Patent Application
20210380995
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format, and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on Apr. 12, 2021, having the file name “P19-012PCTPCT_ST25.txt” and is 495 kb in size.
The present invention relates to a recombinant microbial host cell having improved in vivo conversion of thebaine and/or oripavine to relevant downstream opioid related compounds, wherein the microbial host cell heterologously expresses at least one functional transporter protein. The invention also relates to use of the microbial host cell to make an opioid compound, opioid pathway intermediate, opioid derivative or opioid precursor compound of interest.
Opioids are alkaloid narcotics, natural or synthetic, that act on opioid receptors to produce morphine-like effects. Opioid receptors are found principally in the central and peripheral nervous system and the gastrointestinal tract. The medical uses of various opioids includes pain relief, including anesthesia, as diarrhea suppressors, as cough suppressors, and in replacement therapy for opioid use disorder.
An opiate is a narcotic drug derived from opium. Morphine, the prototypical opiate was first isolated from the flowering opium poppy plant, Papaver somniferum. Other examples of natural opiates also isolated from P. somniferum, include morphine, codeine, thebaine and oripavine.
The term opioid is a broader term for alkaloids that includes opiates and refers to any substance, natural or synthetic, that binds to the brain's opioid receptors.
Today commercially available synthetic opioid medical drug products (e.g. oxycodone, hydrocodone, hydromorphone, oxymorphone, buprenorphine, naltrexone, naloxone, nalbuphine) are obtained by chemical modification of natural opiates (e.g. thebaine) as starting precursor compounds.
For instance, the semi-synthetic opioids buprenorphine, naltrexone, naloxone and/or nalbuphine may be obtained by so-called semi-synthesis from e.g. the natural opiates thebaine or oripavine (Tomas Hudlicky; “Recent advances in process development for opiate-derived pharmaceutical agents”; Can. J. Chem. 93: 492-501 (2015)). As discussed in Hudlicky et al., the natural opiates morphine and codeine are also potential starting materials for semi-synthesis of synthetic opioid medical drug products.
Today commercially available natural and synthetic opioid medical drug products are dependent on industrial opium poppy farming that is susceptible to environmental factors such as pests, disease, and climate, and to geopolitical factors, any of which can introduce instability and variability into this supply chain.
It is therefore desirable to establish a microbial-based manufacturing process for opioids or opioid precursors, as such a controlled, sustainable and scalable system could have the potential to address many of the current challenges associated with the opium poppy plant-based supply chain used to date.
The publication of Galanie et al. (“Complete biosynthesis of opioids in yeast”; Science. 2015 Sep. 4; 349(6252): 1095-1100) describes a complete biosynthesis in engineered yeast of the opioids thebaine and hydrocodone starting from sugar.
WO2018/075670A1 describes biosynthesis in yeast of a number of the herein relevant opioids or opioid precursors, as reproduced here in
Dastmalchi et al. 2019 employs a P450 only as a reductase partner to SalSyn acting on (R)reticuline. Indeed, Dastmalchi 2019 teaches it is preferable to engineer an alternative pathway (In90-99), rather than use a P450 because it is “a perceived bottleneck”.
As known to those skilled in the art, the term “P450”, also known as “cytochrome P450” or simply “CYP”, refers to a broad class of over 50,000 so far identified proteins that function as monooxygenases using heme groups as co-factors tethered by a cysteine-thiolate ligand. Of particular relevance to the current invention are cytochrome P450 enzymes capable of demethylating a reticuline derivative. In some aspects, preferred reticuline derivates include thebaine and oripavine. Individual P450 enzymes in this class may be capable of N-demethylation and/or O-demethylation. Non-limiting examples of activities of P450s capable of demethylating reticuline derivatives include thebaine 6-O-demethylase, thebaine O-demethylase, thebaine N-demethylase, oripavine N-demethylase, oripavine 6-O-demethylase and/or codeine O-demethylase. In some embodiments, a P450 capable of demethylating reticuline derivatives possesses more than one of these activities. The P450s are typically membrane-associated P450s.
Also illustrated in
As discussed in Galanie et al. the overall yield of opioids from engineered microbial-based (e.g. yeast based) manufacturing process for opioids in the art remains inadequate to the extent that such microbial-based processes are to date not the preferred options for industrial commercial production of opioids (such as e.g. buprenorphine).
As used herein, the term “membrane transport protein” (or simply “transporter”) is a membrane-bound or membrane-spanning protein involved in the movement of ions, small molecules, or macro-molecules, such as peptides, across a biological membrane. A variety of transporters have evolved to move the hundreds of thousands of different substrates found in nature across suitable membranes. Of particular relevance to the current invention are transporters capable of transporting opiods and/or opioid pathway intermediates and/or opioid derivatives. An introduction to the topic can be found in Jorgensen et al. (“Origin and evolution of transporter substrate specificity within the NPF family”; eLife 2017; 6:e19466. DOI: 10.7554/eLife.19466).
Without being limited by theory, the present inventors believe that none of the transporter proteins characterized to date are described be capable of transporting opiates like thebaine and/or oripavine into a microbial cell, such as e.g. a yeast cell.
The present invention provides an improved micorbial-based manufacturing process for the production of opioids and/or opioid precursors and/or opioid derivatives.
As discussed above and without being limited by theory, the present inventors believe that none of the transporter proteins characterized to date are described as being capable of transporting opiates like thebaine and/or oripavine into a microbial cell, such as e.g. a yeast cell.
The present inventors tested recombinant expression in Saccharomyces cerevisiae of a number of different transporter proteins to determine whether any of them could have a positive influence on the yield of any of several opioids. The chosen test system was the in vivo bioconversion of thebaine and/or oripavine to relevant downstream opioid biosynthesis compounds and intermediates.
As discussed within the working Examples herein, the inventors identified that a number of transporter proteins had no positive effect on the yields of in vivo bioconversions of thebaine and/or oripavine.
However, the inventors continued their investigations and found that a number of specific transporter proteins could give a surprisingly high improvement in the yield of in vivo bioconversion of thebaine and/or oripavine.
For instance, as discussed in e.g. the Conclusions of Examples 4 and 5 herein, expression of one or more of the transporter genes T14_PsoNPF3_GA, T1_CjaMDR1_GA, T4_EsaGTR_GA, T7_PtrPOT_GA or T97_ScaT14_GA in a yeast strain engineered to be capable of the relevant catalysis, resulted in improved bioconversion of thebaine to northebaine in the range of 22-63% in comparison to the control strain without such transporters. Also, as discussed in e.g. the Conclusion of Example 6, expression of one or more of the transporter genes T65_IjaNPF_GA, T94_EcrPOT_GA and T97_ScaT14_GA resulted in improved bioconversion of thebaine to northebaine in the range of 21.8% to 31.9%. Such increases in yield are objectively a significant improvement.
