Method for producing pyripyropene

1. A method for producing pyripyropene A, comprising: culturing a microorganism in the presence of pyripyropene E to produce pyripyropene A and isolating the pyripyropene A, wherein said microorganism is transformed with at least one heterologous polynucleotide in (I) to (Ill) below or a recombinant vector comprising said heterologous polynucleotide:

(I) a heterologous polynucleotide having at least one nucleotide sequence selected from the nucleotide sequences in (a) to (d) below:

(a) the nucleotide sequence of SEQ ID NO: 266,

(b) a nucleotide sequence which is capable of hybridizing with every nucleotide in the full length complementary sequence in SEQ ID NO: 266 under stringent conditions wherein said stringent conditions comprise washing with 2× saline-sodium citrate (SSC) buffer and 0.5% sodium dodecyl sulfate (SDS) at 60° C. for 20 minutes, and then washing 0.2×SSC and 0.1% SDS at 60° C. for 15 minutes,

(c) the nucleotide sequence of SEQ ID NO: 266 in which one to ten nucleotides are deleted, substituted, inserted or added, and

(d) a nucleotide sequence which has at least 90% identity the nucleotide sequence of SEQ ID NO: 266;

wherein positions of the nucleotide sequence corresponding to positions 3342 to 5158 in SEQ ID NO: 266 encode a protein having CoA ligase activity, positions of the nucleotide sequence corresponding to positions 5382 to 12777 in SEQ ID NO: 266 encode a protein having LovB-like polyketide synthase activity, positions of the nucleotide sequence corresponding to positions 13266 to 15144 in SEQ ID NO: 266 encode a protein having Cytochrome P450 monooxygenase-1 activity, positions of the nucleotide sequence corresponding to positions 16220 to 18018 in SEQ ID NO: 266 encode a protein having Cytochrome P450 monooxygenase-2 activity, positions of the nucleotide sequence corresponding to positions 18506 to 19296 in SEQ ID NO: 266 encode a protein having Cyclase activity, positions of the nucleotide sequence corresponding to positions 19779 to 21389 in SEQ ID NO: 266 encode a protein having FAD-dependent monooxygenase activity, positions of the nucleotide sequence corresponding to positions 21793 to 22877 in SEQ ID NO: 266 encode a protein having UbiA-like prenyltransferase activity, positions of the nucleotide sequence corresponding to positions 23205 to 24773 in SEQ ID NO: 266 encode a protein having Acetyltransferase activity, and positions of the nucleotide sequence corresponding to positions 25824 to 27178 in SEQ ID NO: 266 encode a protein having Acetyltransferase-2 activity;

(II) a heterologous polynucleotide having a nucleotide sequence encoding the amino acid sequences of SEQ ID NOs: 267-275 or an amino acid sequence having one to ten substituted, deleted, added, or inserted residues in the amino acid sequences of SEQ ID NOs: 267-275 and having the same enzymatic activity as the amino acid sequences set forth in SEQ ID NOs: 267-275, respectively,

wherein SEQ ID NO: 267 is a protein having CoA ligase activity, SEQ ID NO: 268 is a protein having LovB-like polyketide synthase activity, SEQ ID NO: 269 is a protein having Cytochrome P450 monooxygenase-1 activity, SEQ ID NO: 270 is a protein having Cytochrome P450 monooxygenase-2 activity, SEQ ID NO: 271 is a protein having cyclase activity, SEQ ID NO: 272 is a protein having FAD-dependent monooxygenase activity, SEQ ID NO: 273 is a protein having UbiA-like prenyltransferase activity, SEQ ID NO: 274 is a protein having Acetyltransferase activity, and SEQ ID NO: 275 is a protein having Acetyltransferase-2 activity;

(III) a heterologous polynucleotide having at least one nucleotide sequence selected from the group consisting of the nucleotide sequences set forth in (1) to (4) below:

(1) the nucleotide sequences set forth in (a) to (i) below:

(a) a nucleotide sequence comprising positions 3342 to 5158 of the nucleotide sequence set forth in SEQ ID NO: 266,

(b) a nucleotide sequence comprising positions 5382 to 12777 of the nucleotide sequence set forth in SEQ ID NO: 266,

(c) a nucleotide sequence comprising positions 13266 to 15144 of the nucleotide sequence set forth in SEQ ID NO: 266,

(d) a nucleotide sequence comprising positions 16220 to 18018 of the nucleotide sequence set forth in SEQ ID NO: 266,

(e) a nucleotide sequence comprising positions 18506 to 19296 of the nucleotide sequence set forth in SEQ ID NO: 266,

