Microbial Cell Factories via MedWorm.com

Evolving thermostability in mutant libraries of ligninolytic oxidoreductases expressed in yeast

Microbial Cell Factories — Thu, 18 Mar 2010 00:00:00 +0100

Conclusions: The existing tradeoff between activity and stability determined from many point mutations discovered by directed evolution and other protein engineering means can be circumvented combining different tools of in vitro evolution. (Source: Microbial Cell Factories)

Fermentation of mixed glucose-xylose substrates by engineered strains of Saccharomyces cerevisiae: role of the coenzyme specificity of xylose reductase, and effect of glucose on xylose utilization

Microbial Cell Factories — Wed, 10 Mar 2010 00:00:00 +0100

Conclusions: Relative improvements in the distribution of fermentation products from xylose that can be directly related to a change in the coenzyme preference of xylose reductase from NADPH in BP000 to NADH in BP10001 increase in response to an increase in the initial concentration of the pentose substrate from 10 to 50 g/L. An inverse relationship between xylose uptake rate and xylitol yield for BP10001 implies that xylitol by-product formation is controlled not only by coenzyme regeneration during two-step oxidoreductive conversion of xylose into xylulose. Although xylose is not detectably utilized at glucose concentrations greater than 4 g/L, the presence of a low residual glucose concentration (< 2 g/L) promotes the uptake of xylose and its conversion into ethanol with only moderate x...

Effect of HXT1 and HXT7 hexose transporter overexpression on wild-type and lactic acid producing Saccharomyces cerevisiae cells

Microbial Cell Factories — Tue, 09 Mar 2010 00:00:00 +0100

Conclusion: Metabolic pathway manipulation for improving the properties and the productivity of microorganisms is a well established concept. A high production relies on a multi-factorial system. We showed that by modulating the first step of the pathway leading to lactic acid accumulation an improvement of about 15% in lactic acid production can be obtained in a yeast strain already developed for industrial application. (Source: Microbial Cell Factories)

Evaluation of different expression systems for the heterologous expression of pyranose 2-oxidase from Trametes multicolor in E.coli

Microbial Cell Factories — Tue, 09 Mar 2010 00:00:00 +0100

The heterologous production of the industrially relevant fungal enzyme pyranose 2-oxidase in the prokaryotic host E. coli was investigated using 3 different expression systems, i.e. the well-studied T7 RNA polymerase based pET21d+, the L-arabinose inducible pBAD and the pCOLD system. Preliminary experiments were done in shaking flasks at 25degreesC and optimized induction conditions to compare the productivity levels of the different expression systems. The pET21d+ and the pCOLD system gave 29 U/L * h and 14 U/L * h of active pyranose 2-oxidase, respectively, whereas the pBAD system only produced 6 U/L * h. Process conditions for batch fermentations were optimized for the pET21d+ and the pCOLD systems in order to reduce the formation of inactive inclusion bodies. The highest productivity r...

A novel fed-batch based cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures

Microbial Cell Factories — Fri, 19 Feb 2010 00:00:00 +0100

Conclusions: We have demonstrated that by applying the novel EnBase(R) Flo cultivation system in shaken cultures high cell densities can be obtained without impairing the productivity per cell. Especially the yield of soluble (correctly folded) proteins was significantly improved in comparison to commonly used LB, Terrific Broth or mineral salt media. This improvement is thought to result from a well controlled physiological state during the whole process. The higher volumetric yields enable the use of lower culture volumes and can thus significantly reduce the amount of time and effort needed for downstream processing or process optimization. We claim that the new cultivation system is widely applicable and, as it is very simple to apply, could widely replace standard shake flask approach...

Bacterial diversity and reductive dehalogenase redundancy in a 1,2-dichloroethane-degrading bacterial consortium enriched from a contaminated aquifer

Microbial Cell Factories — Fri, 19 Feb 2010 00:00:00 +0100

Conclusions: The overall data indicate that the enriched bacterial consortium shares the metabolic functionality between different members of the microbial community and is characterized by a high functional redundancy. These are fundamental features for the maintenance of the community's functionality, especially under stress conditions and suggest the feasibility of a bioremediation treatment with a potential prompt dehalogenation and a process stability over time. (Source: Microbial Cell Factories)

Characterization of two diesel fuel degrading microbial consortia enriched from a non acclimated, complex source of microorganisms

Microbial Cell Factories — Tue, 16 Feb 2010 00:00:00 +0100

Conclusions: ENZ-G1 and ENZ-G2 are very similar highly enriched consortia of bacteria and a fungus capable of extensively degrading a broad range of the hydrocarbons mainly composing diesel fuels. Given their remarkable biodegradation potential, stability and resistance to cryopreservation, both consortia appear very interesting candidates for bioaugmentation operations on Diesel fuel impacted soils and sites. (Source: Microbial Cell Factories)

Reduction of oxidative cellular damage by overexpression of the thioredoxin TRX2 gene improves yield and quality of wine yeast dry active biomass

Microbial Cell Factories — Fri, 12 Feb 2010 00:00:00 +0100

Conclusions: We report several beneficial effects of overexpressing the thioredoxin gene TRX2 in a wine yeast strain. We show that this strain presents an enhanced redox defense. Increased yield of biomass production process in TRX2 overexpressing strain can be of special interest for several industrial applications. (Source: Microbial Cell Factories)

Metabolic regulation of Escherichia coli and its gdhA, glnL, gltB, D mutants under different carbon and nitrogen limitations in the continuous culture

Microbial Cell Factories — Wed, 27 Jan 2010 00:00:00 +0100

Conclusion: The metabolic regulation of E.coli was clarified under both carbon (C)- limitation and nitrogen (N)- limitation based on fermentation, transcriptional mRNA level and enzyme activities. The overall regulation mechanism was proposed. The effects of knocking out N- assimilation pathway genes were also clarified. (Source: Microbial Cell Factories)

Cloning, reassembling and integration of the entire nikkomycin biosynthetic gene cluster into Streptomyces ansochromogeneslead to an improved nikkomycin production

Microbial Cell Factories — Sat, 23 Jan 2010 00:00:00 +0100

Conclusion: A high nikkomycins producing strain (1100 mg L-1 nikkomycins) was obtained by introduction of an extra nikkomycin biosynthetic gene cluster into the genome of S. ansochromogenes. The strategies presented here could be applicable to other bacteria to improve the yields of secondary metabolites. (Source: Microbial Cell Factories)

Isolation, characterization and heterologous expression of a novel chitosanase from Janthinobacterium sp. strain 4239

Microbial Cell Factories — Fri, 22 Jan 2010 00:00:00 +0100

Conclusions: A novel chitosanase enzyme and its corresponding gene was isolated from Janthinobacterium and produced recombinantly in E. coli as a periplasmic enzyme. The Janthinobacterium chitosanase displayed reasonable activity at 10 degrees C to 30 degrees C, temperatures that are preferred in transfection and transformation experiments. (Source: Microbial Cell Factories)

Heterologous expression of glucose oxidase in the yeast Kluyveromyces marxianus

Microbial Cell Factories — Thu, 21 Jan 2010 00:00:00 +0100

Conclusions: Taken together, our results indicate that K. marxianus is indeed a good host for the expression of heterologous proteins, not only for its physiological properties, but also because it correctly secretes and folds these proteins. (Source: Microbial Cell Factories)

The path to next generation biofuels: successes and challenges in the era of synthetic biology

Microbial Cell Factories — Wed, 20 Jan 2010 00:00:00 +0100

Volatility of oil prices along with major concerns about climate change, oil supply security and depleting reserves have sparked renewed interest in the production of fuels from renewable resources. Recent advances in synthetic biology provide new tools for metabolic engineers to direct their strategies and construct optimal biocatalysts for the sustainable production of biofuels. Metabolic engineering and synthetic biology efforts entailing the engineering of native and de-novo pathways for conversion of biomass constituents to short-chain alcohols and advanced biofuels are herewith reviewed. In the foreseeable future, formal integration of functional genomics and systems biology with synthetic biology and metabolic engineering will undoubtedly support the discovery, characterization, and...

