GOVERNMENT RIGHTS

This invention is made with Government support under grant No. 1066100 awarded by the National Science Foundation. The Government has certain rights in this invention.

CROSS-REFERENCE TO RELATED APPLICATION

Pursuant to 35 U.S.C. § 119 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 61/899,498 filed on November 4, 2013; the disclosure of which application is herein incorporated by reference.

INTRODUCTION

Benzylisoquinoline alkaloids (BIAs) are a large group of secondary metabolites from plants and other organisms. These molecules have therapeutic functions in the human body, ranging from the established analgesic and antitussive properties of morphine and codeine, to novel activities against cancer and infection observed for molecules such as berberine and sanguinarine. Supply of ali these BIA molecules so that they are available to researchers and physicians is of interest. The number of synthetic reactions and

requirements for selective stereochemistry means that chemical synthesis of BIAs is low yielding and not a viable means for large-scale production. Instead, for the widely used drugs codeine and morphine, the opium poppy (Papaver somniferum) has been bred and developed as a production crop, intermediates in morphine biosynthesis that find use as drugs and drug precursors do not accumulate because the plant metabolism is evolved to maximize pathway flux to the final opioids. Even for end product metabolites like morphine, accumulation occurs only within specialized cells in the buds and vascular tissue and requires harsh chemical processing of harvested piant materia! during the extraction process, whic may yield less than 2% morphine by dry weight. As such, methods for preparing BIAs are of interest.

SUMMARY

Host cells that are engineered to produce benzySisoqu incline alkaloid (BIA) precursors, such as norcoclaurine (NC) and norlaudanosoline (NL), are provided. The host cells may have one or more modifications selected from: a feedback inhibition alleviating mutation in an enzyme gene; a transcriptional modulation modificatio of a biosynthetic enzyme gene; an inactivating mutation in an enzyme; and a heterologous coding sequence. Also provided are methods of producing a B!A of interest or a precursor thereof using the host cells and compositions, e.g. _s kits, systems etc., that find use in methods of the invention.

BRIEF DESCRIPTION OF THE FIGURES

The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scaie. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

Figure 1 illustrates the biosynthetic pathway from glucose to tyrosine and other BiA precursor molecules.

Figure 2 illustrates the effect of ZWF1 knockout and TKL1 over-expression on the pentose phosphate pathway (PPP). A: native PPP flux, B: Modified PPP flux.

Figure 3 illustrates the synthesis of NC (A) and NL (B) from precursor molecules.

Figure 4 illustrates the effect of four genetic modifications on NC production with varying fed tyrosine.

Figure 5 shows NC production from strains with combinations of genetic

modifications.

Figure 6 shows the levels of ML production in aldehyde oxidoreductase (ALD)/ alcohol dehydrogenase (ADH) gene knockout strains

Figure 7 illustrates the activity of a L-DOPA decarboxylase {DODC} enzyme in vivo. Yeasi strains transformed with DNA to express Papaver somniferum tyrosine/DOPA decarboxylase can convert L-DOPA to dopamine in vivo.

Figure 8 shows the production of norcociaurine (NC) in yeast strains fed 100 m dopamine and varying concentrations of tyrosine.

Figure 9 shows NC production in multiple engineered yeast strains fed 100 mM dopamine and no tyrosine.

Figure 10 shows NC production from dopamine or from L-DOPA in an engineered yeast strain (CSY980) with the additional integration of the L-DOPA decarboxylase

PpDODC.

Figure 11 illustrates a biosynthetic scheme inciuding tyrosine hydroxySation using mammalian tyrosine hydroxylases (TyrHs) with the co-substrate tetrahydrobiopterin (BH4).

Figure 12 shows that tyrosine hydroxylases expressed from yeast cells convert tyrosine to L-DOPA. (A) LC-MS chromaiogram confirms conversion of tyrosine to L-DOPA in the presence of co-substrate, BH4; and (B) L-DOPA ion fragmentation in lysate samples.

Figure 13 shows the co-expression of tyrosine hydroxylase with a BH4 biosynthetic enzyme provides for conversion of tyrosine to L-DOPA.

Figure 14 i!iustrates the synthesis of the B!A precursor molecules cociaurine and N- methy!cociaurine from C.

Figure 5 shows LC- S analysis (A: son counts) of the production of NC-derived B!A precursor molecules including N-methylcoc!aurine (8: m z fragmentation pattern) from L- DOPA in the liquid culture of engineered yeast strains.

DEFINITIONS

Before describing exemplary embodiments in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used in the description.

Unless defined otherwise,, ail technical and scientific terms used herein have the same meaning as commonly understood by one of ordi ary skill in the art to which this invention belongs. Singleton, et aL DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & arkham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with the general meaning of many of the terms used herein. Still, certain terms are defined below for the sake of clarity and ease of reference.

It must be noted that as used herein and i the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a primer" refers to one or more primers, i.e., a single primer and multiple primers. St is further noted that the claims is drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as ''solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

As used herein, the terms "determining," "measuring," and "assessing," and

"assaying ^' ' are used interchangeably and include both quantitative and qualitative determinations.

As used herein, the term "polypeptide" refers to a polymeric form of amino acids of any length, including peptides that range from 2-50 amino acids in length and polypeptides that are greater than 50 amino acids in length. The terms "polypeptide" and "protein" are used interchangeably herein. The term "polypeptide" includes polymers of coded and non- coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones in which the conventional backbone has been replaced with non-natura!ly occurring or synthetic backbones. A polypeptide may be of any convenient length, e.g., 2 or more amino acids, such as 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, 50 or more amino acids, 100 or more amino acids, 300 or more amino acids, such as up to 500 or 1000 or more amino adds. "'Peptides" may be 2 or more amino acids, such as 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, such as up to 50 amino acids. In some embodiments, peptides are between 5 and 30 amino acids in length.

As used herein the term "isolated, ^' refers to an moiety of interest that is at least 80% free, at least 75% free, at ieast 90% free, at least 95% free, at least 98% free, and even at least 99% free from other components with which the moiety is associated with prior to purification.

As used herein, the term "encoded by" refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of 3 or more amino acids, such as 5 or more, 8 or more, 10 or more, 15 or more or 20 or more amino acids from a polypeptide encoded by the nucleic acid sequence. Also encompassed by the term are polypeptide sequences that are immunologically identifiable with a polypeptide encoded by the sequence.

A "vector" is capable of transferring gene sequences to target cells. As used herein, the terms, "vector construct," "expression vector," and "gene transfer vector," are used interchangeably to mean any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target ceils, which is

accomplished by genomic integration of all or a portion of the vector, or transient or inheritable maintenance of the vector as an extrachromosomai element. Thus, the term includes cloning, and expression vehicles, as well as integrating vectors.

An "expression cassette" includes any nucleic acid construct capable of directing the expression of a gene/coding sequence of interest, which is operably linked to a promoter of the expression cassette. Such cassettes is constructed into a "vector," 'Vector construct," "expression vector," or "gene transfer vector," in order to transfer the expression cassette into target cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

A "plurality" contains at Ieast 2 members. I certain cases, a plurality may have 10 or more, suc as 100 or more, 1000 or more, 10,000 or more, 100,000 or more, 10 ⁶ or more, 10 ⁷ or more, 10 ^s or more or 10 ⁹ or more members.

Numeric ranges are inclusive of the numbers defining the range.

The methods described herein include multiple steps. Each step may be performed after a predetermined amount of time has elapsed between steps, as desired. As such, the time between performing each step may be 1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds or more, 5 minutes or more, 10 minutes or more, 60 minutes or more and including 5 hours or more, in certain embodiments, each subsequent step is performed immediately after completion of the previous step, in other embodiments, a step may be performed after an incubation or waiting time after completion of the previous step, e.g., a few minutes to an overnight waiting time.

Other definitions of terms may appear throughout the specification.

DETAILED DESCRIPTION

Host cells that are engineered to produce benzyiisoqu incline alkaloid BIAs) precursors, such as norcociaunne (NC) and noriaudanosoline (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.

Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and Sower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are aiso encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes, in determining whether a number is near to or approximately a specifically recited number, the near or approximating unreciied number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

Unless defined otherwise, al! technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if eac individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the

publications are cited. The citation of any publication is for its disciosure prior to the filing date and should not be construed as an admission thai the present invention is not entitled to antedate suc publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

St is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. St is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method is carried out in the order of events recited or i any other order which is logically possible.

In further describing the subject invention, ber ylisoquino!ine alkaloid precursors of interest are described first in greater detail, followed by host cells for producing the same. Next, methods of interest in which the host cells find use are reviewed. Kits thai may be used in practicing methods of the invention are also described.

BENZYLISOQUINOUNE ALKALOID (BfA)PRECURSQRS

As summarized above, host cells which produce benzylisoquino!ine alkaloid precursors (BIA precursors) are provided. The BIA precursor may be any intermediate or precursor compound in a synthetic pathway {e.g., as described herein) that leads to the production of a 8IA of interest (e.g. , as described herein). In some cases, the BIA precursor has a structure that may be characterized as a BIA or a derivative thereof, in certain cases, the BIA precursor has a structure that may be characterized as a fragment of a BIA. In some cases, the BIA precursor is an early BIA. As used herein, by "early BIA" is meant an early intermediate in the synthesis of a BIA of interest in a ceil, where the eariy BIA is produced by a host ceil from a host eel! feedstock or simple starting compound, in some cases, the eariy BiA is a BIA intermediate that is produced by the subject host ceil solely from a host cell feedstock (e.g., a carbon and nutrient source) without the need for addition of a starting compound to the cells. The term early BIA may refer to a precursor of a BiA end product of interest whether or not the early BiA can be itself he characterized as a benzylisoquinoiine aikaioid.

in some cases, the BiA precursor is an eariy BIA, such as a pre-reticuline benzylisoquinoiine aikaioid. As such, host cells whic produce pre-reticuline benzylisoquinoiine alkaloids (pre-reticuline BIAs) are provided. Reticuline is a major branch point intermediate of interest in the synthesis of downstream BiAs via cell engineering efforts to produce end products such as opioid products. The subject host cells may produce BIA precursors from simple and inexpensive starting materials that may find use in the production of reticuline and downstream BIA end products.

As used herein, the terms "pre-reiicuiine benzylisoquinoiine alkaloid" , "pre- reticuline BIA" and "pre-reticuline BiA precursor" are used inierchangeabiy and refer to a biosynthetic precursor of reticuline whether or not the structure of the reticuline precursor itself is characterized as a benzylisoquinoiine alkaloid. The term pre-reticuiine BIA is meant to include biosynthetic precursors, intermediates and metabolites thereof, of any convenient member of a host cell biosynthetic pathway thai may lead to reticuline. In some cases, the pre-reticuline BIA inciudes a benzylisoquinoiine alkaloid fragment, such as a benzyl fragment, a quinoline fragment or a precursor or derivative thereof, in certain instances, the pre-reticuline BIA has a structure that can be characterized as a benzylisoquinoiine aikaioid or a derivative thereof.

BIA precursors of interest include, but are not limited to, norcoclaurine ( C) and norlaudanosoiine (NL), as well as NC and NL precursors, such as tyrosine, 4- hydroxyphenylacetaSdehyde (4-HPA), 4-hydroxypheny!pyruvic acid {4-HPPA), L~3,4~ dihydroxyphenyialanine(L-DOPA), 3,4-dihydroxyphenyiacetaldehyde 3,4-DHPA), and dopamine, in some embodiments, the one or more BIA precursors are 3,4- dihydroxyphenyiaceta!dehyde (3,4-DHPA) and dopamine, in certain instances, the one or more BIA precursors are 4-hydroxyphenylacetaldehyde (4-HPA) and dopamine. Figures 3A and 3B illustrate the synthesis of NC and NL, respectively from precursor molecules via a Pictet-Speng!er condensation reaction, where the reaction may occur spontaneously or may by catalyzed by any convenient enzymes.

