Software Technology for “Benzylisoquinoline Alkaloids (BIA), producing microbes, as well as methods of making and using them”

Biopharmaceuticals – Christina D. Smolke, Catherine Thodey, Isis Trenchard, Stephanie Galanie, Leland Stanford Junior University

Abstract for “Benzylisoquinoline Alkaloids (BIA), producing microbes, as well as methods of making and using them”

“Aspects include host cells engineered to produce benzylisoquinoline (BIAs) alkaloids. Host cells have heterologous coding sequences that allow for a wide range of enzymes to be involved in the creation of synthetic pathways, starting with starting compounds and ending up as BIAs. Methods for producing the BIAs are also provided. These methods involve cultivating the host cells in culture conditions that promote the expression of enzymes encoded through the heterologous coding sequences. The invention also includes compositions, such as starting compounds, kits, and host cells.

Background for “Benzylisoquinoline Alkaloids (BIA), producing microbes, as well as methods of making and using them”

“Aspects include host cells engineered to produce benzylisoquinoline (BIAs) alkaloids. Host cells have heterologous coding sequences that allow for a wide range of enzymes to be involved in the creation of synthetic pathways, starting with starting compounds and ending up as BIAs. Multiple copies of heterologous sequences can be found in host cells. They may also be derived from different sources than the host cell. In some embodiments, the host cell is selected from a reticuline-producing host cell, a sanguinarine precursor-producing host cell, a protoberberine-producing host cell, a thebaine-producing host cell and an opiate-producing host cell. Methods for producing the BIAs are also provided. These methods involve cultivating the host cells in culture conditions that encourage enzyme activity. The invention also includes compositions, such as starting compounds, kits, and host cells.

“Aspects of the invention include host cell systems that can be engineered to produce benzylisoquinoline (BIAs) alkaloids. Host cells have heterologous coding sequences that allow for a range of enzymes to be involved in the creation of synthetic pathways, starting with starting compounds and ending up as BIAs. In some embodiments, the host cell is selected from a reticuline-producing host cell, a sanguinarine precursor-producing host cell, a protoberberine-producing host cell, a thebaine-producing host cell and an opiate-producing host cell. Methods for producing the BIAs are also provided. These methods involve cultivating the host cells in culture conditions that promote the expression of enzymes encoded from heterologous coding sequences.

“Before we describe the invention in more detail, it is important to understand that this invention does not limit to specific embodiments as these may vary. The terminology used in this invention is intended only to describe particular embodiments. It is not meant to be restrictive. Since the appended claims limit the scope of the invention, it is important to understand that.

“Where a range is given, it is understood that every intervening value between the upper and lowest limits of that range, and any other declared or intervening value within that stated range is included in the invention. These smaller ranges’ upper and lower limits may be included separately and include the invention. However, the exclusions of any limit within the range are not required. The invention includes any ranges that exclude either or both of these limits if the range is stated to include one or both limits.

“Certain ranges of numerical values are presented herein, with the term ‘about.? preceding them. “about” is the term used to indicate that the numerical values are preceded by the term?about. The term?about? is used in this context to indicate literal support for the number it precedes. It can also be used to indicate a number that is close to or approximately that number. When a number is close to or approximately a specific recited one, the nearest or closest unrecited number could be a number that, in its context, provides the substantial equivalent to the specifically recited.

“Unless otherwise stated, all technical terms and scientific terms used in this invention have the same meanings as those commonly understood by an ordinary skilled person of the art to which it belongs. Any method or material similar to the ones described herein may also be used for the practice and testing of the invention. However, the following are representative methods and materials.

“All publications and patents mentioned in this specification are herein included by reference. They are incorporated herein as if each publication or patent was specifically and individually indicated that they were incorporated by referee. Citing any publication does not mean that it is entitled to be disclosed prior to its filing date. The dates of publication may differ from actual publication dates, which could be necessary to be independently verified.

“It should be noted that the singular forms ‘a?, ‘an?, and/or?the are used in this claim and the attached claims. If the context requires otherwise, plural referents may be used. You should also note that claims can be written to exclude any optional elements. This statement serves as an antecedent for the use of exclusive terminology such as “solely”,? ?only? ?only? limitation.”

“As those skilled in the art will see from this disclosure, each embodiment described and illustrated herein contains discrete components and features that can be easily separated from or combined with any other embodiments. This does not alter the scope or spirit the present invention. Any method can be performed in any order that is logically feasible, or in the order in which it was recited.

“Benzylisoquinoline Alkaloids, (BIAs),”

The present invention includes host cells that produce compounds known as benzylisoquinoline (BIAs) as well as biosynthetic intermediates, metabolites, and precursors. A variety of BIAs, biosynthetic precursors, intermediates, and metabolites thereof may be produced by the subject host cells, including but not limited to reticuline, sanguinarine, a protoberberine, berberine, a benzophenanthridine alkaloid, thebaine, an opiate compound, cheilanthifoline, stylopine, cis-N-methylstylopine, salutaridinol, salutaridinol-7-O-acetate, protopine and dihydrosanguinarine, (S)-canadine, oripavine, codeinone, neopine, neomorphine, morphine, codeine, hydromorphone, hydrocodone, oxycodone, oxymorphone, dihydrocodeine, 14-hydroxycodeine and dihydromorphine.”

“The host cell may generate synthetic pathways with any suitable compound. Starting compounds of interest include, but are not limited to, laudanosoline, methyl laudanosoline, norlaudanosoline, methyl norlaudanosoline, norcoclaurine, salutaridine, reticuline, tyramine, dopamine, 4-HPA, 4-HPPA, coclaurine, N-methylcoclaurine, 3?-hydroxy-N-methylcoclaurine, scoulerine, tetrahydrocolumbamine, canadine, laudanine, sanguinarine, thebaine, morphine, codeine, codeinone and dimethyl tetrahydoisoquinoline, e.g., 6,7-dimethyl-1-2-3-4-tetrahydroisoquinoline or another compound that may or may not be normally present in the endogenous BIA pathway. In some embodiments, the starting material is reticuline (orlaudanosoline) or norcoclaurine. The starting material can be either naturally occurring or non-naturally occurring. Based on the synthetic pathway in the host cell, other compounds can also be used to start the desired synthetic pathway. The starting material can be obtained from the host cells, such as tyrosine. Or, the starting material could be supplemented from outside sources. If the host cells are in liquid culture (an in-vivo environment), then the media can be supplemented by the starting material, such as tyrosine and norlaudanosoline. These materials are transported into the cells to make the desired products.

“Host Cells”

“A host cell that produces one or several BIAs is one aspect of the invention, as summarized above. Any type of cell that is suitable for producing the subject BIA-producing cell can be used. See, e.g. US2008/0176754, whose disclosure is incorporated herein in its entirety. Sometimes, the host cell may be yeast. Sometimes, the host cell comes from a yeast strain that has been engineered to produce a BIA. In some embodiments, the host cell is selected from a reticuline-producing host cell, a sanguinarine precursor-producing host cell, a protoberberine-producing host cell, a thebaine-producing host cell and an opiate-producing host cell.”

“Any suitable cells can be used in the subject host cell and method. Sometimes, the host cells may not be plant cells. In some cases, the host cells can be insect cells, mammalian cell, bacterial or yeast cells. Host cells of concern include, but not limited to, bacteria cells like Bacillus subtilis and Escherichiacoli, Streptomyces, Salmonella typhimuium and Streptomyces cells, and insect cells such Drosophila melanogaster and Spodoptera friugiperda Sf9 cell. In some embodiments, host cells can be yeast cells or E.coli cells. In some embodiments, yeast cells may be Saccharomyces cerevisiae (S. cerevisiae). Because cytochrome P450 proteins are involved in certain biosynthetic pathways, yeast is an attractive host cell. They are able fold correctly into the endoplasmic retina membrane, which allows them to continue their activity. The invention uses yeast strains of interest, including those described by Smolke et al. in US2008/0176754, whose disclosure is included by reference in its entirety. ura3-52/ura3-52 trp1-289/trp1-289 leu2-3_112/leu2-3_112 his3 ?1/his3 ?1 MAL2-8C/MAL2-8C SUC2/SUC2), S288C, W303, D273-10B, X2180, A364A, ?1278B, AB972, SK1 and FL100. In some cases, the yeast strain may be any of the S288C (MAT?) ; SUC2 mal-mel gal2 CUP1 hap1 flo8-1 (MAT?), BY4741 ; his3?1; leu2?0; met15?0; ura3?0), BY4742 (MATa; his3?1; leu2?0; lys2?0; ura3?0), BY4743 (MATa/MAT? ; his3??1/his3??1; leu2??0/leu2??0; met15??0/MET15?0/LYS2/lys2??0; and ura3??0/ura3??0, BY4742 (MATa/MAT?). These are derivatives of W303-B strain (MATa/MAT?); ade2-1; his3-11; -15; leu2-3, and -112; ura3-1 and cyr+), which have the yeast NADPH?P450 and NADPH?P450 reductase Another embodiment of the yeast cell is W303alpha. ; his3-11, 15 trp1-1 leu2-3 ura3-1 ade2-1). The identity and genotype of additional yeast strains of interest can be found at EUROSCARF (web.uni-frankfurt.de/fb15/mikro/euroscarf/col_index.html).”

“Host cells” is a term that refers to cells that are able to produce desired BIA(s) from their coding sequences. “Host cells” is a term that refers to cells that contain one or more heterologous coding sequencings that encode activity(ies) that allow the host cells produce desired BIA(s). The heterologous sequences of coding could be integrated into the genome of host cells or transiently added to the cell. The term “heterologous code sequence” is used herein. The term?heterologous coding sequence? is used to refer to any polynucleotide which codes for or eventually codes for a peptide, protein, or equivalent amino acid sequence. As such, ?heterologous coding sequences? Multiple copies of the coding sequences are present in the host cells, so that the cells are expressing additional copies. These heterologous coding sequences may be RNA or any other type of RNA, e.g. DNA, mRNA or any other type, e.g. cDNA or a hybrid RNA/DNA. Full-length transcription units include features such as the coding sequence and introns.

“Heterologous sequences of coding” is the term used herein. The coding portion includes both the peptide and enzyme (i.e. the cDNA sequence or mRNA sequence), as well the coding part of the full-length transcriptal unit (i.e. the gene that contains introns or exons as well as?codon optimized). sequences, truncated or other forms, that code for the enzyme, or code for an equivalent amino acid sequence. As long as the sequence produces a functional proteins, it can be truncated, modified, or any other form of altered sequences. These equivalent amino acid sequences may contain a deletion of one or several amino acids. The deletion can be either C-terminal, N-terminal, or internal. Truncated forms can be envisioned provided they possess the catalytic capabilities indicated in this document. The possibility of fusions between two or more enzymes is possible to facilitate the transfer metabolites within the pathway. However, it must be maintained that their catalytic activities are not lost.

