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PubMed2005

Biotechnological production and applications of coenzyme Q10.

Coenzyme Q10 is widely used as an essential component of ATP generation in the oxidative phosphorylation process and as an antioxidant preventing lipid peroxidation and scavenging superoxide. It is also recommended as a supplement to 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors. Research efforts on the production of coenzyme Q10 by microorganisms focus on the development of potent strains by conventional mutagenesis and metabolic engineering, analysis and modification of the key metabolic pathways and optimization of fermentation strategies. Especially, random mutants with drugs resistance show a high coenzyme Q10 concentration. Metabolic engineering techniques have been applied to improve coenzyme Q10 production. The key enzymes involved in the coenzyme Q10 biosynthesis pathway have been cloned and expressed in Escherichia coli. The rational design of metabolic pathways in combination with engineering optimization of fermentation processes could facilitate the development of viable bioconversion processes.

Antioxidants↗

PubMed2006

Marker and promoter effects on heterologous expression in Aspergillus nidulans.

To study the effects of selection marker, promoter type, and copy number on heterologous expression in Aspergillus nidulans, strains were constructed with single- and multicopy plasmid integrations bearing a reporter gene (lacZ) under the control of either an inducible (alcA) or constitutive (gpdA) promoter and one of three Aspergillus nutritional marker genes (argB, trpC, or niaD). beta-Galactosidase activity in the transformants varied over three orders of magnitude, with the majority of levels in the range of 5x10(3)-1x10(4) U/mg. Significant differences in mean expression levels were found when comparing single-copy transformants with the same promoter but a different marker. Transformants with the argB marker had the highest average expression, approximately threefold over the trpC or niaD clones. For each promoter, maximal expression for the set was seen in the range of the single-copy clones, implying that increasing the copy number does not reliably increase expression in Aspergillus.

Aspergillus nidulans↗

PubMed2006

Functional expression of Candida antarctica lipase B in the Escherichia coli cytoplasm--a screening system for a frequently used biocatalyst.

In this paper, we report for the first time the functional expression of lipase B from the yeast Candida antarctica (CalB) in the Escherichia coli cytoplasm. The enzyme possessing three disulfide bonds was functionally expressed in the strain Origami B. Expression under the control of a lac promoter yielded 2 U mg(-1), whereas expression of a thioredoxin-CalB fusion protein yielded 17 U mg(-1). The native enzyme was most efficiently expressed under control of the cspA promoter (11 U mg(-1)). Coexpression of different chaperones led to a strong increase in active protein formation (up to 61 U mg(-1)). A codon-optimized synthetic variant of calb did not show significant effects on functional protein yield. Functional CalB expression was not only achieved in shake flasks but also in microtiter plate scale. Therefore, this CalB expression system is suitable for high-throughput applications, including the screening of large gene libraries as those derived from directed evolution experiments.

Base Sequence↗

PubMed2006

Growth promoting effect of a transgenic Bacillus mucilaginosus on tobacco planting.

In this study, we have investigated the plant growth promoting effect of Bacillus mucilaginosus strain D(4)B(1), a rhizosphere soil organism, and its transgenic strain NKTS-3 on tobacco planting. The transgenic strain contains a phytase expression cassette that can express high active phytase extracellularly and hydrolyze phytate in the soil to liberate inorganic phosphorus for the growth of tobacco plants. Greenhouse study and field experiments showed that both wild-type B. mucilaginosus and the transgenic strain could promote tobacco plant growth. Moreover, the transgenic strain promoted tobacco plant growth (235% more than control in pot experiments and 125% more than control in field experiments) was higher than the wild-type B. mucilaginosus (183% more than control in pot experiments and 108% more than control in field experiments). In addition, the inoculation with transgenic rhizobacteria could significantly improve root acquisition of phosphorus and increase the phosphorus content of the plant.

6-Phytase↗

PubMed2003

Feedback control for chemostat models.

It is shown that a chemostat with two organisms can be made coexistent by means of feedback control of the dilution rate. Remaining freedom in the feedback law can be used to guarantee robustness or improve particular performance indices. Unfortunately a topological property prevents coexistence by feedback control for chemostats with more than two organisms. We apply our results to control bioreactors aimed at producing commercial products through genetically altered organisms. In all our results the coexistence takes its simplest form: a global asymptotically stable equilibrium point in the interior of the non-negative orthant.

