Microorganisms and methods for production of specific length fatty alcohols and related compounds

Abstract

The invention provides non-naturally occurring microbial organisms containing a fatty alcohol, fatty aldehyde or fatty acid pathway, wherein the microbial organisms selectively produce a fatty alcohol, fatty aldehyde or fatty acid of a specified length. Also provided are non-naturally occurring microbial organisms having a fatty alcohol, fatty aldehyde or fatty acid pathway, wherein the microbial organisms further include an acetyl-CoA pathway. In some aspects, the microbial organisms of the invention have select gene disruptions or enzyme attenuations that increase production of fatty alcohols, fatty aldehydes or fatty acids. The invention additionally provides methods of using the above microbial organisms to produce a fatty alcohol, a fatty aldehyde or a fatty acid.

1 . A non-naturally occurring microbial organism having a malonyl-CoA dependent fatty acyl-CoA elongation (MD-FAE) cycle in combination with a termination pathway,
 wherein said MD-FAE cycle comprises one or more elongase, one or more 3-oxoacyl-CoA reductase, one or more 3-hydroxyacyl-CoA dehydratase, and one or more enoyl-CoA reductase,   wherein said termination pathway comprises a pathway selected from:   (1)  1 H;   (2)  1 K and  1 L;   (3)  1 E and  1 N;   (4)  1 K,  1 J, and  1 N;   (5)  1 E;   (6)  1 K and  1 J;   (7)  1 H and  1 N;   (8)  1 K,  1 L, and  1 N;   (9)  1 E and  1 F;   (10)  1 K,  1 J, and  1 F;   (11)  1 H,  1 N, and  1 F;   (12)  1 K,  1 L,  1 N, and  1 F; and   (13)  1 G,   wherein  1 E is an acyl-CoA reductase (aldehyde forming), wherein  1 F is an alcohol dehydrogenase, wherein  1 G is an acyl-CoA reductase (alcohol forming), wherein  1 H is an acyl-CoA hydrolase, acyl-CoA transferase or acyl-CoA synthase, wherein  1 J is an acyl-ACP reductase, wherein  1 K is an acyl-CoA:ACP acyltransferase, wherein  1 L is a thioesterase, wherein  1 N is an aldehyde dehydrogenase (acid forming) or a carboxylic acid reductase,   wherein an enzyme of the MD-FAE cycle or termination pathway is encoded by at least one exogenous nucleic acid and is expressed in a sufficient amount to produce a compound of Formula (I):   
       
         
           
           
               
               
           
         
         wherein R 1  is C 1-24  linear alkyl; R 2  is CH 2 OH, CHO, or COOH; R 3  is H, OH, or oxo (═O); and   represents a single or double bond with the proviso that the valency of the carbon atom to which R 3  is attached is four, 
         wherein the substrate of each of said enzymes of the MD-FAE cycle and the termination pathway are independently selected from a compound of Formula (II), malonyl-CoA, propionyl-CoA or acetyl-CoA: 
       
       
         
           
           
               
               
           
         
         wherein R 1  is C 1-24  linear alkyl; R 3  is H, OH, or oxo (═O); R 4  is S-CoA, ACP, OH or H; and   represents a single or double bond with the proviso that the valency of the carbon atom to which R 3  is attached is four; 
         wherein said one or more enzymes of the MD-FAE cycle are each selective for a compound of Formula (II) having a number of carbon atoms at R 1  that is no greater than the number of carbon atoms at R 1  of said compound of Formula (I), and 
         wherein said one or more enzymes of the termination pathway are each selective for a compound of Formula (II) having a number of carbon atoms at R 1  that is no less than the number of carbon atoms at R 1  of said compound of Formula (I). 
       
     
     
         2 . The non-naturally occurring microbial organism of  claim 1 , wherein R 1  is C 1-17  linear alkyl, C 9  linear alkyl, C 10  linear alkyl, C 11 , linear alkyl, C 12  linear alkyl or C 13  linear alkyl. 
     