Metabolic engineering of Agrobacterium sp. strain ATCC31749 for production of an alpha-Gal epitope

Microbial Cell Factories — Tue, 12 Jan 2010 00:00:00 +0100

Conclusions: The Agrobacterium biocatalyst developed in this work synthesizes gram-scale quantities of alpha-Gal epitope and does not require expensive cofactors or permeablization, making it an ideal biocatalyst for industrial production of the alpha-Gal epitope. Furthermore, the engineered Agrobacterium, with increased lactose uptake and improved UDP-glucose availability, is a promising host for the production of other medically-relevant oligosaccharides. (Source: Microbial Cell Factories)

Tuning microbial hosts for membrane protein production

Microbial Cell Factories — Tue, 29 Dec 2009 00:00:00 +0100

The last four years have brought exciting progress in membrane protein research. Finally those many efforts that have been put into expression of eukaryotic membrane proteins are coming to fruition and enable to solve an ever-growing number of high resolution structures. In the past, many skilful optimization steps were required to achieve sufficient expression of functional membrane proteins. Optimization was performed individually for every membrane protein, but provided insight about commonly encountered bottlenecks and, more importantly, general guidelines how to alleviate cellular limitations during microbial membrane protein expression. Lately, systems-wide analyses are emerging as powerful means to decipher cellular bottlenecks during heterologous protein production and their use in...

Scale-up from microtiter plate to laboratory fermenter: evaluation by online monitoring techniques of growth and protein expression in Escherichia coli and Hansenula polymorpha fermentations

Microbial Cell Factories — Tue, 22 Dec 2009 00:00:00 +0100

Conclusions: The successful 7000-fold scale-up from a shaken microtiter plate to a stirred tank fermenter was demonstrated in parallel fermentations for standard microbial expression systems. This confirms that the very economical and time efficient platform of microtiter plates can be very easily scaled up to larger stirred tank fermenters under defined engineering conditions. New online monitoring techniques for microtiter plates, such as the BioLector, provide even more real-time kinetic data from fermentations than ever before and at an affordable price. This paves the way for a better understanding of the bioprocess and a more rational process design. (Source: Microbial Cell Factories)

Gene cloning and characterization of a novel esterase from activated sludge metagenome

Microbial Cell Factories — Tue, 22 Dec 2009 00:00:00 +0100

A metagenomic library was prepared using pCC2FOS vector containing about 3.0 Gbp of community DNA from the microbial assemblage of activated sludge. Screening of a part of the un-amplified library resulted in the finding of 1 unique lipolytic clone capable of hydrolyzing tributyrin, in which an esterase gene was identified. This esterase/lipase gene consists of 834 bp and encodes a polypeptide (designated EstAS) of 277 amino acid residuals with a molecular mass of 31 kDa. Sequence analysis indicated that it showed 33% and 31% amino acid identity to esterase/lipase from Gemmata obscuriglobus UQM 2246 (ZP_02733109) and Yarrowia lipolytica CLIB122 (XP_504639), respectively; and several conserved regions were identified, including the putative active site, HSMGG, a catalytic triad (Ser92, His1...

An evolved xylose transporter from Zymomonas mobilis enhances sugar transport in Escherichia coli

Microbial Cell Factories — Tue, 15 Dec 2009 00:00:00 +0100

Conclusions: Through random mutagenesis and partial deletion coupled with high-throughput screening, a mutant of the Glf transporter (2-RD5) was obtained that relieved the inhibition of xylose transport by glucose. The fermentation tests revealed that 2-RD5 was advantageous in xylose and glucose uptakes, while no obvious advantage was seen for xylose co-consumption when co-fermented with glucose. Further efforts could focus on reducing CCR-mediated repression of intracellular metabolism of xylose. Glf should also serve as a useful model to further exploit the molecular mechanism of xylose transport and the CCR-mediated inhibition. (Source: Microbial Cell Factories)

Macromolecular and elemental composition analysis and extracellular metabolite balances of Pichia pastoris growing at different oxygen levels

Microbial Cell Factories — Wed, 09 Dec 2009 00:00:00 +0100

Conclusions: Application of a range of analytical and statistical techniques allowed getting consistent growth parameters and compositional data of P. pastoris cells growing under different oxygenation conditions. The obtained data provides a first view of the effects of oxygen limitation on the physiology of this microorganism, while recombinant Fab production seems to have little or no impact at this level of analysis. Furthermore, the results will be highly useful in other complementary quantitative studies of P. pastoris physiology, such as metabolic flux analysis. (Source: Microbial Cell Factories)

Microbial production host selection for converting second generation feedstocks into bioproducts

Microbial Cell Factories — Fri, 04 Dec 2009 00:00:00 +0100

Conclusions: Based on the results obtained we conclude that a substrate oriented instead of the more commonly used product oriented approach towards the selection of a microbial production host will avoid the requirement for extensive metabolic engineering. Instead of introducing multiple substrate utilization and detoxification routes to efficiently utilize lignocellulosic hydrolysates only one biosynthesis route forming the product of interest has to be engineered. (Source: Microbial Cell Factories)

Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens

Microbial Cell Factories — Thu, 26 Nov 2009 00:00:00 +0100

Conclusion: The production of all of these antibiotic compounds highlights B. amyloliquefaciens GA1 as a good candidate for the development of biocontrol agents. (Source: Microbial Cell Factories)

Functional analysis of conserved aromatic amino acids in the discoidin domain of Paenibacillus beta-1,3-glucanase

Microbial Cell Factories — Wed, 25 Nov 2009 00:00:00 +0100

The 190-kDa Paenibacillus beta-1,3-glucanase (LamA) contains a catalytic module of the glycoside hydrolase family 16 (GH16) and several auxiliary domains. Of these, a discoidin domain (DS domain), present in both eukaryotic and prokaryotic proteins with a wide variety of functions, exists at the carboxyl-terminus. To better understand the bacterial DS domain in terms of its structure and function, this domain alone was expressed in Escherichia coli and characterized. The results indicate that the DS domain binds various polysaccharides and enhances the biological activity of the GH16 module on composite substrates. We also investigated the importance of several conserved aromatic residues in the domain's stability and substrate-binding affinity. Both were affected by mutations of these res...

Selectively improving nikkomycin Z production by blocking the imidazolone biosynthetic pathway of nikkomycin X and uracil feeding in Streptomyces ansochromogenes

Microbial Cell Factories — Mon, 23 Nov 2009 00:00:00 +0100

Conclusion: A nikkomycin Z selectively producing strain was obtained by genetic manipulation combined with precursors feeding .The strategy presented here might be applicable in other bacteria to selectively produce targeted antibiotics. (Source: Microbial Cell Factories)

Overexpression and characterization of medium-chain-length polyhydroxyalkanoate granule bound polymerases from Pseudomonas putida GPo1

Microbial Cell Factories — Thu, 19 Nov 2009 00:00:00 +0100

Conclusion: The kinetic properties and substrate ranges were determined for both granule bound polymerases. PhaC1 and PhaC2 exhibited different characteristics in granule release and activity profiles, suggesting that there are substantial functional differences between granule bound PhaC1 and PhaC2. (Source: Microbial Cell Factories)

Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-beta-mannosidase from Aspergillus niger BK01

Microbial Cell Factories — Fri, 13 Nov 2009 00:00:00 +0100

Conclusions: This study is the first report on the cloning and expression of the thermostable mannan endo-1,4-beta-mannosidase from A. niger in Pichia pastoris. The efficient expression and ease of purification will significantly decrease the production costs of this enzyme. Taking advantage of its acidic pH optimum and high thermostability, this recombinant beta-mannanase will be valuable in various biotechnological applications. (Source: Microbial Cell Factories)

High yield recombinant thermostable alpha-amylase production using an improved Bacillus licheniformis system

Microbial Cell Factories — Sat, 31 Oct 2009 00:00:00 +0100

Conclusions: This production level of BLA by B. licheniformis CBBD302(pHY-amyL) is amongst the highest levels in Gram-positive bacteria reported so far. (Source: Microbial Cell Factories)

Engineering an efficient secretion of leech carboxypeptidase inhibitor in Escherichia coli

Microbial Cell Factories — Thu, 29 Oct 2009 00:00:00 +0100

Conclusions: These results support the hypothesis that heterologous secreted proteins require proper coupling between translation and translocation for optimal high-level production in E. coli. (Source: Microbial Cell Factories)

Yeast cell factory: fishing for the best one or engineering it?

Microbial Cell Factories — Sun, 11 Oct 2009 23:00:00 +0100

When today scientists and bioprocess engineers look at a Microbial Cell Factory for the production of a protein or a metabolite of commercial or research interest, they think at the microorganism of choice first from a (i) molecular, then from a (ii) metabolic and finally from a (iii) process point of view. Analyses and manipulations of the pathway(s) involved in the synthesis of the desired product and how this pathway(s) interacts with the overall cell functions and activities are indeed steps required to obtain high yield of the product (g of compound per g of substrate), high production (g/l) and high productivity (g/l/h). Conceptually, the whole set of biochemical reactions that take place in the microbial cell factory should be considered. This is valid for processes involving natura...