(f) a nucleotide sequence comprising positions 19779 to 21389 of the nucleotide sequence set forth in SEQ ID NO: 266,

(g) a nucleotide sequence comprising positions 21793 to 22877 of the nucleotide sequence set forth in SEQ ID NO: 266,

(h) a nucleotide sequence comprising positions 23205 to 24773 of the nucleotide sequence set forth in SEQ ID NO: 266, and

(i) a nucleotide sequence comprising positions 25824 to 27178 of the nucleotide sequence set forth in SEQ ID NO: 266,

(2) a nucleotide sequence which hybridizes with every nucleotide in the full length complementary sequence in (1) under stringent conditions wherein said stringent conditions comprise washing with 2× saline-sodium citrate (SSC) buffer and 0.5% sodium dodecyl sulfate (SDS) at 60° C. for 20 minutes, and then washing with 0.2×SSC and 0.1% SDS at 60° C. for 15 minutes;

(3) the nucleotide sequence in (1) in which one to ten nucleotides are deleted, substituted, inserted or added, and

(4) a nucleotide sequence with at least 90% sequence identity to the nucleotide sequence in (1),

wherein positions of the nucleotide sequences of (1) to (4) corresponding to positions 3342 to 5158 in SEQ ID NO: 266 encode a protein having CoA ligase activity, positions of the nucleotide sequence corresponding to positions 5382 to 12777 in SEQ ID NO: 266 encode a protein having LovB-like polyketide synthase activity, positions of the nucleotide sequence corresponding to positions 13266 to 15144 in SEQ ID NO: 266 encode a protein having Cytochrome P450 monooxygenase-1 activity, positions of the nucleotide sequence corresponding to positions 16220 to 18018 in SEQ ID NO: 266 encode a protein having Cytochrome P450 monooxygenase-2 activity, positions of the nucleotide sequence corresponding to positions 18506 to 19296 in SEQ ID NO: 266 encode a protein having Cyclase activity, positions of the nucleotide sequence corresponding to positions 19779 to 21389 in SEQ ID NO: 266 encode a protein having FAD-dependent monooxygenase activity, positions of the nucleotide sequence corresponding to positions 21793 to 22877 in SEQ ID NO: 266 encode a protein having UbiA-like prenyltransferase activity, positions of the nucleotide sequence corresponding to positions 23205 to 24773 in SEQ ID NO: 266 encode a protein having Acetyltransferase activity, and positions of the nucleotide sequence corresponding to positions 25824 to 27178 in SEQ ID NO: 266 encode a protein having Acetyltransferase-2 activity.

2. A method for producing pyripyropene A, comprising: culturing a microorganism in the presence of pyripyropene E to produce pyripyropene A and isolating the pyripyropene A, wherein said microorganism is transformed with at least one heterologous polynucleotide in (I) to (II) below or a recombinant vector comprising said heterologous polynucleotide:

(I) a heterologous polynucleotide having a nucleotide sequence encoding polypeptides comprising the amino acid sequences of SEQ ID NOs: 269, 270, and 275 or an amino acid sequence having one to ten substituted, deleted, added, or inserted residues in the amino acid sequences of SEQ ID NOs: 269, 270, and 275 and having the same enzymatic activity as the amino acid sequences set forth in SEQ ID NOs: 269, 270, and 275, respectively;

wherein SEQ ID NO: 269 is a protein having Cytochrome P450 monooxygenase-1 activity, SEQ ID NO: 270 is a protein having Cytochrome P450 monooxygenase-2 activity, and SEQ ID NO: 275 is a protein having Acetyltransferase-2 activity;

(II) a heterologous polynucleotide having a nucleotide sequence selected from the group consisting of the nucleotide sequences in (1) to (4) below:

(1) the nucleotide sequences set forth in (a) to (c) below:

(a) a nucleotide sequence comprising positions 13266 to 15144 of the nucleotide sequence set forth in SEQ ID NO: 266,

(b) a nucleotide sequence comprising positions 16220 to 18018 of the nucleotide sequence set forth in SEQ ID NO: 266, and

(c) a nucleotide sequence comprising positions 25824 to 27178 of the nucleotide sequence set forth in SEQ ID NO: 266,

(2) a nucleotide sequence which is capable of hybridizing with every nucleotide in the full length complementary sequence in (1) under stringent conditions wherein said stringent conditions comprise washing with 2× saline-sodium citrate (SSC) buffer and 0.5% sodium dodecyl sulfate (SDS) at 60° C. for 20 minutes, and then washing with 0.2×SSC and 0.1% SDS at 60° C. for 15 minutes;