The work described herein is believed to be the first time this positive “thebaine and/or oripavine improved bioconversion yield” effect has been demonstrated for a transporter protein.
There are no objective technical reasons to believe that the herein discussed membrane transporter proteins as should directly influence the in vivo bioconversion of thebaine and/or oripavine enzymatic reactions.
Accordingly and without being limited by theory, the improved positive bioconversion yield first demonstrated herein may be related to the herein described transporter proteins increasing the intracellular concentration of thebaine and/or oripavine (i.e. in vivo) in the host microbial cell.
In the working Examples described herein, the inventors noted the positive effect of improvement of the yield of in vivo bioconversion of thebaine and/or oripavine demonstrated for the in vivo conversion of thebaine into northebaine, and for oripavine into nororipavine, and for thevinone into northevinone.
However, since an objectively plausible theory of this identified positive in vivo conversion yield effed relates to “an increased uptake of thebaine and/or oripavine into the host cell and therefore an increased amount of thebaine and/or oripavine present in vivo as such”—there is no reason to believe that this positive yield effect would also not be relevant for the in vivo conversion of thebaine and/or oripavine into other products such as e.g. neopinone or oripavine (see
Further, since thebaine and oripavine are structurally very similar (see e.g.
In many of the working Examples disclosed herein, the positive yield effect was demonstrated using Saccharomyces cerevisiae (S. cerevisiae) as host cell.
However, the herein discussed membrane transporter proteins are not from yeast (see table below). Many are from plants and fungi, and there is objectively no technical reason to believe that they should be optimized to only work positively in S. cerevisiae—to the contrary it is believed that the fact that a positive effect has herein been demonstrated for S. cerevisiae makes it plausible that a similar positive effect would be present for substantially all yeast host cells and many other microbial host cells.
Further, as discussed herein a number of the positively identified transporter are from fungi cells (more precisely from filamentous fungi cells). Prima facie there is no objective reason to believe that these fungi transporter should not work in a fungus cell in general—i.e. the host cell may be a fungus cell, such as e.g. a yeast cell or e.g. a filamentous fungus cell.
As discussed above, downstream to thebaine (i.e. starting from thebaine) may thebaine be converted into neopinone, oripavine or northebaine (see e.g.
As shown in
In working examples herein, fungal N-demethylase genes/enzymes were used that are different from the bacterial N-demethylase (e.g. Bacillus BM3 gene) described in WO2018/075670A1. As discussed in e.g. WO2018/075670A1 and PCT/EP2018/066155 the “conversion of thebaine/oripavine” may also function in fungus host cells in general, such as e.g. a yeast cell or e.g. a filamentous fungus cell.
The PCT/EP2018/066155 application also describes a number of different fungal O-demethylases that are suitable for the thebaine to oripavine conversion. However, PCT/EP2018/066155 does not disclose a microbial host cell, wherein the host cell expresses a P450 capable of demethylase activity on reticuline or its derivatives in combination with heterologous expression of an functional transporter protein.
In short, based on the technical disclosure herein and the prior art knowledge of the skilled person, it is routine work for the skilled person to make a recombinant fungus host cell capable of:
The table below provides both DNA and amino acids sequences of the positive transporter proteins discussed herein—i.e. that in working Examples herein have been recombinantly expressed in Saccharomyces cerevisiae and shown to have a positive influence on the yield of in vivo bioconversion of thebaine to relevant downstream opioid biosynthesis compounds.
Papaver somniferum
Camellia japonica
Eutrema salsugineum
Populus trichocarpa
Argemone mexican
Aquilegia coerulea
Basidiobolus meristosporus
Smittium culicis
Arabidopsis thaliana
Rhizopus microsporus
Chelidonium majus
Mortierella elongate
Lonicera japonica
Emmonsia crescens
Sanguinaria canadensis
Macleaya cordata
Papaver somniferum
Papaver somniferum
Papaver somniferum
Papaver somniferum
Glaucium Flavum
Papaver somniferum
Trema orientale
Cucumis sativus
Helianthus annuus
Musa acuminata subsp.
malaccensis
Nelumbo nucifera
Papaver somniferum
Papaver somniferum
Papaver somniferum
Jatropha curcas
Cucurbita pepo subsp. pepo
Lactuca sativa
Papaver somniferum
Papaver somniferum
Nandina domestica
Papaver bracteatum
Cinnamomum micranthum f.
kanehirae
Papaver somniferum
Papaver somniferum
Rosa chinensis
Erythranthe guttata
Arachis duranensis
Papaver somniferum
Papaver alpinum
Eschscholzia californica
Macleaya cordata
Cinnamomum micranthum f.
kanehirae
Papaver somniferum
Papaver somniferum
Manihot esculenta
Handroanthus impetiginosus
Aquilegia coerulea
Papaver somniferum
Papaver atlanticum
Glaucium Flavum
Papaver somniferum
Cinnamomum micranthum f.
kanehirae
Papaver somniferum
Rosa chinensis
Durio zibethinus
Olea europaea var. sylvestris
Coffea eugenioides
Papaver somniferum
Papaver miyabeanum
Papaver bracteatum
Papaver somniferum
Aquilegia coerulea
Papaver somniferum
Fragaria vesca subsp. vesca
Ziziphus jujuba
Lactuca sativa
Macleaya cordata
Aquilegia coerulea
Papaver nudicale
Papaver bracteatum
Papaver somniferum
Aquilegia coerulea
Papaver somniferum
Papaver somniferum
Prunus yedoensis var.
nudiflora
Macleaya cordata
Helianthus annuus
Carica papaya
Papaver radicatum
Sanguinaria canadensis
Aquilegia coerulea
Papaver somniferum
Malus domestica
Cinnamomum micranthum f.
kanehirae
Artemisia annua
Capsicum chinense
Jatropha curcas
Papaver trinifolium
With reference to gene nomenclature, the “Transporter gene Code” may be seen as an internal code (used in e.g. Examples herein) and in the table below example sequences are connected to public known transporter protein information.
As indicated in the table, the fact that e.g. “T11_AthGTR1_GA” is known to be a transporter protein can be verified by use of the “NPF2.10” entry code in the public known UniProt (https://www.uniprot.org) database.
