ENGINEERED TRANSAMINASE AND METHODS OF MAKING AND USING
Disclosed are transaminase (TA) enzymes and nucleic acids encoding them. In some cases, the transaminase enzymes are non-natural, engineered transaminases. Also disclosed are 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 TA and in some cases engineered TA.
1 - 51 . (canceled)
52 . An engineered transaminase (TA) enzyme comprising at least one alteration of an amino acid of SEQ ID NO:1 or 13 or 31.
53 . The engineered TA of claim 52 , wherein the engineered transaminase enzyme is capable of
a) forming 6-aminocaproic acid from an adipate semialdehyde substrate;
b) forming 6-aminocaproic acid from an adipate semialdehyde substrate at a greater rate as compared to the wild type transaminase;
c) having a higher affinity for an adipate semialdehyde substrate as compared to the wild type transaminase; or
d) any combination of a), b), and c).
54 . The engineered TA of claim 52 , wherein the engineered transaminase comprises one or more amino acid alterations at one or more positions selected from residues V114, S136, T148, P153, 1203, 1204, P206, V207, V111, T216, A237, T264, M265, L386, G19, C22, D70, R94, D99, T109, E112, A113, F137, G144, 1149, K150, Y154, S178, L186, Q208, L234, T242, A315, K318, R338, G336, L386, V390, A406, S416, A421, G17, M21, A50, A76, Y77, Q78, 179, G84, F107, T108, K119, G139, M142, A152, P153, E205, G209, G211, D238, M285, A290, G291, G292, L293, Y297, M353, S387, S388, and G392 or a combination thereof of SEQ ID NO:1.
55 . The engineered TA of claim 52 , wherein the at least one amino acid alteration of the engineered protein is an alteration selected from Table 5 and combinations thereof of SEQ ID NO:1.
56 . The engineered TA of claim 52 , wherein the amino acid alteration comprises at least two, three, four, five, six, seven, eight, or more of an amino acid of SEQ ID NO:1, 13, or 31.
57 . The engineered TA of claim 52 , wherein the engineered TA comprises activity that is at least 20% higher than the activity of the transaminase of SEQ ID NO:1, 13, or 31 with no amino acid alterations.
58 . The engineered TA of claim 52 , further comprising expression of the engineered TA 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 6ACA than a control cell that expresses the wild type TA from which the engineered TA is derived.
59 . An engineered cell comprising the engineered transaminase of claim 52 .
60 . The engineered TA of claim 59 , further comprising expression of the engineered TA 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 6ACA than a control cell that expresses the wild type TA from which the engineered TA is derived
61 . A non-naturally occurring microbial organism comprising at least one exogenous enzyme, the at least one exogenous enzyme comprising a transaminase enzyme converting adipate semialdehyde to 6-amino caproic acid, the transaminase selected from:
(i) a transaminase 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, 3, 4, 5, 9, 12, 13, 26, 27, 30, 31, 38, 50, 52, 64, 74, 78, 79, 81, 91, 106, 108, and 116; or
(ii) an engineered TA of SEQ ID NO: 1, 13 or 31.
62 . The non-naturally occurring microbial organism of claim 61 , wherein the microbial organism further comprises a 6-aminocaproic acid pathway, 6-hydroxy caproic acid pathway, a hexamethylene diamine pathway, caprolactone pathway, or a caprolactam pathway.
63 . The non-naturally occurring microbial organism of claim 61 , wherein the non-naturally occurring microbial organism comprises a species of Acinetobacter, Actinobacillus, Anaerobiospirillum, Aspergillus, Bacillus, Clostridium, Corynebacterium, Escherichia, Gluconobacter, Klebsiella, Kluyveromyces, Lactococcus, Lactobacillus, Mannheimia, Pichia, Pseudomonas, Rhizobium, Rhizopus, Saccharomyces, Schizosaccharomyces, Streptomyces, and Zymomonas.
64 . The non-naturally occurring microbial organism of claim 61 , wherein the TA is derived from Arthrobacter, Paenarthrobacter, Saccharomyces, Clostridium, Gulosibacter, Kocuria, Pseudomonas, and Alcaligenes.
65 . A non-naturally occurring microbial organism or host cell of claim 61 , wherein the non-naturally occurring microbial organism or host cell produces more of one or more of adipate, caproic acid, hexamethylene diamine, caprolactone, or caprolactam than is produced by a control microbial organism or host cell substantially identical to the non-naturally occurring microbial organism or host cell with the exception that the control microbial organism or host cell does not comprise an exogenous transaminase or an exogenous nucleic acid sequence encoding the transaminase.
66 . A method of producing 6-amino caproic acid comprising:
culturing a non-naturally occurring microbial organism or host cell organism comprising at least one exogenous enzyme, the at least one exogenous enzyme comprising a transaminase enzyme converting adipate semialdehyde to 6-amino caproic acid, the transaminase selected from:
(i) a transaminase 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, 3, 4, 5, 9, 12, 13, 26, 27, 30, 31, 38, 50, 52, 64, 74, 78, 79, 81, 91, 106, 108, and 116; or
(ii) an engineered TA of SEQ ID NO: 1, 13 or 31; and producing 6-amino caproic acid.
67 . The method of claim 66 , further comprising recovering 6-amino caproic acid from the non-naturally occurring microbial organism, the fermentation broth, or both.
68 . The method of claim 66 , wherein the microbial organism comprises a 6-aminocaproic acid pathway, 6-hydroxy caproic acid pathway, a hexamethylene diamine pathway, caprolactone pathway, or a caprolactam pathway.
69 . The method of claim 66 , wherein the non-naturally occurring microbial organism comprises a species of Acinetobacter, Actinobacillus, Anaerobiospirillum, Aspergillus, Bacillus, Clostridium, Corynebacterium, Escherichia, Gluconobacter, Klebsiella, Kluyveromyces, Lactococcus, Lactobacillus, Mannheimia, Pichia, Pseudomonas, Rhizobium, Rhizopus, Saccharomyces, Schizosaccharomyces, Streptomyces, and Zymomonas.
70 . The method of claim 66 , wherein the TA is derived from Arthrobacter, Paenarthrobacter, Saccharomyces, Clostridium, Gulosibacter, Kocuria, Pseudomonas, and Alcaligenes.
71 . The method of claim 66 , wherein the non-naturally occurring microbial organism or host cell produces more of one or more of adipate, caproic acid, hexamethylene diamine, caprolactone, or caprolactam than is produced by a control microbial organism or host cell substantially identical to the non-naturally occurring microbial organism or host cell with the exception that the control microbial organism or host cell does not comprise an exogenous transaminase or an exogenous nucleic acid sequence encoding the transaminase.