Heterologous expression of glucose oxidase in the yeast <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis>

Background  

In spite of its advantageous physiological properties for bioprocess applications, the use of the yeast Kluyveromyces marxianus as a host for heterologous protein production has been very limited, in constrast to its close relative Kluyveromyces lactis. In the present work, the model protein glucose oxidase (GOX) from Aspergillus niger was cloned into K. marxianus CBS 6556 and into K. lactis CBS 2359 using three different expression systems. We aimed at verifying how each expression system would affect protein expression, secretion/localization, post-translational modification, and biochemical properties.     

Effects of <Emphasis Type="Italic">crp</Emphasis> deletion in <Emphasis Type="Italic">Salmonella enterica</Emphasis> serotype Gallinarum

Background  

Salmonella enterica serotype Gallinarum (S. Gallinarum) remains an important pathogen of poultry, especially in developing countries. There is a need to develop effective and safe vaccines. In the current study, the effect of crp deletion was investigated with respect to virulence and biochemical properties and the possible use of a deletion mutant as vaccine candidate was preliminarily tested.     

RecA Proteins from <Emphasis Type="Italic">Deinococcus geothermalis</Emphasis> and <Emphasis Type="Italic">Deinococcus murrayi</Emphasis> - Cloning,Purification and Biochemical Characterisation

Background  

Escherichia coli RecA plays a crucial role in recombinational processes, the induction of SOS responses and mutagenic lesion bypasses. It has also been demonstrated that RecA protein is indispensable when it comes to the reassembly of shattered chromosomes in γ-irradiated Deinococcus radiodurans, one of the most radiation-resistant organisms known. Moreover, some functional differences between E. coli and D. radiodurans RecA proteins have also been shown.     

Optimization of the <Emphasis Type="Italic">Lactococcus lactis</Emphasis> nisin-controlled gene expression system NICE for industrial applications

Background  

The nisin-controlled gene expression system NICE of Lactococcus lactis is one of the most widely used expression systems in Gram-positive bacteria. Despite its widespread use, no optimization of the culture conditions and nisin induction has been carried out to obtain maximum yields. As a model system induced production of lysostaphin, an antibacterial protein (mainly against Staphylococcus aureus) produced by S. simulans biovar. Staphylolyticus, was used. Three main areas need optimization for maximum yields: cell density, nisin-controlled induction and protein production, and parameters specific for the target-protein.     

<Emphasis Type="Italic">In silico</Emphasis>, biologically-inspired modelling of genomic variation generation in surface proteins of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Background  

Protozoan parasites improve the likelihood of invading or adapting to the host through their capacity to present a large repertoire of surface molecules. The understanding of the mechanisms underlying the generation of antigenic diversity is crucial to aid in the development of therapies and the study of evolution. Despite advances driven by molecular biology and genomics, there is a need to gain a deeper understanding of key properties that may facilitate variation generation, models for explaining the role of genomic re-arrangements and the characterisation of surface protein families on the basis of their capacity to generate variation. Computer models may be implemented to explore, visualise and estimate the variation generation capacity of gene families in a dynamic fashion. In this paper we report the dynamic simulation of genomic variation using real T. cruzi coding sequences as inputs to a computational simulation system. The effects of random, multiple-point mutations and gene conversions on genomic variation generation were quantitatively estimated and visualised. Simulations were also implemented to investigate the potential role of pseudogenes as a source of antigenic variation in T. cruzi.     

Quorum sensing regulation in <Emphasis Type="Italic">Pseudomonas</Emphasis>

Expression of many bacterial genes is regulated in a cell density-dependent manner via small signal molecules known as autoinducers; this type of regulation is termed quorum sensing (QS). The QS systems that employ N-acyl-homoserine lactones (HSLs) are best un derstood in Gram-negative bacteria. QS regulates expression of various genes, including the genes responsible for the production of virulence factors, synthesis of exoenzymes and antibiotics, antagonistic properties of bacteria, etc. The QS systems of the genus Pseudomonas are linked to other global regulatory networks of the cell, and their functions are controlled by numerous additional regulatory factors. Such regulators and the QS systems together form an intricate multifactorial cascade regulatory network. The review considers the QS systems of several Pseudomonas species, their interaction with other regulatory systems, and their roles in the regulation of cell processes.     

Utilizing high-throughput experimentation to enhance specific productivity of an <Emphasis Type="Italic">E.coli</Emphasis>T7 expression system by phosphate limitation

Background  

The specific productivity of cultivation processes can be optimized, amongst others, by using genetic engineering of strains, choice of suitable host/vector systems or process optimization (e.g. choosing the right induction time). A further possibility is to reduce biomass buildup in favor of an enhanced product formation, e.g. by limiting secondary substrates in the medium, such as phosphate. However, with conventional techniques (e.g. small scale cultivations in shake flasks), it is very tedious to establish optimal conditions for cell growth and protein expression, as the start of protein expression (induction time) and the degree of phosphate limitation have to be determined in numerous concerted, manually conducted experiments.     

