Benzylisoquinoline alkaloids (bia) producing microbes, and methods of making and using the same

Patent metadata

Field	Value
Publication number	US-2017145454-A1
Application number	US-201615360763-A
Country	US
Kind code	A1
Filing date	Nov 23, 2016
Priority date	Mar 15, 2013
Publication date	May 25, 2017
Grant date	—

Abstract

Aspects of the invention include host cells that are engineered to produce benzylisoquinoline alkaloids (BIAs). The host cells include heterologous coding sequences for a variety of enzymes involved in synthetic pathways from starting compounds to BIAs of the host cell. Also provided are methods of producing the BIAs of interest by culturing the host cells under culture conditions that promote expression of enzymes encoded by the heterologous coding sequences of the host cells. Aspects of the invention further include compositions, e.g., host cells, starting compounds and kits, etc., that find use in methods of the invention.

First claim

1 - 130 . (canceled)

131 . A method of preparing a benzylisoquinoline alkaloid product, the method comprising:
culturing an engineered microbial cell under conditions suitable for protein production, wherein the engineered microbial cell is an opioid-producing engineered microbial cell, wherein the engineered microbial cell comprises a plurality of heterologous coding sequences for encoding a plurality of enzymes within a pathway for producing the benzylisoquinoline alkaloid product, and wherein at least one heterologous coding sequence of the plurality of heterologous coding sequences encodes an enzyme that is within a pathway that converts a thebaine to the benzylisoquinoline alkaloid product, wherein the engineered microbial cell is a bacteria cell; adding a starting compound to the cell culture; and recovering the benzylisoquinoline alkaloid product from the cell culture, wherein the benzylisoquinoline alkaloid product is selected from the group consisting of a neopinone, neopine, neomorphine, codeinone, codeine, morphine, oripavine, morphinone, hydrocodone, 14-hydroxycodeinone, dihydrocodeine, oxycodone, 14-hydroxycodeine, hydromorphone, and dihydromorphine.

132 . The method according to claim 131 , wherein the engineered microbial cell comprises:
one or more heterologous coding sequences for encoding one or more enzymes that are selected from the group consisting of Thebaine 6-O demethylase, Codeinone reductase, Codeine O-demethylase, Morphine dehydrogenase, and Morphine reductase.

133 . The method according to claim 132 , wherein the engineered microbial cell comprises heterologous coding sequences for encoding two enzymes that are involved in the pathway that converts a thebaine into the benzylisoquinoline alkaloid product, each of the two enzymes selected from the group consisting of Thebaine 6-O demethylase, Codeinone reductase, Codeine O-demethylase, Morphine dehydrogenase, and Morphine reductase.

134 . The method according to claim 133 , wherein the two enzymes are operably connected along the pathway that converts a thebaine into the benzylisoquinoline alkaloid product.

135 - 136 . (canceled)

137 . The method of claim 131 , wherein the benzylisoquinoline alkaloid product is selected from the group consisting of neopine, neomorphine, codeinone, codeine, morphine, oripavine, morphinone, hydromorphone, dihydromorphine, hydrocodone, dihydrocodeine, 14-hydroxycodeinone, oxycodone, and 14-hydroxycodeine.

138 - 139 . (canceled)

140 . The method of claim 131 , wherein the benzylisoquinoline alkaloid product is produced using one or more of N-methylation, acetalization, hydroxylation, oxidation, reduction, cyclization, dehydration, O-alkylation, demethylation, and isomerization.

141 . The method of claim 131 , wherein at least one heterologous coding sequence of the plurality of heterologous coding sequences encodes an enzyme that is selected from the group consisting of Norcoclaurine 6-O-methyltransferase, Coclaurine-N-methyltransferase, 4′-O-methyltransferase, and Cytochrome P450 80B1.

142 . (canceled)

143 . A method of preparing a benzylisoquinoline alkaloid product, the method comprising:
culturing an engineered microbial cell under conditions suitable for protein production, wherein the engineered microbial cell is an opioid-producing engineered microbial cell, wherein the engineered microbial cell comprises at least three heterologous coding sequences for encoding a first, second, and third enzyme within a pathway for producing the benzylisoquinoline alkaloid product, wherein the first, second, and third enzymes are operably connected along the pathway, and wherein one enzyme of the first, second, and third enzymes is Salutaridine synthase, wherein the engineered microbial cell is a bacteria cell; adding a starting compound to the cell culture; and recovering the benzylisoquinoline alkaloid product from the cell culture, wherein the benzylisoquinoline alkaloid product is selected from the group consisting of a salutaridine, salutaridinol, 7-O-acetylsalutaridinol, thebaine, neopinone, neopine, neomorphine, codeinone, codeine, morphine, oripavine, morphinone, hydrocodone, 14-hydroxycodeinone, dihydrocodeine, oxycodone, 14-hydroxycodeine, hydromorphone, and dihydromorphine.

