Cloning and functional characterization of the germacradienol synthase (spterp13) from Streptomyces peucetius ATCC 27952

Sequence analysis of the metabolically rich genome of Streptomyces peucetius ATCC 27952 revealed a 2,199 bp sesquiterpene alcohol (germacradienol) synthase-encoding gene from the germacradienol synthase/terpene cyclase gene cluster. The gene was named spterp13, and its putative function is as a germacradienol synthase/terpene cyclase. The amino acid sequence of Spterp13 shows 66% identity with SAV2163 (GeoA) from S. avermitilis MA- 4680 and 65% identity with SCO6073 from S. coelicolor A3(2), which produces germacradienol/geosmin. The fulllength recombinant protein was heterologously expressed as a his-tagged fusion protein in Escherichia coli, purified, and shown to catalyze the Mg2+-dependent conversion of farnesyl diphosphate to the germacradienol, which was verified by gas chromatography/mass spectrometry.     

Squalene-hopene cyclase (Spterp25) from Streptomyces peucetius: sequence analysis, expression and functional characterization

Squalene-hopene cyclase, which catalyzes the complex cyclization of squalene to the pentacyclic triterpene, hopene, is a key enzyme in the biosynthesis of hopanoids. The deduced amino acid sequence of the Streptomyces peucetius gene (spterp25) had significant similarity to other prokaryotic squalene-hopene cyclases. Like other triterpene cyclases, the S. peucetius squalene-hopene cyclase contains eight so-called QW-motifs with an aspartate-rich domain. The 2,025-bp squalene-hopene cyclase-encoding gene was expressed in Escherichia coli BL21(DE3)pLySs, and the in vitro activity of the recombinant cyclase was demonstrated using purified membrane protein. The cyclization product hopene was identified by gas chromatography/mass spectrometry (GC/MS).     

Proteomic approach to enhance doxorubicin production in <Emphasis Type="Italic">panK</Emphasis>-integrated <Emphasis Type="Italic">Streptomyces peucetius</Emphasis> ATCC 27952

Biosynthesis of polyketide compounds depends upon the starter and extender units of coenzyme A derivatives of carboxylic acids present in the host organism. To increase the coenzyme A (CoA) pool, pantothenate kinase (panK) gene from Escherichia coli was integrated into S. peucetius ATCC 27952 (panK-integrated strain, BG200), which resulted in increase in aglycone polyketide ε-rhodomycinone (RHO), but decrease in the desired product, i.e., doxorubicin (DXR). To reduce RHO accumulation by synthesizing daunorubicin (DNR) from RHO more efficiently, glycosyltransferase (dnrQS) was overexpressed (pIBR25::dnrQS in panK-integrated strain, BG201). However, DnrQS overexpression still resulted in less production of DXR compared with the parental strain. To understand the results in detail by investigating the proteome changes in the panK-integrated strain, two-dimensional (2D) gel electrophoresis was performed. Among the several proteins that are up- or downregulated in BG200, efflux protein DrrA was our main target of interest, because it is directly related to DXR/DNR production in S. peucetius. DXR transporter DrrAB was additionally introduced in BG200 to enhance secretion of toxic DXR. Compared with S. peucetius ATCC 27952, BG204 (pIBR25::drrAB in panK-integrated strain), produced two times higher amount of DXR, which is 9.4-fold higher than that of panK-integrated strain BG200. The results show that the proteomic approach is quite useful in host development of Streptomyces and understanding cell physiology for antibiotic production.     