(3) the nucleotide sequence in (1) in which one to ten nucleotides are deleted, substituted, inserted or added, and;

(4) a nucleotide sequence comprising at least 90% sequence identity to the nucleotide sequence in (1),

wherein positions of the nucleotide sequence corresponding to positions 13266 to 15144 in SEQ ID NO: 266 encode a protein having Cytochrome P450 monooxygenase-1 activity, positions of the nucleotide sequence corresponding to positions 16220 to 18018 in SEQ ID NO: 266 encode a protein having Cytochrome P450 monooxygenase-2 activity, and positions of the nucleotide sequence corresponding to positions 25824 to 27178 in SEQ ID NO: 266 encode a protein having Acetyltransferase-2 activity.

3. A method for producing pyripyropene A, comprising: culturing a microorganism in the presence of deacetyl pyripyropene E to produce pyripyropene A and isolating the pyripyropene A, wherein said microorganism is transformed with at least one heterologous polynucleotide in (I) and (II) below or a recombinant vector comprising said heterologous polynucleotide:

(VI) a heterologous polynucleotide having a nucleotide sequence encoding the amino acid sequences of SEQ ID NOs: 269, 270, 274, and 275,

wherein SEQ ID NO: 269 is a protein having Cytochrome P450 monooxygenase-1 activity, SEQ ID NO: 270 is a protein having Cytochrome P450 monooxygenase-2 activity, SEQ ID NO: 274 is a protein having Acetyltransferase activity, and SEQ ID NO: 275 is a protein having Acetyltransferase-2 activity;

(VII) a heterologous polynucleotide having a nucleotide sequence selected from the group consisting of the nucleotide sequences in (1) to (4) below:

(1) the nucleotide sequences set forth in (a) to (d) below:

(a) a nucleotide sequence comprising positions 13266 to 15144 of the nucleotide sequence set forth in SEQ ID NO: 266,

(b) a nucleotide sequence comprising positions 16220 to 18018 of the nucleotide sequence set forth in SEQ ID NO: 266,

(c) a nucleotide sequence comprising positions 23205 to 24773 of the nucleotide sequence set forth in SEQ ID NO: 266, and

(d) a nucleotide sequence comprising positions 25824 to 27178 of the nucleotide sequence set forth in SEQ ID NO: 266,

(2) a nucleotide sequence which hybridizes with every nucleotide in the full length complementary sequence in (1) under stringent conditions wherein said stringent conditions comprise washing with 2× saline-sodium citrate (SSC) buffer and 0.5% sodium dodecyl sulfate (SDS) at 60° C. for 20 minutes, and then washing with 0.2×SSC and 0.1% SDS at 60° C. for 15 minutes;

(3) the nucleotide sequence in (1) in which one to ten nucleotides are deleted, substituted, inserted, or added, and;

(4) a nucleotide sequence comprising at least 90% sequence identity to the nucleotide sequence in (1)

wherein positions of the nucleotide sequence corresponding to positions 13266 to 15144 in SEQ ID NO: 266 encode a protein having Cytochrome P450 monooxygenase-1 activity, positions of the nucleotide sequence corresponding to positions 16220 to 18018 in SEQ ID NO: 266 encode a protein having Cytochrome P450 monooxygenase-2 activity, positions of the nucleotide sequence corresponding to positions 23205 to 24773 in SEQ ID NO: 266 encode a protein having Acetyltransferase activity, and positions of the nucleotide sequence corresponding to positions 25824 to 27178 in SEQ ID NO: 266 encode a protein having Acetyltransferase-2 activity.

4. A method for producing 4-hydroxy-6-(pyridine-3-yl)-3-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyl)-2H-pyrane-2-one comprising: culturing a microorganism in the presence of 4-oxo-6-(3-pyridyl)-α-pyrone and isolating 4-hydroxy-6-(pyridine-3-yl)-3-((2E,6E),3,7,11-trimethyldodeca-2,6,10-trienyl)-2H-pyrane-2-one to produce 4-hydroxy-6-(pyridine-3-yl)-3-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyl)-2H-pyrane-2-one and isolating the 4-hydroxy-6-(pyridine-3-yl)-3-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyl)-2H-pyrane-2-one, wherein said microorganism is transformed with at least one heterologous polynucleotide in (I) and (II) below or a recombinant vector comprising said heterologous polynucleotide:

