Microorganisms and Methods in the Fermentation of Benzylisoquinoline Alkaloids

Abstract

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.

We claim: 
     
         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.