Expression of active human sialyltransferase ST6GalNAcI in Escherichia coli

Microbial Cell Factories — Tue, 29 Sep 2009 23:00:00 +0100

Background: The presence of terminal, surface-exposed sialic acid moieties can greatly enhance the in vivo half-life of glycosylated biopharmaceuticals and improve their therapeutic efficacy. Complete and homogeneous sialylation of glycoproteins can be efficiently performed enzymically in vitro but this process requires large amounts of catalytically active sialyltransferases. Furthermore, standard microbial hosts used for large-scale production of recombinant enzymes can only produce small quantities of glycosyltransferases of animal origin, which lack catalytic activity.Results and conclusion. In this work, we have expressed the human sialyltransferase ST6GalNAc I (ST6), an enzyme that sialylates O-linked glycoproteins, in Escherichia coli cells. We observed that wild-type bacterial cell...

Increased expression of the oxidative pentose phosphate pathway and gluconeogenesis in anaerobically growing xylose-utilizing Saccharomyces cerevisiae

Microbial Cell Factories — Wed, 23 Sep 2009 23:00:00 +0100

Conclusions: Co-factor imbalance in the initial twp steps of xylose utilization may reduce ethanol productivity by increasing the need for NADP+ reduction and consequently increase reverse flux in glycolysis. (Source: Microbial Cell Factories)

Surface display of heterologous proteins in Bacillus thuringiensis using a peptidoglycan hydrolase anchor

Microbial Cell Factories — Tue, 15 Sep 2009 23:00:00 +0100

Conclusions: Mbg can be a functional anchor protein to target different heterologous proteins onto the surface of B. thuringiensis cells. Since the LysM domain appears to be universal in Gram-positive bacteria, the strategy presented here could be applicable in other bacteria for developing this type of system. (Source: Microbial Cell Factories)

Complete PHB mobilization in Escherichia coli enhances the stress tolerance: a potential biotechnological application

Microbial Cell Factories — Sun, 30 Aug 2009 23:00:00 +0100

Conclusions: This engineered E. coli with PHB mobilization has a potential biotechnological application as immobilized cell factories for biocatalysis and biotransformation. (Source: Microbial Cell Factories)

Yeast artificial chromosomes employed for random assembly of biosynthetic pathways and production of diverse compounds in Saccharomyces cerevisiae

Microbial Cell Factories — Wed, 12 Aug 2009 23:00:00 +0100

Conclusions: The technology has many potential uses but is particularly suited for generating high numbers of structurally diverse compounds, some of which may not be amenable to chemical synthesis, thus greatly facilitating access to a huge chemical space in the search for new commercially interesting compounds (Source: Microbial Cell Factories)

Comparisons of optically monitored small-scale stirred tank vessels to optically controlled disposable bag bioreactors

Microbial Cell Factories — Tue, 04 Aug 2009 23:00:00 +0100

Conclusions: Similar oxygen delivery rates were achieved in both systems, leading to comparable culture performance (growth and mAb production) across scales and mode of mixing. HTBR model was most fitting when neither system was pH-controlled, providing an information-rich alternative to typically non-monitored mL-scale platforms. (Source: Microbial Cell Factories)

Development and Experimental Verification of a Genome-Scale Metabolic Model for Corynebacterium glutamicum

Microbial Cell Factories — Sun, 02 Aug 2009 23:00:00 +0100

Conclusions: The genome-scale metabolic model provides useful information for the evaluation of the metabolic capabilities and prediction of the metabolic characteristics of C. glutamicum. This can form a basis for the in silico design of C. glutamicum metabolic networks for improved bioproduction of desirable metabolites. (Source: Microbial Cell Factories)

Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain

Microbial Cell Factories — Thu, 23 Jul 2009 23:00:00 +0100

Conclusions: Our results show that the expression of heterologous proteins coded by high RIL codon content coding sequences in a codon bias-adjusted strain is detrimental for their solubility. Our data support the hypothesis that the possible elimination of translational pauses that increase translation rate leads to protein misfolding and aggregation. This stresses the importance of strain selection according to codon content in any scheme where a large amount of biologically active product is desirable. (Source: Microbial Cell Factories)

A novel bacterial isolate Stenotrophomonas maltophilia as living factory for synthesis of gold nanoparticles

Microbial Cell Factories — Sun, 19 Jul 2009 23:00:00 +0100

Conclusions: The process of synthesis of well-dispersed nanoparticles using a noble microorganism isolated from the gold enriched soil sample has been reported in this study, leading to the development of an easy bioprocess for synthesis of GNPs. This is the first study in which an extensive characterisation of the indigenous bacterium isolated from the actual gold enriched soil was conducted. Promising mechanism for the biosynthesis of GNPs by the strain and their stabilization via charge capping suggested involvement of NADPH-dependent reductase enzyme that reduces Au3+ to Au0 through electron shuttle enzymatic metal reduction process. (Source: Microbial Cell Factories)

Bacillus subtilis as potential producer for polyhydroxyalkanoates

Microbial Cell Factories — Sun, 19 Jul 2009 23:00:00 +0100

Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by microbes to overcome environmental stress. Commercial production of PHAs is limited by the high cost of production compared to conventional plastics. Another hindrance is the brittle nature and low strength of polyhydroxybutyrate (PHB), the most widely studied PHA. The needs are to produce PHAs, which have better elastomeric properties suitable for biomedical applications, preferably from inexpensive renewable sources to reduce cost. Certain unique properties of Bacillus subtilis such as lack of the toxic lipo-polysaccharides, expression of self-lysing genes on completion of PHA biosynthetic process - for easy and timely recovery, usage of biowastes as feed enable it to compete as potential candidate for commercial product...

Interactive visualization of clusters in microarray data: an efficient tool for improved metabolic analysis of E. coli

Microbial Cell Factories — Tue, 14 Jul 2009 23:00:00 +0100

Conclusions: It was shown that gcExplorer is a very helpful tool to gain a general overview of microarrayexperiments. Interesting gene expression patterns can easily be found, compared among different experimentsand combined with information about gene function from publicly available databases. (Source: Microbial Cell Factories)

Interactive visualization of clusters in microarray data: an efficient tool for improved metabolic analysis of E. coli

Microbial Cell Factories — Tue, 14 Jul 2009 23:00:00 +0100

Regioselective biooxidation of (+)-valencene by recombinant E. coli expressing CYP109B1 from Bacillus subtilis in a two-liquid-phase system

Microbial Cell Factories — Thu, 09 Jul 2009 23:00:00 +0100

Conclusions: This study demonstrates that the identification of new P450s capable of producing valuable compounds can basically be achieved by screening of recombinant P450 libraries. The biphasic reaction system described in this work presents an attractive way for the production of (+)-nootkatone (4), as it is safe and can easily be controlled and scaled up. (Source: Microbial Cell Factories)

Developing a scalable model of recombinant protein yield from Pichia pastoris: the influence of culture conditions, biomass and induction regime

Microbial Cell Factories — Tue, 30 Jun 2009 23:00:00 +0100

Conclusions: We demonstrate how a rational, stepwise approach to recombinant protein production screens can reduce process development time. (Source: Microbial Cell Factories)

Molecular cloning and expression of a novel trehalose synthase gene from Enterobacter hormaechei

Microbial Cell Factories — Thu, 11 Jun 2009 23:00:00 +0100

Conclusions: In this study, several TreS producing strains were screened from a source of soil bacteria. The characterization of the recombinant TreS of Enterobacter hormaechei suggested its potential application. Consequently, a strategy for isolation of TreS producing strains and cloning of novel treS genes from natural sources was demonstrated. (Source: Microbial Cell Factories)

PimT, an amino-acid exporter controls polyene production via secretion of the quorum-sensing pimaricin-inducer PI-factor in Streptomyces natalensis

Microbial Cell Factories — Mon, 08 Jun 2009 04:00:00 +0100

Conclusion: This report describes the involvement of an amino acid exporter (encoded by pimT in the vicinity of the pimaricin cluster) in modulating the expression of antibiotic biosynthetic genes via secretion of the quorum-sensing pimaricin-inducer PI-factor. The discovery of the participation of amino acid exporters in a signal transduction cascade for the production of polyene macrolides is unexpected, and represents an important step forward towards understanding the regulatory network for polyene regulation. Additionally, this finding constitutes the first detailed characterization of an amino-acid exporter in an Actinomycete, and to our knowledge, the first evidence for the implication of this type of exporters in quorum sensing. (Source: Microbial Cell Factories)

Components of the E. coli envelope are affected by and can react to protein over-production in the cytoplasm

