This is an RSS newsfeed from BioMed Central It is intended to be used with an RSS reader. For more information about RSS newsfeeds from BioMed Central, visit http://www.biomedcentral.com/info/about/rss/ http://www.microbialcellfactories.com The latest articles from Microbial Cell Factories (ISSN 1475-2859) published by BioMed Central Background: The thermotolerant methylotrophic yeast Hansenula polymorpha is capable of alcoholic fermentation of xylose at elevated temperatures (45 - 48 C). Such property of this yeast defines it as a good candidate for the development of an efficient process for simultaneous saccharification and fermentation. However, to be economically viable, the main characteristics of xylose fermentation of H. polymorpha have to be improved. Results: Site-specific mutagenesis of H. polymorpha XYL1 gene encoding xylose reductase was carried out to decrease affinity of this enzyme toward NADPH. The modified version of XYL1 gene under control of the strong constitutive HpGAP promoter was overexpressed on a Delta xyl1 background. This resulted in significant increase in the KM for NADPH in the mutated xylose reductase (K341->R N343->D), while KM for NADH remained nearly unchanged. The recombinant H. polymorpha strain overexpressing the mutated enzyme together with native xylitol dehydrogenase and xylulokinase on Delta xyl1 background was constructed. Xylose consumption, ethanol and xylitol production by the constructed strain were determined for high-temperature xylose fermentation at 48 C. A significant increase in ethanol productivity (up to 7.3 times) was shown in this recombinant strain as compared with the wild type strain. Moreover, the xylitol production by the recombinant strain was reduced considerably to 0.9 mgx(Lxh)-1 as compared to 4.2 mgx(Lxh)-1 for the wild type strain. Conclusions: Recombinant strains of H. polymorpha engineered for improved xylose utilization are described in the present work. These strains show a significant increase in ethanol productivity with simultaneous reduction in the production of xylitol during high-temperature xylose fermentation. http://www.microbialcellfactories.com/content/7/1/21 Olena V Dmytruk, Kostyantyn V Dmytruk, Charles A Abbas, Andriy Y Voronovsky and Andriy A Sibirny Microbial Cell Factories 2008, 7:21 2008-07-23 doi:10.1186/1475-2859-7-21 Microbial Cell Factories 1475-2859 7 21 2008-07-23 Structural characterization of proteins used in biological experiments is largely neglected. In most publications, the information available is totally insufficient to judge the functionality of the proteins used and, therefore, the significance of identified protein-protein interactions (was the interaction specific or due to unspecific binding of misfolded protein regions?) or reliability of kinetic and thermodynamic data (how much protein was in its native form?). As a consequence, the results of single experiments might not only become questionable, but the whole reliability of systems biology, built on these fundaments, would be weakened. The introduction of Minimal Information concerning purified proteins to add as metadata to the main body of a manuscript would render straightforward the assessment of their functional and structural qualities and, consequently, of results obtained using these proteins. Furthermore, accepted standards for protein annotation would simplify data comparison and exchange. This article has been envisaged as a proposal for aggregating scientists who share the opinion that the scientific community needs a platform for Minimum Information for Protein Functionality Evaluation (MIPFE). http://www.microbialcellfactories.com/content/7/1/20 Ario de Marco Microbial Cell Factories 2008, 7:20 2008-07-23 doi:10.1186/1475-2859-7-20 Microbial Cell Factories 1475-2859 7 20 2008-07-23 Background: Selection of an appropriate host organism is crucial for the economic success of biotechnological processes. A generally important selection criterion is a low maintenance energy metabolism to reduce non-productive consumption of substrate. We here investigated, whether various bacilli that are closely related to Bacillus subtilis are potential riboflavin production hosts with low maintenance metabolism. Results: While B. subtilis exhibited indeed the highest maintenance energy coefficient, B. licheniformis and B. amyloliquefaciens exhibited only statistically insignificantly reduced maintenance metabolism. Both B. pumilus and B. subtilis (natto) exhibited irregular growth patterns under glucose limitation such that