of Streptomyces tendae after heat attenuation of restriction

Streptomyces tendae ATCC 31160 produces nikkomycin, a fungicide and insecticide that inhibits chitin synthases. Exposure of S. tendae protoplasts to 50 degrees C for 30 min is required for transformation (10(2) thiostrepton-resistant transformants micrograms of DNA-1) with plasmid pIJ702 or pIJ680 from Streptomyces lividans. pIJ702 and pIJ680 DNA isolated from the S. tendae transformants is efficient (10(6) to 10(7) transformants micrograms of DNA-1) in subsequent transformations of S. tendae protoplasts generated at 30 degrees C. PstI fails to cut the single PstI site in pIJ702 and cuts only one of the two PstI sites in pIJ680 DNA isolated from S. tendae transformants. Digests of plasmid DNA mixtures showed that plasmid DNA from S. tendae does not inhibit PstI activity. pIJ702 and pIJ680 DNA from S. tendae transformants was used to transform S. lividans to show that plasmid DNA remains unchanged, except for modification at some PstI sites in S. tendae, as a consequence of passage through S. tendae. The DNA modification is lost when S. lividans is transformed with plasmid DNA from S. tendae transformants. Since S. tendae modifies only some PstI sites, it appears the modification (presumably restriction activity also) activity in S. tendae recognizes a sequence that includes or overlaps the PstI hexanucleotide recognition sequence.     

Purification and characterization of an endonuclease (E.C. 3.1.30.1) from Streptomyces tendae

A single-strand-specific endonuclease which converted negatively supercoiled DNA to open-circular and linear DNA was purified to homogeneity with Hb-Sepharose 4B, DEAE Trisacryl M, HA-Ultrogel and PBE-94 chromatofocusing from extracts of Streptomyces tendae ATCC 31160. Bio-Gel P-200 chromatography and electrophoresis in SDS-PAGE indicated the native protein was a monomer with a molecular weight of approximately 40-kDa. This enzyme did not hydrolyze double-stranded linear DNA but digested RNA and circular single-strand DNA. Sequence specificity for nicking of negatively supercoiled DNA was not detected.     

Process performance of parallel bioreactors for batch cultivation of Streptomyces tendae

Batch cultivations of the nikkomycin Z producer Streptomyces tendae were performed in three different parallel bioreactor systems (milliliter-scale stirred-tank reactors, shake flasks and shaken microtiter plate) in comparison to a standard liter-scale stirred-tank reactor as reference. Similar dry cell weight concentrations were measured as function of process time in stirred-tank reactors and shake flasks, whereas only poor growth was observed in the shaken microtiter plate. In contrast, the nikkomycin Z production differed significantly between the stirred and shaken bioreactors. The measured product concentrations and product formation kinetics were almost the same in the stirred-tank bioreactors of different scale. Much less nikkomycin Z was formed in the shake flasks and MTP cultivations, most probably due to oxygen limitations. To investigate the non-Newtonian shear-thinning behavior of the culture broth in small-scale bioreactors, a new and simple method was applied to estimate the rheological behavior. The apparent viscosities were found to be very similar in the stirred-tank bioreactors, whereas the apparent viscosity was up to two times increased in the shake flask cultivations due to a lower average shear rate of this reactor system. These data illustrate that different engineering characteristics of parallel bioreactors applied for process development can have major implications for scale-up of bioprocesses with non-Newtonian viscous culture broths.     

Partial deletion of transposon Tn4560 integrated into the genome of Streptomyces tendae

Polymerase chain reaction (PCR), Southern hybridization and DNA sequencing experiments were done to determine whether all of Tn 4560 , a Streptomyces transposon, integrated into the genomes of three nikkomycin non-producing mutants. A deletion of 279 bases occurred at one end of Tn 4560 while present in the genome of one of the mutants.     

Biosynthesis of the earthy odorant geosmin by a bifunctional Streptomyces coelicolor enzyme

Geosmin is responsible for the characteristic odor of moist soil, as well as off-flavors in drinking water and foodstuffs. Geosmin is generated from farnesyl diphosphate (FPP, 2) by an enzyme that is encoded by the SCO6073 gene in the soil organism Streptomyces coelicolor A32 (ref. 3). We have now shown that the recombinant N-terminal half of this protein catalyzes the Mg2+-dependent cyclization of FPP to germacradienol and germacrene D, while the highly homologous C-terminal domain, previously thought to be catalytically silent, catalyzes the Mg2+-dependent conversion of germacradienol to geosmin. Site-directed mutagenesis confirmed that the N- and C-terminal domains each harbor a distinct, independently functioning active site. A mutation in the N-terminal domain of germacradienol-geosmin synthase of a catalytically essential serine to alanine results in the conversion of FPP to a mixture of sesquiterpenes that includes an aberrant product identified as isolepidozene, which was previously suggested to be an enzyme-bound intermediate in the cyclization of FPP to germacradienol.     

Histidine aminotransferase activity in Streptomyces tendae and its correlation with nikkomycin production

Streptomyces tendae Tü901 produces nikkomycins belonging to the nucleoside peptide antibiotics. Mutants defective in histidine catabolism were isolated and characterized with regard to their histidine ammonium-lyase activity and antibiotic synthesis. In the histidine ammonialyase-negative mutant hut-11 which was unimpaired in nikkomycin production histidine aminotransferase activity was detected as an additional histidine metabolizing enzyme. A protein exhibiting histidine aminotransferase activity could be demonstrated on non-denaturing gels of hut-11 crude extracts. Using optimized assay conditions, histidine aminotransferase activity was investigated in the strain hut-11 during growth in nikkomycin production medium. Maximal activity was reached at the end of exponential growth prior to nikkomycin production. In the presence of bromopyruvate, an effective inhibitor of histidine aminotransferase activity in vitro, production of nikkomycin Z and X was markedly reduced in hut-11.     

