Microorganisms and methods for the co-production of isopropanol with primary alcohols, diols and acids

Abstract

The invention provides a non-naturally occurring microbial organism having n-propanol and isopropanol pathways, 1,4-butanediol (14-BDO) and isopropanol pathways, 1,3-butanediol (13-BDO) and isopropanol pathways or methylacrylic acid (MAA) and isopropanol pathways. The microbial organism contains at least one exogenous nucleic acid encoding an enzyme in each of the respective n-propanol, 14-BDO, 13-BDO or MAA and isopropanol pathways. The invention additionally provides a method for co-producing n-propanol and isopropanol, 14-BDO and isopropanol, 13-BDO and isopropanol or MAA and isopropanol. The method can include culturing an n-propanol and an isopropanol co-producing microbial organism, where the microbial organism expresses at least one exogenous nucleic acid encoding an n-propanol, an isopropanol, a 14-BDO, a 13-BDO and/or a MAA pathway enzyme in a sufficient amount to produce each of the respective products, under conditions and for a sufficient period of time to produce each of the respective products.

What is claimed is: 
     
         1 . A non-naturally occurring microbial organism, comprising a microbial organism having an n-propanol pathway and an isopropanol pathway, said n-propanol pathway comprising at least one exogenous nucleic acid encoding an n-propanol pathway enzyme expressed in a sufficient amount to produce n-propanol, said n-propanol pathway comprising:
 a propanol dehydrogenase or a propionaldehyde dehydrogenase,   said isopropanol pathway comprising at least one exogenous nucleic acid encoding an isopropanol pathway enzyme expressed in a sufficient amount to produce isopropanol, said isopropanol pathway comprising:   an isopropanol dehydrogenase, an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase or an acetoacetate decarboxylase.   
     
     
         2 . The non-naturally occurring microbial organism of  claim 1 , further comprising an acetyl-CoA pathway comprising at least one exogenous nucleic acid encoding an acetyl-CoA pathway enzyme expressed in a sufficient amount to produce acetyl-CoA, said acetyl-CoA pathway comprising:
 a pyruvate kinase, a pyruvate dehydrogenase, or a pyruvate ferredoxin oxidoreductase.   
     
     
         3 . The non-naturally occurring microbial organism of  claim 1 , further comprising a propionyl-CoA pathway comprising at least one exogenous nucleic acid encoding a propionyl-CoA pathway enzyme expressed in a sufficient amount to produce propionyl-CoA, said propionyl-CoA pathway comprising:
 a PEP carboxykinase, a PEP carboxylase, a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA transferase, a succinyl-CoA synthetase, a methylmalonyl-CoA mutase, a methylmalonyl-CoA epimerase or a methylmalonyl-CoA decarboxylase.   
     
     
         4 . The non-naturally occurring microbial organism of  claim 1 , wherein said n-propanol pathway comprises a first set of exogenous nucleic acids encoding n-propanol pathway enzymes expressed in a sufficient amount to produce n-propanol, said first set of exogenous nucleic acids encoding:
 a propionaldehyde dehydrogenase and a propanol dehydrogenase, and   wherein said isopropanol pathway comprises a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, said second set of exogenous nucleic acids encoding:   an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA synthetase; an acetoacetate decarboxylase; and an isopropanol dehydrogenase.   
     
     
         5 . The non-naturally occurring microbial organism of  claim 4 , further comprising an acetyl-CoA pathway comprising a third set of exogenous nucleic acids encoding acetyl-CoA pathway enzymes expressed in a sufficient amount to produce acetyl-CoA, said third set of exogenous nucleic acids encoding:
 a pyruvate kinase; and a pyruvate dehydrogenase or a pyruvate ferredoxin oxidoreductase.   
     
     
         6 . The non-naturally occurring microbial organism of  claim 4 , further comprising a propionyl-CoA pathway comprising a third set of exogenous nucleic acids encoding propionyl-CoA pathway enzymes expressed in a sufficient amount to produce propionyl-CoA, said third set of exogenous nucleic acids encoding:
 a PEP carboxykinase or a PEP carboxylase; a malate dehydrogenase; a fumarase; a fumarate reductase; a succinyl-CoA transferase or a succinyl-CoA synthetase; a methylmalonyl-CoA mutase; and a methylmalonyl-CoA decarboxylase.   
     