Synthetic pathways to a BIA precursor may be generated in the host cells, and may start with any convenient starting compound(s) or materials. Figure 1 illustrates a synthetic pathway of interest to BiA precursors starting from glucose. The starting material may be non-naturally occurring or the starting material may be naturally occurring in the host ceil. Any convenient compounds and materials may be used as the starting material, based upon the synthetic pathway present in the host cell. The source of the starting material may foe from the host cell itself, e.g., tyrosine, or the starting material may foe added or supplemented to the host cell from an outside source. As such, in some cases, the starting compound refers to a compound in a synthetic pathway of the cell that is added to the host cell from an outside source that is not part of a growth feedstock or cei! growth media.

Starting compounds of interest inciude, but are not limited to, dopamine, 4-HPA, 4-HPPA, as well as any of the compounds shown in FIG 1. For example, if the host ce!is are growing in liquid culture, the cell media may be supplemented with the starting material, which is transported into the cells and converted into the desired products by the ceii. Starting materials of interest inciude, but are not limited to, inexpensive feedstocks and simple precursor molecules. In some cases, the host ceii utilizes a feedstock including a simple carbon source as the starting material, which the host cell utilizes to produce compounds of the synthetic pathway of the ceil. The host ceii growth feedstock may inciude one or more components, such as a carbon source such as cellulose, starch, free sugars and a nitrogen source, such as ammonium saits or inexpensive amino acids. In some cases, a growth feedstock that finds use as a starting materia! may be derived from a sustainabie source, such as biomass grown on marginal land, including s itchgrass and algae, or biomass waste products from other industrial or farming activities.

HOST CELLS

As summarized above, one aspect of the invention is a host cell that produces one or more BIA precursors. Any convenient ceiis may be utilized in the subject host ceils and methods. In some cases, the host celis are non-piant ceils, in some instances, the host ceils may be characterized as microbial ceils, in certain cases, the host cells are insect cells, mamma!ian cells, bacteria! celis or yeast ceils, Any convenient type of host ceil may be utilized in producing the subject BIA -producing ceiis. see, e.g.. US2008/0178754. and US2G14/0273109 the disclosures of which are incorporated by reference in their entirety. Host celis of interest include, but are not limited to, bacterial ceils, such as Bacillus suhtilis, Escherichia coli, Streptomyces and Salmonella typhi uiu ceiis, insect ceiis such as Drosophila meianogaster S2 and Spodoptera frugiperda Sf9 ceiis and yeast celis such as S. cerevisiae cei!s, Schizosaccharomyces pombe ce!Ss and a Pichia pastoris cells. In some embodiments, the host cells are yeast ceils or E. coli cells. In some cases, the host ceil is a yeast cell. In some instances the host cell is from a strain of yeast engineered to produce a BIA precursor of interest. Any of the host ceils described in US2008/0176754, and US2014/0273109 by Smoike et at may be adapted for use in the subject cells and methods. In certain embodiments, the yeast cells can be of the species Saccharomyces c&r@vi$ia& {$, cerevisiae). in certain embodiments, the yeast ceils can be of the species Schizosaccharomyces po be. In certain embodiments, the yeast ceils can be of the species Pichia pastoris. Yeast is of interest as a host cell because cytochrome P450 proteins, which are involved in some biosynthetic pathways of interest, are able to fold properly into the endoplasmic reticulum membrane so that their activity is maintained. Yeast strains of interest that find use in the invention include, but are not limited to, CEN.PK {Genotype: MATa, ¹ ® ura3-52/ura3-52 trpl-2BWtrpl-289 t u2-3_112fieu2-3__112 his3 M/ is3 Δ? MAL2-8C/MAL2-8C $UC2/$UC2), S288C, W303, D273-10B, X2180, A384A, ∑1278B, AB972, SK1 and FL100, In certain cases, the yeast strain is any of S288C ( ATa; SUC2 mal me! ga!2 CUP1 flo1 fio8-1 hapl ), BY4741 (MATa; his3Al ; leu2A0; met15A0; ura3A0), BY4742 {MATa; his3A1 ; ieu2A0; lys2A0; ura3A0), BY4743 (MATa/ ΑΤα; his3A1 his3A1 ; Ιβυ2Δ0 Ιβυ2Δ0; met15A07 ET15; LYS2/iys2A0; ura3A0/ura3A0), and WAT11 or W(R), derivatives of the W303-B strain (MATa; ade2-1 ; his3-11 , -15; ieu2~3 -112; ura3-1 ; canR; cyr+) which express the Ambidopsis tba!iana NADPH-P450 reductase ATR1 and the yeast NADPH-P450 reductase CPR1 , respectively. I another embodiment, the yeast ceil is W3G3alpba (MATa; his3-1 , 15 ί 1 -1 Seu2-3 ura3-1 ade2-1 ). The identity and genotype of additional yeast strains of interest can be found at EUROSCARF (web.uni- fran kfurt.de/fb 15/mi kro/euroscarf/coijndex. htm I).

The host cells may be engineered to include one or mor modifications (suc as two or more, three or more, four or more, five or more, or even more modifications) that provide for the production of BIA precursors of interest. In some cases, by modification is meant a genetic modification, such as a mutation, addition or deletion of a gene or fragment thereof, or transcription regulation of a gene or fragment thereof. I some cases, the one or more (such as two or more, three or more or four or more) modifications is selected from: a feedback inhibition alleviating mutation in a biosynthetic enzyme gene native to the cell; a transcriptionai modulation modification of a biosynthetic enzyme gene native to the cell; an inactivating mutation in an enzyme native to the cell; and a heterologous coding sequence that encodes an enzyme. A cell that includes one or more modifications may be referred to as a modified cell

A modified cell may overproduce one or more 8i precursor mo!ecuies. By overproduce is meant that the cell has an improved or increased production of a BfA precursor molecule of interest relative to a control cell {e.g., an unmodified cei!). By improved or increased production is meant both the production of some amount of the BiA precursor of interest where the control has no BIA precursor production, as well as an increase of about 10% or more, such as about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or

8 more, such as 2-fold or more, such as 5~fold or more, including 10-fo!d or more in situations where the control has some BSA precursor production.

In some cases, the host celi is capab!e of producing an increased amount of norcoclaunne relative to a control host cell that lacks the one or more modifications (e.g., as described herein). In certain instances, the increased amount of norcociaurine is about 10% or more relative to the control host celi, such as about 20% or more, about 30% or more, about 40% or more _; about 50% or more, about 80% or more, about 80% or more, about 100% or more, 2-fold or more, 5-fold or more, or even 10-fold or more relative to the control host cell.

in some cases, the host celi is capable of producing an increased amount of norlaudonosoline relative to a control host cell that lacks the one or more modifications (e.g., as described herein), in certain instances, the increased amount of norlaudonosoline is about 10% or more relative to the control host cell, such as about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, 2-fold or more, 5-fo!d or more, or even 10-foid or more relative to the control host celi.

In some embodiments, the host celi is capable of producing a 10% or more yield of norcociaurine from a starting compound such as tyrosine, such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or even 90% or more yield of norcociaurine from a starting compound.

In some embodiments, the host cell is capable of producing a 10% or more yield of norlaudonosoline from a starting compound such as tyrosine, such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or even 90% or more yield of norlaudonosoline from a starting compound.

In some embodiments, the host ceil overproduces one or more BIA precursor molecule selected from the group consisting of tyrosine, 4-hydroxypheny!acetaldehyde (4- HPA), L~3,4-dihydroxyphenylaSanine(L-DOPA), 3,4~dihydroxyphenylaceta!dehyde (3,4- DHPA). and dopamine.

Any convenient combinations of the one or more modifications may be included in the subject host ceils, in some cases, two or more (such as two or more, three or more, or four or more) different types of modifications are included. In certain instances, two or more (such as three or more, four or more, five or more, or even more) distinct modifications of the same type of modification are included in the subject cells.

In some embodiments of the host cell, when the cell includes one or more heterologous coding sequences that encode one or more enzymes, it includes at least one additional modification selected from the group consisting of: a feedback inhibition alleviating mutations in a biosynthetic enzyme gene native to the cell; a transcriptional modulalion modification of a biosyntheiic enzyme gene native to the ceil; and an inactivating mutation in an enzyme native to the ce!i. In certain embodiments of the host ceil, when the cell includes one or more feedback inhibition alleviating mutations in one or more biosyntheiic enzyme genes native to the cell, it includes a least one additional modification selected from the group consisting of; a transcriptionai modulation modification of a biosynthetic enzyme gene native to the ceii; an inactivating mutation in an enzyme native to the ceii; and a heterologous coding sequence that encode an enzyme. In some

embodiments of the host ceil, when the ceii inciudes one or more transcriptional modulation modifications of one or more biosyntheiic enzyme genes native to the ceil, it includes at least one additional modification selected from the group consisting of: a feedback inhibition alleviating mutation in a biosynthetic enzyme gene native to the ceii; an inactivating mutation in an enzyme native to the ceii; and a heterologous coding sequence that encodes an enzyme. Sn certain instances of the host cell, when the cell includes one or more inactivating mutations in one or more enzymes native to the ceii, it includes at least one additional modification selected from the group consisting of: a feedback inhibition alleviating mutation in a biosynthetic enzyme gene native to the ceii; a transcriptional modulation modification of a biosynthetic enzyme gene native to the cell; and a

heterologous coding sequence that encodes an enzyme.

Sn certain embodiments of the host cell, the cell includes one or more feedback inhibition ai!eviating mutations in one or more biosynthetic enzyme genes native to the cell; and one or more transcriptional modulation modifications of one or more biosynthetic enzyme gene native to the ceil, in certain embodiments of the host ceii, the ceil inciudes one or more feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes native to the ceii; and one or more inactivating mutations in an enzyme native to the ceii. Sn certai embodiments of the host ceil, the cell includes one or more feedback inhibition aileviating mutations in one or more biosynthetic enzyme genes native to the ceil; and one or more heterologous coding sequences, !n some embodiments, the host cell includes one or more modifications (e.g., as described herein) that include one or more of the genes of interest described in Table i .

Feedback inhibition Ate

in some instances, the host cells are cells that include one or more feedback inhibition aileviating mutations (such as two or more, three or more, four or more, five or more, or even more) in one or more biosynthetic enzyme genes of the cell. In some cases, the one or more biosynthetic enzyme genes are native to the ceil (e.g., is present in an unmodified ceii). As used herein, the term "feedback inhibition alleviating mutation" refers to a mutation that alleviates a feedback inhibition control mechanism of a host cell. Feedback inhibition is a contro! mechanism of the eel! in which an enzyme in the synthetic pathway of a regulated compound is inhibited when that compound has accumulated to a certain level, thereby balancing the amount of the compound in the celi. In some instances, the one or more feedback inhibition alleviating mutations is in an e zyme described in a synthetic pathway of Figure 1 or Figure 2. A mutation that alleviates feedback inhibition reduces the inhibition of a regulated enzyme in the cell of interest relative to a control cell and provides for an increased ievel of the regulated compound or a downstream biosynthetic product thereof. In some cases, by alleviating inhibition of the regulated enzyme is meant that the ICso of inhibition is increased by 2-fold or more, such as by 3-foid or more, 5-fold or more, 10-fold or more, 30-fold or more, 100-fold or more, 300-foid or more, 1000-fold or more, or even more. By increased Ievel is meant a Ievel that is 110% or more of that of the regulated compound in a controi cell or a downstream product thereof, such as 120% or more, 30% or more, 140% or more, 150% or more, 180% or more, 170% or more, 180% or more, 190% or more or 200% or more, such as at least 3-foid or more, at least 5-fold or more, at least 10-foid or more or even more of the regulated compound in the host ceil or a downstream product thereof,

A variety of feedback inhibition control mechanisms and biosynthetic enzymes native to the host cell that are directed to regulation of levels of B!A precursors may be targeted for alleviation in the host cell. The host cell may include one or more feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes native to the cell. The mutation may be located in any convenient biosynthetic enzyme genes native to the host cell where the biosynthetic enzyme is subject to regulatory control, in some

embodiments, the one or more biosynthetic enzyme genes encode one or more enzymes selected from a 3*deoxy-d-arabinose-heptulosonate-7-phosphate (DAHP) synthase and a chorismate mutase. In some embodiments, the one or more biosynthetic enzyme genes encode a 3-deoxy-d-arabinose-heptuiosonate-7-phosphate (DAHP) synthase, in some instances, the one or more biosynthetic enzyme genes encode a chorismate mutase. In certain instances, the one or more feedback inhibition alleviating mutations are present in a biosynthetic enzyme gene selected from AR04 and AR07. in certain instances, the one or more feedback inhibition aiieviating mutations are present in a biosynthetic enzyme gene that is AR04. in certain instances, the one or more feedback inhibition aiieviating mutations are present in a biosynthetic enzyme gene that is AR07. In some embodiments, the host cell includes one or more feedback inhibition aiieviating mutations in one or more biosynthetic enzyme genes such as one of those genes described in Table 1 ,

Any convenient numbers and types of mutations may be utilized to alleviate a feedback inhibition controi mechanism. As used herein, the term "mutation" refers to a deletion, insertion, or substitution of an amino acid(s) residue or nucleotide(s) residue relative to a reference sequence or motif. The mutation may be incorporated as a directed mutation to the native gene at the original locus. In some cases, the mutation may be incorporated as an additional copy of the gene introduced as a genetic integration at a separate locus, or as an additional copy on an episomai vector such as a 2μ or centromeric plasmid. In certain instances, the feedback inhibited copy of the enzyme is under the native cell transcriptional regulation. In some instances, feedback Inhibited copy of the enzyme is introduced with engineered constitutive or dynamic regulation of protein expression fay- placing it under the control of a synthetic promoter.