Microbial Cell Factories — Fri, 05 Jun 2009 04:00:00 +0100

Conclusions: Taking together physiological and biochemical evidence, our work indicates that the E. coli envelope can sense protein over-expression in the cytoplasm and react by modulating the abundance of some membrane proteins, with possible consequences on the membrane traffic of small solutes, i.e. nutrients, drugs and metabolites. Such a response seems to be independent on the nature of the protein being over-expressed. On the other hand both our data reported herein and previous results indicate that membrane lipids may act as a second stress sensor responsive to the aggregation state of the recombinant protein and further contribute to changes in cellular exchanges with the environment. (Source: Microbial Cell Factories)

Overexpression and simple purification of the Thermotoga maritima 6-phosphogluconate dehydrogenase in Escherichia coli and its application for NADPH regeneration

Microbial Cell Factories — Thu, 04 Jun 2009 04:00:00 +0100

Conclusions: We have achieved a more than 500-fold improvement in the expression of soluble T. maritima 6PGDH in E. coli, characterized its basic biochemical properties, and demonstrated its applicability for NADPH regeneration by a new enzyme cocktail. The methodology for over-expression and simple purification of this thermostable protein would be useful for the production of other thermostable proteins in E. coli. (Source: Microbial Cell Factories)

Validation of a high-throughput fermentation system based on online monitoring of biomass and fluorescence in continuously shaken microtiter plates

Microbial Cell Factories — Thu, 04 Jun 2009 04:00:00 +0100

Conclusions: The BioLector technology is a very useful tool to perform quantitative microfermentations under engineered reaction conditions. With this technique, specific yields and rates can be directly deduced from online biomass and product concentrations, which is superior to existing technologies such as microplate readers or optode-based cultivation systems. In particular, applications with strong demand on high-throughput such as clone and media screening and systems biology can benefit from its simple handling, the high quantitative information content and its capacity of automation. (Source: Microbial Cell Factories)

Genome, secretome and glucose transport highlight unique features of the protein production host Pichia pastoris

Microbial Cell Factories — Tue, 02 Jun 2009 04:00:00 +0100

Conclusions: This work highlights significant advantages of using the P. pastoris system with glucose based expression and fermentation strategies. As only few proteins and no proteases are actually secreted on glucose, it becomes evident that cell lysis is the relevant cause of proteolytic degradation of secreted proteins. The endowment with hexose transporters, dominantly of the high affinity type, limits glucose uptake rates and thus overflow metabolism as observed in S. cerevisiae. The presence of 4 genes for glycerol transporters explains the high specific growth rates on this substrate and underlines the suitability of a glycerol/glucose based fermentation strategy. Furthermore, we present an open access web based genome browser (www.pichiagenome.org). (Source: Microbial Cell Factori...

Heme and menaquinone induced electron transport in lactic acid bacteria

Microbial Cell Factories — Fri, 29 May 2009 04:00:00 +0100

Conclusions: We propose that the cyd-genes were present in the common ancestor of lactic acid bacteria, and that multiple gene-loss events best explains the observed distribution of these genes among the species. (Source: Microbial Cell Factories)

Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli

Microbial Cell Factories — Thu, 14 May 2009 04:00:00 +0100

This article reviews the available strategies conceived for exploiting the bacteria physiological mechanisms in order to produce disulfide-bonded proteins with proper folding. (Source: Microbial Cell Factories)

Identification of potentially safe promising fungal cell factories for the production of polyketide natural food colorants using chemotaxonomic rationale

Microbial Cell Factories — Mon, 27 Apr 2009 04:00:00 +0100

Conclusions: The present work brought out that the use of chemotaxonomic tools and a priori knowledge of fungal extrolites is a rational approach towards selection of fungal polyketide pigment producers considering the enormous chemical diversity and biodiversity of ascomycetous fungi. This could very well serve a useful piece of information for the selection of potentially safe fungal cell factories not only for polyketide pigments but also for the other industrially important polyketides, the molecular and genetic basis for the biosynthesis of which has not yet been examined in detail. In addition, 4 out of the 10 chemotaxonomically selected promising Penicillium strains were shown to produce extracellular pigments in the liquid media using a solid support indicating future cell factory ...

Homolactic fermentation from glucose and cellobiose using Bacillus subtilis

Microbial Cell Factories — Tue, 21 Apr 2009 04:00:00 +0100

Conclusion: This study shows that by taking advantage of the cellobiose utilization capability and osmotic stress high resistance of B. subtilis, a robust process for L-lactate production can be developed. (Source: Microbial Cell Factories)

Process development in Hansenula polymorpha and Arxula adeninivorans, a re-assessment

Microbial Cell Factories — Wed, 15 Apr 2009 04:00:00 +0100

A range of industrial H. polymorpha-based processes exist, most of them for the production of pharmaceuticals. The established industrial processes lean on the use of promoters derived from MOX and FMD, genes of the methanol metabolism pathway. In Hansenula polymorpha these promoters are de-repressed upon depletion of a range of carbon sources like glucose and glycerol instead of being induced by methanol as reported for other methylotrophs. Due to these characteristics screening and fermentation modes have been defined for strains harbouring such expression control elements that lean on a limited supplementation of glycerol or glucose to a culture medium. For fermentation of H. polymorpha a synthetic medium (SYN6) has been developed. No industrial processes have been developed so far for...

Optimization of physical factors affecting the production of thermostable organic solvent-tolerant protease from a newly isolated halo tolerant Bacillus subtilis strain Rand

Microbial Cell Factories — Thu, 09 Apr 2009 04:00:00 +0100

Conclusion: Strain Rand has been found to be able to secrete extra-cellular thermostable organic solvent-tolerant protease into the culture medium. The protease exhibited a remarkable stability towards temperature and organic solvent. This unique property makes it attractive and useful to be used in industrial applications. (Source: Microbial Cell Factories)

Medical bioremediation of age-related diseases

Microbial Cell Factories — Thu, 09 Apr 2009 04:00:00 +0100

Catabolic insufficiency in humans leads to the gradual accumulation of a number of pathogenic compounds associated with age-related diseases, including atherosclerosis, Alzheimer's disease, and macular degeneration. Removal of these compounds is a widely researched therapeutic option, but the use of antibodies and endogenous enzymes has failed to produce effective treatments, and may pose risks to cellular homeostasis. Another alternative is "medical bioremediation," or the use of microbial enzymes to augment missing catabolic functions. The genetic diversity in most natural environments provides a resource that can be mined for enzymes capable of degrading just about any energy-rich carbon-based compound. This review discusses targets for degradation, the identification of candidate enzym...

Metabolic engineering for improving anthranilate synthesis from glucose in Escherichia coli

Microbial Cell Factories — Thu, 02 Apr 2009 04:00:00 +0100

Conclusion: This work constitutes the first example of a microbial system for the environmentally-compatible synthesis of anthranilate generated by metabolic engineering. The results presented here, including the characterization of mutation in the trpD gene from strain W3110 trpD9923 and the development of a fermentation strategy, establish a step forward towards the future improvement of a sustainable process for anthranilate production. In addition, the present work provides very useful data regarding the positive and negative consequences of the evaluated metabolic engineering strategies. (Source: Microbial Cell Factories)

Phenotypes and gene expression profiles of Saccharopolyspora erythraea rifampicin-resistant (rif) mutants affected in erythromycin production

Microbial Cell Factories — Mon, 30 Mar 2009 04:00:00 +0100

In this study we have used rifampicin selection as a strategy to genetically improve the erythromycin producer Saccharopolyspora erythraea. Results: Spontaneous rifampicin-resistant (rif) mutants were isolated from the parental strain NRRL2338 and two rif mutations mapping within rpoB, S444F and Q426R, were characterized. With respect to the parental strain, S444F mutants exhibited higher respiratory performance and up to four-fold higher final erythromycin yields; in contrast, Q426R mutants were slow-growing, developmental-defective and severely impaired in erythromycin production. DNA microarray analysis demonstrated that these rif mutations deeply changed the transcriptional profile of S. erythraea. The expression of genes coding for key enzymes of carbon (and energy) and nitrogen centr...

Microbial factories for recombinant pharmaceuticals

Microbial Cell Factories — Tue, 24 Mar 2009 04:00:00 +0100

Most of the hosts used to produce the 151 recombinant pharmaceuticals so far approved for human use by the Food and Drug Administration (FDA) and/or by the European Medicines Agency (EMEA) are microbial cells, either bacteria or yeast. This fact indicates that despite the diverse bottlenecks and obstacles that microbial systems pose to the efficient production of functional mammalian proteins, namely lack or unconventional post-translational modifications, proteolytic instability, poor solubility and activation of cell stress responses, among others, they represent convenient and powerful tools for recombinant protein production. The entering into the market of a progressively increasing number of protein drugs produced in non-microbial systems has not impaired the development of products ...

