Microorganisms and Methods in the Fermentation of Benzylisoquinoline Alkaloids

a technology of benzylisoquinoline and microorganisms, which is applied in the field of microorganisms and methods in the fermentation of benzylisoquinoline alkaloids, can solve the problems of low yield and/or cost, no commercial biosynthetically manufactured bias methods, and thebaine and other morphinan alkaloids and their derivatives, etc., to achieve the effect of increasing the efficiency of bia synthesis

Inactive Publication Date: 2020-10-15
ANTHEIA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes a method for efficiently producing various alkaloids, such as reticuline, salutaridine, salutaridinol, salutaridinol-7-O-acetate, thebaine, and hydromorphone, using microorganisms. The methods involve contacting specific enzymes with their respective substrates for a certain period of time, and can be performed in a cell-free system or with cell culture media. The pH of the medium can be adjusted to optimize the enzymatic reactions. The patent also describes the use of specific substrates and methods for increasing the efficiency of the enzymatic reactions. Overall, the patent provides a way to efficiently produce various alkaloids and their precursors.

Problems solved by technology

The existing manufacturing methods for BIAs, including thebaine and other morphinan alkaloids and their derivatives, suffer from low yields and / or are expensive.
No methods exist to commercially biosynthetically manufacture BIAs, including thebaine and other morphinan alkaloids and their derivatives.
In some cases, the method does not allow salutaridinol or salutaridinol 7-O-acetate to come in contact with water.

Method used

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  • Microorganisms and Methods in the Fermentation of Benzylisoquinoline Alkaloids
  • Microorganisms and Methods in the Fermentation of Benzylisoquinoline Alkaloids
  • Microorganisms and Methods in the Fermentation of Benzylisoquinoline Alkaloids

Examples

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Effect test

example 1

Preparation of Genetically Modified Microorganisms that is Capable of Making High Levels of Salutaridine, Salutaridinol and Thebaine

[0224]The S. cerevisiae strain CEN.PK were transformed with nucleic acids encoding for a variety of enzymes that are capable of or take part in the fermentation of sugar to BIAs, such as thebaine. The genotypes of the strains are found in FIG. 4A. The strains contained up to nine chromosomally integrated plant genes encoding BIA biosynthetic enzymes capable of converting (S)-norlaudanosoline to salutaridine, salutaridinol 7-O-acetate, or thebaine. Strain Sc-2 harbored the first seven biosynthetic genes, resulting in the production of salutaridine. Strain Sc-3 contained two additional genes, encoding SalR and SalAT, leading to the formation of salutaridinol 7-O-acetate, whereas strain Sc-4 also included SalR, SalAT and THS2 gene. The sequences of these constructs are presented as SEQ ID NOs. 58 to 63.

example 2

Hydroxylated Byproducts of Salutaridinol 7-O-acetate

[0225]A longstanding dilemma in thebaine biosynthesis involved the formation of the hydrofuran ring in the precursor intermediate salutaridinol. Although salutaridinol undergoes spontaneous allylic elimination at pH <5 in vitro to yield thebaine (FIG. 2A), the possibility that the cyclization of salutaridinol was an enzymatic process remained to be determined. All indications suggested that the cyclization of salutaridinol into thebaine was a spontaneous reaction. First, it was suggested that the C7 hydroxyl of salutaridinol must be functionalized to furnish a better leaving group for intramolecular SN2′ syn displacement. This prediction was realized with the later discovery of salutaridinol 7-O-acetyltransferase (SalAT), which catalyzes the formation of the unstable intermediate salutaridinol 7-O-acetate At pH 8-9 in vitro, but apparently not at lower pH, thebaine was reported to form spontaneously through allylic rearrangement of...

example 3

Thebaine Synthase

[0229]Kinetic analysis of THS2 was performed using a direct assay with (75)-salutaridinol 7-O-acetate as the substrate. (75)-Salutaridinol 7-O-acetate is relatively stable in chloroform but exposure to aqueous conditions caused the rapid (70%) formation of m / z 330-byproduct. To minimize hydroxylated byproduct formation and maintain linear range parameters, the assay was quenched with chloroform after 30 sec (FIG. 3A). The small amount of thebaine formed spontaneously in the absence of THS2 was subtracted from assays containing active enzyme. THS2 displayed positive cooperativity (η=2.3) with a KA of 20 μM, a Vmax of 4.0 nmol min1 μg−1, and a pH optimum of 8.0 (FIG. 3B, FIG. 3C, and FIG. 5). These results suggested that the THS homodimer exhibits allostery with respect to its substrate. The enzymatic reaction mechanism for thebaine formation likely involves deprotonation of the C4 hydroxyl group of (7S)-salutaridinol 7-O-acetate by a catalytic residue acting as a gen...

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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.

Description

SEQUENCE LISTING[0001]The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on Aug. 16, 2018, is named INX00389_SL.txt and is 210,766 bytes in size.BACKGROUND OF THE DISCLOSURE[0002]Benzylisoquinoline alkaloids (“BIAs”) are a large and structurally diverse family of plant secondary tyrosine metabolites that exhibit a wide range of pharmacological activities. Thebaine, a chemical compound also known as paramorphine and codeine methyl enol ether, belongs to the BIA class of compounds, and within that class, to a BIA subclass of compounds known as morphinan alkaloids, and has long been recognized as a useful feedstock compound in the manufacture of therapeutic agents, including, for example, morphine and codeine. Other BIAs that can be manufactured can include but are not limited to oripavine, oxycodone, hydrocodone, oxymorphone, hydromorphone, naltr...

Claims

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Application Information

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IPC IPC(8): C12P17/12C12N9/04C12N9/10C12N15/81
CPCC12N9/0006C12P17/12C12Y203/0115C12N9/1029C12N15/81C12Y101/01248C12N9/88C12P17/188C12N15/79
Inventor ENQUIST-NEWMAN, MARIAVIDANES, GENEVIEVEHETENYI, KATA ZSUZSANNA
Owner ANTHEIA INC
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