Type II aromatic polyketide biosynthetic tailoring enzymes: diversity and adaptation in Streptomyces secondary metabolism.

Members of the bacterial genus Streptomyces are well known for their ability to produce an exceptionally wide selection of diverse secondary metabolites. These include natural bioactive chemical compounds which have potential applications in medicine, agriculture and other fields of commerce. The outstanding biosynthetic capacity derives from the characteristic genetic flexibility of Streptomyces secondary metabolism pathways: i) Clustering of the biosynthetic genes in chromosome regions redundant for vital primary functions, and ii) the presence of numerous genetic elements within these regions which facilitate DNA rearrangement and transfer between non-progeny species. Decades of intensive genetic research on the organization and function of the biosynthetic routes has led to a variety of molecular biology applications, which can be used to expand the diversity of compounds synthesized. These include techniques which, for example, allow modification and artificial construction of novel pathways, and enable gene-level detection of silent secondary metabolite clusters. Over the years the research has expanded to cover molecular-level analysis of the enzymes responsible for the individual catalytic reactions. In vitro studies of the enzymes provide a detailed insight into their catalytic functions, mechanisms, substrate specificities, interactions and stereochemical determinants. These are factors that are essential for the thorough understanding and rational design of novel biosynthetic routes. The current study is a part of a more extensive research project (Antibiotic Biosynthetic Enzymes; www.sci.utu.fi/projects/biokemia/abe), which focuses on the post-PKS tailoring enzymes involved in various type II aromatic polyketide biosynthetic pathways in Streptomyces bacteria. The initiative here was to investigate specific catalytic steps in anthracycline and angucycline biosynthesis through in vitro biochemical enzyme characterization and structural enzymology. The objectives were to elucidate detailed mechanisms and enzyme-level interactions which cannot be resolved by in vivo genetic studies alone. The first part of the experimental work concerns the homologous polyketide cyclases SnoaL and AknH. These catalyze the closure of the last carbon ring of the tetracyclic carbon frame common to all anthracycline-type compounds. The second part of the study primarily deals with tailoring enzymes PgaE (and its homolog CabE) and PgaM, which are responsible for a cascade of sequential modification reactions in angucycline biosynthesis. The results complemented earlier in vivo findings and confirmed the enzyme functions in vitro. Importantly, we were able to identify the amino acid -level determinants that influence AknH and SnoaL stereoselectivity and to determine the complex biosynthetic steps of the angucycline oxygenation cascade of PgaE and PgaM. In addition, the findings revealed interesting cases of enzyme-level adaptation, as some of the catalytic mechanisms did not coincide with those described for characterised homologs or enzymes of known function. Specifically, SnoaL and AknH were shown to employ a novel acid-base mechanism for aldol condenzation, whereas the hydroxylation reaction catalysed by PgaM involved unexpected oxygen chemistry. Owing to a gene-level fusion of two ancestral reading frames, PgaM was also shown to adopt an unusual quaternary sturucture, a non-covalent fusion complex of two alternative forms of the protein. Furthermore, the work highlighted some common themes encountered in polyketide biosynthetic pathways such as enzyme substrate specificity and intermediate reactivity. These are discussed in the final chapters of the work.Siirretty Doriast

Bacterial hemoglobins and flavohemoglobins: versatile proteins and their impact on microbiology and biotechnology

In response to oxygen limitation or oxidative and nitrosative stress, bacteria express three kinds of hemoglobin proteins: truncated hemoglobins (tr Hbs), hemoglobins (Hbs) and flavohemoglobins (flavo Hbs). The two latter groups share a high sequence homology and structural similarity in their globin domain. Flavohemoglobin proteins contain an additional reductase domain at their C-terminus and their expression is induced in the presence of reactive nitrogen and oxygen species. Flavohemoglobins detoxify NO in an aerobic process, termed nitric oxide dioxygenase reaction, which protects the host from various noxious nitrogen compounds. Only a small number of bacteria express hemoglobin proteins and the best studied of these is from Vitreoscilla sp. Vitreoscilla hemoglobin (VHb) has been expressed in various heterologous hosts under oxygen-limited conditions and has been shown to improve growth and productivity, rendering the protein interesting for biotechnology industry. The close interaction of VHb with the terminal oxidases has been shown and this interplay has been proposed to enhance respiratory activity and energy production by delivering oxygen, the ultimate result being an improvement in growth propertie