For instance, “T14_PsoNPF3_GA” does not have an official (e.g. UniProt) entry code, since it was identified by the present inventors to be a transporter due to e.g. relevant sequence identity to “T11_AthGTR1_GA” and herein presented experimental work.
Accordingly, a first aspect of the invention relates to a recombinant microbial host cell capable of:
As understood by the skilled person in the present context, the term “functional” within the term “functional transporter protein” of the first aspect simply requires that the transporter protein is capable of functioning as a transporter protein within the host cell. In contrast, as known in the art, a protein of interest may be nonfunctional due to e.g. a frameshift mutation or e.g. the insertion of a stop codon, misfolding or immediate degradation in an inappropriate host cell, or for other reasons.
As understood by the skilled person in the present context—the term “thebaine derivative” relates to a compound that thebaine may be converted into, examples of which include but are not limited to neopinone, oripavine and/or northebaine.
Similarly, the term “oripavine derivative” relates to a compound that oripavine ma converted into, examples of which include but are not limited to nororipavine and/or morphinone.
As discussed herein, an embodiment of the first aspect relates to a recombinant microbial (such as a fungus) host cell capable of:
A second aspect of the invention relates to a method of in vivo producing a thebaine derivative or an oripavine derivative in a cell culture, comprising culturing the host cell of the first aspect and/or herein relevant embodiments thereof in the cell culture, under conditions;
A third aspect of the invention relates to a method of producing an opioid compound of interest, comprising first performing in vivo production of a thebaine derivative or an oripavine derivative (such as e.g. neopinone, oripavine, northebaine, nororipavine or morphinone) according to the second aspect and/or herein relevant embodiments thereof, followed by suitable in vivo and/or in vitro synthesis steps on the resulting thebaine derivative or oripavine derivative, in order to obtain the opioid compound of interest.
Embodiments of the present invention are described below by way of examples only.
All definitions of herein relevant terms are in accordance of what would be understood by the skilled person in relation to the herein relevant technical context.
As used herein, the term “opioid pathway” refers to the multi-step synthesis of opioids and/or their derivatives. The natural synthesis of morphine is performed by a series of sequential enzymatic reactions in the opium poppy. At each step in the pathway, the product of the previous (“upstream”) reaction becomes a substrate for the next reaction. However, alternative opioid pathways can be created by substituting different enzymes to carry out a specific catalysis, or by replacing several reactions in the pathway with an alternative multi-step route to achieve the same end product opioid or opioid derivative. Since each reaction product in the pathway soon used as a substrate for the next reaction, all reaction products are known as pathway intermediates until the final opioid or opioid derivative is achieved.
As used herein, the term “opioid transporter” refers to a membrane-bound or membrane-spanning protein involved in the movement across host cell membranes of opioids and/or opioid pathway intermediates and/or opioid derivatives.
An introduction to the NPF family of transporters can be found in Jorgensen et al. (“Origin and evolution of transporter substrate specificity within the NPF family”; eLife 2017; 6:e19466. DOI: 10.7554/eLife.19466).
As used herein, the term “reticuline or a derivative thereof” refers to precursors and intermediates in the production of opioids and opioid derivatives. In some aspects, preferred reticuline derivates of particular relevance to the transporters and enzyme activities disclosed herein include thebaine and/or oripavine.
The term “endogenous” gene refers to a gene that originates from and is produced or synthesized within a particular organism, tissue, or cell and is expressed in the same species, organism, tissue or cell for use in the technologies described herein. Therefore an endogenously expressed gene has the source organism as the host organism.
The term “heterologous” relates to a protein that is genetically engineered (such as through recombinant DNA technologies) into a cell that does not normally make (i.e., express) that protein. Therefore a heterologously expressed gene is present in a host organism that is different from the source organism for that gene.
The term “in vitro” (Latin: in glass) relates to studies that are conducted using components of an organism that have been isolated from their usual biological surroundings. Colloquially, these experiments are commonly called “test tube experiments”. In contrast, in vivo studies are those that are conducted using living organisms in their normal intact state.
The term “in vivo” (Latin for “within the living”) relates to experimentation using a whole living organism, as opposed to a partial or dead organism, or an in vitro (“within the glass”, e.g., in a test tube) controlled environment.
The term “biosynthetic” refers to a means of producing a compound wherein at least one step in the production process for synthesizing the compound is carried out in a recombinant biological host. In some circumstances, preferably the entire synthesis of the desired molecule is carried out in a recombinant host i.e. the entire biosynthetic pathway is present and functional within the recombinant host. In other circumstances, part of the biosynthetic pathway may be present in one host, and another part of the biosynthetic pathway may be present in another host.
The term “biotransformation” refers to the addition of a substrate to isolated cells, such that at least one enzyme endogenously or heterologously expressed in the cells are able to catalyze at least one transformation from said substrate into at least one desired product or biosynthetic pathway intermediate.
The term “recombinant host cell” is a commonly used term in the art. Within the field of genetic engineering, recombinant polynucleotide (e.g. DNA) molecules are polynucleotide (e.g. DNA) molecules that may be formed by methods of genetic recombination (such as molecular cloning) to bring together genetic material from two or more sources, creating DNA sequences that are not naturally found in biological organisms.
The term “Sequence Identity” relates to the relatedness between two amino acid sequences or between two nucleotide sequences.
For purposes of the present invention, the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the—nobrief option) is used as the percent identity and is calculated as follows:
(Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment).
For purposes of the present invention, the degree of sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the—nobrief option) is used as the percent identity and is calculated as follows:
(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps in Alignment).
As broadly known in the art, a microbe is a microscopic organism capable of existing in a single-celled form or in a colony of cells. Typically, microbes are capable of rapidly dividing into a relatively homogenous population and may be cultured by those skilled in the art very effectively under relatively simple conditions to quickly produce high densities of cells. Microbial host cells are such microbes suitable for industrial application which may be engineered (e.g. using recombinant DNA technologies) to produce one or more products of interest (such as opioids, their intermediates or derivatives). Suitable microbial host cells may be eukaryotic or prokaryotic cells. Non-limiting examples of suitable eukaryotes for scalable production of opioids, their intermediates or derivatives, include fungi such as a filamentous fungus cell or a yeast cell. Non-limiting examples of suitable prokaryotes for scalable production of opioids, their intermediates or derivatives, include bacteria, such as E. coli, Pseudomonas sp. or Bacillus subtilis. Non-limiting examples of suitable yeast cells for scalable production of opioids, their intermediates or derivatives, include
As broadly known in the art, a fungus host cell may e.g. be a yeast cell or e.g. a filamentous fungus cell.