     
         7 . The non-naturally occurring microbial organism of  claim 6 , wherein said third set of exogenous nucleic acids further encodes a methylmalonyl-CoA epimerase. 
     
     
         8 . The non-naturally occurring microbial organism of  claim 1 , wherein said n-propanol pathway comprises a first set of exogenous nucleic acids encoding n-propanol pathway enzymes expressed in a sufficient amount to produce n-propanol, said first set of exogenous nucleic acids encoding:
 a PEP carboxykinase or a PEP carboxylase; a malate dehydrogenase; a fumarase; a fumarate reductase; a succinyl-CoA transferase or a succinyl-CoA synthetase; a methylmalonyl-CoA mutase; a methylmalonyl-CoA decarboxylase; a propionaldehyde dehydrogenase and a propanol dehydrogenase, and   wherein said isopropanol pathway comprises a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, said second set of exogenous nucleic acids encoding:   a pyruvate kinase; a pyruvate dehydrogenase or a pyruvate ferredoxin oxidoreductase an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA synthethase; an acetoacetate decarboxylase; and an isopropanol dehydrogenase.   
     
     
         9 . The non-naturally occurring microbial organism of  claim 8 , wherein said first set of exogenous nucleic acids further encodes a methylmalonyl-CoA epimerase. 
     
     
         10 . The non-naturally occurring microbial organism of  claim 1 , wherein said exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         11 . The non-naturally occurring microbial organism of  claim 1 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 
     
     
         12 . A method for producing n-propanol and isopropanol, comprising culturing a non-naturally occurring microbial organism of  claim 1  under conditions and for a sufficient period of time to produce n-propanol and isopropanol. 
     
     
         13 . The method of  claim 12 , wherein said conditions comprise substantially anaerobic culture conditions. 
     
     
         14 . The method of  claim 12 , wherein said exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         15 . A non-naturally occurring microbial organism, comprising a microbial organism having an n-propanol pathway, said n-propanol pathway comprising at least one exogenous nucleic acid encoding an n-propanol pathway enzyme expressed in a sufficient amount to produce n-propanol, said n-propanol pathway comprising:
 a propanol dehydrogenase or a propionaldehyde dehydrogenase.   
     
     
         16 . The non-naturally occurring microbial organism of  claim 15 , wherein said n-propanol pathway comprises a set of exogenous nucleic acids encoding n-propanol pathway enzymes expressed in a sufficient amount to produce n-propanol, said set of exogenous nucleic acids encoding:
 a propionaldehyde dehydrogenase and a propanol dehydrogenase.   
     
     
         17 . The non-naturally occurring microbial organism of  claim 15 , further comprising a propionyl-CoA pathway comprising exogenous nucleic acids encoding propionyl-CoA pathway enzymes expressed in a sufficient amount to produce propionyl-CoA, wherein the propionyl-CoA pathway comprises:
 a PEP carboxykinase, a PEP carboxylase, a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA transferase, a succinyl-CoA synthetase, a methylmalonyl-CoA mutase, a methylmalonyl-CoA epimerase or a methylmalonyl-CoA decarboxylase.   
     
     
         18 . The non-naturally occurring microbial organism of  claim 15 , wherein said n-propanol pathway comprises a first set of exogenous nucleic acids encoding n-propanol pathway enzymes expressed in a sufficient amount to produce n-propanol, said first set of exogenous nucleic acids encoding:
 a PEP carboxykinase or a PEP carboxylase; a malate dehydrogenase; a fumarase; a fumarate reductase; a succinyl-CoA transferase or a succinyl-CoA synthetase; a methylmalonyl-CoA mutase; a methylmalonyl-CoA epimerase; a methylmalonyl-CoA decarboxylase; a propionaldehyde dehydrogenase and a propanol dehydrogenase.   
     
     
         19 . The non-naturally occurring microbial organism of  claim 15 , wherein said exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         20 . The non-naturally occurring microbial organism of  claim 15 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 
     
     
         21 . A method for producing n-propanol, comprising culturing a non-naturally occurring microbial organism of  claim 15  under conditions and for a sufficient period of time to produce n-propanol. 
     
