Methods and materials for improving production of compounds of cyp450 pathways

Abstract

The present invention relates to the field of biosynthesis of compounds of cytochrome P450 (CYP450) pathways. More specifically the invention relates to host cells and methods for improving production of compounds of CYP450 pathways, for example methods and cells for improving titers and / or purity.

Description

[0001] Methods and materials for improving production of compounds of CYP450 pathways

[0002] Technical field

[0003] The present invention relates to the field of biosynthesis of compounds of cytochrome P450 (CYP450) pathways, for example compounds of plant pathways. More specifically, the invention relates to host cells and methods for improving production of compounds of CYP450 pathways, for example methods and cells for improving titers and / or purity.

[0004] Background

[0005] In recent years, engineered microorganisms have become an attractive alternative to chemical synthesis or traditional crop-based manufacturing to produce complex plant- derived natural products. This provides a platform for the sustainable production of specific compounds. However, engineering of multistep pathways in microorganisms is suffering from low titers (Yang et al. (2022)) and release of undesired intermediates as demonstrated by many in the field for the production of isoflavonoids (Liu et al. (2021)), cannabinoids (Luo et al. (2019)), terpenoids (Forman et al. (2022), Hansen et al. (2022)) and alkaloids Srinivasan & Smolke (2020)).

[0006] In plants, biosynthesis of natural products is highly organized and controlled, involving enzymes of the membrane-anchored cytochrome P450 superfamily, cytosolic enzymes, and substrates that may come from various compartments. Cytochrome P450 (CYP450) enzymes are in a large superfamily of heme-containing proteins responsible for catalyzing the oxidation of a wide range of compounds in all kingdoms of life. CYP450 enzymes are mainly anchored to the endoplasmic reticulum (ER). Besides catalytic proteins, additional non-catalytic proteins assist in the organization of metabolic or biosynthetic pathways in complexes termed metabolons. To date, very few non-catalytic proteins have been identified. In the past, cytochrome P450 oxidoreductases (PORs) and cytochrome b5 (CYB5) were the only known interaction partners of CYP450 enzymes.

[0007] In humans, Progesterone Receptor Membrane Component 1 and 2 (PGRMC1 and PGRMC2) are new candidate proteins that interact with CYP450 enzymes (Kabe et al., 2016). Upon interacting with PGRMCs, CYP450 enzymes activities are modulated. More than 13 human P450s have been reported to interact with PRGMC1. Ortholog proteins termed membrane-steroid-binding proteins (MSBPs) have been identified in multiple plant species. In Arabidopsis, MSBPs act as scaffolding hubs to co-localize three P450s involved in monolignol biosynthesis in ER membrane. A decrease in MSBP concentration disrupts monolignol biosynthetic complex formation and causes a reduction in lignin production. The protein scaffolding mediated by MSBPs is essential for directing metabolic flux toward monolignol production.

[0008] Summary

[0009] The present invention provides truncated membrane-steroid-binding protein (MSBP), which is capable of boosting production in microorganisms of products of cytochrome P450 (CYP450) enzyme-containing biosynthetic pathways. In other words, the truncated MSBPs of the invention may boost biosynthetic pathways comprising CYP450s and thereby boost production of products of such pathways. The MSBPs of the invention are generally capable of boosting many different CYP450 enzyme pathways, including pathways for the production of (iso)flavonoids, cannabinoids, terpenoids and alkaloids.

[0010] Nevertheless, the inventors of the present invention have identified and characterized a novel player in plant P450 mediated metabolism, Without being bound by theory, it is believed that the MSBP of the invention act as a general P450 scaffold protein with the potential to increase the flux and substrate channeling towards desired compound(s) for heterologous production in microorganisms.

[0011] Thus, it is a main aspect of the present invention to provide host cells capable of producing a compound of a biosynthetic pathway comprising a cytochrome P450 (CYP450) enzyme, said host cells comprising: i. a heterologous nucleic acid encoding a truncated MSBP according to the invention; and ii. a nucleic acid encoding said CYP450 enzyme.

[0012] A further main aspect of the present invention is to provide methods for producing a compound of a biosynthetic pathway comprising a CYP450 enzyme, said methods comprising the steps of: i. providing a host cell according to the present invention; and ii. cultivating said host cell in a medium, whereby said compound is produced. Another aspect is to provide methods for increasing the titer and / or purity of a compound of a biosynthetic pathway comprising a CYP450 enzyme in a host cell capable of producing said compound, said method comprising the steps of: i. expressing a heterologous nucleic acid encoding a truncated MSBP according to the invention; ii. cultivating said host cell in a medium, whereby the titer and / or purity of said compound is increased, wherein said increase is compared to the titer and / or purity produced by a host cell not expressing said nucleic acid encoding a truncated MSBP, when cultivated in the same conditions.

[0013] Provided herein is also a system of nucleic acids for expression in a host cell, comprising: i. a heterologous nucleic acid encoding a truncated MSBP according to the invention ; and ii. a nucleic acid encoding said CYP450 enzyme.

[0014] Further provided is use of a truncated MSBP lacking at least a part of the C-terminus compared to a corresponding wild-type MSBP in a method for producing a compound of a biosynthetic pathway comprising a CYP450 enzyme, and / or for increasing the production of a compound of a biosynthetic pathway comprising a CYP450 enzyme, such as for increasing the titer of said compound, and / or purity of said compound.

[0015] Description of Drawings

[0016] Figure 1 : Effect of MSBP and MSBPAC on the production level of dhurrin in S. cerevisiae. The impact of MSBP and MSBPAC on the biosynthesis of dhurrin and the intermediates were demonstrated in an engineered S. cerevisiae strain expressing the biosynthetic enzymes of the dhurrin pathway including SbCYP79A1 (SEQ ID NO: 1), SbCYP71 E1 (SEQ ID NO: 2), SbPOR2a (SEQ ID NO: 4) and SbUGT85B1 (SEQ ID NO: 3). Accumulation of dhurrin and intermediates of the biosynthetic pathway in the growth media were analyzed by LC-MS after (A) 8 hours, (B) 12 hours and (C) 24 hours of fermentation. Aldehyde: p-hydroxybenzaldehyde. Oxime: p- hydroxyphenylacetaldoxime. Ox-Gluc: glycosylated p-hydroxyphenylacetaldoxime. Figure 2: Biosynthetic pathway of the cyanogenic glycoside dhurrin. The cyanogenic glycoside dhurrin from Sorghum bicolor is derived from L-tyrosine. The biosynthetic pathway consists of 2 cytochrome P450 enzymes, CYP79A1 (SEQ ID NO: 1) and CYP71E1 (SEQ ID NO: 2), and a UDP-glycosyltransferase UGT85B1 (SEQ ID NO: 3).

[0017] Figure 3: Multiple sequence alignment of MSBPs from Sorgum bicolor (Sorghum_MSBP (SbMSBP, SEQ ID NO: 5)), Oryza sativa spp. japonica (Japonica_Rice_MSBP1 (OsMSBPI, SEQ ID NO: 34) and Japonica_Rice_MSBP2 (OsMSBP2, SEQ ID NO: 35)), Oryza mays (Maize_PredictedMSBP1 (ZmMSBP, SEQ ID NO: 36)), Arabidopsis thaliana (Arabidopsis_MSBP1 (AtMSBPI, SEQ ID NO: 37) and Arabidopsis_MSBP2 (AtMSBP2, SEQ ID NO: 38)), Homo sapiens (Human_PGRMC1 (HsPGRMCI, SEQ ID NO: 39)) and Saccharomyces cerevisiae (Yeast_DAP1 (ScDAPI , SEQ ID NO: 40)). SbMSBPAC (SEQ ID NO: 6) aligns to residues 1-174 of Sorghum_MSBP (SbMSBP, SEQ ID NO: 5). The CYB5-like domains of the MSBPs are highlighted in grey. The CYB5-like domains were obtained from the web-based program InterPro annotation (https: / / www.ebi.ac.uk / interpro / ). * (asterisk) indicates fully conserved residue (same amino acid in all sequences), : (colon) indicates conservation between groups with strongly similar properties (different amino acids but similar properties), e.g. conservative substitutions, . (period) indicates conservation between groups of weakly similar properties (different amino acids, with some, but not high similarity in properties). The sequences were aligned with CLUSTAL 0(1.2.4).

[0018] Figure 4: (A) Biosynthetic pathway of the betalain pigment betanin. Betanin is derived from L-tyrosine and the pathway consists of a cytochrome P450 enzyme CYP76AD1 (SEQ ID NO: 44), a dioxygenase DODA (SEQ ID NO: 45) and a glycosyltransferase CDOPA5GT (SEQ ID NO: 46). (B) Effect of SbMSBP and SbMSBPAC on the production level of betanin in S. cerevisiae. The impact of SbMSBP and SbMSBPAC on the biosynthesis of betanin were demonstrated in an engineered S. cerevisiae strain expressing the biosynthetic enzymes of the betanin pathway including BvCYP76AD1 (SEQ ID NO: 44), BvDODA (SEQ ID NO: 45) a glycosyltransferase MjcDOPA5GT (SEQ ID NO: 46) and AtATRI (SEQ ID NO: 47). Accumulation of betanin in the growth media were analyzed by LC-MS after 24 hours of fermentation. Detailed description

[0019] Definitions

[0020] As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly states otherwise.

[0021] The terms “incubating” and “cultivating” are used interchangeably herein, and refers to maintaining host cells under culture conditions, which allow the cells to grow.

[0022] Preferably, said culture conditions allow expression of the enzyme(s) encoded by the heterologous gene(s) contained in said host cells. In embodiments where the host cell is contained within a multicellular organism (e.g. a fungi), “cultivating” or “incubation” refers to maintaining said multicellular organisms under conditions allowing said multicellular organism to grow. In embodiments where the host cell is a unicellular organism (e.g. a yeast cell), “cultivating” or “incubating” refers to maintaining said unicellular organism under conditions allowing said unicellular organism to grow and / or multiply.

[0023] The term "enzyme" as used herein refers to proteins or polypeptides, which are capable of catalysing biochemical reactions. Further, unless context dictates otherwise, as used herein “enzyme” includes protein fragments that retain the relevant catalytic activity, and may include artificial enzymes synthesized to retain the relevant catalytic activity.

[0024] The term “heterologous nucleic acid” refers to a nucleic acid, which has been inserted into a host cell or into a progenitor of the host cell, e.g. by recombinant or transgenic methods. The respective protein or RNA encoded by a heterologous nucleic acid is also referred to as “heterologous”. The heterologous nucleic acid may be part of a non-integrated nucleic acid, e.g. a vector, including but not limited to a plasmid. The heterologous nucleic acid(s) may be integrated into the host cell genome.

[0025] The term "host cell" refers to a cell, which comprises one or more heterologous nucleic acids.

[0026] The term “polypeptide” as used herein refers a sequential chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length. As is known to those skilled in the art, polypeptides may be processed and / or modified, and the term polypeptide may refer to both unmodified or modified polypeptides.

