Production of fatty acid derivatives

Abstract

The invention relates to compositions and methods, including polynucleotide sequences, amino acid sequences, recombinant host cells and recombinant host cell cultures engineered to produce fatty acid derivative compositions comprising fatty acids, fatty alcohols, fatty aldehydes, fatty esters, alkanes, terminal olefins, internal olefins or ketones. The fatty acid derivative composition is produced extracellularly with a higher titer, yield or productivity than the corresponding wild type or non-engineered host cell.

What is claimed is: 
     
         1 . A cultured genetically engineered host cell comprising:
 (a) a polynucleotide sequence encoding one or more of: (i) an acetyl-CoA carboxylase (EC 6.4.1.2) polypeptide, (ii) a FadR polypeptide, (iii) a heterologous iFAB polypeptide, (iv) a sequence having a transposon insertion in the yijP gene, and (v) a heterologous ACP protein; and   (b) a polynucleotide sequence encoding a fatty acid derivative biosynthetic polypeptide, wherein the genetically engineered host cell produces a fatty acid derivative composition at a higher titer, yield or productivity when cultured in medium containing a carbon source under conditions effective to overexpress the polynucleotide(s) relative to a corresponding wild type host cell propagated under the same conditions as the genetically engineered host cell.   
     
     
         2 . The genetically engineered host cell of  claim 1 , wherein the fatty acid derivative composition comprises a fatty acid derivative selected from the group consisting of a fatty acid, a fatty aldehyde, a fatty alcohol, a fatty ester, an alkane, a terminal olefin, an internal olefin and a ketone. 
     
     
         3 . The genetically engineered host cell of  claim 1  or  2 , wherein the fatty acid derivative composition is produced at a titer that is at least 3 times greater, at least 5 times greater, at least 8 times greater, or at least 10 times greater than the titer of a fatty acid derivative composition produced by a corresponding wild type host cell cultured under the same conditions as the genetically engineered host cell. 
     
     
         4 . The genetically engineered host cell of  claim 1  or  2 , wherein the fatty acid derivative composition is produced at a titer of at least 100 mg/L. 
     
     
         5 . The genetically engineered host cell of  claim 1  or  2 , wherein the fatty acid derivative composition is produced at a titer of from 30 g/L to 250 g/L. 
     
     
         6 . The genetically engineered host cell of  claim 1  or  2 , wherein the fatty acid derivative composition is produced at a yield that is at least 3 times greater, at least 5 times greater, at least 8 times greater, or at least 10 times greater than the yield of a fatty acid derivative composition produced by a corresponding wild type host cell cultured under the same conditions as the genetically engineered host cell. 
     
     
         7 . The genetically engineered host cell of  claim 1  or  2 , wherein the fatty acid derivative composition has a yield of from 10% to 40%. 
     
     
         8 . The genetically engineered host cell of  claim 1  or  2 , wherein the fatty acid derivative composition is produced at a productivity that is at least 3 times greater, at least 5 times greater, at least 8 times greater, or at least 10 times greater than the productivity of a fatty acid derivative composition produced by a corresponding wild type host cell cultured under the same conditions as the genetically engineered host cell. 
     
     
         9 . The genetically engineered host cell of  claim 1  or  2 , wherein the fatty acid derivative composition is produced at a productivity of from 0.7 mg/L/hr to 3 g/L/hr. 
     
     
         10 . The genetically engineered host cell of  claim 1  or  2 , wherein the acetyl-CoA carboxylase (EC 6.4.1.2) polypeptide is overexpressed. 
     
     
         11 . The genetically engineered host cell of  claim 10 , wherein the acetyl-CoA carboxylase (EC 6.4.1.2) polypeptide is accD+. 
     
     
         12 . The genetically engineered host cell of any one of  claims 1  to  11 , wherein the FadR polypeptide is overexpressed. 
     
     
         13 . The genetically engineered host cell of any one of  claims 1  to  12 , wherein the heterologous iFAB polypeptide is overexpressed. 
     
     
         14 . The genetically engineered host cell of  claim 13 , wherein the heterologous iFAB polypeptide is iFAB 138. 
     
     
         15 . The genetically engineered host cell of any one of  claims 1  to  14 , wherein the host cell comprises a transposon insertion in the yijP gene. 
     
     
         16 . The genetically engineered host cell of any one of  claims 1  to  15 , wherein the host cell comprises a heterologous acp sequence. 
     
     
         17 . The genetically engineered host cell of  claim 16 , further comprising an sfp gene. 
     
