Polynucleotides and polypeptides useful for making alkaloid compounds
Inventors
FACCHINI, Peter James • Tucker, Joseph E.
Assignees
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Publication Number
US-11479586-B2
Publication Date
2022-10-25
Expiration Date
Abstract
Polynucleotides and polypeptides useful in the manufacture of a class of chemical compounds known as alkaloids are provided. The polynucleotides and polypeptides may be used to synthesize alkaloids, including reticuline, thebaine and morphine, in vivo and in vitro. The polynucleotides further may be used to examine the presence of the polynucleotides in a cell or a cell extract, and to modulate expression thereof in living cells.
Core Innovation
The invention describes compositions and genetic elements that include isolated polynucleotides and polypeptides, expression vectors, and host cells for producing alkaloids, including reticuline and opiates such as thebaine and morphine. The genetic elements are defined with extensive sequence, variant, hybridization, and complementary nucleic acid sequence relationships using many SEQ ID NOs.
A yeast cell is provided that includes a first polynucleotide encoding a polypeptide having thebaine synthase activity, where the amino acid sequence is at least 90% identical to an amino acid sequence selected from SEQ ID NO: 29 or SEQ ID NO: 900. The invention further provides a second polynucleotide encoding a polypeptide having purine permease activity, and defines additional genetic elements and their encoded activities as polynucleotides and polypeptides for producing the alkaloids.
The document also includes embodiments that use yeast hosts such as Saccharomyces cerevisiae or Yarrowia lipolytica, as well as other host cells including plant cells and microbial hosts. The invention discusses using the polynucleotides to modulate expression and identify modulated levels of thebaine, and it includes hybridization-capable nucleic acid sequences defined under specified stringency conditions, along with alkaloid transport across membranes and inter-alkaloid conversion.
Claims Coverage
The inventive coverage centers on one yeast-cell independent claim and dependent refinements. It requires a first polynucleotide encoding a thebaine synthase activity polypeptide in a yeast cell, and adds optional constraints for a second purine permease polynucleotide, hybridization-defined nucleic acids, tighter sequence-identity thresholds, and silencing of thebaine synthase polynucleotide expression.
Thebaine synthase activity in a yeast cell with defined amino acid sequence identity
A yeast cell comprising a first polynucleotide encoding a polypeptide having thebaine synthase activity comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NO: 29 or SEQ ID NO: 900.
Purine permease activity by adding a second polynucleotide with defined amino acid sequence identity
A yeast cell further including a second polynucleotide encoding a purine permease polypeptide whose amino acid sequence is at least 90% identical to SEQ ID NO: 779 or SEQ ID NO: 901.
Purine permease polypeptide identity threshold
A yeast cell where the second polynucleotide encodes a polypeptide with an amino acid sequence at least 97% identical to SEQ ID NO: 779 or SEQ ID NO: 901.
Hybridization-defined first polynucleotide under moderately stringent conditions
A yeast cell where the first polynucleotide contains a nucleic acid sequence that can hybridize with the nucleic acid sequences of SEQ ID NO: 6 or SEQ ID NO: 372 under at least moderately stringent hybridization conditions.
Tightened thebaine synthase polypeptide identity threshold
A yeast cell specified where the first polynucleotide encodes a polypeptide with an amino acid sequence at least 95% identical to SEQ ID NO: 29 or SEQ ID NO: 900.
Silencing of thebaine synthase polynucleotide expression
A method for reducing expression of a polynucleotide encoding a thebaine synthase polypeptide (with an amino acid sequence at least 90% identical to SEQ ID NO: 29 or SEQ ID NO: 900) by silencing the polynucleotide’s expression in a Papaver somniferum cell (or the cell specified in claim 1).
Overall, the claim coverage centers on yeast cells containing a thebaine synthase activity polypeptide polynucleotide with specified amino acid identity to SEQ ID NO: 29 or SEQ ID NO: 900, with dependent embodiments adding a purine permease polynucleotide with defined sequence identity, introducing hybridization-condition-defined nucleic acids to specified reference sequences, tightening identity thresholds, and reducing thebaine synthase polynucleotide expression via silencing in Papaver somniferum context.
Stated Advantages
Supports alkaloid production including thebaine and morphine-related opiate alkaloids.
Documented Applications
Alkaloid production, conversion, and modulation using heterologous expression and alkaloid transport across membranes in microbial hosts including Saccharomyces cerevisiae and Yarrowia lipolytica, with reference alkaloids including thebaine and related benzylisoquinoline alkaloids.
Producing/exporting alkaloids, including thebaine and morphine-related compounds supported by reticuline pathway precursors and inter-alkaloid conversion.
Using enzymes to mediate inter-alkaloid conversion for opiate alkaloid production.
Expression modulation in plants by mutagenesis or gene silencing including virus induced gene silencing (VIGS).
Detection/genotyping and mapping using labeled polynucleotide probes, including RFLP/molecular marker approaches.
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