(I) a heterologous polynucleotide having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 273 or the amino acid sequence of SEQ ID NO: 273 having one to ten substituted, deleted, added, or inserted residues and having the same enzymatic activity as the amino acid sequence of SEQ ID NO: 273,

wherein SEQ ID NO: 273 is a protein having UbiA-like prenyltransferase activity;

(II) a heterologous polynucleotide having a nucleotide sequence selected from the group consisting of the nucleotide sequences in (1) to (4) below:

(1) a nucleotide sequence comprising positions 21793 to 22877 of the nucleotide sequence set forth in SEQ ID NO: 266, (2) a nucleotide sequence which hybridizes with every nucleotide in the full length complementary sequence in (1) under stringent conditions wherein said stringent conditions comprise washing with 2× saline-sodium citrate (SSC) buffer and 0.5% sodium dodecyl sulfate (SDS) at 60° C. for 20 minutes, and then washing with 0.2×SSC and 0.1% SDS at 60° C. for 15 minutes;

(3) the nucleotide sequence in (1) in which one to ten nucleotides are deleted, substituted, inserted, or added, and;

(4) a nucleotide sequence with at least 90% sequence identity to the nucleotide sequence in (1)

wherein positions of the nucleotide sequence corresponding to positions 21793 to 22877 in SEQ ID NO: 266 encode a protein having UbiA-like prenyltransferase activity.

5. A recombinant vector comprising one or more polynucleotides and a promoter that is heterologous to the one or more polynucleotides, wherein the one or more polynucleotides are selected from the following (a), (b), (c), (d), (e), (f), (g) and (h):

(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 266,

(b) a polynucleotide encoding a polypeptide which consists of an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NOs: 269, 270, 273, 274, and 275,

wherein SEQ ID NO: 269 is a protein having Cytochrome P450 monooxygenase-1 activity, SEQ ID NO: 270 is a protein having Cytochrome P450 monooxygenase-2 activity, SEQ ID NO: 273 is a protein having UbiA-like prenyltransferase activity, SEQ ID NO: 274 is a protein having Acetyltransferase activity, and SEQ ID NO: 275 is a protein having Acetyltransferase-2 activity;

(c) a polynucleotide with at least 90% sequence identity to a polynucleotide encoding the amino acid sequence of SEQ ID NO: 269 and having Cytochrome P450 monooxygenase-1 activity,

(d) a polynucleotide with at least 90% sequence identity to a polynucleotide encoding the amino acid sequence of SEQ ID NO: 270 and having Cytochrome P450 monooxygenase-2 activity,

(e) a polynucleotide with at least 90% sequence identity to a polynucleotide encoding the amino acid sequence of SEQ ID NO: 273 and having UbiA-like prenyltransferase activity,

(f) a polynucleotide with at least 90% sequence identity to a polynucleotide encoding the amino acid sequence of SEQ ID NO: 274 and having Acetyltransferase activity,

(g) a polynucleotide with at least 90% sequence identity to a polynucleotide encoding the amino acid sequence of SEQ ID NO: 275 and having Acetyltransferase-2 activity,

(h) a polynucleotide comprising a nucleotide sequence selected from the group consisting of the nucleotide sequences in (i), (ii), (iii), (iv), and (v):

(i) a nucleotide sequence comprising positions 13266 to 15144 of the nucleotide sequence set forth in SEQ ID NO: 266,

(ii) a nucleotide sequence comprising positions 16220 to 18018 of the nucleotide sequence set forth in SEQ ID NO: 266,

(iii) a nucleotide sequence comprising positions 21793 to 22877 of the nucleotide sequence set forth in SEQ ID NO: 266,

(iv) a nucleotide sequence comprising positions 23205 to 24773 of the nucleotide sequence set forth in SEQ ID NO: 266, and

(v) a nucleotide sequence comprising positions 25824 to 27178 of the nucleotide sequence set forth in SEQ ID NO: 266,

wherein positions of the nucleotide sequence corresponding to positions 13266 to 15144 in SEQ ID NO: 266 encode a protein having Cytochrome P450 monooxygenase-1 activity, positions of the nucleotide sequence corresponding to positions 16220 to 18018 in SEQ ID NO: 266 encode a protein having Cytochrome P450 monooxygenase-2 activity, positions of the nucleotide sequence corresponding to positions 21793 to 22877 in SEQ ID NO: 266 encode a protein having UbiA-like prenyltransferase activity, positions of the nucleotide sequence corresponding to positions 23205 to 24773 in SEQ ID NO: 266 encode a protein having Acetyltransferase activity, and positions of the nucleotide sequence corresponding to positions 25824 to 27178 in SEQ ID NO: 266 encode a protein having Acetyltransferase-2 activity.