Showing papers in "Microbial Cell Factories in 2006"

Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review

Abstract: The methylotrophic yeast Pichia pastoris has been widely reported as a suitable expression system for heterologous protein production. The use of different phenotypes under PAOX promoter, other alternative promoters, culture medium, and operational strategies with the objective to maximize either yield or productivity of the heterologous protein, but also to obtain a repetitive product batch to batch to get a robust process for the final industrial application have been reported. Medium composition, kinetics growth, fermentation operational strategies from fed-batch to continuous cultures using different phenotypes with the most common PAOX promoter and other novel promoters (GAP, FLD, ICL), the use of mixed substrates, on-line monitoring of the key fermentation parameters (methanol) and control algorithms applied to the bioprocess are reviewed and discussed in detail.

Miniature bioreactors: current practices and future opportunities

Abstract: This review focuses on the emerging field of miniature bioreactors (MBRs), and examines the way in which they are used to speed up many areas of bioprocessing. MBRs aim to achieve this acceleration as a result of their inherent high-throughput capability, which results from their ability to perform many cell cultivations in parallel. There are several applications for MBRs, ranging from media development and strain improvement to process optimisation. The potential of MBRs for use in these applications will be explained in detail in this review. MBRs are currently based on several existing bioreactor platforms such as shaken devices, stirred-tank reactors and bubble columns. This review will present the advantages and disadvantages of each design together with an appraisal of prototype and commercialised devices developed for parallel operation. Finally we will discuss how MBRs can be used in conjunction with automated robotic systems and other miniature process units to deliver a fully-integrated, high-throughput (HT) solution for cell cultivation process development.

Rhizobial exopolysaccharides: genetic control and symbiotic functions

Abstract: Specific complex interactions between soil bacteria belonging to Rhizobium, Sinorhizobium, Mesorhizobium, Phylorhizobium, Bradyrhizobium and Azorhizobium commonly known as rhizobia, and their host leguminous plants result in development of root nodules. Nodules are new organs that consist mainly of plant cells infected with bacteroids that provide the host plant with fixed nitrogen. Proper nodule development requires the synthesis and perception of signal molecules such as lipochitooligosaccharides, called Nod factors that are important for induction of nodule development. Bacterial surface polysaccharides are also crucial for establishment of successful symbiosis with legumes. Sugar polymers of rhizobia are composed of a number of different polysaccharides, such as lipopolysaccharides (LPS), capsular polysaccharides (CPS or K-antigens), neutral β-1, 2-glucans and acidic extracellular polysaccharides (EPS). Despite extensive research, the molecular function of the surface polysaccharides in symbiosis remains unclear. This review focuses on exopolysaccharides that are especially important for the invasion that leads to formation of indetermined (with persistent meristem) type of nodules on legumes such as clover, vetch, peas or alfalfa. The significance of EPS synthesis in symbiotic interactions of Rhizobium leguminosarum with clover is especially noticed. Accumulating data suggest that exopolysaccharides may be involved in invasion and nodule development, bacterial release from infection threads, bacteroid development, suppression of plant defense response and protection against plant antimicrobial compounds. Rhizobial exopolysaccharides are species-specific heteropolysaccharide polymers composed of common sugars that are substituted with non-carbohydrate residues. Synthesis of repeating units of exopolysaccharide, their modification, polymerization and export to the cell surface is controlled by clusters of genes, named exo/exs, exp or pss that are localized on rhizobial megaplasmids or chromosome. The function of these genes was identified by isolation and characterization of several mutants disabled in exopolysaccharide synthesis. The effect of exopolysaccharide deficiency on nodule development has been extensively studied. Production of exopolysaccharides is influenced by a complex network of environmental factors such as phosphate, nitrogen or sulphur. There is a strong suggestion that production of a variety of symbiotically active polysaccharides may allow rhizobial strains to adapt to changing environmental conditions and interact efficiently with legumes.

Regulation of methanol utilisation pathway genes in yeasts

Abstract: 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. Furthermore, the role of cis and trans acting factors (e.g. transcription factors, glucose processing enzymes) in the expression of methanol utilisation pathway genes is reviewed both in the context of the native cell environment as well as in heterologous hosts.

Live bacterial vaccines – a review and identification of potential hazards

Abstract: The use of live bacteria to induce an immune response to itself or to a carried vaccine component is an attractive vaccine strategy. Advantages of live bacterial vaccines include their mimicry of a natural infection, intrinsic adjuvant properties and their possibility to be administered orally. Derivatives of pathogenic and non-pathogenic food related bacteria are currently being evaluated as live vaccines. However, pathogenic bacteria demands for attenuation to weaken its virulence. The use of bacteria as vaccine delivery vehicles implies construction of recombinant strains that contain the gene cassette encoding the antigen. With the increased knowledge of mucosal immunity and the availability of genetic tools for heterologous gene expression the concept of live vaccine vehicles gains renewed interest. However, administration of live bacterial vaccines poses some risks. In addition, vaccination using recombinant bacteria results in the release of live recombinant organisms into nature. This places these vaccines in the debate on application of genetically modified organisms. In this review we give an overview of live bacterial vaccines on the market and describe the development of new live vaccines with a focus on attenuated bacteria and food-related lactic acid bacteria. Furthermore, we outline the safety concerns and identify the hazards associated with live bacterial vaccines and try to give some suggestions of what to consider during their development.

