Microbial cell with improved in vivo conversion of thebaine/oripavine
A recombinant microbial host cell having improved in vivo conversion of reticuline and derivatives thereof (such as thebaine and/or oripavine) to relevant downstream opioids (such as neopinone, oripavine, northebaine, nororipavine or morphinone) and related compounds (such as heroin, morphine, codeine, thebaine, oripavine, oxycodone, hydrocodone, hydromorphone, oxymorphone, buprenorphine, naltrexone, naloxone or nalbuphine), wherein the microbial (such as fungal) host cell is heterologously expressing at least one functional transporter protein capable of transporting reticuline or a derivative thereof (such as thebaine and/or oripavine) and a heterologously expressed enzyme capable of acting upon reticuline or a derivative thereof. The invention also relates to uses of the microbial host cells and methods of making an opioid compound and/or opioid precursor compound and/or opioid derivative of interest.
1 . A recombinant yeast cell heterologously expressing a transporter protein, wherein the transporter protein:
(i) comprises an amino acid sequence having at least 70% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 112, 42, 56, 76, 78, 92, 94, 96, 98, 102, 106, 108, 116, 120, 124, 128, 130, 136, 138, 140, 142, 144, 148, 154, 156, 158, 160, 168, 172, 182, amino acids 1 to 607 of SEQ ID NO: 8, amino acids 1 to 1289 of SEQ ID NO:4, amino acids 1 to 654 of SEQ ID NO: 6, and amino acids 1 to 583 of SEQ ID NO: 10; and/or
(ii) comprises an amino acid sequence having at least 70% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 182, 186, 42, 44, 48, 50, 56, 62, 64, 72, 76, 78, 82, 86, 92, 94, 96, 98, 102, 106, 108, 110, 112, 116, 120, 124, 128, 130, 136, 138, 140, 142, 144, 148, 154, 158, 160, 164, 166, 168, 172, 178, 180, and amino acids 1 to 604 of SEQ ID NO: 31; and
wherein heterologous expression of the transporter protein improves the recombinant yeast cell uptake of thebaine or oripavine for in vivo conversion.
2 . The recombinant yeast cell of claim 1 , wherein:
(a) the transporter protein improves the yeast cell uptake of thebaine and comprises an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 112, 42, 56, 76, 78, 92, 94, 96, 98, 102, 106, 108, 116, 120, 124, 128, 130, 136, 138, 140, 142, 144, 148, 154, 156, 158, 160, 168, 172, 182, amino acids 1 to 607 of SEQ ID NO:8, amino acids 1 to 1289 of SEQ ID NO:4, amino acids 1 to 654 of SEQ ID NO:6, and amino acids 1 to 583 of SEQ ID NO: 10; and
(b) the transporter protein improves the yeast cell uptake of oripavine and comprises an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 182, 186, 42, 44, 48, 50, 56, 62, 64, 72, 76, 78, 82, 86, 92, 94, 96, 98, 102, 106, 108, 110, 112, 116, 120, 124, 128, 130, 136, 138, 140, 142, 144, 148, 154, 158, 160, 164, 166, 168, 172, 178, 180, and amino acids 1 to 604 of SEQ ID NO: 31.
3 . The recombinant yeast cell of claim 1 , which further converts thebaine and/or oripavine into an opioid compound of interest due to the in vivo presence of a heterologously expressed demethylase enzyme.
4 . The recombinant yeast cell of claim 3 , wherein the heterologously expressed demethylase enzyme is a P450 capable of demethylase activity on thebaine and/or oripavine, and wherein the recombinant yeast cell:
(1) converts thebaine into neopinone by in vivo heterologous expression of thebaine 6-O-demethylase; or
(2) converts thebaine into oripavine by in vivo heterologous expression of O-demethylase; or
(3) converts thebaine into northebaine by in vivo heterologous expression of N-demethylase; or
(4) converts oripavine into nororipavine by in vivo heterologous expression of heterologously expressed N-demethylase; or
(5) converts oripavine into morphinone by in vivo heterologous expression of oripavine 6-O-demethylase.
5 . The recombinant yeast cell of claim 1 , wherein the transporter protein comprises an amino acid sequence having at least 70% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 10, 12, 14, 16, 20, 22, 27, and 31.
6 . The recombinant yeast cell of claim 1 , wherein the transporter protein comprises an amino acid sequence having at least 70% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, and 188.
7 . The recombinant yeast cell of claim 1 , wherein the transporter protein comprises an amino acid sequence having at least 70% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 42, 50, 96, 106, 136, 140, 164, 180, 182, and 186.
8 . The recombinant yeast cell of claim 1 , wherein the transporter protein comprises an amino acid sequence having at least 70% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 42, 56, 76, 78, 92, 94, 96, 98, 102, 106, 112, 154, 168, 172, and 182.
9 . The recombinant yeast cell of claim 1 , wherein the transporter protein comprises an amino acid sequence having at least 70% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 42, 106, 182, and 186.
10 . The recombinant yeast cell of claim 1 , wherein the recombinant yeast cell is a Saccharomyces cerevisiae cell.
11 . A method of producing in vivo a thebaine derivative or an oripavine derivative in a cell culture, comprising culturing the recombinant yeast cell of claim 1 in the cell culture.
12 . The method of claim 11 , wherein the method further comprises in vivo producing neopinone, oripavine, northebaine, nororipavine or morphinone in the cell culture.
13 . The method of claim 11 , wherein in vivo production a thebaine derivative or an oripavine derivative is increased compared to a control host yeast cell that is not heterologously expressing the transporter protein.
14 . The recombinant yeast cell of claim 3 , wherein the opioid compound of interest is neopinone, oripavine, northebaine, nororipavine, or morphinone.