Microorganisms producing benzylisoquinoline alkaloid (BIA) precursors and methods for making and using the same

Abstract

Provided are host cells engineered to produce benzylisoquinoline alkaloid (BIA) precursors such as higenamine (NC) and holonorhavnine (NL). The host cell may have one or more engineered modifications selected from the following: feedback inhibition mitigation mutations in enzyme genes; transcriptional regulatory modifications of biosynthetic enzyme genes; inactivation mutations in enzymes; and heterologous coding sequences. Also provided are methods for producing a target BIA or its precursor using the host cell and compositions, such as kits, systems, etc., suitable for use in the methods of the present invention.

Description

[0001] The present application is a divisional application of a Chinese patent application (application date (entry date): June 16, 2016, application number: 201480068628.3, and invention name: "Microorganisms producing benzylisoquinoline alkaloid (BIA) precursors and methods for preparing and using the precursors").

[0002] Government Rights

[0003] This invention was made with government support under Grant No. 1066100 awarded by the National Science Foundation. The government has certain rights in this invention.

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims priority under 35 U.S.C. § 119(e) to the filing date of U.S. Provisional Patent Application Serial No. 61 / 899,496, filed on November 4, 2013; the disclosure of which is incorporated herein by reference. Technical Field

[0006] The present invention relates to microorganisms, and in particular to microorganisms that produce benzylisoquinoline alkaloid (BIA) precursors, and methods for preparing and using the precursors. Background Art

[0007] 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 the novel activity observed for molecules such as berberine and sanguinarine for cancer and infection. Supplying all these BIA molecules so that they are available for researchers and physicians is interesting. The number of synthetic reactions and the requirements for selective stereochemistry mean that the chemical synthesis of BIA is low-yield and is not a feasible means for large-scale production. In contrast, for the widely used drugs codeine and morphine, opium poppy (opium poppy / Papaver somniferum) has been cultivated and developed into a production crop. Intermediates in the biosynthesis of morphine, which is suitable for use as a drug and drug precursor, will not accumulate because plant metabolism has evolved to maximize the flux of the pathway to the final opioid. Even for end-product metabolites like morphine, accumulation occurs only within specialized cells in the shoots and vascular tissues and requires harsh chemical processing of the harvested plant material during extraction, which can yield less than 2% morphine by dry weight. Thus, methods for preparing BIA are of interest. Summary of the invention

[0008] Provided are host cells engineered to produce benzylisoquinoline alkaloid (BIA) precursors such as norcoclaurine (NC) and norlaudanosoline (NL). The host cell may have one or more modifications selected from the following: feedback inhibition alleviation mutations in enzyme genes; transcriptional regulation modifications of biosynthetic enzyme genes; inactivation mutations in enzymes; and heterologous coding sequences. Also provided are methods for producing a target BIA or its precursor using the host cell and compositions, such as kits, systems, etc., suitable for use in the methods of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The present invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, as is customary, the various features of the drawings are not to scale. On the contrary, the dimensions of the various features are arbitrarily enlarged or reduced for clarity. The following drawings are included in the drawings.

[0010] Figure 1 The biosynthetic pathway from glucose to tyrosine and other BIA precursor molecules is shown.

[0011] Figure 2 Shown are the effects of ZWF1 knockout and TKL1 overexpression on the pentose phosphate pathway (PPP). A: native PPP flux, B: modified PPP flux.

[0012] Figure 3 The synthesis of NC (A) and NL (B) from precursor molecules is shown.

[0013] Figure 4 Shown are the effects of four genetic modifications on NC production under different feeding conditions of tyrosine.

[0014] Figure 5 The production of NC from strains with combinations of genetic modifications is shown.

[0015] Figure 6 Shown are the levels of NL production in the aldehyde oxidoreductase (ALD) / alcohol dehydrogenase (ADH) knockout strain.

[0016] Figure 7 In vivo L-DOPA decarboxylase (DODC) activity is shown. Yeast strains transformed with DNA to express opium poppy tyrosine / DOPA decarboxylase are able to convert L-DOPA to dopamine in vivo.

[0017] Figure 8 Shown is the production of higenamine (NC) in yeast strains fed 100 mM dopamine and varying concentrations of tyrosine.

[0018] Fig. 9Shown is NC production in various engineered yeast strains fed 100 mM dopamine and without tyrosine.

[0019] Fig.10 The production of NC from dopamine or from L-DOPA in an engineered yeast strain (CSY980) with the additional integration of the L-DOPA decarboxylase PpDODC is shown.

[0020] Fig.11 Shown is a biosynthetic scheme involving tyrosine hydroxylation using mammalian tyrosine hydroxylase (TyrH) with the co-substrate tetrahydrobiopterin (BH4).

[0021] Fig.12 Shown is the conversion of tyrosine to L-DOPA by tyrosine hydroxylase expressed from yeast cells: (A) LC-MS chromatogram confirming the conversion of tyrosine to L-DOPA in the presence of co-substrate BH4; and (B) L-DOPA ion fragmentation in lysate samples.

[0022] Fig.13 Co-expression of tyrosine hydroxylase with BH4 biosynthetic enzymes was shown to provide conversion of tyrosine to L-DOPA.

[0023] Fig.14 The synthesis of the BIA precursor molecules coclaurine and N-methylcoclaurine from NC is shown.

[0024] Fig.15 LC-MS analysis (A: ion counts) (B: ​​m / z fragmentation pattern) showing the production of NC-derived BIA precursor molecules (including N-methylcoclaurine) from L-DOPA in liquid culture of the engineered yeast strain.

[0025] definition

[0026] 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 specification.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994) and Hale & Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide a general meaning of many of the terms used herein to those of skill in the art. In addition, for clarity and ease of reference, certain terms are defined below.

[0028] It must be noted that the singular forms "a", "an", and "the" as used herein and in the appended claims 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. It should also be noted that the claims are drafted to exclude any optional elements. Thus, this statement is intended to serve as a prerequisite for the use of exclusive terms such as "solely", "only", etc., in connection with the recitation of claim elements or the use of a "negative" limitation.

[0029] As used herein, the terms "determine," "measure," and "assess," and "assay" are used interchangeably and include both quantitative and qualitative assays.

[0030] As used herein, the term "polypeptide" refers to a polymeric form of amino acids of any length, including peptides with a length range of 2-50 amino acids and polypeptides with a length greater than 50 amino acids. 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 derived amino acids, and polypeptides with modified peptide backbones, wherein the conventional backbone has been replaced by a non-naturally occurring or synthetic backbone. The polypeptide can have any suitable length, for example, 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 acids." Peptide" can 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, the peptide length is between 5 and 30 amino acids.

[0031] As used herein, the term "isolated" means that the target portion is at least 60% free, at least 75% free, at least 90% free, at least 95% free, at least 98% free, and even at least 99% free of other components with which the portion is associated prior to purification.

[0032] As used herein, the term "encoded by" refers to a nucleic acid sequence encoding a polypeptide sequence, wherein the polypeptide sequence or a portion thereof comprises 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. The term also encompasses polypeptide sequences that can be immunologically identified with a polypeptide encoded by the sequence.

[0033] "Vector" is capable of transferring a gene sequence to a target cell. As used herein, the terms "vector construct," "expression vector," and "gene transfer vector" are used interchangeably to refer to any nucleic acid construct capable of directing the expression of a target gene and capable of transferring a gene sequence to a target cell, either by genomic integration of all or a portion of the vector or by transient or heritable maintenance of the vector as an extrachromosomal element. Thus, the term includes cloning and expression vehicles as well as integration vectors.

[0034] An "expression cassette" includes any nucleic acid construct capable of directing the expression of a target gene / coding sequence, which is operably linked to the promoter of the expression cassette. Such cassettes are constructed into "vectors," "vector constructs," "expression vectors," or "gene transfer vectors" to transfer the expression cassette into target cells. Thus, the term includes cloning and expression vehicles as well as viral vectors.

[0035] A "plurality" includes at least 2 members. In some cases, a plurality can have 10 or more, such as 100 or more, 1000 or more, 10,000 or more, 10 ... 6 or more, 10 7 or more, 10 8 or more or 10 9 or more members.

[0036] Numerical ranges are inclusive of the numbers defining the range.

[0037] The method described herein comprises a plurality of steps. Each step can be performed after a predetermined amount of time has passed between steps as required. Thus, the time between each step can be 1 second or longer, 10 seconds or longer, 30 seconds or longer, 60 seconds or longer, 5 minutes or longer, 10 minutes or longer, 60 minutes or longer and include 5 hours or longer. In certain embodiments, each subsequent step is performed immediately after the previous step is completed. In other embodiments, the step can be performed after incubation or waiting time (e.g., a few minutes to overnight waiting time) after the previous step is completed.

[0038] Additional definitions of terms may appear throughout the specification. DETAILED DESCRIPTION

[0039] Provided are host cells engineered to produce benzylisoquinoline alkaloid (BIA) precursors such as higenamine (NC) and holonorhavnine (NL). The host cell may have one or more engineered modifications selected from the following: feedback inhibition mitigation mutations in enzyme genes; transcriptional regulatory modifications of biosynthetic enzyme genes; inactivation mutations in enzymes; and heterologous coding sequences. Also provided are methods for producing a target BIA or its precursor using the host cell and compositions, such as kits, systems, etc., suitable for use in the methods of the present invention.

[0040] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the specific embodiments described, as such embodiments may of course vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments, and are not intended to be restrictive, as the scope of the present invention will only be limited by the appended claims.

[0041] Where a range of values ​​is provided, it is understood that unless the context clearly indicates otherwise, each intervening value (to one-tenth of the unit of the lower limit) between the upper and lower limits of the range is encompassed within the present invention, as well as any other stated or intervening value within the stated range. The upper and lower limits of these smaller ranges may be independently included within these smaller ranges and are also encompassed within the present invention, subject to any specifically excluded limits within the stated range. Where the range includes one or both of the stated limits, the present invention also includes ranges excluding either or both of those included limits.

[0042] Certain ranges provided herein contain values ​​preceded by the term "about". The term "about" is used herein to indicate exactly the exact number that follows it, as well as numbers that are close to or approximately the number that follows the term. In determining whether a value is close to or approximately a specifically recited value, a close or approximate unrecited value may be a value that, in the context in which it appears, provides a substantial equivalence to the specifically recited value.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used to practice or test the present invention, representative illustrative methods and materials are now described.

[0044] All publications and patents cited in this specification are incorporated herein by reference, just as if each individual publication or patent was specifically and individually indicated as incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials involved in citing the publication. Reference to any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate the publication by virtue of prior invention. In addition, the publication dates provided may be different from the actual publication dates which may need to be independently confirmed.

[0045] It should be 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. It should also be noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as a prerequisite basis for the use of exclusive terminology such as "solely," "only," etc. in connection with the recitation of claim elements or the use of a "negative" limitation.

[0046] It will be apparent to those skilled in the art upon reading this disclosure that each individual embodiment described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any other several embodiments without departing from the scope or spirit of the invention. Any recited method is carried out in the order of events recited or in any other order that is logically possible.

[0047] In further describing the present invention, the target benzylisoquinoline alkaloid precursor is first described in more detail, followed by a host cell for producing the benzylisoquinoline alkaloid precursor. Next, the target method in which the host cell is suitable is reviewed. Kits that can be used to practice the methods of the present invention are also described.

[0048] Benzylisoquinoline alkaloid (BIA) precursor

[0049] As summarized above, a host cell producing a benzylisoquinoline alkaloid precursor (BIA precursor) is provided. A BIA precursor can be any intermediate or precursor compound in a synthetic pathway (e.g., as described herein) that results in the production of a target BIA (e.g., as described herein). In some cases, a BIA precursor has a structure that can be characterized as a BIA or a derivative thereof. In some cases, a BIA precursor has a structure that can be characterized as a fragment of a BIA. In some cases, a BIA precursor is an early BIA. As used herein, "early BIA" means an early intermediate in the synthesis of a target BIA in a cell, wherein the early BIA is produced by a host cell from a host cell raw material or a simple starting compound. In some cases, an early BIA is produced by only a subject host cell from a host cell raw material (e.g., carbon and nutrient sources) without adding a starting compound to a BIA intermediate of the cell. The term early BIA can refer to a precursor of a target BIA final product, whether or not the early BIA itself can be characterized as a benzylisoquinoline alkaloid.

[0050] In some cases, the BIA precursor is an early BIA, such as pre-taurine benzyl isoquinoline alkaloid. Thus, a host cell producing pre-taurine benzyl isoquinoline alkaloid (pre-taurine BIA) is provided. Taurine is a target major branch point intermediate in downstream BIA synthesis that produces final products such as opioid products through cell engineering efforts. The subject host cell can produce BIA precursors from simple and inexpensive starting materials that are suitable for the production of taurine and downstream BIA final products.

