8,638 research outputs found
Genes of different catabolic pathways are coordinately regulated by Dal81 in Saccharomyces cerevisiae
Yeast can use a wide variety of nitrogen compounds. However, the ability to synthesize enzymes and permeases for catabolism of poor nitrogen sources is limited in the presence of a rich one. This general mechanism of transcriptional control is called nitrogen catabolite repression. Poor nitrogen sources, such as leucine, γ-aminobutyric acid (GABA), and allantoin, enable growth after the synthesis of pathway-specific catabolic enzymes and permeases. This synthesis occurs only under conditions of nitrogen limitation and in the presence of a pathway-specific signal. In this work we studied the temporal order in the induction of AGP1, BAP2, UGA4, and DAL7, genes that are involved in the catabolism and use of leucine, GABA, and allantoin, three poor nitrogen sources. We found that when these amino acids are available, cells will express AGP1 and BAP2 in the first place, then DAL7, and at last UGA4. Dal81, a general positive regulator of genes involved in nitrogen utilization related to the metabolisms of GABA, leucine, and allantoin, plays a central role in this coordinated regulation.Fil: Palavecino Ruiz, Marcos Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Correa Garcia, Susana Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Bermudez Moretti, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentin
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Harnessing Yarrowia lipolytica’s potential as a lipid and alkane production platform
Engineering cellular phenotype can enable the in vivo synthesis of renewable fuels, industrial precursors, and pharmaceuticals. Achieving economic viability requires the use of a cellular platform that generates high titers independent of fermentation condition, through either native or imported biosynthetic metabolism. While lacking fully developed genetic tools, the oleaginous yeast Yarrowia lipolytica has the native capacity to produce large titers of lipids and citric acid cycle intermediates. However, unlocking this biosynthetic capacity requires complete rewiring of native metabolism. To this end, this work focuses on the development and engineering of the yeast Y. lipolytica to rewire native metabolism and enable the production of lipids, alkanes, and itaconic acid. Precise control of gene expression is a requisite to enable metabolic and pathway engineering applications for any host organism. However, Y. lipolytica lacks promoter elements strong enough to manipulate intracellular metabolism. Thus, we utilized a hybrid promoter engineering approach to produce libraries of high-expressing, tunable promoters, seven-fold stronger than promoters previously characterized in Y. lipolytica 1,2. We successfully applied this approach to Saccharomyces cerevisiae, expanding transcriptional capacity of the strongest constitutive to highlight our hybrid approach as a generalizable method to increase expression capacity in eukaryotic organisms 3. We utilized our novel Y. lipolytica hybrid promoters to drive intracellular metabolism towards lipid production and to overexpress heterologous enzymes that enable alkane and itaconic acid production. Specifically, we implemented a global rewiring of Y. lipolytica’s native metabolism to increase lipogenesis more than sixty fold to 25.3g/L (the highest lipid production ever reported) and generated cells nearly 90% lipid content. We further expressed a lipoxygenase enzyme to catalyze the novel microbial production of the short-chain n-alkane, pentane. Finally, we exploited Y. lipolytica’s capacity to accumulate citric acid cycle intermediates by expressing a heterologous cis-aconitic acid decarboxylase enzyme to produce itaconic acid. Increasing substrate availability through media optimization and genomic engineering increased pentane and itaconic acid production threefold and eightfold, respectively 4. Collectively, these studies have facilitated the utilization of Y. lipolytica as an industrially relevant microbial platform, and represent a generic approach towards enabling biosynthetic control in microbial hosts will ill-defined gene expression technology.Chemical Engineerin
Expanding the Genetic Toolbox to Improve Metabolic Engineering in the Industrial Oleaginous Yeast, \u3cem\u3eYarrowia lipolytica\u3c/em\u3e
The oleaginous yeast, Yarrowia lipolytica, is becoming a popular host for industrial biotechnology because of its ability to grow on non-conventional feedstocks and naturally accumulate significant amounts of lipids. With new genome editing technologies, engineering novel pathways to produce lipid-derived oleochemicals has become easier. The goal, however, is to expand the genetic toolbox to improve the efficiency of metabolic engineering such that production capacities could expand from proof-of-concept shake flasks to an industrial scale. Building efficient metabolic circuits require controlling strength and timing of several enzymes in a metabolic pathway. One method to do this is through transcription – using suitable promoters to control expression of genes that code for enzymes. Native promoters have limited application because of complex regulation and non-tunable expression. Engineering hybrid promoters alleviates these issues to obtain predictable and tunable gene expression. In Y. lipolytica, how to design these promoters is not fully understood, resulting in only a handful of engineered promoters to date. In this work, we aim to develop tools for gene expression by investigating promoter architecture and designing tunable systems. In addition to Upstream Activating Sequences (UAS), tuning promoter strength can be achieved by varying sequence in the core promoter, TATA motif, and adjacent proximal sequences. UASs can modulate transcription strength and inducibility, enabling controlled timing of expression. A promoter of the acyl-CoA oxidase 2 (POX2) from the β-oxidation pathway was truncated heuristically to identify oleic acid (OA) UAS sequences. By fusing tandem repeats of the OA