[0027] The term "functional homologue” of an amino acid sequence, refers to a polypeptide comprising said amino acid sequence with the proviso that one or more amino acids are substituted, deleted, added, and / or inserted, and which polypeptide has (qualitatively) the same enzymatic functionality for substrate conversion. The term “functional homologue” of a nucleic acid encoding a polypeptide, refers to a nucleic acid comprising said nucleic acid sequence with the proviso that one or more nucleobases are substituted, deleted, added, and / or inserted, and which nucleic acid encodes a polypeptide, which polypeptide has (qualitatively) the same enzymatic functionality for substrate conversion as the polypeptide encoded by said nucleic acid. Nucleic acids or nucleic acid sequence may also be referred to as polynucleotides. Preferably, a functional homologue shares at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to said amino acid sequence.

[0028] The term “alignment” as used herein refers to the arrangement of sequences of DNA, RNA, or polypeptides to identify regions of similarity. By aligning two or more sequences of nucleotides or amino acids, it is possible to identify what residues corresponds to each other in the aligned sequence. In other words, a sequence alignment between two or more sequences can be used to determine what nucleotides or amino acids in the two or more sequences correspond to each other. “Aligning” refers to the act of performing an alignment of two or more sequences. “Corresponding residues” refers to the residues in different sequences that have been identified to correspond to each other after alignment. The skilled person is capable of aligning two or more sequences. Aligning one or more sequences may be performed manually or by bioinformatics computer tools, such as web-based programs. Useful software or webbased programs for aligning sequences and hence identifying corresponding residues in different sequences are Clustal W, Clustal Omega, and MUSCLE. Alignment may also be referred to as “sequence alignment” herein and the terms may be used interchangeably. The term “sequence identity” as used herein describes the relatedness between two amino acid sequences or between two nucleotide sequences, i.e. a candidate sequence (e.g. a mutant sequence) and a reference sequence (such as a wild type sequence) based on their pairwise alignment. For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mo / . 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 5.0.0 or later (available at https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ). The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of 30 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:

[0029] (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0030] The Needleman-Wunsch algorithm is also used to determine whether a given amino acid in a sequence other than the reference sequence corresponds to a given position in a reference sequence.

[0031] For purposes of the present invention, the 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, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the DNAFULL (EMBOSS version of NCBI NLIC4.4) 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:

[0032] (Identical Deoxyribonucleotides x 100) / (Length of Alignment - Total Number of Gaps in Alignment). Sequence identity is calculated over the entire length of the reference sequence.

[0033] The term “C-terminus” refers to carboxyl terminus of a protein, polypeptide, or a fragment thereof. The term “N-terminus” refers to the amino terminus of a protein, polypeptide, or a fragment thereof.

[0034] The term “derivative” refers to a compound derived from another compound either by chemical and / or enzymatic conversion.

[0035] The term “titer” refers to the produced concentration of a compound. With respect to a host cell producing said compound, the term refers to the total concentration of said compound produced by the host cell, or in other words the total amount of the compound divided by the volume of the culture or cultivation medium. Both volatile and non-volatile compounds are included in the titer.

[0036] The term “purity” refers to the percentage or ratio of a compound in relation to all compounds within the same compound group produced by a host cell. For example, the purity of a specific compound of a biosynthetic pathway comprising a CYP450 enzyme is the percentage of said specific compound in relation to all intermediates and / or by-products of said pathway produced by the host cell.

[0037] MSBP and truncated MSBP

[0038] The present invention provides host cells, methods, and polypeptides for production of compounds of biosynthetic pathways comprising one or more CYP450 enzymes.

[0039] Accordingly, production of said compounds may be obtained by expression of one or more CYP450 enzymes, either alone or in combination with one or more additional polypeptides of different activity and / or function.

[0040] The CYP450 enzyme(s) may be capable of catalysing production of one or more plant compounds, precursors, and / or intermediates thereof, for example one or more plant compounds described in the section “CYP450 biosynthetic pathways and plant compounds” herein below or one or more plant compounds belonging to the plant compound classes described therein.

[0041] While expression of the one or more CYP450 enzymes of a biosynthetic pathway can result in the production of a compound of said pathway, the inventors have found that expression of a C-terminal truncated MSBP appear to have a positive effect on the production of said compound, as it results in an increase in titer and / or purity. This is exemplified in Figure 1 for production of dhurrin by engineered yeast using a truncated variant of MSBP from Sorghum bicolor, i.e. SbMSBPAC as set forth in SEQ ID NO: 6.

[0042] The terms “membrane-steroid-binding protein” and “MSBP” will be used interchangeably herein. The term “wild-type MSBP” refers to a MSBP derived from a natural source, such as a plant, fungi, yeast, or human, and that has not been truncated, extended, or otherwise modified. In contrast, the term “truncated MSBP” refers to a polypeptide that is derived from, and thus shares sequence identity with, a wild-type MSBP, but has been truncated. In other words, the term “truncated MSBP” refers to a fragment of a wild-type MSBP. In the context of the present invention, the term “truncated MSBP” refers to a MSBP that has been C-terminal truncated, in other words “truncated MSBP” lacks parts of the C-terminus. Said truncation may not be limited to the C-terminus of the MSBP, but may extend beyond the C-terminus. However, the total length of the amino acid sequence of said truncated MSBP may be both shorter, equal to, or longer than the amino acid sequence of said wild-type MSBP. Thus, said truncated MSBP may comprise a fragment of MSBP as well as other sequences. In preferred embodiments the truncated MSBP however consists of a fragment of MSBP, and consequently the total length of the amino acid sequence of said truncated MSBP is preferably shorter than the amino acid sequence of said wildtype MSBP. The term “C-terminal truncated MSBP” refers to a truncated MSBP lacking at least a part of the C-terminus compared to a corresponding wild-type MSBP.

[0043] Thus, the one or more additional polypeptides expressed in said host cell may preferably be a truncated MSBP lacking at least a part of the C-terminus compared to a corresponding wild-type MSBP according to the present invention. The wild-type MSBP and the truncated MSBP preferably have the structure, activity and / or function as described herein.

[0044] Wild-type MSBP

[0045] As described herein above, the present invention relates to truncated MSBPs lacking at least a part of the C-terminus compared to a corresponding wild-type MSBP. Wild-type MSBPs are cytochrome B5- or Cyt-bs (CYB5)-domain proteins, and without being bound by theory, a wild-type MSBP typically comprises or even consists of the following three domains: i. N-terminus; ii. CYB5-like domain; iii. C-terminus.

[0046] Figure 3 displays a multiple sequence alignment of the amino acid sequence of eight exemplary wild-type MSBPs, including Sorghum bicolorMSBP (SbMSBP, SEQ ID NO: 5). Highlighted in grey in Figure 3 is the CYB5-like domain of each of the MSBPs. The CYB5-like domain of SbMSBP is spanning residues 71-167.

[0047] With respect to the CYB5-like domain of MSBP, said domain is well-conserved across different wild-type MSBPs as also displayed in Figure 3. The CYB5-like domain is downstream the N-terminus and upstream the C-terminus of the MSBP. In other words, the CYB5-like domain is located between the N-terminus and the C-terminus of the MSBP. A person skilled in the art will know how to determine the CYB5-like domain of MSBPs, for example by using a software tool or web-based program available in the field, for example InterPro annotation (https: / / www.ebi.ac.uk / interpro / ). Examples of CYB5-like domains are provided in Figure 3, where they are highlighted in grey, for SbMSBP as set forth in SEQ ID NO: 5 from Sorghum bicolor, OsMSBPI as set forth in SEQ ID NO: 34 and OsMSBP2 as set forth in SEQ ID NO: 35 from Oryza sativa spp. japonica, ZmMSBP as set forth in SEQ ID NO: 36 from Zea mays, AtMSBPI as set forth in SEQ ID NO: 37 and AtMSBP2 as set forth in SEQ ID NO: 38 from Arabidopsis thaliana, HsPGRMCI as set forth in SEQ ID NO: 39 from Homo sapiens, and ScDAPI as set forth in SEQ ID NO: 40 from Saccharomyces cerevisiae.

[0048] Thus, the CYB5-like domain of an MSBP may preferably be the part of said MSBP which aligns with any of the CYB5-like domains highlighted in grey in Figure 3 upon alignment between said MSBPs. In particular, the CYB5-like domain of a given MSBP may preferably be the part of said MSBP which aligns with amino acids 71-167 of SbMSBP of SEQ ID NO: 5, when the sequence of the given MSBP is aligned to the sequence of SbMSBP.

[0049] The C-termini of different wild-type MSBPs differ in their length depending on for example origin and isoform. With respect to the present invention, the C-terminus of a wild-type MSBP may be defined as the residues downstream the CYB5-like domain. In other words, the C-terminus of a wild-type MSBP may be defined as the residues of said wild-type MSBP corresponding to the residues downstream the CYB5-like domain of SbMSBP (SEQ ID NO: 5). Differently worded, the C-terminus of a wild-type MSBP may be defined as any residue of said wild-type MSBP downstream the residue therein corresponding to residue 167 of SbMSBP (SEQ ID NO: 5). In some embodiments, the C-terminus of a wild-type MSBP may be defined as the residues of said wild-type MBSP corresponding to residues 168 to 223 of SEQ ID NO: 5. In other words, the C- terminus of a wild-type MSBP may be defined as the residues downstream the residues corresponding to amino acids 70 to 167 of SbMSBP (SEQ ID NO: 5). The C- terminus of SbMSBP (SEQ ID NO: 5) consists of amino acids 168 to 223 of SEQ ID NO: 5. A person skilled in the art will known how to determine the C-terminus MSBPs, for example by using a software tool available in the field, possibly using SbMSBP (SEQ ID NO: 5) as reference sequence and / or the alignment of different wild-type MSBPs shown in Figure 3.

[0050] With regard to the N-terminus of MSBP, the N-termini of different wild-type MSBPs also differ in their length depending on for example origin and isoform. Some parts of the N- terminus of wild-type MSBP is membrane-bound and thus function as a so-called membrane-anchor of the protein. In other words, the N-terminus of the wild-type MSBP comprises a membrane-anchor. Hence, the truncated MSBP of the present invention preferably also comprises a N-terminus-membrane-anchor. Thus, the truncated MSBP is in general N-terminus-membrane-anchored. With respect to the present invention, the N-terminus of a wild-type MSBP may be defined as the residues upstream the CYB5-like domain. In other words, the N-terminus of a wild-type MSBP may be defined as any residue of said wild-type MSBP upstream the residue therein corresponding to residue 71 of SbMSBP (SEQ ID NO: 5). Indeed, the N-terminus of a wild-type MSBP and / or truncated MSBP may comprise or consist of the 50 to 130 most N-terminal amino acids of the MSBP, such as the 50 to 120 most N-terminal amino acids, for example the 50 to 110 most N-terminal amino acids, such as the 50 to 100 most N- terminal amino acids, for example the 50 to 90 most N-terminal amino acids, such as the 55 to 85 most N-terminal amino acids, for example the 60 to 80 most N-terminal amino acids, such as the 65 to 75 most N-terminal amino acids, for example the 77 to 73 most N-terminal amino acids. In some embodiments, the N-terminus of a wild-type MSBP may be defined as the residues of said wild-type MSBP corresponding to amino acids 1 to 70 of SbMSBP (SEQ ID NO: 5). The N-terminus of SbMSBP (SEQ ID NO: 5) consists of amino acids 1 to 70 of SEQ ID NO: 5. A person skilled in the art will know how to determine the N-terminus MSBPs, for example by using a software tool available in the field, possibly using SbMSBP (SEQ ID NO: 5) as reference sequence and / or the alignment of different wild-type MSBPs shown in Figure 3.