[0051] As used herein, the terms "pre-taurine benzylisoquinoline alkaloid", "pre-taurine BIA" and "pre-taurine BIA precursor" are used interchangeably and refer to the biosynthetic precursor of taurine, regardless of whether the structure of the taurine precursor itself is characterized as a benzylisoquinoline alkaloid. The term pre-taurine BIA means biosynthetic precursors, intermediates and metabolites thereof including any suitable members of the host cell biosynthetic pathway that can produce taurine. In some cases, pre-taurine BIA includes a benzylisoquinoline alkaloid fragment, such as a benzyl fragment, a quinoline fragment or a precursor or derivative thereof. In certain instances, pre-taurine BIA has a structure that can be characterized as a benzylisoquinoline alkaloid or a derivative thereof.

[0052] Target BIA precursors include, but are not limited to, higenamine (NC) and holonorlabdanine (NL) and NC and NL precursors, such as tyrosine, 4-hydroxyphenylacetaldehyde (4-HPA), 4-hydroxyphenylpyruvic acid (4-HPPA), L-3,4-dihydroxyphenylalanine (L-DOPA), 3,4-dihydroxyphenylacetaldehyde (3,4-DHPA) and dopamine. In some embodiments, one or more BIA precursors are 3,4-dihydroxyphenylacetaldehyde (3,4-DHPA) and dopamine. In certain instances, one or more BIA precursors are 4-hydroxyphenylacetaldehyde (4-HPA) and dopamine. Figure 3 A and 3B show the synthesis of NC and NL, respectively, from precursor molecules via a Pictet-Spengler condensation reaction, which may occur spontaneously or may be catalyzed by any suitable enzyme.

[0053] The synthetic pathway for the BIA precursor can be generated in a host cell and can be initiated with any suitable starting compounds or materials. Figure 1 The target synthetic pathway of BIA precursor starting from glucose is shown. The starting material can be non-naturally occurring or the starting material can be naturally occurring in the host cell. Based on the synthetic pathway present in the host cell, any suitable compound and material can be used as the starting material. The source of the starting material can be from the host cell itself (e.g., tyrosine), or the starting material can be added or supplemented to the host cell from an external source. Thus, in some cases, the starting compound refers to a compound in the synthetic pathway of the cell that is added to the host cell from an external source and is not part of the growth feedstock or cell growth medium. The target starting compound includes, but is not limited to, dopamine, 4-HPA, 4-HPPA, and Figure 1 Any compound shown in. For example, if the host cell is grown in a liquid culture medium, the cell culture medium may be supplemented with a starting material, which is transported to the cell and converted into a desired product by the cell. The target starting material includes, but is not limited to, cheap raw materials and simple precursor molecules. In some cases, the host cell utilizes a raw material including a simple carbon source as a starting material, and the host cell utilizes the starting material to produce a compound of the synthetic pathway of the cell. The host cell growth raw material may include one or more compounds, such as a carbon source (such as cellulose, starch, free sugars) and a nitrogen source such as ammonium salts or cheap amino acids. In some cases, the growth raw material suitable for use as a starting material may be derived from a sustainable source, such as biomass grown on marginal land (including switchgrass and algae) or biomass waste products from other industrial or agricultural activities.

[0054] Host cells

[0055] As summarized above, one aspect of the present invention is a host cell that produces one or more BIA precursors. Any suitable cell can be used in the subject host cell and method. In some cases, the host cell is a non-plant cell. In some instances, the host cell can be characterized as a microbial cell. In some cases, the host cell is an insect cell, a mammalian cell, a bacterial cell, or a yeast cell. Any suitable type of host cell can be used to produce a subject BIA cell, see, for example, US2008 / 0176754 and US2014 / 0273109, the disclosures of which are incorporated by reference as a whole. Target host cells include, but are not limited to, bacterial cells, such as Bacillus subtilis cells, Escherichia coli cells, Streptomyces cells, and Salmonella typhimurium cells; insect cells, such as Drosophila melanogaster S2 and Spodoptera frugiperda Sf9 cells; and yeast cells, such as Saccharomyces cerevisiae cells, Schizosaccharomyces pombe cells, and Pichia pastoris cells. In some embodiments, the host cell is a yeast cell or an Escherichia coli cell. In some cases, the host cell is a yeast cell. In some instances, host cells are from yeast strains engineered to produce target BIA precursors. Any host cell described in US2008 / 0176754 and US2014 / 0273109 of Smolke et al. may be suitable for use in subject cells and methods. In certain embodiments, yeast cells may have saccharomyces cerevisiae (S.cerevisiae) species. In certain embodiments, yeast cells may have Schizosaccharomyces pombe species. In certain embodiments, yeast cells may have Pichia pastoris species. Yeast is interesting as a host cell because cytochrome P450 proteins (which participate in some target biosynthetic pathways) can be correctly folded into the endoplasmic reticulum membrane so that their activity is maintained. Target yeast strains suitable for the present invention include, but are not limited to, CEN.PK (genotype: MATa / α ura3-52 / ura3-52 trp1-289 / trp1-289 leu2-3_112 / leu2-3_112 his3 Δ1 / his3 Δ1 MAL2-8C / MAL2-8C SUC2 / SUC2), S288C, W303, D273-10B, X2180, A364A, Σ1278B, AB972, SK1 and FL100.In some cases, the yeast strain is any of the following: S288C (MATα; SUC2 mal mel gal2 CUP1flo1 fllo8-1 hap1), BY4741 (MATα; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0), BY4742 (MATα; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0), BY4743 (MATa / MATα; his3Δ1 / his3Δ1; leu2Δ0 / leu2Δ0; met15Δ0 / MET15; LYS2 / lys2Δ0; ura3Δ0 / ura3Δ0), and WAT11 or W(R), a derivative of the W303-B strain (MATa; ade2-1; his3-11, -15; leu2-3, -112; ura3-1; canR; cyr+), which express the Arabidopsis thaliana NADPH-P450 reductase ATR1 and the yeast NADPH-P450 reductase CPR1, respectively. In another embodiment, the yeast cell is W303α (MATα; his3-11, 15 trp1-1 leu2-3 ura3-1 ade2-1). The identities and genotypes of additional yeast strains of interest can be found at EUROSCARF (web.uni-frankfurt.de / fb15 / mikro / euroscarf / col_index.html).

[0056] Host cell can be engineered to include one or more modifications (such as two or more, three or more, four or more, five or more or even more modifications) that provide the production of target BIA precursor. In some cases, modification means genetic modification, such as mutation, addition or deletion of a gene or its fragment, or transcriptional regulation of a gene or its fragment. In some cases, one or more (such as two or more, three or more or four or more) modifications are selected from: feedback inhibition in the natural biosynthetic enzyme gene of the cell is alleviated mutation; Transcriptional regulation modification of the natural biosynthetic enzyme gene of the cell; Inactivation mutation in the natural enzyme of the cell; And heterologous coding sequence of encoding enzyme. Cells comprising one or more modifications can be referred to as modified cells.

[0057] Modified cells can overproduce one or more BIA precursor molecules.Overproduction means that cells have an increased or increased target BIA precursor molecule production relative to control cells (e.g., unmodified cells).The production of increased or increased means the production of a certain amount of target BIA precursors when the control does not have BIA precursors, and about 10% or more when the control has a certain BIA precursor production, 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, such as 2 times or more, such as 5 times or more, including 10 times or more increase.

[0058] In some cases, the host cell is capable of producing an increased amount of higenamine relative to a control host cell lacking one or more modifications (e.g., as described herein). In certain instances, the increased amount of higenamine is about 10% or more relative to a 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-fold or more, or even 10-fold or more relative to a control host cell.

[0059] In some cases, the host cell is capable of producing an increased amount of holonordanoic acid relative to a control host cell lacking one or more modifications (e.g., as described herein). In certain instances, the increased amount of holonordanoic acid 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-fold or more, or even 10-fold or more relative to the control host cell.

[0060] In some embodiments, the host cell is capable of producing 10% or more yield of higenamine 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 higenamine from a starting compound.

[0061] In some embodiments, the host cell is capable of producing a 10% or more yield of holonorlaudamine 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 holonorlaudamine from a starting compound.

[0062] In some embodiments, the host cell overproduces one or more BIA precursor molecules selected from the group consisting of 4-hydroxyphenylacetaldehyde (4-HPA), L-3,4-dihydroxyphenylalanine (L-DOPA), 3,4-dihydroxyphenylacetaldehyde (3,4-DHPA), and dopamine.

[0063] Any suitable combination of one or more modifications may be included in the subject host cell. 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) different modifications of the same type of modification are included in the subject cell.

[0064] In some embodiments of the host cell, when the cell comprises one or more heterologous coding sequences encoding one or more enzymes, the cell comprises at least one additional modification selected from the group consisting of: a feedback inhibition mitigation mutation in a biosynthetic enzyme gene native to the cell; a transcriptional regulatory modification of a biosynthetic enzyme gene native to the cell; and an inactivating mutation in an enzyme native to the cell. In certain embodiments of the host cell, when the cell comprises one or more feedback inhibition mitigation mutations in one or more biosynthetic enzyme genes native to the cell, the cell comprises at least one additional modification selected from the group consisting of: a transcriptional regulatory 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 encoding an enzyme. In some embodiments of the host cell, when the cell comprises one or more transcriptional regulatory modifications of one or more biosynthetic enzyme genes native to the cell, the cell comprises at least one additional modification selected from the group consisting of: a feedback inhibition mitigation mutation in a biosynthetic enzyme gene native to the cell; an inactivating mutation in an enzyme native to the cell; and a heterologous coding sequence encoding an enzyme. In certain examples of host cells, when the cell comprises one or more inactivating mutations in one or more enzymes native to the cell, the cell 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 regulatory modification of a biosynthetic enzyme gene native to the cell; and a heterologous coding sequence encoding an enzyme.

[0065] In certain embodiments of the host cell, the cell comprises one or more feedback inhibition reduction mutations in one or more biosynthetic enzyme genes native to the cell; and one or more transcriptional regulatory modifications to one or more biosynthetic enzyme genes native to the cell. In certain embodiments of the host cell, the cell comprises one or more feedback inhibition reduction mutations in one or more biosynthetic enzyme genes native to the cell; and one or more inactivation mutations in enzymes native to the cell. In certain embodiments of the host cell, the cell comprises one or more feedback inhibition reduction mutations in one or more biosynthetic enzyme genes native to the cell; and one or more heterologous coding sequences. In some embodiments, the host cell comprises one or more modifications (e.g., as described herein) comprising one or more target genes described in Table 1.

[0066] Feedback inhibition mitigates mutations

[0067] In some examples, the host cell is a cell that contains one or more feedback inhibition mitigating 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, one or more biosynthetic enzyme genes are native to the cell (e.g., present in unmodified cells). As used herein, the term "feedback inhibition mitigating mutation" refers to a mutation that mitigates the feedback inhibition control mechanism of the host cell. Feedback inhibition is a control mechanism of the cell in which an enzyme in the synthetic pathway of a regulated compound is inhibited when the compound has accumulated to a certain level, thereby balancing the amount of the compound in the cell. In some examples, one or more feedback inhibition mitigating mutations are in Figure 1 or Figure 2 Mutations that alleviate feedback inhibition reduce the inhibition of the regulated enzyme in the target cell relative to control cells and provide increased levels of the regulated compound or its downstream biosynthetic product. In some cases, alleviating the inhibition of the regulated enzyme means that the IC 50 Increase by 2 times or more, such as 3 times or more, 5 times or more, 10 times or more, 30 times or more, 100 times or more, 300 times or more, 1000 times or more or even more. The level of improvement means 110% or more of the level of the regulated compound in the control cell or its downstream product, such as 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more or 200% or more, such as at least 3 times or more, at least 5 times or more, at least 10 times or more or even more of the regulated compound in the host cell or its downstream product.

[0068] A variety of feedback inhibition control mechanisms for the regulation of the level of BIA precursors and biosynthetic enzymes native to the host cell can be targeted for alleviation in the host cell. The host cell may contain one or more feedback inhibition alleviation mutations in one or more biosynthetic enzyme genes native to the cell. The mutation may be located in any suitable biosynthetic enzyme gene native to the host cell, wherein 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 3-deoxy-d-arabinose-heptulose-7-phosphate (DAHP) synthase and chorismate mutase. In some embodiments, the one or more biosynthetic enzyme genes encode 3-deoxy-d-arabinose-heptulose-7-phosphate (DAHP) synthase. In some examples, the one or more biosynthetic enzyme genes encode chorismate mutase. In certain examples, one or more feedback inhibition alleviation mutations are present in a biosynthetic enzyme gene selected from ARO4 and ARO7. In certain examples, one or more feedback inhibition alleviation mutations are present in a biosynthetic enzyme gene for ARO4. In certain examples, the one or more feedback inhibition relieving mutations are present in a biosynthetic enzyme gene that is ARO7. In some embodiments, the host cell comprises one or more feedback inhibition relieving mutations in one or more biosynthetic enzyme genes, such as one of those described in Table 1.

