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US 20080038379 A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2008/0038379 A1 Metz et al. (43) Pub. Date: Feb. 14, 2008 (54) PUFA POLYKETIDE SYNTHASE SYSTEMS (60) Provisional application No. 60/457.979, filed on Mar. AND USES THEREOF 26, 2003. Provisional application No. 60/284,066, 75 filed on Apr. 16, 2001. Provisional application No. (75) Inventors: SMt. so S. 60/298.796, filed on Jun. 15, 2001. Provisional appli Willi? ity. Era 8) ); cation No. 60/323,269, filed on Sep. 18, 2001. Siles It flat Colorado Publication Classification Correspondence Address: (51) Int. Cl. SHERIDAN ROSS PC A6IR 36/00 (2006.01) 1560 BROADWAY A23C II/00 (2006.01) SUTE 12OO C07C 53/00 (2006.01) DENVER, CO 80202 A23D 7700 (2006.01) (52) U.S. Cl. ......................... 424/725; 426/580: 426/601; (73) Assignee: MARTEK BIOSCIENCES CORPO- 554/1 RATION, Columbia, MD (US) (21) Appl. No.: 11/778,570 (57) ABSTRACT (22) Filed: Jul. 16, 2007 O O The invention generally relates to polyunsaturated fatty acid Related U.S. Application Data (PUFA) polyketide synthase (PKS) systems, to homologues (60) Continuation of application No. 1 1/676,971, filed on thereof, to isolated nucleic acid molecules and recombinant Feb. 20, 2007, which is a division of application No. nucleic acid molecules encoding biologically active 10/810,352, filed on Mar. 26, 2004, now Pat. No. domains of such a PUFA PKS system, to genetically modi 7.211,418, and which is a continuation-in-part of fied organisms comprising PUFA PKS systems, to methods application No. 10/124,800, filed on Apr. 16, 2002, of making and using such systems for the production of now Pat. No. 7,247.461, and which is a continuation bioactive molecules of interest, and to novel methods for in-part of application No. 09/231,899, filed on Jan. identifying new bacterial and non-bacterial microorganisms 14, 1999, now Pat. No. 6,566,583. having such a PUFA PKS system. Comparison of PKS Orfs/domains: Schizochytrium VS Shewanella Orf 5 Orf 6 Orf 7 Of 8 KS MAT 6X ACP KR Amino acid sequence homology - 20-35% = 35-50% Patent Application Publication Feb. 14, 2008 Sheet 1 of 3 US 2008/0038379 A1 i s s 2 2 Patent Application Publication Feb. 14, 2008 Sheet 2 of 3 US 2008/0038379 A1 Z*OIH US 2008/0038379 A1 Feb. 14, 2008 PUFA POLYKETDE SYNTHASE SYSTEMIS AND detail in U.S. Pat. No. 6,566,583. Finally, the PKS pathways USES THEREOF for PUFA synthesis in eukaryotes such as members of Thraustochytriales, including the complete structural CROSS-REFERENCE TO RELATED description of the PUFA PKS pathway in Schizochytrium APPLICATIONS and the identification of the PUFA PKS pathway in Thraus tochytrium, including details regarding uses of these path 0001. This application is a Continuation of U.S. applica ways, are described in detail in U.S. Patent Application tion Ser. No. 1 1/676,971, filed Feb. 20, 2007, which is a Publication No. 20020194641, published Dec. 19, 2002 Continuation of U.S. application Ser. No. 10/810,352, filed (corresponding to U.S. patent application Ser. No. 10/124, Mar. 26, 2004, now U.S. Pat. No. 7,211,418, which claims 800, filed Apr. 16, 2002). the benefit of priority under 35 U.S.C. S 119(e) from U.S. Provisional Application Ser. No. 60/457.979, filed Mar. 26, 0005 Researchers have attempted to exploit polyketide 2003, entitled “Modification of a Schizochytrium PKS Sys synthase (PKS) systems that have been described in the tem to Facilitate Production of Lipids Rich in Polyunsatu literature as falling into one of three basic types, typically rated Fatty Acids”. U.S. application Ser. No. 10/810,352 is referred to as: Type II, Type I and modular. The Type II also a continuation-in-part of U.S. patent application Ser. system is characterized by separable proteins, each of which No. 10/124,800, filed Apr. 16, 2002, which Claims the carries out a distinct enzymatic reaction. The enzymes work benefit of priority under 35 U.S.C. S 119(e) to: U.S. Provi in concert to produce the end product and each individual sional Application Ser. No. 60/284,066, filed Apr. 16, 2001; enzyme of the system typically participates several times in U.S. Provisional Application Ser. No. 60/298.796, filed Jun. the production of the end product. This type of system 15, 2001; and U.S. Provisional Application Ser. No. 60/323, operates in a manner analogous to the fatty acid synthase 269, filed Sep. 18, 2001. U.S. patent application Ser. No. (FAS) systems found in plants and bacteria. Type I PKS 10/124,800, supra, is also a continuation-in-part of U.S. systems are similar to the Type II system in that the enzymes application Ser. No. 09/231,899, filed Jan. 14, 1999, now are used in an iterative fashion to produce