Modeling and screening can be used to identify operable fragments, mutants, or truncated versions. This can be done by removing, for example N-terminal, or C-terminal regions, from the protein. Then, the derivative is analyzed in order to determine its activity for the desired reaction relative to the original sequence. This is considered an equivalent derivative of enzyme.

“Aspects also relate to heterologous codes that code for amino acids sequences equivalent to native sequences for various enzymes. A sequence of amino acids that is “equivalent”? An amino acid sequence that is?equivalent’ to the original sequence is one that does not contain identical amino acids, but contains at least some amino-acid changes (deletions and substitutions, inversions or insertions). When used for a specific purpose, they do not significantly affect the biological activity of a protein. In the case of a decarboxylase it refers to its catalytic activities. Also, equivalent sequences include sequences that have been engineered or evolved to have different properties than the original amino acids sequence. Mutable properties include selectivity, catalytic activity and stability. An?equivalent’ is possible in certain embodiments. An “equivalent” amino acid sequence is one that has at least 80% to 99% identity at the amino acids level to the specific sequence. In some cases, this may be as low as 85%, 87% or 89%, and in others, less than 92%, 92% and 93%, respectively. Sometimes, though the amino acid sequence might be identical, the DNA sequence has been altered to maximize codon usage in the host organism.

“Host cells can also be altered to have one or more genetic modifications to accommodate heterologous sequences. Modifying the host genome can be used to alter the expression of specific proteins that could interfere with the desired pathway. These native proteins can quickly convert one of the intermediates, or final products, into a metabolite, or another compound that is not useful in the desired pathway. If the native enzyme activity is reduced or absent, then the intermediates produced would be easier to incorporate into the product. If the host cell is a yeast cells and the desired pathway calls for 2-oxoglutarate as a cosubstrate, then the expression of native glutamate/2/oxoglutarate-dehydrogenase enzymes could be decreased or ablated. This would convert the desired cosubstrate (2,oxoglutarate), into succinyl-CoA or glutamate. Sometimes ablation of a protein’s expression may be useful. This could happen in cases where the host cell is a yeast cell. In these cases, the native endogenous glutamate and/or 2-oxoglutarate dehydrogenase enzymes are reduced or ablated. This would allow the desired cosubstrate (2-oxoglutarate) to be converted into succinyl-CoA. These proteins control the export of BIA compounds into the culture media. Host cell gene deletions may be of interest in certain embodiments. These genes are associated with the unfolded proteins response and endoplasmic reticulum(ER) proliferation. These gene deletions could lead to increased BIA production. Cytochrome P450s can induce unfolded protein responses and cause the ER proliferate. Eliminating genes that are associated with stress responses can control or decrease the overall burden on the host cells and improve pathway performance. Genetic alterations can also include altering the promoters endogenous gene expression to increase or introduce additional copies. This could include strains that overexpress the yeast NADPH?P450 reductase, CPR1, to increase the activity of heterologous P450 enzymes. Endogenous enzymes like ARO8, 9 and 10, which directly participate in the synthesis intermediate metabolites, can also be overexpressed.

“Heterologous coding sequencing sequences of interest” include sequences that encode enzymes (either wild-type or equivalent) that are responsible for the production BIAs in plants. The heterologous sequences can code any enzyme in the BIA pathway. In other cases, they can also be from any source. Cheilanthifoline synthase (CFS, EC 1.14) can be found in some cases. 21.2) can be found in Eschscholzia, Papaver somniferum and Argemone myxicana. It is also known to synthesize (S-cheilanthifoline) from (S-scoulerine). Based on the product, the number and choice of enzymes encoded in the heterologous sequences may be made. The present invention can be used to create host cells that contain 1 or many, 2 or 3 or greater, 4 or less, 5 or less, 6 or most, 7 or much more or 9 or all, 10 or mehr, 11 or or, 12 or higher, 13 or better, 14 or extra, or even 15 or so heterologous sequences such as 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, or 15 heterologous sequences.

“Unless otherwise noted the heterologous coding sequences in GENBANK are as reported. Tables 2 and 3 show a list of enzymes of particular interest. Tables 2 and 3 show a list of enzymes of interest. The present invention allows host cells to contain any combination of any of these enzymes. Unless otherwise noted, accession numbers in Table 3 refers to GenBank. Some accession numbers refer to the Saccharomyces genome database (SGD) which is available on the world-wide web at www.yeastgenome.org.”

“In some embodiments, the host cells (e.g. a yeast strain), are engineered to selectively produce a BIA by locating one or more enzymes in a specific compartment of the cell. FIG. 1 illustrates one embodiment of the invention. 18) An enzyme can be targeted to yeast’s endoplasmic reticulum through the fusion of an ER2 targeting sequence with the C-terminus.

“In some cases, the enzyme may be found in the host cell so that the compound that it produces spontaneously rearranges or is converted to a desirable metabolism by another enzyme before reaching a local enzyme that could convert the compound into an unwanted metabolite. To prevent one enzyme from directly acting on a compound and making an undesirable metabolite, or to limit the production of undesirable end product (e.g., a undesirable opioid by-product), it may be possible to select the spatial distance between them. Any of the described enzymes may be located in any suitable compartment within the host cell. This includes, but is not limited to, the organelle, endoplasmic retina, golgi or vacuole as well as the nucleus and plasma membrane. 18).”

“In some embodiments, the host cells may include one or more enzymes that constitute a localization tag. You can use any of the available tags. The localization tag may be a peptidic sequence attached to the N-terminal or C-terminal of an enzyme. You can attach a tag to an enzyme using any method you prefer.

“In some cases the localization tag may be derived from an endogenous yeast proteins. These tags can provide access to a number of yeast organelles, including the endoplasmic reticulum (“ER”), mitochondria (MT), plasma membrane(PM) and vacuole [V].

“In some embodiments, the tag can be an ER routing tag (e.g. ER1). The tag can be a vacuole tag in certain embodiments (e.g. V1). In some embodiments, the tag can be a plasma membrane tag (e.g. P1). The tag may include or be derived from a transmembrane domain within the tail-anchored protein class.

“In certain embodiments, the localization tags locate the enzyme on an organelle’s outside.” The localization tag can be used to locate the enzyme inside an organelle in certain embodiments.

“In certain cases, each type of enzyme can be increased by adding gene copies (i.e. multiple copies), which increases intermediate accumulation, and eventually BIA or BIA precursor manufacturing. The present invention allows for increased BIA production in a host cells by simultaneous expression of multiple species variants or a single enzyme. Sometimes, the host cell may contain additional gene copies of one or more enzymes. Multiple copies of heterologous coding sequences for enzymes can be included in the host cell using any number of methods.

“In some embodiments, multiple copies of heterologous sequences for enzymes are present in the host cell. These could include 2 or more or 3 or more or 4 or more or 5 or more or even 10 or fewer copies. Multiple copies of heterologous sequences are included in some embodiments. This could include multiple copies of one or more of the heterologous codes for an enzyme, or multiple copies of three or more, four, or more. Sometimes, multiple copies of a heterologous sequence for an enzyme may be derived from different sources than the host cell. One example is that the host cell might contain multiple copies of a heterologous sequence of coding, with each copy being derived from a different source. Each copy could include variations in the explicit sequences, based on differences between species of the heterologous sequence that encodes the enzyme of interest.

The engineered host cell media may be taken and monitored to determine the production of BIA compound of interest. Any method can be used to measure and observe the BIA compounds. Methods of interest include, without limitation, LC-MS techniques (e.g. as described herein), where a sample is analyzed using a known amount a standard compound. Identification can be confirmed by m/z or MS/MS fragmentation patterns. Quantitation and measurement of the compound can be done via LC trace peaks with known retention time and/or EIC MS peak analyses by reference to corresponding LC?MS analysis of a standard compound.

“Reticuline-Producing Host Cells”

“Reticuline is a major branch point intermediate in synthesis of BIAs. A high yield of this intermediate is important in engineering efforts to create end products like morphine or sanguinarine. In some cases, to produce reticuline from norlaudanosoline three enzymes are expressed in the host cell: norcoclaurine 6-O-methyltransferase (6OMT; EC 2.1.1.128), coclaurine N-methyltransferase (CNMT; EC 2.1.1.140) and 3?hydroxy-N-methylcoclaurine 4?-O-methylase (4?OMT; EC 2.1.1.116). The enzymes come from different sources than the host cell. An additional cytochrome P450 enzyme (e.g. CYP80B3 and CYP80B1) may be used to produce reticuline using norcoclaurine. Any of the many optimization options available to engineering S. cerevisiae may be used to make reticuline. To produce the highest quality reticuline, it is possible to combine the three methyltransferases of two or more species (e.g. P. somniferum, T. flavum). Another way is to integrate all three methyltransferases from P. somniferum into the yeast genome. The expression of each enzyme can be titrated and not affect the yield of reticuline. You can adjust the expression of the methyltransferase enzymes by adjusting the titration. The methyltransferases can act in concert, sequentially or as a mixture of both on the substrates.

The invention includes strains of S. cerevisiae that have improved reticuline production by overexpression of 6OMT and CNMT genes, as well as 4?OMT genes. The improved or increased production refers to the production of some amount reticuline in comparison to the control. It also includes an increase of 10% or greater, such a about 20%, 30%, 40%, 50%, 60%, or about 80%. This can be 2-fold, 5-fold, or 10-fold, depending on whether the control has any reticuline. Different species of methyltransferases have different substrate specificities [Choi and al. (2002). J Biol Chem 277, 830-835; Liscombe et al. (2009). Plant J 60, 729-74; Morishige et al., (2000) J Biol Chem 275, 23398-23405; Ounaroon et al. (2003) Plant J 36, 808-819; Sato et al., (1994) Eur J Biochem 225, 125-131]. It is possible to combine methyltransferases from different species into one strain. This allows you to benefit from the various substrate specificities and increase flux via multiple methylation pathways. This can increase the yield of reticuline. Some species variants of methyltransferases may be found in certain cases (Table 2). P. somniferum may be used to create species variants of the MMTases. T. flavum is sometimes used to make species variants of the MMTs. C. may be used to derive species variants of the MMTases in some instances. japonica.”

“In some embodiments, the host cells contain two or more heterologous sequences for two or three methyltransferases from 6OMT, CNMMT, and 4?OMT. Sometimes, two or more methyltransferases may be derived from different sources than the host cell.

“In certain cases, the host cell contains heterologous sequences of coding for the methyltransferases 6OMT or CNMT. In some cases, heterologous coding sequences are present in the host cell for the methyltransferases CNMT or 4?OMT.

“In some cases, the host cell may contain heterologous coding sequences of the methyltransferases 6OMT or 4?OMT in certain instances.”