[0069] Any suitable number and type of mutation can be used to alleviate the feedback inhibition control mechanism. As used herein, the term "mutation" refers to the deletion, insertion or substitution of an amino acid residue or nucleotide relative to a reference sequence or motif. The mutation can be incorporated as an orthotopic mutation of a native gene at the original locus. In some cases, the mutation can be incorporated as an additional copy of a gene introduced as genetic integration at a separate locus or as an additional copy on an episomal vector such as 2μ or a centromeric plasmid. In some instances, the copy of the feedback inhibition of the enzyme is under transcriptional regulation of the native cell. In some instances, the copy is introduced under the control of a synthetic promoter under the engineered constitutive or dynamic regulation of protein expression.

[0070] In certain embodiments, one or more feedback inhibition mitigating mutations are present in the ARO4 gene. Target ARO4 mutations include, but are not limited to, substitution of a lysine residue at position 229 with a leucine residue, substitution of a glutamine residue at position 166 with a lysine residue, or mutations as described by Hartmann M, et al. ((2003) Proc Natl Acad Sci USA 100(3):862-867) or Fukuda et al. ((1992) J Ferment Bioeng 74(2):117-119). In some examples, the mutations used to confer feedback inhibition are selected from a mutagenesis library of enzyme mutants. Examples of such selections include growth on o-fluoro-D,L-phenylalanine in the rescue medium with excess tyrosine or growth of an aro3 mutant yeast strain as described by Fukuda et al. ((1990) Breeding of Brewing Yeast Producing a Large Amount of Beta-PhenylethylAlcohol and Beta-Phenylethyl Acetate. Agr Biol Chem Tokyo 54(1):269-271).

[0071] Target ARO7 mutations include, but are not limited to, substitution of the threonine residue at position 226 with isoleucine as described by Schmidheini et al. ((1989), J Bacteriol 171(3): 1245-1253); and additional mutations conferring feedback inhibition selected from a mutagenesis library of microbial chorismate mutase mutants. Examples of such selections include assays for 5-methyltryptophan sensitivity or increased melanin production in strains expressing heterologous tyrosinase (1.9) in the absence of externally fed tyrosine.

[0072] In certain embodiments, a host cell of the invention may comprise 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, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more feedback inhibition reducing mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 feedback inhibition reducing mutations in one or more biosynthetic enzyme genes that are native to the host cell.

[0073] Transcriptional regulatory modifications

[0074] Host cells may comprise one or more transcriptional regulation 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 natural to the cell. Any suitable biosynthetic enzyme gene of the cell can be targeted to transcriptional regulation. Transcriptional regulation means regulating the expression of the target gene in the modified cell relative to a control cell (e.g., an unmodified cell), for example, increasing or decreasing, enhancing or suppressing. In some cases, the transcriptional regulation of the target gene includes increasing or enhancing expression. Increasing or enhancing expression means that the expression level of the target gene increases by 2 times or more, such as 5 times or more and sometimes 25, 50 or 100 times or more, and in certain embodiments 300 times or more or higher, compared to the control, i.e., the expression in the same cell that is unmodified (e.g., by using any suitable gene expression assay), and in certain embodiments 300 times or more or higher. Alternatively, in the case where the expression of the target gene in the cell is so low that it is undetectable, if expression is increased to a level that can be easily detected, the expression level of the target gene is considered to increase. In some instances, the transcriptional regulation of the target gene includes reducing or suppressing expression. Reduce or suppress expression and mean that as compared to the control, the expression level of the target gene is reduced by 2 times or more, such as 5 times or more and sometimes 25, 50 or 100 times or more, and in certain embodiments 300 times or more or higher. In some cases, expression is reduced to an undetectable level. The modification of the target host cell process that can be suitable for use in the subject host cell is described in U.S. Publication No. 20140273109 (14 / 211,611) of Smolke et al., and the disclosed content of the publication is incorporated herein by reference in its entirety.

[0075] Any suitable biosynthetic enzyme gene may be transcriptionally regulated, and includes, but is not limited to, Figure 1 In some embodiments, the host cell comprises one or more transcriptional regulatory modifications of one or more genes, such as those described in Table 1. In some embodiments, the host cell comprises one or more genes, such as those described in Table 1. Figure 1 and 2One or more transcriptional regulatory modifications of one of the genes described in the synthetic pathway of one of the genes.

[0076] In some embodiments, the transcriptional regulation modification includes replacing the natural promoter of one or more biosynthetic enzyme genes with a strong promoter. The promoter driving the expression of the target gene can be a constitutive promoter or an inducible promoter, provided that the promoter can be active in the host cell. The target gene can be expressed from its natural promoter, or a non-natural promoter can be used. Although not required, such promoters should be of medium to high strength in the host using them. The promoter can be regulated or constitutive. In some embodiments, a promoter that is not repressed by glucose or is only slightly repressed when glucose is present in the culture medium is used. There are numerous suitable promoters, examples of which include promoters of glycolytic genes, such as promoters of Bacillus subtilis genes (encoding fructose bisphosphate aldolase) or GAPDH promoters from yeast Saccharomyces cerevisiae (encoding glyceraldehyde-phosphate dehydrogenase) (Bitter GA, Meth. Enzymol. 152: 673684 (1987)). Other strong promoters of interest include, but are not limited to, the ADHI promoter of baker's yeast (Ruohonen L. et al., J. Biotechnol. 39:193203 (1995)); phosphate starvation-induced promoters, such as the PHO5 promoter of yeast (Hinnen, A. et al., Yeast Genetic Engineering, Barr, PJ, et al., eds., Butterworths (1989); alkaline phosphatase promoter from Bacillus licheniformis (Lee. JWK et al., J. Gen. Microbiol. 137: 11271133 (1991)); GPD1 and TEF1. Target yeast promoters include, but are not limited to, inducible promoters such as Gal1-10, Gal1, GalL, GalS; repressible promoters Met25, tetO; and constitutive promoters such as glyceraldehyde 3-phosphate dehydrogenase promoter (GPD), alcohol dehydrogenase promoter (ADH), translation elongation factor-1-α promoter (TEF), cytochrome c-oxidase promoter (CYC1), MRP7 promoter. In some examples, the strong promoter is GPD1. In some examples, the strong promoter is TEF1. Self-replicating yeast expression vectors containing promoters that can be induced by hormones (such as glucocorticoids, steroids and thyroid hormones) are also known and include, but are not limited to, glucocorticoid responsive elements (GRE) and thyroid hormone responsive elements (TRE), see, for example, those promoters described in U.S. Patent No. 7,045,290. Vectors containing constitutive or inducible promoters such as α factor, alcohol oxidase and PGH can be used. In addition, any promoter / enhancer combination (according to the eukaryotic promoter database EPDB) can also be used to drive the expression of the target gene. It should be understood that any suitable promoter specific to the host cell can be selected, such as Escherichia coli.In some cases, promoter selection can be used to optimize transcription, and therefore, enzyme levels to maximize production while minimizing energy resources.

[0077] Inactivating mutation

[0078] The host cell may comprise one or more inactivating mutations (such as two or more, three or more, four or more, five or more or even more) of the enzyme of the cell. The inclusion of one or more inactivating mutations can modify the flux of the synthetic pathway of the host cell to increase the level of the target BIA precursor or the desired enzymatic precursor producing the BIA precursor. In some cases, one or more inactivating mutations are for an enzyme native to the cell. Figure 2 The PPP flux of native pentose phosphate pathway (PPP) and modification is shown, wherein it relates to the inactivation of ZWF1 enzyme.As used herein, " inactivation mutation " means one or more mutations of the gene or regulatory DNA sequence of a cell, wherein the mutation inactivates the biological activity of the protein expressed by the target gene.In some cases, the gene is natural to the cell.In some instances, the gene encodes an inactivated enzyme, and is a part of or is connected to the synthetic pathway of the BIA precursor produced by the host cell.In some instances, the inactivation mutation is located in the regulatory DNA sequence controlling the target gene.In some cases, the inactivation mutation is for the promoter of a gene.Any suitable mutation (e.g., as described herein) can be used to inactivate the target gene or regulatory DNA sequence. "Inactivated" or "inactivation" means that the biological activity of the protein expressed by the mutated gene is reduced by 10% or more, such as 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 relative to a control protein expressed by an unmutated control gene. In some cases, the protein is an enzyme and the inactivating mutation reduces the activity of the enzyme.

[0079] In some embodiments, the cell contains an inactivating mutation in an enzyme that is native to the cell. Any suitable enzyme may be targeted for inactivation. Target enzymes include, but are not limited to, Figure 1 and 2 Those enzymes described in, the action of the enzyme in the synthetic pathway of the host cell tends to reduce the level of the target BIA precursor. In some cases, the enzyme has glucose-6-phosphate dehydrogenase activity. In certain embodiments, the enzyme comprising the inactivating mutation is ZWF1 (see, e.g. Figure 2). In some cases, the enzyme has alcohol dehydrogenase activity. In some embodiments, the enzyme comprising an inactivating mutation is selected from ADH2, ADH3, ADH4, ADH5, ADH6, ADH7 and SFA1. In certain embodiments, the enzyme comprising an inactivating mutation is ADH2. In certain embodiments, the enzyme comprising an inactivating mutation is ADH3. In certain embodiments, the enzyme comprising an inactivating mutation is ADH4. In certain embodiments, the enzyme comprising an inactivating mutation is ADH5. In certain embodiments, the enzyme comprising an inactivating mutation is ADH6. In certain embodiments, the enzyme comprising an inactivating mutation is ADH7. In some cases, the enzyme has aldehyde oxidoreductase activity. In certain embodiments, the enzyme comprising an inactivating mutation is selected from ALD2, ALD3, ALD4, ALD5 and ALD6. In certain embodiments, the enzyme comprising an inactivating mutation is ALD2. In certain embodiments, the enzyme comprising an inactivating mutation is ALD3. In certain embodiments, the enzyme comprising an inactivating mutation is ALD4. In certain embodiments, the enzyme comprising an inactivating mutation is ALD5. In certain embodiments, the enzyme comprising an inactivating mutation is ALD6. In some embodiments, the host cell comprises one or more inactivating mutations of one or more genes described in Table 1.

[0080] Heterologous coding sequence

[0081] In some examples, the host cell is a cell having one or more heterologous coding sequences (such as two or more, three or more, four or more, five or more or even more), which encodes an activity that enables the host cell to produce the desired BIA precursor (e.g., as described herein). As used herein, the term "heterologous coding sequence" is used to indicate any polynucleotide that encodes or ultimately encodes 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 appropriate conditions. Thus, a "heterologous coding sequence" includes multiple copies of a coding sequence that is normally present in a host cell so that the cell expresses additional copies of a coding sequence that is not normally present in the cell. A heterologous coding sequence can be RNA or any type thereof, for example, mRNA, DNA or any type thereof, such as a hybrid of cDNA or RNA / DNA. The target coding sequence includes, but is not limited to, a full-length transcription unit comprising such features as a coding sequence, introns, a promoter region, a 3'-UTR, and an enhancer region.

[0082] In some embodiments, the host cell comprises higenamine (NC) synthase activity. Any suitable NC synthase is suitable for use in the subject host cell. Target NC synthases 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 comprises a heterologous coding sequence for NC synthase or an active fragment thereof. In some instances, the host cell comprises one or more heterologous coding sequences for one or more enzymes or active fragments thereof that convert tyrosine into L-DOPA. In some cases, one or more enzymes are selected 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 comprises one or more heterologous coding sequences for one or more enzymes or active fragments thereof (e.g., EC 4.1.1.28) that convert L-DOPA into dopamine.

[0083] In certain embodiments, the cell comprises one or more heterologous coding sequences for one or more enzymes or active fragments thereof that convert dopamine into 3,4-DHPA. In some cases, the one or more enzymes are monoamine oxidases (MAO) (e.g., EC 1.4.3.4). The one or more heterologous coding sequences may be derived from any suitable species (e.g., as described herein). In some cases, the one or more heterologous coding sequences may be derived from the 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 genes or enzymes described in Table 1.

[0084] In some examples, the one or more heterologous coding sequences include a MAO coding sequence integrated at a genomic locus encoding native ARO10. In certain examples, the one or more heterologous coding sequences include a MAO coding sequence operably linked to an inducible promoter. In some embodiments, the inducible promoter is part of an inducible system that includes a DNA binding protein that targets a promoter of the ARO10 gene. In some embodiments, the host cell includes one or more heterologous coding sequences for one or more enzymes described in the genes of Table 1 or active fragments thereof.

