Benzylisoquinoline Alkaloid (BIA) Precursor Producing Microbes, and Methods of Making and Using the Same

Abstract

Host cells that are engineered to produce benzylisoquinoline alkaloid (BIAs) precursors, such as norcodaurine (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.

What is claimed is: 
     
         1 . A host cell that produces a benzylisoquinoline alkaloid (BIA) precursor, wherein the host cell comprises one or more modifications selected from the group consisting of:
 one or more feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes native to the cell;   one or more transcriptional modulation modifications of one or more biosynthetic enzyme gene native to the cell;   one or more inactivating mutations in one or more enzymes native to the cell; and   one or more heterologous coding sequences that encode one or more enzymes;   wherein when the cell comprises one or more heterologous coding sequences that encode one or more enzymes, it comprises at least one additional modification selected from the group consisting of: a feedback inhibition alleviating mutation in a biosynthetic enzyme gene native to the cell; a transcriptional modulation modification of a biosynthetic enzyme gene native to the cell; and an inactivating mutation in an enzyme native to the cell.   
     
     
         2 . The host cell according to  claim 1 , wherein the BIA precursor is selected from the group consisting of norcoclaurine (NC) and norlaudanosoline (NL). 
     
     
         3 . The host cell according to  claim 1 , wherein the cell comprises one or more feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes native to the cell. 
     
     
         4 . The host cell according to any one of the preceding claims, wherein the cell overproduces one or more BIA precursor molecules. 
     
     
         5 . The host cell according to  claim 4 , wherein the one or more BIA precursor molecules are selected from the group consisting of tyrosine, 4-hydroxyphenylacetaldehyde (4-HPA), L-3,4-dihydroxyphenylalanine (L-DOPA), 3,4-dihydroxyphenylacetaldehyde (3,4-DHPA) and dopamine. 
     
     
         6 . The host cell according to  claim 1 , wherein the cell comprises one or more transcriptional modulation modifications of one or more biosynthetic enzyme genes native to the cell. 
     
     
         7 . The host cell according to  claim 6 , wherein the transcriptional modulation modification is substitution of a strong promoter for a native promoter of the one or more biosynthetic enzyme genes. 
     
     
         8 . The host cell according to  claim 1 , wherein the cell comprises one or more inactivating mutations in one or more enzymes native to the cell. 
     
     
         9 . The host cell according to  claim 8 , wherein the enzyme is a glucose-6-phosphate dehydrogenase, an alcohol dehydrogenase or an aldehyde oxidoreductase. 
     
     
         10 . The host cell according to  claim 1 , wherein the cell comprises one or more heterologous coding sequences that encode one or more enzymes or active fragments thereof. 
     
     
         11 . The host cell according to  claim 1 , wherein the host cell is a eukaryotic cell. 
     
     
         12 . The host cell according to  claim 11 , wherein the eukaryotic cell is a yeast cell. 
     
     
         13 . The host cell according to  claim 12 , wherein the yeast cell is a  S. cerevisiae  cell, a  Schizosaccharomyces pombe  or a  Pichia pastoris  cell. 
     
     
         14 . A method of preparing a benzylisoquinoline alkaloid (BIA), comprising:
 culturing a host cell according to any one of  claims 1  to  13 ;   adding a growth feedstock to the cell culture; and   recovering the BIA from the cell culture.