“In some embodiments, the host cell contains heterologous sequences of coding for all methyltransferases 6OMT and CNMT in certain embodiments.”

“In certain instances, each type of methyltransferase can be increased by adding gene copies (i.e. multiple copies). This increases intermediate accumulation and eventually reticulin production. The present invention allows for increased reticuline production in yeast strains by simultaneous expression of multiple species of a single or several methyltransferases, and incorporation or additional gene copies of one or more methyltransferases.

“In some embodiments, the host cells include multiple copies of amethyltransferase. These could be 2 or more or 3 or more or 4 or more or 5 or more or even 10 or fewer copies. Multiple copies of one or several methyltransferases may be present in certain embodiments. methyltransferases. Multiple copies of the methyltransferase may be derived from different sources than the host cell. One example is that the host cell could contain multiple copies of a heterologous sequence of coding, with each copy being derived from a different source. Each copy could include variations in the explicit sequences, based on differences between species of the enzyme of particular interest encoded by the heterologous code sequence.

“In some cases, multiple copies of a heterologous coding scheme for CNMT are included. Two copies of a heterologous coding scheme for CNMT may be included in certain cases. Multiple copies of a heterologous sequence for 6OMT may be included in some cases. Two copies of the heterologous sequence for 6OMT may be included in certain cases. Multiple copies of a heterologous sequence for 4?OMT may be included in some cases. Two copies of a heterologous sequence for 4?OMT may be included in certain cases.

“In certain cases, the host cell can produce a higher amount of reticuline using norcoclaurine than a control cell that does not have multiple copies of one or more heterologous sequences for the one methyltransferase. The increased amount of reticuline in some cases is 10% higher than the control host cells. This could be as a result of the host cell producing a greater amount of norcoclaurine relative to the control cell.

“In certain cases, the host cell can produce a higher amount of norlaudonosoline from norlaudoline than a control cell that does not have multiple copies of one or more heterologous sequences for the one methyltransferase. The increased amount of reticuline can be as high as 10% relative to the control cell in certain cases. This could include about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, and even 10-fold relative to the control cell.

“In some embodiments, the host cells are capable of producing a 10% to more yield of norcoclaurine reticuline. This includes 20%, 30%, 40%, 50%, 60%, 70%, 70%, 80%, or even 90% yield of norcoclaurine reticuline.

“In some embodiments, reticuline can be produced by the host cell from norlaudonosoline in a yield of 10% or higher, such as 20%, 30%, 40%, 50%, 60%, 70%, 70%, 80%, 85%, 90%, or even more from reticuline.”

“In some embodiments, the host cell includes an engineered strain that includes the biosynthetic pathway. This allows for increased production of reticuline and incorporates any combination the following alternative methylation pathways. In certain instances, the host cell can produce reticuline from norlaudanosoline using a biosynthetic pathway as shown in FIG. 2. In some embodiments, the host cell can produce reticuline from norcoclaurine through a biosynthetic pathway as shown in FIG. 3. Sometimes, norlaudanosoline is used as the starting material for the pathway (FIG. 2, (1), CNMT, and/or 4??OMT could act on this compound to create three distinct methylated intermediates. BIA 2, which was initially methylated initially by 6OMT is then methylated either by CNMT or 6OMT; BIA 3 which was initially methylated initially by CNMT is then methylated either by 6OMT (or both); BIA 4 which has been previously methylated initially by 4OMT is then methylated either by 6OMT or CNMT is methylated to make reticuline.

“In certain cases, when the pathway’s starting material is norcoclaurine (6OMT or CNMT) may react on this compound to create two distinct methylated products (FIG. 3): BIA 10, initially methylated initially by 6OMT is then methylated again by CNMT. BIA 11, initially methylated initially by CNMT is then methylated again by 6OMT. BIA 12, also known by N-methylcoclaurine is previously methylated with 6OMT and CNMT. CYP80B1 and CYP80B3 then sequentially act upon BIA 11 to make reticuline.

“In some cases, the yeast strain is present in the host cell. S. cerevisiae.”

“Sanguinarine & Sanguinarine Precursors-Producing Host cells”

“Aspects of the invention include host cells that produce protoberberine and benzophenanthridine alkaloids, including, but not limited to, cheilanthifoline, stylopine, cis-N-methylstylopine, scoulerine, protopine, dihydrosanguinarine and sanguinarine. FIG. FIG. 4 shows a synthetic pathway found in embodiments host cells as per embodiments according to the present invention. The pathway can be longer and may start from norlaudanosoline or norcoclaurine, but this pathway depiction starts with reticuline. It ends with sanguinarine. If the desired end product is not one of the intermediates of the norlaudanosoline-sinurarine pathway, the pathway may include less enzymes than the ones shown. The invention comprises biosynthetic pathways that include multiple enzymatic steps. For example, CFS (EC 1.14.21.2), CPR, CPR (EC 1.6.2.4), STS(EC 1.14.21.1), STS (EC 1.14.21.1), TNMT, EC 1.14.13.37) and MSH (EC 1.14.13.35) are all catalyzed within an engineered yeast strain to make a variety benzophenanthridine and protoberberine compounds. The present invention also includes tools and methods that optimize the production of benzophenanthridine and protoberberine compounds in the context of an engineered yeast strain.

“In certain embodiments, the host cells are capable of producing sanguinarine, or a sanguinarine precursor. In these cases, the host cell contains one or more heterologous coding sequencings for one or several enzymes from BBE, CFS or CPR, STS or STS. The host cell may also have one or two other heterologous coding sequences. The sanguinarine precursor may be a protoberberine, a benzophenanthridine or an alkaloid.

“In some cases, the source organism may be P. somniferum (E. californica), Arabidopsis.thaliana, Papaver bracteatum, A. mexicana, or E. californica. P. somniferum is sometimes the source organism in certain cases. Sometimes, E. californica is the source organism. A. thaliana is sometimes the source organism. In some cases, P. bracteatum is the source organism. A. is sometimes the source organism in some cases. mexicana.”

“In some cases, the one or more enzymes are two or more enzymes that were derived from different sources than the host cell.”

“In some embodiments, the host cells contain multiple copies of one or more heterologous sequences. Multiple copies of one or more heterologous sequences may be derived from different sources than the host cell in certain embodiments. One example is that the host cell might contain multiple copies of a heterologous sequence of coding, with each copy being derived from a different source. Each copy could include variations in the explicit sequences, based on differences between species of the enzyme of particular interest encoded by the heterologous code sequence.

“In some cases, the host cells may contain more than one, such as three, four, five, six, or more heterologous coding sequences, for two or more enzymes from STS, CFS or CPR, STS or TNMT. P6H, MSH, DBOX, and CFS.

“In some cases, the host cell may also contain one or more gene deletions compared to a native host cells. The one or more deleted genes are selected from IRE1, OPI1, ONO2, INO3, PDR1, PDR3, PDR5, SNQ2, YOR1, IRE1, TPO2, TPO3, PDR10 and PDR15. PDR16. PDR17. QDR1, QDR2, FLR1, QDR3, QDR3, QDR3, QDR3, QDR5, AQR1, AQR2, CIN5, and CIN5.

“In some cases, the host cells are a yeast strain (e.g. as described herein).

“In certain embodiments, the host cells are capable of producing sanguinarine or sanguinarine precursors from norlaudanosoline via the biosynthetic pathway shown in FIG. 4. Sometimes, the host cell can produce the sanguinarine/sinuarine precursor from norlaudanosoline through a biosynthetic pathway as shown in FIG. 11.”

“In some cases, the host cell may contain a heterologous sequence of coding for a BBE protein. A heterologous sequence of coding for the BBE enzyme can be integrated into a host chromosome. Sometimes, the host cell contains a heterologous sequence of coding for a CFS-enzyme. Sometimes, the host cell contains a heterologous sequence of coding for a CPR protein enzyme. Sometimes, the host cell contains a heterologous sequence of coding for a STS protein enzyme. Sometimes, the host cell contains a heterologous coding sequence to encode a TNMT protein. Sometimes, the host cell contains a heterologous sequence of coding for a MSH-enzyme. Sometimes, the host cell contains a heterologous sequence of coding for a P6H protein. Sometimes, the host cell contains a heterologous sequence of coding for a DBOX gene.

“In some cases, the host cells is a yeast strain producing scoulerine. In these cases, the berberine bridge enzyme, (BBE; E. 1.21.3.3)) (e.g. from P. somniferum) is expressed on a low-copy construct (e.g. a low-copy plasmid, or chromosomally or YAC) in the yeast strain with Ps6OMT or PsCNMT and/or Ps4??OMT chromosomally chromosomally chromosomally chromosomally chromosomally or Ps4?OMT. You can use any of the available enzyme variants, such as one or more of those shown in Table 2.

“In certain cases, the sanguinarine precursor may be cheilanthifoline. In some cases, the host cell contains heterologous coding sequences of cheilanthifoline synthase and a cytochrome 450 NADPH reducetase enzyme (CPR; EC 1.6.2.4). Sometimes, the CPR enzyme can be an ATR enzyme (e.g. ATR1). Sometimes, the host cell may be a yeast strain producing cheilanthifoline. In these cases, cheilanthifoline synthase is expressed on a low-copy construct (e.g. a low-copy plasmid, YAC, or chromosomally integrated) in yeast strains with Ps6OMT and PsCNMT and/or Ps4?OMT and/or PsBBE. You can use any of the available enzyme variants, such as one or more of those shown in Table 2.

“In some embodiments, stylopine is produced by the host cell. In these cases, stylopine synthase [EC 1.14.21.1] (e.g. from E.californica (EcSTS), Pa. somniferum(PsSTS), and/or A.mexicana)) is expressed in the host cells on a low-copy construct (e.g. a low copy, YAC, or chromosomally integrated) in cheilanthifoline-producing strain. You can use any of the available enzyme variants, such as one or more of those shown in Table 2.

“In certain cases, the host cells produces cis-N-methylstylopine, where tetrahydroprotoberberine N-methyltransferase (TNMT; EC 2.1.1.122) (e.g., from P. somniferum (PsTNMT) or E. californica (EcTNMT)) is expressed in the host cell from a low copy construct (e.g., a low copy plasmid, YAC or chromosomally integrated) in a stylopine-producing strain. You can use any of the available enzyme variants, such as one or more of those shown in Table 2.

“In certain cases, protopine is produced by host cells. In certain cases, cis-N-methylstylopine 14-hydroxylase (MSH, EC 1.14.13.37) (e.g., from P. somniferum (PsMSH)) is expressed in the host cell from a low copy construct (e.g., a low copy plasmid, YAC or chromosomally integrated) in a cis-N-methylstylopine producing strain. The host cell may contain heterologous coding sequences that encode a TNMT or a MSH enzyme in certain cases. You can use any of the available enzyme variants, such as one or more of those shown in Table 2.