[0085] As used herein, the term "heterologous coding sequence" also includes the coding portion of a peptide or enzyme (i.e., a cDNA or mRNA sequence of the peptide or enzyme) and the coding portion of a full-length transcription unit (i.e., a gene containing introns and exons), as well as "codon-optimized" sequences, truncated sequences or other forms of altered sequences encoding enzymes or encoding equivalent amino acid sequences thereof, provided that the equivalent amino acid sequence produces a functional protein. Such equivalent amino acid sequences may have one or more amino acid deletions, wherein the deletion is N-terminal, C-terminal or internal. Truncated forms are envisioned as long as they have the catalytic ability indicated herein. It is also envisioned that two or more enzymes are fused to facilitate the transfer of metabolites in the pathway, provided that the catalytic activity is maintained.

[0086] Operable fragments, mutants or truncated forms can be identified by modeling and / or screening. This is made possible by deleting, for example, the N-terminus, C-terminus or internal regions of the protein in a stepwise manner, followed by analysis of the derivatives for their activity against the desired reaction compared to the original sequence. If the derivative in question operates in this capacity, it is considered to be an equivalent derivative of the correct enzyme.

[0087] Aspects of the present invention also relate to heterologous coding sequences of amino acid sequences equivalent to the natural amino acid sequences of various enzymes.Amino acid sequences that are "equivalent" are defined as being different from the specific amino acid sequence, but comprising at least some amino acid changes (deletion, substitution, inversion, insertion, etc.), when used for the desired purpose, compared with the similar activity of the specific amino acid sequence, the changes will not substantially affect the biological activity of the protein.In the example of decarboxylase, biological activity refers to its catalytic activity.Equivalent sequences are also intended to include those that have been engineered and / or evolved to have characteristics different from the original amino acid sequence.Target mutagenic properties include catalytic activity, substrate specificity, selectivity, stability, solubility, positioning, etc. In certain embodiments, an "equivalent" amino acid sequence comprises at least 80%-99% identity at the amino acid level to a particular amino acid sequence, in some cases at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and higher, and in some cases at least 95%, 96%, 97%, 98% and 99% identity at the amino acid level. In some cases, the amino acid sequence may be identical, but the DNA sequence is altered, for example, to optimize codon usage for the host organism.

[0088] Host cells may also be modified to have one or more genetic changes to adapt to heterologous coding sequences. Changes in the natural host genome include, but are not limited to, modifying the genome to reduce or eliminate the expression of specific proteins that may interfere with the desired pathway. The presence of such natural proteins may rapidly convert one of the intermediates or final products of the pathway into metabolites or other compounds that are unavailable in the desired pathway. Therefore, if the activity of the natural enzyme is reduced or completely absent, the intermediate produced may be more easily incorporated into the desired product.

[0089] In some examples, in the case where the expression of the elimination protein may be of interest, it is in proteins involved in pleiotropic drug responses, including but not limited to ATP binding cassette (ABC) transporters, multidrug resistance (MDR) pumps and related transcription factors, which are involved in the export of BIA molecules to the culture medium, so the deletion of control compounds is exported to the culture medium, thereby making them more available for incorporation into the desired product. In some embodiments, the target host cell gene deletion includes genes related to the unfolded protein response and endoplasmic reticulum (ER) proliferation. Such gene deletions can produce improved BIA production. The expression of cytochrome P450 can induce the unfolded protein response and can cause ER proliferation. The deletion of genes related to these stress responses can control or reduce the overall burden on the host cell and improve pathway performance. Genetic changes can also include modifying the promoter of endogenous genes to increase expression and / or introduce additional copies of endogenous genes. Examples of this include constructing / using strains that overexpress endogenous yeast NADPH-P450 reductase CPR1 to increase the activity of heterologous P450 enzymes. In addition, endogenous enzymes directly involved in the synthesis of intermediate metabolites, such as ARO8, 9, and 10, can also be overexpressed.

[0090] The target heterologous coding sequence includes but is not limited to the sequence (wild-type sequence or equivalent sequence) of the enzyme that coding is usually responsible for BIA and precursor production in plant. In some cases, the enzyme encoded by heterologous sequence can be any enzyme in BIA approach, and can be from any suitable source. The selection and number of the enzyme encoded by heterologous coding sequence for specific synthetic pathways can be selected based on the desired product. In certain embodiments, host cell of the present invention can comprise 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, 10 or more, 11 or 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, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 heterologous coding sequences.

[0091] In some cases, the peptide sequence encoded by the heterologous coding sequence is as reported in GENBANK. Target enzymes include, but are not limited to, those described herein and those shown in Table 1. The host cell may contain any combination of the listed enzymes from any source. Unless otherwise indicated, the 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 www.yeastgenome.org.

[0092] In some embodiments, host cells (e.g., yeast strains) are engineered to selectively produce target BIA by locating one or more enzymes to the compartment in the cell. In some cases, the enzyme may be located in the host cell so that the compound produced by the enzyme is spontaneously rearranged, or is converted into a desirable metabolite by another enzyme before reaching the positioning enzyme that can convert the compound into an undesirable metabolite. The spatial distance between the two enzymes can be selected to prevent one of the enzymes from directly acting on the compound and producing an undesirable metabolite and limiting the generation of undesirable final products (e.g., undesirable opioid byproducts). In certain embodiments, any enzyme described herein (alone or with a second enzyme) can be located to any suitable compartment in the host cell, including but not limited to organelles, endoplasmic reticulum, Golgi, vacuole, nucleus, plasma membrane or periplasm. In some embodiments, the host cell comprises one or more enzymes, and the enzyme comprises a positioning tag. Any suitable tag can be used. In some cases, the positioning tag is a peptide sequence connected to the N-terminal and or C-terminal ends of the enzyme.

[0093] Any suitable method can be used to connect the label to the enzyme. In some cases, the positioning label is derived from endogenous yeast protein. Such labels can provide paths to various yeast organelles: endoplasmic reticulum (ER), mitochondria (MT), plasma membrane (PM) and vacuole (V). In certain embodiments, the label is an ER path selection label (e.g., ER1). In certain embodiments, the label is a vacuole label (e.g., V1). In certain embodiments, the label is a plasma membrane label (e.g., P1). In certain instances, the label includes or is derived from a transmembrane domain within the tail anchoring category of a protein. In some embodiments, the positioning label positions the enzyme on the outside of an organelle. In certain embodiments, the positioning label positions the enzyme on the inside of an organelle.

[0094] In some instances, the expression of every type of enzyme is increased (that is, multiple copies) by other gene copies, which increases intermediate accumulation and / or BIA precursor production. Embodiments of the present invention include by expressing multiple species variants of single or multiple enzymes simultaneously, and the BIA precursor in the host cell produces an increase. In some cases, the other gene copies of single or multiple enzymes are included in the host cell. Any suitable method can be used, including multiple copies of the heterologous coding sequence for the enzyme in the host cell.

[0095] In some embodiments, host cell comprises multiple copies of heterologous coding sequence for enzyme, such as 2 or more, 3 or more, 4 or more, 5 or more or even 10 or more copies.In certain embodiments, host cell comprises multiple copies of heterologous coding sequence for one or more enzymes, such as multiple copies of two or more, three or more, four or more enzymes etc.In some cases, multiple copies of heterologous coding sequence for enzyme are derived from two or more source organisms different from host cell.For example, host cell can comprise multiple copies of a heterologous coding sequence, wherein each in the copy is derived from different source organisms.Therefore, each copy can comprise some variations in explicit sequence based on the interspecific differences of target enzyme encoded by heterologous coding sequence.

[0096] In some embodiments of the host cell, the heterologous coding sequence is from a source organism selected from the group consisting of: poppy (P. somniferum), yellow thrush (T. flavum) and Japanese golden thread (C. japonica). In some examples, the source organism is poppy, California poppy (E. californica), Japanese golden thread, yellow thrush, creeping barberry (Berberis stolonifer), yellow thrush subspecies glaucum, Coptis chinensis, Thalictrum spp, Coptis spp, Papaver spp, golden flower barberry (Berberis wilsonae), Mexican thorn poppy (A. mexicana) or Berberis spp. In certain examples, the heterologous coding sequence is from a source organism selected from poppy, yellow thrush and Japanese golden thread. In some embodiments, the host cell includes a heterologous coding sequence from one or more source organisms described in Table 1.

[0097] The host cell culture medium of the engineering can be sampled and the production of the target BIA precursor can be monitored. Any suitable method can be used to observe and measure the BIA precursor. The target method includes, but is not limited to, an LC-MS method (e.g., as described herein), wherein the target sample is analyzed by comparison with a known amount of a standard compound. The identity can be confirmed, for example, by m / z and MS / MS fragmentation patterns, and the quantitative or measured compound can be realized by EIC MS peak analysis performed via LC trace peaks of known retention times and / or reference to a known amount of a compound standard substance corresponding to an LC-MS analysis.

[0098] method

[0099] As summarized above, aspects of the invention include methods for preparing a target benzylisoquinoline alkaloid (BIA). Thus, aspects of the invention include culturing host cells under conditions that functionally express one or more host cell modifications (e.g., as described herein) so that the cells convert the target starting compound into a target product BIA or a precursor thereof (e.g., pre-coconutine BIA). The following method is also provided, comprising culturing host cells under conditions suitable for protein production so that one or more heterologous coding sequences are functionally expressed and the target starting compound is converted into the target product BIA. In some examples, the method is a method for preparing a benzylisoquinoline alkaloid (BIA), comprising culturing host cells (e.g., as described herein); adding the starting compound to the cell culture; and recovering BIA from the cell culture. In some embodiments of the method, the starting compound, the BIA product, and the host cell are described by one of the entries in Table 1.

[0100] Any suitable method of cultivating host cells can be used to produce target BIA precursors and downstream BIA. The specific scheme adopted can vary, for example, depending on the host cell, heterologous coding sequence, required BIA precursor, etc. The cell can be present in any suitable environment, such as an environment in which the cell can express one or more functional heterologous enzymes. As used herein, in vitro simply means the outside of living cells, regardless of the position of the cell. As used herein, the term in vivo indicates the inside of living cells, regardless of the position of the cell. In some embodiments, the cell is cultivated under conditions conducive to enzyme expression and with a suitable substrate that can be used to allow the production of BIA precursors in vivo. In some embodiments, the functional enzyme is extracted from the host for the production of BIA under in vitro conditions. In some instances, the host cell is put back into a multicellular host organism. The host cell is in any growth phase, including but not limited to stationary phase and logarithmic growth phase, etc. In addition, the culture itself can be a continuous culture or they can be batch cultures.

[0101] Any suitable cell culture conditions for a particular cell type can be utilized. In certain embodiments, using standard cell culture medium and supplements, the host cell comprising one or more modifications is cultured under standard or easily optimized conditions. As an example, when the selective pressure for plasmid maintenance is not required, the standard growth medium may include 20g / L yeast extract, 10g / L peptone and 20g / L dextrose (YPD). The host cell containing the plasmid is grown in a synthetic complete (SC) medium containing 1.7g / L yeast nitrogen source, 5g / L ammonium sulfate, 20g / L dextrose, supplemented with growth and selection of the appropriate amino acids required. Alternative carbon sources that can be used for inducing enzyme expression include but are not limited to sucrose, raffinose and galactose. In the laboratory, the cell is grown in a container at any suitable temperature (e.g., 30°C) under any suitable rate (e.g., 200rpm) of vibration, such as in a test tube or flask of 1-1000mL or a larger range of volumes. The culture volume can also be scaled up for use in, for example, growing in a larger fermentation vessel as a part of an industrial process.

[0102] Any suitable codon optimization technique for optimizing expression of a heterologous polynucleotide in a host cell may be adapted for use in the subject host cells and methods, see, e.g., Gustafsson, C et al. (2004) Trends Biotechnol, 22, 346-353, which is incorporated by reference in its entirety.

[0103] The subject method can also include adding the starting compound to the cell culture. Any suitable addition method can be suitable for use in the subject method. The cell culture can be supplemented with a sufficient amount of the target starting material (e.g., as described herein), such as mM to μM amount, such as the starting compound between about 1-5mM. It should be understood that the amount of the starting material added, the timing and rate of addition, the form of the material added, etc. can vary according to a variety of factors. The starting material can be added purely or the starting material can be dissolved in a suitable solvent (e.g., cell culture medium, water or organic solvent) in advance. The starting material can be added in a concentrated form (e.g., more than the desired concentration 10x) to minimize the dilution of the cell culture medium when added. The starting material can be added in one or more batches, or added by adding continuously over an extended period of time (e.g., hours or days).

[0104] The subject method can also include reclaiming target BIA precursor or downstream BIA from cell culture.Any suitable separation and isolation method (e.g., chromatographic method or precipitation method) can be suitable for use in the subject method to reclaim target BIA or its precursor from cell culture.Filtration method can be used for separating the soluble part and insoluble part of cell culture.In some cases, liquid chromatography method (e.g., reversed-phase HPLC, size exclusion, normal phase chromatography) is used to separate BIA or precursor from other soluble components of cell culture.In some cases, extraction method (e.g., liquid extraction, purification based on pH, etc.) is used to separate BIA precursor or BIA from other components of cell culture.