144 . The method of claim 143 , wherein the benzylisoquinoline alkaloid product is selected from the group consisting of salutaridine, salutaridinol, 7-O-acetylsalutaridinol, thebaine, neopine, neomorphine, codeinone, codeine, morphine, oripavine, morphinone, hydromorphone, dihydromorphine, hydrocodone, dihydrocodeine, 14-hydroxycodeinone, oxycodone, and 14-hydroxycodeine.

145 . The method of claim 143 , wherein the benzylisoquinoline alkaloid product is selected from the group consisting of neopine, neomorphine, codeinone, codeine, morphine, oripavine, morphinone, hydromorphone, dihydromorphine, 14-hydroxycodeinone, oxycodone, and 14-hydroxycodeine.

146 . (canceled)

147 . The method of claim 143 , wherein the benzylisoquinoline alkaloid product is produced using one or more of N-methylation, acetalization, hydroxylation, oxidation, reduction, cyclization, dehydration, O-alkylation, demethylation, and isomerization.

148 . (canceled)

149 . The method of claim 143 , wherein two enzymes of the first, second, and third enzymes involved in the pathway that produces the benzylisoquinoline alkaloid product are selected from the group consisting of Salutaridine Reductase, Salutaridinol 7-O-acetyltransferase, Norcoclaurine 6-O-methyltransferase, Coclaurine-N-methyltransferase, 4′-O-methyltransferase, Cytochrome P450 80B1, Thebaine 6-O demethylase, Codeinone reductase, Codeine O-demethylase, Morphine dehydrogenase, and Morphine reductase.

150 . (canceled)

151 . A method of preparing a benzylisoquinoline alkaloid product, the method comprising:
culturing an engineered microbial cell under conditions suitable for protein production, wherein the engineered microbial cell is an opioid-producing engineered microbial cell, wherein the engineered microbial cell comprises at least three heterologous coding sequences for encoding a first, second, and third enzyme within a pathway for producing the benzylisoquinoline alkaloid product, wherein the first, second, and third enzymes are operably connected along the pathway, and wherein one enzyme of the first, second, and third enzymes is Salutaridine synthase, wherein the engineered microbial cell is a yeast cell; adding a starting compound to the cell culture; and recovering the benzylisoquinoline alkaloid product from the cell culture, wherein the benzylisoquinoline alkaloid product is selected from the group consisting of a salutaridine, salutaridinol, 7-O-acetylsalutaridinol, thebaine, neopinone, neopine, neomorphine, codeinone, codeine, morphine, oripavine, morphinone, hydrocodone, 14-hydroxycodeinone, dihydrocodeine, oxycodone, 14-hydroxycodeine, hydromorphone, and dihydromorphine.

152 . The method of claim 151 , wherein the benzylisoquinoline alkaloid product is selected from the group consisting of salutaridine, salutaridinol, 7-O-acetylsalutaridinol, thebaine, neopine, neomorphine, codeinone, codeine, morphine, oripavine, morphinone, hydromorphone, dihydromorphine, hydrocodone, dihydrocodeine, 14-hydroxycodeinone, oxycodone, and 14-hydroxycodeine.

153 . The method of claim 151 , wherein the benzylisoquinoline alkaloid product is selected from the group consisting of neopine, neomorphine, codeinone, codeine, morphine, oripavine, morphinone, hydromo

Assignees

• Univ Leland Stanford Junior

Inventors

• Smolke Christina D
• Thodey Catherine
• Trenchard Isis
• Galanie Stephanie

Classifications

• C12P17/18

  containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin {(e.g. Rifamycin C12P17/189)} · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12Y201/01116

  3'-Hydroxy-N-methyl-(S)-coclaurine 4'-O-methyltransferase (2.1.1.116) · CPC title

• C12P17/182 Primary

  Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system (alloxazine or isoalloxazine, e.g. riboflavine C12P25/00) · CPC title

• C12N9/1007 Primary

  Methyltransferases (general) (2.1.1.) · CPC title