Bacteria↗

PubMed2002

Transgenic assessment of CFP-mediated cercosporin export and resistance in a cercosporin-sensitive fungus.

Cercosporin is a toxic polyketide produced by many phytopathogenic members of the fungal genus Cercospora. Cercospora species, themselves, exhibit the highest level of self-resistance to this almost universally toxic photosensitizer. Although the mechanism of cercosporin self-resistance is multi-faceted, partial resistance does appear to be provided by the encoded product of CFP ( cercosporin facilitator protein), a gene recently isolated from the pathogen of soybean, C. kikuchii. CFP has significant similarity to the major facilitator superfamily of integral membrane transport proteins. We expressed CFP in the cercosporin non-producing, cercosporin-sensitive fungus, Cochliobolus heterostrophus, in order to assess the transport activity of CFP and the contribution of CFP to cercosporin resistance in a fungal species free of endogenous toxin production. Expression of the CFP transgene in this fungus results in increased resistance to cercosporin due, apparently, to its export out of the fungus.

Antifungal Agents↗

PubMed2002

Genetic manipulation of the pathogenic yeast Candida parapsilosis.

Candida parapsilosis is an important human pathogen, responsible for severe cases of systemic candidiasis and one of the leading causes of mortality in neonates. In this report, we describe the first system for genetic manipulation of C. parapsilosis. We isolated and subsequently determined DNA sequences of genes encoding galactokinase ( CpGAL1) and orotidine-5'-phosphate decarboxylase ( CpURA3) from a genomic DNA library of C. parapsilosis by functional complementation of corresponding mutations in Saccharomyces cerevisiae. The predicted protein products, Gal1p and Ura3p, displayed a high degree of homology with corresponding sequences of C. albicans and S. cerevisiae, respectively. A collection of galactokinase-deficient ( gal1) strains of C. parapsilosis was prepared using direct selection of mutagenized cells on media containing 2-deoxy-galactose. Additionally, we constructed a plasmid vector carrying CpGAL1 as a selection marker and a genomic DNA fragment with an autonomously replicating sequence activity that transforms the C. parapsilosis gal1 mutant strain with high efficiency. This system for genetic transformation of C. parapsilosis may significantly advance the study of this human pathogen, greatly improving our understanding of its biology and virulence, with implications for drug development.

Candida↗

PubMed2002

Different functions of the class I and class II chitin synthase genes, chsC and chsA, are revealed by repression of chsB expression in Aspergillus nidulans.

The filamentous fungus, Aspergillus nidulans, genome contains at least five chitin synthase-encoding genes. chsB is essential for normal hyphal growth. chsA and chsC are likely to be cooperatively required for hyphal wall integrity. In this study, we constructed chsA chsB and chsC chsB double mutants, in which chsB expression was under a repressible promoter [ alcA(p)]. While chsA or chsC single mutants did not show obvious growth defects, the chsA chsB and chsC chsB double mutants showed different phenotypes from the chsB single mutant and from each other under alcA(p)-repressing conditions. The chsA chsB double mutant produced fewer aerial hyphae and the chsC chsB double mutant showed reduced cell mass. These observations support the idea that chsA and chsC each play a different role in hyphal morphogenesis. In addition, the chitin contents of these double mutants were higher than those of the chsB single mutant. When chsA was expressed ectopically under the chsB promoter in the chsB mutant, the growth defects caused by chsB repression were not remedied at all, although an increased level of chsA mRNA was observed. Thus, it is suggested that the gene products of chsA and chsB themselves have different functions in hyphal morphogenesis.

Aspergillus nidulans↗

PubMed2006

Deletion of RNQ1 gene reveals novel functional relationship between divergently transcribed Bik1p/CLIP-170 and Sfi1p in spindle pole body separation.