[0051] Origin

[0052] The wild-type MSBP wherefrom the truncated MSBP can be derived, may be derived from different sources. Thus, in some embodiments of the present invention, the MSBP is native to a plant, a mammal, such as Homo sapiens, or a yeast.

[0053] In some embodiments, the MSBP is native to a plant, for example the MSBP is derived from Sorghum, such as Sorghum bicolor, Oryza, such as Oryza sativa spp. japonica,Arabidopsis, such as Arabidopsis thaliana, or Zea, such as Zea mays.

[0054] In other embodiments, where the MSBP is a Sorghum bicolor MSBP, said MSBP may be SbMSBP as set forth in SEQ ID NO: 5, or a functional homologue of any of the aforementioned sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity to SEQ ID NO: 5.

[0055] In some further embodiments, where the MSBP is a Oryza sativa spp. japonica MSBP, said MSBP may be OsMSBPI as set forth in SEQ ID NO: 34 or OsMSBP2 as set forth in SEQ ID NO: 35 from Oryza sativa spp. japonica, or a functional homologue of any of the aforementioned sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity to SEQ ID NO: 34 or SEQ ID NO: 35, respectively.

[0056] In other embodiments, where the MSBP is a Zea mays MSBP, said MSBP may be ZmMSBP as set forth in SEQ ID NO: 36, or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity to SEQ ID NO: 36.

[0057] In some other embodiments, where the MSBP is an Arabidopsis thaliana MSBP, said MSBP is AtMSBPI as set forth in SEQ ID NO: 37 or AtMSBP2 as set forth in SEQ ID NO: 38, or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity to SEQ ID NO: 37 or SEQ ID NO: 38, respectively.

[0058] In some further embodiments, where the MSBP is a Homo sapiens MSBP, said MSBP may be HsPGRMCI as set forth in SEQ ID NO: 39, or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity to SEQ ID NO: 39.

[0059] In other embodiments, where the MSBP is a Saccharomyces cerevisiae MSBP, said MSBP may be ScDAPI as set forth in SEQ ID NO: 40, or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity to SEQ ID NO: 40.

[0060] In other embodiments, where the MSBP is a Petunia inflata MSBP, said MSBP may be PinfMSBPI as set forth in SEQ ID NO: 42, or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity to SEQ ID NO: 42.

[0061] Truncated MSBP

[0062] The host cells according to the present invention comprise a heterologous nucleic acid encoding a truncated MSBP. Indeed, in a main aspect of the invention, the host cell is capable of producing a compound of a biosynthetic pathway comprising a cytochrome P450 (CYP450) enzyme, said host cell comprising: i. a heterologous nucleic acid encoding a truncated MSBP lacking at least a part of the C-terminus compared to a corresponding wild-type MSBP, such as at least 10% of the amino acids of the C-terminus of the corresponding wild-type MSBP; and ii. a nucleic acid encoding said CYP450 enzyme.

[0063] The truncated MSBP may for example be any of the polypeptides described herein in this section, and may preferably be defined with respect to the corresponding wild-type MSBP. For example, the heterologous nucleic acid may be any heterologous nucleic acid encoding SbMSBPAC as set forth in SEQ ID NO: 6, or a functional homologue thereof sharing at least 70% sequence identity thereto. The invention also provides such proteins as well as methods using the same. In other embodiments, the truncated MSBP may be SbMSBPACIO as set forth in SEQ ID NO: 92, or a functional homologue thereof sharing at least 70% sequence identity thereto. In some embodiments, the truncated MSBP may be SbMSBPAC20 as set forth in SEQ ID NO: 93, or a functional homologue thereof sharing at least 70% sequence identity thereto. In some embodiments, the truncated MSBP may be SbMSBPAC30 as set forth in SEQ ID NO: 94, or a functional homologue thereof sharing at least 70% sequence identity thereto. In other embodiments, the truncated MSBP may be SbMSBPAC40 as set forth in SEQ ID NO: 95, or a functional homologue thereof sharing at least 70% sequence identity thereto. In further embodiments, the truncated MSBP may be SbMSBPAC50 as set forth in SEQ ID NO: 96, or a functional homologue thereof sharing at least 70% sequence identity thereto. Said functional homologues may also share at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity to SEQ ID NO:92-96, respectively.

[0064] According to the present invention, a truncated MSBP is lacking at least a part of the C- terminus compared to a corresponding wild-type MSBP. The C-terminus of wild-type MSBP may be defined and determined as described herein above. Accordingly, a truncated MSBP is lacking at least one amino acid / residue of the most C-terminal amino acids of the corresponding wild-type MSBP. In some embodiments of the present invention, the truncated MSBP lacks between 1 to 90 amino acids of the most C-terminal amino acids of the corresponding wild-type MSBP, such as between 10 and 80 amino acids, for example between 20 and 70 amino acids, such as between 30 and 60 amino acids, for example between 40 and 55 amino acids, such as between 45 and 50 amino acids of the most C-terminal amino acids of the corresponding wild-type MSBP. In other embodiments, the truncated MSBP lacks at least 1 amino acid of the most C-terminal amino acids of the corresponding wild-type MSBP, such as at least 5 amino acids, for example at least 10 amino acids, such as at least 15 amino acids, for example at least 20 amino acids, such as at least 25 amino acids, for example at least 30 amino acids, for example at least 34 amino acids, such as at least 35 amino acids, for example at least 40 amino acids, such as at least 45 amino acids, for example at least 49 amino acids, such as at least 50 amino acids, for example at least 55 amino acids, such as 56 amino acids, such as at least 60 amino acids, for example at least 70 amino acids, and / or at the most 90 amino acids of the most C-terminal amino acids of the corresponding wild-type MSBP. For example, the wild-type MSBP may be SbMSBP (SEQ ID NO: 5), OsMSBPI (SEQ ID NO: 34), OsMSBP2 (SEQ ID NO: 35), ZmMSBP (SEQ ID NO: 36), AtMSBPI (SEQ ID NO: 37), AtMSBP2 (SEQ ID NO: 38), HsPGRMCI (SEQ ID NO: 39), PinfMSBPI (SEQ ID NO: 42), or ScDAPI (SEQ ID NO: 40), or the wild-type MSBP may be SbMSBP (SEQ ID NO: 5), OsMSBPI (SEQ ID NO: 34), OsMSBP2 (SEQ ID NO: 35), ZmMSBP (SEQ ID NO: 36), AtMSBPI (SEQ ID NO: 37) or AtMSBP2 (SEQ ID NO: 38), or a functional homologue thereof sharing at least 70% sequence identity, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity or 100% sequence identity with any of the aforementioned.

[0065] In some embodiments of the present invention, the truncated MSBP lacks between 1 to 56 amino acids of the most C-terminal amino acids of the corresponding wild-type MSBP, such as between 5 and 56 amino acids, for example between 10 and 56 amino acids, such as between 20 and 53 amino acids, for example between 30 and 53 amino acids, such as between 40 and 50 amino acids, for example between 45 and 49 amino acids of the most C-terminal amino acids of the corresponding wild-type MSBP. In other embodiments, the truncated MSBP lacks at least 10 amino acid of the most C- terminal amino acids of the corresponding wild-type MSBP, such as at least 15 amino acids, for example at least 20 amino acids, such as at least 25 amino acids, for example at least 30 amino acids, such as at least 35 amino acids, for example at least 40 amino acids, such as at least 45 amino acids, for example at least 49 amino acids, such as at least 50 amino acids, for example at least 55 amino acids, such as 56 amino acids of the most C-terminal amino acids of the corresponding wild-type MSBP. Preferably, the corresponding wild-type MSBP is SbMSBP as set forth in SEQ ID NO: 5, or a functional homologue thereof sharing at least 70% sequence identity thereto.

[0066] In some embodiments, said truncated MSBP lacks at least 4%, such as at least 10% of the amino acids of the C-terminus of the corresponding wild-type MSBP, for example at least 15%, such as at least 20%, for example at least 25%, such as at least 30%, for example at least 35%, such as at least 40%, for example at least 45%, such as at least 50%, for example at least 55%, such as at least 60%, for example at least 65%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95%, or 100% of the amino acids of the C-terminus of the corresponding wild-type MSBP. Thus, in other words, in some embodiments the truncated MSBP lacks all the amino acids of the C-terminus of the corresponding wild-type MSBP.

[0067] The skilled person is capable of determining the amino acids of the C-terminus of a polypeptide, such as a MSBP. How to determine the C-terminus of a MSBP is also described herein above in the section “Wild-type MSBP”.

[0068] In some embodiments, the truncated MSBP lacks above-mentioned amino acids of a corresponding wild-type MSBP, where the remainder, i.e. the truncated MSBP shares at least 70% sequence identity with the corresponding N-terminal part of the corresponding wild-type MSBP, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity or 100% sequence identity with the corresponding N-terminal part of the corresponding wild-type MSBP. Preferably, the corresponding wild-type MSBP is SbMSBP as set forth in SEQ ID NO: 5, or a functional homologue thereof sharing at least 70% sequence identity thereto.

[0069] In some embodiments, the truncated MSBP consists of amino acids 1 to n of a wildtype MSBP, wherein n is an integer in the range of 100 to 264, for example in the range 110 to 245, such as in the range of 120 to 230, for example in the range of 130 to 210, such as in the range of 140 to 200, for example in the range of 150 to 190, such as in the range of 160 to 180, for example in the range of 160 to 175. For example, the wildtype MSBP may be SbMSBP (SEQ ID NO: 5), OsMSBPI (SEQ ID NO: 34), OsMSBP2 (SEQ ID NO: 35), ZmMSBP (SEQ ID NO: 36), AtMSBPI (SEQ ID NO: 37), AtMSBP2 (SEQ ID NO: 38), HsPGRMCI (SEQ ID NO: 39), PinfMSBPI (SEQ ID NO: 42), or ScDAPI (SEQ ID NO: 40), or a functional homologue thereof sharing at least 70% sequence identity, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity or 100% sequence identity with any of the aforementioned.