In certain embodiments, the one or more feedback inhibition alleviating mutations are present in the AR04 gene. AR04 mutations of interest include, but are not limited to, substitution of the lysine residue at position 229 with a leucine, a substitution of the giutamine residue at position 166 with a lysine residue, or a mutation as described by Hartmann M, et al. ({2003} Proc Natl Acad Sci U S A 10G(3):882-887) or Fukuda et al.

({1992} J Ferment Bioeng 74(2): 1 17-118). In some instances, mutations for conferring feedback inhibition are selected from a mutagenized library of enzyme mutants. Examples of such selections include rescue of growth of o-fiuoro-D,L-phenySaianine or growth of aro3 mutant yeast strains in media with excess tyrosine as described by Fukuda et al. ((1990) Breeding of Brewing Yeast Producing a Large Amount of Beta-PhenySethyl Alcohol and Beta-Phenyieth l Acetate. Agr Biol Ghern Tokyo 54(1)569-271 ).

AR07 mutations of interest include, but are not limited to, substitution of the threonine residue at position 228 with an isoieueine, as described by Schmidheini et al. ({1989), J Bacterial 17i(3):1245-1253) and additional mutations conferring feedback inhibition selected from a mutagenized library of microbial chorismate mutase mutants. Examples of such; selections include assays for 5-methyliryptophan sensitivity or increased production of melanin pigments in strains expressing heterologous tyrosinase enzymes (1.9) in the absence of externally fed tyrosine.

In certai embodiments, the host cells of the present invention may include 1 or more, 2 or more. 3 or more, 4 or more, 5 or more. 6 or more, 7 or more, 8 or more. 9 or more, 0 or more, 1 or more, 12 or more, 13 or more, 14 or more, or even 5 or more feedback inhibition alleviating mutations, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or 15 feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes native to the host ceil.

Transcn ^

The host cells may include one or more transcriptional modulation modifications (such as two or more, three or more, four or more, five or more, or even more modifications) of one or more biosynthetic enzyme genes of the cell. In some cases, the one or more biosynthetic enzyme genes are native to the cell. Any convenient biosynthetic enzyme genes of the ceii may be targeted for transcription modulation. By transcription modulation is meant that the expression of a gene of interest in a modified cell is modulated, e.g., increased or decreased, enhanced or repressed, relative to a control ceii (e.g., an unmodified ceii). In some cases, transcriptional modulation of the gene of interest includes increasing or enhancing expression. By increasing or enhancing expression is meant that the expression ievel of the gene of interest is increased by 2-foid or more, such as by 5-foid or more and sometimes by 25-, 50-, or 100-fold or more and in certain embodiments 300- fold or more or higher, as compared to a control, i.e., expression in the same cell not modified (e.g., by using any convenient gene expression assay). Alternatively, in cases where expression of the gene of interest in a ceii is so low that it is undetectable, the expression level of the gene of interest is considered to be increased if expression is increased to a Ievel that is easily detectable, in certain instances, transcriptionai modulation of the gene of interest includes decreasing or repressing expression. By decreasing or repressing expression is meant that the expression ievel of the gene of interest is decreased by 2-foid or more, suc as by 5-foid or more and sometimes by 25-, 50-, or 100- fold or more and in certain embodiments 300-fold or more or higher, as compared to a control. In some cases, expression is decreased to a Ievel that is undetectable.

Modifications of host ceii processes of interest that may be adapted for use in the subject host cells are described in U.S. Publication No. 20140273109 (14/21 1 ,611) by Smolke et a!., the disclosure of which is herein incorporated by reference in its entirety.

Any convenient biosynthetic enzyme genes may be transcriptionai ly modulated, and include but are not limited to, those biosynthetic enzymes described in Figure 1, such as A 03, ARQ4, AR01 , AR07, TYR1, TYR, TyrH, DODC, MAO, ARO10, AR09 and TKL. in some instances, the one or more biosynthetic enzyme genes is selected from ARO10, AR09 and TKL. In some cases, the one or more biosynthetic enzyme genes is ARO10. I certain instances, the one or more biosynthetic enzyme genes is AR09. In some embodiments, the one or more biosynthetic enzyme genes is TKL. In some embodiments, the host cell includes one or more transcriptional modulation modifications to one or more genes such as one of those genes described in Table 1. In some embodiments, the host cell includes one or more transcriptionai modulation modifications to one or more genes such as one of those genes described in a synthetic pathway of one of Figures 1 and 2. in some embodiments, the transcriptional modulation modification includes substitution of a strong promoter for a native promoter of the one or more biosynthetic enzyme genes. The promoters driving expression of the genes of interest can be constitutive promoters or inducible promoters, provided thai the promoters can be active in the host cells. The genes of interest may be expressed from their native promoters, or non- native promoters may be used. Although not a requirement, such promoters should be medium to high strength in the host in which they are used. Promoters may be regulated or constitutive. In some embodiments, promoters that are not glucose repressed, or repressed only mildly by the presence of glucose in the culture medium, are used. There are numerous suitable promoters, examples of which include promoters of glycolytic genes such as the promoter of the B. subtilis tsr gene {encoding fructose Diphosphate aidoiase) or GAPDH promoter from yeast S. cerevisiae (coding for glyceraldehyde-phosphate

dehydrogenase) {Bitter G. A., Meth. EnzymoL 152:673 684 (1387)), Other strong promoters of interest include, but are not limited to, the ADHi promoter of baker's yeast (Ruohonen L, et al, J. Biotec noi. 38:183 203 (1995)), the phosphate-starvation induced promoters such as the PH05 promoter of yeast (Hinnen, A., et al, in Yeast Genetic Engineering, Barr, P. J., et al. eds, Butterworths (1989), the alkaline phosphatase promoter from 8. lic eniformis (Lee. J. W. K., et a!., J. Gen. Microbiol. 137:1 127 1133 (1981 )), GPD1 and TEF1. Yeast promoters of interest include, but are not limited to, inducible promoters such as Gal 1-10, Gail , Ga!L, GaiS. repressive promoter Met25, tetO, and constitutive promoters such as glyoeraldeh de 3-phosphate dehydrogenase promoter (GPD), alcohol dehydrogenase promoter (ADH), translation-elongatio factor- -alpha promoter (TEF), cytochrome c- oxidase promoter (GYG1 ), MRP7 promoter, etc. In some instances, the strong promoter is GPD1. In certain instances, the strong promoter is TEF1. Autonomously replicating yeast expression vectors containing promoters inducible by hormones suc as glucocorticoids, steroids, and thyroid hormones are also known and include, but are not limited to, the glucorticoid responsive element (GRE) and thyroid hormone responsive element (TRE), see e.g., those promoters described in U.S. Pat. No. 7,045,290. Vectors containing constitutive or inducibie promoters such as alpha factor, alcohol oxidase, and PGH may be used.

Additionally any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of genes of interest. It is understood that any convenient promoters specific to the host ceil may be selected, e.g., E, coli. in some cases, promoter selectio can be used to optimize transcription, and hence, enzyme levels to maximize production while minimizing energy resources.

The host cells may include one or more inactivating mutations to an enzyme of the cell (such as two or more, three or more, four or more, five or more, or even more). The inclusion of one or more inactivating mutations may modify the flux of a synthetic pathway of a host cell to increase the levels of a BIA precursor of interest or a desirable enzyme or precursor leading to the same. In some cases, the one or more inactivating mutations are to an enzyme native to the cell Figure 2 illustrates a native pentose phosphate pathway (PPP) fiux and modified PPP flux where that involves inactivation of ZWF1 enzyme. As used herein, by "inactivating mutation" is meant one or more mutations to a gene or requlatory DNA sequence of the celi, where the mutation(s) inactivates a bioiogicai activity of the protein expressed by that gene of interest, in some cases, the gene is native to the celi. In some instances _: the gene encodes an enzyme that is inactivated and is part of or connected to the synthetic pathway of a BiA precursor produced by the host ceil, in some instances, an inactivating mutation is located in a regulatory DNA sequence that controis a gene of interest, in certain cases, the inactivating mutation is to a promoter of a gene. Any convenient mutations (e.g., as described herein) may be utilized to inactivate a gene or regulatory DNA sequence of interest. By "inactivated" or "inactivates" is meant that a bioiogicai activity of the protein expressed by the mutated gene is reduced by 10% or more, such as by 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more or 99% or more, re!ative to a control protein expressed by a non-mutated contro! gene. In some cases, the protein is an enzyme and the inactivating mutation reduces the activity of the enzyme.

in some embodiments, the cell includes an inactivating mutatio i a enzyme native to the ceii. Any convenient enzymes may be targeted for inactivation. Enzymes of interest include, but are not iimited to those enzymes, described in Figures 1 and 2 whose action in the synthetic pathway of the host cell tends to reduce the ieveis of a BIA precursor of interest. In some cases, the enzyme has g!ucose-6-phosphate dehydrogenase activity, in certain embodiments, the enzyme that includes an inactivating mutation is ZWF1 (see e.g., Figure 2). in some cases, the enzyme has alcohol dehydrogenase activity, in some embodiments, the enzyme that includes an inactivating mutation is selected from ADH2, ADH3, ADH4, ADH5, ADH6, ADH7 and SFA1. in certain embodiments, the enzyme that includes an inactivating mutaiion(s) is ADH2. in certain embodiments, the enzyme that includes an inactivating mutation(s) is ADH3. in certain embodiments, the enzyme that includes an inactivating mutation(s) is ADH4. In certain embodiments, the enzyme that includes an inactivating mutation(s) is ADH5. In certain embodiments, the enzyme that includes an inactivating mutation(s) is ADH6. In certain embodiments, the enzyme that includes an inactivating mutation(s) is ADH7. in some cases, the enzyme has aldehyde oxidoreductase activity. In certain embodiments, the enzyme that includes an inactivating mutation is selected from ALD2, ALD3, ALD4, ALD5 and ALD6. in certain embodiments, the enzyme that inciudes an inactivating mutation(s) is AID2, In certain embodiments, the enzyme that inciudes an inactivating mutation(s) is AIDS, In certain embodiments, the enzyme that inciudes an inactivating mutation(s) is ALD4. in certain embodiments, the enzyme that includes a inactivating mutation(s) is AIDS. I certain embodiments, the enzyme that includes a inactivating mutation(s is ALD6. In some embodiments, the host eel! includes one or more inactivating mutations to one or more genes described in Table 1 .

