The non-conventional oleaginous yeast Yarrowia lipolytica shows great industrial promise. It naturally produces certain compounds of interest but can also artificially generate non-native ...metabolites, thanks to an engineering process made possible by the significant expansion of a dedicated genetic toolbox. In this review, we present recently developed synthetic biology tools that facilitate the manipulation of Y. lipolytica, including 1) DNA assembly techniques, 2) DNA parts for constructing expression cassettes, 3) genome-editing techniques, and 4) computational tools.
•Due to its metabolic features, Y. lipolytica is a promising cell factory for producing compounds of biotechnological interest.•Fast and efficient synthetic biology tools have been developed, which has boosted engineering strategies for Y. lipolytica.•This review describes the latest advances in synthetic biology for Y. lipolytica.•DNA assembly tools, DNA parts for expression cassettes, genome-editing techniques, and computational tools are covered here.
Although wine yeast gene expression has been thoroughly investigated only few data are available on the evolution the proteome during alcoholic fermentation. This work aimed at specifying the change ...in proteome during fermentation and to assess its connection with transcriptome. The proteome of a wine yeast was monitored by 2-D gel electrophoresis throughout alcoholic fermentation. Proteome was analysed in exponential growth and stationary phase. Among 744 spots, detected we observed significant changes in abundance with 89 spots displaying an increase in intensity and 124 a decrease. We identified 59 proteins among the most regulated and/or the most expressed. Glycolysis and ethanol production, amino acid and sulfur metabolism were the most represented functional categories. We found only a weak correlation between changes in mRNA and protein abundance, which is strongly dependent on the functional category. There are substantial changes in protein abundance during alcoholic fermentation, but they are not directly associated with changes at transcript level suggesting that mRNA is selectively processed and/or translated in stationary phase. These data show that proteome is a relevant level of analysis to gain insight into wine yeast adaptation to alcoholic fermentation.
Postganglionic parasympathetic neurons in guinea-pig cardiac ganglia exhibit choline acetyltransferase (ChAT)-immunoreactivity, and a large fraction (60%) of the ChAT-positive cardiac neurons ...co-express somatostatin-immunoreactivity. This co-expression remained when the cardiac ganglia explants were maintained in culture for 72 h (40% somatostatin-immunoreactive). The guinea-pig cardiac ganglia neurons express the high affinity pituitary adenylate cyclase activating polypeptide (PACAP)-selective PAC
1 receptor, and treatment of the ganglia explants with 20 nM PACAP27 for 72 h to evaluate PACAP regulation of somatostatin expression revealed a dramatic 85% decrease in the number of somatostatin-IR neurons (6% somatostatin-IR neurons) compared with untreated control explant preparations. The decrease in percentage of somatostatin-IR neurons by PACAP27 was time- and concentration-dependent, and selective for PACAP27; PACAP38 and vasoactive intestinal polypeptide were less effective. PACAP6-38, a PACAP antagonist, eliminated the PACAP27-induced change in somatostatin positive neurons. The PACAP-mediated decrease in somatostatin-IR neurons was eliminated in calcium-deficient solutions and by the addition of nifedipine, indicating a requirement for calcium influx through L-type calcium channels. The addition of either the calmodulin inhibitor
N-(4-aminobutyl)-1-naphthalenesulfonamide or the MEK inhibitor PD98059, also eliminated the PACAP27-induced decrease in somatostatin-IR cells. The PACAP27-mediated effect on somatostatin expression was not affected by inhibitors of protein kinase A or phospholipase C, but was reduced by the adenylyl cyclase inhibitor SQ22356, suggesting cAMP involvement. Semiquantitative and quantitative reverse transcription PCR prosomatostatin transcript measurements showed that cardiac ganglia prosomatostatin mRNA levels were not diminished by chronic PACAP27 exposure despite the dramatic decrement in somatostatin-expressing neurons. Neuronal peptide-IR content represents a balance between production and secretion. These results suggested that one of the primary effects of PACAP exposure may be enhanced levels of neuropeptide release that exceeded production levels, resulting in somatostatin depletion and a decrement in the number of identifiable somatostatin-expressing cardiac neurons.
Double-β-decay involves the simultaneous conversion of two neutrons into two protons, and the emission of two electrons and two neutrinos; the neutrinoless process, although not yet observed, is ...thought to involve the emission of the two electrons but no neutrinos. The search for neutrinoless-double-β-decay probes fundamental properties of neutrinos, including whether or not the neutrino and antineutrino are distinct particles. Double-β-decay detectors are large and expensive, so it is essential to achieve the highest possible sensitivity with each study, and removing spurious contributions ('background') from detected signals is crucial. In the nEXO neutrinoless-double-β-decay experiment, the identification, or 'tagging', of the
Ba daughter atom resulting from the double-β decay of
Xe provides a technique for discriminating background. The tagging scheme studied here uses a cryogenic probe to trap the barium atom in a solid xenon matrix, where the barium atom is tagged through fluorescence imaging. Here we demonstrate the imaging and counting of individual barium atoms in solid xenon by scanning a focused laser across a solid xenon matrix deposited on a sapphire window. When the laser irradiates an individual atom, the fluorescence persists for about 30 seconds before dropping abruptly to the background level-a clear confirmation of one-atom imaging. Following evaporation of a barium deposit, the residual barium fluorescence is 0.16 per cent or less. Our technique achieves the imaging of single atoms in a solid noble element, establishing the basic principle of barium tagging for nEXO.