[0070] In other embodiments, the truncated MSBP consists of amino acids 1 to n of a wildtype MSBP, wherein n is an integer selected from the group consisting of 167 to 222, 170 to 231 , 217 to 264, 167 to 211 , 167 to 233, 167 to 233, 171 to 219, 171 to 194, 171 to 240, and 145 to 151. For example, the wild-type MSBP may be SbMSBP (SEQ ID NO: 5), OsMSBPI (SEQ ID NO: 34), OsMSBP2 (SEQ ID NO: 35), ZmMSBP (SEQ ID NO: 36), AtMSBPI (SEQ ID NO: 37), AtMSBP2 (SEQ ID NO: 38), HsPGRMCI (SEQ ID NO: 39), PinfMSBPI (SEQ ID NO: 42), or ScDAPI (SEQ ID NO: 40), respectively, or for example the wild-type MSBP may be SbMSBP (SEQ ID NO: 5), OsMSBPI (SEQ ID NO: 34), OsMSBP2 (SEQ ID NO: 35), ZmMSBP (SEQ ID NO: 36), AtMSBPI (SEQ ID NO: 37) or AtMSBP2 (SEQ ID NO: 38), or a functional homologue thereof sharing at least 70% sequence identity, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity or 100% sequence identity with any of the aforementioned.

[0071] In other embodiments, the truncated MSBP consists of amino acids 1 to n of a wildtype MSBP, wherein n is an integer in the range of 100 to 264, for example wherein the truncated MSBP consists of amino acids 1 to 173 of a wild-type MSBP, or amino acids 1 to 174 of a wild-type MSBP, or amino acids 1 to 183 of a wild-type MSBP, or amino acids 1 to 189 of a wild type MSBP, or amino acids 1 to 193 of a wild-type MSBP, or amino acids 1 to 203 of a wild-type MSBP, or amino acids 1 to 213 of a wild-type MSBP, wherein the wild-type MSBP for example may be SbMSBP (SEQ ID NO: 5), OsMSBPI (SEQ ID NO: 34), OsMSBP2 (SEQ ID NO: 35), ZmMSBP (SEQ ID NO: 36), AtMSBPI (SEQ ID NO: 37), AtMSBP2 (SEQ ID NO: 38), HsPGRMCI (SEQ ID NO: 39), PinfMSBPI (SEQ ID NO: 42), or ScDAPI (SEQ ID NO: 40), or said wild-type MSBP for example may be SbMSBP (SEQ ID NO: 5), OsMSBPI (SEQ ID NO: 34), OsMSBP2 (SEQ ID NO: 35), ZmMSBP (SEQ ID NO: 36), AtMSBPI (SEQ ID NO: 37), or AtMSBP2 (SEQ ID NO: 38) or a functional homologue thereof sharing at least 70% sequence identity, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity or 100% sequence identity with any of the aforementioned.

[0072] In some further embodiments, the truncated MSBP consists of amino acids 1 to n of SEQ ID NO: 5, or a functional homologue thereof sharing at least 70% sequence identity, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity or 100% sequence identity therewith, wherein n is an integer in the range of 167 to 222, such as in the range of 169 to 215, for example in the range of 170 to 200, such as in the range of 170 to 190, for example in the range of 170 to 180, for example n is 174. The truncated MSBPs disclosed herein may be a truncated version of any wild-type MSBP. Examples of wild-type MSBPs are provided herein above in the sections “Wildtype MSBP” and “Origin”. As described herein above, the truncated MSBP may be both shorter than, equal to, or longer than the amino acid sequence of the corresponding wild-type MSBP. For example, the truncated MSBP lacking parts of the C-terminus may have been fused or attached to another polypeptide, for example a signal peptide, or protein, such as a localization tag, for example green-fluorescence protein (GFP). In addition, the truncated MSBP may be glycosylated, acylated, and / or attached to polyethylene glycol (PEG) (PEGylated).

[0073] The present invention discloses improved variants of wild-type MSBPs with improved function and / or activity compared to the activity of said wild-type MSBP.

[0074] Furthermore, the present invention also disclose variants of truncated MSBPs. A variant of a truncated MSBP refers to a functional variant of a truncated MSBP, which retains at least some or all of the function and / or activity of the truncated MSBP, and which has at least 70% identity thereto, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95%, such as at least 100% identity thereto.

[0075] Preferably, a functional homologue of a truncated MSBP retains at least some or all of the function and / or activity of the truncated MSBP. Thus, a functional homologue of a truncated MSBP preferably induces production of one or more compounds of biosynthetic pathways when expressed in a host cell comprising said biosynthetic pathway to the same or similar extend as a truncated MSBP consisting of a consecutive sequence of a wild type MSBP. For example, a functional homologue of a truncated MSBP preferably induces production of dhurrin, betanin or quercetin when expressed in a host cell capable of producing dhurrin, betanin or quercetin, respectively, to the same or similar extend as a truncated MSBP consisting of a consecutive sequence of a wild type MSBP.

[0076] Nucleic acids encoding wild-type and truncated MSBPs

[0077] The heterologous nucleic acid encoding the truncated MSBP may be any heterologous nucleic acid encoding any of the truncated MSBPs described herein. Thus, in some embodiments the invention provides nucleic acids encoding truncated MSBP, wherein said truncated MSBP is SbMSBPAC as set forth in SEQ ID NO: 6, or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity thereto.

[0078] In some embodiments the invention provides nucleic acids encoding truncated MSBP, wherein said truncated MSBP is SbMSBPACIO as set forth in SEQ ID NO: 92, encoded by a nucleic acid such as in SEQ ID NO: 97. In some embodiments truncated MSBP is SbMSBPAC20 as set forth in SEQ ID NO: 93, encoded by a nucleic acid such as in SEQ ID NO: 98. In some embodiments truncated MSBP is SbMSBPAC30 as set forth in SEQ ID NO: 94, encoded by a nucleic acid such as in SEQ ID NO: 99. In some embodiments truncated MSBP is SbMSBPAC40 as set forth in SEQ ID NO: 95, encoded by a nucleic acid such as in SEQ ID NO: 100. In some embodiments truncated MSBP is SbMSBPAC50 as set forth in SEQ ID NO: 96, encoded by a nucleic acid such as in SEQ ID NO: 101.

[0079] In some embodiments, the nucleic acid encoding a truncated MSBP and / or said CYP450 enzyme have been codon optimized for said host cell. In other embodiments the nucleic acid encoding a truncated MSBP and / or said CYP450 enzyme are under control of an inducible promoter. In further embodiments, the nucleic acids encoding are each independently comprised within the genome of said host cell or within a vector comprised in said host cell.

[0080] Use

[0081] Another aspect of the present invention is use of a truncated MSBP lacking at least a part of the C-terminus compared to a corresponding wild-type MSBP in a method for producing a compound of a biosynthetic pathway comprising a CYP450 enzyme. A further aspect is use of a truncated MSBP lacking at least a part of the C-terminus compared to a corresponding wild-type MSBP in a method for increasing the production of a compound of a biosynthetic pathway comprising a CYP450 enzyme, such as for increasing the titer of said compound. Another aspect is use of a truncated MSBP lacking at least a part of the C-terminus compared to a corresponding wild-type MSBP in a method for increasing the purity of said compound. Preferably, said compound is a plant compound, such as a plant compound described in the section “CYP450 biosynthetic pathways and plant compounds” herein. The truncated MSBP may be a truncated as described anywhere herein, in particular in the sections “Truncated MSBP” and “Nucleic acids encoding wild-type and truncated MSBPs”. For example, said truncated MSBP may be SbMSBPAC as set forth in SEQ ID NO: 6, or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity to SEQ ID NO: 6.

[0082] Host cell

[0083] The present disclosure relates to host cells for producing one or more compounds of biosynthetic pathways comprising CYP450 enzymes. The host cells of the invention generally comprise: i. a heterologous nucleic acid encoding a truncated MSBP lacking at least a part of the C-terminus compared to a corresponding wild-type MSBP; and ii. a nucleic acid encoding said CYP450 enzyme.

[0084] The truncated MSBP may be any of the truncated MSBPs described herein, for example as described in the section “Truncated MSBP” herein. For example, the truncated MSBP may lack at least 1% of the C-terminus compared to a corresponding wild-type MSBP, such as at least 5%, for example at least 10%, or more.

[0085] The CYP450 enzyme may be any of the CYP450 enzymes described herein, for example a CYP450 enzyme described in the section “CYP450 biosynthetic pathways and plant compounds”.

[0086] The one or more compounds may be any of the compounds described herein, for example a plant compound, such as dhurrin, or another compound, for example as described in the section “CYP450 biosynthetic pathways and plant compounds” herein below.

[0087] The host cell of the present invention may be a bacterial cell, a microalgae cell, a fungal cell, or a yeast cell. In some embodiments, the host cell is a microalgae cell belonging to the genus of Chlamydomonas, such as Chlamydomonas reinhardtii, Dunaliella, such as Dunaliella salina, Chlorella, such as Chlorella vulgaris, or to the genus of Haematococcus, such as Haematococcus pluvialis.

[0088] In some embodiments, the host cell is comprised within a multicellular organism. In such embodiments, only some of the cells of said multicellular organism may comprise heterologous nucleic acid(s) and / or heterologous polypeptide(s). It is however preferred, that all cells of said multicellular organism are host cells that comprise the same heterologous nucleic acid(s) and / or heterologous polypeptide(s).

[0089] In other embodiments, the host cell is a fungal cell is belonging to the genus of Aspergillus, such as Aspergillus nidulans, Aspergillus niger, or Aspergillus pseudoterreus, or to the genus of Penicillium, such as Penicillium chrysogenum. Said fungal cells may be comprised within a fungi, within part of a fungi or within the spores of said fungi. Preferably, all cells of said fungi or part thereof are host cells comprising the same heterologous nucleic acid(s) and / or heterologous polypeptide(s). The person skilled in the art will appreciate that a fungi or fungal cell(s) as used herein refers to any cell present within or derived from an organism belonging to the Kingdom of Fungi. The methods are applicable to all fungi or fungal cell(s) that are susceptible of genetic modifications.

[0090] The host cell, such as a yeast cell, may be a non-naturally occurring cell, for example a cell which has been engineered to produce said compound of a biosynthetic pathway comprising CYP450 enzyme. The host cell or a progenitor thereof may be prepared by any useful method available to the skilled person. For example, the heterologous nucleic acid(s) may be inserted into a cell by direct uptake, transduction, f-mating, transfection, transformation, bacterial infiltration, or any other methods known in the art useful for creating recombinant host cells.

[0091] In some embodiments, the host cell comprises the nucleic acid constructs described in the section “Nucleic acids” and / or “Dhurrin pathway”.

[0092] The host cell according to the present invention may be comprised in a fermentation liquid and / or broth, a fermentation liquid, and / or a catalytic system. In other words, a fermentation liquid and / or broth, a fermentation system and / or a catalytic system may comprise the host cell according to the present invention.

