Search results for "Heterologous expression."
showing 10 items of 48 documents
Artificial Chromosomes to Explore and to Exploit Biosynthetic Capabilities of Actinomycetes
2012
Actinomycetes are an important source of biologically active compounds, like antibiotics, antitumor agents, and immunosuppressors. Genome sequencing is revealing that this class of microorganisms has larger genomes relative to other bacteria and uses a considerable fraction of its coding capacity (5–10%) for the production of mostly cryptic secondary metabolites. To access actinomycetes biosynthetic capabilities or to improve the pharmacokinetic properties and production yields of these chemically complex compounds, genetic manipulation of the producer strains can be performed. Heterologous expression in amenable hosts can be useful to exploit and to explore the genetic potential of actinom…
Heterologous expression of aRauvolfiacDNA encoding strictosidine glucosidase, a biosynthetic key to over 2000 monoterpenoid indole alkaloids
2002
Strictosidine glucosidase (SG) is an enzyme that catalyses the second step in the biosynthesis of various classes of monoterpenoid indole alkaloids. Based on the comparison of cDNA sequences of SG from Catharanthus roseus and raucaffricine glucosidase (RG) from Rauvolfia serpentina, primers for RT-PCR were designed and the cDNA encoding SG was cloned from R. serpentina cell suspension cultures. The active enzyme was expressed in Escherichia coli and purified to homogeneity. Analysis of its deduced amino-acid sequence assigned the SG from R. serpentina to family 1 of glycosyl hydrolases. In contrast to the SG from C. roseus, the enzyme from R. serpentina is predicted to lack an uncleavable N…
Functional characterization of two human olfactory receptors expressed in the baculovirus Sf9 insect cell system
2005
Olfactory receptors (ORs) are the largest member of the G-protein-coupled receptors which mediate early olfactory perception in discriminating among thousands of odorant molecules. Assigning odorous ligands to ORs is a prerequisite to gaining an understanding of the mechanisms of odorant recognition. The functional expression of ORs represents a critical step in addressing this issue. Due to limitations in heterologous expression, very few mammal ORs have been characterized, and so far only one is from human origin. Consequently, OR function still remains poorly understood, especially in humans, whose genome encodes a restricted chemosensory repertoire compared with most mammal species. In …
Encapsulation of the Bacillus thuringiensis secretable toxins Vip3Aa and Cry1Ia in Pseudomonas fluorescens
2013
Vip3A and Cry1I toxins are secreted during the vegetative growth of Bacillus thuringiensis. Vip3A toxins do not share homology to the crystal (Cry) proteins and are active against a different spectrum of lepidopteran species. Cry1I toxins share similarity with the Cry1 protein group but do not accumulate in the parasporal crystal. Since Vip3A and Cry1I toxins are released from the cell, they are excluded from biological formulates based on spores and crystals of B. thuringiensis. As an approach to obtain novel sprayable insecticides containing Vip3 or Cry1I toxins, Vip3Aa and Cry1Ia proteins were expressed in Pseudomonas fluorescens. This bacterium, non-pathogenic to animals or plants, can …
G protein-coupled odorant receptors underlie mechanosensitivity in mammalian olfactory sensory neurons
2014
Mechanosensitive cells are essential for organisms to sense the external and internal environments, and a variety of molecules have been implicated as mechanical sensors. Here we report that odorant receptors (ORs), a large family of G protein-coupled receptors, underlie the responses to both chemical and mechanical stimuli in mouse olfactory sensory neurons (OSNs). Genetic ablation of key signaling proteins in odor transduction or disruption of OR–G protein coupling eliminates mechanical responses. Curiously, OSNs expressing different OR types display significantly different responses to mechanical stimuli. Genetic swap of putatively mechanosensitive ORs abolishes or reduces mechanical res…
Candida albicans UBI3 and UBI4 promoter regions confer differential regulation of invertase production to Saccharomyces cerevisiae cells in response …
2002
Candida albicans ubiquitin genes UBI3 and UBI4 encode a ubiquitin-hybrid protein involved in ribosome biogenesis and polyubiquitin, respectively. In this work we show that UBI3 and UBI4 promoter regions confer differentialexpr ession consistent with the function of their encoded gene products. Hybrid genes were constructed containing the SUC2 coding region under the controlof UBI3 or UBI4 promoters in the yeast vector pLC7. Invertase production in Saccharomyces cerevisiae transformants was differentially regulated: the UBI4 promoter was induced by stress conditions (thermalupshift and/or starvation) whereas the UBI3 promoter conferred constitutive invertase production in growing yeast cells…
Expression of Phosphofructokinase Is Not Sufficient to Enable Embden-Meyerhof-Parnas Glycolysis in Zymomonas mobilis ZM4
2019
Zymomonas mobilis is a bacterium that produces ethanol from glucose at up to 97% of theoretical efficiency on a carbon basis. One factor contributing to the high efficiency of ethanol production is that Z. mobilis has a low biomass yield. The low biomass yield may be caused partly by the low ATP yield of the Entner-Doudoroff (ED) glycolytic pathway used by Z. mobilis, which produces only one ATP per glucose consumed. To test the hypothesis that ATP yield limits biomass yield in Z. mobilis, we attempted to introduce the Embden-Meyerhof-Parnas (EMP) glycolytic pathway (with double the ATP yield) by expressing phosphofructokinase (Pfk I) from Escherichia coli. Expression of Pfk I caused growth…
Baculovirus infection affects caterpillar chemoperception
2021
International audience; Baculoviruses are double-stranded DNA entomopathogenic viruses that infect predominantly insects of the order Lepidoptera. Research in the last decade has started to disentangle the mechanisms underlying the insect-virus interaction, particularly focusing on the effects of the baculovirus infection in the host's physiology. Among crucial physiological functions, olfaction has a key role in reproductive tasks, food source detection and enemy avoidance. In this work, we describe that Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) induces expression changes in some odorant receptors (ORs)-the centrepiece of insect's olfaction-when infecting larvae from its nat…
Insect Cells for Heterologous Production of Recombinant Proteins
2010
Heterologous gene expression has become an indispensable and powerful tool for the production and subsequent functional analysis of proteins that are difficult to purify from their natural sources. Furthermore, it is the method of choice for the production of variants by introducing site-specific mutations into the DNA encoding the protein of interest. However, many systems are biased by disadvantages. The inability of bacteria to confer important post-translational modifications often results in functional failure of the recombinant protein. In addition, disulfide bonds are not formed properly in bacterial systems. Mammalian cells on the other hand modify properly, but they generally provi…
Molecular cloning and functional bacterial expression of a plant glucosidase specifically involved in alkaloid biosynthesis.
2000
Monoterpenoid indole alkaloids are a vast and structurally complex group of plant secondary compounds. In contrast to other groups of plant products which produce many glycosides, indole alkaloids rarely occur as glucosides. Plants of Rauvolfia serpentina accumulate ajmaline as a major alkaloid, whereas cell suspension cultures of Rauvolfia mainly accumulate the glucoalkaloid raucaffricine at levels of 1.6 g/l. Cell cultures do contain a specific glucosidase. known as raucaffricine-O-beta-D-glucosidase (RG), which catalyzes the in vitro formation of vomilenine, a direct intermediate in ajmaline biosynthesis. Here, we describe the molecular cloning and functional expression of this enzyme in…