Production of Oil in Microorganisms

Abstract

The invention provides methods of manufacturing biodiesel and other oil-based compounds using glycerol and combinations of glycerol and other feedstocks as an energy source in fermentation of oil-bearing microorganisms. Microorganisms disclosed herein contain an exogenous gene encoding lipid pathway modification or hydrocarbon modification enzymes, and can be cultivated using glycerol, including glycerol waste from biodiesel production, as a feedstock.

1 . A microalgae of the genus  Prototheca  containing an exogenous thioesterase gene. 
     
     
         2 . The microalgae of  claim 1 , wherein the gene encodes a protein selected from the group consisting of  Umbellularia californica  fatty acyl-ACP thioesterase (GenBank #AAC49001),  Cinnamomum camphora  fatty acyl-ACP thioesterase (GenBank #Q39473),  Umbellularia californica  fatty acyl-ACP thioesterase (GenBank #Q41635),  Myristica fragrans  fatty acyl-ACP thioesterase (GenBank #AAB71729),  Myristica fragrans  fatty acyl-ACP thioesterase (GenBank #AAB71730),  Elaeis guineensis  fatty acyl-ACP thioesterase (GenBank #ABD83939),  Elaeis guineensis  fatty acyl-ACP thioesterase (GenBank #AAD42220),  Populus tomentosa  fatty acyl-ACP thioesterase (GenBank #ABC47311),  Arabidopsis thaliana  fatty acyl-ACP thioesterase (GenBank #NP — 172327),  Arabidopsis thaliana  fatty acyl-ACP thioesterase (GenBank #CAA85387),  Arabidopsis thaliana  fatty acyl-ACP thioesterase (GenBank #CAA85388),  Gossypium hirsutum  fatty acyl-ACP thioesterase (GenBank #Q9SQI3),  Cuphea lanceolata  fatty acyl-ACP thioesterase (GenBank #CAA54060),  Cuphea hookeriana  fatty acyl-ACP thioesterase (GenBank #AAC72882),  Cuphea calophylla  subsp.  mesostemon  fatty acyl-ACP thioesterase (GenBank #ABB71581),  Cuphea lanceolata  fatty acyl-ACP thioesterase (GenBank #CAC19933),  Elaeis guineensis  fatty acyl-ACP thioesterase (GenBank #AAL15645),  Cuphea hookeriana  fatty acyl-ACP thioesterase (GenBank #Q39513),  Gossypium hirsutum  fatty acyl-ACP thioesterase (GenBank #AAD01982),  Vitis vinifera  fatty acyl-ACP thioesterase (GenBank #CAN81819),  Garcinia mangostana  fatty acyl-ACP thioesterase (GenBank #AAB51525),  Brassica juncea  fatty acyl-ACP thioesterase (GenBank #ABI18986),  Madhuca longifolia  fatty acyl-ACP thioesterase (GenBank #AAX51637),  Brassica napus  fatty acyl-ACP thioesterase (GenBank #ABH11710),  Oryza sativa  ( indica  cultivar-group) fatty acyl-ACP thioesterase (GenBank #EAY86877),  Oryza sativa  ( japonica  cultivar-group) fatty acyl-ACP thioesterase (GenBank #NP — 001068400),  Oryza sativa  ( indica  cultivar-group) fatty acyl-ACP thioesterase (GenBank #EAY99617), and  Cuphea hookeriana  fatty acyl-ACP thioesterase (GenBank #AAC49269). 
     
     
         3 . The microalgae of  claim 1 , wherein the microalgae is selected from the group consisting of  Prototheca stagnora, Prototheca portoricensis, Prototheca wickerhamii, Prototheca moriformis , and  Prototheca zopfii.    
     
     
         4 . The microalgae of  claim 1  that contains a second exogenous gene that encodes a protein selected from a lipid modification enzyme, a hydrocarbon modification enzyme, or a sucrose utilization enzyme. 
     
     
         5 . The microalgae of  claim 4 , wherein the second exogenous gene encodes a sucrose invertase secreted into the extracellular space. 
     
     
         6 . The microalgae of  claim 4 , wherein the second exogenous gene encodes a sucrose invertase targeted to the cytoplasm. 
     
     
         7 . The microalgae of  claim 1  that contains a second exogenous gene that encodes a fatty acyl-CoA/aldehyde reductase, a fatty acyl-CoA reductase, a fatty aldehyde reductase, a fatty aldehyde decarbonylase, or an acyl carrier protein (ACP). 
     
     
         8 . A microalgal culture containing:
 (a) a population of obligate heterotrophic microalgae; and   (b) a culture medium comprising glycerol.   
     
     
         9 . The microalgal culture of  claim 8 , wherein the microalgae are of the genus  Prototheca.    
     
     
         10 . The microalgal culture of  claim 8 , wherein the glycerol is acidulated. 
     
     
         11 . The microalgal culture of  claim 8 , wherein the glycerol is non-acidulated. 
     
     
         12 . The microalgal culture of  claim 8 , wherein the glycerol is provided as a transesterification byproduct from triglyceride produced by microalgae of the same species. 
     