UAS elements, tunable yet inducible fatty acid hybrid promoters were engineered. The current approaches to identify novel UAS elements in Y. lipolytica are laborious. Therefore, we investigated DNA accessibility through nucleosome positioning to determine if a relationship between POX2 UASs and DNA accessibility can be inferred. The goal is to eventually apply this approach develop newer hybrid promoters efficiently. Finally, the hybrid fatty acid inducible promoter we developed was used to rationally engineering a Y. lipolytica strain capable of producing high amounts of free fatty acids. By localizing the fatty acyl / fatty aldehyde reductase in the peroxisome, we compartmentalized fatty alcohol production. This strategy led to upwards of 500 mg/L of fatty alcohols produced. It is a promising route to eventually make short to medium chain fatty alcohols in Y. lipolytica by utilizing the native β-oxidation machinery
Metabolic and Chaperone Gene Loss Marks the Origin of Animals: Evidence for Hsp104 and Hsp78 Sharing Mitochondrial Clients
The evolution of animals involved acquisition of an emergent gene repertoire
for gastrulation. Whether loss of genes also co-evolved with this developmental
reprogramming has not yet been addressed. Here, we identify twenty-four genetic
functions that are retained in fungi and choanoflagellates but undetectable in
animals. These lost genes encode: (i) sixteen distinct biosynthetic functions;
(ii) the two ancestral eukaryotic ClpB disaggregases, Hsp78 and Hsp104, which
function in the mitochondria and cytosol, respectively; and (iii) six other
assorted functions. We present computational and experimental data that are
consistent with a joint function for the differentially localized ClpB
disaggregases, and with the possibility of a shared client/chaperone
relationship between the mitochondrial Fe/S homoaconitase encoded by the lost
LYS4 gene and the two ClpBs. Our analyses lead to the hypothesis that the
evolution of gastrulation-based multicellularity in animals led to efficient
extraction of nutrients from dietary sources, loss of natural selection for
maintenance of energetically expensive biosynthetic pathways, and subsequent
loss of their attendant ClpB chaperones.Comment: This is a reformatted version from the recent official publication in
PLoS ONE (2015). This version differs substantially from first three arXiV
versions. This version uses a fixed-width font for DNA sequences as was done
in the earlier arXiv versions but which is missing in the official PLoS ONE
publication. The title has also been shortened slightly from the official
publicatio
Studies of molecular mechanisms integrating carbon metabolism and growth in plants
Plants use light energy, carbon dioxide and water to produce sugars and other carbohydrates, which serve as stored energy reserves and as building blocks for biosynthetic reactions. Supply of light is variable and plants have evolved means to adjust their growth and development accordingly. An increasing body of evidence suggests that the basic mechanisms for sensing and signaling energy availability in eukaryotes are evolutionary conserved and thus shared between plants, animals and fungi. I have used different experimental approaches that take advantage of findings from other eukaryotes in studying carbon and energy metabolism in plants. In the first part, I developed a novel screening procedure in yeast aimed at isolating cDNAs from other organisms encoding proteins with a possible function in sugar sensing or signaling. The feasibility of the method was confirmed by the cloning of a cDNA from Arabidopsis thaliana encoding a new F-box protein named AtGrh1, which is related to the yeast Grr1 protein that is involved in glucose repression. In the second part of the study, plant homologues of key components in the yeast glucose repression pathway were cloned and characterized in the moss Physcomitrella patens, in which gene function can be studied by gene targeting. We first cloned PpHXK1 which was shown to encode a chloroplast localized hexokinase representing a previously overlooked class of plant hexokinases with an N-terminal chloroplast transit peptide. Significantly, PpHxk1 is the major hexokinase in Physcomitrella, accounting for 80% of the glucose phosphorylating activity. A knockout mutant deleted for PpHXK1 exhibits a complex phenotype affecting growth, development and sensitivities to plant hormones. I also cloned and characterized two closely related Physcomitrella genes, PpSNF1a and PpSNF1b, encoding type 1 Snf1-related kinases. A double knockout mutant for these genes was viable even though it lacks detectable Snf1-like kinase activity. The mutant suffers from pleiotropic phenotypes which may reflect a constitutive high energy growth mode. Significantly, the double mutant requires constant high light and is therefore unable to grow in a normal day/night light cycle. These findings are consistent with the proposed role of the Snf1-related kinases as energy gauges which are needed to recognize and respond to low energy conditions
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Engineering Yarrowia lipolytica for high lipid production
Among potential value-added fuels and chemicals, fatty acid-based chemicals are important due to their wide use in industrial processes and in daily life. Fatty acids produced from microbial systems could provide a sustainable supply to replace the current costly and unsustainable process using plant oil or animal fat. The oleaginous yeast Yarrowia lipolytica naturally possesses moderate lipid production capacity and grows on different kinds of biomass and organic waste. However, fatty acid production from native, un-engineered strains is not economically viable. Therefore, this work develops strategies inspired from synthetic biology and metabolic engineering to expand the engineering potential of Y. lipolytica — helping to establish this organism as a premier platform for industrial-level, high lipid production as well as providing a platform for uncovering novel understanding of lipogenesis.