the maintenance metabolism could not be determined. The sole exception with significantly reduced maintenance energy requirements was the B. licheniformis strain T380B. The frequently used spo0A mutation significantly increased the maintenance metabolism of B. subtilis.At the level of 13C-detected intracellular fluxes, all investigated bacilli exhibited a significant flux through the pentose phosphate pathway, a prerequisite for efficient riboflavin production. Different from all other species, B. subtilis featured high respiratory tricarboxylic acid cycle fluxes in batch and chemostat cultures. In particular under glucose-limited conditions, this led to significant excess formation of NADPH of B. subtilis, while anabolic consumption was rather balanced with catabolic NADPH formation in the other bacilli. Conclusion: Despite its successful commercial production of riboflavin, B. subtilis does not seem to be the optimal cell factory from a bioenergetic point of view. The best choice of the investigated strains is the sporulation-deficient B. licheniformis T380B strain. Beside a low maintenance energy coefficient, this strain grows robustly under different conditions and exhibits only moderate acetate overflow, hence making it a promising production host for biochemicals and riboflavin in particular. http://www.microbialcellfactories.com/content/7/1/19 Simon Tännler, Seraina Decasper and Uwe Sauer Microbial Cell Factories 2008, 7:19 2008-06-18 doi:10.1186/1475-2859-7-19 Microbial Cell Factories 1475-2859 7 19 2008-06-18 Background: Considerable interest in the bioconversion of lignocellulosic biomass into ethanol has led to metabolic engineering of Saccharomyces cerevisiae for fermentation of xylose. In the present study, the transcriptome and proteome of recombinant, xylose-utilising S. cerevisiae grown in aerobic batch cultures on xylose were compared with those of glucose-grown cells both in glucose repressed and derepressed states. The aim was to study at the genome-wide level how signalling and carbon catabolite repression differ in cells grown on either glucose or xylose. The more detailed knowledge whether xylose is sensed as a fermentable carbon source, capable of catabolite repression like glucose, or is rather recognised as a non-fermentable carbon source is important for further engineering this yeast for more efficient anaerobic fermentation of xylose. Results: Genes encoding respiratory proteins, proteins of the tricarboxylic acid and glyoxylate cycles, and gluconeogenesis were only partially repressed by xylose, similar to the genes encoding their transcriptional regulators HAP4, CAT8 and SIP1-2 and 4. Several genes that are repressed via the Snf1p/Mig1p-pathway during growth on glucose had higher expression in the cells grown on xylose than in the glucose repressed cells but lower than in the glucose derepressed cells. The observed expression profiles of the transcription repressor RGT1 and its target genes HXT2-3, encoding hexose transporters suggested that extracellular xylose was sensed by the glucose sensors Rgt2p and Snf3p. Proteome analyses revealed distinct patterns in phosphorylation of hexokinase 2, glucokinase and enolase isoenzymes in the xylose- and glucose-grown cells. Conclusion: The results indicate that the metabolism of yeast growing on xylose corresponds neither to that of fully glucose repressed cells nor that of derepressed cells. This may be one of the major reasons for the suboptimal fermentation of xylose by recombinant S. cerevisiae strains. Phosphorylation of different isoforms of glycolytic enzymes suggests that regulation of glycolysis also occurred at a post-translational level, supporting prior findings. http://www.microbialcellfactories.com/content/7/1/18 Laura Salusjärvi, Matti Kankainen, Rabah Soliymani, Juha-Pekka Pitkänen, Merja Penttilä and Laura Ruohonen Microbial Cell Factories 2008, 7:18 2008-06-04 doi:10.1186/1475-2859-7-18 Microbial Cell Factories 1475-2859 7 18 2008-06-04 Background: Tropane alkaloids, mainly hyoscyamine and scopolamine, are widely used in medicine due to their anticholinergic activity. Scopolamine has a higher demand being the more valuable alkaloid due to its fewer side effects and higher physiological activity. Anisodamine (6β-hydroxyhyoscyamine) is the intermediate in the conversion of hyoscyamine into scopolamine. Current studies report that this alkaloid is potentially applicable in medicine. The gene that codifies for Hyoscyamine 6-β hydroxylase, the enzyme responsible for hyoscyamine hydroxylation