In some circumstances, the fungus host cell is preferably a yeast cell.
The fungal host cell may e.g. be a filamentous fungus cell—such as e.g. an Aspergillus sp. cell, Penicillium sp. cell, Trichoderma sp. cell, Talaromyces sp. cell, Asteromyces sp. cell or Neurospora sp. cell.
A preferred filamentous fungus cell is an Aspergillus sp. cell.
For example, suitable filamentous fungus cell species can be Aspergillus nidulans, Aspergillus sydowii, Aspergillus terreus, Aspergillus oryzae, Aspergillus caelatus, Aspergillus chevalieri, Aspergillus longivesica, Aspergillus parvulus, Aspergillus amylovorus, Aspergillus niger, Aspergillus aculeatus, Aspergillus ellipticus, Aspergillus violaceofuscus, Aspergillus brunneoviolaceus, Aspergillus japonicus, Aspergillus brasiliensis, Aspergillus aculeatinus, Aspergillus thermomutatus, Aspergillus implicatus, Aspergillus acristatus, Penicillium bilaiae, Penicillium rubens, Penicillium chrysogenum, Penicillium expansum, Penicillium antarcticum, Trichoderma reesei, Talaromyces atroroseus, Asteromyces cruciatus, or Neurospora crassa.
The yeast cell may e.g. be Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, Candida albicans, Rhodotorula sp., or Rhodospiridium sp.
In some circumstances, the yeast cell is preferably a Saccharomycete, most preferably, a Saccharomyces cerevisiae cell.
The heterologously expressed enzyme capable of the “conversion of thebaine/oripavine” of item (i) of the first aspect
Preferably, the enzyme capable of converting thebaine/oripavine” into derivatives thereof and/or further intermediates in a pathway for opioid production, is a demethylase.
As discussed above, based on the technical disclosure herein and the prior art knowledge of the skilled person, it is routine work for the skilled person to make a recombinant microbial host (e.g. yeast) capable of:
The skilled person knows from the prior art and/or the technical disclosure herein different suitable examples of an “enzyme capable of converting thebaine/oripavine” into derivatives thereof and/or further intermediates in a pathway for opioid production, which may be heterologously expressed as an enzyme of item (i) of the first aspect such as e.g. the ones explicitly discussed herein.
As discussed above and without being limited by theory, the improved positive yield effect demonstrated herein is probably related to a speculated ability of the herein relevant transporter proteins to transport more thebaine and/or oripavine into the host cell, thereby increasing the intracellular amount of thebaine and/or oripavine (i.e. in vivo) in the host cell.
Consequently, one may obtain the benefit (i.e. improved yield of derivatives “enzyme capable of converting thebaine/oripavine” into derivatives thereof and/or further intermediates in a pathway for opioid production, thereof and/or further intermediates in a pathway for opioid production) of the present invention independently of the specific “enzyme capable of converting thebaine/oripavine/thevinone”into, heterologously enzyme used—i.e. one may in principle use any suitable (e.g. prior art known) “conversion of thebaine/oripavine” heterologously enzyme of interest—i.e. this element of the present invention may be seen as an element based on prior art known knowledge for the skilled person.
Examples of suitable “conversion of thebaine/oripavine” heterologously enzyme may e.g. be:
In working examples herein were used fungal N-demethylase genes/enzymes that are different from the bacterial N-demethylase (e.g. Bacillus BM3 gene) described in WO2018/075670A1.
The fungal N-demethylase based genes/enzymes used in working Example herein are described in international PCT patent application with number PCT/EP2018/066155, which was filed 18 Jun. 2018 and not published at the filing/priority date of the present application.
The PCT/EP2018/066155 application also describes a number of different fungal O-demethylases that are suitable for the thebaine to oripavine conversion.
Accordingly, in a preferred embodiment the N-demethylase is a N-demethylase selected from the group consisting of:
Preferably, the recombinant fungus host cell is capable of:
Most preferably, the recombinant fungus host cell is capable of:
It may also be preferred that the recombinant fungus host cell is capable of:
It some embodiments it may be preferred that the transporter protein capable of transporting reticuline and/or its derivatives is a transporter protein belonging to the NRT1/PTR (NPF) transporter protein family.
The skilled person may routinely determine whether or not a transporter protein capable of transporting reticuline and/or its derivatives is an NPF transporter protein or not.
The two articles:
As discussed in the dated 2015 article of Jorgensen—the Functional EXXEK Motif is essential for NPF—i.e. in accordance with the art, an NPF transporter protein is a protein comprising this EXXEK Motif.
It some embodiments it may be preferred that the transporter protein capable of transporting reticuline and/or its derivatives is a transporter protein belonging to the Purine Uptake Permease (PUP) transporter protein family. The PUP transporters are believed to be a distinct group of a superfamily of drug and metabolite transporters that evolved in terrestrial plant species. Jelesko J. G. 2012 (“An expanding role for purine uptake permease-like transporters in plant secondary metabolism”, Front Pnat Sci 2012; 3:78. As used herein, the term “capable of PUP activity” refers to purine nucleoside transmembrane transporter activity. As used herein, the term PUP transporters refers to uptake transporters capable of enhancing in-vivo concentration of purine nucleobase substrates in the host, and with particular reference to the specific reactions exemplified herein, to increase the uptake of reticuline derivatives, most preferably of thebaine and/or oripavine.
As discussed above, the recombinant host cell of an embodiment of the first aspect is a microbial host cell (such as a yeast cell), wherein the microbial host cell is heterologously expressing at least one functional transporter protein capable of transporting reticuline and/or its derivatives selected from the group consisting of:
It may be preferred that the host cell is a microbial host cell, wherein the microbial host cell is heterologously expressing at least two functional transporter proteins of the first aspect—for instance in working Example 5 the inventors discuss an example of a host cell that is heterologously expressing the six different functional transporter proteins SEQ ID NO:2 (T14_PsoNPF3_GA); SEQ ID NO:4 (T1_CjaMDR1_GA); SEQ ID NO:10 (T60_AmeNPF2_GA); SEQ ID NO:14 (T52_BmePTR2_GA); SEQ ID NO:18 (T11_AthGTR1_GA); SEQ ID NO:22 (T70_CmaNPF_GA).
As discussed in for example the Conclusions of Examples 4 and 5 herein, expression of one of the transporter genes T14_PsoNPF3_GA, T1_CjaMDR1_GA, T4_EsaGTR_GA, T7_PtrPOT_GA or T97_ScaT14_GA in combination with a P450 capable of demethylating reticuline and/or its derivatives in a yeast strain was shown to improve bioconversion of thebaine to northebaine in the range of 22-63% in comparison to the control strain. This is objectively a significant improvement.