Applications of cell sorting in biotechnology

Abstract: Due to its unique capability to analyze a large number of single cells for several parameters simultaneously, flow cytometry has changed our understanding of the behavior of cells in culture and of the population dynamics even of clonal populations. The potential of this method for biotechnological research, which is based on populations of living cells, was soon appreciated. Sorting applications, however, are still less frequent than one would expect with regard to their potential. This review highlights important contributions where flow cytometric cell sorting was used for physiological research, protein engineering, cell engineering, specifically emphasizing selection of overproducing cell lines. Finally conclusions are drawn concerning the impact of cell sorting on inverse metabolic engineering and systems biology.

Enzymes: An integrated view of structure, dynamics and function

Abstract: Microbes utilize enzymes to perform a variety of functions. Enzymes are biocatalysts working as highly efficient machines at the molecular level. In the past, enzymes have been viewed as static entities and their function has been explained on the basis of direct structural interactions between the enzyme and the substrate. A variety of experimental and computational techniques, however, continue to reveal that proteins are dynamically active machines, with various parts exhibiting internal motions at a wide range of time-scales. Increasing evidence also indicates that these internal protein motions play a role in promoting protein function such as enzyme catalysis. Moreover, the thermodynamical fluctuations of the solvent, surrounding the protein, have an impact on internal protein motions and, therefore, on enzyme function. In this review, we describe recent biochemical and theoretical investigations of internal protein dynamics linked to enzyme catalysis. In the enzyme cyclophilin A, investigations have lead to the discovery of a network of protein vibrations promoting catalysis. Cyclophilin A catalyzes peptidyl-prolyl cis/trans isomerization in a variety of peptide and protein substrates. Recent studies of cyclophilin A are discussed in detail and other enzymes (dihydrofolate reductase and liver alcohol dehydrogenase) where similar discoveries have been reported are also briefly discussed. The detailed characterization of the discovered networks indicates that protein dynamics plays a role in rate-enhancement achieved by enzymes. An integrated view of enzyme structure, dynamics and function have wide implications in understanding allosteric and co-operative effects, as well as protein engineering of more efficient enzymes and novel drug design.

Co-utilization of L-arabinose and D-xylose by laboratory and industrial Saccharomyces cerevisiae strains

Abstract: Background: Fermentation of lignocellulosic biomass is an attractive alternative for the production of bioethanol. Traditionally, the yeast Saccharomyces cerevisiae is used in industrial ethanol fermentations. However, S. cerevisiae is naturally not able to ferment the pentose sugars D-xylose and L-arabinose, which are present in high amounts in lignocellulosic raw materials. Results: We describe the engineering of laboratory and industrial S. cerevisiae strains to coferment the pentose sugars D-xylose and L-arabinose. Introduction of a fungal xylose and a bacterial arabinose pathway resulted in strains able to grow on both pentose sugars. Introduction of a xylose pathway into an arabinose-fermenting laboratory strain resulted in nearly complete conversion of arabinose into arabitol due to the L-arabinose reductase activity of the xylose reductase. The industrial strain displayed lower arabitol yield and increased ethanol yield from xylose and arabinose. Conclusion: Our work demonstrates simultaneous co-utilization of xylose and arabinose in recombinant strains of S. cerevisiae. In addition, the co-utilization of arabinose together with xylose significantly reduced formation of the by-product xylitol, which contributed to improved ethanol production.

Brucella spp noncanonical LPS: structure, biosynthesis, and interaction with host immune system.

Abstract: Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and non-professional phagocytes, and cause abortion in domestic animals and undulant fever in humans. Several species are recognized within the genus Brucella and this classification is mainly based on the difference in pathogenicity and in host preference. Brucella strains may occur as either smooth or rough, expressing smooth LPS (S-LPS) or rough LPS (R-LPS) as major surface antigen. This bacterium possesses an unconventional non-endotoxic lipopolysaccharide that confers resistance to anti-microbial attacks and modulates the host immune response. The strains that are pathogenic for humans (B. abortus, B. suis, B. melitensis) carry a smooth LPS involved in the virulence of these bacteria. The LPS O-chain protects the bacteria from cellular cationic peptides, oxygen metabolites and complement-mediated lysis and it is a key molecule for Brucella survival and replication in the host. Here, we review i) Brucella LPS structure; ii) Brucella genome, iii) genes involved in LPS biosynthesis; iv) the interaction between LPS and innate immunity.