Plasmid selection in <Emphasis Type="Italic">Escherichia coli</Emphasis> using an endogenous essential gene marker

Background  

Antibiotic resistance genes are widely used for selection of recombinant bacteria, but their use risks contributing to the spread of antibiotic resistance. In particular, the practice is inappropriate for some intrinsically resistant bacteria and in vaccine production, and costly for industrial scale production. Non-antibiotic systems are available, but require mutant host strains, defined media or expensive reagents. An unexplored concept is over-expression of a host essential gene to enable selection in the presence of a chemical inhibitor of the gene product. To test this idea in E. coli, we used the growth essential target gene fabI as the plasmid-borne marker and the biocide triclosan as the selective agent.     

Control of <Emphasis Type="Italic">gag-pol</Emphasis> gene expression in the <Emphasis Type="Italic">Candida albicans</Emphasis> retrotransposon Tca2

Background  

In the C. albicans retrotransposon Tca2, the gag and pol ORFs are separated by a UGA stop codon, 3' of which is a potential RNA pseudoknot. It is unclear how the Tca2 gag UGA codon is bypassed to allow pol expression. However, in other retroelements, translational readthrough of the gag stop codon can be directed by its flanking sequence, including a 3' pseudoknot.     

Growth and recombinant protein expression with <Emphasis Type="Italic">Escherichia coli</Emphasis> in different batch cultivation media

Parallel operated milliliter-scale stirred tank bioreactors were applied for recombinant protein expression studies in simple batch experiments without pH titration. An enzymatic glucose release system (EnBase), a complex medium, and the frequently used LB and TB media were compared with regard to growth of Escherichia coli and recombinant protein expression (alcohol dehydrogenase (ADH) from Lactobacillus brevis and formate dehydrogenase (FDH) from Candida boidinii). Dissolved oxygen and pH were recorded online, optical densities were measured at-line, and the activities of ADH and FDH were analyzed offline. Best growth was observed in a complex medium with maximum dry cell weight concentrations of 14 g L⁻¹. EnBase cultivations enabled final dry cell weight concentrations between 6 and 8 g L⁻¹. The pH remained nearly constant in EnBase cultivations due to the continuous glucose release, showing the usefulness of this glucose release system especially for pH-sensitive bioprocesses. Cell-specific enzyme activities varied considerably depending on the different media used. Maximum specific ADH activities were measured with the complex medium, 6 h after induction with IPTG, whereas the highest specific FDH activities were achieved with the EnBase medium at low glucose release profiles 24 h after induction. Hence, depending on the recombinant protein, different medium compositions, times for induction, and times for cell harvest have to be evaluated to achieve efficient expression of recombinant proteins in E. coli. A rapid experimental evaluation can easily be performed with parallel batch operated small-scale stirred tank bioreactors.     

High-Level Expression of Heme-Dependent Catalase Gene <Emphasis Type="Italic">katA</Emphasis> from <Emphasis Type="Italic">Lactobacillus Sakei</Emphasis> Protects <Emphasis Type="Italic">Lactobacillus Rhamnosus</Emphasis> from Oxidative Stress

Lactic acid bacteria (LAB) are generally sensitive to hydrogen peroxide (H₂O₂), Lactobacillus sakei YSI8 is one of the very few LAB strains able to degrade H₂O₂ through the action of a heme-dependent catalase. Lactobacillus rhamnosus strains are very important probiotic starter cultures in meat product fermentation, but they are deficient in catalase. In this study, the effect of heterologous expression of L. sakei catalase gene katA in L. rhamnosus on its oxidative stress resistance was tested. The recombinant L. rhamnosus AS 1.2466 was able to decompose H₂O₂ and the catalase activity reached 2.85 μmol H₂O₂/min/10⁸ c.f.u. Furthermore, the expression of the katA gene in L. rhamnosus conferred enhanced oxidative resistance on the host. The survival ratios after short-term H₂O₂ challenge were increased 600 and 10⁴-fold at exponential and stationary phase, respectively. Further, viable cells were 100-fold higher in long-term aerated cultures. Simulation experiment demonstrated that both growth and catalase activity of recombinant L. rhamnosus displayed high stability under environmental conditions similar to those encountered during sausage fermentation.     

Regulation of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> body size and male tail development by the novel gene <Emphasis Type="Italic">lon-8</Emphasis>

Background  

In C. elegans and other nematode species, body size is determined by the composition of the extracellular cuticle as well as by the nuclear DNA content of the underlying hypodermis. Mutants that are defective in these processes can exhibit either a short or a long body size phenotype. Several mutations that give a long body size (Lon) phenotype have been characterized and found to be regulated by the DBL-1/TGF-β pathway, that controls post-embryonic growth and male tail development.     