     
         3 . (canceled) 
     
     
         4 . The non-naturally occurring microbial organism of  claim 1 , wherein said microbial organism comprises two, three, or four exogenous nucleic acids each encoding an enzyme of said MD-FAE cycle, or wherein said microbial organism comprises two, three, or four exogenous nucleic acids each encoding an enzyme of said termination pathway, or wherein said microbial organism comprises exogenous nucleic acids encoding each of the enzymes of at least one of the pathways selected from (1)-(13). 
     
     
         5 - 8 . (canceled) 
     
     
         9 . The non naturally occurring microbial organism of  claim 1 , wherein said enzyme of the MD-FAE cycle or termination pathway is expressed in a sufficient amount to produce a compound selected from the Formulas (III)-(VI): 
       
         
           
           
               
               
           
         
       
       wherein R 1  is C 1-17  linear alkyl, C 9  linear alkyl, C 10  linear alkyl, C 11 , linear alkyl, C 12  linear alkyl or C 13  linear alkyl. 
     
     
         10 . (canceled) 
     
     
         11 . The non-naturally occurring microbial organism of  claim 1 , wherein said microbial organism further comprises an acetyl-CoA pathway and at least one exogenous nucleic acid encoding an acetyl-CoA pathway enzyme expressed in a sufficient amount to produce acetyl-CoA, wherein said acetyl-CoA pathway comprises a pathway selected from:
 (1)  2 A and  2 B;   (2)  2 A,  2 C, and  2 D;   (3)  2 H;   (4)  2 G and  2 D;   (5)  2 E,  2 F and  2 B;   (6)  2 E and  2 I;   (7)  2 J,  2 F and  2 B;   (8)  2 J and  2 I;   (9)  3 A,  3 B, and  3 C;   (10)  3 A,  3 B,  3 J,  3 K, and  3 D;   (11)  3 A,  3 B,  3 G, and  3 D;   (12)  3 A,  3 F, and  3 D;   (13)  3 N,  3 H,  3 B and  3 C;   (14)  3 N,  3 H,  3 B,  3 J,  3 K, and  3 D;   (15)  3 N,  3 H,  3 B,  3 G, and  3 D;   (16)  3 N,  3 H,  3 F, and  3 D;   (17)  3 L,  3 M,  3 B and  3 C;   (18)  3 L,  3 M,  3 B,  3 J,  3 K, and  3 D;   (19)  3 L,  3 M,  3 B,  3 G, and  3 D;   (20)  3 L,  3 M,  3 F, and  3 D;   (21)  4 A,  4 B,  4 D,  4 H,  4 I, and  4 J;   (22)  4 A,  4 B,  4 E,  4 F,  4 H,  4 I, and  4 J;   (23)  4 A,  4 B,  4 E,  4 K,  4 L,  4 H,  4 I, and  4 J;   (24)  4 A,  4 C,  4 D,  4 H, and  4 J;   (25)  4 A,  4 C,  4 E,  4 F,  4 H, and  4 J;   (26)  4 A,  4 C,  4 E,  4 K,  4 L,  4 H, and  4 J;   (27)  5 A,  5 B,  5 D, and  5 G;   (28)  5 A,  5 B,  5 E,  5 F, and  5 G;   (29)  5 A,  5 B,  5 E,  5 K,  5 L, and  5 G;   (30)  5 A,  5 C, and  5 D;   (31)  5 A,  5 C,  5 E, and  5 F; and   (32)  5 A,  5 C,  5 E,  5 K, and  5 L,   wherein  2 A is a pyruvate oxidase (acetate-forming), wherein  2 B is an acetyl-CoA synthetase, an acetyl-CoA ligase or an acetyl-CoA transferase, wherein  2 C is an acetate kinase, wherein  2 D is a phosphotransacetylase, wherein  2 E is a pyruvate decarboxylase, wherein  2 F is an acetaldehyde dehydrogenase, wherein  2 G is a pyruvate oxidase (acetyl-phosphate forming), wherein  2 H is a pyruvate dehydrogenase, a pyruvate:ferredoxin oxidoreductase, a pyruvate:NAD(P)H oxidoreductase or a pyruvate formate lyase, wherein  2 I is an acetaldehyde dehydrogenase (acylating), wherein  2 J is a threonine aldolase, wherein  3 A is a phosphoenolpyruvate (PEP) carboxylase or a PEP carboxykinase, wherein  3 B is an oxaloacetate decarboxylase, wherein  3 C is a malonate semialdehyde dehydrogenase (acetylating), wherein  3 D is an acetyl-CoA carboxylase or a malonyl-CoA decarboxylase, wherein  3 F is an oxaloacetate dehydrogenase or an oxaloacetate oxidoreductase, wherein  3 G is a malonate semialdehyde dehydrogenase (acylating), wherein  3 H is a pyruvate carboxylase, wherein  3 J is a malonate semialdehyde dehydrogenase, wherein  3 K is a malonyl-CoA synthetase or a malonyl-CoA transferase, wherein  3 L is a malic enzyme, wherein  3 M is a malate dehydrogenase or a malate oxidoreductase, wherein  3 N is a pyruvate kinase or a PEP phosphatase, wherein  4 A is a citrate synthase, wherein  4 B is a citrate transporter, wherein  4 C is a citrate/malate transporter, wherein  4 D is an ATP citrate lyase, wherein  4 E is a citrate lyase, wherein  4 F is an acetyl-CoA synthetase or an acetyl-CoA transferase, wherein  4 H is a cytosolic malate dehydrogenase, wherein  4 I is a malate transporter, wherein  4 J is a mitochondrial malate dehydrogenase, wherein  4 K is an acetate kinase, wherein  4 L is a phosphotransacetylase, wherein  5 A is a citrate synthase, wherein  5 B is a citrate transporter, wherein  5 C is a citrate/oxaloacetate transporter, wherein  5 D is an ATP citrate lyase, wherein  5 E is a citrate lyase, wherein  5 F is an acetyl-CoA synthetase or an acetyl-CoA transferase, wherein  5 G is an oxaloacetate transporter, wherein  5 K is an acetate kinase, and wherein  5 L is a phosphotransacetylase.   
     
