Microorganisms and Methods in the Fermentation of Benzylisoquinoline Alkaloids
Disclosed herein are methods that may be used for the synthesis of benzylisoquinoline alkaloids (“BIAs”) such as thebaine and morphine and their derivatives. The methods disclosed can be used to produce thebaine, oripavine, codeine, morphine, oxycodone, hydrocodone, oxymorphone, hydromorphone, naltrexone, naloxone, hydroxycodeinone, neopinone, and/or buprenorphine. Compositions and organisms useful for the synthesis of BIAs, including thebaine synthases and polynucleotides encoding the same, are provided. Further, methods of adjusting pH to optimize the reaction are disclosed.
1 . A method of making salutaridine comprising contacting salutaridine synthase with reticuline within a medium to convert reticuline to salutaridine, wherein the pH of said medium is between 6 and 13.
2 . The method of claim 1 , wherein said medium does not contain any cells.
3 . The method of claim 1 or 2 , wherein said conversion of reticuline to salutaridine occurs outside of a cell.
4 . The method of claim 1 , wherein said medium is cell culture media and optionally contains lysed cells.
5 . The method of claim 1 or 4 , wherein said conversion of reticuline to salutaridine occurs within a cell.
6 . The method of any one of claims 1 to 5 , wherein said reticuline is R-reticuline.
7 . The method of claims 1 to 6 , wherein pH of said media is greater than 6.
8 . The method of claims 1 to 6 , wherein pH of said media is between 7 to 7.4.
9 . The method of claims 1 to 6 , wherein pH of said media is between 7.5 to 7.9.
10 . The method of claims 1 to 6 , wherein pH of said media is between 8 to 8.4.
11 . The method of any one of claims 1 to 10 , wherein said pH is adjusted or maintained by supplementing said medium with an acidic or alkali or buffering substance.
12 . The method of claim 11 , wherein said pH is maintained by supplementing said medium with an alkali substance.
13 . The method of claim 12 , wherein said alkali substance is NH 4 OH or NaOH.
14 . The method of any one of claims 1 to 13 , wherein said contacting is for at least 24 hours.
15 . The method of any one of claims 1 to 14 , wherein said contacting is for at least 48 hours.
16 . The method of any one of claims 1 to 14 , wherein said contacting is between 24 and 48 hours.
17 . The method of any one of claims 1 to 16 , wherein salutaridine is present within said medium at a concentration of at least 75 μg/L.
18 . The method of any one of claims 1 to 17 , wherein salutaridine is present within said medium at a concentration of at least 200 μg/L.
19 . The method of any one of claims 5 to 18 , wherein the cell is a yeast cell.
20 . The method of claim 19 , wherein said yeast cell from the genus Saccharomyces.
21 . The method of claim 20 , wherein said yeast cell from the species Saccharomyces cerevisiae.
22 . The method of any one of claims 5 to 21 , wherein said cell further comprises a salutaridine reductase, a salutaridinol 7-O-acetyltransferase, and/or a thebaine synthase.
23 . The method of any one of claims 5 to 22 , wherein said cell further comprises a purine permease and/or a cytochrome p450 reductase.
24 . The method of any one of claims 5 to 23 , wherein said salutaridine synthase is heterologous to said cell.
25 . The method of any one of claims 22 to 24 , wherein said salutaridine reductase, salutaridinol 7-O-acetyltransferase, and/or thebaine synthase is heterologous to said cell.
26 . The method of any one of claims 23 to 25 , wherein said purine permease and/or cytochrome p450 reductase is heterologous to said cell.
27 . A method of making salutaridinol comprising contacting salutaridine reductase with salutaridine within a medium to convert salutaridine to salutaridinol, wherein the pH of said medium is between 5 and 13.
28 . The method of claim 27 , wherein said medium does not contain any cells.
29 . The method of claim 27 or 28 , wherein said conversion of salutaridine to salutaridinol occurs outside of a cell.
30 . The method of claim 27 , wherein said medium is cell culture media and optionally contains lysed cells.