[0093] Bacterial cell

[0094] In some embodiments of the present disclosure, the host cell is a bacterial cell, e.g. a bacterial cell belonging to the genus of Escherichia, such as E. coli, Pseudomonas, such as Pseudomonas putida, Corynebacterium, such as Cory nebacteri urn glutamicum, Streptomyces, such as Streptomyces coelicolor, or Bacillus, such as Bacillus subtilis.

[0095] The person skilled in the art will appreciate that a bacterial cell includes prokaryotic cells that may be propagated in culture. The bacterial cell may act as a host cell for the recombinant expression of heterologous polypeptide(s). The bacterial cell may be transformed, transfected or infected with a vector for expression of a nucleic acid sequence inserted into the vector.

[0096] Yeast cell

[0097] The person skilled in the art will appreciate that a “yeast cell” is herein defined to include the group consisting of small, unicellular organisms capable of growth and reproduction through budding or direct division (fission), or by growth as simple irregular filaments (mycelium). The yeast cell may be transformed or transfected with a heterologous vector for expression of a nucleic acid inserted into the heterologous vector. Examples of a yeast cell include, but are not limited to Saccharomyces cerevisiae, Pichia pastoris (Komagataella phaffii), Yarrowia lipolytica, Kluyveromyces lactis, and Candida tropicalis, commonly used for transformation, transfection and expression of heterologous polypeptides.

[0098] Thus, in some embodiments, the yeast cell belongs to the genus of Saccharomyces, such as S. cerevisiae, Pichia, such as Pichia pastoris, Yarrowia, such as Yarrowia lipolytica, Kluyveromyces, such as Kluyveromyces lactis, or Candida such as Candida tropicalis.

[0099] Modifications

[0100] In addition to any one of the above polypeptides and / or nucleic acids, such as the nucleic acid encoding a truncated MSBP and the nucleic acid encoding a CYP450 enzyme, the host cell may further comprise additional modifications, such as one or more mutations, for example mutations of the native genome of said host cell. Such modifications or mutations may be, but are not limited to, deletion, overexpression of endogenous or heterologous genes and / or nucleic acids or point-mutations.

[0101] For example, the host cell may further comprise a nucleic acid encoding a cytochrome b5 (CYB5).

[0102] In some embodiments, the host cell further comprises a nucleic acid encoding a cytochrome P450 oxidoreductase (POR).

[0103] In other embodiments, the host cell may further comprise a nucleic acid encoding a glycosyltransferase.

[0104] In other embodiments, the host cell may further comprise a deletion, such as a total or partial deletion, of a gene encoding a glucan 1,3-beta-glucosidase, optionally a glucan 1 ,3-beta-glucosidase with EC number 3.2.1.58. In some embodiments, the host cell comprises a deletion, such as a total or partial deletion, of a gene encoding EXG1 as set forth in SEQ ID NO: 43, or a functional homologue thereof sharing at least 70% sequence identity thereto. In other embodiments, the gene encoding said glucan 1,3- beta-glucosidase may be EXG1 as set forth in SEQ ID NO: 41 , or a functional homologue thereof sharing at least 70% sequence identity thereto.

[0105] Indeed, the host cell may further comprise at least one nucleic acid encoding one or more of polypeptides of said biosynthetic pathway comprising a CYP450 enzyme, optionally said at least one nucleic acid is codon-optimised for said host cell.

[0106] The host cell may further be modified to produce or overproduce a precursor of said compounds of a biosynthetic pathway comprising a CYP450 enzyme.

[0107] CYP450 biosynthetic pathways and plant compounds

[0108] The present invention provides host cells, methods, and polypeptides for production of compounds of biosynthetic pathways comprising one or more CYP450 enzymes. Thus, it is a main aspect of the invention to provide host cells expressing a nucleic acid encoding a truncated MSBP and a nucleic acid encoding a CYP450 enzyme, preferably of a biosynthetic pathway comprising said CYP450 enzyme. Said host cells are preferably capable of producing a compound of a biosynthetic pathway comprising said cytochrome P450 (CYP450) enzyme. Said compound is preferably a product of said biosynthetic pathway.

[0109] In some embodiments, said nucleic acid encoding a CYP450 enzyme is a heterologous nucleic acid. In other embodiments, said CYP450 enzyme is a heterologous CYP450 enzyme, for example said CYP450 enzyme is native to a plant, a yeast, a fungus, or a mammal.

[0110] Indeed, the present invention may be useful in the field of overproduction of plant compounds, such as plant metabolites of various bioactivity. Thus, the CYP450 biosynthetic pathway may be a plant pathway, such as a cyanogenic glucoside pathway, a anthocyanin pathway, a flavonoid pathway, an isoflavonoid pathway, a cannabinoid pathway, an alkaloid pathway, a betalain pathway, a betaxanthin pathway, or a terpenoid pathway. Thus, the host cells are preferably capable of producing a compound, wherein the compound is the product of any of the aforementioned pathways. In some embodiments, the plant compound is an alkaloid. Thus, in some embodiments, the compound is a plant compound selected from the group consisting of cyanogenic glucosides, anthocyanins, flavonoids, isoflavonoids, cannabinoids, alkaloids, betalains, betaxanthins, and terpenoids. CYP450 biosynthetic pathways where the present invention may be useful for overproduction of plant compounds, such as plant metabolites of various bioactivity, are reviewed in Yang et al. (2022).

[0111] Dhurrin pathway

[0112] For example, the plant compound of the biosynthetic pathway comprising one or more CYP450 enzymes may be a cyanogenic glucoside, for example dhurrin. The dhurrin pathway comprises two CYP450 enzymes, a UDP-glucuronosyltransferase (UGT), as well as a POR assisting said CYP450 enzymes. Figure 2 is a schematic illustration of the dhurrin pathway.

[0113] Thus, in some embodiments said CYP450 enzyme is a Sorghum CYP450 enzyme, such as a Sorghum CYP450 enzyme, for example SbCYP79A1 as set forth in SEQ ID NO: 1 and / or SbCYP71 E1 as set forth in SEQ ID NO: 2, or functional homologues thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity to any of the aforementioned.

[0114] In some embodiments, the host cell further comprises a nucleic acid encoding a UDP- glucuronosyltransferase (UGT), such as a Sorghum UGT, for example a Sorghum bicolor UGT , such as SbllGT85B1 as set forth in SEQ ID NO: 3, or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity thereto.

[0115] In other embodiments, the host cell further comprises a POR, for example a Sorghum POR, such as a Sorghum bicolor POR, for example said POR is SbPOR2a as set forth in SEQ ID NO: 4, or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity thereto.

[0116] The host cell capable of producing a cyanogenic glycoside, for example dhurrin and comprising one or more of the polypeptides described in this section, may be a host cell as described in the section “Host cell”, preferably a yeast cell as described in the section ’’Yeast cell”, for example a S. cerevisiae cell. The host cell may comprise one or more nucleic acids encoding polypeptides of the dhurrin pathway as described in section “Nucleic acids of the dhurrin pathway” herein.

[0117] Betalain

[0118] For example, the plant compound of the biosynthetic pathway comprising one or more CYP450 enzymes may be a betalain, for example a betacyanin or a betaxanthin. For, example, said compound may be betanin. The betalain pathway comprises one CYP450 enzyme, a CYP76AD1. Figure 4a is a schematic illustration of the betalain pathway.

[0119] Thus, in some embodiments said CYP450 enzyme is a Beta CYP450 enzyme, such as a Beta vulgaris CYP450 enzyme, for example BvCYP76AD1 as set forth in SEQ ID NO: 44, or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity to any of the aforementioned. In some embodiments, the host cell further comprises a nucleic acid encoding a dioxygenase (DODA), such as a Beta DODA, for example a Beta vulgaris DODA, such as BvDODA as set forth in SEQ ID NO: 45, or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity thereto.

[0120] In some embodiments, the host cell further comprises a glycosyltransferase, for example a Mirabilis glycosyltransferase, such as a Mirabilis jalapa glycosyltransferase, for example said glycosyltransferase is MjcDOPA5GT as set forth in SEQ ID NO: 46, or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity thereto.

[0121] In some embodiments, the host cell further comprises a cytochrome p450 reductase (CYP450 reductase), for example an Arabidopsis CYP450 reductase, such as an Arabidopsis thaliana CYP450 reductase, for example said CYP450 reductase is AtATRI as set forth in SEQ ID NO: 47, or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity thereto.

[0122] The host cell capable of producing betalain, for example betanin and comprising one or more of the polypeptides described in this section, may be a host cell as described in the section “Host cell”, preferably a yeast cell as described in the section ’’Yeast cell”, for example a S. cerevisiae cell. The host cell may comprise one or more nucleic acids encoding polypeptides of the betalain pathway as described in section “Nucleic acids of the dhurrin pathway” herein.

[0123] Flavonoid pathway

[0124] For example, the plant compound of the biosynthetic pathway comprising one or more CYP450 enzymes may be a flavonoid, for example a flavonol, a flavone, a flavonone, an anthocyamin or an isoflavone. For example, the compound may be a flavonol, for example quercetin. The flavonoid pathway comprises one CYP450 enzyme.

[0125] Thus, in some embodiments said CYP450 enzyme is a Petunia CYP450 enzyme, such as a Petunia hybrida CYP450 enzyme, for example PhF3’H as set forth in SEQ ID NO: 74, or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity to any of the aforementioned.

[0126] In some embodiments, the host cell further comprises a nucleic acid encoding a flavanone 3-hydroxylase (F3H), such as a Petunia F3H, for example a Petunia inflata F3H, such as PinfF3H as set forth in SEQ ID NO: 72, or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity thereto.

[0127] In some embodiments, the host cell further comprises a flavonol synthase (FLS), for example a Petunia FLS, such as a Petunia inflata FLS, for example said FLS is PinfFLS as set forth in SEQ ID NO: 75, or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity thereto.

[0128] In some embodiments, the host cell further comprises a cytochrome p450 reductase (CYP450 reductase), for example a Petunia CYP450 reductase, such as a Petunia inflata CYP450 reductase, for example said CYP450 reductase is PinfPORa as set forth in SEQ ID NO: 73, or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity thereto.

[0129] The host cell capable of producing a flavonoid, such as quercetin and comprising one or more of the polypeptides described in this section, may be a host cell as described in the section “Host cell”, preferably a yeast cell as described in the section ’’Yeast cell”, for example a S. cerevisiae cell. The host cell may comprise one or more nucleic acids encoding polypeptides of the flavonoid pathway as described in section “Nucleic acids of the dhurrin pathway” herein.

[0130] Methods

[0131] Provided herein is a method for producing a compound of a biosynthetic pathway comprising a cytochrome P450 (CYP450) enzyme, said method comprising the steps of: i. providing a host cell according to any one of the preceding claims; and ii. cultivating said host cell in a medium, whereby said compound is produced.

