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 The progressive solving of the conformation of aggregated proteins and the conceptual understanding of the biology of inclusion bodies in recombinant bacteria is providing exciting insights on protein folding and quality. Interestingly, newest data also show an unexpected functional and structural complexity of soluble recombinant protein species and picture the whole bacterial cell factory scenario as more intricate than formerly believed. http://www.microbialcellfactories.com/content/8/1/4 Monica Martinez-Alonso, Nuria Gonzalez-Montalban, Elena Garcia-Fruitos and Antonio Villaverde Microbial Cell Factories 2009, 8:4 2009-01-08 doi:10.1186/1475-2859-8-4 Microbial Cell Factories 1475-2859 8 4 2009-01-08 Class IIa bacteriocins from lactic acid bacteria are small, cationic proteins with antilisterial activity. Within this class, the pediocins are those bacteriocins that share a highly conserved hydrophilic and charged N-terminal part harboring the consensus sequence -YGNGV- and a more variable hydrophobic and /or amphiphilic C-terminal part. Several pediocins have been isolated and characterized. Despite the structural similarities, their molecular weight varies, as well as their spectrum of antimicrobial activity. They exhibit important technological properties, e.g. thermostability and retaining of activity at a wide pH range, which along with the bactericidal action against Gram-positive food spoilage and pathogenic bacteria, make them an important class of biopreservatives. Much new information regarding the pediocins has emerged during the last years. In this review, we summarize and discuss all the available information regarding the sources of pediocins, the characteristics of their biosynthesis and production in fermentation systems, the characteristics of the known pediocin molecules, and their antibacterial action. The advances made by genetic engineering in improving the features of pediocins are also discussed, as well as their perspectives for future applications. http://www.microbialcellfactories.com/content/8/1/3 Maria Papagianni and Sofia Anastasiadou Microbial Cell Factories 2009, 8:3 2009-01-08 doi:10.1186/1475-2859-8-3 Microbial Cell Factories 1475-2859 8 3 2009-01-08 Background: Deletion of large blocks of nonessential genes that are not needed for metabolic pathways of interest can reduce the production of unwanted by-products, increase genome stability, and streamline metabolism without physiological compromise. Researchers have recently constructed a reduced-genome Escherichia coli strain MDS42 that lacks 14.3% of its chromosome. Results: Here we describe the reengineering of the MDS42 genome to increase the production of the essential amino acid L-threonine. To this end, we over-expressed a feedback-resistant threonine operon (thrA*BC), deleted the genes that encode threonine dehydrogenase (tdh) and threonine transporters (tdcC and sstT), and introduced a mutant threonine exporter (rhtA23) in MDS42. The resulting strain, MDS-205, shows an ~83% increase in L-threonine production when cells are grown by flask fermentation, compared to a wild-type E. coli strain MG1655 engineered with the same threonine-specific modifications described above. And transcriptional analysis revealed the effect of the deletion of non-essential genes on the central metabolism and threonine pathways in MDS-205. Conclusions: This result demonstrates that the elimination of genes unnecessary for cell growth can increase the productivity of an industrial strain, most likely by reducing the metabolic burden and improving the metabolic efficiency of cells. http://www.microbialcellfactories.com/content/8/1/2 Jun Hyoung Lee, Bong Hyun Sung, Mi Sun Kim, Frederick R Blattner, Byoung Hoon Yoon, Jung Hoe Kim and Sun Chang Kim Microbial Cell Factories 2009, 8:2 2009-01-07 doi:10.1186/1475-2859-8-2 Microbial Cell Factories 1475-2859 8 2 2009-01-07 Although many secondary metabolites with diverse biological activities have been isolated from myxobacteria, most strains of these biotechnologically important gliding prokaryotes remain difficult to handle genetically. In this study we describe the new fast growing myxobacterial thermophilic isolate GT-2 as a heterologous host for the expression of natural product biosynthetic pathways isolated from other myxobacteria. According to the results of sequence analysis of the 16S rDNA, this moderately thermophilic isolate