“In some cases, the host cells produce dihydrosanguinarine. In these cases, protopine 6-hydroxylase, (EC 1.14.13.55) is expressed in the host cells from a low-copy construct (e.g. a low copyplasmid, or YAC) in a protopine-producing strain. You can use any of the available enzyme variants, such as one or more of those shown in Table 2.

“In some cases, the host cell produces sanguinarine, where dihydrobenzophenanthridine oxidase (DBOX; EC 1.5.3.12) (e.g., from P. somniferum (PsDBOX)) is expressed in the host cell from a low copy construct (e.g., a low copy plasmid, YAC or chromosomally integrated) in a dihydrosanguinarine producing strain. You can use any of the available enzyme variants, such as one or more of those shown in Table 2.

“In some embodiments, the host cells are engineered to produce more cheilanthifoline that a control strain. Additional copies of CFS can be found in the engineered strain. More refers to both the production or a decrease in cheilanthifoline from the control strain, and also the increase of approximately 10% or greater. This is because the engineered strain has more copies of CFS than the control strain. Variation in the expression of a cytochrome P450 target compound may increase its production by changing gene copy number, promoter strength, or regulator regulation. Sometimes, expression of a high-copy plasmid doesn’t result in measurable cheilanthifoline. Cheilanthifoline production can be higher in certain cases, where multiple copies of CFS gene are expressed by host cells from low copy constructs. Some embodiments of the CFS gene include more copies in the host cell. This increases the production of cheilanthifoline.

“In some cases, in order to produce more stylopine variants of enzymes CFS/STS from different species can be combined in a host cell. An engineered host cell can produce higher levels of stylopine if it is compared to a control cell lacking the desired enzymes. The production of some stylopine in the engineered host cell is increased, but not the production of all stylopine. It can also be observed that the control cells have some stylopine, and the increase is 2-fold or greater. FIG. 13(c) shows the measurement of stylopine from different CFS versions, expressed with different STS variants. 13(c). To increase the levels of stylopine, any combination of enzymes can be used.

“In certain instances, in order to produce more cheilanthifoline host cells (e.g. yeast strains) may be engineered to contain chromosomally-integrated NADPH cytochrome P450 reducetases from various species to maximize the activity of cytochrome P450s. It is possible to measure the cheilanthifoline produced by different cheilanthifoline-synthese enzymes and cytochrome P450 NADPH reducer enzymes. To produce higher levels of cheilanthifoline than a control, any combination of these enzymes can be used.

“In some cases, in order to produce more cheilanthifoline and stylopine, host cell (e.g. yeast strains) with high levels of cytochrome B5 can be used to maximize the activity of cytochromeP450s. It is possible to measure the cheilanthifoline and stylopine levels in host cells that have cytochrome B5 overexpressed or not. Sometimes, the host cell may overexpress cytochrome B5 and produce more cheilanthifoline and stylopine than a control.

“In certain embodiments, host cells produce more protoberberinealkaloids. The host cell’s optimization is achieved by the deletion genes associated with unfolded protein responses and endoplasmic reticulum proliferation to increase BIA production. Gene deletions that are of interest include OPI1, HAC1, OPI1, INO1, and INO3 (e.g. Table 3). Sometimes, cytochrome P450s expression can induce the unfolded proteins response and cause the ER proliferate. The elimination of genes that are associated with stress responses can control or reduce the overall burden on the host cells and improve pathway performance. More refers to the production of some quantity of protoberberinealkaloids in cases where the control does not have any. It also includes an increase of approximately 10%, such a about 20%, 30%, 40%, 50%, 60%, about 80%, about 100%, or 2-fold or greater, such a 5-fold, 10-fold, or even more for situations where there is some protoberberinealkaloids production.

“In some cases, the host cell may contain one or more heterologous coding sequences that encode for one or several proteins involved in the transport of compounds across cell membranes. PDR1, PDR5, SQ2, YOR1, PDR3, PDR3, CIN5, or PDR3 are some of the proteins that transport compounds across cell membranes.

“In some embodiments, genes that are involved in the pleiotropic drugs response, such as ATP-binding cassette transporters (ABC) pumps, multidrug resistance pump (MDR) pumps, and associated transcription factors, can be deleted from the host cell in order to decrease the export of BIA molecules to the culture medium. PDR1, STB5, PDR3, PDR5, SNQ2, YOR1, TPO1, TPO2, TPO3, TPO4, PDR10 and PDR15 are all examples of genes. Multiple deletions can occur in any combination. Sometimes, the host cell may have one or more gene mutations that cause lower levels of reticuline and scoulerine or cheilanthifoline in some cases.

“Another embodiment places a gene that is involved in the pleiotropic drugs response in host cells. This promoter can be either inducible or growth-stage dependent and will control temporal control of BIA transportation. A stationary phase promoter can be used to control a transporter gene that causes a BIA of particular interest to remain in the cell until it reaches stationary phase. This may increase the conversion of starting materials into the desired end products in host cells.

“Protoberberine-Producing Host Cells”

“Aspects include engineered host cells capable of producing protoberberine alkaloids. The protoberberinealkaloids may have one of these structures in some cases:

“Where R1-R14 can be selected independently from?H (e.g. a lower alkyl like methyl (CH3), or ethyl), or a hydroxyl, or an alkoxy(OR) (e.g. a lower Alkoxy such methoxy, or ethoxy).

In certain cases, protoberberine alkaloids may be produced from reticuline, other analogs thereof, and derivatives thereof. In certain cases, the host cells may include one or more heterologous coding sequences to express one or more of, or any combination of, the following enzymes: berberine bridge enzyme (BBE), scoulerine 9?-O-methyltransferase (S9OMT), canadine synthase (CAS), and (S)-tetrahydroprotoberberine oxidase (STOX), wherein the one or more enzymes is derived from a different source organism as compared to the host cell.”

“In some cases, the source organism may be P. somniferum or E. californica or C. japonica, T. Flavum, Berberis subsp. glaucum, Coptis chinensis, Thalictrum spp, Coptis spp, Papaver spp, Berberis wilsonae, A. mexicana, or Berberis spp.”

“In some embodiments, the host cell may contain multiple copies of one or more heterologous sequences. Sometimes, multiple copies of one or more heterologous sequences may be derived from different sources than the host cell. One example is that the host cell could contain multiple copies of a heterologous sequence of coding, each copy being derived from a different source. Each copy could include variations in the explicit sequences, based on differences between species of the enzyme of particular interest encoded by the heterologous code sequence.

“In some cases, the host cell may contain two or more heterologous coding sequencings for two or three enzymes from BBE, CAS, CAS, and STOX. Sometimes, the host cell contains three or more heterologous coding sequencings for more than three enzymes, including S9OMT and CAS. Sometimes, the host cell contains heterologous sequences of coding for each enzyme, BBE, S9OMT and CAS.

“In certain embodiments, the host cell includes the heterologous sequence for CAS and the heterologous sequence for ATR1. In some embodiments, the heterologous sequence for STOX is found in the host cell.

“In some embodiments, the host cells (e.g. an engineered yeast strain), support a biosynthetic pathway, as shown in FIG. 5.”

“In some instances, the host cell includes the gene STOX that shares 75% or more (e.g., 78%) nucleic acid sequence identity with the naturally occurring gene B. wilsonae (S)-tetrahydroprotoberberine oxidase (Table 3). This gene could be a non-natural sequence of nucleotides, codon-optimized to yeast expression.

“Aspects” of the invention include the functional expression in yeast culture of STOX and its homologues. In some embodiments, host cells can be engineered to make berberine using its precursor (S-canadine).

“In one embodiment, the expression levels of enzymes are relatively low (e.g. CEN/ARS vector, genomic expression) for BBE and CAS (see, for example, FIG. 6a ) and high (e.g. 2?m vector, multiple genomic copies) S9OMT (see e.g. FIG. 6b ). Any method can be used to alter the expression levels. These methods include, but are not limited, altering the strength of a constitutive promoter, using a inducible promoter and changing the number of genes episomally or genomeally (see FIG. 6c), changing the selection marker and/or culture conditions to correspond with promoter activity.

“In some embodiments, one or more enzymes can be recombinantly derived from a yeast artificial genome (e.g. FIG. 10).”

“Aspects include the functional expression in live yeast culture of CAS or its homologues as part of larger biosynthetic pathways. In some embodiments, host strains can be engineered to make berberine from norlaudanosoline (or its precursors) in certain embodiments. 5, 7, or Table 2. In certain cases, the host cell can produce berberine by reticuline using a biosynthetic pathway that is FIG. 5. Another embodiment of the invention includes a cytochrome P450 partner for CAS in the host cell. This is ATR1, and co-expression may result in higher activity than E.californica CPR or A.thaliana ATR2, P. somiferum CPR or endogenous yeasts CPR (e.g. FIG. 6d ).”

“Aspects include the functional expression in yeast culture of STOX and its homologues. In some embodiments, host cells can be engineered to make (S)-canadine from norlaudanosoline, which is the precursor of Berberine. Some embodiments allow the host cell to produce (S)-canadine using norlaudanosoline.

Heterologous transporters may be expressed in engineered strains to increase the BIA accumulation. This includes, but is not limited to, plant ATP-binding cassette protein from BIA-producing species. To accumulate berberine in the host cell, some embodiments may include one or more heterologous sequences of coding for transporters from CjABCB1, CjABCB2, or CjABCB2.

“In some cases, the yeast strain is used as the host cell.”

“Thebaine-Producing host Cells”

The invention includes engineered host cells that can produce thebaine from reticuline and its precursors. A strain of reticuline, or one of its precursors, may be used. It can also be added to culture media or a fraction of cell lysate. In some instances, the host cells may include one or more heterologous coding sequences to express for one or more enzymes selected from salutaridine synthase (SalSyn), cytochrome P450 2D6 (CYP2D6), cytochrome P450 2D2 (CYP2D2), salutaridine reductase (Sal R), and/or salutaridinol 7-O-acetyltransferase (SalAT). One or more of the enzymes could be derived from another source organism than the host cell.

“In some cases, the source organism may be P. somniferum or P. bracteatum. Papaver orientale, Papaver species, Homo sapiens, Rattus norvegicus, Papaver spp., Papaver spp.

“In some cases, host cells may be engineered yeast strains that support biosynthetic pathways in a host, as shown in FIG. 8.”

“In some embodiments, the host cell may contain multiple copies of one or more heterologous sequences. Sometimes, multiple copies of one or more heterologous sequences may be derived from more than one source organism.

“In certain cases, the host cell may contain two heterologous sequences of coding. The two heterologous sequences of coding are in some cases for the enzymes SalR or SalAT.

“In some cases, the host cell contains the genes CYP2D6, CYP2D2, SalSyn, and/or SalSyn. These genes can be engineered or natural P450s that can produce salutaridine from reticuline. It could be a non-natural or natural nucleotide sequence that is codon-optimized to yeast expression.