Synthesis of rhamnolipid biosurfactant and mode of hexadecane uptake by Pseudomonas species

Microbial Cell Factories — Wed, 11 Mar 2009 04:00:00 +0100

Conclusions: This study throws more light on the uptake mechanism of hydrocarbon by Pseudomonas aeruginosa. We report here a new and exciting line of research for hydrocarbon uptake involving internalization of biosurfactant covered hydrocarbon inside cell for subsequent breakdown. (Source: Microbial Cell Factories)

Bioreactor mixing efficiency modulates the activity of a prpoS::GFP reporter gene in E. coli

Microbial Cell Factories — Wed, 25 Feb 2009 05:00:00 +0100

Conclusion: The generation of extracellular perturbations (in the present case, at the level of the sugar concentration and the dissolved oxygen level) has led to a drop at the level of the rpoS expression level. This drop has to be attributed to a segregation phenomenon in microbial population, with a major sub-population exhibiting a low expression level and a minor sub-population keeping its initial elevated expression level. The intensity of the segregation, as well as its time of appearance during the culture can be related to the bioreactor mixing efficiency. (Source: Microbial Cell Factories)

Bioreactor mixing efficiency modulates the activity of a prpoS::GFP reporter gene in E. coli

Microbial Cell Factories — Wed, 25 Feb 2009 05:00:00 +0100

Conclusion The generation of extracellular perturbations (in our case, at the level of the sugar concentration and the dissolved oxygen level) has led to a drop at the level of the rpoS expression level. This drop has been mainly attributed to a segregation phenomenon at the level of the microbial population, with a major sub-population exhibiting a low expression level and a minor sub-population keeping its initial elevated expression level. The intensity of the segregation, as well as its time of appearance during the culture can be related to the bioreactor mixing efficiency. (Source: Microbial Cell Factories)

Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of Hepatitis B surface antigen

Microbial Cell Factories — Tue, 10 Feb 2009 05:00:00 +0100

Conclusion: In comparison to the highest yields reported so far, our simple cultivation process resulted in an ~7 fold enhancement in total HBsAg production with more than 30% of soluble protein competent for assembly into VLPs. This work opens up the possibility of significantly reducing the cost of vaccine production with implications for expanding hepatitis B vaccination in resource-poor countries. (Source: Microbial Cell Factories)

Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of Hepatitis B surface antigen

Microbial Cell Factories — Tue, 10 Feb 2009 05:00:00 +0100

Altering the ribosomal subunit ratio in yeast maximizes recombinant protein yield

Microbial Cell Factories — Thu, 29 Jan 2009 05:00:00 +0100

Conclusions: This work demonstrates the power of a rational approach to recombinant protein production by using the results of transcriptome analysis to engineer improved strains, thereby revealing the underlying biological events involved. (Source: Microbial Cell Factories)

Export of recombinant proteins in Escherichia coli using ABC transporter with an attached lipase ABC transporter recognition domain (LARD)

Microbial Cell Factories — Thu, 29 Jan 2009 05:00:00 +0100

Conclusion: The LARDs or whole TliA were attached to C-termini of model proteins and enabled the export of the model proteins such as GFP and EGF in E. coli supplemented with ABC transporter. These results open the possibility for the extracellular production of recombinant proteins in Pseudomonas using LARDs or TliA as a C-terminal signal sequence. (Source: Microbial Cell Factories)

Pediocins: The bacteriocins of Pediococci. Sources, production, properties and applications

Microbial Cell Factories — Thu, 08 Jan 2009 05:00:00 +0100

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 inf...

Learning about protein solubility from bacterial inclusion bodies

Microbial Cell Factories — Thu, 08 Jan 2009 05:00:00 +0100

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. (Source: Microbial Cell Factories)

Metabolic engineering of a reduced-genome strain of Escherichia coli for L-threonine production

Microbial Cell Factories — Wed, 07 Jan 2009 05:00:00 +0100

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. (Source: Microbial Cell Factories)

Molecular cloning and characterization of a novel pyrethroid-hydrolyzing esterase originating from the Metagenome

Microbial Cell Factories — Tue, 30 Dec 2008 05:00:00 +0100

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. (Source: Microbial Cell Factories)

Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol

Microbial Cell Factories — Wed, 03 Dec 2008 05:00:00 +0100

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 isozy...

The scientific impact of microbial cell factories

Microbial Cell Factories — Mon, 01 Dec 2008 05:00:00 +0100

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 C...

Engineering inclusion bodies for non denaturing extraction of functional proteins

Microbial Cell Factories — Mon, 01 Dec 2008 05:00:00 +0100

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. (Source: Microbial Cell Factories)

Norvaline is accumulated after a down-shift of oxygen in Escherichia coli W3110

Microbial Cell Factories — Tue, 21 Oct 2008 04:00:00 +0100

Conclusion: Norvaline synthesis has been so far mainly related to an imbalance of the synthesis of the branched chain amino acids under conditions were pyruvate level is high. Here we show that simply a downshift of oxygen is sufficient to cause norvaline accumulation at a high glucose concentration as a consequence of the accumulation of pyruvate and its direct chain elongation over α-ketobutyrate and α-ketovalerate.Although the flux to norvaline is low, millimolar concentrations are accumulated in the cultivation broth, which is far above the level which has been discussed for being relevant for misincorporation of norvaline into recombinant proteins. Therefore we believe that our finding is relevant for recombinant protein production but also may even have implications for the physiol...

Norvaline is accumulated after a down shift of oxygen in Escherichia coli W3110

Microbial Cell Factories — Tue, 21 Oct 2008 04:00:00 +0100

Conclusions: Norvaline synthesis has been so far mainly related to an imbalance of the synthesis of the branched chain amino acids under conditions were pyruvate level is high. Here we show that simply a downshift of oxygen is sufficient to cause norvaline accumulation at a high glucose concentration as a consequence of the accumulation of pyruvate and its direct chain elongation over alpha-ketobutyrate and alpha-ketovalerate. Although the flux to norvaline is low, millimolar concentrations are accumulated in the cultivation broth, which is far above the level which has been discussed for being relevant for misincorporation of norvaline into recombinant proteins. Therefore we believe that our finding is relevant for recombinant protein production but also may even have implications for the...

Sample processing for DNA chip array-based analysis of enterohemorrhagic Escherichia coli (EHEC)

Microbial Cell Factories — Mon, 13 Oct 2008 04:00:00 +0100

Conclusions: In order to implement the DNA chip array-based analysis for direct EHEC detection the sample processing was established in course of this work. However, this sample preparation mode may also be applied to other types of EHEC DNA-based sensing systems. (Source: Microbial Cell Factories)

Marine metagenomics: strategies for the discovery of novel enzymes with biotechnological applications from marine environments

Microbial Cell Factories — Thu, 21 Aug 2008 04:00:00 +0100

Metagenomic based strategies have previously been successfully employed as powerful tools to isolate and identify enzymes with novel biocatalytic activities from the unculturable component of microbial communities from various terrestrial environmental niches. Both sequence based and function based screening approaches have been employed to identify genes encoding novel biocatalytic activities and metabolic pathways from metagenomic libraries. While much of the focus to date has centred on terrestrial based microbial ecosystems, it is clear that the marine environment has enormous microbial biodiversity that remains largely unstudied. Marine microbes are both extremely abundant and diverse; the environments they occupy likewise consist of very diverse niches. As culture-dependent methods h...

Marine metagenomics: Strategies for the discovery of novel enzymes with biotechnological applications from marine ecosystems

Microbial Cell Factories — Thu, 21 Aug 2008 04:00:00 +0100

Metagenomic based strategies have previously been successfully employed as powerful tools to isolate and identify enzymes with novel biocatalytic activities from the unculturable component of microbial communities from various terrestrial environmental niches. Both sequence based and function based screening approaches have been employed to identify genes encoding novel biocatalytic activities and metabolic pathways from metagenomic libraries. While much of the focus to date has centred on terrestrial based microbial ecosystems, it is clear that the marine environment has enormous microbial biodiversity that remains largely unstudied. Marine microbes are both extremely abundant and diverse; the environment they occupy likewise consist of very diverse niches. As culture-dependent methods ha...