Heterologous Coding Sequences

In some instances, the host cells are cells that harbor one or more heterologous coding sequences (such as two or more, three or more, four or more, five or more, or even more) which encode activity(ies) that enable the host cells to produce desired BIA precursor(s), e.g., as described herein. As used herein, the term "heterologous coding sequence" is used to indicate any polynucleotide that codes for, or ultimately codes for, a peptide or protein or its equivalent amino acid sequence, e.g., an enzyme, that is not normally present in the host organism and can be expressed in the host cell under proper conditions. As such, "heterologous coding sequences" includes multiple copies of coding sequences that are normally present in the host cell, such that the ceii is expressing additional copies of a coding sequence that are not normally present in the cells. The heterologous coding sequences can be RNA or any type thereof, e.g., mRNA, DNA or any type thereof, e.g., cDNA, or a hybrid of RNA/DNA. Coding sequences of interest include, but are not limited to, full-length transcription units that include such features as the coding sequence, introns, promoter regions, S'-UTRs and enhancer regions.

In some embodiments, the host cell includes norcoclaurine (NC) synthase activity. Any convenient NC synthase enzymes find use in the subject host ceils. NC synthase enzymes of interest include, but are not limited to, enzymes such as EC 4.2.1.78, as described in Table 1. In certain embodiments, the host cell includes a heterologous coding sequence for an NC synthase or an active fragment thereof. In some instances, the host cell includes one or more heterologous coding sequences for one or more enzymes or active fragments thereof that convert tyrosi e to L-DOPA. i certain cases, the one or more enzymes is seiected from bacterial tyrosinases, eukaryotic tyrosinases (e.g., EC 1.14.18.1 ) and tyrosine hydroxylases (e.g., EC 1 .14.16.2.) In some instances, the host cell includes one or more heterologous coding sequences for one or more enzymes or active fragments thereof that convert L-DOPA to dopamine (e.g., EC 4.1.1.28).

In certain embodiments, the cell includes one or more heterologous coding sequences for one or more enzymes or active fragments thereof that convert dopamine to 3,4-DHPA. I certain cases, the one or more enzymes is a monoamine oxidase (MAO) (e.g., EC 1.4.3.4). The one or more heterologous coding sequences may be derived from any convenient species {e.g., as described herein). In some cases, the one or more heterologous coding sequences may be derived from a species described in Table 1. in some cases, the one or more heterologous coding sequences are present in a gene or enzyme selected from those described in Table 1.

In some instances, the one or more heterologous coding sequences include a MAO coding sequence integrated at a genomic locus encoding native ARO10. In certain instances, the one o more heterologous coding sequences include a MAO coding sequence operabiy linked to an inducible promoter, in some embodiments, the inducible promoter is part of an inducible system including a DNA binding protein targeted to a promoter regulating the AR010 gene, in some embodiments, the host cell includes one or heterologous coding sequences for one or more enzymes or active fragments thereof described in the genes of Table 1.

As used herein, the term "heterologous coding sequences" aiso includes the coding portion of the peptide or enzyme, i.e., the cDNA or mRNA sequence, of the peptide or enzyme, as well as the coding portion of the full-length transcriptional unit, i.e., the gene including introns and exons, as well as "codon optimized" sequences, truncated sequences or other forms of altered sequences that code for the enzyme or code for its equivalent amino acid sequence, provided that the equivalent amino acid sequence produces a functional protein. Such equivalent amino acid sequences can have a deletion of one or more amino acids, with the deletion being N-termina!, C-terminal or interna!. Truncated forms are envisioned as long as they have the catalytic capability indicated herein. Fusions of two or more enzymes are also envisioned to facilitate the transfer of metabolites in the pathway, provided that catalytic activities are maintained.

Operable fragments, mutants or truncated forms may be identified by modeling and/or screening. This is made possible by deletion of, for example, N-terminal, C-terminal or internal regions of the protein in a step-wise fashion, followed by analysis of the resulting derivative with regard to its activity for the desired reaction compared to the original sequence. If the derivative in question operates in this capacity, it is considered to constitute an equivalent derivative of the enzyme proper.

Aspects of the present invention also relate to heterologous coding sequences that code for amino acid sequences that are equivalent to the native amino acid sequences for the various enzymes. An amino acid sequence that is "equivalent" is defined as an amino acid sequence that is not identical to the specific amino acid sequence, but rather contains at least some amino acid changes {deletions, substitutions, inversions, insertions, etc.) that do not essentially affect the biological activity of the protein as compared to a similar activity of the specific amino acid sequence, when used for a desired purpose. The biological activity refers to, in the example of a decarboxylase, its catalytic activity. Equivalent sequences are also meant to include those which have been engineered and/or evolved to have properties different from the original amino acid sequence. Mutable properties of interest include catalytic activity, substrate specificity, selectivity, stability, solubility, localization, etc. in certain embodiments, an "equivalent" amino acid sequence contains at least 80%-99% identity at the amino acid level to the specific amino acid sequence, in some cases at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and more in certain cases, at least 95%, 98%, 97%, 98% and 99% identity, at the amino acid level. In some cases, the amino acid sequence may be identical but the DMA sequence is altered such as to optimize codon usage for the host organism, for example.

The host cells may also be modified to possess one or more genetic alterations to accommodate the heterologous coding sequences. Alterations of the native host genome include, but are not limited to, modifying the genome to reduce or ablate expression of a specific protein that may interfere with the desired pathway. The presence of such native proteins may rapidly convert one of the intermediates or final products of the pathway into a metabolite or other compound that is not usable in the desired pathway. Thus, if the activity of the native enzyme were reduced or altogether absent, the produced intermediates would be more readily available for incorporation into the desired product.

In some instances, where ablation of expression of a protein may be of interest, is in proteins involved in the pSeiotropic drug response, including, but not limited to, ATP-binding cassette {ABC} transporters, multidrug resistance {MDR} pumps and associated transcription factors, These proteins are involved in the export of BiA molecules into the culture medium, thus deletion controls the export of the compounds into the media, making them more available for incorporation into the desired product. In some embodiments, host cell gene deletions of interest include genes associated with the unfolded protein response and endoplasmic reticulum (ER) proliferation. Such gene deletions may lead to improved BIA production. The expression of cytochrome P450s may induce the unfolded protein response and may cause the ER to proliferate. Deletion of genes associated with these stress responses may control or reduce overall burden on the host cell and improve pathway performance. Genetic alterations may also include modifying the promoters of endogenous genes to increase expression and/or introducing additional copies of endogenous genes. Examples of this include the construction/use of strains which overexpress the endogenous yeast NADPH-P450 reductase CPR1 to increase activity of heterologous P450 enzymes. In addition, endogenous enzymes such as ARQ8, 9, and 10, which are directly involved in the synthesis of intermediate metabolites, may also be overexpressed.

Heterologous coding sequences of interest include but are not limited to sequences that encode enzymes, either wild-type or equivalent sequences, that are normally responsible for the production of BIAS and precursors in plants. In some cases, the enzymes for which the heterologous sequences code can be any of the enzymes in the BIA pathway, and can be from any convenient source. The choice and number of enzymes encoded by the heterologous coding sequences for the particular synthetic pathway may be selected based upon the desired product. In certain embodiments, the host cells of the present invention may include 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 8 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 o more, 12 or more, 13 or more, 14 or more, or even 15 or more heterologous coding sequences, such as 1 , 2, 3, 4, 5, 8, 7, 8, 9, 10, 1 1, 12, 13, 14 or 15 heterologous coding sequences.

in some cases, peptide sequences encoded b the heterologous coding sequences are as reported in GEN8ANK. Enzymes of interest include, but are not limited to, those enzymes described herein and those shown in Table . The host cells may include any combination of the listed enzymes, from any source. Unless otherwise indicated, accession numbers in Table 1 refer to GenBank. Some accession numbers refer to the

Saccharomyces genome database (SGD), which is available on the world-wide web at www.yeastgenome.org.

In some embodiments, the host cell (e.g., a yeast strain) is engineered for selective production of a BSA of interest by localizing one or more enzymes to a compartment in the cell, in some cases, an enzyme may be located in the host cell such that the compound produced by this enzyme spontaneously rearranges, or is converted by another enzyme to a desirable metabolite before reaching a localized enzyme that may convert the compound into an undesirable metabolite. The spatiai distance between two enzymes may be selected to prevent one of the enzymes from acting directly on a compound to make an undesirable metabolite, and restrict production of undesirable end products {e.g., an undesirable opioid by-product). In certain embodiments, any of the enzymes described herein, either singularly or together with a second enzyme, may be localized to any convenient compartment in the host cell, including but not limited to, an organelle, endoplasmic reticulum, golgi, vacuole, nucleus, plasma membrane or the periplasm. In some embodiments, the host cell includes one or more of the enzymes that include a localization tag. Any convenient tags may be utilized. In some cases, the localization tag is a peptidic sequence that is attached at the N- terminal and or G erminaS of the enzyme.

Any convenient methods may be utilized for attaching a tag to the enzyme. In some cases, the localization tag is derived from an endogenous yeast protein. Such tags may provide route to a variety of yeast organelles; the endoplasmic reticulum (ER), mitochondria (MT), plasma membrane (PM), and vacuole (V). In certain embodiments, the tag is an ER routing tag (e.g., ER1), I certain embodiments, the tag is a vacuole tag (e.g., V1). I certain embodiments, the tag is a plasma membrane tag (e.g., P1). In certain instances, the tag includes or is derived from, a transmembrane domain from within the tail-anchored class of proteins. In some embodiments, the localization tag locates the enzyme on the outside of an organelle. In certain embodiments,, the localization tag locates the enzyme on the inside of an organelle.

in some instances, the expression of each type of enzyme is increased through additional gene copies (i.e., multiple copies), which increases intermediate accumulation and/or BIA precursor production. Embodiments of the present invention include increased BSA precursor production in a host cell throug simultaneous expression of multiple species variants of a single or multiple enzymes. In some cases, additional gene copies of a single or multiple enzymes are included in the host cell. Any convenient methods may be utilized including multiple copies of a heterologous coding sequence for an enzyme in the host cell

!n some embodiments, the host cell includes multiple copies of a heterologous coding sequence for an enzyme, such as 2 or more, 3 or more, 4 or more, 5 or more, or even 10 or more copies. In certain embodiments, the host cell include multiple copies of heterologous coding sequences for one or more enzymes, such as multiple copies of two or more, three or more, four or more, etc. In some cases, the multiple copies of the

heterologous coding sequence for an enzyme are derived from two or more different source organisms as compared to the host cell. For example, the host ceil may include multiple copies of one heterologous coding sequence, where each of the copies is derived from a different source organism. As such, each copy may include some variations in explicit sequences based o inter-species differences of the enzyme of interest that is encoded by the heterologous coding sequence.

In some embodiments of the host ceil, the heteroiogous coding sequence is from a source organism selected from the group consisting of P somniferum, T, flavum and C. japonica. In some instances, the source organism is P. somniferum, E, califomica, C.

japonica, T. flavum, Berberis stolonifer, T. flavum subsp. giaucum, Coptis cninensis, Thaiictrum spp, Coptis spp, Papaver spp, Berberis wilsonae, A, mexicana, or Berberis spp. In certain instances, the heteroiogous coding sequence is from a source organism selected from P. somniferum, T. f!avu and C. japonica, In some embodiments, the host cell includes a heteroiogous coding sequence from one or more of the source organisms described in Table 1.

The engineered host cell medium may be sampled and monitored for the production of BIA precursors of interest. The BIA precursors may be observed and measured using any convenient methods. Methods of interest include, but are not limited to, LC- S methods (e.g., as described herein) where a sample of interest is analyzed by comparison with a known amount of a standard compound. Identity may be confirmed, e.g., by m/z and MS/MS fragmentation patterns, and quantitation or measurement of the compound may be achieved via LC trace peaks of know retention time and/or EIC MS peak analysis by reference to corresponding LC-MS analysis of a known amount of a standard of the compound. METHODS

As summarized above, aspects of the invention include methods of preparing a benzyiisoquinoiine alkaloid {BiA) of interest. As such, aspects of the invention include cuituring a host cell under conditions in which the one or more host cell mod tficat ions (e.g., as described herein) are functionally expressed such thai the cell converts starting compounds of interest into product BIAs of interest or precursors thereof {e.g., pre-reticuline B!As). Aiso provided are methods that include cuituring a host ceil under conditions suitable for protein production such that one or more heterologous coding sequences are functionally expressed and convert starting compounds of interest into product BIAs of interest. In some instances, the method is a method of preparing a benzyiisoquinoiine alkaloid (BIA), include cuituring a host celi (e.g., as described herein); adding a starting compound to the ceil culture; and recovering the BIA from the ceil culture. In some embodiments of the method, the starting compound, BIA product and host cell are described by one of the entries of Table 1 ,

Any convenient methods of cuituring host cells may be employed for producing the B!A precursors and downstream BIAs of interest. The particular protocol that is employed may vary, e.g., depending on host cell, the heterologous coding sequences, the desired BIA precursors, etc. The cells may be present in any convenient environment, such as an environment in which the cells are capable of expressing one or more functional

heterologous enzymes. In vitro, as used herein, simply means outside of a living ceil, regardless of the location of the celi. As used herein, the term in vivo indicates inside a living cell, regardless of the location of the celi. In some embodiments, the ceils are cultured under conditions that are conducive to enzyme expression and with appropriate substrates available to allow production of B!A precursors in vivo. In some embodiments, the functional enzymes are extracted from the host for production of BiAs under in vitro conditions. In some instances, the host cells are placed back into a multicellular host organism. The host cells are in any phase of growth, including, but not limited to, stationary phase and iog~ growth phase, etc. In addition, the cultures themselves may be continuous cultures or they may be batch cultures.

