Compositions and methods for making benzylisoquinoline alkaloids, morphinan alkaloids, thebaine, and derivatives thereof
Inventors
FACCHINI, Peter James • Chen, Xue • COLBECK, Jeffrey C. • Tucker, Joseph
Assignees
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Publication Number
US-11142780-B2
Publication Date
2021-10-12
Expiration Date
Abstract
Disclosed herein are methods that may be used for the synthesis of benzylisoquinoline alkaloids (BIAs) such as alkaloid morphinan. 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 synthesis polypeptides, purine permeases, and polynucleotides encoding the same, are provided.
Core Innovation
The invention centers on producing thebaine by contacting salutaridinol-7-O-acetate with a thebaine synthase (THS) polypeptide or with a genetically modified cell comprising a polynucleotide encoding a heterologous THS. The THS has thebaine synthase activity and an amino acid sequence at least 90% sequence identical to SEQ ID NOs: 6, 31, 32, 80, or 81, with some embodiments specifying 95% identity.
In related embodiments, the conversion is performed in vitro or under conditions permitting conversion in a genetically modified cell. The disclosure also includes upstream enzymatic formation of salutaridinol-7-O-acetate from salutaridinol by salutaridinol-7-O-acetyltransferase and formation of salutaridinol from salutaridine by salutaridine reductase, each with defined sequence-identity constraints.
The document further describes inclusion of purine permease activity in the engineered cell and reports engineered expression or co-expression of THS/Betv1-1 with SalAT/SalR and purine permeases to increase thebaine titers and reduce an m/z 330 by-product.
Claims Coverage
The document provides coverage across two independent claims, one cell-based and one in vitro, centered on converting salutaridinol-7-O-acetate into thebaine using a THS polypeptide with defined sequence identity constraints. Dependent claims further specify higher identity thresholds, upstream conversion steps to form salutaridinol-7-O-acetate and salutaridinol, and purine permease activity to increase thebaine titers.
Cell-based thebaine production using heterologous thebaine synthase
A method for producing thebaine by contacting salutaridinol-7-O-acetate with a genetically modified cell comprising a polynucleotide encoding a heterologous enzyme having thebaine synthase activity, where the amino acid sequence is at least 90% sequence identical to SEQ ID NOs 6, 31, 32, 80, or 81, and growing the cell under conditions permitting conversion into thebaine.
In vitro thebaine production using heterologous thebaine synthase polypeptide
A method for producing thebaine by contacting salutaridinol-7-O-acetate in vitro with a polypeptide comprising an amino acid sequence that is at least 90% sequence identical to any one of SEQ ID NOs 6, 31, 32, 80, or 81 having thebaine synthase activity.
Purine permease activity to increase thebaine titers
The genetically modified cell includes a polynucleotide encoding a heterologous enzyme with purine permease activity and an amino acid sequence at least 90% sequence identical to specified SEQ ID NOs, including SEQ ID NOs 35, 37, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, or 63.
Salutaridinol-7-o-acetyltransferase to form salutaridinol-7-O-acetate
Producing salutaridinol-7-O-acetate by contacting salutaridinol with a polypeptide having salutaridinol-7-O-acetyltransferase activity and an amino acid sequence at least 90% identical to SEQ ID NO: 2.
Salutaridine reductase to form salutaridinol
Producing salutaridinol by contacting salutaridine with a salutaridine reductase polypeptide having an amino acid sequence at least 90% sequence identical to SEQ ID NO: 4.
Overall, the claims coverage centers on producing thebaine by converting salutaridinol-7-O-acetate via a THS polypeptide with defined sequence identity, with dependent claim coverage further specifying upstream enzymatic steps and the addition of purine permease activity to increase thebaine titers.
Stated Advantages
The engineered expression/co-expression of THS/Betv1-1 with SalAT/SalR and purine permeases increases thebaine titers.
The engineered approach can reduce formation of the m/z 330 by-product.
Documented Applications
Producing thebaine via in vitro BIA synthesis by converting morphinan substrates using expressed or purified thebaine synthase polypeptides, optionally combined with purine permease.
Producing thebaine from salutaridinol-7-O-acetate using genetically modified cells.
Producing thebaine from salutaridinol-7-O-acetate using in vitro polypeptide contacting.
Eliminating or reducing m/z 330 by-products in SalAT-coupled assays.
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