Nisin inducible production of Listeriolysin O in Lactococcus lactis NZ9000

Microbial Cell Factories — Tue, 29 Jul 2008 04:00:00 +0100

Conclusion: The LLO production method described in this work provides an approach to efficient LLO production in the Gram-positive Lactococcus bacterium to yield a significant source of the protein for research and diagnostic applications. Expression of LLO in L. lactis has a number of benefits over E. coli which may facilitate both in vivo and in vitro applications of this system. (Source: Microbial Cell Factories)

Overexpression of the riboflavin biosynthetic pathway in Pichia pastoris

Microbial Cell Factories — Tue, 29 Jul 2008 04:00:00 +0100

Conclusions: The regulation of the pathway is distributed over more than one gene. High cell density cultivations of a P. pastoris strain overexpressing all six RIB genes allow the accumulation of 175 mg/L riboflavin in the supernatant. The basis for rational engineering of riboflavin production in P. pastoris has thus been established. (Source: Microbial Cell Factories)

Biological treatment of tannery wastewater by using salt-tolerant bacterial strains

Microbial Cell Factories — Tue, 29 Apr 2008 04:00:00 +0100

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. (Source: Microbial Cell Factories)

Biological treatment of tannery wastewater by using salt-tolerant bacterial strains

Microbial Cell Factories — Tue, 29 Apr 2008 04:00:00 +0100

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. (Source: Microbial Cell Factories)

Simple generation of site-directed point mutations in the Escherichia coli chromosome using Red/ET Recombination

Microbial Cell Factories — Thu, 24 Apr 2008 04:00:00 +0100

Conclusions: 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. (Source: Microbial Cell Factories)

Towards the development of Bacillus subtilis as a cell factory for membrane proteins and protein complexes

Microbial Cell Factories — Fri, 04 Apr 2008 04:00:00 +0100

Conclusions: 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. (Source: Microbial Cell Factories)

Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview

Microbial Cell Factories — Fri, 04 Apr 2008 04:00:00 +0100

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, pharmacologic 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 ...

Folding and assembly of large macromolecular complexes monitored by hydrogen-deuterium exchange and mass spectrometry

Microbial Cell Factories — Fri, 04 Apr 2008 04:00:00 +0100

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. (Source: Microbial Cell Factories)

Metabolic responses to pyruvate kinase deletion in lysine producing Corynebacterium glutamicum

Microbial Cell Factories — Thu, 13 Mar 2008 04:00:00 +0100

Conclusion: The metabolic flux analysis performed illustrates the high flexibility of the metabolic network of C. glutamicum to compensate for external perturbation. The organism could almost maintain its growth and production performance through a local redirection of the metabolic flux, thereby fulfilling all anabolic and catabolic needs. The formation of the undesired overflow metabolites dihydroxyacetone and glycerol in the deletion mutant, however, indicates a limiting capacity of the metabolism down-stream of their common precursor glyceraldehyde 3-phosphate and opens possibilities for further strain engineering. (Source: Microbial Cell Factories)

Expression in Escherichia coli, purification, refolding and antifungal activity of an osmotin from Solanum nigrum

Microbial Cell Factories — Tue, 11 Mar 2008 04:00:00 +0100

Conclusions: Since SnOLP displays activity against economically important plant pathogenic fungi and oomycete, it represents a novel PR-5 protein with promising utility for biotechnological applications. (Source: Microbial Cell Factories)

Improved determination of plasmid copy number using quantitative real-time PCR for monitoring fermentation processes

Microbial Cell Factories — Fri, 07 Mar 2008 05:00:00 +0100

Conclusion: Using whole cells as a template source and relative quantification considering different PCR amplification efficiencies are significant improvements of the qPCR method for PCN determination. Due to the approaches used, the method is suitable for PCN determination in fermentation processes using various media and conditions. (Source: Microbial Cell Factories)

A review on slurry bioreactors for bioremediation of soils and sediments

Microbial Cell Factories — Fri, 29 Feb 2008 05:00:00 +0100

The aim of this work is to present a critical review on slurry bioreactors (SB) and their application to bioremediation of soils and sediments polluted with recalcitrant and toxic compounds. The scope of the review encompasses the following subjects: (i) process fundamentals of SB and analysis of advantages and disadvantages; (ii) the most recent applications of SB to laboratory scale and commercial scale soil bioremediation, with a focus on pesticides, explosives, polynuclear aromatic hydrocarbons, and chlorinated organic pollutants; (iii) trends on the use of surfactants to improve availability of contaminants and supplementation with degradable carbon sources to enhance cometabolism of pollutants; (iv) recent findings on the utilization of electron acceptors other than oxygen; (v) bioau...

Switching the mode of sucrose utilization by Saccharomyces cerevisiae

Microbial Cell Factories — Wed, 27 Feb 2008 05:00:00 +0100

Conclusions: Higher cell densities during batch cultures on 20 g/L sucrose were achieved by using a S. cerevisiae strain engineered in the sucrose uptake system. Such result was accomplished by effectively reducing sucrose uptake by the yeast cells, avoiding overflow metabolism, with the concomitant reduction in ethanol production. The use of this modified yeast strain in simpler batch culture mode can be a viable option to more complicated traditional sucrose-limited fed-batch cultures for biomass-directed processes of S. cerevisiae. (Source: Microbial Cell Factories)

Conversion of beta-carotene into astaxanthin: Two separate enzymes or a bifunctional hydroxylase-ketolase protein?

Microbial Cell Factories — Wed, 20 Feb 2008 05:00:00 +0100

Astaxanthin is a xanthophyl of great interest in animal nutrition and human health. The market prospect in the nutraceutics industries for this health-protective molecule is very promising. Astaxanthin is synthesized by several bacteria, algae and plants from beta-carotene by the sequential action of two enzymes: a beta-carotene, 3,3'-hydroxylase that introduces an hydroxyl group at the 3 (and 3') positions of each of the two beta-ionone rings of beta-carotene, and a beta-carotene ketolase that introduces keto groups at carbons 4 and 4' of the beta-ionone rings. Astaxhanthin is also produced by the yeast-like basidiomycete Xanthophyllomyces dendrorhous. A gene crtS involved in the conversion of beta-carotene to astaxanthin has been cloned simultaneously by two research groups. Complementat...

Development of an improved Pseudoalteromonas haloplanktis TAC125 strain for recombinant protein secretion at low temperature

Microbial Cell Factories — Thu, 07 Feb 2008 05:00:00 +0100

Conclusion: Here we report a cell engineering approach to the construction of a P. haloplanktis TAC125 strain with reduced extra-cellular protease activity. The improved strain is able to secrete the psychrophilic alpha-amylase (the carrier of our recombinant secretion system), while it displays a significant reduction of protease content in the culture medium. These features make the gspE mutant an improved host with a remarkable biotechnological potential in recombinant protein secretion at low temperature. Moreover this work demonstrates that P. haloplanktis TAC125 is a versatile psychrophilic host for recombinant protein production since it can be easily improved by a directed engineering approach. To the best of our knowledge, this is the first described example of a strain improvemen...

Coutilization of glucose and glycerol enhances the production of aromatic compounds in an Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system

Microbial Cell Factories — Tue, 22 Jan 2008 05:00:00 +0100

Conclusions: Due to the lack of catabolite repression, PB12 strain carrying multicopy plasmids containing tktA and aroGfbr genes is capable of coutilizing glucose and other carbon sources; this capacity, reduces its m but increases the production of aromatic compounds. (Source: Microbial Cell Factories)

Production of single chain Fab (scFab) fragments in Bacillus megaterium

Microbial Cell Factories — Tue, 27 Nov 2007 05:00:00 +0100

Conclusion: B. megaterium allows the secretory production of antibody fragments including the novel scFab antibody format. The yield and quality of functional antibody fragment is comparable to the periplasmic production in E. coli. (Source: Microbial Cell Factories)

Complete gene expression profiling of Saccharopolyspora erythraea using GeneChip DNA microarrays

Microbial Cell Factories — Mon, 26 Nov 2007 05:00:00 +0100

Conclusions: The use of a S. erythraea DNA microarray improved specificity and sensitivity of gene expression analysis, allowing a global and at the same time detailed picture of how S. erythraea genes are modulated. This work underlines the importance of using DNA microarrays, coupled with an exhaustive statistical and bioinformatic analysis of the results, to understand the transcriptional organization of the chromosomes of micro-organisms producing natural products. (Source: Microbial Cell Factories)

Systems biology and biological systems diversity for the engineering of microbial cell factories

Microbial Cell Factories — Tue, 20 Nov 2007 05:00:00 +0100

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Expression of human alpha-1-proteinase inhibitor in Aspergillus niger