     
         18 . The genetically engineered host cell of any one of  claims 1  to  17 , wherein the polynucleotide sequence encoding a fatty acid derivative biosynthetic polypeptide is selected from the group consisting of a polypeptide:
 (a) having thioesterase activity, wherein the recombinant host cell synthesizes fatty acids; 
 (b) having thioesterase activity and carboxylic acid reductase (“CAR”) activity, wherein the recombinant host cell synthesizes fatty aldehydes and fatty alcohols; 
 (c) having thioesterase activity, carboxylic acid reductase activity and alcohol dehydrogenase activity wherein the recombinant host cell synthesizes fatty alcohols; 
 (d) having acyl-CoA reductase (“AAR”) activity wherein the recombinant host cell synthesizes fatty aldehydes and fatty alcohols; 
 (e) having acyl-CoA reductase (“AAR”) activity and alcohol dehydrogenase activity wherein the recombinant host cell synthesizes fatty alcohols; 
 (f) having fatty alcohol forming acyl-CoA reductase (“FAR”) activity, wherein the recombinant host cell synthesizes fatty alcohols; 
 (g) having thioesterase activity, carboxylic acid reductase activity and aldehyde decarbonylase activity, wherein the recombinant host cell synthesizes alkanes; 
 (h) having acyl-CoA reductase (“AAR”) activity and aldehyde decarbonylase activity, wherein the recombinant host cell synthesizes alkanes; 
 (i) having ester synthase activity wherein the recombinant host cell synthesizes fatty esters; 
 (j) having thioesterase activity, acyl-CoA synthase activity and ester synthase activity wherein the recombinant host cell synthesizes fatty esters; 
 (k) having OleA activity, wherein the recombinant host cell synthesizes aliphatic ketones; 
 (l) having OleABCD activity, wherein the recombinant host cell synthesizes internal olefins; and 
 (m) having thioesterase activity and decarboxylase activity, wherein the recombinant host cell synthesizes terminal olefins. 
 
     
     
         19 . The genetically engineered host cell of any one of  claims 1  to  16 , wherein the fatty acid derivative composition is produced extracellularly. 
     
     
         20 . A cell culture comprising the genetically engineered host cell of any one of  claims 1  to  19 . 
     
     
         21 . The cell culture of  claim 20 , wherein the culture medium comprises a fatty acid derivative composition. 
     
     
         22 . The cell culture of  claim 20 , wherein the fatty acid derivative composition comprises at least one fatty acid derivative selected from the group consisting of a fatty acid, a fatty aldehyde, a fatty alcohol, a fatty ester, an alkane, a terminal olefin, an internal olefin and a ketone. 
     
     
         23 . The cell culture of  claim 20 , wherein the fatty acid derivative is a C6, C8, C10, C12, C13, C14, C15, C16, C17, or C18 fatty acid derivative. 
     
     
         24 . The cell culture of  claim 20 , wherein the fatty acid derivative is a C10:1, C12:1, C14:1, C16:1, or C18:1 unsaturated fatty acid derivative. 
     
     
         25 . The cell culture of  claim 20 , wherein the fatty acid derivative composition comprises one or more of C 8 , C 10 , C 12 , C 14 , C 16 , and C 18  fatty acid derivatives. 
     
     
         26 . The cell culture of  claim 22 , wherein the fatty acid derivative composition comprises fatty acids. 
     
     
         27 . The cell culture of  claim 22 , wherein the fatty acid derivative composition comprises fatty aldehydes. 
     
     
         28 . The cell culture of  claim 22 , wherein the fatty acid derivative composition comprises fatty alcohols. 
     
     
         29 . The cell culture of  claim 22 , wherein the fatty acid derivative composition comprises fatty esters. 
     
     
         30 . The cell culture of  claim 22 , wherein the fatty acid derivative composition comprises an alkane. 
     
     
         31 . The cell culture of  claim 22 , wherein the fatty acid derivative composition comprises a terminal olefin. 
     
     
         32 . The cell culture of  claim 22 , wherein the fatty acid derivative composition comprises an internal olefin. 
     
     
         33 . The cell culture of  claim 22 , wherein the fatty acid derivative composition comprises a ketone. 
     
     
         34 . The cell culture of  claim 20 , wherein the fatty acid derivative composition comprises a fatty acid derivative having a double bond at position 7 in the carbon chain (between C 7  and C 8 ) from the reduced end of the fatty alcohol. 
     
     
         35 . The cell culture of  claim 20 , wherein the fatty acid derivative composition comprises unsaturated fatty acid derivatives. 
     
