Microorganisms, Compositions, and Methods for Producing Fatty Acids

Abstract

Methods of producing an unsaturated free fatty acid comprising at least 18 carbon atoms are provided. In some embodiments, the methods comprise culturing an engineered microorganism in a culture medium, wherein the engineered microorganism comprises at least one recombinant enzyme selected from acyl-lipid desaturase delta-9 (EC:1.14.19.1), acyl-lipid desaturase delta-12 (EC:1.14.19.6), acyl-lipid desaturase delta-15 (EC:1.14.19.-), and thioesterase (EC:3.1.2.14). Engineered microorganisms comprising at least one of those recombinant enzymes are also provided. The methods and organisms can be used to produce at least one free fatty acid selected from oleic acid, linoleic acid and α-linolenic acid.

1 . An unsaturated fatty acid composition formulated for oral administration to a human, wherein the unsaturated fatty acid composition comprises an isolated linolenic acid in a proportion approximate to that of palm oil, wherein the an isolated linolenic acid is separated from saturated fatty acids, and wherein the isolated linolenic acid is produced by a method comprising culturing an engineered microorganism in a culture medium, wherein the engineered microorganism comprises at least one recombinant acyl-lipid desaturase specific for a substrate comprising at least 18 carbon atoms, and wherein production of acyl-ACP synthetase (EC:6.2.1.20) is reduced or eliminated in the engineered microorganism, and wherein the amount of the linolenic acid produced by the engineered microorganism is increased relative to the amount of the linolenic acid produced by an otherwise identical microorganism lacking the at least one recombinant acyl-lipid desaturase. 
     
     
         2 . The unsaturated fatty acid composition of  claim 1 , wherein the unsaturated fatty acid composition further comprises oleic acid in a proportion approximate to that of palm oil. 
     
     
         3 . The unsaturated fatty acid composition of  claim 2 , wherein the linolenic acid and the oleic acid are produced by the same engineered microorganism. 
     
     
         4 . The unsaturated fatty acid composition of  claim 2 , wherein the linolenic acid and the oleic acid are produced by the different engineered microorganisms and combined. 
     
     
         5 . The unsaturated fatty acid composition of  claim 1 , wherein the unsaturated fatty acid composition further comprises at least one of octadecatetraenoic acid, eicosatetraenoic acid, eicosapentaenoic acid (EPA), docosapentaenoic acid, or docosahexaenoic acid (DHA). 
     
     
         6 . The unsaturated fatty acid composition of  claim 1 , wherein the unsaturated fatty acid composition comprises linoleic acid, alpha-linolenic acid, or a combination thereof. 
     
     
         7 . An unsaturated fatty acid composition formulated for oral administration to a human, wherein the unsaturated fatty acid composition comprises an isolated linolenic acid in a proportion approximate to that of soybean oil, wherein the an isolated linolenic acid is separated from saturated fatty acids, and wherein the isolated linolenic acid is produced by a method comprising culturing an engineered microorganism in a culture medium, wherein the engineered microorganism comprises at least one recombinant acyl-lipid desaturase specific for a substrate comprising at least 18 carbon atoms, and wherein production of acyl-ACP synthetase (EC:6.2.1.20) is reduced or eliminated in the engineered microorganism, and wherein the amount of the linolenic acid produced by the engineered microorganism is increased relative to the amount of the linolenic acid produced by an otherwise identical microorganism lacking the at least one recombinant acyl-lipid desaturase. 
     
     
         8 . The unsaturated fatty acid composition of  claim 7 , wherein the unsaturated fatty acid composition further comprises oleic acid in a proportion approximate to that of palm oil. 
     
     
         9 . The unsaturated fatty acid composition of  claim 8 , wherein the linolenic acid and the oleic acid are produced by the same engineered microorganism. 
     
     
         10 . The unsaturated fatty acid composition of  claim 8 , wherein the linolenic acid and the oleic acid are produced by the different engineered microorganisms and combined. 
     
     
         11 . The unsaturated fatty acid composition of  claim 7 , wherein the unsaturated fatty acid composition further comprises at least one of octadecatetraenoic acid, eicosatetraenoic acid, eicosapentaenoic acid (EPA), docosapentaenoic acid, or docosahexaenoic acid (DHA). 
     