31 . The method of claim 27 or 30 , wherein said conversion of salutaridine to salutaridinol occurs within a cell.
32 . The method of claims 27 to 31 , wherein pH of said media is greater than 5.
33 . The method of claims 27 to 32 , wherein pH of said media is between 5.5 to 5.9.
34 . The method of claims 27 to 33 , wherein pH of said media is between 6 to 6.4.
35 . The method of claims 27 to 34 , wherein pH of said media is between 6.5 to 7.0.
36 . The method of any one of claims 27 to 35 , wherein said pH is adjusted or maintained by supplementing said medium with an acidic or alkali substance.
37 . The method of claim 36 , wherein said pH is maintained by supplementing said medium with an alkali substance.
38 . The method of claim 37 , wherein said alkali substance is NH 4 OH or NaOH.
39 . The method of any one of claims 27 to 38 , wherein said contacting is for at least 24 hours.
40 . The method of any one of claims 27 to 39 , wherein said contacting is for at least 48 hours.
41 . The method of any one of claims 27 to 40 , wherein said contacting is between 24 and 48 hours.
42 . The method of any one of claims 27 to 41 , wherein salutaridinol is present within said medium at a concentration of at least 25 μg/L.
43 . The method of any one of claims 27 to 42 , wherein salutaridinol is present within said medium at a concentration of at least 100 μg/L.
44 . The method of any one of claims 31 to 43 , wherein the cell is a yeast cell.
45 . The method of claim 44 , wherein said yeast cell from the genus Saccharomyces.
46 . The method of claim 45 , wherein said yeast cell from the species Saccharomyces cerevisiae.
47 . The method of any one of claims 31 to 46 , wherein said cell further comprises a salutaridine synthase, salutaridinol 7-O-acetyltransferase, and/or a thebaine synthase.
48 . The method of any one of claims 31 to 47 , wherein said cell further comprises a purine permease and/or a cytochrome p450 reductase.
49 . The method of any one of claims 31 to 48 , wherein said salutaridine reductase is heterologous to said cell.
50 . The method of any one of claims 47 to 49 , wherein said salutaridine synthase, salutaridinol 7-O-acetyltransferase, and/or thebaine synthase is heterologous to said cell.
51 . The method of any one of claims 48 to 50 , wherein said purine permease and/or cytochrome p450 reductase is heterologous to said cell.
52 . A method of making salutaridinol-7-0-acetate comprising contacting salutaridinol 7-O-acetyltransferase with salutaridinol within a medium to convert salutaridinol to salutaridinol-7-O-acetate, wherein the pH of said medium is between 6 and 13.
53 . The method of claim 52 , wherein said medium does not contain any cells.
54 . The method of claim 52 or 53 , wherein said conversion of salutaridinol to salutaridinol-7-O-acetate occurs outside of a cell.
55 . The method of claim 52 , wherein said medium is cell culture media and optionally contains lysed cells.
56 . The method of claim 52 or 55 , wherein said conversion of salutaridinol to salutaridinol-7-O-acetate occurs within a cell.
57 . The method of claims 52 to 56 , wherein pH of said media is greater than 6.
58 . The method of claims 52 to 57 , wherein pH of said media is between 6.5 to 6.9.
59 . The method of claims 52 to 57 , wherein pH of said media is between 7 to 7.4.
60 . The method of claims 52 to 57 , wherein pH of said media is between 7.5 to 7.9.
61 . The method of any one of claims 52 to 60 , wherein said pH is adjusted or maintained by supplementing said medium with an acidic or alkali substance.
62 . The method of claim 61 , wherein said pH is maintained by supplementing said medium with an alkali substance.
63 . The method of claim 62 , wherein said alkali substance is NH 4 OH or NaOH.
64 . The method of any one of claims 52 to 63 , wherein said contacting is for at least 24 hours.
65 . The method of any one of claims 52 to 64 , wherein said contacting is for at least 48 hours.
66 . The method of any one of claims 52 to 65 , wherein said contacting is between 24 and 48 hours.
