Materials and Methods for Producing Alkaloids

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. The host cell of claim 1 that 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.