“In some embodiments, the host cells include a cytochrome P450 reducetase partner to CYP2D6, CYP2D2, or SalSyn. This may lead to higher CYP2D6, CYP2D2, or SalSyn activity than E. californica, A. thaliana ATR2, P. somiferum CPR, and endogenous yeast CPR.”

Summary for “Benzylisoquinoline Alkaloids (BIA), producing microbes, as well as methods of making and using them”

“Aspects include host cells engineered to produce benzylisoquinoline (BIAs) alkaloids. Host cells have heterologous coding sequences that allow for a wide range of enzymes to be involved in the creation of synthetic pathways, starting with starting compounds and ending up as BIAs. Multiple copies of heterologous sequences can be found in host cells. They may also be derived from different sources than the host cell. In some embodiments, the host cell is selected from a reticuline-producing host cell, a sanguinarine precursor-producing host cell, a protoberberine-producing host cell, a thebaine-producing host cell and an opiate-producing host cell. Methods for producing the BIAs are also provided. These methods involve cultivating the host cells in culture conditions that encourage enzyme activity. The invention also includes compositions, such as starting compounds, kits, and host cells.

“Aspects of the invention include host cell systems that can be engineered to produce benzylisoquinoline (BIAs) alkaloids. Host cells have heterologous coding sequences that allow for a range of enzymes to be involved in the creation of synthetic pathways, starting with starting compounds and ending up as BIAs. In some embodiments, the host cell is selected from a reticuline-producing host cell, a sanguinarine precursor-producing host cell, a protoberberine-producing host cell, a thebaine-producing host cell and an opiate-producing host cell. Methods for producing the BIAs are also provided. These methods involve cultivating the host cells in culture conditions that promote the expression of enzymes encoded from heterologous coding sequences.

“Before we describe the invention in more detail, it is important to understand that this invention does not limit to specific embodiments as these may vary. The terminology used in this invention is intended only to describe particular embodiments. It is not meant to be restrictive. Since the appended claims limit the scope of the invention, it is important to understand that.

“Where a range is given, it is understood that every intervening value between the upper and lowest limits of that range, and any other declared or intervening value within that stated range is included in the invention. These smaller ranges’ upper and lower limits may be included separately and include the invention. However, the exclusions of any limit within the range are not required. The invention includes any ranges that exclude either or both of these limits if the range is stated to include one or both limits.

“Certain ranges of numerical values are presented herein, with the term ‘about.? preceding them. “about” is the term used to indicate that the numerical values are preceded by the term?about. The term?about? is used in this context to indicate literal support for the number it precedes. It can also be used to indicate a number that is close to or approximately that number. When a number is close to or approximately a specific recited one, the nearest or closest unrecited number could be a number that, in its context, provides the substantial equivalent to the specifically recited.

“Unless otherwise stated, all technical terms and scientific terms used in this invention have the same meanings as those commonly understood by an ordinary skilled person of the art to which it belongs. Any method or material similar to the ones described herein may also be used for the practice and testing of the invention. However, the following are representative methods and materials.

“All publications and patents mentioned in this specification are herein included by reference. They are incorporated herein as if each publication or patent was specifically and individually indicated that they were incorporated by referee. Citing any publication does not mean that it is entitled to be disclosed prior to its filing date. The dates of publication may differ from actual publication dates, which could be necessary to be independently verified.

“It should be noted that the singular forms ‘a?, ‘an?, and/or?the are used in this claim and the attached claims. If the context requires otherwise, plural referents may be used. You should also note that claims can be written to exclude any optional elements. This statement serves as an antecedent for the use of exclusive terminology such as “solely”,? ?only? ?only? limitation.”

“As those skilled in the art will see from this disclosure, each embodiment described and illustrated herein contains discrete components and features that can be easily separated from or combined with any other embodiments. This does not alter the scope or spirit the present invention. Any method can be performed in any order that is logically feasible, or in the order in which it was recited.

“Benzylisoquinoline Alkaloids, (BIAs),”

The present invention includes host cells that produce compounds known as benzylisoquinoline (BIAs) as well as biosynthetic intermediates, metabolites, and precursors. A variety of BIAs, biosynthetic precursors, intermediates, and metabolites thereof may be produced by the subject host cells, including but not limited to reticuline, sanguinarine, a protoberberine, berberine, a benzophenanthridine alkaloid, thebaine, an opiate compound, cheilanthifoline, stylopine, cis-N-methylstylopine, salutaridinol, salutaridinol-7-O-acetate, protopine and dihydrosanguinarine, (S)-canadine, oripavine, codeinone, neopine, neomorphine, morphine, codeine, hydromorphone, hydrocodone, oxycodone, oxymorphone, dihydrocodeine, 14-hydroxycodeine and dihydromorphine.”

“The host cell may generate synthetic pathways with any suitable compound. Starting compounds of interest include, but are not limited to, laudanosoline, methyl laudanosoline, norlaudanosoline, methyl norlaudanosoline, norcoclaurine, salutaridine, reticuline, tyramine, dopamine, 4-HPA, 4-HPPA, coclaurine, N-methylcoclaurine, 3?-hydroxy-N-methylcoclaurine, scoulerine, tetrahydrocolumbamine, canadine, laudanine, sanguinarine, thebaine, morphine, codeine, codeinone and dimethyl tetrahydoisoquinoline, e.g., 6,7-dimethyl-1-2-3-4-tetrahydroisoquinoline or another compound that may or may not be normally present in the endogenous BIA pathway. In some embodiments, the starting material is reticuline (orlaudanosoline) or norcoclaurine. The starting material can be either naturally occurring or non-naturally occurring. Based on the synthetic pathway in the host cell, other compounds can also be used to start the desired synthetic pathway. The starting material can be obtained from the host cells, such as tyrosine. Or, the starting material could be supplemented from outside sources. If the host cells are in liquid culture (an in-vivo environment), then the media can be supplemented by the starting material, such as tyrosine and norlaudanosoline. These materials are transported into the cells to make the desired products.

“Host Cells”

“A host cell that produces one or several BIAs is one aspect of the invention, as summarized above. Any type of cell that is suitable for producing the subject BIA-producing cell can be used. See, e.g. US2008/0176754, whose disclosure is incorporated herein in its entirety. Sometimes, the host cell may be yeast. Sometimes, the host cell comes from a yeast strain that has been engineered to produce a BIA. In some embodiments, the host cell is selected from a reticuline-producing host cell, a sanguinarine precursor-producing host cell, a protoberberine-producing host cell, a thebaine-producing host cell and an opiate-producing host cell.”

“Any suitable cells can be used in the subject host cell and method. Sometimes, the host cells may not be plant cells. In some cases, the host cells can be insect cells, mammalian cell, bacterial or yeast cells. Host cells of concern include, but not limited to, bacteria cells like Bacillus subtilis and Escherichiacoli, Streptomyces, Salmonella typhimuium and Streptomyces cells, and insect cells such Drosophila melanogaster and Spodoptera friugiperda Sf9 cell. In some embodiments, host cells can be yeast cells or E.coli cells. In some embodiments, yeast cells may be Saccharomyces cerevisiae (S. cerevisiae). Because cytochrome P450 proteins are involved in certain biosynthetic pathways, yeast is an attractive host cell. They are able fold correctly into the endoplasmic retina membrane, which allows them to continue their activity. The invention uses yeast strains of interest, including those described by Smolke et al. in US2008/0176754, whose disclosure is included by reference in its entirety. ura3-52/ura3-52 trp1-289/trp1-289 leu2-3_112/leu2-3_112 his3 ?1/his3 ?1 MAL2-8C/MAL2-8C SUC2/SUC2), S288C, W303, D273-10B, X2180, A364A, ?1278B, AB972, SK1 and FL100. In some cases, the yeast strain may be any of the S288C (MAT?) ; SUC2 mal-mel gal2 CUP1 hap1 flo8-1 (MAT?), BY4741 ; his3?1; leu2?0; met15?0; ura3?0), BY4742 (MATa; his3?1; leu2?0; lys2?0; ura3?0), BY4743 (MATa/MAT? ; his3??1/his3??1; leu2??0/leu2??0; met15??0/MET15?0/LYS2/lys2??0; and ura3??0/ura3??0, BY4742 (MATa/MAT?). These are derivatives of W303-B strain (MATa/MAT?); ade2-1; his3-11; -15; leu2-3, and -112; ura3-1 and cyr+), which have the yeast NADPH?P450 and NADPH?P450 reductase Another embodiment of the yeast cell is W303alpha. ; his3-11, 15 trp1-1 leu2-3 ura3-1 ade2-1). The identity and genotype of additional yeast strains of interest can be found at EUROSCARF (web.uni-frankfurt.de/fb15/mikro/euroscarf/col_index.html).”

“Host cells” is a term that refers to cells that are able to produce desired BIA(s) from their coding sequences. “Host cells” is a term that refers to cells that contain one or more heterologous coding sequencings that encode activity(ies) that allow the host cells produce desired BIA(s). The heterologous sequences of coding could be integrated into the genome of host cells or transiently added to the cell. The term “heterologous code sequence” is used herein. The term?heterologous coding sequence? is used to refer to any polynucleotide which codes for or eventually codes for a peptide, protein, or equivalent amino acid sequence. As such, ?heterologous coding sequences? Multiple copies of the coding sequences are present in the host cells, so that the cells are expressing additional copies. These heterologous coding sequences may be RNA or any other type of RNA, e.g. DNA, mRNA or any other type, e.g. cDNA or a hybrid RNA/DNA. Full-length transcription units include features such as the coding sequence and introns.

“Heterologous sequences of coding” is the term used herein. The coding portion includes both the peptide and enzyme (i.e. the cDNA sequence or mRNA sequence), as well the coding part of the full-length transcriptal unit (i.e. the gene that contains introns or exons as well as?codon optimized). sequences, truncated or other forms, that code for the enzyme, or code for an equivalent amino acid sequence. As long as the sequence produces a functional proteins, it can be truncated, modified, or any other form of altered sequences. These equivalent amino acid sequences may contain a deletion of one or several amino acids. The deletion can be either C-terminal, N-terminal, or internal. Truncated forms can be envisioned provided they possess the catalytic capabilities indicated in this document. The possibility of fusions between two or more enzymes is possible to facilitate the transfer metabolites within the pathway. However, it must be maintained that their catalytic activities are not lost.

Modeling and screening can be used to identify operable fragments, mutants, or truncated versions. This can be done by removing, for example N-terminal, or C-terminal regions, from the protein. Then, the derivative is analyzed in order to determine its activity for the desired reaction relative to the original sequence. This is considered an equivalent derivative of enzyme.