     
         22 . The method of  claim 21 , wherein said conditions comprise substantially anaerobic culture conditions. 
     
     
         23 . The method of  claim 21 , wherein said exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         24 . A non-naturally occurring microbial organism, comprising a microbial organism having a 1,4-butanediol pathway and an isopropanol pathway, said 1,4-butanediol pathway comprising at least one exogenous nucleic acid encoding a 1,4-butanediol pathway enzyme expressed in a sufficient amount to produce 1,4-butanediol, said 1,4-butanediol pathway comprising:
 a 4-hydroxybutyraldehyde reductase, a succinyl-CoA reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA synthetase, or a 4-hydroxybutyryl-CoA reductase (aldehyde-forming),   said isopropanol pathway comprising at least one exogenous nucleic acid encoding an isopropanol pathway enzyme expressed in a sufficient amount to produce isopropanol, said isopropanol pathway comprising:   an isopropanol dehydrogenase, an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, or an acetoacetate decarboxylase.   
     
     
         25 . A non-naturally occurring microbial organism, comprising a microbial organism having a 1,3-butanediol pathway and an isopropanol pathway, said 1,3-butanediol pathway comprising at least one exogenous nucleic acid encoding a 1,3-butanediol pathway enzyme expressed in a sufficient amount to produce 1,3-butanediol, said 1,3-butanediol pathway comprising:
 a 3-hydroxybutyraldehyde reductase, a succinyl-CoA reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA synthetase, a 4-hydroxybutyryl-CoA dehydratase, a crotonase, or a 3-hydroxybutyryl-CoA reductase (aldehyde forming),   said isopropanol pathway comprising at least one exogenous nucleic acid encoding an isopropanol pathway enzyme expressed in a sufficient amount to produce isopropanol, said isopropanol pathway comprising:   an isopropanol dehydrogenase, an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, or an acetoacetate decarboxylase.   
     
     
         26 . A non-naturally occurring microbial organism, comprising a microbial organism having a methylacrylic acid pathway and an isopropanol pathway, said methylacrylic acid pathway comprising at least one exogenous nucleic acid encoding a methylacrylic acid pathway enzyme expressed in a sufficient amount to produce methylacrylic acid, said methylacrylic acid pathway comprising:
 a 4-hydroxybutyryl-CoA mutase, a succinyl-CoA reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA synthetase, a 3-hydroxyisobutyryl-CoA dehydratase, a methacrylyl-CoA transferase, a methacrylyl-CoA synthetase, a methacrylyl-CoA hydrolase,   said isopropanol pathway comprising at least one exogenous nucleic acid encoding an isopropanol pathway enzyme expressed in a sufficient amount to produce isopropanol, said isopropanol pathway comprising:   an isopropanol dehydrogenase, an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, or an acetoacetate decarboxylase.   
     
     
         27 . The non-naturally occurring microbial organism of any one of  claims 24 - 26 , further comprising an acetyl-CoA pathway comprising at least one exogenous nucleic acid encoding an acetyl-CoA pathway enzyme expressed in a sufficient amount to produce acetyl-CoA, said acetyl-CoA pathway comprising:
 a pyruvate kinase, a pyruvate dehydrogenase, or a pyruvate ferredoxin oxidoreductase.   
     
     
         28 . The non-naturally occurring microbial organism of any one of  claims 24 - 26 , further comprising a succinyl-CoA pathway comprising at least one exogenous nucleic acid encoding a succinyl-CoA pathway enzyme expressed in a sufficient amount to produce succinyl-CoA, said succinyl-CoA pathway comprising:
 a PEP carboxykinase, a PEP carboxylase, a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA transferase or a succinyl-CoA synthetase.   
     
     
         29 . The non-naturally occurring microbial organism of  claim 24 , wherein said 1,4-butanediol pathway comprises a first set of exogenous nucleic acids encoding 1,4-butanediol pathway enzymes expressed in a sufficient amount to produce 1,4-butanediol, said first set of exogenous nucleic acids encoding:
 a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA reductase (aldehyde-forming); and a 4-hydroxybutyraldehyde reductase, and   wherein said isopropanol pathway comprises a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, said second set of exogenous nucleic acids encoding:   an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA synthetase; an acetoacetate decarboxylase; and an isopropanol dehydrogenase.   
     