and epoxidation, leading to scopolamine was isolated from Brugmansia candida. Results: The h6hcDNA was cloned into pYES2.1 and pYES2.1/V5-His-TOPO vectors to produce an untagged and a tagged protein, respectively. The H6H enzyme was produced in Saccharomyces cerevisiae in order to obtain a biological catalyst for potential industrial applications. Protein extracts of the induced yeast were analyzed by Western blot. The expression was detected 4 h after induction and no degradation was observed during the period assayed. The tagged and the untagged proteins were able to transform hyoscyamine, showing a functional expression of the h6hcDNA. Conclusion: The strains obtained in this work are promising and potentially applicable in biocatalytic processes. http://www.microbialcellfactories.com/content/7/1/17 Alejandra B Cardillo, Julián Rodríguez Talou and Ana M Giulietti Microbial Cell Factories 2008, 7:17 2008-05-27 doi:10.1186/1475-2859-7-17 Microbial Cell Factories 1475-2859 7 17 2008-05-27 Background: Lipolysis is an important process of cheese ripening that contributes to the formation of flavour. Propionibacterium freudenreichii is the main agent of lipolysis in Emmental cheese; however, the enzymes involved produced by this species have not yet been identified. Lipolysis is performed by esterases (carboxylic ester hydrolases, EC 3.1.1.-) which are able to hydrolyse acylglycerols bearing short, medium and long chain fatty acids. The genome sequence of P. freudenreichii type strain CIP103027T was recently obtained in our laboratory.The aim of this study was to identify as exhaustively as possible the potential esterases in P. freudenreichii that could be involved in the hydrolysis of acylglycerols in Emmental cheese. The proteins identified were produced in a soluble and active form by heterologous expression in Escherichia coli for further study of their activity and specificity of hydrolysed substrates. Results: The approach chosen was a genomic search approach that combined and compared four methods based on automatic and manual searches of homology and motifs among P. freudenreichii CIP103027T predicted proteins. Twenty-three putative esterases were identified in this step. Then a selection step permitted to focus the study on the 12 most probable esterases, according to the presence of the GXSXG motif of the α/β hydrolase fold family. The 12 corresponding coding sequences were cloned in expression vectors, containing soluble N-terminal fusion proteins. The best conditions to express each protein in a soluble form were found thanks to an expression screening, using an incomplete factorial experimental design. Eleven out of the 12 proteins were expressed in a soluble form in E. coli and six showed esterase activity on 1-naphthyl acetate and/or propionate, as demonstrated by a zymographic method. Conclusion: We were able to demonstrate that our genomic search approach was efficient to identify esterases from the genome of a P. freudenreichii strain, more exhaustively than classical approaches. This study highlights the interest in using the automatic search of motifs, with the manual search of homology to previously characterised enzymes as a complementary method. Only further characterisations would permit the identification of the esterases of P. freudenreichii involved in the lipolysis in Emmental cheese. http://www.microbialcellfactories.com/content/7/1/16 Julien Dherbécourt, Hélène Falentin, Stéphane Canaan and Anne Thierry Microbial Cell Factories 2008, 7:16 2008-05-22 doi:10.1186/1475-2859-7-16 Microbial Cell Factories 1475-2859 7 16 2008-05-22 Background: High salinity (1–10% w/v) of tannery wastewater makes it difficult to be treated by conventional biological treatment. Salt tolerant microbes can adapt to these saline conditions and degrade the organics in saline wastewater. Results: Four salt tolerant bacterial strains isolated from marine and tannery saline wastewater samples were identified as Pseudomonas aeruginosa, Bacillus flexus, Exiguobacterium homiense and Staphylococcus aureus. Growth factors of the identified strains were optimized. Tannery saline wastewater obtained from a Common Effluent Treatment Plant (CETP) near Chennai (southern India) was treated with pure and mixed consortia of four salt tolerant bacterial strains. Experiments with optimized conditions and varying salt content (between 2 and 10% (w/v) were conducted. Salt inhibition effects on COD removal rate were noted. Comparative analysis was made by treating the tannery saline wastewater with