Impact of the small RNA RyhB on growth, physiology and heterologous protein expression in Escherichia coli

The small noncoding RNA RyhB is a regulator of iron homeostasis in Escherichia coli. During iron limitation, it downregulates the expression of a number of iron-containing proteins, including enzymes of the tricarboxylic acid cycle and the respiratory chain. Because this infers a potential for RyhB to limit energy metabolism and biosynthetic capacity, the effect of knocking out ryhB on the physiology and heterologous protein productivity of E. coli has been analyzed. During iron limitation, induced either through insufficient extracellular supply or through overexpression of an iron-containing protein, ryhB mutants showed unaltered growth and substrate consumption. They did, however, exhibit significantly lowered acetate production rates. Plasmid-based expression of green fluorescent protein and the heterologous Vitreoscilla hemoglobin VHb was negatively affected by the ryhB knock-ou

Vitreoscilla hemoglobin promoter is not responsive to nitrosative and oxidative stress in Escherichia coli

The Vitreoscilla hemoglobin gene (vhb) is expressed under oxygen-limited conditions via an FNR-dependent mechanism. Furthermore, cAMP-CRP has been implicated in its regulation. Recently, VHb protein has been reported to protect a heterologous host from nitrosative stress. In this study we analyzed the regulation of the Vitreoscilla hemoglobin promoter (Pvhb) in Escherichia coli under nitrosative and oxidative stress conditions. Our results show unambiguously that expression of neither VHb nor chloramphenicol acetyltransferase under the control of Pvhb is induced under the experimental conditions used. Thus, a clear discrepancy between in vivo function, i.e. protection against nitrosative stress, and regulation of gene expression is obvious. The regulation of Pvhb reported here is in clear contrast to the expression pattern of flavohemoglobins from various microorganisms, which are generally induced by nitrosative stress. However, the length of Pvhb is only 146 bp and therefore, we cannot rule out that additional regulatory sequences may be located in the upstream region of Pvh

Endogenous PttHb1 and PttTrHb, and heterologous Vitreoscilla vhb haemoglobin gene expression in hybrid aspen roots with ectomycorrhizal interaction

Present knowledge on plant non-symbiotic class-1 (Hb1) and truncated (TrHb) haemoglobin genes is almost entirely based on herbaceous species while the corresponding tree haemoglobin genes are not well known. The function of these genes has recently been linked with endosymbioses between plants and microbes. In this work, the coding sequences of hybrid aspen (Populus tremula×tremuloides) PttHb1 and PttTrHb were characterized, indicating that the key residues of haem and ligand binding of both genes were conserved in the deduced amino acid sequences. The expression of PttHb1 and PttTrHb was examined in parallel with that of the heterologous Vitreoscilla haemoglobin gene (vhb) during ectomycorrhiza/ectomycorrhizal (ECM) interaction. Both ECM fungi studied, Leccinum populinum and Xerocomus subtomentosus, enhanced root formation and subsequent growth of roots of all hybrid aspen lines, but only L. populinum was able to form mycorrhizas. Real-time PCR results show that the dual culture with the ECM fungus, with or without emergence of symbiotic structures, increased the expression of both PttHb1 and PttTrHb in the roots of non-transgenic hybrid aspens. PttHb1 and PttTrHb had expression peaks 5 h and 2 d after inoculation, respectively, pointing to different functions for these genes during interaction with root growth-improving fungi. In contrast, ECM fungi were not able to enhance the expression of hybrid aspen endogenous haemoglobin genes in the VHb lines, which may be a consequence of the compensating action of heterologous haemoglobi

Intrinsic non-symbiotic and truncated haemoglobins and heterologous Vitreoscilla haemoglobin expression in plants