Accordingly, preferably the recombinant host cell of the first aspect is a microbial host cell, wherein the microbial host cell (such as a yeast host cell) is heterologously expressing a P450 capable of demethylating reticuline and/or its derivatives and also heterologously expressing at least one functional transporter protein capable of transporting reticuline and/or its derivatives selected from the group consisting of:
and
As discussed above and in working examples 4 and 5 herein, “T14_PsoNPF3_GA” and T97_ScaT14_GA are demonstrated to have a positive in vivo conversion effect for both thebaine and oripavine.
Accordingly, in some embodiments, the recombinant microbial host cell of the first aspect is a microbial cell, wherein the microbial host cell (such as yeast host cell) is heterologously expressing at least one functional transporter protein selected from the group consisting of:
As discussed above, a second aspect of the invention relates to a method of in vivo producing a thebaine derivative or an oripavine derivative in a cell culture, comprising culturing the microbial host cell of the first aspect and/or herein relevant embodiments thereof in the cell culture, under conditions;
A preferred embodiment of the second aspect relates to a method of in vivo producing neopinone, oripavine, northebaine, nororipavine or morphinone in a cell culture, comprising culturing the host cell of the first aspect and/or herein relevant embodiments thereof in the cell culture, under conditions;
In relation to item (B)—thebaine and/or oripavine may be present in vivo in the host cell via e.g.:
As discussed herein—thebaine and/or oripavine in vivo biosynthesis within a microbial host cell (such as a yeast host cell) is well known in the art.
It is also well known (see e.g. working examples herein) how to prepare thebaine and/or oripavine supplemented cell culture medium or reaction medium to be taken up by the yeast host cell in order for the thebaine and/or oripavine to be present in vivo in the microbial host cell.
As discussed above, the improved positive yield effect demonstrated herein is probably related to that the herein relevant transporter proteins increase the intracellular amount of thebaine and/or oripavine (i.e. in vivo) in the fungus host cell because more thebaine and/or oripavine is transported into the host yeast cell. This advantageous effect is also relevant in relation to in vivo biosynthesis within the host cell of thebaine/oripavine, since some of the thebaine/oripavine may be exported out of the host cell and herein relevant transporter proteins can then transport the thebaine/oripavine back into the host cell again.
Using methods known in the art, the in item (C) produced neopinone, oripavine, northebaine, nororipavine or morphinone may be isolated in order to get a substantially pure (e.g. at least 20%, 30%, 50%, 60% or at least 90% pure w/w) composition of the compound(s). Alternatively, they may e.g. in vivo be converted to further herein relevant downstream compounds (see e.g.
In short, based on the technical disclosure herein and the prior art knowledge of the skilled person—it is routine work for the skilled person to perform the method of the second aspect and/or herein relevant embodiments thereof.
In relation to the second aspect—it may be preferred that it is a method for producing neopinone, oripavine or northebaine, wherein
In relation to the second aspect—it may be preferred that it is a method for producing oripavine or northebaine, wherein
Most preferably is a method for producing northebaine, wherein
Further, and in relation to the second aspect, it may be preferred that it is a method for producing oripavine, northebaine or nororipavine, wherein
Preferably, the method of the second aspect and/or herein relevant embodiments thereof is a method, wherein there in item (C) of the second aspect is an increased in vivo conversion of thebaine and/or oripavine due to that the cultured host cell is heterologously expressing at least one functional transporter protein (e.g. T14_PsoNPF3_GA) of the first aspect and/or herein relevant embodiments thereof; and
wherein the “increased in vivo conversion of thebaine and/or oripavine” is understood to be relative to an otherwise identical performed method using an otherwise identical control host cell that is not heterologously expressing at least one functional transporter protein (e.g. T14_PsoNPF3_GA) of the first aspect and/or herein relevant embodiments thereof.
The “increased in vivo conversion of thebaine and/or oripavine” is understood to be relative to an otherwise identical control host cell, which is not heterologously expressing at least one functional transporter protein (e.g. T14_PsoNPF3_GA) of the first aspect.
The skilled person knows or can easily identify (by e.g. routine genome sequencing) an “otherwise identical control host cell” with no heterologously expressing of at least one functional transporter protein (e.g. T14_PsoNPF3_GA) of the first aspect.
If for instance the yeast host cell is heterologously expressing e.g. T14_PsoNPF3_GA—then is the method of the second aspect and/or herein relevant embodiments thereof simply performed with the host cell heterologously expressing T14_PsoNPF3_GA and the otherwise identical control host cell with no expressing of T14_PsoNPF3_GA and the amount of in vivo conversion of thebaine and/or oripavine is then measured (e.g. by LC-MS)—if the use of the host cell with expressing of T14_PsoNPF3_GA is giving increased in vivo conversion as compared to the control host cell then it is understood that there is an increased in vivo conversion of thebaine and/or oripavine due to that the host cell is heterologously expressing T14_PsoNPF3_GA.
As discussed above, a third aspect of the invention relates to a method of producing an opioid compound of interest, comprising first performing in vivo production of a thebaine derivative or an oripavine derivative (such as e.g. neopinone, oripavine, northebaine, nororipavine or morphinone) according to the second aspect and/or herein relevant embodiments thereof followed by suitable in vivo and/or in vitro synthesis steps in order to obtain the opioid compound of interest.
A preferred embodiment of the third aspect relates to a method of producing an opioid compound of interest, comprising first performing in vivo production of neopinone, oripavine, northebaine, nororipavine or morphinone according to the second aspect and/or herein relevant embodiments thereof followed by suitable in vivo and/or in vitro synthesis steps in order to obtain the opioid compound of interest.
In short, based on the technical disclosure herein and the prior art knowledge of the skilled person—it is routine work for the skilled person to perform the method of the third aspect and/or herein relevant embodiments thereof.
As discussed herein, starting from neopinone, oripavine, northebaine, nororipavine or morphinone—suitable in vivo and/or in vitro synthesis steps in order to obtain the opioid compound of interest are well known in the art. See for example WO2018/211331 and Sipos et al. (2009).