[0132] Methods for increasing titer

[0133] Further provided herein is a method for increasing the titer of a compound of a biosynthetic pathway comprising a cytochrome P450 (CYP450) enzyme in a host cell capable of producing said compound, said method comprising the steps of: i. expressing a heterologous nucleic acid encoding a truncated membrane-steroid-binding protein (MSBP)(truncated MSBP) lacking at least 10% of the amino acids of the C-terminus of the corresponding wild-type MSBP in said host cell; ii. cultivating said host cell in a medium, whereby the titer of said compound is increased, wherein said increase is compared to the titer produced by a host cell not expressing said nucleic acid encoding a truncated MSBP, when cultivated in the same conditions.

[0134] In some embodiments, the titer of said compound is increased compared to the titer of said compound produced in the absence of the truncated MSBP, when the production is performed under the same conditions, and the titer is measured under the same conditions.

[0135] In other embodiments, the increased titer of said compound is compared to the titer of said compound produced in the presence of the wild-type MSBP.

[0136] The titer of said compound may be increased at least 1-fold, for example at least 1.1- fold, such as at least 1.2-fold, for example at least 1.3-fold, such as at least 1.5-fold, for example at least 1.7-fold, such as at least 2-fold, for example at least 2.3-fold, such as at least 2.5-fold, for example at least 3-fold, or more.

[0137] In some embodiments, the titer of said compound is increased at least 1% compared to the titer of said compound produced in the absence of the nucleic acid encoding a truncated MSBP, such as at least 5%, for example at least 10%, such as at least 15%, for example at least 20%, such as at least 20%, for example at least 25%, such as at least 30%, for example at least 35%, such as at least 40%, for example at least 45%, such as at least 50%, for example at least 55%, such as at least 60%, for example at least 65%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 100%, such as at least 125%, for example at least 150%, or more.

[0138] The titer of said compound may be defined as the total titer of said compound, precursors, pathway intermediates, and / or derivatives thereof.

[0139] In some embodiments, wherein the host cell is capable of producing dhurrin, dhurrin is produced with a titer of at least 120 mg / L, such as at least 130 mg / L, such as at least 140 mg / L, such as at least 150 mg / L, such as at least 160 mg / L, such as at least 165 mg / L, such as at least 170 mg / L, such as at least 175 mg / L, such as at least 180 mg / L, such as at least 190 mg / L, such as at least 200 mg / L, or more. Host cells capable of producing dhurrin are described herein, for example in the section “Dhurrin pathway”.

[0140] Methods for determining the titer are known in the art.

[0141] Methods for increasing purity

[0142] Also provided herein is a method for increasing the purity of a compound of a biosynthetic pathway comprising a cytochrome P450 (CYP450) enzyme in a host cell capable of producing said compound, said method comprising the steps of: i. expressing a heterologous nucleic acid encoding a truncated membrane- steroid-binding protein (MSBP)(truncated MSBP) lacking at least 10% of the amino acids of the C-terminus of the corresponding wild-type MSBP in said host cell; ii. cultivating said host cell in a medium, whereby the purity of said compound is increased, wherein said increase is compared to the purity of said compound obtained from a host cell not expressing said nucleic acid encoding a truncated MSBP, when cultivated in the same conditions.

[0143] In some embodiments, the purity of said compound is increased compared to the purity of said compound obtained in the absence of the truncated MSBP, when the production is performed under the same conditions, and the purity is measured under the same conditions. For example, the increased purity of said compound may be compared to the purity of said compound produced in the presence of the wild-type MSBP. The purity of said compound may be defined as the total purity of said compound, precursors, intermediates, and / or derivatives thereof.

[0144] The purity of said compound may be increased by at least 1% compared to the purity of the same compound obtained from a host cell not expressing said nucleic acid encoding a truncated MSBP, such as at least 5%, for example at least 10%, such as at least 15%, for example at least 20%, such as at least 20%, for example at least 25%, such as at least 30%, for example at least 35%, such as at least 40%, for example at least 45%, such as at least 50%, for example at least 55%, such as at least 60%, for example at least 65%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 100%, such as at least 125%, for example at least 150%, or more.

[0145] Methods for determining the purity are known in the art.

[0146] Further methods

[0147] The host cell may be a host cell as described in the section “Host cell” herein. In some embodiments, the host cell is a yeast cell as described in the section “Yeast cell”. In other embodiments, the host cell is a bacterial cell as described in the section “Bacterial cell”.

[0148] The wild-type MSBP, such as the corresponding wild-type MSBP, may be a MSBP as described in the section “MSBP and truncated MSBP” herein, in particular in the sections “Wild-type MSBP”, “Origin”, and “Nucleic acids encoding wild-type and truncated MSBPs”. The truncated MSBP may be a truncated MSBP as described in the section “MSBP and truncated MSBP” herein, in particular in the sections “Truncated MSBP” and “Nucleic acids encoding wild-type and truncated MSBPs”.

[0149] It may be desirable to recover and / or isolate the compounds obtained by the methods disclosed herein. Thus, any of the methods disclosed herein may further comprise a step of isolating said compound of a biosynthetic pathway comprising a cytochrome P450 (CYP450) enzyme. For example, the method may further comprise a step of isolating said compound, wherein the isolation comprises liquid-chromatography (LC), and / or two-phase partitioning during cultivation, such as during fermentation. Other methods for recovering and / or isolating the compounds obtained by the present invention are known in the art.

[0150] The compounds, such as the isolated compounds, may be modified further, for example the compounds may be converted to derivatives thereof. Hence, any of the methods disclosed herein may further comprise a step of converting said compound into a derivative thereof. In some embodiments, the step of converting said compound into a derivative thereof is performed in vitro, for example the conversion of said compound to a derivative thereof comprises chemical or enzymatic conversion. In other embodiments, the step of converting said compound into a derivative thereof is performed in vivo. For example, the conversion of said compound into a derivative thereof may be performed in a host cell, such as a yeast cell, for example as described in the section “Host cell”.

[0151] In addition, the compounds, such as the isolated compounds, and / or derivatives thereof may be formulated into a composition. The composition may further comprise one or more additional compounds. In all simplicity, said composition may be a fermentation liquid obtained from any of the methods described herein.

[0152] Nucleic acids

[0153] In addition to the host cells and methods provided herein, the present invention also provide nucleic acids and nucleic acid constructs for expression in a host cell.

[0154] Thus, provided herein is a system of nucleic acids for expression in a host cell, said system comprising: i. a heterologous nucleic acid encoding a truncated MSBP lacking at least a part of the C-terminus of the corresponding wild-type MSBP, such as at least 10% of the C-terminus of the corresponding wild-type MSBP; and ii. a nucleic acid encoding said CYP450 enzyme.

[0155] In preferred embodiments, the nucleic acid encoding a truncated MSBP is as described herein, in particular in the sections “Truncated MSBP” and “Nucleic acids encoding wild-type and truncated MSBPs”. The CYP450 enzyme may be as described herein in the section “CYP450 biosynthetic pathways and plant compounds”, and the wild-type MSBP may be as described herein in the sections “Wild-type MSBP” and “Origin”. Examples of a system of nucleic acids is a vector or a plasmid comprising one or more nucleic acids, for example circular or linear vectors for expression in a host cell.

[0156] In further embodiments, the system of nucleic acids may further comprise a promotor capable of inducing expression of the heterologous nucleic acid in the host cell. The skilled person will be able to select suitable promoters for a given host cell. In some embodiment, the promoter is an inducible promoter, operably linked to any one or more of the nucleic acids provided herein. The system of nucleic acids may further comprise additional element for expression of said nucleic acids in a host cell, for example regulatory elements, such as terminators, 5’llTRs, enhancers, or silencers, and introns.

[0157] Nucleic acids of the dhurrin pathway

[0158] Thus, in some embodiments, the host cell may be capable of producing a cyanogenic glycoside, for example dhurrin, said host cell comprises one or more of the following, preferably all of the following: i. a nucleic acid encoding a truncated MSBP, for example SbMSBPAC as set forth in SEQ ID NO: 6, such as SEQ ID NO: 12; ii. a nucleic acid encoding a CYP450 enzyme, for example SbCYP79A1 as set forth in SEQ ID NO: 1 , such as SEQ ID NO: 7, and / or SbCYP71 E1 as set forth in SEQ ID NO: 2, such as SEQ ID NO: 8; and further comprises: iii. a nucleic acid encoding a POR, for example SbPOR2a as set forth in SEQ ID NO: 4, such as SEQ ID NO: 10; and / or iv. a nucleic acid encoding a UDP-glucuronosyltransferase, for example SbUGT85B1 as set forth in SEQ ID NO: 3, such as SEQ ID NO: 9, or functional homologues thereof sharing at least 70% sequence identity to any of the aforementioned.

[0159] In other embodiments, the host cell may be capable of producing a cyanogenic glycoside, for example dhurrin, said host cell comprising one or more of the following, preferably all of the following: i. a nucleic acid encoding SbMSBPAC as set forth in SEQ ID NO: 6, such as SEQ ID NO: 12; ii. a nucleic acid encoding SbCYP79A1 as set forth in SEQ ID NO: 1, such as SEQ ID NO: 7, and SbCYP71E1 as set forth in SEQ ID NO: 2, such as SEQ ID NO: 8; iii. a nucleic acid encoding SbPOR2a as set forth in SEQ ID NO: 4, such as SEQ ID NO: 10; and iv. a nucleic acid encoding SbllGT85B1 as set forth in SEQ ID NO: 3, such as SEQ ID NO: 9, or functional homologues thereof sharing at least 70% sequence identity to any of the aforementioned.

[0160] Nucleic acids of the betalain pathway

[0161] Thus, in some embodiments, the host cell may be capable of producing a betalain, for example betanin, said host cell comprises one or more of the following, preferably all of the following: v. a nucleic acid encoding a truncated MSBP, for example SbMSBPAC as set forth in SEQ ID NO: 6, such as SEQ ID NO: 12; vi. a nucleic acid encoding a CYP450 enzyme, for example BvCYP45076AD1 as set forth in SEQ ID NO: 44, such as SEQ ID NO: 48; and further comprises: vii. a nucleic acid encoding a cytochrome P450 reductase, for example AtATRI as set forth in SEQ ID NO: 47, such as SEQ ID NO: 51 ; viii. a nucleic acid encoding a dioxygenase, for example BvDODA as set forth in SEQ ID NO: 45, such as SEQ ID NO: 49; and / or ix. a nucleic acid encoding a glycosyltransferase, for example MjcDOPA5GT as set forth in SEQ ID NO: 46, such as SEQ ID NO: 50, or functional homologues thereof sharing at least 70% sequence identity to any of the aforementioned.