The complex <Emphasis Type="Italic">whiJ</Emphasis> locus mediates environmentally sensitive repression of development of <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> A3(2)

A segment of DNA was isolated that complemented several poorly characterised sporulation-defective white-colony mutants of Streptomyces coelicolor A3(2) from an early collection (Hopwood et al., J Gen Microbiol 61: 397–408, 1970). Complementation was attributable to a gene, SCO4543, named whiJ, encoding a likely DNA-binding protein. Surprisingly, although some mutations in whiJ had a white colony phenotype, complete deletion of the wild-type or mutant gene gave a wild-type morphology. The whiJ gene is a member of a large paralogous set of S. coelicolor genes including abaAorfA, which regulates antibiotic production; and genes flanking whiJ are paralogues of other gene classes that are often associated with whiJ-like genes (Gehring et al., Proc Natl Acad Sci USA 97: 9642–9647, 2000). Thus, the small gene SCO4542 encodes a paralogue of the abaAorfD gene product, and SCO4544 encodes a paralogue of a family of likely anti-sigma factors (including the product of abaAorfB). Deletion of SCO4542 resulted in a medium-dependent bald- or white-colony phenotype, which could be completely suppressed by the simultaneous deletion of whiJ. A model is proposed in which WhiJ binds to operator sequences to repress developmental genes, with repression being released by interaction with the WhiJ-associated SCO4542 protein. It is suggested that this activity of SCO4542 protein is prevented by an unknown signal.     

Effects of temperature and light on the growth and geosmin production of Lyngbya kuetzingii (Cyanophyta)

The effects of temperature and light on the growth and geosmin production of Lyngbya kuetzingii were determined. Of the three temperatures tested, 10, 25 and 35°C, the maximal geosmin concentration and geosmin productivity were yielded at 10°C, while the highest chl a production was observed at 25°C. In the studies on light intensity, the maximal geosmin concentration and geosmin productivity were observed at 10 μmol m⁻² s⁻¹, while the highest chl a production was at 20 μmol m⁻² s⁻¹. It was suggested that more geosmin was synthesized with lower chl a demand. Meanwhile, the relative amounts of extra- and intracellular geosmin were investigated. Under optimum growth conditions (20 μmol m⁻² s⁻¹, 25°C; BG-11 medium), the amounts of extracellular geosmin increased as the growth progressed and reached the maximum in the stationary phase, while the intracellular geosmin reached its maximum value in the late exponential phase, and then began to decline. However, under the low temperature (10°C) or light (10 μmol m⁻² s⁻¹) conditions, more intracellular geosmin was synthesized and mainly accumulated in the cells. The proportions of extracellular geosmin were high, to 33.33 and 32.27%, respectively, during the stationary phase at 35°C and 20 μmol m⁻² s⁻¹. It was indicated that low temperature or light could stimulate geosmin production and favor the accumulation of geosmin in cells, while more intracellular geosmin may be released into the medium at higher temperatures or optimum light intensity.     

Inactivation of the extracytoplasmic function sigma factor Sig6 stimulates avermectin production in <Emphasis Type="Italic">Streptomyces avermitilis</Emphasis>

The role of the extracytoplasmic function (ECF) σ factor Sig6 (SAV663) in avermectin production by Streptomyces avermitilis was investigated by gene-deletion, complementation and over-expression experiments. Inactivation of Sig6 had no major effect on growth, stress responses, or morphology. Avermectin yield was increased 2- to 2.7-fold (~680 μg/ml) relative to the wild-type strain by deletion of the sig6 gene, and was restored to the wild-type level by introduction of a single copy of sig6. Introduction of extra multi-copy or integrative sig6 vectors into the wild-type decreased avermectin yield by 56–63%. Taken together, these findings indicate that Sig6 plays a negative regulatory role in avermectin production in S. avermitilis. RT-PCR analysis demonstrated that this role of Sig6 is mediated by the pathway-specific activator gene aveR.     

Geosmin production and polyphasic characterization of Oscillatoria limosa Agardh ex Gomont isolated from the open canal of a large drinking water system in Tianjin City,China