“Aspects also relate to heterologous codes that code for amino acids sequences equivalent to native sequences for various enzymes. A sequence of amino acids that is “equivalent”? An amino acid sequence that is?equivalent’ to the original sequence is one that does not contain identical amino acids, but contains at least some amino-acid changes (deletions and substitutions, inversions or insertions). When used for a specific purpose, they do not significantly affect the biological activity of a protein. In the case of a decarboxylase it refers to its catalytic activities. Also, equivalent sequences include sequences that have been engineered or evolved to have different properties than the original amino acids sequence. Mutable properties include selectivity, catalytic activity and stability. An?equivalent’ is possible in certain embodiments. An “equivalent” amino acid sequence is one that has at least 80% to 99% identity at the amino acids level to the specific sequence. In some cases, this may be as low as 85%, 87% or 89%, and in others, less than 92%, 92% and 93%, respectively. Sometimes, though the amino acid sequence might be identical, the DNA sequence has been altered to maximize codon usage in the host organism.

“Host cells can also be altered to have one or more genetic modifications to accommodate heterologous sequences. Modifying the host genome can be used to alter the expression of specific proteins that could interfere with the desired pathway. These native proteins can quickly convert one of the intermediates, or final products, into a metabolite, or another compound that is not useful in the desired pathway. If the native enzyme activity is reduced or absent, then the intermediates produced would be easier to incorporate into the product. If the host cell is a yeast cells and the desired pathway calls for 2-oxoglutarate as a cosubstrate, then the expression of native glutamate/2/oxoglutarate-dehydrogenase enzymes could be decreased or ablated. This would convert the desired cosubstrate (2,oxoglutarate), into succinyl-CoA or glutamate. Sometimes ablation of a protein’s expression may be useful. This could happen in cases where the host cell is a yeast cell. In these cases, the native endogenous glutamate and/or 2-oxoglutarate dehydrogenase enzymes are reduced or ablated. This would allow the desired cosubstrate (2-oxoglutarate) to be converted into succinyl-CoA. These proteins control the export of BIA compounds into the culture media. Host cell gene deletions may be of interest in certain embodiments. These genes are associated with the unfolded proteins response and endoplasmic reticulum(ER) proliferation. These gene deletions could lead to increased BIA production. Cytochrome P450s can induce unfolded protein responses and cause the ER proliferate. Eliminating genes that are associated with stress responses can control or decrease the overall burden on the host cells and improve pathway performance. Genetic alterations can also include altering the promoters endogenous gene expression to increase or introduce additional copies. This could include strains that overexpress the yeast NADPH?P450 reductase, CPR1, to increase the activity of heterologous P450 enzymes. Endogenous enzymes like ARO8, 9 and 10, which directly participate in the synthesis intermediate metabolites, can also be overexpressed.

“Heterologous coding sequencing sequences of interest” include sequences that encode enzymes (either wild-type or equivalent) that are responsible for the production BIAs in plants. The heterologous sequences can code any enzyme in the BIA pathway. In other cases, they can also be from any source. Cheilanthifoline synthase (CFS, EC 1.14) can be found in some cases. 21.2) can be found in Eschscholzia, Papaver somniferum and Argemone myxicana. It is also known to synthesize (S-cheilanthifoline) from (S-scoulerine). Based on the product, the number and choice of enzymes encoded in the heterologous sequences may be made. The present invention can be used to create host cells that contain 1 or many, 2 or 3 or greater, 4 or less, 5 or less, 6 or most, 7 or much more or 9 or all, 10 or mehr, 11 or or, 12 or higher, 13 or better, 14 or extra, or even 15 or so heterologous sequences such as 1, 2, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, or 15 heterologous sequences.

“Unless otherwise noted the heterologous coding sequences in GENBANK are as reported. Tables 2 and 3 show a list of enzymes of particular interest. Tables 2 and 3 show a list of enzymes of interest. The present invention allows host cells to contain any combination of any of these enzymes. Unless otherwise noted, accession numbers in Table 3 refers to GenBank. Some accession numbers refer to the Saccharomyces genome database (SGD) which is available on the world-wide web at www.yeastgenome.org.”

“In some embodiments, the host cells (e.g. a yeast strain), are engineered to selectively produce a BIA by locating one or more enzymes in a specific compartment of the cell. FIG. 1 illustrates one embodiment of the invention. 18) An enzyme can be targeted to yeast’s endoplasmic reticulum through the fusion of an ER2 targeting sequence with the C-terminus.

“In some cases, the enzyme may be found in the host cell so that the compound that it produces spontaneously rearranges or is converted to a desirable metabolism by another enzyme before reaching a local enzyme that could convert the compound into an unwanted metabolite. To prevent one enzyme from directly acting on a compound and making an undesirable metabolite, or to limit the production of undesirable end product (e.g., a undesirable opioid by-product), it may be possible to select the spatial distance between them. Any of the described enzymes may be located in any suitable compartment within the host cell. This includes, but is not limited to, the organelle, endoplasmic retina, golgi or vacuole as well as the nucleus and plasma membrane. 18).”

“In some embodiments, the host cells may include one or more enzymes that constitute a localization tag. You can use any of the available tags. The localization tag may be a peptidic sequence attached to the N-terminal or C-terminal of an enzyme. You can attach a tag to an enzyme using any method you prefer.

“In some cases the localization tag may be derived from an endogenous yeast proteins. These tags can provide access to a number of yeast organelles, including the endoplasmic reticulum (“ER”), mitochondria (MT), plasma membrane(PM) and vacuole [V].

“In some embodiments, the tag can be an ER routing tag (e.g. ER1). The tag can be a vacuole tag in certain embodiments (e.g. V1). In some embodiments, the tag can be a plasma membrane tag (e.g. P1). The tag may include or be derived from a transmembrane domain within the tail-anchored protein class.

“In certain embodiments, the localization tags locate the enzyme on an organelle’s outside.” The localization tag can be used to locate the enzyme inside an organelle in certain embodiments.

“In certain cases, each type of enzyme can be increased by adding gene copies (i.e. multiple copies), which increases intermediate accumulation, and eventually BIA or BIA precursor manufacturing. The present invention allows for increased BIA production in a host cells by simultaneous expression of multiple species variants or a single enzyme. Sometimes, the host cell may contain additional gene copies of one or more enzymes. Multiple copies of heterologous coding sequences for enzymes can be included in the host cell using any number of methods.

“In some embodiments, multiple copies of heterologous sequences for enzymes are present in the host cell. These could include 2 or more or 3 or more or 4 or more or 5 or more or even 10 or fewer copies. Multiple copies of heterologous sequences are included in some embodiments. This could include multiple copies of one or more of the heterologous codes for an enzyme, or multiple copies of three or more, four, or more. Sometimes, multiple copies of a heterologous sequence for an enzyme may be derived from different sources than the host cell. One example is that the host cell might contain multiple copies of a heterologous sequence of coding, with each copy being derived from a different source. Each copy could include variations in the explicit sequences, based on differences between species of the heterologous sequence that encodes the enzyme of interest.

The engineered host cell media may be taken and monitored to determine the production of BIA compound of interest. Any method can be used to measure and observe the BIA compounds. Methods of interest include, without limitation, LC-MS techniques (e.g. as described herein), where a sample is analyzed using a known amount a standard compound. Identification can be confirmed by m/z or MS/MS fragmentation patterns. Quantitation and measurement of the compound can be done via LC trace peaks with known retention time and/or EIC MS peak analyses by reference to corresponding LC?MS analysis of a standard compound.

“Reticuline-Producing Host Cells”

“Reticuline is a major branch point intermediate in synthesis of BIAs. A high yield of this intermediate is important in engineering efforts to create end products like morphine or sanguinarine. In some cases, to produce reticuline from norlaudanosoline three enzymes are expressed in the host cell: norcoclaurine 6-O-methyltransferase (6OMT; EC 2.1.1.128), coclaurine N-methyltransferase (CNMT; EC 2.1.1.140) and 3?hydroxy-N-methylcoclaurine 4?-O-methylase (4?OMT; EC 2.1.1.116). The enzymes come from different sources than the host cell. An additional cytochrome P450 enzyme (e.g. CYP80B3 and CYP80B1) may be used to produce reticuline using norcoclaurine. Any of the many optimization options available to engineering S. cerevisiae may be used to make reticuline. To produce the highest quality reticuline, it is possible to combine the three methyltransferases of two or more species (e.g. P. somniferum, T. flavum). Another way is to integrate all three methyltransferases from P. somniferum into the yeast genome. The expression of each enzyme can be titrated and not affect the yield of reticuline. You can adjust the expression of the methyltransferase enzymes by adjusting the titration. The methyltransferases can act in concert, sequentially or as a mixture of both on the substrates.

The invention includes strains of S. cerevisiae that have improved reticuline production by overexpression of 6OMT and CNMT genes, as well as 4?OMT genes. The improved or increased production refers to the production of some amount reticuline in comparison to the control. It also includes an increase of 10% or greater, such a about 20%, 30%, 40%, 50%, 60%, or about 80%. This can be 2-fold, 5-fold, or 10-fold, depending on whether the control has any reticuline. Different species of methyltransferases have different substrate specificities [Choi and al. (2002). J Biol Chem 277, 830-835; Liscombe et al. (2009). Plant J 60, 729-74; Morishige et al., (2000) J Biol Chem 275, 23398-23405; Ounaroon et al. (2003) Plant J 36, 808-819; Sato et al., (1994) Eur J Biochem 225, 125-131]. It is possible to combine methyltransferases from different species into one strain. This allows you to benefit from the various substrate specificities and increase flux via multiple methylation pathways. This can increase the yield of reticuline. Some species variants of methyltransferases may be found in certain cases (Table 2). P. somniferum may be used to create species variants of the MMTases. T. flavum is sometimes used to make species variants of the MMTs. C. may be used to derive species variants of the MMTases in some instances. japonica.”

“In some embodiments, the host cells contain two or more heterologous sequences for two or three methyltransferases from 6OMT, CNMMT, and 4?OMT. Sometimes, two or more methyltransferases may be derived from different sources than the host cell.

“In certain cases, the host cell contains heterologous sequences of coding for the methyltransferases 6OMT or CNMT. In some cases, heterologous coding sequences are present in the host cell for the methyltransferases CNMT or 4?OMT.

“In some cases, the host cell may contain heterologous coding sequences of the methyltransferases 6OMT or 4?OMT in certain instances.”

“In some embodiments, the host cell contains heterologous sequences of coding for all methyltransferases 6OMT and CNMT in certain embodiments.”

“In certain instances, each type of methyltransferase can be increased by adding gene copies (i.e. multiple copies). This increases intermediate accumulation and eventually reticulin production. The present invention allows for increased reticuline production in yeast strains by simultaneous expression of multiple species of a single or several methyltransferases, and incorporation or additional gene copies of one or more methyltransferases.

