Microorganisms and Methods in the Fermentation of Benzylisoquinoline Alkaloids
AssigneeIntrexon Corporation
InventorsMaria Enquist-Newman, Genevieve Vidanes, Kata Zsuzsanna Hetenyi
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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.
Claims129
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.
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.
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.
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.
A vector comprising a nucleotide sequence that is substantially identical to any one of SEQ ID NOs. 58 to 63.
A vector comprising a nucleotide sequence that is substantially identical to SEQ ID NO. 64.
A vector comprising a nucleotide sequence that is substantially identical to SEQ ID NO. 65.
A method of making a hydroxylated product comprising contacting (7S)-salutaridinol 7-O-acetate with water where (7S)-salutaridinol 7-O-acetate is hydroxylated.
The method of claim 85109 wherein said hydroxylated product is any hydroxylated product presented in FIG. 2A.
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 SN2′ mechanism takes place.
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.
Dependencies are inferred from the claim text.