Biosynthesis of isoprenoids, polyunsaturated fatty acids and flavonoids in Saccharomyces cerevisiae

Abstract: Industrial biotechnology employs the controlled use of microorganisms for the production of synthetic chemicals or simple biomass that can further be used in a diverse array of applications that span the pharmaceutical, chemical and nutraceutical industries. Recent advances in metagenomics and in the incorporation of entire biosynthetic pathways into Saccharomyces cerevisiae have greatly expanded both the fitness and the repertoire of biochemicals that can be synthesized from this popular microorganism. Further, the availability of the S. cerevisiae entire genome sequence allows the application of systems biology approaches for improving its enormous biosynthetic potential. In this review, we will describe some of the efforts on using S. cerevisiae as a cell factory for the biosynthesis of high-value natural products that belong to the families of isoprenoids, flavonoids and long chain polyunsaturated fatty acids. As natural products are increasingly becoming the center of attention of the pharmaceutical and nutraceutical industries, the use of S. cerevisiae for their production is only expected to expand in the future, further allowing the biosynthesis of novel molecular structures with unique properties.

Morphological development of Aspergillus niger in submerged citric acid fermentation as a function of the spore inoculum level. Application of neural network and cluster analysis for characterization of mycelial morphology.

Abstract: Although the citric acid fermentation by Aspergillus niger is one of the most important industrial microbial processes and various aspects of the fermentation appear in a very large number of publications since the 1950s, the effect of the spore inoculum level on fungal morphology is a rather neglected area. The aim of the presented investigations was to quantify the effects of changing spore inoculum level on the resulting mycelial morphology and to investigate the physiology that underlines the phenomena. Batch fermentations were carried out in a stirred tank bioreactor, which were inoculated directly with spores in concentrations ranging from 104 to 109 spores per ml. Morphological features, evaluated by digital image analysis, were classified using an artificial neural network (ANN), which considered four main object types: globular and elongated pellets, clumps and free mycelial trees. The significance of the particular morphological features and their combination was determined by cluster analysis. Cell volume fraction analysis for the various inoculum levels tested revealed that by rising the spore inoculum level from 104 to 109 spores per ml, a clear transition from pelleted to dispersed forms occurs. Glucosamine formation and release by the mycelium appears to be related to spore inoculum level. Maximum concentrations detected in fermentations inoculated with 104 and 105 spores/ml, where pellets predominated. At much higher inoculum levels (108, 109 spores/ml), lower dissolved oxygen levels during the early fermentation phase were associated with slower ammonium ions uptakes and significantly lower glucosamine concentrations while the mycelium developed in dispersed morphologies. A big increase in the main and total hyphal lengths and branching frequency was observed in mycelial trees as inoculum levels rise from 104 to 109 spores/ml, while in aggregated forms particle sizes and their compactness decreased. The methods used in this study, allowed for the detailed quantification of the transition between the two extreme morphological forms. The impact of spore inoculum level on the detailed characteristics of the particular morphological forms produced was high. Control of mycelial morphology is often regarded as a prerequisite to ensure increased productivities in industrial applications. The research described here demonstrates that adjusting the spore inoculum level controls effectively mycelial morphology.

Improvement of bioprocess monitoring: development of novel concepts

Abstract: The advancement of bioprocess monitoring will play a crucial role to meet the future requirements of bioprocess technology. Major issues are the acceleration of process development to reduce the time to the market and to ensure optimal exploitation of the cell factory and further to cope with the requirements of the Process Analytical Technology initiative. Due to the enormous complexity of cellular systems and lack of appropriate sensor systems microbial production processes are still poorly understood. This holds generally true for the most microbial production processes, in particular for the recombinant protein production due to strong interaction between recombinant gene expression and host cell metabolism. Therefore, it is necessary to scrutinise the role of the different cellular compartments in the biosynthesis process in order to develop comprehensive process monitoring concepts by involving the most significant process variables and their interconnections. Although research for the development of novel sensor systems is progressing their applicability in bioprocessing is very limited with respect to on-line and in-situ measurement due to specific requirements of aseptic conditions, high number of analytes, drift, and often rather low physiological relevance. A comprehensive survey of the state of the art of bioprocess monitoring reveals that only a limited number of metabolic variables show a close correlation to the currently explored chemical/physical principles. In order to circumvent this unsatisfying situation mathematical methods are applied to uncover "hidden" information contained in the on-line data and thereby creating correlations to the multitude of highly specific biochemical off-line data. Modelling enables the continuous prediction of otherwise discrete off-line data whereby critical process states can be more easily detected. The challenging issue of this concept is to establish significant on-line and off-line data sets. In this context, online sensor systems are reviewed with respect to commercial availability in combination with the suitability of offline analytical measurement methods. In a case study, the aptitude of the concept to exploit easily available online data for prediction of complex process variables in a recombinant E. coli fed-batch cultivation aiming at the improvement of monitoring capabilities is demonstrated. In addition, the perspectives for model-based process supervision and process control are outlined.