67 . The method of any one of claims 56 to 66 , wherein the cell is a yeast cell.
68 . The method of claim 67 , wherein said yeast cell from the genus Saccharomyces.
69 . The method of claim 68 , wherein said yeast cell from the species Saccharomyces cerevisiae.
70 . The method of any one of claims 56 to 69 , wherein said cell further comprises a salutaridine reductase, a salutaridine synthase, and/or a thebaine synthase.
71 . The method of any one of claims 56 to 70 , wherein said cell further comprises a purine permease and/or a cytochrome p450 reductase.
72 . The method of any one of claims 52 to 71 , wherein said salutaridinol 7-O-acetyltransferase is heterologous to said cell.
73 . The method of any one of claims 70 to 72 , wherein said salutaridine reductase, salutaridine synthase and/or thebaine synthase is heterologous to said cell.
74 . The method of any one of claims 71 to 73 , wherein said purine permease and/or cytochrome p450 reductase is heterologous to said cell.
75 . A method of making thebaine comprising contacting thebaine synthase with to salutaridinol-7-O-acetate within a medium to convert to salutaridinol-7-O-acetate to thebaine, wherein the pH of said medium is between 5 and 13.
76 . The method of claim 75 , wherein said medium does not contain any cells.
77 . The method of claim 75 or 76 , wherein said conversion of salutaridine to salutaridinol occurs outside of a cell.
78 . The method of claim 76 , wherein said medium is cell culture media and optionally contains lysed cells.
79 . The method of claim 76 or 78 , wherein said conversion of salutaridinol-7-O-acetate to thebaine occurs within a cell.
80 . The method of claims 76 to 79 , wherein pH of said media is greater than 5.
81 . The method of claims 76 to 80 , wherein pH of said media is between 7 to 7.4.
82 . The method of claims 76 to 80 , wherein pH of said media is between 7.5 to 7.9.
83 . The method of claims 76 to 80 , wherein pH of said media is between 8 to 8.4.
84 . The method of any one of claims 76 to 83 , wherein said pH is adjusted or maintained by supplementing said medium with an acidic or alkali or buffering substance.
85 . The method of claim 84 , wherein said pH is maintained by supplementing said medium with an alkali substance.
86 . The method of claim 85 , wherein said alkali substance is NH 4 OH or NaOH.
87 . The method of any one of claims 75 to 86 , wherein said contacting is for at least 30 seconds.
88 . The method of claim 87 , wherein thebaine is present within said medium at a concentration of at least 750 pmol μg protein −1 .
89 . The method of claim 87 or 88 , wherein thebaine is present within said medium at a concentration of at least 900 pmol μg protein −1 .
90 . The method of any one of claims 75 to 89 , wherein said contacting is for at least 60 seconds.
91 . The method of claim 90 , wherein thebaine is present within said medium at a concentration of at least 1500 pmol μg protein −1 .
92 . The method of claim 91 , wherein thebaine is present within said medium at a concentration of at least 1900 pmol μg protein −1 .
93 . The method of any one of claims 75 to 92 , wherein said contacting is for at least 24 hours.
94 . The method of any one of claims 75 to 93 , wherein said contacting is for at least 48 hours.
95 . The method of any one of claims 75 to 93 , wherein said contacting is between 24 and 48 hours.
96 . The method of any one of claims 75 to 95 , wherein thebaine is present within said medium at a concentration of at least 15 μg/L.
97 . The method of any one of claims 27 to 42 , wherein thebaine is present within said medium at a concentration of at least 25 μg/L.
98 . The method of any one of claims 79 to 97 , wherein the cell is a yeast cell.
99 . The method of claim 98 , wherein said yeast cell from the genus Saccharomyces.
100 . The method of claim 99 , wherein said yeast cell from the species Saccharomyces cerevisiae.
101 . The method of any one of claims 79 to 100 , wherein said cell further comprises a salutaridine reductase, a salutaridine synthase, and/or a salutaridinol 7-O-acetyltransferase.