Microbial Cell Factories — Mon, 29 Oct 2007 04:00:00 +0100

Conclusion: We examined the suitability of the filamentous fungus A. niger for the expression of the human gene for alpha1-PI, a medium size glycoprotein of high therapeutic value. The heterologous expression of the human gene for alpha1-PI in A. niger was successfully achieved to produce the secreted mature human r-alpha1-PI in A. niger as a biologically active glycosylated protein with improved stability and with yields of up to 12 mg/L in shake-flask growth. (Source: Microbial Cell Factories)

Recombinant protein production in the new Millennium

Microbial Cell Factories — Sat, 20 Oct 2007 04:00:00 +0100

Recombinant protein production encounters great progress due to novel high throughput analytical tools and advanced cell engineering approaches. The Microbial Physiology Section of the European Federation of Biotechnology accompanies this development in a successful series of conferences, supported by Microbial Cell Factories. (Source: Microbial Cell Factories)

Saccharomyces cerevisiae: a versatile eukaryotic system in virology

Microbial Cell Factories — Wed, 10 Oct 2007 04:00:00 +0100

The yeast Saccharomyces cerevisiae is a well-established model system for understanding fundamental cellular processes relevant to higher eukaryotic organisms. Less known is its value for virus research, an area in which Saccharomyces cerevisiae has proven to be very fruitful as well. The present review will discuss the main achievements of yeast-based studies in basic and applied virus research. These include the analysis of the function of individual proteins from important pathogenic viruses, the elucidation of key processes in viral replication through the development of systems that allow the replication of higher eukayotic viruses in yeast, and the use of yeast in antiviral drug development and vaccine production. (Source: Microbial Cell Factories)

Production of L-carnitine by secondary metabolism of bacteria

Microbial Cell Factories — Tue, 02 Oct 2007 04:00:00 +0100

The increasing commercial demand for L-carnitine has led to a multiplication of efforts to improve its production with bacteria. The use of different cell environments, such as growing, resting, permeabilized, dried, osmotically stressed, freely suspended and immobilized cells, to maintain enzymes sufficiently active for L-carnitine production is discussed in the text. The different cell states of enterobacteria, such as Escherichia coli and Proteus sp., which can be used to produce L-carnitine from crotonobetaine or D-carnitine as substrate are analyzed. Moreover, the combined application of both bioprocess and metabolic engineering has allowed a deeper understanding of the main factors controlling the production process, such as energy depletion and the alteration of the acetyl-CoA/CoA r...

Metabolic transcription analysis of engineered Escherichia coli strains that overproduce L-phenylalanine

Microbial Cell Factories — Wed, 19 Sep 2007 04:00:00 +0100

Conclusions: The identification of potential rate-limiting steps and the detection of transcriptional responses in overproducing microorganisms may suggest "reverse engineering" strategies for the further improvement of L-Phe production strains. (Source: Microbial Cell Factories)

Production of Recombinant Peanut Allergen Ara h 2 using Lactococcus lactis

Microbial Cell Factories — Tue, 21 Aug 2007 04:00:00 +0100

Conclusions: Recombinant production of Ara h 2 using L. lactis can offer high yields of secreted, full length and immunologically active allergen. The L. lactis expression system can support recombinant allergen material for immunotherapy and component resolved allergen diagnostics (Source: Microbial Cell Factories)

Microbial proteomics: a mass spectrometry primer for biologists

Microbial Cell Factories — Wed, 15 Aug 2007 04:00:00 +0100

It is now more than 10 years since the publication of the first microbial genome sequence and science is now moving towards a post genomic era with transcriptomics and proteomics offering insights into cellular processes and function. The ability to assess the entire protein network of a cell at a given spatial or temporal point will have a profound effect upon microbial science as the function of proteins is inextricably linked to phenotype. Whilst such a situation is still beyond current technologies rapid advances in mass spectrometry, bioinformatics and protein separation technologies have produced a step change in our current proteomic capabilities. Subsequently a small, but steadily growing, number of groups are taking advantage of this cutting edge technology to discover more about ...

Redox stress proteins are involved in adaptation response of the hyperthermoacidophilic archaeon Sulfolobus solfataricus to nickel challenge

Microbial Cell Factories — Sun, 12 Aug 2007 04:00:00 +0100

Conclusions: These findings suggest a series of preferential gene expression pathways activated in adaptation response to metal challenge. (Source: Microbial Cell Factories)

Hitting Bacteria at the Heart of the Central Dogma: Sequence-Specific Inhibition

Microbial Cell Factories — Fri, 10 Aug 2007 04:00:00 +0100

An important objective in developing new drugs is the achievement of high specificity to maximize curing effect and minimize side-effects, and high specificity is an integral part of the antisense approach. The antisense techniques have been extensively developed from the application of simple long regular antisense RNA (asRNA) molecules to highly modified versions conferring resistance to nucleases, stability of hybrid formation and other beneficial characteristics, though still preserving the specificity of the original nucleic acids. These new and improved second- and third-generation antisense molecules have shown promising results. The first antisense drug has been approved and more are in clinical trials. However, these antisense drugs are mainly designed for the treatment of differe...

Transcriptional response of Pichia pastoris in fed-batch cultivations to Rhizopus oryzae lipase production reveals UPR induction

Microbial Cell Factories — Mon, 16 Jul 2007 04:00:00 +0100

Conclusions: The bead-based sandwich hybridization assay has shown its potential as a reliable instrument for quantification of specific mRNA species in P. pastoris cells grown in fed-batch cultures. As a proof-of-principle, the influence of the carbon and nitrogen sources, the specific growth rate, as well as the ROL overexpression on the transcriptional levels of a reduced set of bioprocess-relevant genes has been quantitatively studied, revealing that ROL overexpression and secretion seems to trigger the UPR in P. pastoris, resulting in a physiological bottleneck for the production process. (Source: Microbial Cell Factories)

Homologous high-throughput expression and purification of highly conserved E. coli Proteins

Microbial Cell Factories — Wed, 06 Jun 2007 04:00:00 +0100

Conclusions: Here, we report a cost-efficient procedure to produce around 200 soluble recombinant E. coli proteins in large-scale, including removal of LPS by polymyxin B coated beads for subsequent use of the proteins in downstream immunological studies. (Source: Microbial Cell Factories)

Glycolysis and the regulation of glucose transport in Lactococcus lactis spp. lactis in batch and fed-batch culture

Microbial Cell Factories — Thu, 24 May 2007 04:00:00 +0100

Conclusion: Determination of intracellular metabolites pools showed that FBP cannot be regarded as a direct regulator of product formation, since almost identical concentrations were obtained at both low (13.75mM) and high (138 mM) glucose levels, at which neither the glucose uptake rates and the glycolytic flux, nor the fermentation patterns were similar (mixed acids vs homolactic, respectively). Glucostat data showed instead that the control of the flux through the glycolytic pathway under the examined conditions, resides to a large extent in processes outside the pathway, like the ATP consuming reactions and glucose transport. A regulation mechanism is proposed governed by the energy state of the cell by which L. lactis can handle the glycolytic flux through the allosteric properties of...

Development of expression vectors for Esherichia.coli based on the pCR2 replicon.

Microbial Cell Factories — Thu, 10 May 2007 04:00:00 +0100

Conclusions: We have constructed a series of expression vectors based on the pCR2 replicon and demonstrated its high stability and sustained expression capacity, in the absence of selection pressure which will make it an efficient tool for metabolic engineering and co-expression studies, as well as for scale up of expression. (Source: Microbial Cell Factories)

Efficient production and secretion of bovine β-lactoglobulin by Lactobacillus casei

Microbial Cell Factories — Fri, 06 Apr 2007 04:00:00 +0100

Conclusion: BLG production and secretion in L. casei were significantly improved by fusions to a propeptide enhancer and a carrier protein. The resulting recombinant strains will be further tested for their ability to modulate the immune response against BLG via mucosal delivery in a cow's milk allergy model in mice. (Source: Microbial Cell Factories)

Evidence for glycosylation on a DNA-binding protein of Salmonella enterica

Microbial Cell Factories — Mon, 02 Apr 2007 04:00:00 +0100

Conclusions: Taken together, these results indicate that Dps undergoes post-translational modifications in the pre- and early stationary phases of bacterial growth. There is also evidence that a small mannose-containing oligosaccharide is linked to this bacterial protein. (Source: Microbial Cell Factories)

An improved system for estradiol-dependent regulation of gene expression in yeast

Microbial Cell Factories — Tue, 20 Mar 2007 04:00:00 +0100

Conclusion: The feedback regulatory loop that we have engineered allows a 250-fold induction of the regulated gene, without increasing the basal activity of the target promoter, and achieving a 12-fold higher regulation efficiency than the previous configuration. (Source: Microbial Cell Factories)

Potential and utilization of thermophiles and thermostable enzymes in biorefining

Microbial Cell Factories — Thu, 15 Mar 2007 04:00:00 +0100

In todays world, there is an increasing trend towards the use of renewable, cheap and readily available biomass in the production of a wide variety of fine and bulk chemicals in different biorefineries. Biorefineries utilize the activities of microbial cells and their enzymes to convert biomass into target products. Many of these processes require enzymes which are operationally stable at high temperature thus allowing e.g. easy mixing, better substrate solubility, high mass transfer rate, and lowered risk of contamination. Thermophiles have often been proposed as sources of industrially relevant thermostable enzymes. Here we discuss existing and potential applications of thermophiles and thermostable enzymes with focus on conversion of carbohydrate containing rawmaterials. Their importan...