[0105] Also included is a method for engineering host cells for the purpose of producing target BIA or its precursor. Any suitable method can be used to insert DNA into the host cell. The method is used to insert a heterologous coding sequence into the host cell so that the host cell functionally expresses the enzyme and converts the target starting compound into the target product BIA.

[0106] Any suitable promoter can be used in the subject host cell and method. The promoter driving the expression of the heterologous coding sequence can be a constitutive promoter or an inducible promoter, provided that the promoter is active in the host cell. The heterologous coding sequence can be expressed from its native promoter, or a non-natural promoter can be used. Such promoters should be low to high strength in the host using them. The promoter can be regulated or constitutive. In certain embodiments, a promoter that is not inhibited by glucose or is only slightly inhibited when glucose is present in the culture medium is used. Target promoters include, but are not limited to, promoters of glycolytic genes, such as the promoter of the Bacillus subtilis gene (the promoter region of the fructose bisphosphate aldolase gene encoding) or the promoter of the glyceraldehyde 3-phosphate dehydrogenase gene from the yeast Saccharomyces cerevisiae (GPD, GAPDH or TDH3); the ADH1 promoter of baker's yeast; phosphate starvation-induced promoters, such as the PHO5 promoter of yeast; the alkaline phosphatase promoter from Bacillus licheniformis; yeast inducible promoters, such as Gal1-10, Gal1, GalL, GalS; repressible promoters Met25, tetO; and constitutive promoters, such as the glyceraldehyde 3-phosphate dehydrogenase promoter (GPD), the alcohol dehydrogenase promoter (ADH), the translation elongation factor-1-α promoter (TEF), the cytochrome c-oxidase promoter (CYC1), the MRP7 promoter, etc. Self-replicating yeast expression vectors containing promoters that can be induced by hormones (such as glucocorticoids, steroids and thyroid hormones) can also be used and include but are not limited to glucocorticoid responsive elements (GRE) and thyroid hormone responsive elements (TRE). These and other examples are described in U.S. Patent No. 7,045,290, which is incorporated by reference, including the references cited therein. Other vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH can be used. In addition, any promoter / enhancer combination (according to the eukaryotic promoter database EPDB) can also be used to drive the expression of genes. Any suitable appropriate promoter can be selected for host cells, such as Escherichia coli. Promoter selection can also be used to optimize transcription, and therefore, optimize enzyme levels to maximize production while minimizing energy resources.

[0107] Any suitable vector can be used in the subject host cells and methods. Target vectors include those for use in yeast and other cells. The types of yeast vectors can be divided 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 (YAC). Vector DNA is introduced into prokaryotic or eukaryotic cells via any suitable transformation or transfection technique.

[0108] utility

[0109] The host cells and methods of the present invention (e.g., as described above) are suitable for a variety of applications. Target applications include, but are not limited to, research applications and therapeutic applications. The methods of the present invention are suitable for a variety of different applications, including any suitable application in which the production of BIA is of interest.

[0110] The subject host cells and methods are suitable for a variety of therapeutic applications. Target therapeutic applications include those applications in which the preparation of pharmaceutical products containing BIA is of interest. The host cells described herein produce benzylisoquinoline alkaloid precursors (BIA precursors). Cynocarpine is a target major branch point intermediate in the synthesis of BIAs including engineering efforts to produce final products such as opioid products. The subject host cells can be used to produce BIA precursors from simple and inexpensive starting materials, which can be used to produce cynocarpine and BIA final products. Therefore, the subject host cells are suitable for supplying therapeutically active BIA or its precursors.

[0111] In some instances, host cells and methods are suitable for producing commercial-scale quantities of BIA or its precursors, where chemical synthesis of these compounds is low-yield and is not a feasible means for large-scale production. In some cases, host cells and methods are used in fermentation facilities that will include, for example, bioreactors (fermenters) of 5,000-200,000 liters of capacity, thereby allowing rapid production of target BIA or its precursors for therapeutic products. Such applications may include industrial-scale production of target BIA from fermentable carbon sources such as cellulose, starch, and free sugars.

[0112] The subject host cell and method are applicable to multiple research applications. The subject host cell and method can be used for analyzing the effect of multiple enzymes on the biosynthetic pathway of multiple target BIA or its precursor. In addition, host cell can be engineered to produce BIA or its precursor, and described BIA or its precursor is applicable to test the target biological activity in terms of unconfirmed therapeutic function. In some cases, engineered host cell illustrates the high-yield biosynthetic pathway for target BIA or its precursor with a variety of heterologous coding sequences comprising encoding multiple enzymes. In some cases, research application includes producing precursors for target therapeutic molecules, and described precursor can be further chemically modified or derivatized to desired products or screened for increased target therapeutic activity subsequently. In some instances, host cell strains are used to screen for interested enzymatic activity in this type of pathway, which can result in finding enzymes via the conversion of the BIA metabolites produced in these strains.

[0113] The subject host cell and method can be used as the production platform of plant specialized metabolites.The subject host cell and method can be used as the platform of drug library development and plant enzyme discovery.For example, the subject host cell and method can be applicable to produce interesting scaffold molecules (such as protopine) by adopting yeast strain and develop drug library based on natural product by combinatorial biosynthesis or by chemical means further functionalization compound structure.By producing drug library in this way, any potential drug hit is relevant to the production host that is easy to large-scale culture and production.As another example, these subject host cells and methods can be applicable to plant enzyme discovery.The subject host cell provides the clean background of limiting metabolites to express plant EST library to identify new enzyme activity.The subject host cell and method provide the expression method and culture condition of functional expression and activity increase of plant enzyme in yeast.

[0114] Kits and Systems

[0115] Aspects of the invention also include kits and systems, wherein the kits and systems may include one or more components employed in the methods of the invention, e.g., host cells, starting compounds, heterologous coding sequences, vectors, culture media, etc. (as described herein). In some embodiments, the subject kits include host cells (e.g., as described herein), and one or more components selected from the following: starting compounds, heterologous coding sequences and / or vectors comprising the heterologous coding sequences, vectors, growth materials, components suitable for expression systems (e.g., cells, cloning vectors, multiple cloning sites (MCS), bidirectional promoters, internal ribosome entry sites (IRES), etc.), and culture media.

[0116] Any components described herein can be provided in a kit, for example, a host cell comprising one or more modifications, a starting compound, a culture medium, etc. A variety of components, cloning vectors, and expression systems suitable for preparing and using heterologous coding sequences can be applied to the subject kit. The kit can also include tubes, buffers, etc., and instructions for use. As desired, the various reagent components of the kit can be present in separate containers, or some or all of the components can be pre-combined in a reagent mixture in a single container.

[0117] Also provided are systems for producing a BIA of interest, wherein the systems may include engineered host cells comprising one or more modifications (e.g., as described herein), starting compounds, culture media, fermentors, and fermentation equipment, e.g., equipment suitable for maintaining growth conditions for the host cells, sampling and monitoring equipment and components, etc. Various components suitable for large-scale fermentation of yeast cells may be suitable for use in the subject systems.

[0118] In some cases, the system includes large-scale fermentation of the host cell for engineering and monitoring and purification of the BIA compound produced by the fermented host cell. In certain embodiments, the host cell of the engineering in the fermentor produces one or more required BIA products or conditions of its precursor by one or more starting compounds (for example, as described herein) added to the system. In some instances, the host cell produces a target BIA precursor (for example, as described herein). In some cases, the target BIA product is an opioid product, such as codeine, neopine (neopine), morphine, new morphine, hydrocodone, oxycodone, hydromorphone, dihydrocodeine, 14-hydroxycodeine or dihydromorphine.

[0119] In some cases, the system includes a device for monitoring and or analyzing one or more BIA compounds or their precursors produced by the subject host cell. For example, an LC-MS analysis system, a chromatographic system or any suitable system as described herein, wherein a sample can be analyzed or compared with a standard (for example, as described herein). The fermentation medium can be monitored at any suitable time before and during fermentation by sampling and analysis. When the starting compound is converted into a target BIA product or precursor and completed, fermentation can stop and the purification of the BIA product can be carried out. Therefore, in some cases, the subject system includes a purification component suitable for purifying the target BIA product or precursor of the host culture medium produced therein. The purification component can include any suitable mode for the BIA product or precursor that can be used for purifying fermentation, including but not limited to, silica chromatography, reverse phase chromatography, ion exchange chromatography, HIC chromatography, size exclusion chromatography, liquid extraction and pH extraction method. In some cases, the subject system provides the generation and separation of the target BIA fermentation product after one or more starting compounds are input to the system.

[0120] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of the invention as viewed by the inventors, nor are they intended to represent that the following experiments are all or the only experiments performed. Although every effort has been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), some experimental errors and deviations should still be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is degrees Celsius, and pressure is at or near atmospheric pressure.

[0121] experiment

[0122] Embodiment 1

[0123] A series of specific genetic modifications provide biosynthetic processes in Saccharomyces cerevisiae for producing BIAs from simple, inexpensive raw materials or precursor molecules. Methods for constructing novel strains capable of producing early BIA molecules, norhicladin (NC) and holonorhavnine (NL), from non-BIA precursors or simple raw materials are described. NC has never been reported as a product of microbial synthesis and is a natural precursor of 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.

[0124] A. Yeast strains overproducing tyrosine and related BIA precursors

[0125] For the purpose of increasing the intracellular concentration of BIA precursor molecules, strains of Saccharomyces cerevisiae were developed at improved flux through the aromatic amino acid biosynthetic pathway, including tyrosine, 4-hydroxyphenylacetaldehyde (4-HPA), L-3,4-dihydroxyphenylalanine (L-DOPA), 3,4-dihydroxyphenylacetaldehyde (3,4-DHPA) and dopamine. These strains generally combine genetic modifications to yeast strains for the purpose of increasing carbon flux from central metabolism for aromatic amino acid synthesis, and include the introduction of key heterologous enzymes for the production of BIA precursor molecules that are not naturally produced by yeast. Genetic modifications were used, including the introduction of feedback inhibition mitigation mutations into genes encoding natural biosynthetic enzymes, adjusting the transcriptional regulation of natural biosynthetic enzymes, deleting genes encoding enzymes that divert precursor molecules away from the intended pathway, and introducing heterologous enzymes for the conversion of natural endogenous molecules into non-natural BIA precursor molecules.

[0126] Specific description:

[0127] 1.1) Biosynthetic pathways in engineered strains Feedback inhibition mitigation mutations (1.1.1) were incorporated into the native yeast gene ARO4, which encodes 3-deoxy-d-arabinose-heptulose-7-phosphate (DAHP) synthase, either alone or in combination. This mutation (ARO4 FBR ) as a directed mutation of the native gene at the original locus, as an additional copy introduced as a genetic integration at a separate locus, or incorporated as an additional copy on an episomal vector such as 2μ or a centromeric plasmid. FBR refers to feedback resistant mutants and mutations. A feedback inhibited copy of DAHP synthase is introduced under native yeast transcriptional regulation or by placing the feedback inhibited copy under the control of a synthetic promoter under engineered constitutive or dynamic regulation of protein expression.

[0128] 1.1.1) ARO4 FBRThe mutation may include, for example, substitution of the lysine residue at position 229 with leucine (see, e.g., Hartmann M, et al. (2003) Evolution of feedback-inhibited beta / alpha barrel isoenzymes by gene duplication and a single mutation. Proc Natl Acad Sci US A 100(3):862-867), substitution of the glutamine residue at position 166 with a lysine residue (see, e.g., Fukuda K et al. (1992) Feedback-Insensitive Mutation of 3-Deoxy-D-Arabino-Hepturosonate-7-Phosphate Synthase Caused by a Single Nucleotide Substitution of Aro4 Structural Gene in Saccharomyces-Cerevisiae. J Ferment Bioeng 74(2):117-119), or an additional mutation conferring feedback inhibition selected from a mutagenesis library of microbial DHAP synthase mutants. Examples of such selections include growth on o-fluoro-D,L-phenylalanine on media rescued with excess tyrosine (see, e.g., Fukuda et al. (1990) Breeding of Brewing Yeast Producing a Large Amount of Beta-PhenylethylAlcohol and Beta-Phenylethyl Acetate. Agr Biol Chem Tokyo 54(1):269-271) or growth of an aro3 mutant yeast strain.

[0129] 1.2) Biosynthetic pathways in engineered strains A feedback inhibition alleviating mutation (1.2.1) was incorporated into the native yeast gene ARO7, which encodes the enzyme chorismate mutase. This mutation (ARO7 FBR ) as a directed mutation of 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 episomal vector such as 2μ or a centromeric plasmid. A feedback-inhibited copy of the chorismate mutase is introduced under native yeast transcriptional regulation or by placing the feedback-inhibited copy under the control of a synthetic promoter under engineered constitutive or dynamic regulation of protein expression.