102 . The method of any one of claims 79 to 101 , wherein said cell further comprises a purine permease and/or a cytochrome p450 reductase.
103 . The method of any one of claims 75 to 102 , wherein said THS is heterologous to said cell.
104 . The method of any one of claims 101 to 103 , wherein said salutaridine reductase, salutaridine synthase and/or salutaridinol 7-O-acetyltransferase is heterologous to said cell.
105 . The method of any one of claims 102 to 104 , wherein said purine permease and/or cytochrome p450 reductase is heterologous to said cell.
106 . A vector comprising a nucleotide sequence that is substantially identical to any one of SEQ ID NOs. 58 to 63.
107 . A vector comprising a nucleotide sequence that is substantially identical to SEQ ID NO. 64 .
108 . A vector comprising a nucleotide sequence that is substantially identical to SEQ ID NO. 65 .
109 . A method of making a hydroxylated product comprising contacting (7S)-salutaridinol 7-O-acetate with water where (7S)-salutaridinol 7-O-acetate is hydroxylated.
110 . The method of claim 85109 wherein said hydroxylated product is any hydroxylated product presented in FIG. 2A .
111 . The method of claim 109 or 110 , wherein said method is performed within a cell.
112 . The method of claim 111 , wherein said cell (i) does not comprise thebaine synthase; (ii) comprises an inactive thebaine synthase; or (iii) comprises a thebaine synthase having reduced activity compared to a wild-type thebaine synthase.
113 . The method of claim 111 or 112 , wherein said cell comprises a heterologous salutaridinol 7-O-acetyltransferase.
114 . The method of claim 113 , wherein said (7S)-salutaridinol 7-O-acetate is produced by said heterologous salutaridinol 7-O-acetyltransferase.
115 . The method of any one of claims 109 to 114 , wherein said (7S)-salutaridinol 7-O-acetate does not come into contact with thebaine synthase.
116 . The method of any one of claims 111 to 115 , wherein said cell further comprises an gene that is a tyrosine hydroxylase (TYR); DOPA decarboxylase (DODC); norcoclaurine synthase (NCS); 6-0-Methyltransferase (6OMT); coclaurine N-methyltransferase (CNMT), cytochrome P450 N-methylcoclaurine hydroxylase (NMCH), and 4-0-methyltransferase (4OMT); cytochrome P450 reductase (CPR), salutaridine synthase (SAS); salutaridine reductase (SalR); or any combination thereof.
117 . The method of claim 116 , wherein said gene is heterologous to said cell.
118 . A method of making thebaine comprising placing salutaridinol or salutaridinol 7-O-acetate in a pH of greater than 7.5 and maintaining said pH at greater than 7.5 until an S N 2′ mechanism takes place.
119 . The method of claim 118 , wherein said method uses salutaridinol 7-O-acetate.
120 . The method of claim 118 or 119 , wherein said method takes place within a cell.
121 . The method of any one of claims 118 to 120 , wherein said pH is greater than 8.0.
122 . The method of any one of claims 118 to 121 , wherein the method does not allow salutaridinol or salutaridinol 7-O-acetate to come in contact with water.
123 . The method of claim 122 , wherein the method occurs within an enzyme.
124 . The method of claim 123 , wherein said enzyme is thebaine synthase.
125 . A method of making a BIA comprising contacting (7S)-salutaridinol 7-O-acetate with an enzyme that is capable of converting (7S)-salutaridinol 7-O-acetate into a BIA, wherein said enzyme has a Vmax of greater than 2.0 nmol min −1 μg −1 .
126 . The method of claim 125 , wherein said Vmax is between 1.5 to 4.0 nmol min −1 μg −1 .
127 . The method of claim 125 or 126 , wherein said Vmax is greater than 4.0 nmol min −1 μ −1 .
128 . The method of any one of claims 125 to 127 , wherein said BIA is thebaine.
129 . The method of any one of claims 125 to 128 , wherein said pH is maintained at greater than 7.5.