As understood—the term “in vitro synthesis steps” may e.g. relate to suitable chemical synthesis steps as e.g. illustrated for buprenorphine in
Preferably, the opioid compound of interest is heroin, morphine, codeine, thebaine, oripavine, oxycodone, hydrocodone, hydromorphone, oxymorphone, buprenorphine, naltrexone, naloxone, nalmefene, noroxymorphone or nalbuphine. In some embodiments, most preferably the opioid compound of interest is buprenorphine, nalmefene or noroxymorphone.
As discussed above, the amino acid sequence for P450 CYPDN8 N-demethylase from Rhizopus microspores is shown as SEQ ID NO. 9 herein and discussed in international PCT patent application with number PCT/EP2018/066155, which was filed 18 Jun. 2018.
PCT patent application with number PCT/EP2018/066155 also describes other herein relevant technical details such as e.g. further details in relation to herein referred pOD75 and pOD13 plasmids. Accordingly, based on the technical disclosure herein and the technical disclosure of PCT patent application with number PCT/EP2018/066155—the skilled person can routinely carry out the relevant technical matter of the present invention—such as e.g. the relevant working Examples herein.
Saccharomyces cerevisiae yeast strains were constructed in strain background EVST25898 (genotype MATalpha his3Δ0 leu2Δ0 ura3Δ0 aro3Δ::pTEF1-ARO4(K229L)-tCYC1::pPGK1-ARO7(T266L)tADH1::KI CAT5-91Met GAL2 ho MIP1-661Thr SAL1-1 YORWΔ22::npBIO1nt20npBIO6nt).
The EVST25898 with the genotype above corresponds to S288C (genotype MATalpha his3Δ0 leu2Δ0 ura3Δ0). S288C is a publicly available widely used laboratory strain (see the Saccharomyces Genome Database (SGD)). As is known from other works, one would get similar results by use of EVST25898 with genotype above or by use of S288C (genotype MATalpha his3Δ0 leu2Δ0 ura3Δ0) as background/control strains, since these two host phenotypes are substantially identical.
Strain was transformed with relevant plasmids using the lithium acetate method (Gietz et al. 2002. Methods Enzymol. Vol 350, p 87-96).
For testing the impact of possible transporter proteins on the bioconversion of Thebaine to Northebaine, the host yeast strain was transformed with a plasmid containing cytochrome P450 gene CYPDN8 N-demethylase from Rhizopus microspores (pOD75) along with a plasmid containing Cel_CPR (co) from Cunninghamella elegans (pOD13) in combination with the various possible transporter proteins. Genes were inserted and expressed using either P413TEF, P415TEF or p416TEF, all described by Mumberg et al., 1995. Gene. Apr. 14; 156(1):119-22.
The control strain was constructed by transforming strain EVST25898 with pOD75, pOD13 as well as an empty plasmid: p416TEF.
Table 1 describes the plasmids that were expressed with the yeast strains. Transformants were selected in synthetic complete (SC) agar plates lacking histidine, leucine and uracil. Transformation plates were incubated for 3-4 days at 30° C. until visible colonies were obtained.
Cunninghamella elegans
Rhizopus microsporus
Strain EVST25898 was modified by genomic integration using the Saccharomyces cerevisiae gene integration and expression system developed by Mikkelsen, M D et al. (Metab. Eng. 14, Issue 2, 104-111 (2012)). The cytochrome P450 gene CYPDN8, N-demethylase from Rhizopus microspores was expressed using the well-known Saccharomyces cerevisiae TEF1 promoter, and the Cel_CPR (co) from Cunninghamella elegans was expressed using the Saccharomyces cerevisiae PGK1 promoter. The expression cassette was integrated in site XII-5 using the Kluyveromyces lactis URA3 marker as selection marker for growth on media lacking uracil (described by Mikkelsen, M D et al. (Metab. Eng. 14, Issue 2, 104-111 (2012)). Subsequently, the transporter genes T11_AthGTR1_GA (SEQ ID NO: 17), T52_BmePTR2_GA (SEQ ID NO: 13), T14_PsoNPF3_GA (SEQ ID NO: 1), T60_AmeNPF2_GA (SEQ ID NO: 9), T1_CjaMDR1_GA (SEQ ID NO: 3) and T70_CmaNPF_GA (SEQ ID NO: 21) were integrated into the site XI-5 of the Saccharomyces cerevisiae strain using the Saccharomyces cerevisiae TEF1, PGK1, TEF2, TDH3, TPI1, and FOCI promoters respectively. Selection for transformants was done using the well-known Kluyveromyces lactis LEU2 marker available e.g. from EUROSCARF (http://www.euroscarf.de) and growth on media lacking leucine. After that, plasmid pOD13 (see Table 1) was transformed with the resulting strain in order to make the strain prototrophic by selecting on media lacking histidine. Transformation plates were incubated for 3-4 days at 30° C. until visible colonies were obtained.
Cultivation. Yeast strains were cultivated in 96-deep-well-plate (DWP) format. Cells were grown in 0.5 ml SC-His-Leu-Ura medium at 30° C. with shaking at 250 rpm in ISF1-X Kuhner shaker for 20-24 hours and utilized as precultures for in vivo bioconversion assays.
50 μl of the overnight cell cultures were grown in 450 μl Synthetic complete (SC)-His-Leu-Ura medium (pH 7) or DELFT minimal medium (pH 7) containing 0.5 mM thebaine or oripavine. Both media contain 0.1 M potassium phosphate buffer.
Thebaine (or Oripavine) were added via a 25 mM stock solution in DMSO. Cells were grown for 72 hours with shaking at 250 rpm.
Harvest. 50 μl of cell cultures were transferred to a new 96-deep-well-plate containing 50 μl of MilliQ water. The harvested 96 well plate was incubated at 80° C. for 10 minutes. Plate was then centrifugated for 10 minutes at 4000 rpm. The supernatants were then diluted in MilliQ water to reach a final dilution of 1:100. Thebaine, northebaine, oripavine and nororipavine contents were analyzed by LC-MS.
For all compounds (Thebaine, Northebaine, Oripavine and Nororipavine) stock solutions were prepared in DMSO at a concentration of 10 mM. Standard solutions were prepared at concentrations of 6 μM, 4 μM, 2 μM, 1 μM, 500 μM, 200 nM, 100 nM, 50 nM, 20 nM and 10 nM from the stock solutions. Samples were injected into the Agilent 1290 UPLC coupled to an Ultivo Triple Quadrupole. The LC-MS method was as follows: Mobile Phase A. H2O+0.1% Formic acid; Mobile Phase B: Acetonitrile+0.1% Formic acid; Column: Phenornenex Kinetex 1.7 μm XB-C18 100 Å, 2.1×100 mm. The elution gradient is shown hi Table 2 and the LC-MS conditions are given in Table 3. Table 4 shows the mass spectrometer source and detector parameters and Table 5 shows the target compounds, their retention times, their parent on, transition ions (MRM) as well as dwell times, cone voltages and collision energies used.