[0130] 1.2.1) ARO7 FBRMutant alleles may include, for example, substitution of the threonine residue at position 226 with isoleucine (see, e.g., Schmidheini et al. (1989) A Single Point Mutation Results in aConstitutively Activated and Feedback-Resistant Chorismate Mutase ofSaccharomyces-Cerevisiae. J Bacteriol 171(3): 1245-1253) or additional mutations conferring feedback inhibition selected from a mutagenesis library of microbial chorismate mutase mutants. Examples of such selections include assays for 5-methyltryptophan sensitivity or increased melanin production in strains expressing heterologous tyrosinase (1.9) in the absence of externally fed tyrosine.

[0131] 1.3) Biosynthetic pathways in engineered strains incorporate the introduction of strong promoter elements (such as GPD1, TEF1, etc.) for overexpression of the native yeast gene ARO10, which encodes an enzyme with hydroxyphenylpyruvate decarboxylase activity. This genetic modification is incorporated as a directed exchange of the native promoter DNA sequence at the original 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 episomal vector such as 2μ or a centromeric plasmid.

[0132] 1.4) Biosynthetic pathways in engineered strains incorporate the introduction of strong promoter elements (such as GPD1, TEF1, etc.) for overexpression of the native yeast gene ARO9, which encodes an enzyme with hydroxyphenylpyruvate / glutamate transaminase activity. This genetic modification is incorporated as a directed exchange of the native promoter DNA sequence at the original 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 episomal vector such as 2μ or a centromeric plasmid.

[0133] 1.5) Biosynthetic pathways in engineered strains incorporate the introduction of strong promoter elements (such as GPD1, TEF1, etc.) for overexpression of the native yeast gene TKL, which encodes an enzyme with transketolase activity. This genetic modification is incorporated as a directed exchange of the native promoter DNA sequence at the original 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 episomal vector such as 2μ or a centromeric plasmid.

[0134] 1.6) The biosynthetic pathway in the engineered strain was improved by incorporating a deletion or inactivating mutation of the native yeast gene ZWF1, which encodes an enzyme with glucose-6-phosphate dehydrogenase activity.

[0135] 1.7) The biosynthetic pathway in the engineered strain is improved by incorporating one or more deletions or inactivating mutations of a known native alcohol dehydrogenase, such as the enzyme encoded by the genes ADH2, ADH3, ADH4, ADH5, ADH6, ADH7 or SFA1.

[0136] 1.8) The biosynthetic pathway in the engineered strain is improved by incorporating one or more deletions or inactivating mutations of known native aldehyde oxidoreductases (eg, ALD2, ALD3, ALD4, ALD5 or ALD6).

[0137] 1.9) Biosynthetic pathways incorporate heterologous enzymes for converting tyrosine to L-DOPA. The enzyme may be from one of the classes including, but not limited to, bacterial tyrosinase, eukaryotic tyrosinase, and tyrosine hydroxylase (Table 1). The gene for the enzyme is incorporated as a genetic integration or on an episomal vector such as 2μ or a centromeric plasmid. This L-DOPA-producing enzyme is introduced by placing the enzyme under the control of a synthetic promoter under engineered constitutive or dynamic regulation of protein expression.

[0138] 1.9.1) In the biosynthetic pathway using tyrosine hydroxylase for the conversion of tyrosine to L-DOPA, additional expression of genes encoding enzymes for the synthesis and recycling of the pterin cofactor tetrahydrobiopterin (BH4) and its derivatives is incorporated into the engineered strain to support the activity of tyrosine hydroxylase. These enzymes include GTP cyclohydrolase, 6-pyruvyl-tetrahydropterin synthase, septopterin reductase, 4a-hydroxytetrahydrobiopterin dehydratase, and quinone-type dihydropteridine reductase (Table 1). The genes for these enzymes are incorporated as genetic integrations or on episomal vectors such as 2μ or centromeric plasmids. These BH4 synthesis and recycling enzymes are introduced by placing the enzymes under the control of synthetic promoters in engineered constitutive or dynamic regulation of protein expression.

[0139] 1.10) The biosynthetic pathway incorporates heterologous enzymes for decarboxylating L-DOPA to produce dopamine. Enzymes with this activity are encoded by genes from a variety of organisms, including bacteria, plants, and mammals. Examples include: Pseudomonas putida DOPA decarboxylase (PpDODC), Rattus norvegicus DOPA decarboxylase (RnDODC), and Papaver somniferum tyrosine / DOPA decarboxylase (PsTYDC) (Table 1). The genes for the enzymes are incorporated as genetic integrations or on episomal vectors such as 2μ or centromeric plasmids. Such dopamine-producing enzymes are introduced by placing the enzyme under the control of a synthetic promoter in engineered constitutive or dynamic regulation of protein expression.

[0140] 1.11) 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 such enzymes that can be used in strains include human monoamine oxidase A (hMAOA), Escherichia coli monoamine oxidase (EcMAO), and Micrococcus luteus monoamine oxidase (MIMAO) (Table 1). The gene for the enzyme is incorporated as a genetic integration or incorporated on an episomal vector such as 2μ or a centromeric plasmid. This 3,4-DHPA-producing enzyme is introduced by placing the enzyme under the control of a synthetic promoter under engineered constitutive or dynamic regulation of protein expression.

[0141] 1.11.1) The strain used to produce NC requires dopamine and 4-HPA, while the strain used to produce NL requires dopamine and 3,4-DHPA, but not 4-HPA. The specific modification used to convert the strain producing NC into the strain producing NL is the integration of the MAO gene into the yeast genome at the locus encoding the native yeast gene ARO10. This combination is responsible for the deletion of the native yeast enzyme for converting the tyrosine biosynthetic precursor to 4-HPA and the introduction of an enzyme capable of converting dopamine to 3,4-DHPA.

[0142] 1.11.2) A yeast strain was constructed with a gene expressing the MAO enzyme (1.11) under the control of an inducible promoter. When the strain is grown in the presence of an inducer, it catalyzes the conversion of dopamine to 3,4-DHPA, and in the absence of an inducer, the strain produces only 4-HPA.

[0143] 1.11.2.1) A yeast strain was constructed with inducible MAO expression (1.11.2), wherein the inducible system also contains a DNA binding protein (1.3) that targets the promoter of the ARO10 gene. The synthetic promoter controlling ARO10 is thus repressed when the promoter controlling the MAO gene is activated, and ARO10 is only expressed when the MAO gene is not transcriptionally active. This system allows the construction of a single strain that conditionally produces only the precursors of NC or NL.

[0144] B. Yeast strains producing NC

[0145] Methods for producing the BIA molecule NC in yeast were developed and the first system for microbial synthesis of NC was demonstrated. Using the engineered strains described herein, NC was produced and accumulated for its own value or combined with the biosynthetic pathway of additional heterologous enzymes for completing the synthesis of downstream BIAs.

[0146] Specific description:

[0147] 2.1) Yeast strains are grown in liquid culture to high cell concentrations, then diluted back to intermediate concentrations (as measured by optical density or OD) in defined medium containing high concentrations of dopamine. The medium components need only satisfy the conditions for growth of the strain; various growth feedstocks (e.g., different sugars, nitrogen sources) are used. NC produced by these yeast strains is excreted by the yeast cells and can be measured in the spent medium. Additional NC retained by the cells is recovered via cell lysis and extraction from the lysate.

[0148] 2.2) Yeast strains containing various combinations of modifications as described in (1.1-1.8) substantially improve the measurable production of NC from unmodified strains in a fed dopamine assay, as described above (2.1). The modifications (1.1-1.8) provide for production of NC from fed dopamine under conditions where no extracellular tyrosine is available in the yeast culture medium; NC production from unmodified yeast strains under these conditions is most often undetectable.

[0149] 2.3) Yeast strains producing NC when containing the modifications as described in (1.10) were grown as described in (2.1), when the additional BIA precursor added to the culture medium was L-DOPA instead of dopamine.

[0150] 2.3.1) Yeast strains as described in (2.3) containing different combinations of modifications as described in (1.1-1.8) substantially improve the production of NC.

[0151] 2.4) Yeast strains producing NC when containing heterologous enzymes for converting tyrosine to dopamine (1.9, 1.10) together with different combinations of the modifications (1.1-1.8) described above are grown in a medium not supplemented with tyrosine, L-DOPA or dopamine. This specific example constitutes complete synthesis of NC by the strain from a simple carbon and nitrogen source.

[0152] 2.5) The yeast strain is modified and cultured as described above (2.1-2.4), wherein the biosynthetic pathway includes the incorporation of a heterologous enzyme NCS, or a truncated version of the NCS enzyme, for stereospecific catalysis of the reaction to condense dopamine and 4-HPA for S-NC production. This enzyme can be derived from one of several plants, such as opium poppy, Japanese goldenseal, and yellow thrush (Table 1). The gene for the enzyme is incorporated as a genetic integration or incorporated on an episomal vector such as 2μ or a centromeric plasmid. This enzyme that produces S-NC is introduced by placing the enzyme under the control of a synthetic promoter under engineered constitutive or dynamic regulation of protein expression. The final NC produced will be a mixture of enantiomers, with a preference for the S-stereoisomer.

[0153] C. NL-producing yeast strains

[0154] Methods were developed to produce the BIA molecule NL from yeast. Using the engineered strains described herein, NL was produced and accumulated for its own value or combined with biosynthetic pathways of other heterologous enzymes for use in completing the synthesis of downstream BIAs.

[0155] Specific description:

[0156] 3.1) Yeast strains containing modifications as described in (1.11, 1.11.1-1.11.2, 1.11.2.1) are grown in liquid culture as described in (2.1) to produce NL.

[0157] 3.2) Yeast strains containing various combinations of gene deletions as described in (1.7, 1.8) improved NL production in the fed dopamine assay over that measurable in the unmodified strain, as described above (3.1).

[0158] 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) are grown as described in (3.1), when the additional BIA precursor added to the medium is L-DOPA instead of dopamine.

[0159] 3.3.1) Yeast strains as described in (3.3) containing combinations of gene deletions as described in (1.7, 1.8) improve NL production.

[0160] 3.4) Yeast strains producing NL when containing heterologous enzymes for converting tyrosine to dopamine (1.9, 1.10) and dopamine to 3,4-HPA (1.11) together with different combinations of modifications described above (1.1-1.8, 1.11.1) are grown in medium not supplemented with tyrosine, L-DOPA or dopamine. This specific example constitutes complete synthesis of NL by the strain from simple carbon and nitrogen sources.

[0161] 3.5) The yeast strain is modified and cultured as described above (3.1-3.4), wherein the biosynthetic pathway includes the incorporation of a heterologous enzyme NCS, or a truncated version of the NCS enzyme (Table 1), for stereospecific catalysis of the reaction to condense dopamine and 3,4-HPA for S-NL production. This enzyme can be derived from one of several plants, such as opium poppy, Japanese goldenseal, and yellow thrush (Table 1). The gene for the enzyme is incorporated as a genetic integration or incorporated on a free-type vector such as 2μ or a centromeric plasmid. This S-NL-producing enzyme is introduced by placing the enzyme under the control of a synthetic promoter under engineered constitutive or dynamic regulation of protein expression. The NC ultimately produced is a mixture of enantiomers, with a preference for the S-stereoisomer.

[0162] Figure 1 :Biosynthesis of tyrosine and BIA precursor molecules

[0163] A schematic diagram of the biosynthetic pathway from glucose to tyrosine and other BIA precursors is shown. Aromatic amino acid intermediates present in natural yeast metabolism are written in black. Endogenous yeast enzymes are written in gray (except TYR, TyrH, DODC and MAO). Heterologous enzymes and non-natural BIA precursor molecules include TYR, TyrH, DODC and MAO. As described in (1.1, 1.2), the wild-type yeast enzymes encoded by ARO4 and ARO7 are allosterically inhibited by tyrosine, indicated here by gray dotted lines. The individual steps in the pentose phosphate pathway and glycolysis are not explicitly detailed in this figure, but the genes TKL1 and ZWF1 (targeted in 1.5, 1.6) are involved in the pentose phosphate pathway, as indicated.

[0164] Figure 2 :Effects of ZWF1 knockout and TKL1 overexpression on the pentose phosphate pathway (PPP)

[0165] Schematic diagrams detailing how modifications to TKL1 (1.5) and ZWF1 (1.6) affect total carbon flow through the pentose phosphate pathway in yeast when glucose is the primary carbon source. Panel A shows wild-type carbon flow; Panel B shows relative changes in carbon flow in modified strains.