is closely related to Corallococcus macrosporus and was therefore named C. macrosporus GT-2. Fast growth of moderately thermophilic strains results in shorter fermentation and generation times, aspects which are of significant interest for molecular biological work as well as production of secondary metabolites. Development of a genetic manipulation system allowed the introduction of the complete myxochromide biosynthetic gene cluster, located on a transposable fragment, into the chromosome of GT-2. Genetic engineering of the biosynthetic gene cluster by promoter exchange leads to much higher production of myxochromides in the heterologous host C. macrosporus GT-2 in comparison to the original producer Stigmatella aurantiaca and to the previously described heterologous host Pseudomonas putida (600 mg/L versus 8 mg/L and 40 mg/L, respectively). http://www.microbialcellfactories.com/content/8/1/1 Olena Perlova, Klaus Gerth, Silvia Kuhlmann, Youming Zhang and Rolf Muller Microbial Cell Factories 2009, 8:1 2009-01-06 doi:10.1186/1475-2859-8-1 Microbial Cell Factories 1475-2859 8 1 2009-01-06 Background: Pyrethroids and pyrethrins are widely used insecticides. Extensive applications not only result in pest resistance to these insecticides, but also may lead to environmental issues and human exposure. Numerous studies have shown that very high exposure to pyrethroids might cause potential problems to man and aquatic organisms. Therefore, it is important to develop a rapid and efficient disposal process to eliminate or minimize contamination of surface water, groundwater and agricultural products by pyrethroid insecticides. Bioremediation is considered to be a reliable and cost-effective technique for pesticides abatement and a major factor determining the fate of pyrethroid pesticides in the environment, and suitable esterase is expected to be useful for potential application for detoxification of pyrethroid residues. Soil is a complex environment considered as one of the main reservoirs of microbial diversity on the planet. However, most of the microorganisms in nature are inaccessible as they are uncultivable in the laboratory. Metagenomic approaches provide a powerful tool for accessing novel valuable genetic resources (novel enzymes) and developing various biotechnological applications. Results: The pyrethroid pesticides residues on foods and the environmental contamination are a public safety concern. Pretreatment with pyrethroid-hydrolyzing esterase has the potential to alleviate the conditions. To this end, a pyrethroid-hydrolyzing esterase gene was successfully cloned using metagenomic DNA combined with activity-based functional screening from soil, sequence analysis of the DNA responsible for the pye3 gene revealed an open reading frame of 819 bp encoding for a protein of 272 amino acid residues. Extensive multiple sequence alignments of the deduced amino acid of Pye3 with the most homologous carboxylesterases revealed moderate identity (45-49%).The recombinant Pye3 was heterologously expressed in E. coli BL21(DE3), purified and characterized. The molecular mass of the native enzyme was approximately 31 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of the Pye3 indicated molecular mass of 31 kDa and 31.5 kDa, respectively, suggesting that the Pye3 is a monomer. The purified Pye3 not only degraded all pyrethroid pesticides tested, but also hydrolyzed rho-nitrophenyl esters of medium-short chain fatty acids, indicating that the Pye3 is an esterase with broader specificity. The Km values for trans-Permethrin and cis-permethrin are 0.10 muM and 0.18 muM, respectively, and these catalytic properties were superior to carboxylesterases from resistant insects and mammals. The catalytic activity of the Pye3 was strongly inhibited by Hg2+, Ag+, rho-chloromercuribenzoate, whereas less pronounced effect was observed in the presence of divalent cations, the chelating agent EDTA and phenanthroline. Conclusion: A novel pyrethroid-hydrolyzing esterase gene was successfully cloned using metagenomic DNA combined with activity-based functional screening from soil, the broader substrate specificities and higher activity of the pyrethroid-hydrolyzing esterase (Pye3) make it an ideal candidate for in situ for detoxification of pyrethroids where they cause environmental contamination problems. Consequently, metagenomic DNA clone library offers possibilities to discover novel bio-molecules through the expression