Taste and odor (T & O) episodes always cause strong effects on drinking water supply system. Luanhe River diversion into Tianjin City in China is an important drinking water resource. Massive growth of a benthic filamentous cyanobacterium with geosmin production in the open canal caused a strong earthy odor episode in Tianjin. On the basis of the morphological and molecular identification of this cyanobacterium as Oscillatoria limosa Agardh ex Gomont, the genetic basis for geosmin biosynthesis and factors influencing growth and geosmin production of O. limosa CHAB 7000 were studied in this work. A 2268-bp open reading frame, encoding 755 amino acids, was amplified and characterized as the geosmin synthase gene (geo), followed by a cyclic nucleotide-binding protein gene (cnb). Phylogenetic analysis implied that the evolution of the geosmin genes in O. limosa CHAB 7000 might involve a horizontal gene transfer event. Examination on the growth and geosmin production of O. limosa CHAB 7000 at different light intensities showed that the maximum geosmin production was observed at 10 μmol photons m⁻² s⁻¹, while the optimum growth was at 60 μmol photons m⁻² s⁻¹. Under three temperature conditions (15 °C, 25 °C, and 35 °C), the maximum growth and geosmin production were observed at 25 °C. Most amounts of geosmin were retained in cells during the growth phase, but high temperature and low light intensity increased the release of geosmin into the medium, implying that O. limosa CHAB 7000 had a high potential harm for the release of geosmin from its cells at these adverse conditions.     

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.     

Tryptophan catabolism via kynurenine production in <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis>: identification of three genes coding for the enzymes of tryptophan to anthranilate pathway

Most enzymes involved in tryptophan catabolism via kynurenine formation are highly conserved in Prokaryotes and Eukaryotes. In humans, alterations of this pathway have been related to different pathologies mainly involving the central nervous system. In Bacteria, tryptophan and some of its derivates are important antibiotic precursors. Tryptophan degradation via kynurenine formation involves two different pathways: the eukaryotic kynurenine pathway, also recently found in some bacteria, and the tryptophan-to-anthranilate pathway, which is widespread in microorganisms. The latter produces anthranilate using three enzymes also involved in the kynurenine pathway: tryptophan 2,3-dioxygenase (TDO), kynureninase (KYN), and kynurenine formamidase (KFA). In Streptomyces coelicolor, where it had not been demonstrated which genes code for these enzymes, tryptophan seems to be important for the calcium- dependent antibiotic (CDA) production. In this study, we describe three adjacent genes of S. coelicolor (SCO3644, SCO3645, and SCO3646), demonstrating their involvement in the tryptophan-to-anthranilate pathway: SCO3644 codes for a KFA, SCO3645 for a KYN and SCO3646 for a TDO. Therefore, these genes can be considered as homologous respectively to kynB, kynU, and kynA of other microorganisms and belong to a constitutive catabolic pathway in S. coelicolor, which expression increases during the stationary phase of a culture grown in the presence of tryptophan. Moreover, the S. coelicolor ΔkynU strain, in which SCO3645 gene is deleted, produces higher amounts of CDA compared to the wild-type strain. Overall, these results describe a pathway, which is used by S. coelicolor to catabolize tryptophan and that could be inactivated to increase antibiotic production.     

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     

Sugar uptake and sensitivity to carbon catabolite regulation in Streptomyces peucetius var. caesius

Streptomyces peucetius var. caesius produces a family of secondary metabolites called anthracyclines. Production of these compounds is negatively affected in the presence of glucose, galactose, and lactose, but the greatest effect is observed under conditions of excess glucose. Other carbon sources, such as arabinose or glutamate, show either no effect or stimulate production. Among the carbon sources that negatively affect anthracycline production, glucose is consumed in greater concentrations. We determined glucose and galactose transport in S. peucetius var. caesius and in a mutant of this strain whose anthracycline production is insensitive to carbon catabolite repression (CCR). In the original strain, incorporation of glucose and galactose was stimulated when the microorganism was grown in media containing these sugars, although we also observed basal galactose incorporation. Both the induced and the basal incorporation of galactose were suppressed when the microorganism was grown in the presence of glucose. Furthermore, adding glucose directly during the transport assay also inhibited galactose incorporation. In the mutant strain, we observed a reduction in both glucose (48%) and galactose (81%) incorporation compared to the original. Galactose transport in this mutant showed reduced sensitivity to the negative effect of glucose; however, it was still sensitive to inhibition. The deficient transport of these sugars, as well as CCR sensitivity to glucose in this mutant was corrected when the mutant was transformed with the SCO2127 region of the Streptomyces coelicolor genome. Our results support a role for glucose as the most easily utilized carbon source capable of exerting the greatest repression on anthracycline biosynthesis. In consequence, glucose also prevented the repressive effect of galactose by suppressing its incorporation. This suggests the participation of an integral regulatory system, which is initiated by an increase in incorporation of repressive sugars and their metabolism as a prerequisite for establishing the phenomenon of CCR in S. peucetius var. caesius.     