[0162] In other embodiments, the host cell may be capable of producing a betalain, for example betanin, said host cell comprising one or more of the following, preferably all of the following: v. a nucleic acid encoding SbMSBPAC as set forth in SEQ ID NO: 6, such as SEQ ID NO: 12; vi. a nucleic acid encoding BvCYP45076AD1 as set forth in SEQ ID NO: 44, such as SEQ ID NO: 48; vii. a nucleic acid encoding AtATRI as set forth in SEQ ID NO: 47, such as SEQ ID NO: 51 ; viii. a nucleic acid encoding BvDODA as set forth in SEQ ID NO: 45, such as SEQ ID NO: 49; and ix. a nucleic acid encoding MjcDOPA5GT as set forth in SEQ ID NO: 46, such as SEQ ID NO: 50, or functional homologues thereof sharing at least 70% sequence identity to any of the aforementioned.

[0163] Nucleic acids of the flavonoid pathway

[0164] Thus, in some embodiments, the host cell may be capable of producing a flavonoid, for example quercetin, said host cell comprises one or more of the following, preferably all of the following one or more of the following, preferably all of the following: x. a nucleic acid encoding SbMSBPAC as set forth in SEQ ID NO: 6, such as SEQ ID NO: 12; xi. a nucleic acid encoding PhF3’H as set forth in SEQ ID NO: 70, such as SEQ ID NO: 74; xii. a nucleic acid encoding PinfPORa as set forth in SEQ ID NO: 69, such as SEQ ID NO: 73; xiii. a nucleic acid encoding PinfF3H as set forth in SEQ ID NO: 68, such as SEQ ID NO: 72; and xiv. a nucleic acid encoding PinfFLS as set forth in SEQ ID NO: 71, such as SEQ ID NO: 75, or functional homologues thereof sharing at least 70% sequence identity to any of the aforementioned.

[0165] Examples

[0166] Example 1 - Methods and Materials

[0167] Construction of a vector for genomic integration

[0168] All vectors generated and used are listed in table 4. An assembler vector system consisting of three vectors, pX-3-Ass1, pAss2 and pX-3-Ass3 was used for genome integration of gene constructs in S. cerevisiae (Hansen et al (2022)). The vectors contained an AsiSI / Nb.Bsml USER cassette used for insertion of gene fragments by USER cloning (Nour-Eldin et al. (2010)). Primers used for PCR amplification of gene fragments are listed in table 3. All PCR amplifications were carried out using PfuX7 polymerase (Norholm (2010)).

[0169] Table 3. List of primers

[0170] Table 4. List of vectors and plasmids. The SEQ ID NOs of heterologous nucleic acids comprised in the vectors are provided.

[0171] Yeast growth media

[0172] Yeast Extract-Peptone-Dextrose (YPD) media: 10 g / L Bacto™ Yeast extract, 20 g / L Bacto™ peptone and 20 g / L D-glucose Synthetic complete (SC) media without uracil: 1 .92 g / L Yeast Synthetic Drop-out Medium Supplements without uracil (Y1501 ; Sigma-Aldrich), 6.7 g / L Yeast Nitrogen Base without amino acids (Y2025; US Biological Life Sciences) and 20 g / L D-glucose. Synthetic complete (SC) media: 1.92 g / L Yeast Synthetic Drop-out Medium Supplements without uracil (Y1501 ; Sigma-Aldrich), 20 mg / L uracil, 6.7 g / L Yeast Nitrogen Base without amino acids (Y2025; US Biological Life Sciences) and 20 g / L D- glucose.

[0173] YPD and SC without uracil agar plates were prepared with 20 g / L agar.

[0174] Incorporation of the dhurrin Pathway in the Yeast S. cerevisiae

[0175] Parental yeast strain was S. cerevisiae strain Y05210 (BY4741 ; MATa; his3A1; leu2A0; met15A0; ura3A0; YLR300w::kanMX4)(Euroscarf). Generated yeast strains are listed in table 5, and were made using a lithium acetate transformation method (Gietz & Woods (2002)).

[0176] To make competent yeast cells, the parental yeast strain was first inoculated from a glycerol stock in 1 ml YPD medium and grown overnight at 30°C with shake (200 rpm). Overnight culture was diluted 1:20 in water and 100 pL was plated on YPD agar plates and grown for 18 hours at 28°C. A 50 pL scrape of yeast culture was washed in 1 ml water and pelleted by centrifugation (6000 x g for 1 min). Pellet was resuspended in 1 ml 0.1 M Lithium Acetate and incubated at 7 min at 28°C. Finally, competent yeast cells were harvested by centrifugation (6000 x g for 1 min) and supernatant was removed.

[0177] Competent yeast cells were transformed by solubilizing the pellets in 240 pL PEG 3350 (50% w / v), 36 pL LiAc (1.0 M), 50 pL boiled SS-Carrier DNA (2 mg / ml) and 50 pL Notl digested plasmids (1.5 pg). Transformation mixture was incubated at 42 °C for 40 min before the yeast cells were harvested by centrifugation (6000 x g for 2 min).

[0178] Transformed yeast cells were resuspended in 100 pL sterile water and plated on SC without uracil agar plates. Plates were incubated for 3 days at 28 °C and transformants with correctly integrated gene fragments were identified by colony PCR as described by Jensen et al. (2014) using primers specific for the insert and genome.

[0179] Table 5. List of S. cerevisiae strains generated. The SEQ ID NO of heterologous nucleic acids are provided as well as the polypeptides encoded by said nucleic acids. Fermentation with Engineered Yeast Strains in culture tubes

[0180] Engineered S. cerevisiae strains were inoculated in 1 ml SC media in 13 ml polypropylene tubes (65.515.006; Sarstedt) and grown overnight at 30 °C with shake (200 rpm) to obtain precultures. Precultures were inoculated in 2 ml SC media in 13 ml polypropylene tubes to reach an optical density at 600 nm of 0.05 and incubated at 30 °C with shake (200 rpm). After 8, 12 and 24 hours of incubation, the optical density at 600 nm was measured and the supernatant of the yeast cultures was obtained by centrifugation (6000 x g for 2 min) and stored at -70 °C prior to LC-MS analysis.

[0181] Metabolite profiles of Engineered S. cerevisiae strains

[0182] The metabolite profiles of the engineered yeast strains were guantified by LC-MS by the following procedure. Previously obtained supernatant of the yeast cultures was diluted 1:100 in water and filtrated using a 0.22 pm 96 well filter plates (MSGVN22; Merck Millipore). Samples were analyzed by TripleQuad LC-MS and metabolite profiles can be seen in Figure 1.

[0183] Example 2 - Results

[0184] Here we show the impact of co-expressing the truncated version of MSBP, MSBPAC (truncated MSBP), in a yeast cell expressing a plant biosynthetic pathway to demonstrate its effect on the production levels of a plant compound. The biosynthetic pathway of the cyanogenic glucoside dhurrin from Sorghum (Figure 2) was used as proof of concept. The dhurrin pathway consisting of two cytochrome P450 enzymes (SbCYP79A1 (SEQ ID NO: 1) and SbCYP71 E1 (SEQ ID NO: 2)), a UDP- glycosyltransferase (SbUGT85B1 (SEQ ID NO: 3)) and a cytochrome P450 reductase (SbPOR2a (SEQ ID NO: 4) were expressed in S. cerevisiae alone or together with either the full-length MSBP (SEQ ID NO: 5) or the truncated version, MSBPAC (SEQ ID NO: 6).

[0185] The engineered S. cerevisiae strain co-expressing SbMSBPAC (SEQ ID NO: 6) together with the other dhurrin biosynthetic pathway proteins accumulated 38000 nM / ODeoo dhurrin after 8 hours of fermentation, which is a 2.3 fold increase compared to the strain co-expressing full-length SbMSBP (SEQ ID NO: 5) and a 2.5 fold increase compared to the strain expressing the dhurrin pathway alone. After 12 hours of fermentation, the strain co-expressing SbMSBPAC had accumulated 2 fold more dhurrin compared to the two other strains. After 24 hours of incubation, the strain expressing SbMSBPAC accumulated 1.6 fold more dhurrin than the strain expressing the dhurrin pathway alone and 1.3 fold more dhurrin than the strain co-expressing the full-length SbMSBP. In addition to the increased production of the final product of the dhurrin biosynthetic pathway, the purity of the final product dhurrin also increased when the SbMSBPAC was expressed in the S. cerevisiae strain compared to the strain coexpressing SbMSBP. Indeed, the co-expression of SbMSBPAC resulted in less pathway intermediates compared to final product as can also be seen in Figure 1.

[0186] This illustrates that co-expressing MSBPAC in yeast cells together with a plant biosynthetic pathway both enhances the production over a long fermentation and remarkably increases the production, in particular within the first 24 hours.

[0187] Example 3 - Materials and Methods - Betalain

[0188] Construction of a vector for genomic integration

[0189] All vectors generated and used are listed in table 7. The same assembler vector system as described in Example 1 was used for genome integration of gene constructs in S. cerevisiae. Primers used for PCR amplification of gene fragments are listed in table 6. The gene fragments were amplified and inserted into vectors as described in Example 1.

[0190] Table 6. List of primers used in Example 3

[0191] Table 7. List of vectors and plasmids used in Example 3. The SEQ ID NOs of heterologous nucleic acids comprised in the vectors are provided. Yeast growth media

[0192] The same yeast growth media from Example 1 was used except the addition of 20 mM ascorbic acid to synthetic complete (SC) media used for fermentations. Incorporation of the betanin pathway in the yeast S. cerevisiae

[0193] The betanin pathway was introduced in the same parental yeast strain (Y05210) as described in Example 1 using the same lithium acetate transformation method. Generated yeast strains are listed in table 8.

[0194] Table 8. List of engineered yeast strains for Example 3 and Example 4

[0195] Fermentation with Engineered Yeast Strains in culture tubes

[0196] Engineered yeast strains were cultivated as described in Example 1 , except the addition of 20 mM ascorbic acid in the growth media. Yeast strains were grown in triplicates for 24 hours at 30°C.

[0197] Quantification of produced betanin in Engineered Yeast Strains

[0198] Yeast cells were pelleted by centrifugation (6000 x g for 2 min) and the supernatants were diluted 1:10 with 5 pH 20 mM Na-acetate buffer in a Nunc MicroWell 96-Well Optical-Bottom plate (Thermo Fischer) to a volume of 100 pL. Betanin production was guantified using absorbance measurements taken at a wavelength of 535 nm with microplate reader (SpectraMax M5). The Lambert-Beer law was applied to calculate betanin concentration, utilizing a molar extinction coefficient of 65,600 L mol-1cm-1.

[0199] Example 4 - Results - Betalain

[0200] Here we show that we get similar results as Example 2 when co-expressing the truncated version of SbMSBP, SbMSBPAC (truncated SbMSBP), in a yeast cell expressing another plant biosynthetic pathway. Here the betanin pathway was used as a proof of concept to demonstrate the versatile impact of MSBPAC to increase production titers of plant natural products in engineered yeast. Betanin is betalain pigment naturally found in plants such as Beta vulgaris. The betanin pathway is commonly used as a reporter system and consists of one cytochrome P450 enzyme (BvCYP76AD1 (SEQ ID NO: 44)), a dioxygenase (BvDODA (SEQ ID NO: 45)) a glycosyltransferase (MjcDOPA5GT (SEQ ID NO: 46)) and a cytochrome P450 reductase (AtATRI (SEQ ID NO: 47) (Figure 4a). The pathway was expressed in S. cerevisiae alone or together with either the full-length SbMSBP (SEQ ID NO: 5) or the truncated version, SbMSBPAC (SEQ ID NO: 6).