Bioconversion. Expression of transporter genes in a strain containing cytochrome P450 gene CYPDN8 and cytochrome P450 reductase Cel_CPR (co) gave remarkable improvement in bioconversion of thebaine to northebaine for some of the transporter genes, where some exhibited a significant improved bioconversion when strains were grown in presence of 0.5 mM thebaine.
10.0
2
9.8
0
9.7
−1
11.7
19
10.9
11
11.7
19
11.9
21
9.8
0
8.8
−10
Control DELFT
9.8
0.0
DELFT minimal medium
Expression of one of the transporter genes T14_PsoNPF3_GA, T1_CjaMDR1_GA, T4_EsaGTR_GA or T7_PtrPOT_GA in a yeast strain that contains cytochrome P450 gene CYPDN8 and cytochrome P450 reductase Cel_CPR (co), results in improved bioconversion of thebaine to northebaine in the range of 24-63% in comparison to the control strain.
Further, significant improvement was also seen for the transporter genes T60_AmeNPF2_GA, T57_AcoNPF_GA, T52_BmePTR2_GA, T38_ScuPTR2_GA, T11_AthGTR1_GA, T19_RmiPTR2_GA, T70_CmaNPF_GA or T54_MelPOT_GA.
The results of this Example demonstrate expression of one of the transporter genes T14_PsoNPF3_GA, T1_CjaMDR1_GA, T4_EsaGTR_GA or T7_PtrPOT_GA in a yeast strain that contains cytochrome P450 gene CYPDN8 and cytochrome P450 reductase Cel_CPR (co), results in improved bioconversion of thebaine to northebaine in the range of 24-63% in comparison to the control strain.
Further, significant improvement was also seen for the transporter genes T60_AmeNPF2_GA, T57_AcoNPF_GA, T52_BmePTR2_GA, T38_ScuPTR2_GA, T11_AthGTR1_GA, T19_RmiPTR2_GA, T70_CmaNPF_GA or T54_MelPOT_GA.
Further, Transporters were Tested for Improvement in Conversion of the Thebaine Derivative Oripavine to Nororipavine
Bioconversion. Expression of transporter gene T14_PsoNPF3_GA from Papaver somniferum in a strain containing cytochrome P450 gene CYPDN8 and cytochrome P450 reductase Cel_CPR (co) showed remarkable improvement in bioconversion of oripavine to nororipavine. In an assay where a strain was grown in presence of 0.5 mM oripavine, the strain containing T14_PsoNPF3_GA exhibited 2.3% bioconversion of the oripavine to nororipavine, which corresponds to an improvement in bioconversion of oripavine to nororipavine by 64% in comparison to the control strain.
The result of this Example demonstrate that expression of transporter gene T14_PsoNPF3_GA gave around 64% more bioconversion of oripavine to nororipavine—which is a remarkable yield improvement.
This Example 5 discusses transporter genes that are not explicitly mentioned in corresponding Example 4 above.
Bioconversion. In bioconversion experiments similar to Example 4 above—3 additional transporters have shown to improve bioconversion of thebaine to northebaine.
As shown in Table 8 below, T65_IjaNPF_GA, T94_EcrPOT_GA and T97_ScaT14_GA are able to improve bioconversion of thebaine to northebaine by 29.9%, 31.9% and 21.8%, respectively, when compared to a control strain.
Table 8 also shows a yeast strain which genes CYPDN8 from Rhizopus microspores and Cel_CPR_co from Cunninghamella elegans have been integrated into host strain EVST25898 (Example 1) at Chromosome XII-5 with URA3 from Kluyveromyces lactis as selection marker. Subsequently, 6 different transporters T11_AthGTR1_GA, T52_BmePTR2_GA, T14_PsoNPF3_GA, T60_AmeNPF2_GA, T1_CjaMDR1_GA, and T70_CmaNPF_GA were expressed in the same strain at Chromosome XI-5 with LEU2 from Kluyveromyces lactis as selection marker. Plasmid p0013 (Table 1) was also expressed in the same strain to make the strain prototrophic. An indication of improvement in the bioconversion of thebaine to northebaine when multiple copies of various transporters were expressed in the same strain.
When multiple of different genes were expressed in the yeast cell, it is referred to as gene1+gene2, etc.
In bioconversion experiments similar to Example 4 above—the results of this Example demonstrate that three additional transporters have shown to improve bioconversion of thebaine to northebaine. As shown in Table 8, T65_IjaNPF_GA, T94_EcrPOT_GA and T97_ScaT14_GA are able to improve bioconversion of thebaine to northebaine by 29.9%, 31.9% and 21.8%, respectively, when compared to a control strain.
Further, a strain comprising a combination of 6 transporter proteins discussed in Example 4 gave a very good improvement of thebaine to northebaine.
Further, Transporters were Tested for Improvement in Conversion of the Thebaine Derivative Oripavine to Nororipavine
Bioconversion. In bioconversion experiments similar to Example 4 above—an additional transporter that is able to help improving bioconversion of oripavine to nororipavine has been identified. As shown in Table 9 below, T97_ScaT14_GA from Sanguinaria canadensis is able to convert close to 5% of oripavine to nororipavine when fed with 0.5 mM oripavine. In comparison to the control strain, expression of T97_ScaT14_GA improves the bioconversion of oripavine to nororipavine by 254.4%.
In bioconversion experiments similar to Example 4 above, the results of this Example demonstrate an additional transporter able to help in improving bioconversion of oripavine to nororipavine has been identified.
As shown in Table 9, T97_ScaT14_GA from Sanguinaria canadensis is able to convert close to 5% of oripavine to nororipavine when fed with 0.5 mM oripavine. In comparison to the control strain, expression of T97_ScaT14_GA improves the bioconversion of oripavine to nororipavine by 254.4%.
Bioconversion. The impact of purine uptake permease transporter proteins on bioconversion of thebaine to northebaine was studied by transforming yeast strain with a plasmid containing a cytochrome P450 comparable to the above examples that was capable of acting on reticuline derivatives such as thebaine and/or oripavine using the backbone plasmid p415TEF. A plasmid containing cytochrome P450 reductase (p0013 from Example 1) was also expressed in combination with various candidate transporter proteins. Yeast strain construction and method of screening for PUP transporters were as previously described in Example 1. Table 10 shows the result of percentage bioconversion from thebaine to northebaine with the expression of various PUP transporters. Table 10 also presents the percentage improvement in the bioconversion when normalized for a control strain expressing P450 but not expressing any heterologous transporter.