Spindle pole body (SPB; the microtubule organizing center in yeast) duplication is essential to form a bipolar spindle. The duplicated SPBs must then separate and migrate to opposite sides of the nucleus. We identified a novel functional relationship in SPB separation between the microtubule stabilizing protein Bik1p/CLIP-170 and the SPB half-bridge protein Sfi1p. A genetic interaction between BIK1 and SFI1 was discovered in a synthetic lethal screen using a strain deficient in the prion protein gene RNQ1. RNQ1 deletion reduced expression from the divergently transcribed BIK1, allowing us to identify genetic interactors with bik1. The sfi1-1 bik1 synthetic lethality was suppressed by over-expression of CIK1, KAR1, and PPH21. Genetic analysis indicated that the sfi1-1 bik1 synthetic lethality was unlikely related to the function of Bik1p in the dynein pathway or to defects in spindle position. Furthermore, a sfi1-1 Deltakip2 mutant was viable, suggesting that the Bik1p pool at the cytoplasmic microtubule plus-ends may not be required in sfi1-1. Microscopic examination indicated the sfi1-1 mutant was delayed in SPB duplication, SPB separation, or spindle elongation and the sfi-1 Deltabik1 double mutant arrested with duplicated but unseparated SPBs. These results suggest that Bik1p has a previously uncharacterized function in the separation of duplicated SPBs.

Alleles↗

PubMed2004

Nodulin 22 from Phaseolus vulgaris protects Escherichia coli cells from oxidative stress.

Plant genes that are induced during the formation and function of a root nodule are called nodulin genes. Cloning and functional analysis of nodule-specific gene products are of valuable help in establishing the role and requirements of the host plant for the specificity and effectiveness of the symbiosis. A cDNA clone (nod22) was isolated from Phaseolus vulgaris L. (common bean) cDNA library derived from Rhizobium-infected roots. Nodulin 22 (Nod22) transcripts are accumulated from early to late stages in root nodule development. RT-PCR in situ studies indicated that Nod22 transcripts are highly accumulated in cortical, vascular bundle and infected cells. The deduced Nod22 protein contains a highly hydrophobic N-terminus, with signal peptide characteristics, and a C-terminal extension with high identity to the alpha-crystallin domains found in alpha-crystallin lens chaperone, and other small heat-shock proteins. These domains have not been previously described in other known nodulins, but have been observed in small heat-shock proteins found in plant tissues exposed to elevated temperature and oxidative stress. Nod22, when it is over-expressed in Escherichia coli, cells confers protection against oxidative stress suggesting its possible role in plant host protection from oxidative toxicity during the Rhizobium-legume symbiosis.

Amino Acid Sequence↗

PubMed2004

Localization and function of SulP, a nuclear-encoded chloroplast sulfate permease in Chlamydomonas reinhardtii.

Recent work [H.-C. Chen et al. (2003) Planta 218:98-106] reported on the genomic, proteomic, phylogenetic and evolutionary aspects of a putative nuclear gene ( SulP) encoding a chloroplast sulfate permease in the model green alga Chlamydomonas reinhardtii. In this article, evidence is provided for the envelope localization of the SulP protein and its function in the uptake and assimilation of sulfate by the chloroplast. Localization of the SulP protein in the chloroplast envelope was concluded upon isolation of C. reinhardtii chloroplasts, followed by fractionation into envelope and thylakoid membranes and Western blotting of these fractions with specific polyclonal antibodies raised against the recombinant SulP protein. The function of the SulP protein was probed in antisense transformants of C. reinhardtii having lower expression levels of the SulP gene. Results showed that cellular sulfate uptake capacity was lowered as a consequence of attenuated SulP gene expression in the cell, directly affecting rates of de novo protein biosynthesis in the chloroplast. The antisense transformants exhibited phenotypes of sulfate-deprived cells, displaying slow rates of light-saturated oxygen evolution, low levels of Rubisco in the chloroplast and low steady-state levels of the photosystem-II D1 reaction-center protein. The role of the chloroplast sulfate transport in the uptake and assimilation of sulfate in C. reinhardtii is discussed along with its impact on the repair of photosystem-II from a frequently occurring photo-oxidative damage and potential use for the elucidation of the H(2)-evolution-related metabolism in this green alga.

Animals↗

PubMed2002

Multiple environmental signals determine the transcriptional activation of the mycoparasitism related gene prb1 in Trichoderma atroviride.