Metabolic engineering of microbes for oligosaccharide and polysaccharide synthesis

Abstract: Metabolic engineering has recently been embraced as an effective tool for developing whole-cell biocatalysts for oligosaccharide and polysaccharide synthesis. Microbial catalysts now provide a practical means to derive many valuable oligosaccharides, previously inaccessible through other methods, in sufficient quantities to support research and clinical applications. The synthesis process based upon these microbes is scalable as it avoids expensive starting materials. Most impressive is the high product concentrations (up to 188 g/L) achieved through microbe-catalyzed synthesis. The overall cost for selected molecules has been brought to a reasonable range (estimated $ 30–50/g). Microbial synthesis of oligosaccharides and polysaccharides is a carbon-intensive and energy-intensive process, presenting some unique challenges in metabolic engineering. Unlike nicotinamide cofactors, the required sugar nucleotides are products of multiple interacting pathways, adding significant complexity to the metabolic engineering effort. Besides the challenge of providing the necessary mammalian-originated glycosyltransferases in active form, an adequate uptake of sugar acceptors can be an issue when another sugar is necessary as a carbon and energy source. These challenges are analyzed, and various strategies used to overcome these difficulties are reviewed in this article. Despite the impressive success of the microbial coupling strategy, there is a need to develop a single strain that can achieve at least the same efficiency. Host selection and the manner with which the synthesis interacts with the central metabolism are two important factors in the design of microbial catalysts. Additionally, unlike in vitro enzymatic synthesis, product degradation and byproduct formation are challenges of whole-cell systems that require additional engineering. A systematic approach that accounts for various and often conflicting requirements of the synthesis holds the key to deriving an efficient catalyst. Metabolic engineering strategies applied to selected polysaccharides (hyaluronan, alginate, and exopolysaccharides for food use) are reviewed in this article to highlight the recent progress in this area and similarity to challenges in oligosaccharide synthesis. Many naturally occurring microbes possess highly efficient mechanisms for polysaccharide synthesis. These mechanisms could potentially be engineered into a microbe for oligosaccharide and polysaccharide synthesis with enhanced efficiency.

A general method for selection of riboflavin-overproducing food grade micro-organisms

Abstract: This study describes a strategy to select and isolate spontaneous riboflavin-overproducing strains of Lactobacillus (Lb.) plantarum, Leuconostoc (Lc.) mesenteroides and Propionibacterium (P.) freudenreichii. The toxic riboflavin analogue roseoflavin was used to isolate natural riboflavin-overproducing variants of the food grade micro-organisms Lb. plantarum, Lc. mesenteroides and P. freudenreichii strains. The method was successfully employed for strains of all three species. The mutation(s) responsible for the observed overproduction of riboflavin were identified for isolates of two species. Selection for spontaneous roseoflavin-resistant mutants was found to be a reliable method to obtain natural riboflavin-overproducing strains of a number of species commonly used in the food industry. This study presents a convenient method for deriving riboflavin-overproducing strains of bacterial starter cultures, which are currently used in the food industry, by a non-recombinant methodology. Use of such starter strains can be exploited to increase the vitamin content in certain food products.

Versatile modeling and optimization of fed batch processes for the production of secreted heterologous proteins with Pichia pastoris

Abstract: Background Secretion of heterologous proteins depends both on biomass concentration and on the specific product secretion rate, which in turn is not constant at varying specific growth rates. As fed batch processes usually do not maintain a steady state throughout the feed phase, it is not trivial to model and optimize such a process by mathematical means.

Heterologous expression of lcc1 gene from Trametes trogii in Pichia pastoris and characterization of the recombinant enzyme

Abstract: Background: Fungal laccases are useful enzymes for industrial applications; they exhibit broad substrate specificity and thus are able to oxidize a variety of xenobiotic compounds including chlorinated phenolics, synthetic dyes, pesticides and polycyclic aromatic hydrocarbons. Unfortunately, the biotechnological exploitation of laccases can be hampered by the difficulties concerning the enzyme production by the native hosts. Results: In order to obtain a simple and efficient source of laccase, the lcc1 cDNA isolated from the white-rot fungus Trametes trogii has been successfully expressed in the methylotrophic yeast Pichia pastoris under the control of the methanol induced alcohol oxidase promoter P AOX1 . The recombinant Lcc1 was produced as a secreted protein with the native N-terminal prepropeptide for signal trafficking, and thus easily recovered from the culture medium. At the 1-liter scale, as calculated on the basis of the specific activity, the recombinant protein was produced at a yield of 17 mg/l. The highest production level obtained in fed-batch culture was 2520 U/l, corresponding to a specific productivity of 31.5 U/g biomass. The purified recombinant laccase exhibited a behaviour similar to the main laccase produced by T. trogii. Lcc1 showed high activity in the presence of organic solvents and a high decolourization capacity towards azo, triarylmethane, indigo carmine and anthraquinonic dyes, that could be significantly enhanced in the presence of the redox mediators 1-hydroxybenzotriazole and violuric acid. Conclusion: Heterologous expression of T. trogii laccase lcc1 in the methylotrophic yeast P. pastoris was successfully achieved. The biochemical and kinetic characterization of the recombinant protein suggests potential technological applications for this enzyme.