Any convenient celi culture conditions for a particular cell type may be utilized. In certain embodiments, the host cells that includes one or more modifications is cultured under standard or readily optimized conditions, with standard cell culture media and supplements. As one example, standard growth media when selective pressure for piasmid maintenance is not required may contain 20 g/L yeast extract, 10 g/L peptone, and 20 g/L dextrose (YPD). Host cells containing plasmids is grown in synthetic complete (SC) media containing 1 J g/L yeast nitroge base, 5 g/L ammonium sulfate, and 20 g/L dextrose supplemented with the appropriate amino acids required for growth and selection. Alternative carbon sources which may be useful for inducible enzyme expression include, but are not limited to, sucrose, raffinose, and galactose. Cells is grown at any convenient temperature (e.g., 30°C) with shaking at any convenient rate (e.g.. 200 rpm) in a vessel, e.g., in test tubes or flasks in volumes ranging from 1-1000 ml, or larger, in the laboratory. Culture volumes can also be scaled up for growth in larger fermentation vessels, for example, as part of an industrial process.

Any convenient codon optimization techniques for optimizing the expression of heterologous polynucleotides in host cells may be adapted for use in the subject host ceils and methods, see e.g., Gustafsson, C. et a!. (2004) Trends Biotec nol, 22, 346-353, which is incorporated by reference in its entirety.

The subject method may also include adding a starting compound to the ceil culture. Any convenient methods of addition may be adapted for use in the subject methods. The cell culture may be supplemented with a sufficient amount of the starting materials of interest (e.g., as described herein), e.g., a mM to pSVI amount such as between about 1-5 m of a starting compound, it is understood that the amount of starting materia! added, the timing and rate of addition, the form of material added, etc., may vary according to a variety of factors. The starting material may be added neat or pre-dissolved in a suitable solvent (e.g., cell culture media, water or an organic solvent). The starting material may be added in concentrated form (e.g., 10x over desired concentration) to minimize dilution of the cell culture medium upon addition. The starting material may be added in one or more batches, or by continuous addition over an extended period of time (e.g., hours or days).

The subject methods may also include recovering the BIA precursor or downstream B!A of interest from the cell culture. Any convenient methods of separation and isolation (e.g., chromatography methods or precipitation methods) may be adapted for use in the subject methods to recover the B!A of interest or precursor thereof from the cell culture. Filtration methods may be used to separate soluble from insoluble fractions of the cell culture. In some cases, liquid chromatography methods (e.g., reverse phase HPLC, size exclusion, normal phase chromatography) are used to separate the BIA or precursor from other soluble components of the cell culture. In some cases, extraction methods (e.g., liquid extraction, pH based purification, etc.) are used to separate the BIA precursor or BIA from other components of the cell culture.

Also included are methods of engineering host cells for the purpose of producing BiAs of interest or precursors thereof, inserting DNA into host cells may be achieved using any convenient methods. The methods are used to insert the heterologous coding sequences into the host cells such that the host cells functionally express the enzymes and convert starting compounds of interest into product BIAs of interest.

Any convenient promoters may be utilized in the subject host cells and methods. The promoters driving expression of the heterologous coding sequences may be

constitutive promoters or inducible promoters, provided that the promoters are active in the host cells. The heterologous coding sequences may be expressed from their native promoters, or non-native promoters may be used. Suc promoters may be low to high strength in the host in which they are used. Promoters may foe regulated or constitutive, in certain embodiments, promoters that are not glucose repressed, or repressed only mildly by the presence of glucose in the culture medium, are used. Promoters of interest include but are not limited to, promoters of glycolytic genes such as the promoter of the B. subtilis tsr gene (encoding the promoter region of the fructose bisphosphate aldolase gene) or the promoter from yeast S. cerevisiae gene coding for glyceraldehyde 3-phosphate

dehydrogenase (GPD, GAPDH, or TDH3), the ADH1 promoter of baker's yeast, the phosphate-starvation induced promoters such as the PH05 promoter of yeast, the aikaline phosphatase promoter from B. licheniformis, yeast inducible promoters such as Gal1-10, Gall , GalL, Ga!S, repressible promoter et25, tetO, and constitutive promoters such as giyceraldehyde 3-phosphate dehydrogenase promoter (GPD), alcohol dehydrogenase promoter (ADH), translation-elongation factor-1 -α promoter (TEF), cytochrome e-oxidase promoter (CYC1 ), RP7 promoter, etc. Autonomously replicating yeast expression vectors containing promoters inducible by hormones such as glucocorticoids, steroids, and thyroid hormones may aiso be used and include, but are not limited to, the glucorticoid responsive element (GRE) and thyroid hormone responsive element (TRE). These and other examples are described U.S. Pat. No. 7,045,290, which is incorporated by reference, including the references cited therein. Additional vectors containing constitutive or inducible promoters such as a factor, alcohol oxidase, and PGH may be used. Additionally any

promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of genes. Any convenient appropriate promoters may be selected for the host cell, e.g. , E. colt. One can aiso use promoter selection to optimize transcript, and hence, enzyme levels to maximize production while minimizing energy resources.

Any convenient vectors may be utilized in the subject host cells and methods.

Vectors of interest inciude vectors for use in yeast and other cells. The types of yeast vectors ca be broke up into 4 general categories: integrative vectors (Yip), autonomously replicating high copy-number vectors (YEp or 2μ plasmids), autonomously replicating low copy-number vectors (YCp or centromeric plasmids) and vectors for cloning large fragments (YACs). Vector DNA is introduced into prokaryotic or eukaryotic ceils via any convenient transformation or transfection techniques. UTIL!TY

The host cells and methods of the invention, e.g., as described above, find use in a variety of appiications. Appiications of interest include, but are not limited to: research applications and therapeutic applications. Methods of the invention find use in a variety of different appiications induding any convenient application where the production of BiAs is of interest.

The subject host ceiis and methods find use in a variety of therapeutic appiications. Therapeutic appiications of interest include those applications in which the preparation of pharmaceutical products that include BiAs is of interest. The host cel!s described herein produce benzyl isoquinoiine a!kaioid precursors (BIA precursors). Reticuiine is a major branch point intermediate of interest in the synthesis of BiAs inciuding engineering efforts to produce end products such as opioid products. The subject host ceils may be utilized to produce BIA precursors from simple and inexpensive starting materials that may find use in the production of reticuiine and BIA end products. As such, the subject host ceiis find use in the supply of therapeutically active BIAs or precursors thereof.

In some instances, the host ceiis and methods find use in the production of commercial scale amounts of BIAs or precursors thereof where chemical synthesis of these compounds is low yielding and not a viabie means for large-scale production. In certain cases, the host ceiis and methods are utilized in a fermentation facility that would include bioreaciors (fermenters) of e.g., 5,000-200,000 liter capacity allowing for rapid production of BIAs of interest or precursors thereof for therapeutic products. Such applications may include the industriai-scale production of BiAs of interest from fermentable carbon sources such as cellulose, starch, and free sugars.

The subject host ceiis and methods find use in a variety of research applications. The subject host cells and methods may be used to analyze the effects of a variety of enzymes on the biosynthetic pathways of a variety of BIAs of interest or precursors thereof. In addition, the host cells may be engineered to produce BSAs or precursors thereof that find use in testing for bioactivity of interest in as yet unprove therapeutic functions. In some cases, the engineering of host cells to include a variety of heterologous coding sequences that encode for a variety of enzymes elucidates the high yielding biosynthetic pathways towards BIAs of interest, or precursors thereof. I certain cases, research applications include the production of precursors for therapeutic molecules of interest that can then be further chemicai!y modified or derivatized to desired products or for screening for increased therapeutic activities of interest. In some instances, host cell strains are used to screen for enzyme activities that are of interest in such pathways, which may lead to enzyme discovery via conversion of BIA metabolites produced in these strains.

The subject host cells and methods may be used as a production platform for plant spectalized metabolites. The subject host eel is and methods may be used as a platform for drug library development as well as plant enzyme discovery. For example, the subject host cells and methods may find use in the development of natural product based drug iibraries by taking yeast strains producing interesting scaffold molecules, suc as protopine, and further funetionaSizing the compound structure through combinatorial biosynthesis or by chemical means. By producing drug iibraries in this way, any potentiai drug hits are already associated with a production host that is amenable to large-scale culture and production. As another example, these subject host ceiis and methods may find use in plant enzyme discovery. The subject host celis provide a clean background of defined metabolites to express p!ant EST iibraries to identify new enzyme activities. The subject host cells and methods provide expression methods and culture conditions for the functional expression and increased activity of plant enzymes in yeast.

KITS AND SYSTEMS

Aspects of the invention further include kits and systems, where the kits and systems may include one or more components employed i methods of the invention, e.g., host cells, starting compounds, heterologous coding sequences, vectors, culture medium, etc., as described herein. In some embodiments, the subject kit includes a host ceii (e.g., as described herein), and one or more components selected from the foi!owing; starting compounds, a heterologous coding sequence and/or a vector including the same, vectors, growth feedstock, components suitable for use in expression systems (e.g., cells, cloning vectors, multiple cloning sites (MCS), bi-directional promoters, an interna! ribosome entry site (IRES), etc.) and a culture medium.

An of the components described herein ma be provided in the kits, e.g., host cells including one or more modifications, starting compounds, culture medium, etc, A variety of components suitable for use in making and using heterologous coding sequences, cloning vectors and expression systems may find use in the subject kits. Kits may also include tubes, buffers, etc., and instructions for use. The various reagent components of the kits may be present in separate containers, or some or ail of them may be pre-combined into a reagent mixture in a single container, as desired.

Also provided are systems for producing a BiA of interest, where the systems may include engineered host ceiis including one or more modifications (e.g., as described herein), starting compounds, culture medium, a fermenter and fermentation equipment, e.g., an apparatus suitable for maintaining growth conditions for the host cells, sampling and monitoring equipment and components, and the like. A variety of components suitable for use in large scale fermentation of yeast ceils may find use in the subject systems.

In some cases, the system includes components for the large scale fermentation of engineered host cells, and the monitoring and purification of BIA compounds produced by the fermented host ceils, in certain embodiments, one or starting compounds (e.g., as described herein) are added to the system, under conditions by which the engineered host cells in the fermenter produce one or more desired 8IA products or precursors thereof, in some instances, the host cells produce a BIA precursor of interest (e.g., as described herein). In certain cases, the B!A products of interest are opioid products, such as codeine, neopine, morphine, neomorphine, hydrocodone, oxycodone, hydromorphone,

dihydrocodeine, 14-hydroxycodeine, or dihydroniorphine.

in some cases, the system includes means for monitoring and or analyzing one or more BIA compounds or precursors thereof produced by the subject host cells. For example, a LC-SV1S anaiysis system as described herein, a chromatography system, or any convenient system where the sample may be analyzed and compared to a standard, e.g., as described herein. The fermentation medium may be monitored at any convenient times before and during fermentation by sampling and analysis. When the conversion of starting compounds to BIA products or precursors of interest is complete, the fermentation may be haited and purification of the Bi A products may be done. As such, in some cases, the subject system includes a purification component suitable for purifying the B!A products or precursors of interest from the host ce!l medium into which it is produced. The purification component may include any convenient means that may be used to purify the BIA products or precursors of fermentation, including but not limited to, silica chromatography, reverse- phase chromatography, ion exchange chromatography, HIC chromatography, size exciusion chromatography, liquid extraction and pH extraction methods. In some cases, the subject system provides for the production and isolation of B!A fermentation products of interest fo!iowing the input of one or more starting compounds to the system.