Global expression studies in baker's yeast reveal target genes for the improvement of industrially-relevant traits: the cases of <Emphasis Type="Italic">CAF16</Emphasis> and <Emphasis Type="Italic">ORC2</Emphasis>

Background  

Recent years have seen a huge growth in the market of industrial yeasts with the need for strains affording better performance or to be used in new applications. Stress tolerance of commercial Saccharomyces cerevisiae yeasts is, without doubt, a trait that needs improving. Such trait is, however, complex, and therefore only in-depth knowledge of their biochemical, physiological and genetic principles can help us to define improvement strategies and to identify the key factors for strain selection.     

Use of the <Emphasis Type="Italic">piggyBac</Emphasis> transposon to create HIV-1 <Emphasis Type="Italic">gag</Emphasis> transgenic insect cell lines for continuous VLP production

Background  

Insect baculovirus-produced Human immunodeficiency virus type 1 (HIV-1) Gag virus-like-particles (VLPs) stimulate good humoral and cell-mediated immune responses in animals and are thought to be suitable as a vaccine candidate. Drawbacks to this production system include contamination of VLP preparations with baculovirus and the necessity for routine maintenance of infectious baculovirus stock. We used piggyBac transposition as a novel method to create transgenic insect cell lines for continuous VLP production as an alternative to the baculovirus system.     

Influence of <Emphasis Type="Italic">Daphnia</Emphasis> infochemicals on functional traits of <Emphasis Type="Italic">Microcystis</Emphasis> strains (Cyanobacteria)

We conducted a laboratory experiment to investigate the influence of Daphnia infochemicals on growth rate, microcystin production, colony formation and cell size of eight Microcystis strains isolated from two lakes. The strains were characterized genetically by their 16S-23S rDNA ITS sequence. The experiment was composed of four treatments: (1) a control using filtered WC medium, (2) addition of Scenedesmus obliquus culture medium filtrate, (3) addition of Daphnia magna culture medium filtrate and (4) addition of sodium octyl sulphate, a commercially available Daphnia infochemical. Our results showed that sympatric strains differed strongly for the measured functional traits, while no correlations between traits were found. Between-strain differences in growth rate, microcystin production, colony formation and cell size were generally larger than the differences in phenotypes observed between treatments. Despite this, several strains reacted to the infochemicals by changing functional trait values. Daphnia culture medium filtrate and, to a lesser extent, sodium octyl sulphate had a negative influence on the growth rate of half of the strains and stimulated microcystin production in one strain, but the latter effect was not Daphnia-specific as Scenedesmus culture medium filtrate had the same effect. Daphnia culture medium filtrate also induced colony formation in one strain. Our data suggest that Daphnia infochemicals generally have a weak influence on growth rate, microcystin production and colony formation of Microcystis strains as compared to the inter-strain variability, while existing inducible effects are highly strain-specific.     

Over-expression,purification and functional characterization of <Emphasis Type="Italic">Celosia</Emphasis> ClpS as a fused protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A ClpS homologue from Celosia cristata was expressed as maltose-binding fusion protein under the control of strong inducible tac promoter of pMALc2X vector in TB1 strain of Escherichia coli. SDS-PAGE analysis showed that fused ClpS is produced as about 63 kDa protein in recombinant bacteria. Expressed product was purified to homogeneity with a yield of about 31 mg/l of bacterial culture. The results indicated that heterologous expression of Celosia ClpS does not affect bacterial growth under different induced conditions. Total cellular antioxidant assessment results revealed that the induction of ClpS activates the bacterial antioxidative system. Since, the purified ClpS did not exhibit antioxidant activity in vitro, we speculated a functional corelation between bacterial proteolytic apparatus and its anti-oxidative system. This prediction may contribute to our better understanding of functional relationship between proteolytic and antioxidative systems in biological worlds in the future investigations.     

Development of a <Emphasis Type="Italic">Bacillus subtilis</Emphasis> expression system using the improved P<Emphasis Type="Italic">glv</Emphasis> promoter

Background  

B. subtilis is an important organism in the biotechnological application. The efficient expression system is desirable in production of recombinant gene products in B. subtilis. Recently, we developed a new inducible expression system in B. subtilis, which directed by B. subtilis maltose utilization operon promoter P _glv . The system demonstrated high-level expression for target proteins in B. subtilis when induced by maltose. However, the system was markedly repressed by glucose. This limited the application of the system as a high-expression tool in biotechnology field. The aim of this study was to further improve the P _glv promoter system and enhance its expression strength.     

Genome-scale metabolic reconstruction and <Emphasis Type="Italic">in silico</Emphasis> analysis of methylotrophic yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis> for strain improvement

Background  

Pichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensive in silico model of P. pastoris can be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeast's metabolism.