ENGINEERED TRANS-ENOYL COA REDUCTASE AND METHODS OF MAKING AND USING
Disclosed are trans-enoyl CoA reductase (TER) enzymes and nucleic acids encoding them. In some cases, the TER enzymes are non-natural, engineered trans-enoyl CoA reductase. TER enzymes were shown to catalyse the conversion of 5-carboxy-2-pentenoyl-CoA into adipyl-CoA for improved adipate production and the conversion of crotonyl-CoA into 6-aminocaproate. The enzymes can be used in biosynthetic methods and engineered microorganisms that enhance or improve the biosynthesis of 6-aminocaproate, hexamethylenediamine, caproic acid, caprolactone, or caprolactam. The engineered microorganisms include exogenous TER and in some cases engineered TER.
1 - 72 . (canceled)
73 . An engineered trans-enoyl CoA reductase (TER) enzyme comprising one or more alteration of an amino acid of SEQ ID NO:1 or SEQ ID NO: 24.
74 . The engineered TER of claim 73 , wherein the one or more amino acid alterations is selected from one or more positions corresponding to residues Q11, A39, S48, Q52, S59, G97, S103, H104, V105, N106, F107, I147, S148, V149, L152, T153, L156, R200, D201, R202, V301, T302, E303, K306, N307, N308, L316, or combinations thereof of SEQ ID NO:1.
75 . The engineered TER of claim 73 , wherein the alterations of the amino acid is selected from positions corresponding to S96, L98, M274, T275 or combinations thereof of SEQ ID NO: 24.
76 . The engineered TER of claim 73 , wherein the amino acid sequence comprises at least 60% identity to at least 25 contiguous amino acids of SEQ ID NO:1 or SEQ ID NO: 24.
77 . The engineered TER of claim 73 , wherein the engineered trans-enoyl CoA reductase reacts with 5-carboxyl-2-pentenoyl-CoA to form adipyl-CoA.
78 . The engineered TER of claim 73 , wherein the engineered TER comprises activity that is at least 20% higher than the activity of the trans-enoyl CoA reductase of SEQ ID NO:1 or SEQ ID NO: 24 with no amino acid alterations.
79 . The engineered TER of claim 73 , further comprising expression of the engineered TER in a cell that comprises genes encoding a 3-oxoadipyl-CoA thiolase, a 3-oxoadipyl-CoA dehydrogenase, and a 3-oxoadipyl-CoA dehydratase is able to produce at least 10% more adipate than a control cell that expresses the wild type TER from which the engineered TER is derived.
80 . The engineered TER of claim 73 , wherein the engineered TER can further use crotonyl-CoA as a substrate.
81 . The engineered TER of claim 73 , wherein the engineered TER has greater catalytic efficiency for 5-carboxyl-2-pentenoyl-CoA as the substrate compared to crotonyl-CoA as the substrate.
82 . A recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an engineered TER of claim 73 .
83 . A non-naturally occurring microbial organism comprising at least one exogenous enzyme, the at least one exogenous enzyme comprising a TER converting 5-carboxyl-2-pentenoyl-CoA to adipyl CoA, the TER selected from:
(i) a TER comprising the amino acid sequence of SEQ ID NO:1, 4-9, 11, 14, 15, or 17-26;
(ii) a TER comprising an amino acid sequence having at least 50% sequence identity to at least 25 or more contiguous amino acids of any of SEQ ID NO:1, 4-9, 11, 14, 15, or 17-26; or
(iii) An engineered TER comprising the amino acid sequence of SEQ ID NO: 1 or 24.
84 . The non-naturally occurring microbial organism of claim 83 , wherein the TER is derived from a bacterial, yeast, fungal, algal, animal, or plant species.
85 . The non-naturally occurring microbial organism of claim 83 , wherein the non-naturally occurring microbial organism comprises two, three, four, five, six, or seven exogenous nucleic acids each encoding an enzyme for the 6-aminocaproic acid pathway, hexamethylenediamine pathway, caprolactam pathway, 1,6-hexanediol pathway, caprolactone pathway, or a combination of two or more pathways.
86 . The non-naturally occurring microbial organism of claim 83 , wherein the 6-aminocaproic acid pathway comprises: (i) transaminase, (ii) 6-aminocaproate dehydrogenase, or (iii) transaminase and 6-aminocaproate dehydrogenase enzymes.
87 . A method of producing adipyl-CoA comprising:
culturing a non-naturally occurring microbial organism or host cell comprising at least one exogenous enzyme, the at least one exogenous enzyme comprising a TER converting 5-carboxyl-2-pentenoyl-CoA to adipyl CoA, the TER selected from:
a. a TER comprising the amino acid sequence of SEQ ID NO:1, 4-9, 11, 14, 15, or 17-26;
b. a TER comprising an amino acid sequence having at least 50% sequence identity to at least 25 or more contiguous amino acids of any of SEQ ID NO:1, 4-9, 11, 14, 15, or 17-26; or
c. an engineered TER comprising the amino acid sequence of SEQ ID NO: 1 or 24;
for a sufficient time period and conditions for producing adipyl-CoA.
88 . The method of claim 87 , further comprising recovering adipyl-CoA from the non-naturally occurring microbial organism, the fermentation broth, or both.
89 . The method of claim 87 , wherein the TER is derived from a bacterial, yeast, fungal, algal, animal, or plant species.
90 . The method of claim 87 , wherein the non-naturally occurring microbial organism further comprises two, three, four, five, six, or seven exogenous nucleic acids each encoding an enzyme for the 6-aminocaproic acid pathway, hexamethylenediamine pathway, caprolactam pathway, 1,6-hexanediol pathway, caprolactone pathway, or a combination of two or more pathways.
91 . The method of claim 90 , comprising producing hexamethylenediamine.
92 . The method of claim 90 , wherein the 6-aminocaproic acid pathway comprises: (i) transaminase, (ii) 6-aminocaproate dehydrogenase, or (iii) transaminase and 6-aminocaproate dehydrogenase enzymes.