     
         36 . The cell culture of  claim 20 , wherein the fatty acid derivative composition comprises saturated fatty acid derivatives. 
     
     
         37 . The cell culture of  claim 20 , wherein the fatty acid derivative composition comprises branched chain fatty acid derivatives. 
     
     
         38 . The cell culture of  claim 20 , wherein the fatty acid derivative has a fraction of modern carbon of about 1.003 to about 1.5. 
     
     
         39 . The cell culture of  claim 20 , wherein the fatty acid derivative has a δ 13 C of from about −10.9 to about −15.4. 
     
     
         40 . A cultured recombinant host cell, engineered to increase the production of malonyl CoA, comprising:
 a polynucleotide sequence encoding one or more of: (i) an acetyl-CoA carboxylase (EC 6.4.1.2) polypeptide, (ii) a FadR polypeptide, (iii) a heterologous iFAB polypeptide, or (iv) a sequence having a transposon insertion in the yijP gene, wherein the engineered host cells produces a fatty acid derivative composition at a higher titer, yield or productivity when cultured in medium containing a carbon source under conditions effective to overexpress the polynucleotide(s) relative to a corresponding wild type host cell propagated under the same conditions as the genetically engineered host cell.   
     
     
         41 . The cultured recombinant host cell of  claim 40 , wherein the host cell further comprises a polynucleotide sequence encoding a fatty acid derivative biosynthetic polypeptide. 
     
     
         42 . The cultured recombinant host cell of  claim 40 , wherein the fatty acid derivative composition produced by the cultured genetically engineered host cell has a titer that is at least 3 times greater, at least 5 times greater, at least 8 times greater, or at least 10 times greater than the titer of a fatty acid derivative composition produced by a corresponding wild type host cell cultured under the same conditions as the genetically engineered host cell. 
     
     
         43 . The cultured recombinant host cell of  claim 40 , wherein the host cell has a titer of at least 100 mg/L. 
     
     
         44 . The cultured recombinant host cell of  claim 40 , wherein the fatty acid derivative composition produced by the cultured genetically engineered host cell has a titer of from 30 g/L to 250 g/L. 
     
     
         45 . The cultured recombinant host cell of  claim 40 , wherein the fatty acid derivative composition produced by a cultured genetically engineered host cell has a yield that is at least 3 times greater, at least 5 times greater, at least 8 times greater, or at least 10 times greater than the yield of a fatty acid derivative composition produced by a corresponding wild type host cell cultured under the same conditions as the genetically engineered host cell. 
     
     
         46 . The cultured recombinant host cell of  claim 40 , wherein the fatty acid derivative composition produced by the cultured genetically engineered host cell has a yield of from 10% to 40%. 
     
     
         47 . The cultured recombinant host cell of  claim 40 , wherein the fatty acid derivative composition produced by a cultured genetically engineered host cell has a productivity that is at least 3 times greater, at least 5 times greater, at least 8 times greater, or at least 10 times greater than the productivity of a fatty acid derivative composition produced by a corresponding wild type host cell cultured under the same conditions as the genetically engineered host cell. 
     
     
         48 . The cultured recombinant host cell of  claim 40 , wherein the fatty acid derivative composition produced by a cultured genetically engineered host cell has a productivity of from 0.7 mg/L/hr to 3 g/L/hr. 
     
     
         49 . The cultured recombinant host cell of any one of  claims 40  to  49 , wherein the acetyl-CoA carboxylase (EC 6.4.1.2) polypeptide is overexpressed. 
     
     
         50 . The cultured recombinant host cell of  claim 49 , wherein the acetyl-CoA carboxylase (EC 6.4.1.2) polypeptide is accD+. 
     
     
         51 . The cultured recombinant host cell of any one of  claims 40  to  50 , wherein the FadR polypeptide is overexpressed. 
     
     
         52 . The cultured recombinant host cell of any one of  claims 40  to  51 , wherein the heterologous iFAB polypeptide is overexpressed. 
     
     
         53 . The cultured recombinant host cell of  claim 52  wherein the heterologous iFAB polypeptide is iFAB 138. 
     
     
         54 . The cultured recombinant host cell of any one of  claims 40  to  53 , wherein the host cell comprises a transposon insertion in the yijP gene. 
     
