Beta-alanine/alpha-ketoglutarate aminotransferase for 3-hydroxypropionic acid production

Abstract

The present disclosure provides novel beta-alanine/alpha ketoglutarate aminotransferase nucleic acid and protein sequences having increased biological activity. Also provided are cells containing such enzymes, as well as methods of their use, for example to produce malonyl semialdehyde and downstream products thereof, such as 3-hydroxypropionic acid and derivatives thereof.

We claim: 
     
         1 . A method of making 3-hydroxypropionic acid (3-HP), comprising:
 culturing a recombinant cell comprising an exogenous nucleic acid sequence encoding a protein variant that comprises at least 95% sequence identity to SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19 and has beta-alanine aminotransferase (BAAT) activity, under conditions that permit the cell to make 3-HP.   
     
     
         2 . The method of  claim 1 , wherein the protein comprises a sequence having at least 97% sequence identity to SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19, wherein the protein has BAAT activity. 
     
     
         3 . The method of  claim 1 , wherein the protein comprises a sequence having at least 98% sequence identity to SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19, wherein the protein has BAAT activity. 
     
     
         4 . The method of  claim 1 , wherein the protein comprises a sequence having at least 99% sequence identity to SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19, wherein the protein has BAAT activity. 
     
     
         5 . The method of  claim 1 , wherein the protein comprises the amino acid sequence shown in SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19. 
     
     
         6 . The method of  claim 1 , wherein the protein comprises 1-10 conservative amino acid substitutions and has BAAT activity. 
     
     
         7 . The method of  claim 1 , wherein the protein comprises at least 50 contiguous amino acids of the amino acid sequence shown in any of SEQ ID NOS: 4, 6, 10, 12, 14, 17 or 19, and wherein the protein has BAAT activity. 
     
     
         8 . The method of  claim 1 , wherein the protein comprises a substitution at the amino acid corresponding to position 3 of SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19, a substitution at the amino acid corresponding to position 24 of SEQ ID NO: 4, 6, 10, 12, 14, 17 or 9, a substitution at the amino acid corresponding to position 110 of SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19, a substitution at the amino acid corresponding to position 113 of SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19, a substitution at the amino acid corresponding to position 133 of SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19, or combinations thereof, and wherein the protein has BAAT activity. 
     
     
         9 . The method of  claim 8 , wherein the substitution at the amino acid corresponding to position 3 of SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19 is His or a conservative substitution of His, the substitution at the amino acid corresponding to position 24 of SEQ ID NO: 4, 6, 10, 12, 14, 17 or 9 is Thr or a conservative substitution of Thr, the substitution at the amino acid corresponding to position 110 of SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19 is Asn or a conservative substitution of Asn, the substitution at the amino acid corresponding to position 113 of SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19 is Leu or a conservative substitution of Leu, the substitution at the amino acid corresponding to position 133 of SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19 is Thr or a conservative substitution of Thr, or combinations thereof, and wherein the protein has BAAT activity. 
     
     
         10 . The method of  claim 1 , wherein the nucleic acid sequence encodes a protein that comprises at least 95% sequence identity to SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19, wherein the amino acid corresponding to position 3 of SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19 is His, the amino acid corresponding to position 24 of SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19 is Thr, the amino acid corresponding to position 110 of SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19 is Asn, the amino acid corresponding to position 113 of SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19 is Leu, the amino acid corresponding to position 133 of SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19 is Thr, or combinations thereof, and wherein the protein has BAAT activity. 
     
     
         11 . The method of  claim 8 , wherein the amino acid corresponding to position 3 of SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19 is substituted with His or a conservative substitution of His and the protein has BAAT activity. 
     
     
         12 . The method of  claim 8 , wherein the amino acid corresponding to position 24 of SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19 is substituted with Thr or a conservative substitution of Thr and the protein has BAAT activity. 
     
     
         13 . The method of  claim 8 , wherein the amino acid corresponding to position 110 of SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19 is substituted with Asn or a conservative substitution of Asn and the protein has BAAT activity. 
     
     
         14 . The method of  claim 8 , wherein the amino acid corresponding to position 113 of SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19 with Leu or a conservative substitution of Leu is substituted and the protein has BAAT activity. 
     
     
         15 . The method of  claim 8 , wherein the amino acid corresponding to position 133 of SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19 is substituted with Thr or a conservative substitution of Thr and the protein has BAAT activity. 
     
     
         16 . The method of  claim 8 , wherein the amino acid corresponding to position 3 of SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19 is His and the protein has BAAT activity. 
     
     
         17 . The method of  claim 8 , wherein the amino acid corresponding to position 24 of SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19 is Thr the protein has BAAT activity. 
     
     
         18 . The method of  claim 8 , wherein the amino acid corresponding to position 110 of SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19 is Asn the protein has BAAT activity. 
     
     
         19 . The method of  claim 8 , wherein the amino acid corresponding to position 113 of SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19 is Leu the protein has BAAT activity. 
     
     
         20 . The method of  claim 8 , wherein the amino acid corresponding to position 133 of SEQ ID NO: 4, 6, 10, 12, 14, 17 or 19 is Thr the protein has BAAT activity. 
     
     
         21 . The method of  claim 1 , wherein the nucleic acid sequence comprises at least 95% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 3, 5, 7, 9, 11, 13, 16 or 18. 
     
     
         22 . The method of  claim 1 , wherein the nucleic acid sequence comprises at least 98% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 3, 5, 7, 9, 11, 13, 16 or 18. 
     
     
         23 . The method of  claim 1 , wherein the nucleic acid sequence comprises at least 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 3, 5, 7, 9, 11, 13, 16 or 18. 
     
     
         24 . The method of  claim 1 , wherein the nucleic acid sequence comprises SEQ ID NO: 3, 5, 7, 9, 11, 13, 16 or 18. 
     
     
         25 . The method of  claim 1 , wherein the nucleic acid sequence is part of a vector. 
     
     
         26 . The method of  claim 1 , wherein the nucleic acid sequence is operably linked to a promoter sequence. 
     
     
         27 . The method of  claim 1 , wherein the recombinant cell is a prokaryotic cell. 
     
     
         28 . The method of  claim 1 , wherein the recombinant cell is a yeast cell. 
     
     
         29 . The method of  claim 1 , wherein the cell comprises a ΔpoxB mutation, an ΔadhE, ΔatpFH mutation, or combinations thereof. 
     
     
         30 . The method of  claim 1 , wherein the cell further comprises an exogenous nucleic acid molecule comprising at least 95% sequence identity to SEQ ID NO: 52. 
     
     
         31 . The method of  claim 1 , wherein the cell further comprises an exogenous or an endogenous nucleic acid molecule encoding a 3-HP dehydrogenase. 
     
     
         32 . The method of  claim 1 , wherein the method is a method of making an ester of 3-HP, and the cell further comprises an exogenous nucleic acid molecule encoding a lipase or esterase and the cell produces an ester of 3-HP. 
     
     
         33 . The method of  claim 1 , wherein the method is a method of making polymerized 3-HP, and the cell further comprises an exogenous nucleic acid molecule encoding an esterase and the cell produces polymerized 3-HP. 
     
     
         34 . The method of  claim 1 , wherein method is a method of making 1,3-propanediol, and the cell further comprises an exogenous or an endogenous nucleic acid molecule encoding an alcohol dehydrogenase, an aldehyde dehydrogenase, or both, and wherein the cell produces 1,3-propanediol.