Analysis of neutral lipid biosynthesis in Streptomyces avermitilis MA-4680 and characterization of an acyltransferase involved herein

The physiology of lipid production in Streptomyces avermitilis MA-4680 with regard to the fatty acid composition of the accumulated lipids and their cellular distribution was analyzed. Cells were able to accumulate about ten to 30 lipid granules with diameters between 100 and 500 nm filling about 70–80% of the cell cytoplasm. Gas chromatography/mass spectrometry analyses of total cellular lipids and from isolated triacylglycerols (TAG) confirmed a similar fatty acid composition with a large portion of iso- and anteiso-methyl-branched fatty acids. De novo biosynthesis of wax esters (WE) appeared only during cocultivation on glucose and hexadecanol as carbon source. Homology alignments with the wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT; AtfA) from Acinetobacter baylyi strain ADP1 yielded one open reading frame in the genome databases of S. avermitilis MA-4680 referred to as SAV7256 with 25.3% homology. The highly conserved HHAxxDG active site motif found in AtfA, which is present in SAV7256, as well as the similar hydrophobicity profiles of AtfA and SAV7256 indicate a similar structure and function of both proteins. High acyl-CoA:diacylglycerol acyltransferase activity (DGAT; 143 pmol (mg min)⁻¹) but low wax ester synthase activity (WS; 1.3 pmol (mg min)⁻¹) were detected in crude extracts of S. avermitilis, which were consistent with the high TAG and negligible WE content of the cells. This indicates that TAG accumulation in S. avermitilis MA-4680 is mediated by the classical acyl-CoA-dependent DGAT pathway. Heterologous expression experiments in recombinant Escherichia coli BL21(DE3) demonstrated both WS and DGAT enzyme activity of SAV7256. Furthermore, substrate specificities of the acyltransferase SAV7256 will be discussed. Chlud Kaddor and Karolin Biermann contributed equally to this work.     

Expression profiling of <Emphasis Type="Italic">Streptomyces peucetius</Emphasis> metabolic genes using DNA microarray analysis

Doxorubicin (DXR) and daunorubicin (DNR) are anthracycline antibiotics produced by Streptomyces peucetius and widely used as cancer chemotherapeutic agents. To improve their productivity, regulation of DXR/DNR synthesis genes as well as central metabolic pathway genes must be understood more clearly. So far, studies have focused on DXR/DNR gene regulation. To investigate the correlation between the central metabolic pathway genes and DXR/DNR productivity, we selected 265 genes involved in glycolysis, fermentation, the citric acid cycle, butanoate metabolism, etc., and searched for their sequences in the S. peucetius genome by comparing gene sequences to those of Streptomyces coelicolor. The homologous genes were amplified by PCR and arrayed on glass microarray slides. Gene expression was monitored under two different growth media conditions, R2YE and NDYE. Genes involved in the production of malonyl-CoA and propionyl-CoA, the main precursors for doxorubicin synthesis, were mainly upregulated in NDYE media. Genes related to acetyl-CoA and the urea cycle were also upregulated. These changes in gene expression were confirmed by real-time RT-PCR.     