     
         55 . A cell culture comprising the cultured recombinant host cell of any one of  claims 40  to  54   
     
     
         56 . A method of making a fatty acid derivative composition, comprising the steps of:
 (a) engineering a parental host cell to obtain a recombinant host cell which comprises an acetyl-CoA carboxylase (EC 6.4.1.2) polypeptide, (ii) a FadR polypeptide, (iii) a heterologous iFAB polypeptide, and (iv) a sequence having a transposon insertion in the yijP gene;   (b) further engineering the cell to comprise polynucleotide sequence encoding a fatty acid derivative biosynthetic polypeptide;   (c) culturing the recombinant host cell in the presence of a carbon source under conditions effective to result in a yield, titer or productivity of the fatty acid derivative composition that is at least 3 times the yield, titer or productivity of fatty acid derivative composition produced by the parental microbial cell cultured under the same conditions; and   (d) optionally isolating the fatty acid derivative composition.   
     
     
         57 . The method of  claim 56 , wherein the host cell is further engineered to comprise a polynucleotide sequence encoding a heterologous acp protein. 
     
     
         58 . The method of  claim 57 , wherein the host cell is further engineered to comprise an sfp gene. 
     
     
         59 . The method of  claim 56 , wherein the fatty acid derivative biosynthetic polypeptide is selected from the group consisting of a polypeptide:
 (a) having thioesterase activity, wherein the recombinant host cell synthesizes fatty acids;   (b) having thioesterase activity and carboxylic acid reductase (“CAR”) activity, wherein the recombinant host cell synthesizes fatty aldehydes and fatty alcohols;   (c) having thioesterase activity, carboxylic acid reductase activity and alcohol dehydrogenase activity wherein the recombinant host cell synthesizes fatty alcohols;   (d) having acyl-CoA reductase (“AAR”) activity wherein the recombinant host cell synthesizes fatty aldehydes and fatty alcohols;   (e) having acyl-CoA reductase (“AAR”) activity and alcohol dehydrogenase activity wherein the recombinant host cell synthesizes fatty alcohols;   (f) having fatty alcohol forming acyl-CoA reductase (“FAR”) activity, wherein the recombinant host cell synthesizes fatty alcohols;   (g) having thioesterase activity, carboxylic acid reductase activity and aldehyde decarbonylase activity, wherein the recombinant host cell synthesizes alkanes;   (h) having acyl-CoA reductase (“AAR”) activity and aldehyde decarbonylase activity, wherein the recombinant host cell synthesizes alkanes;   (i) having ester synthase activity wherein the recombinant host cell synthesizes fatty esters;   (j) having thioesterase activity, acyl-CoA synthase activity and ester synthase activity wherein the recombinant host cell synthesizes fatty esters;   (k) having OleA activity, wherein the recombinant host cell synthesizes aliphatic ketones;   (l) having OleABCD activity, wherein the recombinant host cell synthesizes internal olefins; and   (m) having thioesterase activity and decarboxylase activity, wherein the recombinant host cell synthesizes terminal olefins.   
     
     
         60 . The method of  claim 56 , where in the fatty acid derivative is selected from the group consisting of a fatty acid, a fatty alcohol, a fatty aldehyde, a fatty acid ester, a hydrocarbon, a ketone, and an olefin. 
     
     
         61 . The method of  claim 56 , where in the fatty acid derivative is a C6, C8, C10, C12, C13, C14, C15, C16, C17, or C18 fatty acid derivative. 
     
     
         62 . The method of  claim 56 , where in the fatty acid derivative is a C10:1, C12:1, C14:1, C16:1, or C18:1 unsaturated fatty acid derivative. 
     
     
         63 . A method of making a fatty acid derivative composition with a higher titer, yield or productivity of fatty acid derivatives than produced by a parental host cell, the method comprising:
 (a) engineering a parental host cell to obtain a recombinant host cell which comprises one or more of: (i) a polynucleotide encoding an acetyl-CoA carboxylase (EC 6.4.1.2) polypeptide, (ii) a polynucleotide encoding a FadR polypeptide, (iii) a polynucleotide encoding a heterologous iFAB polypeptide, (iv) a sequence having a transposon insertion in the yijP gene, and (v) a polynucleotide encoding a heterologous ACP protein;   (b) further engineering the cell to comprise a polynucleotide sequence encoding a fatty acid derivative biosynthetic polypeptide;   (c) culturing the recombinant host cell in the presence of a carbon source under conditions effective to result in a yield, titer or productivity of fatty acid derivatives that is at least 3 times the yield, titer or productivity of fatty acid derivatives produced by the parental microbial cell cultured under the same conditions; and   (d) optionally isolating the fatty acid derivative composition.