US2020325465A1PendingUtilityA1

Enhancing microbial metabolism of c5 organic carbon

Assignee: MARA RENEWABLES CORPPriority: Jul 13, 2015Filed: Apr 30, 2020Published: Oct 15, 2020

Est. expiryJul 13, 2035(~9 yrs left)· nominal 20-yr term from priority

Inventors:Alexandra Merkx-Jacques David Woodhall Mark Scaife Roberto E. Armenta Denise Muise Holly Rasmussen Jeremy Benjamin

C12P 7/64C07K 14/38C12N 1/10C07K 2319/21C12N 15/52C12N 9/1205C12N 15/63C12Y 207/01017C12P 7/6445C12Y 503/01005C12N 1/12C07K 14/40C12N 15/65C12N 9/92C12P 7/6427Y02E50/10C12P 7/6434C12P 7/6432C12P 7/6431

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Claims

Abstract

Provided herein are recombinant microorganisms having two or more copies of a nucleic acid sequence encoding xylose isomerase, wherein the nucleic acid encoding the xylose isomerase is an exogenous nucleic acid. Optionally, the recombinant microorganisms include at least one nucleic acid sequence encoding a xylulose kinase and/or at least one nucleic acid sequence encoding a xylose transporter. The provided recombinant microorganisms are capable of growing on xylose as a carbon source.

Claims

exact text as granted — not AI-modified

What is claimed is: 
     
         1 . A recombinant microorganism comprising two or more copies of a nucleic acid sequence encoding xylose isomerase, wherein the nucleic acid encoding xylose isomerase is an exogenous nucleic acid. 
     
     
         2 . The recombinant microorganism of  claim 1 , further comprising at least one nucleic acid sequence encoding a xylulose kinase. 
     
     
         3 . The recombinant microorganism of  claim 2 , further comprising at least one nucleic acid sequence encoding a xylose transporter. 
     
     
         4 . The recombinant microorganism of  claim 1 , wherein the microorganism comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of the exogenous nucleic acid sequence encoding xylose isomerase. 
     
     
         5 . The recombinant microorganism of  claim 1 , wherein the 6 nucleic acid sequence encoding the xylose isomerase is at least 90% identical to SEQ ID NO:2. 
     
     
         6 . The recombinant microorganism of  claim 2 , wherein the microorganism comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of the nucleic acid sequence encoding the xylulose kinase. 
     
     
         7 . The recombinant microorganism of  claim 6 , wherein the nucleic acid sequence encoding the xylulose kinase is at least 90% identical to SEQ ID NO:5. 
     
     
         8 . The recombinant microorganism of  claim 3 , wherein the microorganism comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of the nucleic acid sequence encoding the xylose transporter. 
     
     
         9 . The recombinant microorganism of  claim 8 , wherein the xylose transporter is GXS1 from  Candida intermedia.    
     
     
         10 . The recombinant microorganism of  claim 8 , wherein the nucleic acid sequence encoding the xylose transporter is at least 90% identical to SEQ ID NO:23. 
     
     
         11 . The recombinant microorganism of  claim 3 , wherein the recombinant microorganism has increased xylose transport activity as compared to a non-recombinant control microorganism, increased xylose isomerase activity as compared to a non-recombinant control microorganism, increased xylulose kinase activity as compared to a non-recombinant control microorganism, or any combination thereof. 
     
     
         12 . The recombinant microorganism of  claim 3 , wherein the recombinant microorganism grows with xylose as the sole carbon source. 
     
     
         13 . The recombinant microorganism of  claim 3 , wherein the nucleic acid sequence encoding the xylose isomerase, the xylulose kinase and/or the xylose transporter is operably linked to a promoter. 
     
     
         14 . The recombinant microorganism of  claim 13 , wherein the promoter is a tubulin promoter that is at least 80% identical to SEQ ID NO:25 or SEQ ID NO:26. 
     