Enhanced doxorubicin production by Streptomyces peucetius using a combination of classical strain mutation and medium optimization

Abstract

Doxorubicin (DXR), which is produced by Streptomyces peucetius, is an important anthracycline-type antibiotic used for the treatment of various cancers. However, due to the low DXR productivity of wild-type S. peucetius, it is difficult to produce DXR by one-step fermentation. In this study, a DXR-resistance screening method was developed to screen for DXR high-producing mutants. Then, S. peucetius SIPI-11 was treated several times with UV and ARTP (atmospheric and room temperature plasma) to induce mutations. Treated strains were screened by spreading on a DXR-containing plate, isolating a mutant (S. peucetius 33-24) with enhanced DXR yield (570?mg/L vs. 119?mg/L for the original strain). The components of the fermentation medium, including the carbon and nitrogen sources, were optimized to further enhance DXR yield (to 850?mg/L). The pH of the fermentation medium and culture temperature were also optimized for effective DXR production. Finally, DXR production by S. peucetius 33-24 was investigated in flask culture and a fermenter. The yield of DXR was as high as 1100?mg/L in a 5-L fermenter, which is the highest DXR productivity reported thus far, suggesting that S. peucetius 33-24 has the potential to produce DXR by direct fermentation.     

Fish and land use influence <Emphasis Type="Italic">Gammarus lacustris</Emphasis> and <Emphasis Type="Italic">Hyalella azteca</Emphasis> (Amphipoda) densities in large wetlands across the upper Midwest

Gammarus lacustris and Hyalella azteca (hereafter G. lacustris and H. azteca, respectively) are important components of secondary production in wetlands and shallow lakes of the upper Midwest, USA. Within the past 50 years, amphipod densities have decreased while occurrences of fish and intensity of agricultural land use have increased markedly across this landscape. We investigated influences of fish, sedimentation, and submerged aquatic vegetation (SAV) on densities of G. lacustris and H. azteca in semipermanent and permanent wetlands and shallow lakes (n = 283) throughout seven eco-physiographic regions of Iowa, Minnesota, and North Dakota during 2004–2005. G. lacustris and H. azteca densities were positively correlated with densities of SAV (P < 0.001 and P < 0.001, respectively). Both species were negatively correlated with densities of large fish (non-Cyprinidae; P = 0.01 and P = 0.013, respectively) and with high densities of fathead minnows (Pimephales promelas; P < 0.001 and P = 0.033, respectively). H. azteca densities also were negatively correlated with densities of small fish (e.g., other minnows [Cyprinidae] and sticklebacks [Gasterosteidae]; P = 0.048) and common carp (Cyprinus spp.; P = 0.022). G. lacustris densities were negatively correlated with high levels of suspended solids (an index for sedimentation; P < 0.001). H. azteca densities were positively correlated with the width of upland-vegetation buffers (P = 0.004). Our results indicate that sedimentation and fish reduce amphipod densities and may contribute to the current low densities of amphipods in the upper Midwest. Thus, removing/excluding fish, and providing a thick buffer of upland vegetation around wetlands may help restore amphipod densities and wetland and water quality within this landscape.     

Effects of light and temperature on the odor production of 2-methylisoborneol-producing Pseudanabaena sp. and geosmin-producing Anabaena ucrainica (cyanobacteria)

Frequent off-flavor events caused by geosmin and 2-methylisoborneol (MIB) have attracted research on the main producers, cyanobacteria. This study evaluated the effects of light and temperature on the odor production of MIB-producing Pseudanabaena sp. Lauterborn and geosmin-producing Anabaena ucrainica (Schhorb.) Watanabe. The maximum MIB production and lowest growth rate (indicated by the chlorophyll a (Chl a)) were observed at 35 °C compared with that at 10 °C and 25 °C. Cultures grown under a light intensity of 60 μmol photons m⁻² s⁻¹ demonstrated the highest MIB production and minimum growth rate, whereas the minimum MIB production and maximum growth rate were obtained under 10 μmol photons m⁻² s⁻¹. Similar patterns were observed for geosmin production. A. ucrainica had the highest geosmin production and lowest Chl a concentration under 10 °C and 60 μmol photons m⁻² s⁻¹. Moreover, greater proportions of geosmin and MIB were released into extracellular under growth-inhibiting temperatures and light intensities. An inverse correlation between odor production and the cell growth rate was suggested, and this relationship may reflect the competition for substrates of odor and Chl a synthesis. Thus, the accumulation of geosmin and MIB was probably the result of decreased cellular metabolic activity in cyanobacteria.