of genes from uncultivated bacteria. http://www.microbialcellfactories.com/content/7/1/38 Gang Li, Kui Wang and Yu H Liu Microbial Cell Factories 2008, 7:38 2008-12-30 doi:10.1186/1475-2859-7-38 Microbial Cell Factories 1475-2859 7 38 2008-12-30 Background: Whole cell-catalyzed biotransformation is a clear process option for the production of chiral alcohols via enantioselective reduction of precursor ketones. A wide variety of synthetically useful reductases are expressed heterologously in Escherichia coli to a high level of activity. Therefore, this microbe has become a prime system for carrying out whole-cell bioreductions at different scales. The limited capacity of central metabolic pathways in E. coli usually requires that reductase coenzyme in the form of NADPH or NADH be regenerated through a suitable oxidation reaction catalyzed by a second NADP+ or NAD+ dependent dehydrogenase that is co-expressed. Candida tenuis xylose reductase (CtXR) was previously shown to promote NADH dependent reduction of aromatic alpha-keto esters with high Prelog-type stereoselectivity. We describe here the development of a new whole-cell biocatalyst that is based on an E. coli strain co-expressing CtXR and formate dehydrogenase from Candida boidinii (CbFDH). The bacterial system was evaluated for the synthesis of ethyl R-4-cyanomandelate under different process conditions and benchmarked against a previously described catalyst derived from Saccharomyces cerevisiae expressing CtXR. Results: Gene co-expression from a pETDuet-1 vector yielded about 260 and 90 units of intracellular CtXR and CbFDH activity per gram of dry E. coli cell mass (gCDW). The maximum conversion rate (rS) for ethyl 4-cyanobenzoylformate by intact or polymyxin B sulphate-permeabilized cells was similar (2 mmol/gCDWh), suggesting that the activity of CbFDH was partly rate-limiting overall. Uncatalyzed ester hydrolysis in substrate as well as inactivation of CtXR and CbFDH in the presence of the alpha-keto ester constituted major restrictions to the yield of alcohol product. Using optimized reaction conditions (100 mM substrate; 40 gCDW/L), we obtained ethyl R-4-cyanomandelate with an enantiomeric excess (e.e.) of 97.2% in a yield of 82%. By increasing the substrate concentration to 500 mM, the e.e. could be enhanced to [almost equal to]100%, however, at the cost of a 3-fold decreased yield. A recombinant strain of S. cerevisiae converted 100 mM substrate to 45 mM ethyl R-4-cyanomandelate with an e.e. of [greater than or equal to] 99.9%. Modifications to the recombinant E. coli (cell permeabilisation; addition of exogenous NAD+) and addition of a water immiscible solvent (e.g. hexane or 1-butyl-3-methylimidazolium hexafluorophosphate) were not useful. To enhance the overall capacity for NADH regeneration in the system, we supplemented the original biocatalyst after permeabilisation with also permeabilised E. coli cells that expressed solely CbFDH (410 U/gCDW). The positive effect on yield (18% -> 62%; 100 mM substrate) caused by a change in the ratio of FDH to XR activity from 2 to 20 was invalidated by a corresponding loss in product enantiomeric purity from 86% to only 71%. Conclusions: A whole-cell system based on E. coli co-expressing CtXR and CbFDH is a powerful and surprisingly robust biocatalyst for the synthesis of ethyl R-4-cyanomandelate in high optical purity and yield. A clear requirement for further optimization of the specific productivity of the biocatalyst is to remove the kinetic bottleneck of NADH regeneration through enhancement ([greater than or equal to] 10-fold) of the intracellular level of FDH activity. http://www.microbialcellfactories.com/content/7/1/37 Regina Kratzer, Matej Pukl, Sigrid Egger and Bernd Nidetzky Microbial Cell Factories 2008, 7:37 2008-12-10 doi:10.1186/1475-2859-7-37 Microbial Cell Factories 1475-2859 7 37 2008-12-10 Background: Increasing energy costs and environmental concerns have motivated engineering microbes for the production of "second generation" biofuels that have better properties than ethanol. Results and conclusionsSaccharomyces cerevisiae was engineered with an n-butanol biosynthetic pathway, in which isozymes from a number of different organisms (S. cerevisiae, Escherichia coli, Clostridium beijerinckii, and Ralstonia eutropha) were substituted for the Clostridial enzymes and their effect on n-butanol production was compared. By choosing the appropriate isozymes, we were able to