Improvement of bioconversion. When compared to a control strain without a heterologous transporter, several strains engineered with PUP transporters exhibited at least 50% greater bioconversion of the 0.5 mM thebaine fed in this assay. Amongst the PUP transporters examined, PUP transporters T152_GfIPUP3_87, T149_AcoPUP3_59, T109_GfIPUP3_83, T142_McoPUP3_4, T144_PsoPUP3_19, T141_EcaPUP3_88, T182_CpaPUP3_62, T193_AanPUP3_55 and T122_PsoPUP3_17 exhibited improvements in bioconversion of thebaine to northebaine in the range of 48-94% in comparison to the control strain without a heterologous transporter (Table 10). Expression of some PUP transporters, such as T152_GfIPUP3_87 from Glaucium flavum, T149_AcoPUP3_59 from Aquilegia coerulea, and T142_McoPUP3_4 from Macleaya cordata, gave remarkable improvements in the P450-mediated bioconversion of thebaine to northebaine.
Glaucium flavum
Macleaya cordata
Papaver somniferum
Aquilegia coerulea
Glaucium flavum
Eschscholzia californica
Carica papaya
Artemisia annua
Cinnamomum micranthum f. kanehirae
Sanguinaria canadensis
Papaver somniferum
Papaver somniferum
Table 11 shows some of the PUP transporters that have been herein demonstrated for the first time to shown very considerable improvements in the bioconversion from Thebaine to Northebaine by P450s. In particular, the results of this Example demonstrate that expression of PUP transporters T152_GflPUP3_87 from Glaucium flavum, T149_AcoPUP3_59 from Aquilegia coerulea, T109_GfIPUP3_83 from Glaucium flavum, T142_McoPUP3_4 from Macleaya cordata, T144_PsoPUP3_19 from Papaver somniferum, T141_EcaPUP3_88 from Eschscholzia californica, T182_CpaPUP3_62 from Carica papaya, T193_AanPUP3_55 from Artemisia annua, T132_CmiPUP3_10 from Cinnamomum micranthum f. kanehirae, T186_ScaPUP3_84 from Sanguinaria canadensis, T175_PsoPUP3_6 from Papaver somniferum and T122_PsoPUP3_17 from Papaver somniferum, each stimulated somewhere in the range of 48-94% more bioconversion of thebaine to northebaine. The improvements in yield shown herein are both unexpected and highly valuable given the nature of the opioid-related compounds produced.
Bioconversion. The impact of purine uptake permease transporter proteins on bioconversion of oripavine to nororipavine was studied by transforming yeast with a plasmid containing a comparable cytochrome P450 that was capable of acting on reticuline derivatives such as thebaine and/or oripavine using the backbone plasmid p415TEF. A plasmid containing cytochrome P450 reductase (pOD13 from Example 1) was also expressed in combination with various possible transporter proteins. Yeast strain construction and method of screening for PUP transporters were as previously described in Example 1. Table 12 shows the result of percentage bioconversion from oripavine to nororipavine with the expression of various PUP transporters. Table 12 also presents the percentage improvement in the bioconversion when normalized for a control strain expressing P450 but not expressing any heterologous transporter.
Improvement of bioconversion. The percentage bioconversion of strains displayed by several PUP transporters exhibited as high as 1600% and greater bioconversion of the 0.5 mM oripavine fed to the assay when compared to a control strain expressing P450 but not expressing transporter. Amongst the transporters examined in this example, PUP transporters T149_AcoPUP3_59, T168_FvePUP3_37, T116_HanPUP3_56, T192_CmiPUP3_47, T109_GfIPUP3_83, T180_McoPUP3_46, T193_AanPUP3_55, T165_AcoPUP3_13, T195_JcuPUP3_71 and T143_CmiPUP3_11 exhibited improvements in the P450-mediated bioconversion of oripavine to nororipavine in the range of 1400-1662% in comparison to the control strain expressing P450 but not expressing a heterologous transporter (Table 12). Expression of some PUP transporters, such as T149_AcoPUP3_59 from Aquilegia coerulea, T168_FvePUP3_37 from Fragaria vesca subsp. vesca, and T116_HanPUP3_56 from Helianthus annuus gave particularly remarkable improvements in the P450-mediated bioconversion of oripavine to nororipavine.
Aquilegia coerulea
Fragaria vesca subsp. vesca
Helianthus annuus
Cinnamomum micranthum f. kanehirae
Glaucium Flavum
Macleaya cordata
Artemisia annua
Aquilegia coerulea
Jatropha curcas
Cinnamomum micranthum f. kanehirae
Table 13 shows some of the PUP transporters that have been demonstrated herein for the first time to shown particularly high improvements in the P450-mediated bioconversion of oripavine to nororipavine. Amongst the transporters examined in this example, PUP transporters T149_AcoPUP3_59 from Aquilegia coerulea, T168_FvePUP3_37 from Fragaria vesca subsp. vesca, T116_HanPUP3_56 from Helianthus annuus, T192_CmiPUP3_47 from Cinnamomum micranthum f. kanehirae, T109_GfIPUP3_83 from Glaucium flavum, T180_McoPUP3_46 from Macleaya cordata, T193_AanPUP3_55 from Artemisia annua, T165_AcoPUP3_13 from Aquilegia coerulea, T195_JcuPUP3_71 from Jatropha curcas and T143_CmiPUP3_11 from Cinnamomum micranthum f. kanehirae, exhibited improvements in the range of 1400-1662% more P450-mediated bioconversion of thebaine to northebaine in comparison to the control strain expressing P450 but not expressing a heterologous transporter. Such improvements in yield are particularly remarkable and represent a significant step forward towards a sustainable, secure, and scalable biosynthetic means of producing these compounds.
| Number | Date | Country | Kind |
|---|---|---|---|
| 18200911.8 | Oct 2018 | EP | regional |
| 19197480.7 | Sep 2019 | EP | regional |
This application is a U.S. national phase application under 35 U.S.C. § 371 of International Application No. PCT/EP2019/077548, filed Oct. 10, 2019, which claims the benefit of European Patent Application No. 18200911.8, filed Oct. 17, 2018, and European Patent Application No. 19197480.7, filed Sep. 16, 2019, the disclosures of each of which are explicitly incorporated by reference herein in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/077548 | 10/10/2019 | WO | 00 |
