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

Abstract

Methods and engineered yeast cells for generating a benzylisoquinoline alkaloid product are provided herein. A method comprises providing engineered yeast cells and a feedstock to a reactor. In the reactor, the engineered yeast cells are subjected to fermentation by incubating the engineered yeast cells for a time period to produce a solution comprising the BIA product and cellular material. The solution comprises not more than one class of molecule selected from the group of protoberberine, morphinan, isopavine, aporphine, and benzylisoquinoline. Additionally, at least one separation unit is used to separate the BIA product from the cellular material to provide the product stream comprising the BIA product.

1 .- 15 . (canceled) 
     
     
         16 . An engineered non-plant cell having modifications that result in overproduction of a derivative of tyrosine, along a pathway to produce the benzylisoquinoline alkaloid product, with respect to a non-engineered non-plant cell, wherein the modifications comprise at least three modifications, wherein at least one modification of the at least three modifications is a transcriptional modulation mutation or an inactivating mutation, and wherein each modification of the at least three modifications is selected from the group consisting of: a feedback inhibition alleviating mutation, a transcriptional modulation mutation, and an inactivating mutation, wherein a first modification of the at least three modifications is associated with a first enzyme, a second modification of the at least three modifications is associated with a second enzyme, and a third modification of the at least three modifications is associated with a third enzyme, and wherein each enzyme of the first, second, and third enzymes is distinct from any other enzyme of the first, second, and third enzymes, and wherein each enzyme of the first, second, and third enzyme is selected from the group consisting of: transketolase, glucose-6-phosphate dehydrogenase, pentafunctional AROM protein, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, chorismate mutase, tyrosinase, tyrosine hydroxylase, L-DOPA decarboxylase, monoamine oxidase, aromatic/aminoadipate aminotransferase, aromatic aminotransferase, phenylpyruvate decarboxylase, alcohol dehydroxylase 2, alcohol dehydroxylase 3, alcohol dehydroxylase 4, alcohol dehydroxylase 5, alcohol dehydroxylase 6, alcohol dehydroxylase 7, aldehyde oxidase 2, aldehyde oxidase 3, aldehyde oxidase 4, aldehyde oxidase 5, and aldehyde oxidase 6. 
     
     
         17 . The engineered non-plant cell of  claim 16 , wherein the engineered non-plant cell is an engineered fungal cell. 
     
     
         18 . The engineered non-plant cell of  claim 17 , wherein the engineered fungal cell comprises one or more feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes that encode 3-deoxy-d-arabinose-heptulosonate-7-phosphate synthase. 
     
     
         19 . The engineered non-plant cell of  claim 17 , wherein the engineered fungal cell comprises one or more feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes that encode chorismate mutase. 
     
     
         20 . The engineered non-plant cell of  claim 17 , wherein the engineered fungal cell further comprises at least one transcriptional modulation modification in a biosynthetic enzyme gene. 
     
     
         21 . The engineered non-plant cell of  claim 16 , wherein at least one process parameter of the batch reactor is modifiable to alter a resultant benzylisoquinoline alkaloid product composition, wherein the at least one process parameter that is modifiable comprises at least one of dissolved oxygen, pH, stirring speed, aeration rate, and cell density. 
     
     
         22 . The engineered non-plant cell of  claim 16 , wherein the benzylisoquinoline alkaloid product is selected from the group of norcoclaurine, norlaudanosoline, tyramine, 4-hydroxyphenylacetaldehyde, 4-hydroxyphenylpyruvic acid, L-3, 4-dihydroxyphenylalanine, 3,4-dihydroxyphenylacetaldehyde, and dopamine. 
     
     
         23 . The engineered non-plant cell of  claim 16 , wherein the benzylisoquinoline alkaloid product is a benzylisoquinoline alkaloid that has a structural class that is selected from the group of benzyisoquinolines, protoberberines, protopines, benzophenanthridines, promorphinans, morphinans, secoberbines, phthalideisoquinolines, aporphines, and bisbenzylisoquinolines. 
     
     
         24 . The engineered non-plant cell of  claim 16 , wherein the product stream does not contain more than 5 ppm of a molecule selected from the group of lignin, pigments, flavonoids, phenanthreoids, latex, rubisco, meconic acid, pseudomorphine, narceine, thebaol, and pollen. 
     
     
         25 . The engineered non-plant cell of  claim 16 , wherein the product stream does not contain a detectable amount of a substance selected from the group consisting of pesticides, fungicides, or herbicides. 
     
     
         26 . The engineered non-plant cell of  claim 17 , wherein the engineered fungal cell comprises two or more heterologous coding sequences, wherein the two or more heterologous coding sequences encode at least a first enzyme and a second enzyme that are involved in a metabolic pathway that converts the tyrosine into the benzylisoquinoline alkaloid product, wherein the first enzyme and second enzyme are operably connected along the metabolic pathway. 
     
     
         27 . The engineered non-plant cell of  claim 17 , wherein the engineered fungal cell comprises three heterologous coding sequences, wherein the three heterologous coding sequences encode a first enzyme, second enzyme, and third enzyme that are involved in a metabolic pathway that converts the tyrosine into the benzylisoquinoline alkaloid product, wherein the first enzyme, second enzyme, and third enzyme are operably connected along the metabolic pathway. 
     
     
         28 . A pharmaceutical opioid formulation that comprises a pharmaceutical opioid compound, wherein the mixture does not contain a detectable amount of a compound selected from the group consisting of silicon, polysaccharides, lignin, pigments, flavonoids, phenanthreoids, latex, gum, wax, rubisco, meconic acid, pseudomorphine, narceine, thebaol, pesticides, fungicides, herbicides, and pollen. 
     
     
         29 . The pharmaceutical opioid formulation of  claim 16 , wherein the pharmaceutical opioid formulation is within a medicament. 
     
     
         30 . The pharmaceutical opioid formulation of  claim 16 , wherein the pharmaceutical opioid formulation is within a pharmaceutical delivery vehicle.