Benzylisoquinoline alkaloid (BIA) precursor producing microbes, and methods of making and using the same
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorsMichael Shareef Siddiqui, Christina D. Smolke
Life of this patent
Estimating the term…
Abstract
Host cells that are engineered to produce benzylisoquinoline alkaloid (BIAs) precursors, such as norcoclaurine (NC) and norlaudanosoline (NL), are provided. The host cells may have one or more engineered modifications selected from: a feedback inhibition alleviating mutation in a enzyme gene; a transcriptional modulation modification of a biosynthetic enzyme gene; an inactivating mutation in an enzyme; and a heterologous coding sequence. Also provided are methods of producing a BIA of interest or a precursor thereof using the host cells and compositions, e.g., kits, systems etc., that find use in methods of the invention.
Claims25
A method of preparing a metabolite that is a precursor benzylisoquinoline alkaloid product, the method comprising:
- culturing an engineered non-plant cell with a feed stock, said engineered non-plant cell comprising three coding sequence modifications, wherein the three coding sequence modifications are modifications to coding sequences that encode a first, second, and third enzyme, respectively, that are involved in a metabolic pathway that produces the precursor benzylisoquinoline alkaloid product,
- wherein the precursor benzylisoquinoline alkaloid product is selected from the group consisting of a tyrosine, 4-hydroxyphenylacetaldehyde, 4-hydroxyphenylpyruvic acid, L-3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenylacetaldehyde, dopamine, norcoclaurine, and norlaudanosoline, and
- wherein the first enzyme involved in the metabolic pathway that produces the precursor benzylisoquinoline alkaloid product is selected from the group consisting of Transketolase (TKL1), Glucose-6-phosphate dehydrogenase (ZWF1), Pentafunctional AROM protein (ARO1), Bifunctional chorismate synthase (ARO2), 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (ARO3), 3-deoxy-d-arabinose-heptulosonate-7-phosphate synthase (ARO4), Chorismate mutase (ARO7), tyrosinase (TYR1), and tyrosinase (TYR),
- wherein the second enzyme involved in the metabolic pathway that produces the precursor benzylisoquinoline alkaloid product is selected from the group consisting of TKL1, ZWF1, ARO1, ARO2, ARO3, ARO4, ARO7, TYR1, NCS, TYR, Aromatic aminotransferase (ARO9), Phenylpyruvate decarboxylase (ARO10), TyrH, DODC, and MAO, and
- wherein the third enzyme involved in the metabolic pathway that produces the precursor benzylisoquinoline alkaloid product is selected from the group consisting of TKL1, ZWF1, ARO1, ARO2, ARO3, ARO4, ARO7, TYR1, and TYR.
Dependencies are inferred from the claim text.