Trichoderma atroviride parasitizes a large variety of phytopathogenic fungi. This characteristic has allowed its use as a biological control agent. The production of hydrolytic enzymes appears to be a key element in the parasitic process. Among the enzymes released by Trichoderma, the proteinase Prb1 plays a major role. We show here that the corresponding gene ( prb1) is subject to nitrogen catabolite repression. Accordingly, induction of prb1 transcription by Rhizoctonia solani cell walls and by osmotic stress requires release from a repressed condition, which is determined by nitrogen availability. Furthermore, the transcription pattern of the prb1 gene was not affected when an inhibitor of p38-Hog1, a regulator of the response to osmotic shock, was used. In contrast, a MEK1/2 (MAPK/ERK) inhibitor blocked prb1 transcription in response to nitrogen limitation, indicating that the pathway employed in the nitrogen response involves proteins similar to p42-p44. Fusion of the prb1 promoter to the gfp reporter gene allowed the detection of a novel regulatory element, providing an initial insight into the nature of the sites that control prb1 expression.

Blotting, Northern↗

PubMed2006

The transcription factor Gcr1 stimulates cell growth by participating in nutrient-responsive gene expression on a global level.

Transcriptomic reprogramming is critical to the coordination between growth and cell cycle progression in response to changing extracellular conditions. In Saccharomyces cerevisiae, the transcription factor Gcr1 contributes to this coordination by supporting maximum expression of G1 cyclins in addition to regulating both glucose-induced and glucose-repressed genes. We report here the comprehensive genome-wide expression profiling of gcr1Delta cells. Our data show that reduced expression of ribosomal protein genes in gcr1Delta cells is detectable both 20 min after glucose addition and in steady-state cultures of raffinose-grown cells, showing that this defect is not the result of slow growth or growth on a repressing sugar. However, the large cell phenotype of the gcr1Delta mutant occurs only in the presence of repressing sugars. GCR1 deletion also results in aberrant derepression of numerous glucose repressed loci; glucose-grown gcr1Delta cells actively respire, demonstrating that this global alteration in transcription corresponds to significant changes at the physiological level. These data offer an insight into the coordination of growth and cell division by providing an integrated view of the transcriptomic, phenotypic, and metabolic consequences of GCR1 deletion.

Carbon↗

PubMed2004

A comparative, two-dimensional 14N ESEEM characterization of reduced [2Fe-2S] clusters in hyperthermophilic archaeal high- and low-potential Rieske-type proteins.

Proteins of the Rieske and Rieske-type family contain a [2Fe-2S] cluster with mixed ligation by two histidines and two cysteines, and play important roles in various biological electron transfer reactions. We report here the comparative orientation-selected ESEEM and HYSCORE studies of the reduced clusters from two hyperthermophilic Rieske-type proteins; a high-potential, archaeal Rieske protein called sulredoxin (SDX) from Sulfolobus tokodaii with weak homology to the cytochrome bc-associated Rieske proteins, and a low-potential, archaeal homolog of an oxygenase-associated Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus. (14)N ESEEM and HYSCORE spectra of SDX and ARF show well-defined variations, which are primarily determined by changes of quadrupole couplings (up to 50% depending on the selected orientation) of the two coordinated nitrogens. These are due to variations in coordination geometry of the histidine imidazole ligands rather than to variations of hyperfine couplings of these nitrogens, which do not exceed 8-10%. The measured quadrupole couplings and their differences in the two proteins are consistent with those calculated using the reported crystal structures of high- and low-potential Rieske proteins. These results suggest that exploration of quadrupole tensors might provide a more accurate method for characterization of the histidine coordination in different proteins and mutants than hyperfine tensors, and might have potential applications in a wider range of biological systems.

Electron Spin Resonance Spectroscopy↗

PubMed2003

Evaluation of a high temperature immobilised enzyme reactor for production of non-reducing oligosaccharides.

