Compositions and methods for producing benzylisoquinoline alkaloids
AssigneeCalifornia Institute of Technology
InventorsD. Christina Smolke, Kristy Hawkins
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Abstract
The present invention relates to host cells that produce compounds that are characterized as benzylisoquinolines, as well as select precursors and intermediates thereof. The host cells comprise one, two or more heterologous coding sequences wherein each of the heterologous coding sequences encodes an enzyme involved in the metabolic pathway of a benzylisoquinoline, or its precursors or intermediates from a starting compound. The invention also relates to methods of producing the benzylisoquinoline, as well as select precursors and intermediates thereof by culturing the host cells under culture conditions that promote expression of the enzymes that produce the benzylisoquinoline or precursors or intermediates thereof.
Claims37
A host cell that produces norcoclaurine, the host cell comprising at least a first, second, third and fourth heterologous coding sequence, wherein the first, second, third and fourth heterologous coding sequences encode a first, second, third and fourth enzyme, respectively, wherein the first, second, third and fourth enzymes are involved in a metabolic pathway that converts tyrosine in the host cell into the norcoclaurine.
A host cell that produces reticuline from norlaudanosoline, the host cell comprising a first second and third heterologous coding sequence, wherein the first, second and third heterologous coding sequences encode a first, second and third enzyme that are involved in a metabolic pathway that converts the norlaudanosoline into the reticuline.
A host cell that produces dopamine, the host cell comprising a first and second heterologous coding sequence, wherein the first and second heterologous coding sequences encode a first and second enzyme, respectively, that are involved in a metabolic pathway that converts tyrosine naturally produced in the host cell into the dopamine.
A host cell that produces 4-HPA, the host cell comprising a first and second heterologous coding sequence, wherein the first and second heterologous coding sequences encode a first and second enzyme, respectively, that are involved in a metabolic pathway that converts tyrosine naturally produced in the host cell into the 4-HPA.
A method of preparing a metabolite of tyrosine comprising
- a) culturing a host cell under conditions suitable for protein production, said host cell comprising a first and second heterologous coding sequence, wherein the first and second heterologous coding sequences encode a first and second enzyme, respectively, that are involved in a metabolic pathway that converts the tyrosine into the metabolite,
- b) optionally adding tyrosine to the cell culture, and
- c) recovering the metabolite from the cell culture.
A method of preparing a metabolite of norlaudanosoline comprising
- a) culturing a host cell under conditions suitable for protein production, said host cell comprising a first and second heterologous coding sequence, wherein the first and second heterologous coding sequences encode a first and second enzyme, respectively, that are involved in a metabolic pathway that converts the norlaudanosoline into the metabolite,
- b) adding norlaudanosoline to the cell culture, and
- c) recovering the metabolite from the cell culture.
A method of screening a nucleotide library for at least one nucleotide sequence that codes for at least one peptide with enzymatic activity directed towards benzylisoquinolines, the method comprising
- a) providing a nucleic acid library to a culture of host cells, the host cells being engineered to produce a first benzylisoquinoline and the library being constructed for peptide expression in the host cell,
- b) optionally providing a starting material to the host cells,
- c) culturing the host cells under conditions appropriate for peptide expression,
- d) determining the presence or absence of a second benzylisoquinoline,
- wherein the presence of a second benzylisoquinoline indicates the presence of at least one nucleotide sequence in the nucleotide library that codes for at least one peptide with enzymatic activity directed towards benzylisoquinolines.
Dependencies are inferred from the claim text.