To do so, first, novel synthetic promoters and high expression plasmid were necessary to achieve the ability to tune gene expression levels inside the cell. We developed a hybrid promoter engineering strategy to create a promoter library exhibiting a range of more than 400-fold in terms of mRNA levels as well as engineered plasmids with regulated centromeric function to achieve a 2.7 fold expression range. Next, a rational and evolutionary metabolic engineering approach was coupled with genomic and transcriptomic studies to both engineer and understand underlying lipogenesis in this organism. Through the engineering efforts, we successfully increased the lipid production titer to over 40 g/L in bioreactor as well as identified novel lipogenic enhancers and mechanisms. In addition, we identified and characterized a mutant mga2 protein with superior lipogenesis enhancing capacity, which can regulate fatty acid desaturation and carbon flux inside the cells. Collectively, these studies have facilitated the utilization of Y. lipolytica as an industrially relevant microbial lipid production platform and supplied novel understanding of its lipogenesis process. The methods and concepts developed here can also be adapted to other oleaginous microbes and serve as a template for enabling value-added chemical production in other nonconventional organism.Biochemistr
The Regulation of NAP4 in Saccharomyces cerevisiae
The CCAAT binding-factor (CBF) is a transcriptional activator conserved in eukaryotes. The CBF in Saccharomyces cerevisiae is a multimeric heteromer termed the Hap2/3/4/5 complex. Hap4, which contains the activation domain of the complex, is also the regulatory subunit and is known to be transcriptionally controlled by carbon sources. However, little is known about Hap4 regulation. In this report, I identify mechanisms by which Hap4 is regulated, including: (1) transcriptional regulation via two short upstream open reading frames (uORFs) in the 5\u27 leader sequence of HAP4 mRNA; (2) proteasome-dependent degradation of Hap4; and (3) identification of two negative regulators of HAP4 expression, CYC8 and SIN4. I also report differential patterns of Hap4 cellular localization which depends on (1) carbon sources, (2) abundance of Hap4 protein, and (3) presence or absence of mitochondrial DNA (mtDNA)
Transcriptional Regulation of Metabolic Genes by the Basic Leucine Zipper Transcription Factor Hac1ip and Nutrient Stimuli
Saccharomyces cerevisiae cells respond to nutrients in their environment by altering their metabolic and transcriptional state in order to optimise the use of available nutrients and decide which of the several developmental pathways to pursue. In the yeast S. cerevisiae, meiosis and pseudohyphal
growth are two major differentiation outcomes in response to nitrogen starvation. A central component of unfolded protein response pathway, the bZIP transcription factor Hac1ip, negatively regulates meiosis and pseudohyphal growth. The present study investigates this negative regulatory mechanism at early meiotic genes by Hac1ip in nitrogen-rich conditions. Regulation of transcription by Ume6p transcriptional regulator, Rpd3p-Sin3p histone deacetylase complex and Isw2p-Itc1p chromatin remodelling complex at URS1 was also investigated here. We also tested for induction of pseudohyphal growth in diploids from SK1 genetic background in response to nitrogen starvation conditions known to induce meiosis. I constructed destabilised β-galactosidase reporters expressed from URS1-
CYC1-Ub-X-lacZ reporters to analyze transcriptional activity at URS1 site of early meiotic genes in nutrient rich conditions. The data presented here successfully demonstrates Hac1ip-mediated repression at URS1 sites in
nitrogen-rich conditions. URS1-CYC1-Ub-X-lacZ reporters were expressed in mitotic repression machinery mutants (ume6Δ, rpd3Δ, sin3Δ, isw2Δ and itc1Δ) under nitrogen rich conditions. The data presented here from these experiments not only corroborates their known role in repression at URS1 but also suggested regulation at additional sites in the minimal CYC1 promoter. Deletion of Sin3p suggested independent repression function separable from Rpd3p. Isw2p also acts independently of Itc1p at sites other than URS1. We also show that pseudohyphal growth was stimulated by non-fermentable carbon sources in sporulation efficient SK1 genetic background. The data also indicates that stimulation of pseudohyphal growth by non-fermentable carbon sources does not require respiration function or functional mitochondrial RTG pathway
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