the end product. U.S. Pat. No. 6,566,583. Each of the above-identified patent The Type I differs from Type II in that enzymatic activities, applications is incorporated herein by reference in its instead of being associated with separable proteins, occur as entirety. domains of larger proteins. This system is analogous to the 0002 This application does not claim the benefit of Type I FAS systems found in animals and fungi. priority from U.S. application Ser. No. 09/090,793, filed Jun. 0006. In contrast to the Type I and II systems, in modular 4, 1998, now U.S. Pat. No. 6,140,486, although U.S. appli PKS systems, each enzyme domain is used only once in the cation Ser. No. 09/090,793 is incorporated herein by refer production of the end product. The domains are found in ence in its entirety. very large proteins and the product of each reaction is passed on to another domain in the PKS protein. Additionally, in all FIELD OF THE INVENTION of the PKS systems described above, if a carbon-carbon 0003. This invention relates to polyunsaturated fatty acid double bond is incorporated into the end product, it is always (PUFA) polyketide synthase (PKS) systems from microor in the trans configuration. ganisms, including eukaryotic organisms, such as Thraus 0007. In the Type I and Type II PKS systems described tochytrid microorganisms. More particularly, this invention above, the same set of reactions is carried out in each cycle relates to nucleic acids encoding non-bacterial PUFA PKS until the end product is obtained. There is no allowance for systems, to non-bacterial PUFA PKS systems, to genetically the introduction of unique reactions during the biosynthetic modified organisms comprising non-bacterial PUFA PKS procedure. The modular PKS systems require huge proteins systems, and to methods of making and using the non that do not utilize the economy of iterative reactions (i.e., a bacterial PUFA PKS systems disclosed herein. This inven distinct domain is required for each reaction). Additionally, tion also relates to genetically modified microorganisms and as stated above, carbon-carbon double bonds are introduced methods to efficiently produce lipids (triacylglyerols (TAG), in the trans configuration in all of the previously described as well as membrane-associated phospholipids (PL)) PKS systems. enriched in various polyunsaturated fatty acids (PUFAs) and particularly, eicosapentaenoic acid (C20:5, co-3; EPA) by 0008 Polyunsaturated fatty acids (PUFAs) are critical manipulation of a PUFA polyketide synthase (PKS) system. components of membrane lipids in most eukaryotes (Lau ritzen et al., Prog. Lipid Res. 401 (2001); McConn et al., BACKGROUND OF THE INVENTION Plant J. 15, 521 (1998)) and are precursors of certain hormones and signaling molecules (Heller et al., Drugs 55. 0004 Polyketide synthase (PKS) systems are generally 487 (1998); Creelman et al., Annu. Rev. Plant Physiol. Plant known in the art as enzyme complexes derived from fatty Mol. Biol. 48, 355 (1997)). Known pathways of PUFA acid synthase (FAS) systems, but which are often highly synthesis involve the processing of saturated 16:0 or 18:0 modified to produce specialized products that typically show fatty acids (the abbreviation X;Y indicates an acyl group little resemblance to fatty acids. It has now been shown, containing X carbon atoms and Y double bonds (usually cis however, that polyketide synthase systems exist in marine in PUFAs); double-bond positions of PUFAs are indicated bacteria and certain microalgae that are capable of synthe relative to the methyl carbon of the fatty acid chain (c)3 or sizing PUFAs from malonyl-CoA. The PKS pathways for (O6) with systematic methylene interruption of the double PUFA synthesis in Shewanella and another marine bacteria, bonds) derived from fatty acid synthase (FAS) by elongation Vibrio marinus, are described in detail in U.S. Pat. No. and aerobic desaturation reactions (Sprecher, Curr. Opin. 6,140,486. The PKS pathways for PUFA synthesis in the Clin. Nutr. Metab. Care 2, 135 (1999); Parker-Barnes et al., eukaryotic Thraustochytrid, Schizochytrium is described in Proc. Natl. Acad. Sci. USA97, 8284 (2000); Shanklin et al., US 2008/0038379 A1 Feb. 14, 2008 Annu. Rev. Plant Physiol. Plant Nol. Biol. 49, 611 (1998)). ciated with fish oils. However, the PUFAs found in com Starting from acetyl-CoA, the synthesis of docosahexaenoic mercially developed plant oils are typically limited to acid (DHA) requires approximately 30 distinct enzyme linoleic acid (eighteen carbons with 2 double bonds, in the activities and nearly 70 reactions including the four repeti delta 9 and 12 positions—18:2 delta 9, 12) and linolenic acid tive steps of the fatty acid synthesis cycle.