Quantifying the metabolic capabilities of engineered Zymomonas mobilis using linear programming analysis

Microbial Cell Factories — Fri, 09 Mar 2007 05:00:00 +0100

Conclusions: Applying LP to study the Z. mobilis physiology enabled the identification of the main factors influencing the accomplishment of certain biological objectives due to metabolic network connectivity only. This first-level metabolic analysis model forms the basis for the incorporation of more complex regulatory mechanisms and the formation of more realistic models for the accurate simulation of the in vivo Z.mobilis physiology. (Source: Microbial Cell Factories)

Molecular and bioengineering strategies to improve alginate and polydydroxyalkanoate production by Azotobacter vinelandii

Microbial Cell Factories — Fri, 16 Feb 2007 07:00:00 +0100

Several aspects of alginate and PHB synthesis in Azotobacter vinelandii at a molecular level have been elucidated in articles published during the last ten years. It is now clear that alginate and PHB synthesis are under a very complex genetic control. Genetic modification of A. vinelandii has produced a number of very interesting mutants which have particular traits for alginate production. One of these mutants has been shown to produce the alginate with the highest mean molecular mass so far reported. Nevertheless, all these mutants produce less alginate (in volumetric terms) than the parental strain. Recent work has also shed light on the factors determining molecular mass distribution; the most important of these being identified as; dissolved oxygen tension and specific growth rate. R...

Molecular and bioengineering strategies to improve alginate and polydydroxyalkanoate production by Azotobacter vinelandii

Microbial Cell Factories — Fri, 16 Feb 2007 05:00:00 +0100

Several aspects of alginate and PHB synthesis in Azotobacter vinelandii at a molecular level have been elucidated in articles published during the last ten years. It is now clear that alginate and PHB synthesis are under a very complex genetic control. Genetic modification of A. vinelandii has produced a number of very interesting mutants which have particular traits for alginate production. One of these mutants has been shown to produce the alginate with the highest mean molecular mass so far reported. Recent work has also shed light on the factors determining molecular mass distribution; the most important of these being identified as; dissolved oxygen tension and specific growth rate. The use of specific mutants has been very useful for the correct analysis and interpretation of the fac...

Fluxome analysis using GC-MS

Microbial Cell Factories — Wed, 07 Feb 2007 07:00:00 +0100

Fluxome analysis aims at the quantitative analysis of in vivo carbon fluxes in metabolic networks, i. e. intracellular activities of enzymes and pathways. It allows investigating the effects of genetic or environmental modifications and thus precisely provides a global perspective on the integrated genetic and metabolic regulation within the intact metabolic network. The experimental and computational approaches developed in this area have revealed fascinating insights into metabolic properties of various biological systems. Most of the comprehensive approaches for metabolic flux studies today involve isotopic tracer studies and GC-MS for measurement of the labeling pattern of metabolites. Initially developed and applied mainly in the field of biomedicine these GC-MS based metabolic flux a...

Comparison of the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant Saccharomyces cerevisiae

Microbial Cell Factories — Mon, 05 Feb 2007 07:00:00 +0100

Conclusions: Despite by-product formation, the XR-XDH xylose utilization pathway resulted in faster ethanol production than using the best presently reported XI pathway in the strain background investigated. The need for robust industrial yeast strains for fermentation of undetoxified spruce hydrolysates was also confirmed. (Source: Microbial Cell Factories)

A perspective on microarrays: current applications, pitfalls, and potential uses

Microbial Cell Factories — Thu, 25 Jan 2007 07:00:00 +0100

With advances in robotics, computational capabilities, and the fabrication of high quality glass slides coinciding with increased genomic information being available on public databases, microarray technology is increasingly being used in laboratories around the world. Fields as varied as: toxicology, evolutionary biology, drug development and production, disease characterization, diagnostics development, cellular physiology and stress responses, and forensics have benefited from its use. However, for many researchers not familiar with microarrays, current articles and reviews often address neither the fundamental principles behind the technology nor the proper designing of experiments. Although, microarray technology is relatively simple, conceptually, its practice does require careful pl...

Identification of novel immunogens in Pasteurella multocida

Microbial Cell Factories — Thu, 18 Jan 2007 07:00:00 +0100

Conclusions: These data show the range of bacterial immunogens recognized by the chicken immune system, including 6 novel immunoreactive proteins. (Source: Microbial Cell Factories)

Polymyxin B as inhibitor of LPS contamination of Schistosoma mansoni recombinant proteins in human cytokine analysis

Microbial Cell Factories — Wed, 03 Jan 2007 07:00:00 +0100

Conclusion: This study showed that Polymyxin B was able to neutralize the effect of endotoxin, as contaminant in S. mansoni recombinant antigens produced in E. coli, in inducing TNF-α and IL-10 production. (Source: Microbial Cell Factories)

Secretion and properties of a hybrid Kluyveromyces lactis-Aspergillus niger β-galactosidase

Microbial Cell Factories — Mon, 18 Dec 2006 07:00:00 +0100

Conclusion: A hybrid protein between K. lactis and A. niger β-galactosidases was constructed that increases the yield of the protein released to the growth medium. Modifications introduced in the construction, besides to improve secretion, conferred to the protein biochemical characteristics of biotechnological interest. (Source: Microbial Cell Factories)

Regulation of methanol utilisation pathway genes in yeasts

Microbial Cell Factories — Thu, 14 Dec 2006 07:00:00 +0100

Methylotrophic yeasts such as Candida boidinii, Hansenula polymorpha, Pichia methanolica and Pichia pastoris are an emerging group of eukaryotic hosts for recombinant protein production with an ever increasing number of applications during the last 30 years. Their applications are linked to the use of strong methanol-inducible promoters derived from genes of the methanol utilisation pathway. These promoters are tightly regulated, highly repressed in presence of non-limiting concentrations of glucose in the medium and strongly induced if methanol is used as carbon source. Several factors involved in this tight control and their regulatory effects have been described so far. This review summarises available data about the regulation of promoters from methanol utilisation pathway genes. Furth...

A novel genetic system for recombinant protein secretion in the Antarctic Pseudoalteromonas haloplanktis TAC125

Microbial Cell Factories — Thu, 14 Dec 2006 07:00:00 +0100

Conclusion: Data presented here demonstrate that the "cold" gene-expression system is efficient since the secretion yield of tested chimeras is always above 80%. These secretion performances place the α-amylase derived secretion system amongst the best heterologous secretion systems in Gram-negative bacteria reported so far. As for the quality of the secreted passenger proteins, data presented suggest that the system also allows the correct disulphide bond formation of chimera components, secreting a fully active passenger. (Source: Microbial Cell Factories)

Antisense RNA based down-regulation of RNaseE in E.coli

Microbial Cell Factories — Tue, 12 Dec 2006 07:00:00 +0100

Conclusion: In difference to eukaryotic cells, where the RNAi technology is widely used, this technology is rather unexplored in bacteria, although different natural systems use antisense RNA-based silencing, and a few studies have earlier indicated the potential of this technology also in prokaryotes. Our results show that even complicated self-regulatory systems such as RNaseE may be controlled by antisense RNA technology, indicating that systems based on antisense RNA expression may have a potential for controlling detrimental factors with plasmid-based constructs in arbitrary strains while maintaining their beneficial characteristics. The study also proved that the RNA sandwich hybridisation technique is directly applicable to quantify small RNA molecules in crude cell extracts, which ...

High yield recombinant penicillin G amidase production and export into the growth medium using Bacillus megaterium

Microbial Cell Factories — Tue, 28 Nov 2006 07:00:00 +0100

Conclusion: The combination of genetic and medium optimization led to an overall 7-fold improvement of PGA production and export in B. megaterium. The exclusion of certain amino acids from the minimal medium led for the first time to higher volumetric PGA activities than obtained for complex medium cultivations. (Source: Microbial Cell Factories)