Lactate production yield from engineered yeasts is dependent from the host background, the lactate dehydrogenase source and the lactate export

Abstract: Metabolic pathway manipulation for improving the properties and the productivity of microorganisms is becoming a well established concept. For the production of important metabolites, but also for a better understanding of the fundamentals of cell biology, detailed studies are required. In this work we analysed the lactate production from metabolic engineered Saccharomyces cerevisiae cells expressing a heterologous lactate dehydrogenase (LDH) gene. The LDH gene expression in a budding yeast cell introduces a novel and alternative pathway for the NAD+ regeneration, allowing a direct reduction of the intracellular pyruvate to lactate, leading to a simultaneous accumulation of lactate and ethanol. Four different S. cerevisiae strains were transformed with six different wild type and one mutagenised LDH genes, in combination or not with the over-expression of a lactate transporter. The resulting yield values (grams of lactate produced per grams of glucose consumed) varied from as low as 0,0008 to as high as 0.52 g g-1. In this respect, and to the best of our knowledge, higher redirections of the glycolysis flux have never been obtained before without any disruption and/or limitation of the competing biochemical pathways. In the present work it is shown that the redirection of the pathway towards the lactate production can be strongly modulated by the genetic background of the host cell, by the source of the heterologous Ldh enzyme, by improving its biochemical properties as well as by modulating the export of lactate in the culture media.

Effects of the presence of ColE1 plasmid DNA in Escherichia coli on the host cell metabolism

Abstract: Although understanding of physiological interactions between plasmid DNA and its host is important for vector design and host optimization in many biotechnological applications, to our knowledge, global studies on plasmid-host interactions have not been performed to date even for well-characterized plasmids. Escherichia coli cells, either devoid of plasmid DNA or bearing plasmid pOri1 (with a single ColE1 replication origin) or plasmid pOri2 (with double ColE1 replication origins), were cultured in a chemostat. We used a combination of metabolic flux analysis, DNA microarray and enzyme activity analysis methods to explore differences in the metabolism between these strains. We found that the presence of plasmids significantly influenced various metabolic pathways in the host cells, e.g. glycolysis, the tricarboxylic acid (TCA) cycle and the pentose phosphate (PP) pathway. Expression of rpiA, a gene coding for ribose-5-phosphate isomerase A, was considerably decreased in E. coli carrying a high copy number plasmid relative to E. coli carrying a low copy number plasmid and plasmid-free E. coli. The rpiA gene was cloned into an expression vector to construct plasmid pETrpiA. Following induction of pETrpiA-bearing E. coli, which harbored either pOri1 or pOri2, with isopropyl-β-D-thiogalactopyranoside (IPTG), the copy number of pOri1 and pOri2 was sigificantly higher than that measured in a host devoid of pETrpiA. The presence of plasmids can significantly influence some metabolic pathways in the host cell. We believe that the results of detailed metabolic analysis may be useful in optimizing host strains, vectors and cultivation conditions for various biotechnological purposes.

Identification of anthranilate and benzoate metabolic operons of Pseudomonas fluorescens and functional characterization of their promoter regions

Abstract: In an effort to identify alternate recombinant gene expression systems in Pseudomonas fluorescens, we identified genes encoding two native metabolic pathways that were inducible with inexpensive compounds: the anthranilate operon (antABC) and the benzoate operon (benABCD). The antABC and benABCD operons were identified by homology to the Acinetobacter sp. anthranilate operon and Pseudomonas putida benzoate operon, and were confirmed to be regulated by anthranilate or benzoate, respectively. Fusions of the putative promoter regions to the E. coli lacZ gene were constructed to confirm inducible gene expression. Each operon was found to be controlled by an AraC family transcriptional activator, located immediately upstream of the first structural gene in each respective operon (antR or benR). We have found the anthranilate and benzoate promoters to be useful for tightly controlling recombinant gene expression at both small (< 1 L) and large (20 L) fermentation scales.

Revisiting the Cellulosimicrobium cellulans yeast-lytic β-1,3-glucanases toolbox: A review

Abstract: Cellulosimicrobium cellulans (also known with the synonyms Cellulomonas cellulans, Oerskovia xanthineolytica, and Arthrobacter luteus) is an actinomycete that excretes yeast cell wall lytic enzyme complexes containing endo-β-1,3-glucanases [EC 3.2.1.39 and 3.2.1.6] as key constituents. Three genes encoding endo-β-1,3-glucanases from two C. cellulans strains have been cloned and characterised over the past years. The βglII and βglII A genes from strain DSM 10297 (also known as O. xanthineolytica LL G109) encoded proteins of 40.8 and 28.6 kDa, respectively, whereas the β-1,3-glucanase gene from strain ATCC 21606 (also known as A. luteus 73–14) encoded a 54.5 kDa protein. Alignment of their deduced amino acid sequences reveal that βglII and βglII A have catalytic domains assigned to family 16 of glycosyl hydrolases, whereas the catalytic domain from the 54.5 kDa glucanase belongs to family 64. Notably, both βglII and the 54.5 kDa β-1,3-glucanase are multidomain proteins, having a lectin-like C-terminal domain that has been assigned to family 13 of carbohydrate binding modules, and that confers to β-1,3-glucanases the ability to lyse viable yeast cells. Furthermore, βglII may also undergo posttranslational proteolytic processing of its C-terminal domain, resulting in a truncated enzyme retaining its glucanase activity but with very low yeast-lytic activity. In this review, the diversity in terms of structural and functional characteristics of the C. cellulans β-1,3-glucanases has been compiled and compared.