The following examples are put forth so as to provide those of ordinary skiii in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have bee made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.), but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

EXPERIMENTAL

Exam pie I

A series of specific genetic modifications provide a biosynthetic process in Sacc aromyces cerevisiae for the production of Bl As from simple, inexpensive feedstocks or precursor molecules. Methods for constructing novel strains capable of producing the early BIA molecules norcocSaurine (NC) and norlaudanoso!ine (NL) from non-BSA

precursors or simple feedstocks are described. NC has never been reported as a product of microbial synthesis and is the natural precursor to all known BIA molecules. Methods for manipulating the regulation of yeast biosynthetic pathways and for optimizing the production of aromatic amino acids and related BIA precursors are also described.

A-.Tyroji ^

Strains of S. cerevisiae are developed with improved flu through the aromatic amino acid biosynthesis pathway for the purposes of increasing intracellular concentrations of BIA precursor molecules including tyrosine, 4-hydroxyphenylaceta!dehyde (4-HPA), L- 3,4-dihydraxyphenylaSanine (L-DOPA), 3,4-dihydroxyphenyiacetaidehyde (3,4-DHPA), and dopamine. These strains combine genetic modifications to the yeast strain for the purpose of increasing carbon flux from central metabolism towards aromatic amino acid synthesis in general, towards tyrosine in particular, and include the introduction of key heterologous enzymes for the production of BIA precursor molecules not naturally produced by yeast. Genetic modifications are employed including the introduction of feedback inhibition alleviating mutations to genes encoding native biosynthetic enzymes, tuning of

transcriptional regulation of native biosynthetic enzymes, deletion of genes encoding enzymes that divert precursor molecules away from the intended pathway, and introduction of heterologous enzymes for the conversion of naturally endogenous molecules into non- native B!A precursor molecules.

Specific description:

1.1 ) The biosynthetic pathway in the engineered strain incorporates feedback inhibition alleviating mutations ( .1. } to the native yeast gene AR04, which encodes a 3- deoxy-d-arabinose-heptulosonate-7-phosphate (DAHP) synthase, alone or in combination. This mutation (AR04 ^8R ) is incorporated as a directed mutation to the native gene at the original locus, as an additional copy introduced as a genetic integration at a separate locus, or as an additional copy on an episoma! vector such as a 2μ or centromeric plasmid. FBR refers to feedback resistant mutants and mutations. The feedback inhibited copy of the DAHP synthase enzyme is under the native yeast transcriptional regulation or is introduced with engineered constitutive or dynamic regulation of protein expression by placing it under the control of a synthetic promoter.

1.1.1 ) AR04 ^FSfi mutations may include, for example, a substitution of the lysine residue at position 229 with a leucine (see e.g., Hartmann , et al. (2003) Evolution of feedback-inhibited beta /alpha barrel isoenzymes by gene duplication and a single mutation. Proc Natl Acad Sci U S A 100{3):862-867), a substitution of the giutamine residue at position 188 with a lysine residue (see e.g., Fukuda K et al. (1992) Feedback-Insensitive Mutation of 3-Deoxy-D-Arabino-Hepturosonate-7-Phosphafe Synthase Caused by a Single Nucleotide Substitution of Aro4 Structural Gene in Saccharomyces-Cerevisiae. J Ferment Bioeng 74(2): 117-119), or an additionai mutation conferring feedback inhibition selected from a muiagenized library of microbial DHAP synthase mutants. Examples of such selections include rescue of growth on o-fluoro-D,L-phenylaianine {see e.g., Fukuda et al. (1990) Breeding of Brewing Yeast Producing a Large Amount of Bata-Prtenylethyl Alcohol and Beta-Phenylethyl Acetate. Agr Bioi Chem Tokyo 54(1):289-271} or growth of aro3 mutant yeast strains on media with excess tyrosine.

1.2) The foiosynthetic pathway in the engineered strain incorporates a feedback inhibition alleviating mutation ( .2.1 ) to the native yeast gene ARG7, which encodes the enzyme chorismate mutase. This mutation (AR07 ^FSR ) is incorporated as a directed mutation to the native gene at the original iocus, as an additional copy introduced as a genetic integration at a separate Iocus, or as an additional copy on an episomai vector such as a 2μ or centromeric plasmid. The feedback inhibited copy of the chorismate mutase enzyme is under the native yeast transcriptional regulatio or is introduced with engineered

constitutive or dynamic regulation of protein expression by placing it under the control of a synthetic promoter.

1.2. ) AR07 ^FSR mutant alleles may include, for example, a substitution of the threonine residue at position 228 with an isoleucine (see e.g., Schmidheini et al. (1989) A Single Point Mutation Results in a Constitutive!y Activated and Feedback-Resistant Chorismate Mutase of Saccharomyces-Cerevisiae. J Bacteriol 171 (3): 1245-1253) or an additional mutation conferring feedback inhibition selected from a muiagenized library of microbial chorismate mutase mutants. Examples of such selections include assays for 5- methyltryptophan sensitivity or increased production of melanin pigments in strains expressing heterologous tyrosinase enzymes (1.9) in the absence of externally fed tyrosine.

1.3) The biosynthetic pathway in the engineered strain incorporates the introduction of a strong promoter element {such as GPD1 , TEF1 , etc) for the

overexpression of the native yeast gene ARO10, which encodes an enzyme with hydroxyphenyipyruvate decarboxylase activity. This genetic modification is incorporated as a directed swapping of the native promoter DNA sequence at the original Iocus, as an additional copy of the gene under new transcriptional regulatio introduced as a genetic integration at a separate iocus, or as an additionai copy on an episomai vector such as a 2μ or centromeric piasmid.

1.4) The biosynthetic pathway in the engineered strain incorporates the introduction of a strong promoter element {such as GPDi , TEF1 , etc) for the overexpression of the native yeasf gene AR09, which encodes an enzyme with

hydroxypheny!pyruvate/giutarnie acid transaminase activity. This genetic modification is incorporated as a directed swapping of the native promoter DMA sequence at the originai locus, as an additional copy of the gene under new transcriptional regulation introduced as a genetic integration at a separate locus, or as an additional copy on an episoma! vector such as a 2μ or centromeric plasmid.

1.5) The biosynthetic pathway in the engineered strain incorporates the introduction of a strong promoter element (suc as GPD1 , TEF1 , etc) for the

overexpression of the native yeast gene T L _; which encodes an enzyme with transketo!ase activity. This genetic modification is incorporated as a directed swapping of the native promoter DNA sequence at the original focus, as an additional copy of the gene under new transcriptionai regulation introduced as a genetic integration at a separate iocus, or as an additional copy on an episoma! vector such as a 2μ or centromeric piasmid.

1.6} The biosynthetic pathway in the engineered strain is improved by the incorporation of a deletion or inactivating mutation of the native yeasi gene ZWF1 , which encodes an enzyme with glucose-6-phosphate dehydrogenase activity,

1.7 The biosynthetic pathway in the engineered strain is improved by the incorporation of one or more deietion(s) or inactivating mutation(s) of known native alcohol dehydrogenase enzymes, such as the enzymes encoded by the genes ADH2, ADH3, ADH4, ADH5, ADH8, ADH7, or SFA1.

1.8} The biosynthetic pathway in the engineered strain is improved by the incorporation of one or more deietion(s) or inactivating mutation (s) of known native aldehyde oxidoreductases, suc as ALD2, ALD3, ALD4, ALD5, or ALD6.

1.9) The biosynthetic pathway incorporates a heterologous enzyme for the conversion of tyrosine to L-DOPA. This enzyme may be from one of several classes, including, but not iimited to bacteria! tyrosinases, eukaryotic tyrosinases, and tyrosine hydroxylases (Table 1). The gene for this enzyme is incorporated as a genetic integration or o a episomal vector such as a 2μ or centromeric plasmid. This L-DOPA producing enzyme is introduced with engineered constitutive or dynamic regulation of protein expression by placing it under the control of a synthetic promoter.

1.9.1 } In a biosynthetic pathway using a tyrosine hydroxylase enzyme for the conversion of tyrosine to L-DOPA, additional expression of genes encoding enzymes for the synthesis and recycling of the pterin cofactor tetrahydrobiopterin (8H4) and its derivatives are incorporated into the engineered strai in support of the activity of the tyrosine hydroxylase enzyme. These enzymes include GTP eyclohydroSase, 6-pyruvoyl- tetrahydropterin synthase, sepiapteri reductase, 4a-hydroxyietrahydrobiopteri

dehydratase, and quinoid dihydropteridine reductase (Table i ). The genes for these enzymes are incorporated as a genetic integration or on an episomai vector such as a 2μ o centromeric plasmid. These BH4 synthesis and recycling enzymes are introduced with engineered constitutive or dynamic regulation of protein expression by piacing it under the control of a synthetic promoter.

1.10) The biosynthetic pathway incorporates a heterologous enzyme for the decarboxylation of L-DOPA to produce dopamine. Enzymes with this activity are encoded by a genes from a variety of organisms including bacteria, plants, and mammals. Examples include Pseudomonas p tida DOPA decarboxylase (PpDODC), Rattus norvegic s OOP A decarboxylase (RnDODC), and Papaver so nif&rum tyrosine/DOPA decarboxylase (PsTYDC) (Table 1 ). The gene for this enzyme is incorporated as a genetic integration or on an episomai vector such as a 2μ or centromeric plasmid. This dopamine producing enzyme is introduced with engineered constitutive or dynamic regulation of protein expression by piacing it under the control of a synthetic promoter.

1.1 1 ) A biosynthetic pathway for the production of 3,4-DHPA incorporates a heterologous enzyme for the oxidation of dopamine to 3,4-DHPA. Examples of genes encoding this enzyme that may be used in the strain include human monoamine oxidase A (hMAOA), £. co// ^' monoamine oxidase (Ec AO), and Micrococcus luteus monoamine oxidase ( I AO) (Tabie 1 ). The gene for this enzyme is incorporated as a genetic integration or on an episomai vector such as a 2μ or centromeric plasmid. This 3,4-DHPA producing enzyme is introduced with engineered constitutive or dynamic regulation of protein expression by placing it under the control of a synthetic promoter.

1.1 1.1 ) Strains for the production of NC require dopamine and 4-HPA, while strains for the production of NL require dopamine and 3,4-DHPA, but not 4-HPA. A specific modification for the conversion of an NC producing strain into an NL producing strai is the i tegration of a MAO gene into the yeast genome at the locus encodi g the native yeast gene ARO10. This combines a deletion of the native yeast enzyme responsible for converting a tyrosine biosynthetic precursor to 4-HPA with the introduction of the enzyme capable of converting dopamine to 3,4-DHPA.

1.11.2) Yeast strains are constructed with a gene that expresses a MAO enzyme (1.1 1 ) under the control of an inducible promoter. When the strain is grown in the presence of the inducer it can catalyze the conversion of dopamine to 3,4-DHPA, in the absence of inducer the strain only produces 4-HPA.

1 ,11 ,2.1 ) Yeast strains are constructed with inducible MAO expression

(1 ,11 ,2), where the inducible system also contains a DNA binding protein targeted to the promoter regulating the ARO10 gene (1.3). The synthetic promoter controlling ARO10 is therefore repressed when the promoter controlling the MAO gene is activated and ARO10 is only expressed whe the MAO gene is not transcriptionally active. This system allows for the construction of a single strain that conditionally only produces the precursors for NC or NL.

B. NC-pfoducing yeast strains

Methods are developed to produce the 8IA molecule NC in yeast and demonstrate a first system for microbial synthesis of NC. With the engineered strains described herein, NC is produced and accumulated for its own value or combined with a biosynthetic pathway of additional heterologous enzymes for the complete synthesis of downstream BiAs.

