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      Engineering of a high lipid producing Yarrowia lipolytica strain

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          Abstract

          Background

          Microbial lipids are produced by many oleaginous organisms including the well-characterized yeast Yarrowia lipolytica, which can be engineered for increased lipid yield by up-regulation of the lipid biosynthetic pathway and down-regulation or deletion of competing pathways.

          Results

          We describe a strain engineering strategy centered on diacylglycerol acyltransferase (DGA) gene overexpression that applied combinatorial screening of overexpression and deletion genetic targets to construct a high lipid producing yeast biocatalyst. The resulting strain, NS432, combines overexpression of a heterologous DGA1 enzyme from Rhodosporidium toruloides, a heterlogous DGA2 enzyme from Claviceps purpurea, and deletion of the native TGL3 lipase regulator. These three genetic modifications, selected for their effect on lipid production, enabled a 77 % lipid content and 0.21 g lipid per g glucose yield in batch fermentation. In fed-batch glucose fermentation NS432 produced 85 g/L lipid at a productivity of 0.73 g/L/h.

          Conclusions

          The yields, productivities, and titers reported in this study may further support the applied goal of cost-effective, large -scale microbial lipid production for use as biofuels and biochemicals.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s13068-016-0492-3) contains supplementary material, which is available to authorized users.

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          Most cited references47

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          Molecular Cloning : A Laboratory Manual

          <p>The first two editions of this manual have been mainstays of molecular biology for nearly twenty years, with an unrivalled reputation for reliability, accuracy, and clarity.<br>In this new edition, authors Joseph Sambrook and David Russell have completely updated the book, revising every protocol and adding a mass of new material, to broaden its scope and maintain its unbeatable value for studies in genetics, molecular cell biology, developmental biology, microbiology, neuroscience, and immunology.<br>Handsomely redesigned and presented in new bindings of proven durability, this three–volume work is essential for everyone using today’s biomolecular techniques.<br>The opening chapters describe essential techniques, some well–established, some new, that are used every day in the best laboratories for isolating, analyzing and cloning DNA molecules, both large and small.<br>These are followed by chapters on cDNA cloning and exon trapping, amplification of DNA, generation and use of nucleic acid probes, mutagenesis, and DNA sequencing.<br>The concluding chapters deal with methods to screen expression libraries, express cloned genes in both prokaryotes and eukaryotic cells, analyze transcripts and proteins, and detect protein–protein interactions.<br>The Appendix is a compendium of reagents, vectors, media, technical suppliers, kits, electronic resources and other essential information.<br>As in earlier editions, this is the only manual that explains how to achieve success in cloning and provides a wealth of information about why techniques work, how they were first developed, and how they have evolved. </p>
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            Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets.

            Lipid droplets (LDs) store metabolic energy and membrane lipid precursors. With excess metabolic energy, cells synthesize triacylglycerol (TG) and form LDs that grow dramatically. It is unclear how TG synthesis relates to LD formation and growth. Here, we identify two LD subpopulations: smaller LDs of relatively constant size, and LDs that grow larger. The latter population contains isoenzymes for each step of TG synthesis. Glycerol-3-phosphate acyltransferase 4 (GPAT4), which catalyzes the first and rate-limiting step, relocalizes from the endoplasmic reticulum (ER) to a subset of forming LDs, where it becomes stably associated. ER-to-LD targeting of GPAT4 and other LD-localized TG synthesis isozymes is required for LD growth. Key features of GPAT4 ER-to-LD targeting and function in LD growth are conserved between Drosophila and mammalian cells. Our results explain how TG synthesis is coupled with LD growth and identify two distinct LD subpopulations based on their capacity for localized TG synthesis. Copyright © 2013 Elsevier Inc. All rights reserved.
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              Discovery of functional elements in 12 Drosophila genomes using evolutionary signatures.

              Sequencing of multiple related species followed by comparative genomics analysis constitutes a powerful approach for the systematic understanding of any genome. Here, we use the genomes of 12 Drosophila species for the de novo discovery of functional elements in the fly. Each type of functional element shows characteristic patterns of change, or 'evolutionary signatures', dictated by its precise selective constraints. Such signatures enable recognition of new protein-coding genes and exons, spurious and incorrect gene annotations, and numerous unusual gene structures, including abundant stop-codon readthrough. Similarly, we predict non-protein-coding RNA genes and structures, and new microRNA (miRNA) genes. We provide evidence of miRNA processing and functionality from both hairpin arms and both DNA strands. We identify several classes of pre- and post-transcriptional regulatory motifs, and predict individual motif instances with high confidence. We also study how discovery power scales with the divergence and number of species compared, and we provide general guidelines for comparative studies.
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                Author and article information

                Contributors
                friedlanderj@gmail.com
                vtsakraklides@novogyinc.com
                akamineni@novogyinc.com
                emily.greenhagen@gmail.com
                aconsiglio@novogyinc.com
                kmacewen@novogyinc.com
                dcrabtree@novogyinc.com
                jafshar@novogyinc.com
                dr.rlnugent@gmail.com
                mhamilton@novogyinc.com
                jshaw@novogyinc.com
                Colin.r.south@gmail.com
                gregstep@MIT.EDU
                contact@novogyinc.com
                Journal
                Biotechnol Biofuels
                Biotechnol Biofuels
                Biotechnology for Biofuels
                BioMed Central (London )
                1754-6834
                31 March 2016
                31 March 2016
                2016
                : 9
                : 77
                Affiliations
                [ ]Total New Energies, 5858 Horton Street, Emeryville, CA 94610 USA
                [ ]Novogy, Inc., 85 Bolton Street, Cambridge, MA 02140 USA
                [ ]Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139 USA
                [ ]Twist Bioscience, 455 Mission Bay Blvd South, San Francisco, CA 94158 USA
                [ ]Evelo Therapeutics, 620 Memorial Dr., Cambridge, MA 02139 USA
                Article
                492
                10.1186/s13068-016-0492-3
                4815080
                27034715
                89d1082a-6a76-4db6-bfbd-4379979cac54
                © Friedlander et al. 2016

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 23 December 2015
                : 21 March 2016
                Funding
                Funded by: Novogy, Inc.
                Categories
                Research
                Custom metadata
                © The Author(s) 2016

                Biotechnology
                yarrowia lipolytica,lipid accumulation,oleaginous yeast,metabolic engineering
                Biotechnology
                yarrowia lipolytica, lipid accumulation, oleaginous yeast, metabolic engineering

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