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

Abstract: During the last years B. megaterium was continuously developed as production host for the secretion of proteins into the growth medium. Here, recombinant production and export of B. megaterium ATCC14945 penicillin G amidase (PGA) which is used in the reverse synthesis of β-lactam antibiotics were systematically improved. For this purpose, the PGA leader peptide was replaced by the B. megaterium LipA counterpart. A production strain deficient in the extracellular protease NprM and in xylose utilization to prevent gene inducer deprivation was constructed and employed. A buffered mineral medium containing calcium ions and defined amino acid supplements for optimal PGA production was developed in microscale cultivations and scaled up to a 2 Liter bioreactor. Productivities of up to 40 mg PGA per L growth medium were reached. 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.

A new wireless system for decentralised measurement of physiological parameters from shake flasks.

Abstract: Background Shake flasks are widely used because of their low price and simple handling. Many researcher are, however, not aware of the physiological consequences of oxygen limitation and substrate overflow metabolism that occur in shake flasks. Availability of a wireless measuring system brings the possibilities for quality control and design of cultivation conditions.

Quantification of the fractal nature of mycelial aggregation in Aspergillus niger submerged cultures

Abstract: Fractal geometry estimates have proven useful in studying the growth strategies of fungi in response to different environments on soil or on agar substrates, but their use in mycelia grown submerged is still rare. In the present study, the effects of certain important fermentation parameters, such as the spore inoculum level, phosphate and manganese concentrations in the medium, on mycelial morphology of the citric acid producer Aspergillus niger were determined by fractal geometry. The value of employing fractal geometry to describe mycelial structures was examined in comparison with information from other descriptors including classic morphological parameters derived from image analysis. Fractal analysis of distinct morphological forms produced by fermentation conditions that influence fungal morphology and acid production, showed that the two fractal dimensions DBS (box surface dimension) and DBM (box mass dimension) are very sensitive indexes, capable of describing morphological differences. The two box-counting methods applied (one applied to the whole mass of the mycelial particles and the other applied to their surface only) enabled evaluation of fractal dimensions for mycelial particles in this analysis in the region of DBS = 1.20–1.70 and DBM = 1.20–2.70. The global structure of sufficiently branched mycelia was described by a single fractal dimension D, which did not exceed 1.30. Such simple structures are true mass fractals (DBS = DBM = D) and they could be young mycelia or dispersed forms of growth produced by very dense spore inocula (108–109 spores/ml) or by addition of manganese in the medium. Mycelial clumps and pellets were effectively discriminated by fractal analysis. Fractal dimension values were plotted together with classic morphological parameters derived from image analysis for comparisons. Their sensitivity to treatment was analogous to the sensitivity of classic morphological parameters suggesting that they could be equally used as morphological descriptors. Starting from a spore, the mycelium develops as a mass fractal and, depending on culture conditions, it either turns to a surface fractal or remains a mass fractal. Since fractal dimensions give a measure of the degree of complexity and the mass filling properties of an object, it may be possible that a large number of morphological parameters which contribute to the overall complexity of the particles, could be replaced by these indexes effectively.

Determination of bacteriocin activity with bioassays carried out on solid and liquid substrates: assessing the factor "indicator microorganism"

Abstract: Successful application of growth inhibition techniques for quantitative determination of bacteriocins relies on the sensitivity of the applied indicator microorganism to the bacteriocin to which is exposed. However, information on indicator microorganisms' performance and comparisons in bacteriocin determination with bioassays is almost non-existing in the literature. The aim of the present work was to evaluate the parameter "indicator microorganism" in bioassays carried out on solid -agar diffusion assay- and liquid -turbidometric assay- substrates, applied in the quantification of the most studied bacteriocin nisin. The performance of characterized microorganisms of known sources, belonging to the genera of Lactobacillus, Pediococcus, Micrococcus and Leuconostoc, has been assessed in this work in the assays of plate agar diffusion and turbidometry. Dose responses and sensitivities were examined and compared over a range of assay variables in standard bacteriocin solutions, fermentation broth filtrates and processed food samples. Measurements on inhibition zones produced on agar plates were made by means of digital image analysis. The data produced were analyzed statistically using the ANOVA technique and pairwise comparisons tests. Sensitivity limits and linearity of responses to bacteriocin varied significantly among different test-microorganisms in both applied methods, the lower sensitivity limits depending on both the test-microorganism and the applied method. In both methods, however, only two of the nine tested microorganisms (Lactobacillus curvatus ATCC 51436 and Pediococcus acidilactici ATCC 25740) were sensitive to very low concentrations of the bacteriocin and produced a linear-type of response in all kinds of samples used in this work. In all cases, very low bacteriocin concentrations, e.g. 1 IU/ml nisin, were more accurately determined in the turbidometric assay. The present work shows that in growth inhibition techniques used in bacteriocin quantification, the choice of the indicator microorganism is critical. Evaluation of sensitivity levels and type of produced responses showed that they can vary widely among different test-microorganisms and different applied methods, indicating that not all microorganisms can be used successfully as indicators and that measurements of growth inhibition in liquid media produce more reliable results.