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    ARTICLE Received 10 May 2013 | Accepted 10 Oct 2013 | Published 8 Nov 2013 DOI: 10.1038/ncomms3751 Probing single- to multi-cell level charge transport in Geobacter sulfurreducens DL-1 Xiaocheng Jiang1,*, Jinsong Hu2,*, Emily R. Petersen3, Lisa A. Fitzgerald4, Charles S. Jackan1, Alexander M. Lieber1, Bradley R. Ringeisen4, Charles M. Lieber1,5 & Justin C. Biffinger4 Microbial fuel cells, in which living microorganisms convert chemical energy into electricity, represent a potentially sustainable energy technology for the future. Here we report the single-bacterium level current measurements of Geobacter sulfurreducens DL-1 to elucidate the fundamental limits and factors determining maximum power output from a microbial fuel cell. Quantized stepwise current outputs of 92(±33) and 196(±20) fA are generated from microelectrode arrays confined in isolated wells. Simultaneous cell imaging/tracking and current recording reveals that the current steps are directly correlated with the contact of one or two cells with the electrodes. This work establishes the amount of current generated by an individual Geobacter cell in the absence of a biofilm and highlights the potential upper limit of microbial fuel cell performance for Geobacter in thin biofilms. 1 Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA. 2 CAS Key Laboratory of Molecular Nanostructure and Nanotechnology, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China. 3 Nova Research, Inc., 1900 Elkin Street, Suite 230, Alexandria, Virginia 22308, USA. 4 Chemistry Division, US Naval Research Laboratory, 4555 Overlook Avenue, SW, Washington, District of Columbia 20375, USA. 5 School of Engineering and Applied Science, Harvard University, Cambridge, Massachusetts 02138, USA.
  • Seagrass Communities of the Gulf Coast of Florida: Status and Ecology

    Seagrass Communities of the Gulf Coast of Florida: Status and Ecology

    CLINTON J. DAWES August 2004 RONALD C. PHILLIPS GEROLD MORRISON CLINTON J. DAWES University of South Florida Tampa, Florida, USA RONALD C. PHILLIPS Institute of Biology of the Southern Seas Sevastopol, Crimea, Ukraine GEROLD MORRISON Environmental Protection Commission of Hillsborough County Tampa, Florida, USA August 2004 COPIES This document may be obtained from the following agencies: Tampa Bay Estuary Program FWC Fish and Wildlife Research Institute 100 8th Avenue SE 100 8th Avenue SE Mail Station I-1/NEP ATTN: Librarian St. Petersburg, FL 33701-5020 St. Petersburg, FL 33701-5020 Tel 727-893-2765 Fax 727-893-2767 Tel 727-896-8626 Fax 727-823-0166 www.tbep.org http://research.MyFWC.com CITATION Dawes, C.J., R.C. Phillips, and G. Morrison. 2004. Seagrass Communities of the Gulf Coast of Florida: Status and Ecology. Florida Fish and Wildlife Conservation Commission Fish and Wildlife Research Institute and the Tampa Bay Estuary Program. St. Petersburg, FL. iv + 74 pp. AUTHORS Clinton J. Dawes, Ph.D. Distinguished University Research Professor University of South Florida Department of Biology Tampa, FL 33620 [email protected] Ronald C. Phillips, Ph.D. Associate Institute of Biology of the Southern Seas 2, Nakhimov Ave. Sevastopol 99011 Crimea, Ukraine [email protected] Gerold Morrison, Ph.D. Director, Environmental Resource Management Environmental Protection Commission of Hillsborough County 3629 Queen Palm Drive Tampa, FL 33619 813-272-5960 ext 1025 [email protected] ii TABLE of CONTENTS iv Foreword and Acknowledgements 1 Introduction 6 Distribution, Status, and Trends 15 Autecology and Population Genetics 28 Ecological Roles 42 Natural and Anthropogenic Effects 49 Appendix: Taxonomy of Florida Seagrasses 55 References iii FOREWORD The waters along Florida’s Gulf of Mexico coastline, which stretches from the tropical Florida Keys in the south to the temperate Panhandle in the north, contain the most extensive and diverse seagrass meadows in the United States.