“In some embodiments, the host cells include multiple copies of amethyltransferase. These could be 2 or more or 3 or more or 4 or more or 5 or more or even 10 or fewer copies. Multiple copies of one or several methyltransferases may be present in certain embodiments. methyltransferases. Multiple copies of the methyltransferase may be derived from different sources than the host cell. One example is that the host cell could contain multiple copies of a heterologous sequence of coding, with each copy being derived from a different source. Each copy could include variations in the explicit sequences, based on differences between species of the enzyme of particular interest encoded by the heterologous code sequence.

“In some cases, multiple copies of a heterologous coding scheme for CNMT are included. Two copies of a heterologous coding scheme for CNMT may be included in certain cases. Multiple copies of a heterologous sequence for 6OMT may be included in some cases. Two copies of the heterologous sequence for 6OMT may be included in certain cases. Multiple copies of a heterologous sequence for 4?OMT may be included in some cases. Two copies of a heterologous sequence for 4?OMT may be included in certain cases.

“In certain cases, the host cell can produce a higher amount of reticuline using norcoclaurine than a control cell that does not have multiple copies of one or more heterologous sequences for the one methyltransferase. The increased amount of reticuline in some cases is 10% higher than the control host cells. This could be as a result of the host cell producing a greater amount of norcoclaurine relative to the control cell.

“In certain cases, the host cell can produce a higher amount of norlaudonosoline from norlaudoline than a control cell that does not have multiple copies of one or more heterologous sequences for the one methyltransferase. The increased amount of reticuline can be as high as 10% relative to the control cell in certain cases. This could include about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, and even 10-fold relative to the control cell.

“In some embodiments, the host cells are capable of producing a 10% to more yield of norcoclaurine reticuline. This includes 20%, 30%, 40%, 50%, 60%, 70%, 70%, 80%, or even 90% yield of norcoclaurine reticuline.

“In some embodiments, reticuline can be produced by the host cell from norlaudonosoline in a yield of 10% or higher, such as 20%, 30%, 40%, 50%, 60%, 70%, 70%, 80%, 85%, 90%, or even more from reticuline.”

“In some embodiments, the host cell includes an engineered strain that includes the biosynthetic pathway. This allows for increased production of reticuline and incorporates any combination the following alternative methylation pathways. In certain instances, the host cell can produce reticuline from norlaudanosoline using a biosynthetic pathway as shown in FIG. 2. In some embodiments, the host cell can produce reticuline from norcoclaurine through a biosynthetic pathway as shown in FIG. 3. Sometimes, norlaudanosoline is used as the starting material for the pathway (FIG. 2, (1), CNMT, and/or 4??OMT could act on this compound to create three distinct methylated intermediates. BIA 2, which was initially methylated initially by 6OMT is then methylated either by CNMT or 6OMT; BIA 3 which was initially methylated initially by CNMT is then methylated either by 6OMT (or both); BIA 4 which has been previously methylated initially by 4OMT is then methylated either by 6OMT or CNMT is methylated to make reticuline.

“In certain cases, when the pathway’s starting material is norcoclaurine (6OMT or CNMT) may react on this compound to create two distinct methylated products (FIG. 3): BIA 10, initially methylated initially by 6OMT is then methylated again by CNMT. BIA 11, initially methylated initially by CNMT is then methylated again by 6OMT. BIA 12, also known by N-methylcoclaurine is previously methylated with 6OMT and CNMT. CYP80B1 and CYP80B3 then sequentially act upon BIA 11 to make reticuline.

“In some cases, the yeast strain is present in the host cell. S. cerevisiae.”

“Sanguinarine & Sanguinarine Precursors-Producing Host cells”

“Aspects of the invention include host cells that produce protoberberine and benzophenanthridine alkaloids, including, but not limited to, cheilanthifoline, stylopine, cis-N-methylstylopine, scoulerine, protopine, dihydrosanguinarine and sanguinarine. FIG. FIG. 4 shows a synthetic pathway found in embodiments host cells as per embodiments according to the present invention. The pathway can be longer and may start from norlaudanosoline or norcoclaurine, but this pathway depiction starts with reticuline. It ends with sanguinarine. If the desired end product is not one of the intermediates of the norlaudanosoline-sinurarine pathway, the pathway may include less enzymes than the ones shown. The invention comprises biosynthetic pathways that include multiple enzymatic steps. For example, CFS (EC 1.14.21.2), CPR, CPR (EC 1.6.2.4), STS(EC 1.14.21.1), STS (EC 1.14.21.1), TNMT, EC 1.14.13.37) and MSH (EC 1.14.13.35) are all catalyzed within an engineered yeast strain to make a variety benzophenanthridine and protoberberine compounds. The present invention also includes tools and methods that optimize the production of benzophenanthridine and protoberberine compounds in the context of an engineered yeast strain.

“In certain embodiments, the host cells are capable of producing sanguinarine, or a sanguinarine precursor. In these cases, the host cell contains one or more heterologous coding sequencings for one or several enzymes from BBE, CFS or CPR, STS or STS. The host cell may also have one or two other heterologous coding sequences. The sanguinarine precursor may be a protoberberine, a benzophenanthridine or an alkaloid.

“In some cases, the source organism may be P. somniferum (E. californica), Arabidopsis.thaliana, Papaver bracteatum, A. mexicana, or E. californica. P. somniferum is sometimes the source organism in certain cases. Sometimes, E. californica is the source organism. A. thaliana is sometimes the source organism. In some cases, P. bracteatum is the source organism. A. is sometimes the source organism in some cases. mexicana.”

“In some cases, the one or more enzymes are two or more enzymes that were derived from different sources than the host cell.”

“In some embodiments, the host cells contain multiple copies of one or more heterologous sequences. Multiple copies of one or more heterologous sequences may be derived from different sources than the host cell in certain embodiments. One example is that the host cell might contain multiple copies of a heterologous sequence of coding, with each copy being derived from a different source. Each copy could include variations in the explicit sequences, based on differences between species of the enzyme of particular interest encoded by the heterologous code sequence.

“In some cases, the host cells may contain more than one, such as three, four, five, six, or more heterologous coding sequences, for two or more enzymes from STS, CFS or CPR, STS or TNMT. P6H, MSH, DBOX, and CFS.

“In some cases, the host cell may also contain one or more gene deletions compared to a native host cells. The one or more deleted genes are selected from IRE1, OPI1, ONO2, INO3, PDR1, PDR3, PDR5, SNQ2, YOR1, IRE1, TPO2, TPO3, PDR10 and PDR15. PDR16. PDR17. QDR1, QDR2, FLR1, QDR3, QDR3, QDR3, QDR3, QDR5, AQR1, AQR2, CIN5, and CIN5.

“In some cases, the host cells are a yeast strain (e.g. as described herein).

“In certain embodiments, the host cells are capable of producing sanguinarine or sanguinarine precursors from norlaudanosoline via the biosynthetic pathway shown in FIG. 4. Sometimes, the host cell can produce the sanguinarine/sinuarine precursor from norlaudanosoline through a biosynthetic pathway as shown in FIG. 11.”

“In some cases, the host cell may contain a heterologous sequence of coding for a BBE protein. A heterologous sequence of coding for the BBE enzyme can be integrated into a host chromosome. Sometimes, the host cell contains a heterologous sequence of coding for a CFS-enzyme. Sometimes, the host cell contains a heterologous sequence of coding for a CPR protein enzyme. Sometimes, the host cell contains a heterologous sequence of coding for a STS protein enzyme. Sometimes, the host cell contains a heterologous coding sequence to encode a TNMT protein. Sometimes, the host cell contains a heterologous sequence of coding for a MSH-enzyme. Sometimes, the host cell contains a heterologous sequence of coding for a P6H protein. Sometimes, the host cell contains a heterologous sequence of coding for a DBOX gene.

“In some cases, the host cells is a yeast strain producing scoulerine. In these cases, the berberine bridge enzyme, (BBE; E. 1.21.3.3)) (e.g. from P. somniferum) is expressed on a low-copy construct (e.g. a low-copy plasmid, or chromosomally or YAC) in the yeast strain with Ps6OMT or PsCNMT and/or Ps4??OMT chromosomally chromosomally chromosomally chromosomally chromosomally or Ps4?OMT. You can use any of the available enzyme variants, such as one or more of those shown in Table 2.

“In certain cases, the sanguinarine precursor may be cheilanthifoline. In some cases, the host cell contains heterologous coding sequences of cheilanthifoline synthase and a cytochrome 450 NADPH reducetase enzyme (CPR; EC 1.6.2.4). Sometimes, the CPR enzyme can be an ATR enzyme (e.g. ATR1). Sometimes, the host cell may be a yeast strain producing cheilanthifoline. In these cases, cheilanthifoline synthase is expressed on a low-copy construct (e.g. a low-copy plasmid, YAC, or chromosomally integrated) in yeast strains with Ps6OMT and PsCNMT and/or Ps4?OMT and/or PsBBE. You can use any of the available enzyme variants, such as one or more of those shown in Table 2.

“In some embodiments, stylopine is produced by the host cell. In these cases, stylopine synthase [EC 1.14.21.1] (e.g. from E.californica (EcSTS), Pa. somniferum(PsSTS), and/or A.mexicana)) is expressed in the host cells on a low-copy construct (e.g. a low copy, YAC, or chromosomally integrated) in cheilanthifoline-producing strain. You can use any of the available enzyme variants, such as one or more of those shown in Table 2.

“In certain cases, the host cells produces cis-N-methylstylopine, where tetrahydroprotoberberine N-methyltransferase (TNMT; EC 2.1.1.122) (e.g., from P. somniferum (PsTNMT) or E. californica (EcTNMT)) is expressed in the host cell from a low copy construct (e.g., a low copy plasmid, YAC or chromosomally integrated) in a stylopine-producing strain. You can use any of the available enzyme variants, such as one or more of those shown in Table 2.

“In certain cases, protopine is produced by host cells. In certain cases, cis-N-methylstylopine 14-hydroxylase (MSH, EC 1.14.13.37) (e.g., from P. somniferum (PsMSH)) is expressed in the host cell from a low copy construct (e.g., a low copy plasmid, YAC or chromosomally integrated) in a cis-N-methylstylopine producing strain. The host cell may contain heterologous coding sequences that encode a TNMT or a MSH enzyme in certain cases. You can use any of the available enzyme variants, such as one or more of those shown in Table 2.

“In some cases, the host cells produce dihydrosanguinarine. In these cases, protopine 6-hydroxylase, (EC 1.14.13.55) is expressed in the host cells from a low-copy construct (e.g. a low copyplasmid, or YAC) in a protopine-producing strain. You can use any of the available enzyme variants, such as one or more of those shown in Table 2.