Surface display of proteins by Gram-negative bacterial autotransporters

Abstract: Expressing proteins of interest as fusions to proteins of the bacterial envelope is a powerful technique with many biotechnological and medical applications. Autotransporters have recently emerged as a good tool for bacterial surface display. These proteins are composed of an N-terminal signal peptide, followed by a passenger domain and a translocator domain that mediates the outer membrane translocation of the passenger. The natural passenger domain of autotransporters can be replaced by heterologous proteins that become displayed at the bacterial surface by the translocator domain. The simplicity and versatility of this system has made it very attractive and it has been used to display functional enzymes, vaccine antigens as well as polypeptides libraries. The recent advances in the study of the translocation mechanism of autotransporters have raised several controversial issues with implications for their use as display systems. These issues include the requirement for the displayed polypeptides to remain in a translocation-competent state in the periplasm, the requirement for specific signal sequences and "autochaperone" domains, and the influence of the genetic background of the expression host strain. It is therefore important to better understand the mechanism of translocation of autotransporters in order to employ them to their full potential. This review will focus on the recent advances in the study of the translocation mechanism of autotransporters and describe practical considerations regarding their use for bacterial surface display.

Developing nucleic acid-based electrical detection systems.

Abstract: Development of nucleic acid-based detection systems is the main focus of many research groups and high technology companies The enormous work done in this field is particularly due to the broad versatility and variety of these sensing devices From optical to electrical systems, from label-dependent to label-free approaches, from single to multi-analyte and array formats, this wide range of possibilities makes the research field very diversified and competitive New challenges and requirements for an ideal detector suitable for nucleic acid analysis include high sensitivity and high specificity protocol that can be completed in a relatively short time offering at the same time low detection limit Moreover, systems that can be miniaturized and automated present a significant advantage over conventional technology, especially if detection is needed in the field Electrical system technology for nucleic acid-based detection is an enabling mode for making miniaturized to micro- and nanometer scale bio-monitoring devices via the fusion of modern micro- and nanofabrication technology and molecular biotechnology The electrical biosensors that rely on the conversion of the Watson-Crick base-pair recognition event into a useful electrical signal are advancing rapidly, and recently are receiving much attention as a valuable tool for microbial pathogen detection Pathogens may pose a serious threat to humans, animal and plants, thus their detection and analysis is a significant element of public health Although different conventional methods for detection of pathogenic microorganisms and their toxins exist and are currently being applied, improvements of molecular-based detection methodologies have changed these traditional detection techniques and introduced a new era of rapid, miniaturized and automated electrical chip detection technologies into pathogen identification sector In this review some developments and current directions in nucleic acid-based electrical detection are discussed

Intensification of the aerobic bioremediation of an actual site soil historically contaminated by polychlorinated biphenyls (pcbs) through bioaugmentation with a non acclimated, complex source of microorganisms

Abstract: The biotreatability of actual-site polychlorinated biphenyl (PCB)-contaminated soils is often limited by their poor content of autochthonous pollutant-degrading microorganisms. In such cases, inoculation might be the solution for a successful bioremediation. Some pure and mixed cultures of characterized PCB degrading bacteria have been tested to this purpose. However, several failures have been recorded mostly due to the inability of inoculated microbes to compete with autochthonous microflora and to face the toxicity and the scarcity of nutrients occurring in the contaminated biotope. Complex microbial systems, such as compost or sludge, normally consisting of a large variety of robust microorganisms and essential nutrients, would have better chances to succeed in colonizing degraded contaminated soils. However, such sources of microorganisms have been poorly applied in soil bioremediation and in particular in the biotreatment of soil with PCBs. Thus, in this study the effects of Enzyveba, i.e. a consortium of non-adapted microorganisms developed from composted material, on the slurry- and solid-phase aerobic bioremediation of an actual-site, aged PCB-contaminated soil were studied. A slow and only partial biodegradation of low-chlorinated biphenyls, along with a moderate depletion of initial soil ecotoxicity, were observed in the not-inoculated reactors. Enzyveba significantly increased the availability and the persistence of aerobic PCB- and chlorobenzoic acid-degrading cultivable bacteria in the bioreactors, in particular during the earlier phase of treatment. It also markedly enhanced PCB-biodegradation rate and extent (from 50 to 100%) as well as the final soil detoxification, in particular under slurry-phase conditions. Taken together, data obtained suggest that Enzyveba enhanced the biotreatability of the selected soil by providing exogenous bacteria and fungi able to remove inhibitory or toxic intermediates of PCB biodegradation and/or exogenous nutrients able to sustain microorganisms in charge for PCB mineralization. Enzyveba appears a promising agent for bioaugmenting actual-site PCB-polluted soils with a native low content of indigenous specialized microflora. This not only for its positive effects on the soil biotreatability but also for its availability on the market at a relatively low cost.