[0166] Figure 3 A: Synthesis of NC from precursor molecules

[0167] NC is synthesized from one molecule of dopamine and one molecule of 4-HPA via the Pickett-Spengler condensation reaction. The reaction can occur spontaneously to produce a racemic mixture of R- and S-NC. The reaction can alternatively be catalyzed by the plant enzyme NCS, which produces S-NC.

[0168] Figure 3 B: Synthesis of NL from precursor molecules

[0169] NL is synthesized from one molecule of dopamine and one molecule of 3,4-DHPA via the Pickett-Spengler condensation reaction. The reaction can occur spontaneously to produce a racemic mixture of R- and S-NL. Although 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)-Norlaudanosoline Synthase-the 1st Enzyme in the Benzylisoquinoline Biosynthetic-Pathway. Febs Lett 129(1):5-9).

[0170] Measurement of BIA molecules was performed by LC-MS molecules, 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 consistent with standard and published detection methods (see, e.g., Schmidt et al. (2007) Poppy alkaloid profiling by electrospraytandem mass spectrometry and electrospray FT-ICR mass spectrometry after [ring-13C6]-tyramine feeding. Phytochemistry 68(2): 189-202).

[0171] Figure 4 : Effects of four genetic modifications on NC production under different tyrosine feeding conditions.

[0172] NC production was demonstrated in strains with targeted genetic modifications at several concentrations of fed tyrosine, including no fed tyrosine. The wild-type strain CEN.PK2 was integrated with a construct conferring one of the four genetic changes (as described in 1.1-1.4): TEF1 Promoter replacement to overexpress ARO10, by using P TEF1 Promoter replacement to overexpress ARO9 and ARO4 FBR Chromosomal integration of alleles and ARO7 FBR When incorporated individually, only P TEF1 -ARO10 and ARO4 FBR While these modifications increased NC production at all tyrosine concentrations, ARO FBR The integrated strains improved most significantly under zero-fed tyrosine.

[0173] Figure 5 : NC production in combination with genetic modifications

[0174] Some of the genetic modifications described above (1.5, 1.6) are only associated with ARO4 FBR Integrated combinations of mutants improve NC production (1.1). The figure shows four strains P engineered with a single genetic modification. TEF1 -AR010(1.3),P GPD -TKL1 (1.5), ZWF1 knockout (1.6), and ARO4 FBR (1.1), together with three strains constructed by genetic modification combinations: strain A (P GPD -TKL1, ARO4 FBR ), strain B (ZWF1 knockout, ARO4 FBR) and strain C(P GPD -TKL1, ZWF1 knockout, ARO4 FBR ). NC production is shown normalized to the WT strain, with strain C showing a five-fold increase in NC production.

[0175] Figure 6 :NL production in ALD / ADH knockout strains

[0176] NL production was improved by deleting competing yeast enzymes (1.7, 1.8) in a strain expressing human MAOA on a 2μ plasmid (1.11) and growing in medium containing dopamine. NL production is shown as titers measured in spent medium normalized to WT (with hMAO, but without deletions). The improvement in production was more than ten-fold over WT NL production.

[0177] Figure 7 :Activity of DODC enzyme in vivo

[0178] Yeast strains transformed with DNA to express opium poppy tyrosine / DOPA decarboxylase are able to convert L-DOPA to dopamine in vivo. Strains with the 2μ plasmid were grown in selective medium and then diluted back into medium containing L-DOPA. The spent medium was then measured for concentrations of L-DOPA (retention time 4.8 minutes, m / z = +198) and dopamine (retention time 4.2 minutes, m / z = +154).

[0179] Figure 8

[0180] NCs were produced in various wild-type yeast laboratory strains at different tyrosine concentrations as described in (2.1). Specifically, each yeast strain was inoculated into a separate liquid culture and grown overnight to an OD of 600 ~10, and then diluted back to OD in YNB minimal medium without tyrosine 600~1 and grown for 3 hours. 100 μl of each culture was mixed into 400 μl YNB medium containing 100 mM dopamine and different concentrations of tyrosine; each strain was grown in triplicate samples in each medium condition. NC titers were measured from culture supernatants on an LC-MS instrument, detecting m / z+272 ion counts in the peak as described by (see, e.g., Schmidt et al. (2007) Poppy alkaloid profiling by electrospray tandem mass spectrometry and electrospray FT-ICR massspectrometry after [ring-13C6]-tyramine feeding. Phytochemistry 68(2): 189-202). The area of ​​each peak was integrated to calculate the relative quantification of NC in each sample, and the results were normalized to the ion count area in CEN.PK2 yeast culture with 0 mg / L tyrosine.

[0181] Fig. 9

[0182] NCs were produced as described in (2.1) in various engineered yeast strains fed 100 mM dopamine and without tyrosine. Figure 5 The strains CSY977-981 were engineered to contain a combination of the genetic modifications described in (1.1-1.6); the labels below each strain name indicate which modification was incorporated into each strain. Strain CSY981 contains five genetic modifications to the native metabolism of yeast and exhibits a twenty-fold increase in NC titer over wild-type strain CEN.PK2.

[0183] Fig.10

[0184] NCs were produced as described in (2.1) (for black diamonds) and as described in (2.3) (for gray circles). Here, NCs were produced in an engineered yeast strain (CSY980) that additionally incorporated the L-DOPA decarboxylase PpDODC (1.10). In separate liquid cultures, this yeast strain was grown in YNB minimal medium containing different concentrations of dopamine (black diamonds) and YNB minimal medium containing L-DOPA (gray circles; cultures not fed with dopamine). The solid black line represents the linear regression of the relationship between measured NCs and fed dopamine. The peak area measurements for the L-DOPA-fed samples were plotted along the regression line for the dopamine-fed samples to show the "equivalent fed dopamine" amount for the cultures fed with L-DOPA. The L-DOPA medium was mixed to achieve a target concentration of 10 mM L-DOPA, however L-DOPA was not completely soluble at that concentration and the effective concentration of dissolved L-DOPA was estimated to be approximately 6 mM. Based on the average NC titers of the two L-DOPA-fed yeast cultures, the "equivalent fed dopamine" concentration was approximately 50 mM or 8 times the fed L-DOPA concentration (indicated by the grey dashed line).

[0185] Fig.11

[0186] Mammalian tyrosine hydroxylase (TyrH) is capable of hydroxylating tyrosine, but is dependent on the co-substrate tetrahydrobiopterin (BH4) for activity, as described in (1.9.1). During the catalysis of tyrosine to L-DOPA by TyrH, molecular oxygen is split and transferred to tyrosine and BH4, as shown by reaction 1. BH4 is oxidized to BH4-4α-methanolamine (4αOH-BH4). Two heterologous enzymes are expressed in yeast to synthesize BH4 from dihydroneopterin triphosphate, an intermediate in the folate synthesis pathway. First, 6-pyruvyltetrahydropterin synthase (PTPS) converts dihydroneopterin to PTP (reaction 2), which is then reduced to BH4 by Seppterin reductase (SepR, reaction 3). Both enzymes are responsible for regenerating BH4 from its 4α-methanolamine form. First, pterin-4a-methanolamine dehydratase (PCD) catalyzes a water loss reaction to form dihydrobiopterin (reaction 4). Dihydrobiopterin is subsequently reduced to tetrahydrobiopterin by quinone-type dihydropteridine reductase (QDHPR, reaction 5).

[0187] Fig.12

[0188] Tyrosine hydroxylase expressed from yeast cells converts tyrosine to L-DOPA. Yeast strains transformed with plasmids carrying tyrosine hydroxylases from humans (hTH2) and rats (RnTyrH) were grown in liquid culture medium and then dissolved in a buffer containing tyrosine and co-substrate BH4. After incubation at 30°C for 6 hours, L-DOPA in the lysate mixture was measured by LC-MS. (A) LC-MS chromatograms confirm that tyrosine is converted to L-DOPA depending on the presence of co-substrate BH4. (B) Fragmentation of the +198 m / z ion peak further confirms the presence of L-DOPA in the lysate sample. (See, e.g., Lv et al. (2010) LC-MS-MS Simultaneous Determination of L-Dopa and its prodrug n-PentylHydrochloride in Rat Plasma. Chromatographia, 72 (3 / 4), 239-243).

[0189] Fig.13

[0190] Co-expression of tyrosine hydroxylase with BH4 biosynthetic enzymes enables conversion of tyrosine to L-DOPA in yeast cell lysates. An engineered yeast strain incorporating constructs expressing rat tyrosine hydroxylase (RnTyrH) and rat septopterin reductase (RnSepR) was grown in liquid culture and then lysed in a buffer containing tyrosine, NADPH, and the BH4 biosynthetic precursor septopterin. Co-expression of TyrH with BH4 biosynthetic genes provides activity of tyrosine hydroxylase in the absence of BH4, but in the presence of the BH4 precursor septopterin.

[0191] Fig.14

[0192] The BIA molecules coclaurine and N-methylcoclaurine are synthesized from NC. The conversion of NC into downstream BIA molecules by plant methyltransferases extends microbial BIA synthesis. These specific BIA molecules can only be synthesized via a NC-dependent biosynthetic scheme.

[0193] Fig.15

[0194] The engineered yeast strain produces NC-derived BIA molecules from L-DOPA in liquid culture. A copy of PpDODC was integrated into the engineered yeast strain CSY979 (e.g. Fig. 9), thereby providing the production of NC from L-DOPA (Figure A, lower chromatogram). Next, a copy of the opium poppy 6-O-methyltransferase (Ps6OMT) gene was integrated into this yeast strain to achieve the production of lacunarine from L-DOPA (Figure A, middle chromatogram). Finally, copies of the Ps6OMT and opium poppy lacunarine-N-methyltransferase (PsCNMT) genes were integrated into the CSY979 yeast strain carrying the PpDODC gene to achieve the production of N-methyllacunarine from L-DOPA (Figure A, upper chromatogram). Both NC and lacunarine measurements matched the chromatogram from the chemical standard. The production of N-methyllacunarine was further confirmed by matching the fragmentation pattern of the +300 m / z ion peak with the pattern in the open literature (Figure B). (See, eg, Schmidt et al. (2007) Poppy alkaloid profiling by electrospraytandem mass spectrometry and electrospray FT-ICR mass spectrometry after [ring-13C6]-tyramine feeding. Phytochemistry 68(2):189-202).

[0195]

[0196]

[0197]

[0198]

[0199] Notwithstanding the accompanying terms, the disclosure set forth herein is defined by the following terms:

[0200] 1. A host cell producing a benzylisoquinoline alkaloid (BIA) precursor, wherein the host cell comprises one or more modifications selected from the group consisting of:

[0201] one or more feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes native to the cell;

[0202] one or more transcriptional regulatory modifications to one or more biosynthetic enzyme genes native to the cell;

[0203] one or more inactivating mutations in one or more enzymes native to the cell; and

[0204] one or more heterologous coding sequences encoding one or more enzymes;

[0205] Wherein when the cell comprises one or more heterologous coding sequences encoding one or more enzymes, the cell 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 regulatory modification to a biosynthetic enzyme gene native to the cell; and an inactivating mutation in an enzyme native to the cell.

[0206] 2. The host cell according to clause 1, wherein the BIA precursor is selected from the group consisting of higenamine (NC) and holonorlabdanine (NL).

[0207] 3. A host cell according to clause 2, wherein the BIA precursor is higenamine (NC).

[0208] 4. A host cell according to clause 2, wherein the BIA precursor is holonorlabdanine (NL).

[0209] 5. A 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, the cell comprises at least one additional modification selected from the group consisting of: transcriptional regulatory modifications to the biosynthetic enzyme genes native to the cell; inactivating mutations in enzymes native to the cell; and heterologous coding sequences encoding enzymes.

[0210] 6. A host cell according to clause 1, wherein when the cell comprises one or more transcriptional regulatory modifications to one or more biosynthetic enzyme genes native to the cell, the cell 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; an inactivating mutation in an enzyme native to the cell; and a heterologous coding sequence encoding an enzyme.

[0211] 7. A host cell according to clause 1, wherein when the cell comprises one or more inactivating mutations in one or more enzymes native to the cell, the cell 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 regulatory modification of a biosynthetic enzyme gene native to the cell; and a heterologous coding sequence encoding an enzyme.

[0212] 8. A host cell according to clause 1, wherein the cell comprises one or more feedback inhibition reducing mutations in one or more biosynthetic enzyme genes native to the cell.

[0213] 9. A host cell according to clause 8, wherein the one or more biosynthetic enzyme genes encode one or more enzymes selected from 3-deoxy-d-arabino-heptulosonic acid-7-phosphate (DAHP) synthase and chorismate mutase.

[0214] 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 ARO4 and ARO7.

[0215] 11. The host cell according to clause 10, wherein the one or more feedback inhibition alleviating mutations are present in the ARO4 gene.