Heterologous expression of the alpha-amylase inhibitor gene cloned from an amplified genomic sequence of Streptomyces tendae.

The coding region for a secreted proteinaceous inhibitor of the human alpha-amylase (tendamistat; HOE 467) was identified by using a synthetic oligonucleotide probe. The gene is part of a 37-kilobase amplified genomic sequence found in an overproducing mutant of Streptomyces tendae. After subcloning, sequence analysis revealed an open reading frame of 312 base pairs preceded by a putative ribosome-binding site. The reading frame is 30 codons longer than necessary for the mature protein. This sequence coded for an amino-terminal extension of tendamistat and shows typical features of a signal peptide. After being cloned into Streptomyces vector plasmids and transformed to the heterologous host, Streptomyces lividans TK24, the gene was expressed, and the alpha-amylase inhibitor was correctly processed and secreted into the culture medium. The amount of secreted protein was dependent on the gene dosage and on the promoter arrangement.     

Nutritional control of nikkomycin and juglomycin production by Streptomyces tendae in continuous culture

Summary Continuous cultures with Streptomyces tendae revealed some interesting facts. In a continuous culture running for more than 2500 h the production of either nikkomycins or juglomycins could be selected by varying the feed composition. Decreasing the phosphate supply in the feed broth from the initial concentration of 2.5 mm to 1.0 mm enhanced the productivity of nikkomycins and decreased the productivity of juglomycins. When switching back to the initial conditions of the experiment after 2000 h nearly the same production behaviour as at the beginning of the fermentation could be observed. This indicated a stable behaviour of the population with regard to nikkomycin productivity. The long continuous fermentation showed the ability of S. tendae Tü 901/8c to produce nikkomycin at a high level for at least 1500 h. In a second continuous culture it was shown that the productivity of the nikkomycins and juglomycins decreased and increased, respectively, with increasing dilution rate. Comparing batch cultures with continuous fermentations, higher juglomycin productivity was found in the latter. These facts indicate that the strain responds to complex interacting physiological controls, by producing either nikkomycins or juglomycins in a higher amount. Offprint requests to: D. Hege-Treskatis     

Effects of carbon source, phosphorus concentration, and several micronutrients on biomass and geosmin production by Streptomyces halstedii

The effects of various carbon sources, phosphorus concentration, and different concentrations of the micronutrients calcium, cobalt, copper, iron, manganese, potassium, and zinc were determined on biomass dry weight production, geosmin production, and geosmin/biomass (G/B) values for Streptomyces halstedii, a geosmin-producing actinomycete isolated from the sediment of an aquaculture pond. Of the substrates tested, maltose as a sole carbon source promoted maximal growth by S. halstedii while mannitol promoted maximal geosmin production, and galactose yielded the highest G/B values. Fish-food pellets and galactose were poor substrates for growth. Increasing phosphorus concentrations enhanced geosmin production and G/B values. Of the seven micronutrients tested, zinc, iron, and copper had the most profound effects on biomass and geosmin production. Increasing zinc concentrations promoted biomass production while inhibiting geosmin production and G/B values; increasing concentrations of copper and iron inhibited biomass and geosmin production. Increased copper concentrations had the greatest effect in preventing growth and geosmin production by S. halstedii. Journal of Industrial Microbiology & Biotechnology (2001) 26, 241–247. Received 20 September 2000/ Accepted in revised form 17 January 2001     

Use of Tn4560 to generate nikkomycin non-producing mutants of Streptomyces tendae

Abstract Transposon Tn 4560 was used to generate three nikkomycin non-producing mutants in Streptomyces tendae ATCC 31160 Southern hybridization confirmed that Tn 4560 was present in 10–12-kb Bam HI fragments of the chromosomes of the mutants. Biologically active nikkomycins were not detected in culture broths of the mutants as determined by bioassays and HPLC. Differences in the HPLC profiles of culture broths suggest that Tn 4560 inserted into different genes in the mutants.     

Modelling and parameter identification for batch fermentations with Streptomyces tendae under phosphate limitation

Summary A simple structured model for the dynamics of phosphate-limited batch fermentations with Streptomyces tendae is presented. The model describes the influence of intracellular phosphate storage upon the growth behav our of the culture. The development of the model takes into account the possible internal regulatory processes of phosphate metabolism. These complex biochemical pathways are summarized with regard to rate-limiting steps to obtain relatively simple model equations. The model parameters are fitted to the experimental data with an identification programme based on the sequential quadratic programming algorithm. Modifications in this algorithm yield a good performance for this application. With respect to the sensitivity of the model parameters, a feedback on the modelling is given. After several loops of modelling and identification, a model was achieved that fits to a set of batch fermentations. Furthermore the simulations show that RNA measurements of some recent fermentations can be interpreted by the simulated internal state variable and that there is evidence for RNA as an intracellular phosphate reserve. Offprint requests to: K.-P. Kuhn     

Characterization of pellet morphology during submerged growth of Streptomyces tendae by image analysis

Many filamentous bacteria and fungi tend to form pellets, or mixtures of dispersed mycelium and pellets in liquid fermentation broths. In some cases, a specific kind of morphology is required for optimum product yield. When quantitative analysis and characterization of the pellet morphology are needed, an image processing system can be used. It allows a fast and reproducible analysis of the frequency distribution of pellet size, mean pellet size, contents of pellets, or their shape. The use of such a system allows for an on-line analysis. For a demonstration of the method, results of two fermentations of Streptomyces tendae are shown.