Application of a wide-range yeast vector (CoMed™) system to recombinant protein production in dimorphic Arxula adeninivorans, methylotrophic Hansenula polymorpha and other yeasts

Abstract: Yeasts provide attractive expression platforms in combining ease of genetic manipulation and fermentation of a microbial organism with the capability to secrete and to modify proteins according to a general eukaryotic scheme However, early restriction to a single yeast platform can result in costly and time-consuming failures It is therefore advisable to assess several selected systems in parallel for the capability to produce a particular protein in desired amounts and quality A suitable vector must contain a targeting sequence, a promoter element and a selection marker that function in all selected organisms These criteria are fulfilled by a wide-range integrative yeast expression vector (CoMed™) system based on A adeninivorans- and H polymorpha- derived elements that can be introduced in a modular way The vector system and a selection of modular elements for vector design are presented Individual single vector constructs were used to transform a range of yeast species Various successful examples are described A vector with a combination of an rDNA sequence for genomic targeting, the E coli- derived hph gene for selection and the A adeninivorans-derived TEF1 promoter for expression control of a GFP (green fluorescent protein) gene was employed in a first example to transform eight different species including Hansenula polymorpha, Arxula adeninivorans and others In a second example, a vector for the secretion of IL-6 was constructed, now using an A adeninivorans-derived LEU2 gene for selection of recombinants in a range of auxotrophic hosts In this example, differences in precursor processing were observed: only in A adeninivorans processing of a MFα1/IL-6 fusion was performed in a faithful way rDNA targeting provides a tool to co-integrate up to 3 different expression plasmids by a single transformation step Thus, a versatile system is at hand that allows a comparative assessment of newly introduced metabolic pathways in several organisms or a comparative co-expression of bottleneck genes in cases where production or secretion of a certain product is impaired

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

Abstract: Background The β-galactosidase from Kluyveromyces lactis is a protein of outstanding biotechnological interest in the food industry and milk whey reutilization. However, due to its intracellular nature, its industrial production is limited by the high cost associated to extraction and downstream processing. The yeast-system is an attractive method for producing many heterologous proteins. The addition of a secretory signal in the recombinant protein is the method of choice to sort it out of the cell, although biotechnological success is not guaranteed. The cell wall acting as a molecular sieve to large molecules, culture conditions and structural determinants present in the protein, all have a decisive role in the overall process. Protein engineering, combining domains of related proteins, is an alternative to take into account when the task is difficult. In this work, we have constructed and analyzed two hybrid proteins from the β-galactosidase of K. lactis, intracellular, and its Aspergillus niger homologue that is extracellular. In both, a heterologous signal peptide for secretion was also included at the N-terminus of the recombinant proteins. One of the hybrid proteins obtained has interesting properties for its biotechnological utilization.

The chaperone DnaK controls the fractioning of functional protein between soluble and insoluble cell fractions in inclusion body-forming cells

Abstract: The molecular mechanics of inclusion body formation is still far from being completely understood, specially regarding the occurrence of properly folded, protein species that exhibit natural biological activities. We have here comparatively explored thermally promoted, in vivo protein aggregation and the formation of bacterial inclusion bodies, from both structural and functional sides. Also, the status of the soluble and insoluble protein versions in both aggregation systems have been examined as well as the role of the main molecular chaperones GroEL and DnaK in the conformational quality of the target polypeptide. While thermal denaturation results in the formation of heterogeneous aggregates that are rather stable in composition, protein deposition as inclusion bodies renders homogenous but strongly evolving structures, which are progressively enriched in the main protein species while gaining native-like structure. Although both type of aggregates display common features, inclusion body formation but not thermal-induced aggregation involves deposition of functional polypeptides that confer biological activity to such particles, at expenses of the average conformational quality of the protein population remaining in the soluble cell fraction. In absence of DnaK, however, the activity and conformational nativeness of inclusion body proteins are dramatically impaired while the soluble protein version gains specific activity. The chaperone DnaK controls the fractioning of active protein between soluble and insoluble cell fractions in inclusion body-forming cells but not during thermally-driven protein aggregation. This cell protein, probably through diverse activities, is responsible for the occurrence and enrichment in inclusion bodies of native-like, functional polypeptides, that are much less represented in other kind of protein aggregates.