Future ton-scale liquefied noble gas detectors depend on efficient light detection in the vacuum ultraviolet (VUV) range. In the past years, silicon photomultipliers (SiPMs) have emerged as a valid ...alternative to standard photomultiplier tubes or large-area avalanche photodiodes. The next-generation double-beta decay experiment, nEXO, with a 5-ton liquid xenon time projection chamber will use SiPMs for detecting the 175-nm xenon scintillation light, in order to achieve an energy resolution of <inline-formula> <tex-math notation="LaTeX">\boldsymbol {\sigma }/\boldsymbol {Q_{\beta \beta }} = 1 </tex-math></inline-formula>%. This paper presents recent measurements of the VUV-HD generation SiPMs from Fondazione Bruno Kessler, Trento, Italy, in two complementary setups. It includes measurements of the photon-detection efficiency (PDE) with gaseous xenon scintillation light in a vacuum setup and dark measurements in a dry nitrogen gas setup. We report improved PDE at 175 nm compared to previous generation devices that would meet the criteria of nEXO. Furthermore, we present the projected nEXO detector light collection and energy resolution that could be achieved by using these SiPMs.
Characterization of the vacuum ultraviolet (VUV) reflectance of silicon photomultipliers (SiPMs) is important for large-scale SiPM-based photodetector systems. We report the angular dependence of the ...specular reflectance in vacuum of SiPMs manufactured by Fondazionc Bruno Kessler (FBK) and Hamamatsu Photonics K.K. (HPK) over wavelengths ranging from 120 to 280 nm. Refractive index and extinction coefficient of the thin silicon-dioxide film deposited on the surface of the FBK SiPMs are derived from reflectance data of an FBK silicon wafer with the same deposited oxide film as SiPMs. The diffuse reflectance of SiPMs is also measured at 193 nm. We use the VUV spectral dependence of the optical constants to predict the reflectance of the FBK silicon wafer and FBK SiPMs in liquid xenon.
Alternative pre-mRNA splicing is a major mechanism contributing to the proteome complexity of most eukaryotes, especially mammals. In less complex organisms, such as yeasts, the numbers of genes that ...contain introns are low and cases of alternative splicing (AS) with functional implications are rare. We report the first case of AS with functional consequences in the yeast Yarrowia lipolytica. The splicing pattern was found to govern the cellular localization of malate dehydrogenase, an enzyme of the central carbon metabolism. This ubiquitous enzyme is involved in the tricarboxylic acid cycle in mitochondria and in the glyoxylate cycle, which takes place in peroxisomes and the cytosol. In Saccharomyces cerevisiae, three genes encode three compartment-specific enzymes. In contrast, only two genes exist in Y. lipolytica. One gene (YlMDH1, YALI0D16753g) encodes a predicted mitochondrial protein, whereas the second gene (YlMDH2, YALI0E14190g) generates the cytosolic and peroxisomal forms through the alternative use of two 3'-splice sites in the second intron. Both splicing variants were detected in cDNA libraries obtained from cells grown under different conditions. Mutants expressing the individual YlMdh2p isoforms tagged with fluorescent proteins confirmed that they localized to either the cytosolic or the peroxisomal compartment.
In Candida albicans, the quorum-sensing molecule farnesol inhibits the transition from yeast to hyphae but has no effect on cellular growth. We show that the addition of exogenous farnesol to ...cultures of Candida parapsilosis causes the cells to arrest, but not at a specific stage in the cell cycle. The cells are not susceptible to additional farnesol. However, the cells do eventually recover from arrest. Unlike in C. albicans, in C. parapsilosis sterols are localized to the tips of budding cells, and this polarization is disrupted by the addition of farnesol. We used the results of a genome sequence survey to design and manufacture partial genomic microarrays that were applied to determining the transcriptional response of C. parapsilosis to the presence of exogenous farnesol. In both C. albicans and C. parapsilosis, exposure to farnesol results in increased expression of the oxidoreductases GRP2 and ADH7 and altered expression of genes involved in sterol metabolism. There is no effect on expression of C. parapsilosis orthologs of genes involved in hyphal growth in C. albicans. Farnesol therefore differs significantly in its effects on C. parapsilosis and C. albicans.
Understanding reflective properties of materials and photodetection efficiency (PDE) of photodetectors is important for optimizing energy resolution and sensitivity of the next generation ...neutrinoless double beta decay, direct detection dark matter, and neutrino oscillation experiments that will use noble liquid gases, such as nEXO, DARWIN, DarkSide-20k, and DUNE . Little information is currently available about reflectivity and PDE in liquid noble gases, because such measurements are difficult to conduct in a cryogenic environment and at short enough wavelengths. Here we report a measurement of specular reflectivity and relative PDE of Hamamatsu VUV4 silicon photomultipliers (SiPMs) with 50 μm micro-cells conducted with xenon scintillation light (∼175 nm) in liquid xenon. The specular reflectivity at 15ˆ incidence of three samples of VUV4 SiPMs is found to be 30.4±1.4%, 28.6±1.3%, and 28.0±1.3%, respectively. The PDE at normal incidence differs by ±8% (standard deviation) among the three devices. The angular dependence of the reflectivity and PDE was also measured for one of the SiPMs. Both the reflectivity and PDE decrease as the angle of incidence increases. This is the first measurement of an angular dependence of PDE and reflectivity of a SiPM in liquid xenon.
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