There is interest in the production of non-reducing carbohydrates due to their potential application in various industrial fields, particularly the food industry. In this paper, we describe the development of an immobilised cell bioprocess for the synthesis of non-reducing maltodextrins at high temperatures. The trehalosyl-dextrins-forming enzyme (TDFE) isolated from the thermoacidophilic archaeon Sulfolobus solfataricus (strain MT4), was recently expressed at high yields in Escherichia coli (strain Rb-791). Here, we evaluate different matrices, such as polyacrylamide gel, crude egg white, chitosan and calcium alginate for their effectiveness in immobilising whole recombinant E. coli cells subjected to prior thermal permeabilisation. Calcium-alginate based gels formed a solid biocatalyst with a good activity yield and the best enzymatic stability at the operating temperature (75 degrees C). Therefore, these beads were used to pack a glass column reactor to perform the bioconversion of interest. Optimal operating parameters were defined in relation to the substrate stream flow-rate and the substrate-to-biocatalyst ratio. The production of trehalosylmaltotetraose from maltohexaose reached equilibrium with a constant of about 2.6 at 75 degrees C. The bioreactor was exploited for production of trehalosylmaltodextrins from a commercial mixture of maltodextrins, achieving a productivity of 106.5 mg ml(-1) h(-1) (g biocatalyst)(-1) with ~40% conversion when using a 30% (w/v) solution.

Bioreactors↗

PubMed2003

Biosynthesis of medium-chain-length poly(hydroxyalkanoates) with altered composition by mutant hybrid PHA synthases.

Pseudomonas resinovorans harbors two isogenic poly(hydroxyalkanoates) (PHAs) synthase genes ( phaC1(Pre), phaC2(Pre)) responsible for the production of intracellular medium-chain-length (mcl-)PHAs. Sequence analysis showed that the putative gene-products of these genes contain a conserved alpha/beta-hydrolase fold in the carboxy-terminal half of the proteins. Hybrid genes pha7 and pha8 were constructed by exchanging the alpha/beta-hydrolase-fold coding portions of phaC1(Pre) and phaC2(Pre) at the 3' terminal. When grown with decanoate as carbon source, the pha7- or pha8-transformed Escherichia coli LS1298 produced PHAs containing 73-75% beta-hydroxydecanoate (beta-HD) and 25-27% beta-hydroxyoctanoate (beta-HO). Deletion mutants, Delta pha7 and Delta pha8, were isolated during the PCR-based construction of pha7 and pha8, respectively. Cells harboring these mutants produced PHAs containing 55-60 mol% beta-HD and 40-45 mol% beta-HO. These results demonstrate the feasibility of generating active hybrid mcl-PHA synthase genes and their mutants with the potential of producing polymers having a varied repeat-unit composition.

Acyltransferases↗

PubMed2006

Improved production of ethanol by deleting FPS1 and over-expressing GLT1 in Saccharomyces cerevisiae.

To improve ethanol production in Saccharomyces cerevisiae, two yeast strains were constructed. In the mutant KAM-3, the FPS1 gene, which encodes a channel protein responsible for glycerol export, was deleted. The mutant KAM-11 had the GLT1 gene (encoding glutamate synthase) placed under the PGK1 promoter while having the FPS1 deletion. Growth rate and biomass concentration remained virtually unchanged with the mutant KAM-11, compared to that of the parent. Over-expression of GLT1 by the PGK1 promoter along with FPS1 deletion resulted in a 14% higher ethanol production and a 30% lower glycerol formation compared to the parental strain under anaerobic fermentation conditions. Furthermore, acetate and pyruvic acid formation was also reduced in order for cells to maintain redox balance.

Efficiency↗

PubMed2006

Protein biosensors based on the principle of fluorescence resonance energy transfer for monitoring cellular dynamics.

Genetically-coded, fluorescence resonance energy transfer (FRET) biosensors are widely used to study molecular events from single cells to whole organisms. They are unique among biosensors because of their spontaneous fluorescence and targeting specificity to both organelles and tissues. In this review, we discuss the theoretical basis of FRET with a focus on key parameters responsible for designing FRET biosensors that have the highest sensitivity. Next, we discuss recent applications that are grouped into four common biosensor design patterns--intermolecular FRET, intramolecular FRET, FRET from substrate cleavage and FRET using multiple colour fluorescent proteins. Lastly, we discuss recent progress in creating fluorescent proteins suitable for FRET purposes. Together these advances in the development of FRET biosensors are beginning to unravel the interconnected and intricate signalling processes as they are occurring in living cells and organisms.

Animals↗