improve production of n-butanol ten-fold to 2.5 mg/L. The most productive strains harbored the C. beijerinckii 3-hydroxybutyryl-CoA dehydrogenase, which uses NADH as a co-factor, rather than the R. eutropha isozyme, which uses NADPH, and the acetoacetyl-CoA transferase from S. cerevisiae or E. coli rather than that from R. eutropha. Surprisingly, expression of the genes encoding the butyryl-CoA dehydrogenase from C. beijerinckii (bcd and etfAB) did not improve butanol production significantly as previously reported in E. coli. Using metabolite analysis, we were able to determine which steps in the n-butanol biosynthetic pathway were the most problematic and ripe for future improvement. http://www.microbialcellfactories.com/content/7/1/36 Eric J. Steen, Rossana Chan, Nilu Prasad, Samuel Myers, Christopher J. Petzold, Alyssa Redding, Mario Ouellet and Jay D. Keasling Microbial Cell Factories 2008, 7:36 2008-12-03 doi:10.1186/1475-2859-7-36 Microbial Cell Factories 1475-2859 7 36 2008-12-03 Background: Escherichia coli induces heat shock genes to the temperature up-shift, and changes the metabolism by complicated mechanism. The heat shock response is of practical importance for the variety of applications such as temperature-induced heterologous protein production, simultaneous saccharification and fermentation (SSF) etc. However, the effect of heat shock on the metabolic regulation is not well investigated. It is strongly desired to understand the metabolic changes and its mechanism upon heat shock in practice for the efficient metabolite production by temperature up-shift. In the present research, therefore, we investigated the effect of temperature up-shift from 37degreesC to 42degreesC on the metabolism in view of gene expressions. Results: The results of aerobic batch and continuous cultivations of E. coli BW25113 indicate that more acetate was accumulated with lower biomass yield and less glucose consumption rate at 42degreesC as compared to the case at 37degreesC. The down- regulation of the glucose uptake rate corresponds to the down-regulation of ptsG gene expression caused by the up-regulation of mlc gene expression. In accordance with up-regulation of arcA, which may be caused by the lower oxygen solubility at 42degreesC, the expressions of the TCA cycle-related genes and the respiratory chain gene cyoA were down-regulated. The decreased activity of TCA cycle caused more acetate formation at higher temperature, which is not preferred in heterologous protein production etc. This can be overcome by the arcA gene knockout to some extent. The time courses of gene expressions revealed that the heat shock genes such as groEL, dnaK, htpG and ibpB as well as mlc were expressed in much the same way as that of rpoH during the first 10-20 minutes after temperature up-shift. Under microaerobic condition, the fermentation changed in such a way that formate and lactate were more produced due to up-regulation of pflA and ldhA genes while ethanol was less produced due to down-regulation of adhE gene at higher temperature as compared to the case at 37degreesC. Conclusion: The present result clarified the mechanism of metabolic changes upon heat shock from 37degreesC to 42degreesC based on gene expressions of heat shock genes, global regulators, and the metabolic pathway genes. It is recommended to use arcA gene knockout mutant to prevent higher acetate production upon heat shock, where it must be noted that the cell yield may be decreased due to TCA cycle activation by arcA gene knockout. http://www.microbialcellfactories.com/content/7/1/35 Chowdhury MORINUL Hasan and Kazuyuki Shimizu Microbial Cell Factories 2008, 7:35 2008-12-02 doi:10.1186/1475-2859-7-35 Microbial Cell Factories 1475-2859 7 35 2008-12-02 1 Abstract Background: For a long time IBs were considered to be inactive deposits of accumulated target proteins. In our previous studies, we discovered IBs containing a high percentage of correctly folded protein that can be extracted under non-denaturing conditions in biologically active form without applying any renaturation steps. In order to widen the concept of correctly folded protein inside IBs, G-CSF (granulocyte colony stimulating factor) and three additional proteins were chosen for this study: GFP (Green fluorescent protein), His7dN6TNF-alpha (Truncated form of Tumor necrosis factor alpha with an N-terminal histidine tag) and dN19 LT-alpha (Truncated form of Lymphotoxin alpha). Results: Four structurally different proteins that accumulate in the bacterial cell in the form of IBs were studied, revealing that distribution of each target protein between the soluble fraction (cytoplasm) and insoluble fraction (IBs) depends on the nature of the target protein. Irrespective of the folding pattern of each protein, spectroscopy studies have shown that proteins in IBs exhibit similar structural characteristics to the biologically active pure protein when produced at low temperature. In the case of the three studied proteins, G-CSF, His7dN6TNF-alpha, and GFP, a significant amount of protein could be extracted from IBs with 0.2 % N-lauroyl sarcosine (NLS) and the proteins retained biological activity although no renaturation procedure was applied. Conclusion: This study shows that the presence of biologically active proteins inside IBs is more general than usually believed. A large amount of properly folded protein is trapped inside IBs prepared at lower temperatures. This protein can be released from IBs with mild detergents under non-denaturing conditions. Therefore, the active protein can be obtained from such IBs without any renaturation procedure. This is of great importance for the biopharmaceutical industry. Furthermore, such IBs composed of active proteins could also be used as pure nanoparticles in diagnostics, as biocatalysts in enzymatic processes, or even as biopharmaceuticals. http://www.microbialcellfactories.com/content/7/1/34 Spela Peternel, Joze Grdadolnik, Vladka Gaberc-Porekar and Radovan Komel Microbial Cell Factories 2008, 7:34 2008-12-01 doi:10.1186/1475-2859-7-34 Microbial Cell Factories 1475-2859 7 34 2008-12-01 Microbial Cell Factories was launched in 2002 under an Open Access policy, to cover a gap in the current offer of the scientific literature in Biotechnology and Applied Microbiology areas. The microbial cell factory concept, although present as a side topic within the scope of many journals in the field, deserves a specific attention as a particular, well defined issue in the microbial production and transformation of biotechnologically relevant substances. Intriguingly, the Cell Factory concept stresses the relevance of host cell genetics and metabolism in the context of the production process, and focus on the physiological aspects of the productive event. Since 2002, the journal has published more than 170 relevant manuscripts in form of Research articles, Technical notes, Reviews and Commentaries, highlighting the role of the hosting cell from both biological and technological sides. http://www.microbialcellfactories.com/content/7/1/33 Maurilio De Felice, Diethard Mattanovich, Maria Papagianni, Grzegorz Wegrzyn and Antonio Villaverde Microbial Cell Factories 2008, 7:33 2008-12-01 doi:10.1186/1475-2859-7-33 Microbial Cell Factories 1475-2859 7 33 2008-12-01 Laccases are multi-copper containing oxidases (EC 1.10.3.2), widely distributed in fungi, higher plants and bacteria. Laccase catalyses the oxidation of phenols, polyphenols and anilines by one-electron abstraction, with the concomitant reduction of oxygen to water in a four-electron transfer process. In the presence of small redox mediators, laccase offers a broader repertory of oxidations including non-phenolic substrates. Hence, fungal laccases are considered as ideal green catalysts of great biotechnological impact due to their few requirements (they only require air, and they produce water as the only by-product) and their broad substrate specificity, including direct bioelectrocatalysis.Thus, laccases and/or laccase-mediator systems find potential applications in bioremediation, paper pulp bleaching, finishing of textiles, bio-fuel cells and more. Significantly, laccases can be used in organic synthesis, as they can perform exquisite transformations ranging from the oxidation of functional groups to the heteromolecular coupling for production of new antibiotics derivatives, or the catalysis of key steps in the synthesis of complex natural products. In this review, the application of fungal laccases and their engineering by rational design and directed evolution for organic synthesis purposes are discussed. http://www.microbialcellfactories.com/content/7/1/32 Adinarayana Kunamneni, Susana Camarero, Carlos García-Burgos, Francisco J Plou, Antonio Ballesteros and Miguel Alcalde Microbial Cell Factories 2008, 7:32 2008-11-20 doi:10.1186/1475-2859-7-32 Microbial Cell Factories 1475-2859 7 32 2008-11-20