Microorganisms having increased lipid productivity

Abstract

The present invention provides mutant microorganism that have higher lipid productivity than the wild type microorganisms from which they are derived while biomass at levels that are within approximately 50% of wild type biomass productivities under nitrogen replete conditions. Particular mutants produce at least twice as much FAME lipid as wild type while producing at least 75% of the biomass produced by wild type cells under nitrogen replete conditions. Also provided are methods of producing lipid using the mutant strains.

What is claimed is: 
     
         1 . A classically-derived or genetically engineered mutant algal or heterokont microorganism that produces at least 50% more fatty acid methyl ester-derivatizable lipids (FAME lipids) than a control microorganism and at least 70% of the amount of biomass produced by the control microorganism when the mutant microorganism and control microorganism are cultured under identical conditions under which the control microorganism is producing biomass, wherein the mutant microorganism has attenuated expression of a gene encoding a polypeptide comprising an amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO:15, and SEQ ID NO: 16. 
     
     
         2 . A mutant algal or heterokont microorganism according to  claim 1 , wherein the mutant microorganism and control microorganism are cultured under identical conditions which are nitrogen replete with respect to the control microorganism. 
     
     
         3 . A mutant algal or heterokont microorganism according to  claim 1 , wherein the control microorganism is a wild type microorganism. 
     
     
         4 . A mutant algal or heterokont microorganism according to  claim 1 , wherein the mutant microorganism produces at least 50% more FAME lipids than a control microorganism while accumulating at least 70% the amount of biomass accumulated by the control microorganism over a culture period of at least five days. 
     
     
         5 . A mutant algal or heterokont microorganism according to  claim 4 , wherein the mutant microorganism produces at least 50% more FAME lipids than a control microorganism while accumulating at least 70% the amount of biomass accumulated by the control microorganism over a culture period of at least ten days. 
     
     
         6 . A mutant algal or heterokont microorganism according to  claim 4 , wherein the mutant microorganism accumulates at least 80% the amount of biomass accumulated by the control microorganism. 
     
     
         7 . A mutant algal or heterokont microorganism according to  claim 6 , wherein the mutant microorganism accumulates at least 90% the amount of biomass accumulated by the control microorganism. 
     
     
         8 . A mutant algal or heterokont microorganism according to  claim 7 , wherein the mutant microorganism accumulates at least 95% the amount of biomass accumulated by the control microorganism. 
     
     
         9 . A mutant algal or heterokont microorganism according to  claim 4 , wherein the mutant microorganism produces at least 75% more FAME lipids than the wild type microorgamsm. 
     
     
         10 . A mutant algal or heterokont microorganism according to  claim 9 , wherein the mutant microorganism produces at least 100% more FAME lipids than the control microorganism. 
     
     
         11 . A mutant algal or heterokont microorganism according to  claim 1 , wherein the mutant microorganism exhibits a F AME/TOC ratio at least 30% higher than the FAME/TOC ratio of the control microorganism. 
     
     
         12 . A mutant algal or heterokont microorganism according to  claim 1 , wherein the polypeptide further comprises a PAS3 domain. 
     
     
         13 . A mutant algal or heterokont microorganism according to  claim 2 , wherein the P AS3 domain has at least 65% identity to a sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25. 
     
     
         14 . A mutant heterokont microorganism according to  claim 1 , wherein the heterokont microorganism is a Labyrinthulomycite species of  Labryinthula, Labryinthuloides, Thraustochytrium, Schizochytrium, Aplanochytrium, Aurantiochytrium, Oblongichytrium, Japonochytrium, Diplophrys , or  Ulkenia.    
     
     
         15 . A mutant algal microorganism according to  claim 1 , wherein the algal microorganism is an algal species of  Achnanthes, Amphiprora, Amphora, Ankistrodesmus, Asteromonas, Boekelovia, Bolidomonas, Borodinella, Botrydium, Botryococcus, Bracteococcus, Chaetoceros, Carteria, Chlamydomonas, Chlorococcum, Chlorogonium, Chlorella, Chroomonas, Chrysosphaera, Cricosphaera, Crypthecodinium, Cryptomonas, Cyclotella, Desmodesmus, Dunaliella, Elipsoidon, Emiliania, Eremosphaera, Ernodesmius, Euglena, Eustigmatos, Franceia, Fragilaria, Fragilaropsis, Gloeothamnion, Haematococcus, Hantzschia, Heterosigma, Hymenomonas, Isochrysis, Lepocinclis, Micractinium, Monodus, Monoraphidium, Nannochloris, Nannochloropsis, Navicula, Neochloris, Nephrochloris, Nephroselmis, Nitzschia, Ochromonas, Oedogonium, Oocystis, Ostreococcus, Parachlorella, Parietochloris, Pascheria, Pavlova, Pelagomonas, Phceodactylum, Phagus, Picochlorum, Platymonas, Pleurochrysis, Pleurococcus, Prototheca, Pseudochlorella, Pseudoneochloris, Pseudostaurastrum, Pyramimonas, Pyrobotrys, Scenedesmus, Schizochlamydella, Skeletonema, Spyrogyra, Stichococcus, Tetrachlorella, Tetraselmis, Thalassiosira, Tribonema, Vaucheria, Viridiella, Vischeria,  or  Volvox.    
     
     
         16 . A method of producing lipid, comprising culturing the microorganism of  claim 1  and isolating lipid from the microorganism, the culture medium, or both. 
     
     
         17 . A method of producing lipid, comprising culturing a microorganism according to  claim 1  under conditions in which the FAME to TOC ratio of the microorganism is maintained between 0.3 and 0.9, and isolating lipid from the microorganism, the culture medium, or both. 
     
     
         18 . A method according to  claim 17 , wherein the FAME to TOC ratio is maintained between about 0.4 and about 0.8. 
     
     
         19 . An RNAi construct comprising a sequence homologous to at least a portion of a gene that encodes a polypeptide having at least 80% identity to SEQ ID NO:2, SEQ ID NO: 17, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. 
     
     
         20 . A method of producing lipid, comprising culturing a classical mutagenesis derived or genetically engineered mutant algal or heterokont microorganism that produces at least 30% more fatty acid methyl ester-derivatizable lipids (FAME lipids) than a control algal or heterokont microorganism and at least 45% of the amount of biomass produced by the control microorganism when the mutant microorganism and control microorganism are cultured under identical conditions under which the control microorganism is producing biomass, wherein the mutant microorganism has attenuated expression of a gene encoding a polypeptide comprising an amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO:15, and SEQ ID NO: 16.