     
         12 . The unsaturated fatty acid composition of  claim 7 , wherein the unsaturated fatty acid composition comprises linoleic acid, alpha-linolenic acid, or a combination thereof. 
     
     
         13 . An fatty acid composition formulated for oral administration to a human, wherein the fatty acid composition comprises i) an isolated unsaturated fatty acid selected from linolenic acid, alpha-linolenic acid and oleic acid, wherein the isolated unsaturated fatty acid is present in the composition in a proportion approximate to that of palm oil, and ii) an isolated fatty acid selected from palmitic acid, stearic acid, lauric acid, myristic acid, margaric acid, and arachidic acid, wherein the unsaturated fatty acid and the saturated fatty acid are produced by a method comprising culturing an engineered microorganism in a culture medium, wherein the engineered microorganism comprises at least one recombinant acyl-lipid desaturase specific for a substrate comprising at least 18 carbon atoms, and wherein production of acyl-ACP synthetase (EC:6.2.1.20) is reduced or eliminated in the engineered microorganism, and wherein the amount of the unsaturated fatty acid produced by the engineered microorganism is increased relative to the amount of the unsaturated fatty acid produced by an otherwise identical microorganism lacking the at least one recombinant acyl-lipid desaturase. 
     
     
         14 . The fatty acid composition of  claim 13 , comprising two or more of palmitic acid, stearic acid, oleic acid, linoleic acid, lauric acid, myristic acid, palmitoleic acid, margaric acid, linolenic acid, arachidic acid, eicosenoic acid, and docosanoic acid in proportions that approximate that of palm oil. 
     
     
         15 . The fatty acid composition of  claim 13 , comprising three or more of palmitic acid, stearic acid, oleic acid, linoleic acid, lauric acid, myristic acid, palmitoleic acid, margaric acid, linolenic acid, arachidic acid, eicosenoic acid, and docosanoic acid in proportions that approximate that of palm oil. 
     
     
         16 . The fatty acid composition of  claim 13 , comprising two or more of palmitic acid, stearic acid, oleic acid, linoleic acid, lauric acid, myristic acid, palmitoleic acid, margaric acid, linolenic acid, arachidic acid, eicosenoic acid, and docosanoic acid in proportions that approximate that of soybean oil. 
     
     
         17 . The fatty acid composition of  claim 13 , comprising three or more of palmitic acid, stearic acid, oleic acid, linoleic acid, lauric acid, myristic acid, palmitoleic acid, margaric acid, linolenic acid, arachidic acid, eicosenoic acid, and docosanoic acid in proportions that approximate that of soybean oil. 
     
     
         18 . The unsaturated fatty acid composition of  claim 1 , wherein the linolenic acid comprises alpha-linolenic acid, wherein the alpha-linolenic acid is present in the composition in an amount effective to prevent coronary heart disease or stroke. 
     
     
         19 . The unsaturated fatty acid composition of  claim 1 , wherein the linolenic acid comprises alpha-linolenic acid, wherein the alpha-linolenic acid is present in the composition in an amount effective to have a neuroprotective effect. 
     
     
         20 . A method of preventing a cardiovascular disease selected from coronary heart disease and stroke, comprising administering to a human subject in need thereof an unsaturated fatty acid composition formulated for oral administration to the human, wherein the unsaturated fatty acid composition comprises an isolated alpha-linolenic acid in a proportion approximate to that of palm oil, wherein the an isolated alpha-linolenic acid is separated from saturated fatty acids, and wherein the isolated alpha-linolenic acid is produced by a method comprising culturing an engineered microorganism in a culture medium, wherein the engineered microorganism comprises at least one recombinant acyl-lipid desaturase specific for a substrate comprising at least 18 carbon atoms, and wherein production of acyl-ACP synthetase (EC:6.2.1.20) is reduced or eliminated in the engineered microorganism, and wherein the amount of the alpha-linolenic acid produced by the engineered microorganism is increased relative to the amount of the alpha-linolenic acid produced by an otherwise identical microorganism lacking the at least one recombinant acyl-lipid desaturase, wherein the alpha-linolenic acid is present in an amount effective to prevent the cardiovascular disease.