Specific description:

2.1 Yeast strains are grown in liquid culture to a high cell concentration before back diluting to intermediate concentrations (as measured by optical density or OD) in defined media containing high concentrations of dopamine. The media components only need to satisfy conditions for growth of the strains; various growth feedstocks are used (for example, different sugars, nitrogen sources). The NC produced by these yeast strains is excreted by the yeast cells and is measurable in the spent media. Additional NC retained by cells is recovered via cell lysis and extraction from the lysate.

2.2 Yeast strains containing various combinations of the modifications as described in (1.1-1.8) substantially improve NC production from that measurable in unmodified strains in fed dopamine assays as described above (2.1 ). In conditions where no extracellular tyrosine is available in the yeast media, modifications described (1.1-18) provide for production of NC from fed dopamine; under these conditions the NC production from unmodified yeast strains is most often undetectable,

2.3) Yeast strains that produce NC when containing the modification as described in (1.10) and grown as described in (2.1 ) when the additional BIA precursor added to media is L-DOPA instead of dopamine,

2.3.1 ) Yeast strains as described in (2.3) containing various combinations of the modifications as described in (1.1-1.8) substantially improve production of NC.

2.4) Yeast strains that produce NC when containing both the heterologous enzymes for conversion of tyrosine to dopamine ( 1.9, 1.10) alongside various combinations of modifications described above (1.1 -1.8) and grown in media without supplementation of tyrosine, L-DOPA, or dopamine. This specific example constitutes complete synthesis of NC by the strain from simple carbon and nitrogen sources.

2.5) Yeast strains are modified and cultured as described above (2.1 -2.4) where the biosynthetic pathway includes the incorporation of the heterologous enzyme NCS, or truncated versions of the NCS enzyme, for the stereospecific catalysis of the reaction condensing dopamine and 4-HPA for S-NC production. This enzyme may originate from one of several plants , such as Papaver somntferum, Coptis japomca, and Thaiicitum fiavu (Table 1), The gene for this enzyme is incorporated as a genetic integration or o an episomaS vector such as a 2μ or centromeric plasmid. This S~NC producing enzyme is introduced with engineered constitutive or dynamic regulation of protein expression by placing it under the controi of a synthetic promoter. The NC ultimately produced wili be an enantiomeric mixture with bias towards the S-stereoisomer.

Methods are developed to produce the BIA molecule NL from yeast. With the engineered strains described herein, NL is produced and accumulated for its own value or combined with a biosyniheiic pathway of further heterologous enzymes for the complete synthesis of downstream BIAs,

Specific description:

3.1 } Yeast strains containing modifications as described in (1.11, 1 ,11 ,1-1,1 .2, 1.1 1.2.1} are grown in liquid culture as described in (2.1 ) produce L.

3.2) Yeast strains containing various combinations of gene deletions as described in (1.7,1.8) improve NL production from that measurable in unmodified strains in fed dopamine assays as descnbed above (3,1).

3.3) Yeast strains that produce NL when containing the modifications as described in (1.10, 1.11 ,1.11.1-1.11.2, 1.11.2.1} and grown as described in (3.1) when the additional BfA precursor is added to media is L-DOPA instead of dopamine.

3.3.1 } Yeast strains as described in (3,3) containing combinations of gene deletions described in (1 ,7,1.8) improve production of NL,

3.4} Yeast strains that produce NL when containing both the heterologous enzymes for conversion of tyrosine to dopamine (1.9, 1.10) and dopamine fo 3,4-HPA (1.11) alongside various combinations of modifications described above (1.1- .8, 1.11.1 ) are grown in media without supplementation of tyrosine, L-DOPA, or dopamine. This specific example constitutes complete synthesis of NL by the strain from simpie carbon and nitrogen sources.

3.5) Yeast strains modified and cultured as described above (3,1-3.4) where the biosynthetic pathway includes the incorporation of the heterologous enzyme NCS, or truncated versions of the NCS enzyme (Table 1), for the stereospecific catalysis of the reaction condensing dopamine and 3,4-HPA for S-NL production. This enzyme may originate from one of several plants, such as Papaver somniferum, Coptis japonica, and Thaiicitum fiavum (labia 1), The gene for this enzyme is incorporated as a genetic integration or on an episomai vector such as a 2μ or centromeric plasmid. This S-NL producing enzyme is introduced with engineered constitutive or dynamic regulation of protein expressio by placing it under the control of a synthetic promoter. The NC ultimately produced is an enantiomeric mixture with bias towards the S-stereoisomer.

Figure 1 : Biosynthesis of tyrosine and BIA precursor moiecuies

Schematic showing the biosynthetic pathway from glucose to tyrosine and other BIA precursors. Aromatic amino acid intermediates present in native yeast metabolism are written in black. Endogenous yeast enzymes are written in grey {apart from TYR _; TyrH, DODC and MAO). Heterologous enzymes and non-natural BIA precursor moiecuies include TYR, TyrH, DODC and MAO. As described in (1 .1 , 12) wild-type yeast enzymes encoded by AR04 and AR07 are allostericaS!y inhibited by tyrosine, indicated here by the dotted grey line, individual steps in the pentose phosphosphate pathway and giyco!ysis are not explicitly detaiied in this figure, although the genes TKL1 and ZWF1 (targeted in 1 .5, 1 .6) are involved in the pentose phosphate pathway, as indicated.

Figure 2; Effect of ZWF1 knockout and TKL1 over-expression on pentose phosphate pathway (PPP)

Schematic detailing how modifications to TKL {1 .5} and ZWF1 (1.6) affect the overaii carbon flow throug the pentose phosphate pathway in yeast when glucose is the primary carbon source. Panel A represents wild-type carbon flow; Panel B represents the relative change in carbon flow in a modified strain.

Figure 3Ai Synthesis of NC f om precursor moiecuies

NC is synthesized from one molecule of dopamine and one molecule of 4-HPA via a Pictet-Spengler condensation reaction . This reaction can occur spontaneously to produce a racemic mixture of R- and S- NC. This reaction can alternatively be catalyzed by the plant enzyme NCS, which produces S-NC.

Figure 3B: Synthesis of NL from precursor molecules

NL is synthesized from one molecule of dopamine and one molecule of 3,4-DHPA via a Pictet-Spengler condensation reaction. This reaction ca occur spontaneously to produce a racemic mixture of R- and S~ NL. While the natural product of NCS is NC, the enzyme has been shown to catalyze the stereospecific production of S-NL (see e.g., Rueffer et al. (1981 } (S)-Norlaudanosoiine Synthase - the 1 st E zyme in the

Benzy!isoquinoline Biosynthetic-Pathway. Febs Lett 129(1 ):5-9).

Measurement of the BIA molecules is performed by LC-MS analysis, where NC production ( m/z ^™ +272, 19.2 min retention time) and NL production {m/z-+288, 18.9 min retention time) were observed, with ion MS2 fragmentation agreeing with both standards and published detection methods (see e.g., Schmidt et al. (2007) Poppy alkaloid profiling by eiectrospray tandem mass spectrometry and electrospray FT-iCR mass spectrometry after [ring-13C6Hyramine feeding. Photochemistry 68(2): 189-202). NC production was demonstrated at several concentrations of fed tyrosine, including no fed tyrosine, in strains with targeted genetic modifications. Wild-type strain, CEN.P 2, was integrated with constructs conferring one of four genetic changes (as described in 1.1- 1.4): overexpression of ARO10 by promoter replacement with PT _E F _I , overexpression of A 09 by promoter replacement with PTEP _I , chromosomal integration of an AR04 ^F8R allele, and chromosomal integration of an AR07 ^FBR allele. When incorporated a!one _; only the P _TE p ARO10 and AR04 ^f:SR increase production of NC. Whi!e both these modifications increased NC production at all tyrosine concentrations, the ARO*" integrated strain improved most drastically at zero fed tyrosine.

Figure 5; JSIC prodyctjon w

Some genetic modifications as described above (1,5, 1 ,6) improve NC production only in combination wit the integration of the AR04 ^F8R mutant (1 .1 ), This figure shows four strains engineered wit single genetic modifications, P _TSF1 -ARO10 (1 .3), P _GP D-TKL1 (1.5), ZWF1 knockout (1.6), and AR04 ^F8R (1 ,1 ), alongside three strains constructed with combinations of genetic modifications: Strain A (P _GF > _D -TKL1 , AR04 ^F8R ), Strain B (ZWF1 knockout, AR04 ^FSR ), and Strain C (PG _P D- KL , ZWF1 knockout, AR04 ^FSR ). NC productio is shown normalized to the WT strain, with Strain C exhibiting a five-fold increase in NC production.

Figure 6: N L production in ALD/APH knockout strains

NL production is improved by the deletion of competing yeast enzymes (1.7,1,8 in a strain expressing human MAOA on a 2μ plasmid (1.1 1 ) and grown in media containing dopamine. NL production is shown as titer measured in spent media normalized to the WT (with hlvAO, but with no deietions). Improvements in production is as much as ten times WT

Yeast strains transformed with DNA to express Papaver somniferum tyrosine/DOPA decarboxylase can convert L-DOPA to dopamine in vivo. Strains harboring a 2μ plasmid were grown in selective media and then back-diluted into media containing L-DOPA. Spent media was then measured for concentrations of L-DOPA (retention time 4.8 min, m/z=+198) and dopamine (retention time 4.2 min, m/z=+154).

Figure 8

NC is produced as described in (2.1 ) in multiple wild-type yeast lab strains at varying tyrosine concentrations. Specifically, each yeast strain is inoculated into separate liquid cultures and grown overnight to OD _w - 0, then back-diiuted in YN8 minimal media without tyrosine to an OD ₅₀₀ ^■ - 1 and grown for 3 hours. 100 pi of each culture was mixed into 400 μΙ YNB media containing 100 mM dopamine and varying concentrations of tyrosine; each strain was grown in each media condition in triplicate samples. NC titer was measured from culture supernatant on an LC-SV1S instrument detecting m/z +272 ion count in peaks as described by (see e.g., Schmidt ei a!. (2007) Poppy alkaloid profiling by eiectrospray tandem mass spectrometry and electrospray FT-SCR mass spectrometry after [ring-13C63-tyramine feeding. Phytochemistry 68{2): 189-202). The area of each peak was integrated to calculate a relative quantity of the NC in each sampie and the results were normalized to the ion count area in CEN.PK2 yeast culture with 0 mg/L tyrosine.

Figure 9

NC is produced as described in (2,1 in multiple engineered yeast strains at fed 100 mM dopamine and no tyrosine. These data were generated in a separate experiment from those in Figure 5. The strains CSY977-981 were engineered to contain combinations of the genetic modifications described in (1.1-1.6); the labels underneath each strain name indicate which modifications were incorporated into eac strain. Strain CSY981 contains five genetic modifications to yeasi native metabolism and exhibits a twelve-fold increase in NC titer above the wild-type yeast strain CE .PK2.

Figure 10

NC production as described in (2.1 ) for black diamonds and as described in (2.3) for gray circles. Here NC was produced in an engineered yeasi strain (CSY980) wit the additional integration of the L-DOPA decarboxylase PpDODC (1.10). In separate liquid cultures this yeasi strain was grown in Y B minimal media containing varying

concentrations of dopamine (black diamonds) and YNB minimal media containing L-DOPA (gray circles; cultures not fed dopamine). The solid black line represents a linear regression of the relationship between the measured NC and fed dopamine. The peak area

measurements for the L-DOPA fed samples were plotted along the regression line for dopamine fed samples to show an "equivalent fed dopamine" quantity for the cultures fed L- DOPA, L-DOPA media was mixed to achieve a target concentratio of 10 mM L-DOPA, however L-DOPA was not fully soiuble at that concentration, and the effective concentration of dissolved L-DOPA is estimated to be approximately 6 mM. Based on the average NC titers of the two L-DOPA fed yeasi cultures the "equivalent fed dopamine" concentration is approximately 50 mM or 8x the fed L-DOPA concentration (indicated by the gray dotted lines).