activated sludge obtained from CETP and with natural habitat microbes present in raw tannery saline wastewater. Conclusion: Salt tolerant bacterial mixed consortia showed appreciable biodegradation at all saline concentrations (2%, 4%, 6%, 8% and 10% w/v) with 80% COD reduction in particular at 8% salinity level the consortia could be used as suitable working cultures for tannery saline wastewater treatment. http://www.microbialcellfactories.com/content/7/1/15 Senthilkumar Sivaprakasam, Surianarayanan Mahadevan, Sudharshan Sekar and Susheela Rajakumar Microbial Cell Factories 2008, 7:15 2008-04-29 doi:10.1186/1475-2859-7-15 Microbial Cell Factories 1475-2859 7 15 2008-04-29 Background: Introducing point mutations into bacterial chromosomes is important for further progress in studies relying on functional genomics, systems- and synthetic biology, and for metabolic engineering. For many investigations, chromosomal systems are required rather than artificial plasmid based systems. Results: Here we describe the introduction of a single point mutation into the Escherichia coli chromosome by site-directed mutagenesis without leaving any selection marker. We used Red®/ET® Recombination in combination with rpsL counter-selection to introduce a single point mutation into the E. coli MG1655 genome, one of the widely used bacterial model strains in systems biology. The method we present is rapid and highly efficient. Since single-stranded synthetic oligonucleotides can be used for recombination, any chromosomal modification can be designed. Conclusion: Chromosomal modifications performed by rpsL counter-selection may also be used for other bacteria that contain an rpsL homologue, since Red®/ET® Recombination has been applied to several enteric bacteria before. http://www.microbialcellfactories.com/content/7/1/14 Ralf Heermann, Tim Zeppenfeld and Kirsten Jung Microbial Cell Factories 2008, 7:14 2008-04-24 doi:10.1186/1475-2859-7-14 Microbial Cell Factories 1475-2859 7 14 2008-04-24 Background: Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells. Results: Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3) host cells in the absence of isopropyl-β-D-thiogalactopyranoside (IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture. Conclusion: The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community. http://www.microbialcellfactories.com/content/7/1/13 Ana LS Vanz, Gaby Renard, Mario S Palma, Jocelei M Chies, Sérgio L Dalmora, Luiz A Basso and Diógenes S Santos Microbial Cell Factories 2008, 7:13 2008-04-04 doi:10.1186/1475-2859-7-13 Microbial Cell Factories 1475-2859 7 13 2008-04-04 Recent advances in protein mass spectrometry (MS) have enabled determinations of hydrogen deuterium exchange (HDX) in large macromolecular complexes. HDX-MS became a valuable tool to follow protein folding, assembly and aggregation. The methodology has a wide range of applications in biotechnology ranging from quality control for over-expressed proteins and their complexes to screening of potential ligands and inhibitors. This review provides an introduction to protein folding and assembly followed by the principles of HDX and MS detection, and concludes with selected examples of applications that might be of interest to the biotechnology community. http://www.microbialcellfactories.com/content/7/1/12 Bohumila Suchanova and Roman Tuma Microbial Cell Factories 2008, 7:12 2008-04-04 doi:10.1186/1475-2859-7-12 Microbial Cell Factories 1475-2859 7 12 2008-04-04 Different species of microorganisms including yeasts, filamentous fungi and bacteria have been used in the past 25 years for the controlled production of foreign proteins of scientific, pharmacological or industrial interest. A major obstacle for protein production processes and a limit to overall success has been the abundance of misfolded polypeptides, which fail to reach their native conformation. The presence of misfolded or folding-reluctant protein species causes considerable stress in host cells. The characterization of such adverse conditions and the elicited cell responses have permitted to better understand the physiology and molecular biology of conformational stress. Therefore, microbial cell factories for recombinant protein production are depicted here as a source of knowledge that has considerably helped to picture the extremely rich landscape of in vivo protein folding, and the main cellular players of this complex process are described for the most important cell factories used for biotechnological