     
         12 . The non-naturally occurring microbial organism of  claim 11 , wherein said microbial organism comprises two, three, four, five, six, seven or eight exogenous nucleic acids each encoding an acetyl-CoA pathway enzyme, or wherein said microbial organism comprises exogenous nucleic acids encoding each of the acetyl-CoA pathway enzymes of at least one of the pathways selected from (1)-(32). 
     
     
         13 . (canceled) 
     
     
         14 . The non-naturally occurring microbial organism of  claim 1 , further comprising one or more gene disruptions, said one or more gene disruptions occurring in endogenous genes encoding proteins or enzymes involved in: native production of ethanol, glycerol, acetate, formate, lactate, CO 2 , fatty acids, or malonyl-CoA by said microbial organism; transfer of pathway intermediates to cellular compartments other than the cytosol; or native degradation of a MD-FAE cycle intermediate or a termination pathway intermediate by said microbial organism, wherein said one or more gene disruptions confer increased production of the compound of Formula (I) in said microbial organism. 
     
     
         15 . (canceled) 
     
     
         16 . The non-naturally occurring microbial organism of  claim 1 , wherein one or more enzymes of the MD-FAE cycle or the termination pathway preferentially react with an NADH cofactor or have reduced preference for reacting with an NAD(P)H cofactor, wherein said one or more enzymes of the MD-FAE cycle are a 3-ketoacyl-CoA reductase or an enoyl-CoA reductase, and wherein said one or more enzymes of the termination pathway are selected from an acyl-CoA reductase (aldehyde forming), an alcohol dehydrogenase, an acyl-CoA reductase (alcohol forming), an aldehyde decarbonylase, an acyl-ACP reductase, an aldehyde dehydrogenase (acid forming) and a carboxylic acid reductase. 
     