“In some cases, the host cell produces sanguinarine, where dihydrobenzophenanthridine oxidase (DBOX; EC 1.5.3.12) (e.g., from P. somniferum (PsDBOX)) is expressed in the host cell from a low copy construct (e.g., a low copy plasmid, YAC or chromosomally integrated) in a dihydrosanguinarine producing strain. You can use any of the available enzyme variants, such as one or more of those shown in Table 2.

“In some embodiments, the host cells are engineered to produce more cheilanthifoline that a control strain. Additional copies of CFS can be found in the engineered strain. More refers to both the production or a decrease in cheilanthifoline from the control strain, and also the increase of approximately 10% or greater. This is because the engineered strain has more copies of CFS than the control strain. Variation in the expression of a cytochrome P450 target compound may increase its production by changing gene copy number, promoter strength, or regulator regulation. Sometimes, expression of a high-copy plasmid doesn’t result in measurable cheilanthifoline. Cheilanthifoline production can be higher in certain cases, where multiple copies of CFS gene are expressed by host cells from low copy constructs. Some embodiments of the CFS gene include more copies in the host cell. This increases the production of cheilanthifoline.

“In some cases, in order to produce more stylopine variants of enzymes CFS/STS from different species can be combined in a host cell. An engineered host cell can produce higher levels of stylopine if it is compared to a control cell lacking the desired enzymes. The production of some stylopine in the engineered host cell is increased, but not the production of all stylopine. It can also be observed that the control cells have some stylopine, and the increase is 2-fold or greater. FIG. 13(c) shows the measurement of stylopine from different CFS versions, expressed with different STS variants. 13(c). To increase the levels of stylopine, any combination of enzymes can be used.

“In certain instances, in order to produce more cheilanthifoline host cells (e.g. yeast strains) may be engineered to contain chromosomally-integrated NADPH cytochrome P450 reducetases from various species to maximize the activity of cytochrome P450s. It is possible to measure the cheilanthifoline produced by different cheilanthifoline-synthese enzymes and cytochrome P450 NADPH reducer enzymes. To produce higher levels of cheilanthifoline than a control, any combination of these enzymes can be used.

“In some cases, in order to produce more cheilanthifoline and stylopine, host cell (e.g. yeast strains) with high levels of cytochrome B5 can be used to maximize the activity of cytochromeP450s. It is possible to measure the cheilanthifoline and stylopine levels in host cells that have cytochrome B5 overexpressed or not. Sometimes, the host cell may overexpress cytochrome B5 and produce more cheilanthifoline and stylopine than a control.

“In certain embodiments, host cells produce more protoberberinealkaloids. The host cell’s optimization is achieved by the deletion genes associated with unfolded protein responses and endoplasmic reticulum proliferation to increase BIA production. Gene deletions that are of interest include OPI1, HAC1, OPI1, INO1, and INO3 (e.g. Table 3). Sometimes, cytochrome P450s expression can induce the unfolded proteins response and cause the ER proliferate. The elimination of genes that are associated with stress responses can control or reduce the overall burden on the host cells and improve pathway performance. More refers to the production of some quantity of protoberberinealkaloids in cases where the control does not have any. It also includes an increase of approximately 10%, such a about 20%, 30%, 40%, 50%, 60%, about 80%, about 100%, or 2-fold or greater, such a 5-fold, 10-fold, or even more for situations where there is some protoberberinealkaloids production.

“In some cases, the host cell may contain one or more heterologous coding sequences that encode for one or several proteins involved in the transport of compounds across cell membranes. PDR1, PDR5, SQ2, YOR1, PDR3, PDR3, CIN5, or PDR3 are some of the proteins that transport compounds across cell membranes.

“In some embodiments, genes that are involved in the pleiotropic drugs response, such as ATP-binding cassette transporters (ABC) pumps, multidrug resistance pump (MDR) pumps, and associated transcription factors, can be deleted from the host cell in order to decrease the export of BIA molecules to the culture medium. PDR1, STB5, PDR3, PDR5, SNQ2, YOR1, TPO1, TPO2, TPO3, TPO4, PDR10 and PDR15 are all examples of genes. Multiple deletions can occur in any combination. Sometimes, the host cell may have one or more gene mutations that cause lower levels of reticuline and scoulerine or cheilanthifoline in some cases.

“Another embodiment places a gene that is involved in the pleiotropic drugs response in host cells. This promoter can be either inducible or growth-stage dependent and will control temporal control of BIA transportation. A stationary phase promoter can be used to control a transporter gene that causes a BIA of particular interest to remain in the cell until it reaches stationary phase. This may increase the conversion of starting materials into the desired end products in host cells.

“Protoberberine-Producing Host Cells”

“Aspects include engineered host cells capable of producing protoberberine alkaloids. The protoberberinealkaloids may have one of these structures in some cases:

“Where R1-R14 can be selected independently from?H (e.g. a lower alkyl like methyl (CH3), or ethyl), or a hydroxyl, or an alkoxy(OR) (e.g. a lower Alkoxy such methoxy, or ethoxy).

In certain cases, protoberberine alkaloids may be produced from reticuline, other analogs thereof, and derivatives thereof. In certain cases, the host cells may include one or more heterologous coding sequences to express one or more of, or any combination of, the following enzymes: berberine bridge enzyme (BBE), scoulerine 9?-O-methyltransferase (S9OMT), canadine synthase (CAS), and (S)-tetrahydroprotoberberine oxidase (STOX), wherein the one or more enzymes is derived from a different source organism as compared to the host cell.”

“In some cases, the source organism may be P. somniferum or E. californica or C. japonica, T. Flavum, Berberis subsp. glaucum, Coptis chinensis, Thalictrum spp, Coptis spp, Papaver spp, Berberis wilsonae, A. mexicana, or Berberis spp.”

“In some embodiments, the host cell may contain multiple copies of one or more heterologous sequences. Sometimes, multiple copies of one or more heterologous sequences may be derived from different sources than the host cell. One example is that the host cell could contain multiple copies of a heterologous sequence of coding, each copy being derived from a different source. Each copy could include variations in the explicit sequences, based on differences between species of the enzyme of particular interest encoded by the heterologous code sequence.

“In some cases, the host cell may contain two or more heterologous coding sequencings for two or three enzymes from BBE, CAS, CAS, and STOX. Sometimes, the host cell contains three or more heterologous coding sequencings for more than three enzymes, including S9OMT and CAS. Sometimes, the host cell contains heterologous sequences of coding for each enzyme, BBE, S9OMT and CAS.

“In certain embodiments, the host cell includes the heterologous sequence for CAS and the heterologous sequence for ATR1. In some embodiments, the heterologous sequence for STOX is found in the host cell.

“In some embodiments, the host cells (e.g. an engineered yeast strain), support a biosynthetic pathway, as shown in FIG. 5.”

“In some instances, the host cell includes the gene STOX that shares 75% or more (e.g., 78%) nucleic acid sequence identity with the naturally occurring gene B. wilsonae (S)-tetrahydroprotoberberine oxidase (Table 3). This gene could be a non-natural sequence of nucleotides, codon-optimized to yeast expression.

“Aspects” of the invention include the functional expression in yeast culture of STOX and its homologues. In some embodiments, host cells can be engineered to make berberine using its precursor (S-canadine).

“In one embodiment, the expression levels of enzymes are relatively low (e.g. CEN/ARS vector, genomic expression) for BBE and CAS (see, for example, FIG. 6a ) and high (e.g. 2?m vector, multiple genomic copies) S9OMT (see e.g. FIG. 6b ). Any method can be used to alter the expression levels. These methods include, but are not limited, altering the strength of a constitutive promoter, using a inducible promoter and changing the number of genes episomally or genomeally (see FIG. 6c), changing the selection marker and/or culture conditions to correspond with promoter activity.

“In some embodiments, one or more enzymes can be recombinantly derived from a yeast artificial genome (e.g. FIG. 10).”

“Aspects include the functional expression in live yeast culture of CAS or its homologues as part of larger biosynthetic pathways. In some embodiments, host strains can be engineered to make berberine from norlaudanosoline (or its precursors) in certain embodiments. 5, 7, or Table 2. In certain cases, the host cell can produce berberine by reticuline using a biosynthetic pathway that is FIG. 5. Another embodiment of the invention includes a cytochrome P450 partner for CAS in the host cell. This is ATR1, and co-expression may result in higher activity than E.californica CPR or A.thaliana ATR2, P. somiferum CPR or endogenous yeasts CPR (e.g. FIG. 6d ).”

“Aspects include the functional expression in yeast culture of STOX and its homologues. In some embodiments, host cells can be engineered to make (S)-canadine from norlaudanosoline, which is the precursor of Berberine. Some embodiments allow the host cell to produce (S)-canadine using norlaudanosoline.

Heterologous transporters may be expressed in engineered strains to increase the BIA accumulation. This includes, but is not limited to, plant ATP-binding cassette protein from BIA-producing species. To accumulate berberine in the host cell, some embodiments may include one or more heterologous sequences of coding for transporters from CjABCB1, CjABCB2, or CjABCB2.

“In some cases, the yeast strain is used as the host cell.”

“Thebaine-Producing host Cells”

The invention includes engineered host cells that can produce thebaine from reticuline and its precursors. A strain of reticuline, or one of its precursors, may be used. It can also be added to culture media or a fraction of cell lysate. In some instances, the host cells may include one or more heterologous coding sequences to express for one or more enzymes selected from salutaridine synthase (SalSyn), cytochrome P450 2D6 (CYP2D6), cytochrome P450 2D2 (CYP2D2), salutaridine reductase (Sal R), and/or salutaridinol 7-O-acetyltransferase (SalAT). One or more of the enzymes could be derived from another source organism than the host cell.

“In some cases, the source organism may be P. somniferum or P. bracteatum. Papaver orientale, Papaver species, Homo sapiens, Rattus norvegicus, Papaver spp., Papaver spp.

“In some cases, host cells may be engineered yeast strains that support biosynthetic pathways in a host, as shown in FIG. 8.”

“In some embodiments, the host cell may contain multiple copies of one or more heterologous sequences. Sometimes, multiple copies of one or more heterologous sequences may be derived from more than one source organism.

“In certain cases, the host cell may contain two heterologous sequences of coding. The two heterologous sequences of coding are in some cases for the enzymes SalR or SalAT.

“In some cases, the host cell contains the genes CYP2D6, CYP2D2, SalSyn, and/or SalSyn. These genes can be engineered or natural P450s that can produce salutaridine from reticuline. It could be a non-natural or natural nucleotide sequence that is codon-optimized to yeast expression.

“In some embodiments, the host cells include a cytochrome P450 reducetase partner to CYP2D6, CYP2D2, or SalSyn. This may lead to higher CYP2D6, CYP2D2, or SalSyn activity than E. californica, A. thaliana ATR2, P. somiferum CPR, and endogenous yeast CPR.”

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