purposes. http://www.microbialcellfactories.com/content/7/1/11 Brigitte Gasser, Markku Saloheimo, Ursula Rinas, Martin Dragosits, Escarlata Rodríguez-Carmona, Kristin Baumann, Maria Giuliani, Ermenegilda Parrilli, Paola Branduardi, Christine Lang, Danilo Porro, Pau Ferrer, Maria Luisa Tutino, Diethard Mattanovich and Antonio Villaverde Microbial Cell Factories 2008, 7:11 2008-04-04 doi:10.1186/1475-2859-7-11 Microbial Cell Factories 1475-2859 7 11 2008-04-04 Background: The Gram-positive bacterium Bacillus subtilis is an important producer of high quality industrial enzymes and a few eukaryotic proteins. Most of these proteins are secreted into the growth medium, but successful examples of cytoplasmic protein production are also known. Therefore, one may anticipate that the high protein production potential of B. subtilis can be exploited for protein complexes and membrane proteins to facilitate their functional and structural analysis. The high quality of proteins produced with B. subtilis results from the action of cellular quality control systems that efficiently remove misfolded or incompletely synthesized proteins. Paradoxically, cellular quality control systems also represent bottlenecks for the production of various heterologous proteins at significant concentrations. Conclusion: While inactivation of quality control systems has the potential to improve protein production yields, this could be achieved at the expense of product quality. Mechanisms underlying degradation of secretory proteins are nowadays well understood and often controllable. It will therefore be a major challenge for future research to identify and modulate quality control systems of B. subtilis that limit the production of high quality protein complexes and membrane proteins, and to enhance those systems that facilitate assembly of these proteins. http://www.microbialcellfactories.com/content/7/1/10 Jessica C Zweers, Imrich Barák, Dörte Becher, Arnold JM Driessen, Michael Hecker, Vesa P Kontinen, Manfred J Saller, L'udmila Vavrová and Jan Maarten van Dijl Microbial Cell Factories 2008, 7:10 2008-04-04 doi:10.1186/1475-2859-7-10 Microbial Cell Factories 1475-2859 7 10 2008-04-04 Background: Metabolic engineering of Saccharomyces cerevisiae for xylose fermentation into fuel ethanol has oftentimes relied on insertion of a heterologous pathway that consists of xylose reductase (XR) and xylitol dehydrogenase (XDH) and brings about isomerization of xylose into xylulose via xylitol. Incomplete recycling of redox cosubstrates in the catalytic steps of the NADPH-preferring XR and the NAD+-dependent XDH results in formation of xylitol by-product and hence in lowering of the overall yield of ethanol on xylose. Structure-guided site-directed mutagenesis was previously employed to change the coenzyme preference of Candida tenuis XR about 170-fold from NADPH in the wild-type to NADH in a Lys274→Arg Asn276→Asp double mutant which in spite of the structural modifications introduced had retained the original catalytic efficiency for reduction of xylose by NADH. This work was carried out to assess physiological consequences in xylose-fermenting S. cerevisiae resulting from a well defined alteration of XR cosubstrate specificity. Results: An isogenic pair of yeast strains was derived from S. cerevisiae Cen.PK 113-7D through chromosomal integration of a three-gene cassette that carried a single copy for C. tenuis XR in wild-type or double mutant form, XDH from Galactocandida mastotermitis, and the endogenous xylulose kinase (XK). Overexpression of each gene was under control of the constitutive TDH3 promoter. Measurement of intracellular levels of XR, XDH, and XK activities confirmed the expected phenotypes. The strain harboring the XR double mutant showed 42% enhanced ethanol yield (0.34 g/g) compared to the reference strain harboring wild-type XR during anaerobic bioreactor conversions of xylose (20 g/L). Likewise, the yields of xylitol (0.19 g/g) and glycerol (0.02 g/g) were decreased 52% and 57% respectively in the XR mutant strain. The xylose uptake rate per gram of cell dry weight was identical (0.07 ± 0.02 h-1) in both strains. Conclusion: Integration of enzyme and strain engineering to enhance utilization of NADH in the XR-catalyzed conversion of xylose results in notably improved fermentation capabilities of recombinant S. cerevisiae. http://www.microbialcellfactories.com/content/7/1/9 Barbara Petschacher and Bernd Nidetzky Microbial Cell Factories 2008, 7:9 2008-03-17 doi:10.1186/1475-2859-7-9 Microbial Cell Factories 1475-2859 7 9 2008-03-17