Figure 11

Mammalian tyrosine hydroxylases (TyrHs) are capable of hydroxy!ating tyrosine, but are dependent on the co-substrate tetrahydrobiopterin (8H4) for activity, as described in (1 ,9,1). During the catalysis of tyrosine to L-DOPA b TyrH, molecular oxygen is split and transferred to tyrosine and BH4, as shown by reaction 1. BH4 is oxidized to BH4~4o carbinolamine (4aOH-BH4). Two heterologous enzymes are expressed i yeasi to synthesize BH4 from the folate synthesis pathway intermediate, dihydroneopteri triphosphate. First, 6~pyruvoylfetrahydropferin synthase (PTPS) converts dihydroneopterin to PTP (reaction 2), which is then reduced to BH4 by sepiapterin reductase (SepR, reaction 3). Two enzymes are responsible for the regeneration of BH4 from its 4a-carbinoiamine form. First, ptenn-4a-carbino!a ine dehydratase (PCD) catalyzes a loss of water reaction to form dihydrobioterin (reaction 4). Dihydrobiopterin is then reduced to tetrahydrobiopterin by quinoid dihydropteridine reductase (QDHP , reaction 5).

Figure 12

Tyrosine hydroxylases expressed from yeast cells convert tyrosine to L-DOPA. Yeast strains transformed with p!asmids carrying tyrosine hydroxyiases from human (hTH2) and rat RnTyrH) were grown in liquid media and then lysed in buffer containing tyrosine and the co-substrate BH4. After 6-hour incubations at 30°C, L-DOPA was measured in the lysate mixture by LC-MS. (A) LC-MS chromatogram confirms conversion of tyrosine to L- DOPA dependent on the presence of the co-substrate, BH4. (B) Fragmentation of the +198 m/z ion peak further confirms the presence of L-DOPA in lysate samples. (See e.g. , Lv et al. (2010) LC-MS- S Simultaneous Determination of L-Dopa and its prodrug n-Penty!

Hydrochloride in Rat Plasma. C romatographia, 72(3/4), 239-243).

Co-expression of tyrosine hydroxylase with a BH4 biosynthetic enzyme enables conversion of tyrosine to L-DOPA in yeast eel! iysates. Engineered yeast strains integrated with constructs expressing rat tyrosine hydroxylase (RnTyrH) and rat sepiapterin reductase (RnSepR) were grown in liquid media and then lysed in buffer containing tyrosine, NADPH, and the BH4 biosynthetic precursor, sepiapterin. Co-expression of a TyrH with the BH4 biosynthesis gene provides for activity of the tyrosine hydroxylase in the absence of BH4, but in the presence of the BH4 precursor, sepiapterin.

Synthesis of the BIA molecules coclaurine and N-methylcoclaurine from MC

Conversion of NC to downstream BI A molecules by plant methyitransferase enzymes extends microbial BIA synthesis. These particular BIA molecules can only be synthesized via a NC-dependent biosynthesis scheme.

Figure 15

Engineered yeast strains produce NC-derived BIA molecules from L-DOPA in liquid culture. A copy of PpDODC was integrated into the engineered yeast strain, CSY979 (as described in Figure 9), providing for the production of NC from L-DOPA {panel A, bottom chromatogram). Next a copy of the opium poppy 8-O-methyltransferase (Ps60MT) gene was integrated into this yeast strain to enable the production of coclaurine from L-DOPA (panel A, middle chromatogram). Finally, a copy of both Ps60MT and the opium poppy coclaurine-N-methyltransferase (PsCN T) genes were integrated into the CSY979 yeast strain carrying the PpDODC gene to enable the production of N-methy!cociaurine from L~ DOPA (panel A, top chromatogram). Both the NC and coc!auhne measurements matched chromatograms from chemical standards. The production of N-methyicociaurine was further confirmed by matching the fragmentation pattern of the +300 m/z ion peak to patterns in published literature (Panel B). (see e.g., Schmidt et a!. (2007) Popp aikaioid profiiing by eJectrospray tandem mass spectrometry and electrospray FT-SCR mass spectrometry after [ring-13C63-tyramine feeding. Phytochernsstry 88(2): 189-202).

Atty. Docket: STAN-1078WO (S 13-438}

Table 1 : Genes of interest as components of the engineered metabolic pathways

Atty. Docket: STAN-1078WO (S 13-438}

Atty. Docket: STAN-1078WO (S 13-438}

Notwithstanding the appended clauses, the disclosure set forth herein is also defined b the following clauses:

1. A host ceil that produces a benzylisoquinoline alkaloid (B!A) precursor, wherein the host cell comprises one or more modifications selected from the grou 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 ceil; and

one o 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 th cell.

2. Th host cell according to Clause 1 , wherein the BIA precursor is selected from the group consisting of norcoclaurine (NC) and norlaudanosoline (NL).

3, The host cell according to Clause 2, wherein the BiA precursor is norcoclaurine (NC).

4, The host cell according to Clause 2, wherein the B!A precursor is

norlaudanosoline (NL),

5. The host cell according to Clause 1 , wherein when the cell comprises one or more feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes native to the cell, it comprises a least one additional modification selected from the group consisting of: a transcriptional modulation modification of a biosynthetic en2yme gene nativ to the cell; an inactivating mutation in an enzyme native to the cell; and a heterologous coding sequence that encode an enzyme.

6, The host cell according to Clause 1 , wherein when the ceil comprises one or more transcriptional modulation modifications of one or more biosynthetic enzyme genes native to the cell, 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 ceii; an inactivating mutation in an enzyme native to the cell; and a heterologous coding sequence thai encodes an enzyme.

7. The host cell according to Clause 1 , wherein when the ceil comprises one or more inactivating mutations in one or more enzymes native to the ceil it comprises at least one additional modification selected from the grou consisting of: a feedback inhibition alleviating mutation in a biosynthetic enzyme gene native to the cell; a transcriptional modulation modiftcation of a biosynthetic enzyme gene native to the ceii; and a heterologous coding sequence that encodes an enzyme.

8. The host cell according to Clause 1 , wherein the ceil comprises one or more feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes native to the cell.

9. The host ceii according to Clause 8, wherein the one or more biosynthetic enzyme genes encode one or more enzymes selected from a 3-deoxy-d-arabinose- heptulosonate-7-phosphate (DAHP) synthase and a chonsmate mutase,

10. The host cell according to Clause 9, wherein the one or more feedback inhibition alleviating mutations are present in a biosynthetic enzyme gene selected from AR04 and AR07.

11. The host cell according to Clause 10, wherein the one or more feedback inhibition alleviating mutations are present in the AR04 gene.

12. The host cell according to Clause 9, wherein the mutations are selected from mutation of position 229 and mutation of position 166 of the DAHP.

13. The host cell according to any one of the preceding clauses, wherein the cell overproduces one or more B!A precursor molecules.

14. Th host ceii according to Clause 13, wherein the one or mor Bl A precursor molecules are selected from the group consisting of tyrosine, 4- hydroxyphenyfacetaidehyde (4-HPA), L-S^-dihydroxyphenylafanine {L-DOPA), 3,4- dihydroxyphenyiaeetaldehyd (3 _r 4-DHPA) and dopamine.

15. The host cell according to Clause 14, wherein the one or more BIA precursor molecules are 3,4-dihydroxyphenylacetaldehyde (3,4-DHPA) and dopamine.

16. The host cell according to Clause 14, wherein the one or more BIA precursor molecules are 4-hydroxyphenylacetaldehyde (4-HPA) and dopamine.

17. The host ceii according to Clause 1 , wherein the ceil comprises one or more transcriptional modulation modifications of one or more biosynthetic enzyme genes native to the cell. 18. The host cell according to Clause 17, wherein the transcriptional modulation modification is substitution of a strong promoter for a native promoter of the one or more biosynthetic enzyme genes.

19. The host ceil according to Clause 18, wherein the one or more biosynthetic enzyme genes is selected from the group consisting of ARO10, AR09 and T L

20. The host cell according to Clause , wherein the ceil comprises one or more inactivating mutations in one or more enzymes native to the ceii.

21. The host cell according to Clause 20, wherein the enzyme is a giucose-6- phosphate dehydrogenase.

22. The host cell according to Clause 21 , wherein the enzyme is ZWF1.

23. The host cell according to Clause 20, wherein the ceil comprises one or more inactivating mutations in one or more enzymes native to the cell comprising alcohol dehydrogenase activity.

24. The host cell according to Clause 23, wherein the one or more enzymes is selected from the group consisting of ADH2, ADH3, ADH4, ADH5, ADH6, ADH7 and SFA1.

25. The host cell according to Clause 20, wherein the cell comprises one or more inactivating mutations in one or more enzymes native to the eel! comprising aldehyde oxidoreductase activity.

26. The host cell according to Clause 25, wherein the one or more enzymes is selected from the group consisting of ALD2 AIDS, ALD4, ALD5, and ALD6.

27. The host cell according to Clause 1 , wherein the ceil comprises one or more heterologous coding sequences that encode one or more enzymes or active fragments thereof.

28. The host cell according to Clause 27, wherein the host eel! comprises an NC synthase activity.

29. The host cell according to Clause 27, wherein the host ceii comprises a heterologous coding sequence for an NC synthase or active fragment thereof.

30. The host ceil according to Clause 29, wherein the one or more enzymes or active fragments thereof convert tyrosine to L-DOPA.

31. The host cell according to Clause 27, wherein the one or more enzymes or active fragments thereof is selected from the grou consisting of bacterial tyrosinases, eukaryotic tyrosinases and tyrosine hydroxylases.

32. Th host cell according to Clause 27, wherein the ceil comprises on or more heterologous coding sequences for one or more enzymes or active fragments thereof that convert L-DOPA to dopamine.

33. The host cell according to Clause 27, wherein the cell comprises one or more heterologous coding sequences for one or more enzymes or active fragments ihereof that convert dopamine to 3,4~DHPA.

34. The host cell according to Clause 33, wherein the one or more enzymes is a monoamine oxidase (MAO).

35. The host cell according to Clause 34, wherein the one or more heterologous coding sequences comprises a Iv!AO coding sequence integrated at a genomic locus encoding native ARO10.

38. The host cell according to Clause 34, wherein the one or more heterologous coding sequences comprises a IviAG coding sequence operabiy linked to an inducsbie promoter.

37. The host ceil according to Clause 38, wherein the inducible promoter is part of an inducible system comprising a DNA binding protein targeted to a promoter regulating the ARO10 gene.

38. The host cell according to Clause 27, wherein the heterologous coding sequence is from a source organism selected from the group consisting of P. somniferum, T.

flavum and C aponica,

39. The host cell according to Clause 1 , wherein the host cell is a eukaryotic ceil.

40. The host cell according to Clause 39, wherein the eukaryotic ceil is a yeast ceil.

41. The host cell according to Clause 40, wherein the yeast cell is a S.cerevisiae cell, a Schizosaccharomyces pombe or a Pichia pastoris celi.

42. The host cell according to Clause 41 , wherein the yeast ceil is a S.cerevisiae ceil.

43. A method of preparing a benzySisoquinoline alkaloid (B!A), comprising;

cuituring a host ce!i according to any one of Clauses 1 to 42;

adding a growth feedstock to the ceil culture; and

recovering the B!A from the cell culture.

44. The method according to Clause 43, wherein the 8IA is a B!A precursor selected from noriaudanosoline and norcociaurine.

45. The method according to Clause 43, further comprising adding a starting compound to the ceil culture.

46. The method according to Clause 43, further comprising producing a BIA precursor selected from reticuiine, cociaurine, N-methyicoclaurine and norreticuiine. 47. The method according to Clause 43, further comprising producing one or more opiate compounds.

48. The method according to Clause 45, wherein the one or more opiate compounds is selected from oripavine, morphine, codeine, hydromorphone, hydrocodone, oxycodone and oxymorphone from thebaine.

49. A kit comprising:

a host ceil according to any one of Clauses 1 to 42; and

one or more components selected from a starting compound, a growth Feedstock, a heterologous coding sequence, cloning vectors, muitipie cloning sites (MCS), bidirectional promoters, an interna! ribosome entry site (IRES) and a eel! culture media.

Although the foregoing invention has been described in some detail by way of illustration and exampie fo purposes of clarity of understanding, it is readiiy apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currentfy known equivalents and equivalents deveioped in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention Is embodied by the appended claims.