[0216] 12. The host cell according to clause 9, wherein the mutation is selected from a mutation at position 229 and a mutation at position 166 of the DAHP.

[0217] 13. A host cell according to any of the preceding clauses, wherein the cell overproduces one or more BIA precursor molecules.

[0218] 14. A host cell according to clause 13, wherein the one or more BIA precursor molecules are selected from the group consisting of: tyrosine, 4-hydroxyphenylacetaldehyde (4-HPA), L-3,4-dihydroxyphenylalanine (L-DOPA), 3,4-dihydroxyphenylacetaldehyde (3,4-DHPA) and dopamine.

[0219] 15. A host cell according to clause 14, wherein the one or more BIA precursor molecules are 3,4-dihydroxyphenylacetaldehyde (3,4-DHPA) and dopamine.

[0220] 16. A host cell according to clause 14, wherein the one or more BIA precursor molecules are 4-hydroxyphenylacetaldehyde (4-HPA) and dopamine.

[0221] 17. A host cell according to clause 1, wherein the cell comprises one or more transcriptional regulatory modifications to one or more biosynthetic enzyme genes native to the cell.

[0222] 18. The host cell according to clause 17, wherein the transcriptional regulatory modification is replacement of the native promoter of the one or more biosynthetic enzyme genes with a strong promoter.

[0223] 19. The host cell according to clause 18, wherein the one or more biosynthetic enzyme genes are selected from the group consisting of ARO10, ARO9 and TKL.

[0224] 20. A host cell according to clause 1, wherein the cell comprises one or more inactivating mutations in one or more enzymes native to the cell.

[0225] 21. The host cell according to clause 20, wherein the enzyme is glucose-6-phosphate dehydrogenase.

[0226] 22. A host cell according to clause 21, wherein the enzyme is ZWF1.

[0227] 23. A host cell according to clause 20, wherein the cell comprises one or more inactivating mutations in one or more enzymes comprising alcohol dehydrogenase activity that are native to the cell.

[0228] 24. A host cell according to clause 23, wherein the one or more enzymes are selected from the group consisting of ADH2, ADH3, ADH4, ADH5, ADH6, ADH7 and SFA1.

[0229] 25. A host cell according to clause 20, wherein the cell comprises one or more inactivating mutations in one or more enzymes comprising aldehyde oxidoreductase activity that are native to the cell.

[0230] 26. The host cell according to clause 25, wherein the one or more enzymes are selected from the group consisting of ALD2, ALD3, ALD4, ALD5 and ALD6.

[0231] 27. A host cell according to clause 1, wherein the cell comprises one or more heterologous coding sequences encoding one or more enzymes or active fragments thereof.

[0232] 28. The host cell according to clause 27, wherein the host cell comprises NC synthase activity.

[0233] 29. The host cell according to clause 27, wherein the host cell comprises a heterologous coding sequence for NC synthase or an active fragment thereof.

[0234] 30. The host cell according to clause 29, wherein the one or more enzymes or active fragments thereof convert tyrosine into L-DOPA.

[0235] 31. The host cell according to clause 27, wherein the one or more enzymes or active fragments thereof are selected from the group consisting of bacterial tyrosinase, eukaryotic tyrosinase and tyrosine hydroxylase.

[0236] 32. The host cell of clause 27, wherein the cell comprises one or more heterologous coding sequences for one or more enzymes or active fragments thereof that convert L-DOPA into dopamine.

[0237] 33. The host cell of clause 27, wherein the cell comprises one or more heterologous coding sequences for one or more enzymes or active fragments thereof that convert dopamine into 3,4-DHPA.

[0238] 34. A host cell according to clause 33, wherein the one or more enzymes is monoamine oxidase (MAO).

[0239] 35. The host cell of clause 34, wherein the one or more heterologous coding sequences comprises a MAO coding sequence integrated at a genomic locus encoding native ARO10.

[0240] 36. A host cell according to clause 34, wherein the one or more heterologous coding sequences comprises a MAO coding sequence operably linked to an inducible promoter.

[0241] 37. The host cell according to clause 36, wherein the inducible promoter is part of an inducible system comprising a DNA binding protein targeted to regulate the promoter of the ARO10 gene.

[0242] 38. The host cell of clause 27, wherein the heterologous coding sequence is from a source organism selected from the group consisting of opium poppy, goldenseal and Coptis chinensis.

[0243] 39. A host cell according to clause 1, wherein the host cell is a eukaryotic cell.

[0244] 40. The host cell according to clause 39, wherein the eukaryotic cell is a yeast cell.

[0245] 41. The host cell according to clause 40, wherein the yeast cell is a Saccharomyces cerevisiae cell, Schizosaccharomyces pombe cell or a Pichia pastoris cell.

[0246] 42. A host cell according to clause 41, wherein the yeast cell is a Saccharomyces cerevisiae cell.

[0247] 43. A method for preparing benzylisoquinoline alkaloid (BIA), the method comprising:

[0248] Cultivating a host cell according to any one of clauses 1 to 42:

[0249] adding growth material to the cell culture; and

[0250] The BIA is recovered from the cell culture.

[0251] 44. The method according to clause 43, wherein the BIA is a BIA precursor selected from holonorlaudanine and higenamine.

[0252] 45. The method according to clause 43, further comprising adding a starting compound to the cell culture.

[0253] 46. ​​The method according to clause 43, further comprising producing a BIA precursor selected from the group consisting of taurocholine, lagenaria, N-methyllagenaria, and nortaurocholine.

[0254] 47. The method of clause 43, further comprising producing one or more opioid compounds.

[0255] 48. The method of clause 45, wherein the one or more opioid compounds are selected from oripavine, morphine, codeine, hydromorphone, hydrocodone, oxycodone, and oxymorphone from thebaine.

[0256] 49. A kit comprising:

[0257] A host cell according to any one of clauses 1 to 42; and

[0258] One or more components selected from the group consisting of: a starting compound, a growth material, a heterologous coding sequence, a cloning vector, a multiple cloning site (MCS), a bidirectional promoter, an internal ribosome entry site (IRES), and a cell culture medium.

[0259] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art that certain changes and modifications may be made thereto in accordance with the teachings of the invention without departing from the spirit or scope of the appended claims.

[0260] Therefore, the above only illustrates the principle of the present invention.It should be understood that those skilled in the art will be able to envision various arrangements, although these arrangements are not explicitly described or shown in this article, but they embody the principle of the present invention and are included in its spirit and scope.In addition, all embodiments and conditional language cited herein are mainly intended to help readers understand the principle of the present invention and the conception of promoting technology provided by the inventor, and should be interpreted as not limiting such particularly cited embodiments and conditions.In addition, all statements citing the principles, aspects and embodiments of the present invention and its specific embodiments herein are intended to cover their structural equivalents and functional equivalents.In addition, it is hoped that such equivalents include currently known equivalents and equivalents developed in the future, that is, regardless of the structure, any element developed to perform the same function.Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein.On the contrary, the scope and spirit of the present invention are embodied by the appended claims.

Claims

1. A host cell for producing a benzylisoquinoline alkaloid (BIA) precursor, wherein the host cell is a microbial cell, the BIA precursor is selected from the group consisting of: tyrosine, 4-hydroxyphenylacetaldehyde, 4-hydroxyphenylpyruvate, L-3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenylacetaldehyde, dopamine, norhiclaudine (NC) and holonordane (NL), the host cell comprises the following modifications: a feedback inhibition alleviation mutation in an enzyme gene, a transcriptional regulation modification of a biosynthetic enzyme gene, an inactivating mutation in an enzyme, and a heterologous coding sequence encoding an enzyme, wherein the enzyme is involved in a metabolic pathway for producing the precursor benzylisoquinoline alkaloid product, and wherein The feedback inhibition alleviating mutation in the enzyme gene affects one or more enzyme genes selected from ARO4 and ARO7; The transcriptional regulatory modification of the biosynthetic enzyme gene increases the expression of one or more enzyme genes selected from ARO10 and TKL1; The inactivating mutation in the enzyme involves one or more enzymes selected from the group consisting of ZWF1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, SFA1, ALD2, ALD3, ALD4, ALD5 and ALD6; and The enzyme-encoding heterologous coding sequence encodes one or more enzymes selected from the group consisting of TYR, TyrH, FOL2, PTPS, SepR, PCD, QDHPR, DODC, TYDC, MAO, NCS, 6OMT, CNMT and 4'OMT.

2. The host cell of claim 1, wherein the feedback inhibition alleviating mutation is present in the ARO7 gene.

3. The host cell of claim 1, wherein the feedback inhibition alleviating mutation is present in the ARO4 gene. 4 . The host cell of claim 1 , wherein the feedback inhibition alleviating mutation is selected from a mutation at position 229 and a mutation at position 166 of the ARO4.

5. The host cell of claim 1, wherein the inactivating mutation in the enzyme involves ZWF1.

6. The host cell of claim 1, wherein the inactivating mutation in the enzyme involves one or more enzymes selected from the group consisting of ALD2, ALD3, ALD4, ALD5, ALD6, ALD7 and SFA1.

7. The host cell of claim 1, wherein the inactivating mutation in the enzyme involves one or more enzymes selected from the group consisting of ALD2, ALD3, ALD4, ALD5 and ALD6.

8. The host cell of claim 1, wherein the host cell comprises higenamine (NC) synthase activity.

9. The host cell of claim 1, wherein the host cell comprises a heterologous coding sequence for NC synthase or an active fragment thereof.

10. The host cell of claim 1, wherein the heterologous enzyme-encoding sequence comprises a MAO coding sequence integrated at a genomic locus encoding native ARO10.

11. The host cell of claim 10, wherein the heterologous coding sequence encoding an enzyme comprises a MAO coding sequence operably linked to an inducible promoter.

12. The host cell of claim 11, wherein the inducible promoter is part of an inducible system comprising a DNA binding protein that targets and regulates the promoter of the ARO10 gene.

13. The host cell of claim 1, wherein the heterologous coding sequence is from a source organism selected from the group consisting of: opium poppy (P. somniferum), yellow thrush (T. flavum), and Japanese goldenseal (C. japonica).

14. The host cell of claim 1, wherein the transcriptional regulatory modification is replacement of the native promoter of the biosynthetic enzyme gene with a strong promoter.

15. The host cell of claim 1, wherein the biosynthetic enzyme gene is ARO10.

16. The host cell of claim 1, wherein the biosynthetic enzyme gene is TKL1.

17. The host cell of claim 14, wherein the strong promoter is selected from the group consisting of: GAPDH promoter, ADHI promoter, Gal1-10 promoter, Gal1 promoter, GalL promoter, GalS promoter, GPD promoter, ADH promoter, TEF promoter, CYC1 promoter and MRP7 promoter.

18. The host cell of any one of claims 1-17, wherein the cell overproduces one or more BIA precursors.

19. The host cell of claim 18, wherein the one or more BIA precursors are selected from the group consisting of 3,4-dihydroxyphenylacetaldehyde and dopamine.

20. The host cell of claim 18, wherein the one or more BIA precursors are selected from the group consisting of 4-hydroxyphenylacetaldehyde and dopamine.

21. The host cell of claim 18, wherein the one or more BIA precursors are selected from the group consisting of higenamine and holonorlabdanine.

22. The host cell of claim 18, wherein the BIA precursor is higenamine.

23. The host cell of claim 18, wherein the BIA precursor is holonorlabdanine.

24. The host cell of any one of claims 1-23, wherein the host cell is a eukaryotic cell.

25. The host cell of claim 24, wherein the eukaryotic cell is a yeast cell.

26. The host cell of claim 25, wherein the yeast cell is a S. cerevisiae cell, a Schizosaccharomyces pombe cell, or a Pichia pastoris cell.

27. The host cell of claim 25, wherein the yeast cell is a Saccharomyces cerevisiae cell.

28. A method for preparing benzylisoquinoline alkaloid (BIA), the method comprising: Cultivating the host cell according to any one of claims 1 to 27: adding growth material to the cell culture; as well as The BIA is recovered from the cell culture.

29. The method of claim 28, wherein the BIA is a BIA precursor selected from holonorlaudanine and higenamine.

30. The method of claim 28, further comprising adding a starting compound to the cell culture.

31. The method of claim 28, further comprising producing a BIA precursor selected from the group consisting of taurocholine, coclaurine, N-methylcoclaurine, and nortaurocholine.

32. The method of claim 28, further comprising producing one or more opioid compounds.

33. The method of claim 32, wherein the one or more opioid compounds are selected from oripavine, morphine, codeine, hydromorphone, hydrocodone, oxycodone, and oxymorphone from thebaine.

34. A kit comprising: The host cell according to any one of claims 1 to 27; as well as One or more components selected from the group consisting of: a starting compound, a growth material, a heterologous coding sequence, a cloning vector, a multiple cloning site (MCS), a bidirectional promoter, an internal ribosome entry site (IRES), and a cell culture medium.

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