     
         17 . The non-naturally occurring microbial organism of  claim 1 , further comprising one or more gene disruptions, said one or more gene disruptions occurring in genes encoding proteins or enzymes that result in an increased ratio of NAD(P)H to NAD(P) present in the cytosol of said microbial organism following said disruptions. 
     
     
         18 - 19 . (canceled) 
     
     
         20 . The non-naturally occurring microbial organism of  claim 1 , wherein said microbial organism is Crabtree positive and is in culture medium comprising excess glucose, thereby increasing the ratio of NAD(P)H to NAD(P) present in the cytosol of said microbial organism. 
     
     
         21 . The non-naturally occurring microbial organism of  claim 1 , further comprising at least one exogenous nucleic acid encoding an extracellular transporter or an extracellular transport system for the compound of Formula (I). 
     
     
         22 . The non-naturally occurring microbial organism of  claim 1 , wherein one or more endogenous enzymes involved in: native production of ethanol, glycerol, acetate, formate, lactate, CO 2 , fatty acids, or malonyl-CoA by said microbial organism; transfer of pathway intermediates to cellular compartments other than the cytosol; or native degradation of a MD-FAE cycle intermediate or a termination pathway intermediate by said microbial organism, has attenuated enzyme activity or expression levels. 
     
     
         23 . (canceled) 
     
     
         24 . The non-naturally occurring microbial organism of  claim 1 , wherein one or more endogenous enzymes involved in the oxidation of NAD(P)H or NADH, has attenuated enzyme activity or expression levels. 
     
     
         25 . (canceled) 
     
     
         26 . A method for producing a compound of Formula (I): 
       
         
           
           
               
               
           
         
         wherein R 1  is C 1-24  linear alkyl; R 2  is CH 2 OH, CHO, or COOH; R 3  is H, OH, or oxo (═O); and   represents a single or double bond with the proviso that the valency of the carbon atom to which R 3  is attached is four, comprising culturing the non-naturally occurring microbial organism of  claim 1  under conditions and for a sufficient period of time to produce said compound of Formula (I). 
       
     
     
         27 - 28 . (canceled) 
     
     
         29 . Culture medium comprising bioderived compound of Formula (I): 
       
         
           
           
               
               
           
         
         wherein R 1  is C 1-24  linear alkyl; R 2  is CH 2 OH, CHO, or COOH; R 3  is H, OH, or oxo (═O); and   represents a single or double bond with the proviso that the valency of the carbon atom to which R 3  is attached is four, wherein said bioderived compound has a carbon-12, carbon-13 and carbon-14 isotope ratio that reflects an atmospheric carbon dioxide uptake source. 
       
     
     
         30 . (canceled) 
     
     
         31 . A bioderived compound of Formula (I): 
       
         
           
           
               
               
           
         
         wherein R 1  is C 1-24  linear alkyl; R 2  is CH 2 OH, CHO, or COOH; R 3  is H, OH, or oxo (═O); and   represents a single or double bond with the proviso that the valency of the carbon atom to which R 3  is attached is four, having a carbon-12, carbon-13 and carbon-14 isotope ratio that reflects an atmospheric carbon dioxide uptake source. 
       
     
     
         32 - 33 . (canceled) 
     
     
         34 . A composition comprising said bioderived compound of  claim 31  and a compound other than said bioderived compound. 
     
     
         35 . (canceled) 
     
     
         36 . A composition comprising the bioderived compound of  claim 31 , or a cell lysate or culture supernatant thereof. 
     
     
         37 . A biobased product comprising said bioderived compound of  claim 31 , wherein said biobased product is a biofuel, chemical, polymer, surfactant, soap, detergent, shampoo, lubricating oil additive, fragrance, flavor material or acrylate. 
     
     
         38 - 39 . (canceled) 
     
     
         40 . A molded product obtained by molding a biobased product of  claim 37 , biobased product is a polymer. 
     
     
         41 . A process for producing a biobased product of  claim 37  comprising chemically reacting said bioderived compound with itself or another compound in a reaction.