     
         30 . The non-naturally occurring microbial organism of  claim 25 , wherein said 1,3-butanediol pathway comprises a first set of exogenous nucleic acids encoding 1,3-butanediol pathway enzymes expressed in a sufficient amount to produce 1,3-butanediol, said first set of exogenous nucleic acids encoding:
 a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA dehydratase; a crotonase; and a 3-hydroxybutyraldehyde reductase, and   wherein said isopropanol pathway comprises a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, said second set of exogenous nucleic acids encoding:   an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA synthetase; an acetoacetate decarboxylase; and an isopropanol dehydrogenase.   
     
     
         31 . The non-naturally occurring microbial organism of  claim 26 , wherein said methylacrylic acid pathway comprises a first set of exogenous nucleic acids encoding methylacrylic acid pathway enzymes expressed in a sufficient amount to produce methylacrylic acid, said first set of exogenous nucleic acids encoding:
 a succinyl-CoA reductase; a 4-hydroxybutyrate dehydrogenase; a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase; a 4-hydroxybutyryl-CoA mutase; a 3-hydroxyisobutyryl-CoA dehydratase; and a methacrylyl-CoA transferase, a methacrylyl-CoA synthetase or a methacrylyl-CoA hydrolase, and   wherein said isopropanol pathway comprises a second set of exogenous nucleic acids encoding isopropanol pathway enzymes expressed in a sufficient amount to produce isopropanol, said second set of exogenous nucleic acids encoding:   an acetyl-CoA acetyl thiolase; an acetoacetyl-CoA transferase or an acetoacetyl-CoA hydrolase or an acetoacetyl-CoA synthetase; an acetoacetate decarboxylase; and an isopropanol dehydrogenase.   
     
     
         32 . The non-naturally occurring microbial organism of any one of  claims 29 - 31 , further comprising an acetyl-CoA pathway comprising a third set of exogenous nucleic acids encoding acetyl-CoA pathway enzymes expressed in a sufficient amount to produce acetyl-CoA, said third set of exogenous nucleic acids encoding:
 a pyruvate kinase; and a pyruvate dehydrogenase or a pyruvate ferredoxin oxidoreductase.   
     
     
         33 . The non-naturally occurring microbial organism of any one of  claims 29 - 31 , further comprising a succinyl-CoA pathway comprising a third set of exogenous nucleic acids encoding succinyl-CoA pathway enzymes expressed in a sufficient amount to produce succinyl-CoA, said third set of exogenous nucleic acids encoding:
 a PEP carboxykinase, a PEP carboxylase, a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA transferase and a succinyl-CoA synthetase.   
     
     
         34 . The non-naturally occurring microbial organism of any one of  claims 24 - 26 , wherein said exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         35 . The non-naturally occurring microbial organism of any one of  claims 24 - 26 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 
     
     
         36 . A method for producing 1,4-butanediol and isopropanol, comprising culturing a non-naturally occurring microbial organism of  claim 24  under conditions and for a sufficient period of time to produce 1,4-butanediol and isopropanol. 
     
     
         37 . The method of  claim 36 , wherein said conditions comprise substantially anaerobic culture conditions. 
     
     
         38 . The method of  claim 36 , wherein said exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         39 . A method for producing 1,3-butanediol and isopropanol, comprising culturing a non-naturally occurring microbial organism of  claim 25  under conditions and for a sufficient period of time to produce 1,3-butanediol and isopropanol. 
     
     
         40 . The method of  claim 39 , wherein said conditions comprise substantially anaerobic culture conditions. 
     
     
         41 . The method of  claim 39 , wherein said exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         42 . A method for producing methylacrylic acid and isopropanol, comprising culturing a non-naturally occurring microbial organism of  claim 26  under conditions and for a sufficient period of time to produce methylacrylic acid and isopropanol. 
     
     
         43 . The method of  claim 42 , wherein said conditions comprise substantially anaerobic culture conditions. 
     
     
         44 . The method of  claim 42 , wherein said exogenous nucleic acid is a heterologous nucleic acid.