Materials and Methods for Producing Alkaloids

Abstract

The invention generally relates to methods of producing loline alkaloids or precursors thereof, expression constructs, and host cells useful for producing loline alkaloids or precursors thereof, and methods for producing loline alkaloids or precursors thereof in a host cell.

20 . A host cell able to produce more of at least one loline alkaloid or precursor thereof, than does a control cell, as a result of the host cell being transformed or modified to comprise at least one polynucleotide selected from the group consisting of:
 i) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:1, 12, 13 and 61 or a variant thereof with at least 40% identity to any one of SEQ ID NO:1, 12, 13 and 61 with at least one of activity of a gamma-class PLP enzyme and an activity substantially equivalent to that of a lolC gene product,   ii) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:2, 14, 15 and 62 or a variant thereof with at least 40% identity to any one of SEQ ID NO:2, 14, 15 and 62 with at least one of activity of an alpha-class PLP enzyme and activity substantially equivalent to that of the lolD gene product,   iii) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:3, 16, 17 and 63 or a variant thereof with at least 40% identity to of any one of SEQ ID NO:3, 16, 17 and 63 with at least one of monooxygenase activity and activity substantially equivalent to that of the lolF gene product, iv) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:4, 18 and 19 or a variant thereof with at least 40% identity of any one of SEQ ID NO:4, 18 and 19 with at least one of amino acid bridging activity and activity substantially equivalent to that of the lolA gene product,   v) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:5, 20, 21 and 64 or a variant thereof with at least 40% identity to of any one of SEQ ID NO:5, 20, 21 and 64 with at least one of activity of an alpha-class PLP enzyme and activity substantially equivalent to that of the lolT gene product,   vi) a polynucleotide encoding a polypeptide comprising the sequence of of any one of SEQ ID NO:6, 22, 23 and 65 or a variant thereof with at least 40% identity to of any one of SEQ ID NO:6, 22, 23 and 65 with at least one of activity of a non-heme iron dioxygenase and activity substantially equivalent to that of the lolE gene product,   vii) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:7, 24, 25 and 66 or a variant thereof with at least 40% identity to any one of SEQ ID NO:7, 24, 25 and 66 with at least one of activity of a non-heme iron dioxygenase and activity substantially equivalent to that of the lolO gene product,   viii) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:8, 26 and 27 or a variant thereof with at least 40% identity to any one of SEQ ID NO:8, 26 and 27 with activity substantially equivalent to that of the lolU gene product,   ix) a polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:9 or 28 or a variant thereof with at least 40% identity to SEQ ID NO:9 or 28 with at least one of N-Methyltransferase activity and activity substantially equivalent to that of the lolM gene product,   x) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:10, 29 and 67 or a variant thereof with at least 40% identity to any one of SEQ ID NO:10, 29 and 67 with at least one of acetamidase activity and activity substantially equivalent to that of the lolN gene product, and   xi) a polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:11 or 30 or a variant thereof with at least 40% identity to SEQ ID NO:11 or 30 with at least one of cytochrome P450 monooxygenase activity and activity substantially equivalent to that of the lolP gene product.   
     
     
         21 . The host cell of  claim 20  wherein the host cell is able to produce more of at least one loline alkaloid, than does a control cell, as a result of the host cell being transformed or modified to comprise the at least one polynucleotide. 
     
     
         22 . The host cell of  claim 20  wherein the host cell is tolerant of endogenous (3-amino-3-carboxypropyl) proline (ACPP) production. 
     
     
         23 . The host cell of  claim 22  wherein the host cell has been pre-selected for tolerance to cellular ACPP. 
     
     
         24 . The host cell of  claim 20  wherein the host cell prior to modification or transformation, is able to convert exo-1-aminopyrrolizidine (1-AP) to exo-1-acetamido-pyrrolizidine (AcAP). 
     
     
         25 . The host cell of  claim 24  wherein the host cell prior to modification or transformation, has been pre-selected for the ability to convert 1-AP to AcAP. 
     
     
         26 . The host cell of  claim 20  wherein the host cell is transformed or modified to comprise at least the polynucleotides of i), ii), iii), v) and vii). 
     
     
         27 . The host cell of  claim 20  wherein the host cell is not transformed or modified to comprise the polynucleotide of vi). 
     
     
         28 . The host cell of  claim 20  wherein the host cell is not transformed or modified to comprise the polynucleotide of viii). 
     
     
         29 . A method for producing a host cell that produces at least one loline alkaloid or precursor thereof, the method comprising modifying or transforming a host cell to comprise at least one polynucleotide as defined in  claim 20 . 
     
     
         30 . The method of  claim 29  wherein the host cell produces more of at least one loline alkaloid, than does a control cell, as a result of the host cell being transformed or modified to comprise the at least one polynucleotide. 
     
     
         31 . The method of  claim 29  wherein the host cell is tolerant of endogenous (3-amino-3-carboxypropyl) proline (ACPP) production. 
     
     
         32 . The method of  claim 29  further comprising the step of pre-selecting the host cell for tolerance to cellular ACPP. 
     
     
         33 . The method of  claim 29  wherein the host cell prior to modification or transformation, is able to convert exo-1-aminopyrrolizidine (1-AP) to exo-1-acetamido-pyrrolizidine (AcAP). 
     
     
         34 . The method of  claim 29  wherein the host cell prior to modification or transformation, has been pre-selected for the ability to convert 1-AP to AcAP. 
     
     
         35 . The method of  claim 29  further comprising the step of pre-selecting the host cell for said ability to convert 1-AP to AcAP. 
     
     
         36 . The method of  claim 29  wherein the host cell is transformed or modified to comprise at least the polynucleotides of i), ii), iii), v) and vii). 
     
     
         37 . A method for producing at least one loline alkaloid or a precursor thereof, the method comprising culturing the host cells of claim  1  under conditions conducive to the production of the at least one loline alkaloid or precursor thereof, by the host cells. 
     
     
         38 . The method of  claim 37  wherein the host cell produces at least one loline alkaloid.