     
         13 . The microalgal culture of  claim 9 , wherein the microalgae is selected from the group consisting of  Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, Prototheca wickerhamii , and  Prototheca zopfii.    
     
     
         14 . The microalgal culture of  claim 8 , wherein the culture medium is essentially free of carbon sources other than glycerol. 
     
     
         15 . The microalgal culture of  claim 8 , wherein the culture is axenic. 
     
     
         16 . The microalgal culture of  claim 8 , wherein the microalgae are at least 50% lipid by dry weight. 
     
     
         17 . A method of cultivating obligate heterotrophic microalgae, comprising:
 (a) providing an obligate heterotrophic microalgae cell;   (b) placing the microalgae cell in culture medium comprising glycerol as a carbon source; and   (c) culturing the microalgae cell under heterotrophic growth conditions in the presence of a sucrose invertase enzyme until the microalgae cell accumulates at least 10% of its dry cell weight as lipid.   
     
     
         18 . The method of  claim 17 , wherein the microalgae cell is of the genus  Prototheca.    
     
     
         19 . The method of  claim 18 , wherein the microalgae cell is selected from the group consisting of  Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, Prototheca wickerhamii , and  Prototheca zopfii.    
     
     
         20 . The method of  claim 17 , wherein the culture is axenic. 
     
     
         21 . The method of  claim 17 , wherein the culture medium is essentially free of carbon sources other than glycerol. 
     
     
         22 . The method of  claim 17 , wherein the sucrose is provided by a material selected from acidulated reagent-grade glycerol, non-acidulated reagent-grade glycerol, acidulated biodiesel waste glycerol, non-acidulated biodiesel waste glycerol, glycerol byproduct of a transesterification reaction, or purified glycerol. 
     
     
         23 . The method of  claim 17 , wherein the culture medium further comprises at least one additional carbon source selected from glucose, xylose, fructose, arabinose, mannose, galactose, acetate, depolymerized cellulosic material, sugarcane bagasse, rice hulls, corn stover, wheat straw, rice straw, sugar beet pulp, citrus pulp, citrus peels, hardwood thinnings, softwood thinnings, wood chips, sawdust, pulp mill waste, urban paper waste, grass clippings, switchgrass, hybrid poplar wood,  miscanthus , fiber cane or fiber sorghum. 
     
     
         24 . The method of  claim 17 , wherein the lipid comprises at least 50% C18:1. 
     
     
         25 . The method of  claim 17 , wherein the microalgae cell is at least 50% lipid by dry weight. 
     
     
         26 . The method of  claim 17 , wherein the culture medium comprises at least 1% sucrose. 
     
     
         27 . The method of  claim 17 , wherein the culturing step occurs primarily in an absence of light. 
     
     
         28 . The method of  claim 17 , wherein the microalgae cell has a lipid profile comprising at least 3% C:18:0 and at least 8% C:16:0. 
     
     
         29 . The method of  claim 17 , further comprising isolating the lipid from the microalgae cell. 
     
     
         30 . The method of  claim 29 , further comprising subjecting the lipid to a chemical reaction that changes the chemical structure of the lipid. 
     
     
         31 . The method of  claim 30 , wherein the chemical reaction is selected from cracking, transesterification, hydrogenation, deoxygenation, or isomerization. 
     
     
         32 . The method of  claim 31 , wherein the lipid is subjected to hydrogenation, deoxygenation and isomerization to produce a renewable diesel fuel that meets ASTM D975 specifications. 
     
     
         33 . The method of  claim 31 , wherein the lipid is subjected to transesterification to produce a biodiesel fuel that meets ASTM D6751 specifications. 
     
     
         34 . The method of  claim 17 , wherein the culturing is continued until the microalgae cell accumulates at least 60% of its dry cell weight as lipid. 
     
     
         35 . A method of cultivating microalgae, comprising culturing a recombinant microalgae cell of the genus  Prototheca  (a) in a culture medium containing glycerol as a primary carbon source; (b) under heterotrophic growth and limited nitrogen conditions, and (c) until the microalgae cell accumulates at least 10% of its dry cell weight as lipid. 
     
     
         36 . The method of  claim 35 , wherein the culture is axenic. 
     
     
         37 . The method of  claim 35 , wherein the culture medium is essentially free of carbon sources other than glycerol. 
     
     
         38 . The method of  claim 35 , wherein the sucrose is provided by a material selected from acidulated reagent-grade glycerol, non-acidulated reagent-grade glycerol, acidulated biodiesel waste glycerol, non-acidulated biodiesel waste glycerol, glycerol byproduct of a transesterification reaction, or purified glycerol. 
     
     
         39 . The method of  claim 35 , wherein the recombinant microalgae is cultured without selecting for a selectable marker other than glycerol utilization. 
     
     
         40 . The method of  claim 35 , wherein the culturing is continued until the microalgae cell accumulates at least 60% of its dry cell weight as lipid. 
     
     
         41 . The method of  claim 36 , wherein said culturing is continued until the microalgae cell accumulates at least 60% of its dry cell weight as lipid. 
     
     
         42 . The method of  claim 37 , wherein said culturing is continued until the microalgae cell accumulates at least 60% of its dry cell weight as lipid.