To date, haemoglobins (Hbs) have been shown to exist in all kingdoms of life. The least studied and understood groups are plant non-symbiotic haemoglobins (nsHbs) and the recently found plant truncated Hbs (trHbs). From a biotechnological point of view, the best characterized and almost exclusively applied Hb is the bacterial Vitreoscilla haemoglobin (VHb). In this review, the present state of knowledge of structural features and ligand binding kinetics of plant nsHbs and trHbs and their proposed roles as oxygen carriers, oxygen sensors, and for oxygen storage, in nitric oxide (NO) detoxification, and in peroxidase activity are described. Furthermore, in order to predict the functioning of plant Hbs, their characteristics will be compared with those of the better known bacterial globins. In this context, the effects of heterologous applications of VHb on plants are reviewed. Finally, the challenging future of plant Hb research is discusse

Aikuisopiskelun henkilökohtaistaminen ja ohjaaminen

Personalizing and guiding adult learningOppilaitoksilla on velvoite laatia tutkintoon valmistavassa aikuiskoulutuksessa henkilökohtaiset opintosuunnitelmat (hops). Velvoite on ollut voimassa vuodesta 1992 lähtien. Vuosi sitten hopsista annettiin yhtenevät periaatteet. Niihin sisällytettiin aiemman työ- ja koulutuskokemuksella hankitun lähtötason arviointi, opiskeluohjelman laatiminen yhdessä opiskelijan kanssa, mahdollisten oppimisvaikeuksien ja niiden edellyttämän tuen huomioonottaminen sekä opiskeluohjelman tarkentaminen opiskelun kuluessa. Hopsin on näin ollen määrä sopeuttaa aikuisopinnot aikuisuuteen

Expressing creatine kinase in transgenic tobacco - a first step towards introducing an energy buffering system in plants

Creatine kinase a key enzyme in cellular energy homeostasis of vertebrates offers the promise of engineering plants with enhanced stress tolerance. In order to provide plants with such an energy buffering system, tobacco was transformed with a cDNA, encoding the cytosolic brain-type isoform of chicken creatine kinase (BB-CK), the expression of which was under the control of the cauliflower mosaic virus 35S (CaMV 35S) promoter. Transgenic tobacco plants were selected and suspension cultures generated. Both transgenic plants and suspension cultures were shown to stably express enzymatically active BB-CK in vitro and in vivo, and in most cases for three successive generations (T0-T2). Exogenously supplied creatine was shown to enter the plant cells and resulted in only a slight reduction in root growth at concentrations up to 10 mM. Furthermore, the BB-CK expressing tobacco plants and cell suspension cultures were able to convert creatine into phosphocreatin

Transcriptional activity of Pseudomonas aeruginosa fhp promoter is dependent on two regulators in addition to FhpR

The regulation of flavohemoglobin expression is complex and depending on its host organism requires a wide variety of different transcriptional regulators. In Pseudomonas aeruginosa, the flavohemoglobin (Fhp) and its cognate regulator FhpR form an NO-sensing and detoxifying system regulated by their common bidirectional promoter P fhp/P fhpR. The intergenic fhp-fhpR region of P. aeruginosa PAO1 was used as a bait to isolate proteins affecting the transcription of fhp and fhpR. In addition to the FhpR, we identified two previously uncharacterized P. aeruginosa proteins, PA0779 and PA3697. Both PA0779 and PA3697 were found to be essential for NO3 − and NO2 − induced P fhp activity under aerobic and low-oxygen conditions, and needed for the full function of P fhp/P fhpR as NO responsive regulatory circuit under aerobic conditions. In addition, we show that the transcriptional activity of P fhpR is highly inducible upon addition of SNP under aerobic conditions, but not by NO3 −, NO2 − or under low-oxygen conditions, supporting the findings that FhpR is not the only factor affecting flavohemoglobin expression in P. aeruginos

SRM dataset of the proteome of inactivated iron-sulfur cluster biogenesis regulator SufR in Synechocystis sp. PCC 6803

This article contains SRM proteomics data related to the research article entitled ”Inactivation of iron-sulfur cluster biogenesis regulator SufR in Synechocystis sp. PCC 6803 induces unique iron-dependent protein-level responses”[1]. The data described here provide comprehensive information on the applied SRM assays, together with the results of quantifying 94 Synechocystis sp. PCC 6803 proteins.  The data has been deposited in Panorama public  (https://panoramaweb.org/labkey/SufR) and from PASSEL under the PASS00765 identifier (http://www.peptideatlas.org/PASS/PASS00765).  </p