     
         15 . The recombinant microorganism of  claim 3 , wherein the nucleic acid sequence encoding the xylose isomerase, the xylulose kinase and/or the xylose transporter comprises a terminator. 
     
     
         16 . The recombinant microorganism of  claim 15 , wherein the terminator is a tubulin terminator that is at least 80% identical to SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:30. 
     
     
         17 . The recombinant microorganism of  claim 1 , wherein the microorganism is either a  Thraustochytrium  or a  Schizochytrium  microorganism. 
     
     
         18 . The recombinant microorganism of  claim 17 , wherein the microorganism is ONC-T18. 
     
     
         19 . A method of making a recombinant xylose-metabolizing microorganism comprising:
 providing one or more nucleic acid constructs comprising a nucleic acid sequence encoding a xylose isomerase, a nucleic acid sequence encoding a xylulose kinase and a nucleic acid sequence encoding a xylose transporter;   transforming the microorganism with the one or more nucleic acid constructs; and   isolating microorganisms comprising at least two or more copies of the nucleic acid sequences encoding the xylose isomerase.   
     
     
         20 . The method of  claim 19 , further comprising isolating microorganisms comprising at least one copy of the nucleic acid sequence encoding the xylulose kinase. 
     
     
         21 . The method of  claim 20 , isolating microorganisms comprising at least one copy of the xylose transporter. 
     
     
         22 . The method of  claim 19 , wherein the providing comprises providing a first nucleic acid construct comprising a nucleic acid sequence encoding a xylose isomerase, a second nucleic acid construct comprising a nucleic acid sequence encoding a xylulose kinase and a third nucleic acid construct comprising a nucleic acid sequence encoding a xylose transporter; 
     
     
         23 . The method of  claim 22 , wherein the first, second, and/or third nucleic acid construct comprises a promoter, a selectable marker, a nucleic acid sequence encoding a 2A peptide, the nucleic acid sequence encoding the xylose isomerase, and a terminator. 
     
     
         24 . The method of  claim 23 , wherein the promoter is a tubulin promoter that is at least 80% identical to SEQ ID NO:25 or SEQ ID NO:26. 
     
     
         25 . The method of  claim 23 , wherein the terminator is a tubulin terminator that is at least 80% identical to SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:30. 
     
     
         26 . The method of  claim 19 , wherein the isolated recombinant microorganism comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of the exogenous nucleic acid sequence encoding xylose isomerase. 
     
     
         27 . The method of  claim 19 , wherein the nucleic acid sequence encoding the xylose isomerase is at least 90% identical to SEQ ID NO:2. 
     
     
         28 . The method of  claim 19 , wherein the isolated recombinant microorganism comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of the nucleic acid sequence encoding the xylulose kinase. 
     
     
         29 . The method of  claim 19 , wherein the nucleic acid sequence encoding the xylulose kinase is at least 90% identical to SEQ ID NO:5. 
     
     
         30 . The method of  claim 19 , wherein the isolated recombinant microorganism comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of the nucleic acid sequence encoding the xylose transporter. 
     
     
         31 . The method of  claim 30 , wherein the xylose transporter is GXS1 from  Candida intermedia.    
     
     
         32 . The method of  claim 30 , wherein the nucleic acid sequence encoding the xylose transporter is at least 90% identical to SEQ ID NO:23. 
     
     
         33 . The method of  claim 19 , wherein the isolated recombinant microorganism has increased xylose transport activity as compared to a control non-recombinant microorganism, increased xylose isomerase activity as compared to a control non-recombinant microorganism, increased xylulose kinase activity as compared to a control non-recombinant microorganism, or a combination thereof. 
     
     
         34 . The method of  claim 19 , wherein the isolated recombinant microorganism grows with xylose as the sole carbon source. 
     
     
         35 . The method of  claim 19 , wherein the microorganism is either a  Thraustochytrium  or a  Schizochytrium  microorganism. 
     
     
         36 . The method of  claim 19 , wherein the microorganism is ONC-T18.

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