[0201] The engineered S. cerevisiae strain co-expressing SbMSBPAC (SEQ ID NO: 6) together with the betanin pathway produced 1613 pg / L betanin, which is a 2.9 fold increase compared to the strain expressing the betanin pathway alone (Figure 4b). The engineered S. cerevisiae strain co-expressing SbMSBPAC (SEQ ID NO: 6) together with the betanin pathway also produced a higher level of betanin compared to the S. cerevisiae strain co-expressing SbMSBP (SEQ ID NO: 5) (see Figure 4b).

[0202] Example 5 - Serial Truncation Construction of a vector for genomic integration

[0203] The same assembler vector system from Example 1 was used for genome integration of serially truncated SbMSBPI in S. cerevisiae. Forward primer P#156 (SEQ ID NO: 31) from Example 1 together with primers listed in Table 12. was used to amplify all serially truncated SbMSBPI fragments. PCR amplification and insertion into assembler vectors were done as described in Example 1. All vectors generated and used are listed in table 13.

[0204] Table 12. List of primers Table 13. List of vectors and plasmids. The SEQ ID NOs of heterologous nucleic acids comprised in the vectors are provided.

[0205] Yeast growth media

[0206] The same yeast growth media from Example 1 are used. Incorporation of the dhurrin Pathway in the Yeast S. cerevisiae

[0207] The same transformation protocol as described in Example 1 was used. Generated yeast strains are listed in table 14.

[0208] Table 14. List of S. cerevisiae strains generated. The SEQ ID NO of heterologous nucleic acids are provided as well as the polypeptides encoded by said nucleic acids.

[0209] Fermentation with Engineered Yeast Strains in culture tubes is performed essentially as described in Example 1. Metabolite profiles of Engineered S. cerevisiae strains is performed essentially as described in Example 1.

[0210] Sequence overview SEQ ID NO: 7 to SEQ ID NO: 12 and SEQ ID NO:97 to SEQ ID NO: 101 were codon- optimized for S. cerevisiae.

[0211] References

[0212] Gietz & Woods (2002). Transformation of yeast by lithium acetate / single-stranded carrier DNA / polyethylene glycol method. In Methods in enzymology (Vol. 350, pp. 87- 96). Academic Press.

[0213] Jensen et al. (2014). EasyClone: method for iterative chromosomal integration of multiple genes Saccharomyces cerevisiae. FEMS yeast research, 14(2), 238-248. N0rholm (2010). A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC biotechnology, 10, 1-7.

[0214] Nour-Eldin et al. (2010). USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories. Plant secondary metabolism engineering: methods and applications, 185-200.

[0215] Hansen et al. (2022). Tripterygium wilfordii cytochrome P450s catalyze the methyl shift and epoxidations in the biosynthesis of triptonide. Nature Communications, 13(1), 5011.

[0216] Liu et al. (2021). De novo biosynthesis of bioactive isoflavonoids by engineered yeast cell factories. Nature Communications 12, 6085.

[0217] Luo et al. (2019). Complete biosynthesis of cannabinoids and their unnatural analogues in yeast. Nature 567, 123-126.

[0218] Forman et al. (2022). A gene cluster in Ginkgo biloba encodes unique multifunctional cytochrome P450s that initiate ginkgolide biosynthesis. Nature Communications 13, 5143.

[0219] Srinivasan & Smolke (2020). Biosynthesis of medicinal tropane alkaloids in yeast. Nature 585, 614-619.

[0220] Kabe et al. (2016). Haem-dependent dimerization of PGRMC1 / Sigma-2 receptor facilitates cancer proliferation and chemoresistance. Nature Communications 7, 11030.

[0221] Yang et al. (2022). Metabolic and cellular engineering for the production of natural products. Current Opinion in Biotechnology 77, 102760.

[0222] Items

[0223] 1. A host cell capable of producing a compound of a biosynthetic pathway comprising a cytochrome P450 (CYP450) enzyme, said host cell comprising: i. a heterologous nucleic acid encoding a truncated membrane-steroid- binding protein (MSBP)(truncated MSBP) lacking a part of the C- terminus of the corresponding wild-type MSBP, such as at least 10% of the amino acids of the C-terminus of the corresponding wild-type MSBP; and ii. a nucleic acid encoding said CYP450 enzyme.

[0224] 3. The host cell according to any one of the preceding items, wherein the C- terminus of the wild-type MSBP is defined as the residues downstream of the CYB5-like domain.

[0225] 4. The host cell according to any one of the preceding items, wherein the C- terminus of the wild-type MSBP is defined as the residues downstream of the CYB5-like domain marked in grey in figure 3.

[0226] 5. The host cell according to any one of the preceding items, wherein the C- terminus of the wild-type MSBP is defined as the residues downstream of the residues corresponding to amino acids 70 to 167 of SbMSBP as set forth in SEQ ID NO: 5.

[0227] 6. The host cell according to any one of the preceding items, wherein the C- terminus is defined as the residues downstream of the residue corresponding to amino acid 167 of SbMSBP as set forth in SEQ ID NO: 5.

[0228] 7. The host cell according to any one of the preceding items, wherein the truncated MSBP is N-terminus-membrane-anchored.

[0229] 8. The host cell according to any one of the preceding items, wherein said truncated MSBP lacks at least 4% of the amino acids of the C-terminus of the corresponding wild-type MSBP, such as at least 5%, such as at least 10%, such as at least 15%, such as at least 20%, for example at least 25%, such as at least 30%, for example at least 35%, such as at least 40%, for example at least 45%, such as at least 50%, for example at least 55%, such as at least 60%, for example at least 65%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% of the amino acids of the C-terminus of the corresponding wild-type MSBP, or more. The host cell according to any one of the preceding items, wherein said truncated MSBP lacks at least 4% of the amino acids of the C-terminus of the corresponding wild-type MSBP, such as at least 5%, such as at least 10%, such as at least 15%, such as at least 20%, for example at least 25%, such as at least 30%, for example at least 35%, such as at least 40%, for example at least 45%, such as at least 50%, for example at least 55%, such as at least 60%, for example at least 65%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% of the amino acids of the C-terminus of the corresponding wild-type MSBP, wherein said C-terminus is defined as the residues downstream of the residue corresponding to amino acid 167 of SbMSBP as set forth in SEQ ID NO: 5. The host cell according to any one of the preceding items, wherein the wildtype MSBP is native to a plant, a mammal, such as Homo sapiens (human), or a yeast. The host cell according to any one of the preceding items, wherein the wild-type MSBP is derived from Sorghum, such as Sorghum bicolor, Arabidopsis, such as Arabidopsis thaliana, Zea, such as Zea mays, Oryza, such as Oryza sativa spp. japonica, or Petunia, such as P. inflata. The host cell according to any one of the preceding items, wherein the wild-type MSBP is a Sorghum bicolor MSBP, such as SbMSBP as set forth in SEQ ID NO: 5, or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity thereto. The host cell according to any one of the preceding items, wherein said truncated MSBP lacks between 10 to 56 amino acids of the most C-terminal amino acids of the corresponding wild-type MSBP, such as between 20 and 53 amino acids, for example between 30 and 53 amino acids, such as between 40 and 50 amino acids, for example between 45 and 49 amino acids of the most C-terminal amino acids of the corresponding wild-type MSBP, preferably wherein the corresponding wild-type MSBP is SbMSBP as set forth in SEQ ID NO: 5, or a functional homologue thereof sharing at least 70% sequence identity thereto. The host cell according to any one of the preceding items, wherein said truncated MSBP lacks between 10 to 56 amino acids of the most C-terminal amino acids of the corresponding wild-type MSBP, such as between 10 to 50 amino acids, such as between 10 to 40 amino acids, such as between 10 to 30 amino acids, such as between 10 to 20 amino acids, such as between 20 to 50 amino acids, such as between 30 to 50 amino acids, such as between 40 to 50 amino acids of the most C-terminal amino acids of the corresponding wild-type MSBP, preferably wherein the corresponding wild-type MSBP is SbMSBP as set forth in SEQ ID NO: 5, OsMSBPI (SEQ ID NO: 34), OsMSBP2 (SEQ ID NO: 35), ZmMSBP (SEQ ID NO: 36), AtMSBPI (SEQ ID NO: 37), AtMSBP2 (SEQ ID NO: 38), HsPGRMCI (SEQ ID NO: 39), PinfMSBPI (SEQ ID NO: 42), or ScDAPI (SEQ ID NO: 40), or a functional homologue thereof sharing at least 70% sequence identity thereto. The according to any one of the preceding items, wherein said truncated MSBP lacks at least 10 amino acids of the most C-terminal amino acids of the corresponding wild-type MSBP, such as at least 20 amino acids, such as at least 30 amino acids, such as at least 34 amino acids, such as at least 40 amino acids, such as at least 50 amino acids of the corresponding wild-type MSBP, preferably wherein the corresponding wild-type MSBP is SbMSBP as set forth in SEQ ID NO: 5, or a functional homologue thereof sharing at least 70% sequence identity thereto. The host cell according to any one of the preceding claims, wherein the truncated MSBP consists of amino acids 1 to n of a wild-type MSBP, wherein n is an integer in the range of 100 to 264, for example in the range 110 to 245, such as in the range of 120 to 230, for example in the range of 130 to 210, such as in the range of 140 to 200, for example in the range of 150 to 190, such as in the range of 160 to 180, for example in the range of 160 to 175, and wherein the wild-type MSBP may be SbMSBP (SEQ ID NO: 5), or a functional homologue thereof sharing at least 70% sequence identity, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity or 100% sequence identity with any of the aforementioned. The host cell according to any one of the preceding claims, wherein the truncated MSBP consists of amino acids 1 to n of a wild-type MSBP, wherein n is an integer in the range of 100 to 264, for example in the range 110 to 245, such as in the range of 120 to 230, for example in the range of 130 to 210, such as in the range of 140 to 200, for example in the range of 150 to 190, such as in the range of 160 to 180, for example in the range of 160 to 175, for example wherein the truncated MSBP consists of amino acids 1 to 173 of a wild-type MSBP, or amino acids 1 to 174 of a wild-type MSBP, or amino acids 1 to 183 of a wild-type MSBP, or amino acids 1 to 189 of a wild type MSBP, or amino acids 1 to 193 of a wild-type MSBP, or amino acids 1 to 203 of a wild-type MSBP, or amino acids 1 to 213 of a wild-type MSBP and wherein the wild-type MSBP may be SbMSBP (SEQ ID NO: 5), OsMSBPI (SEQ ID NO: 34), OsMSBP2 (SEQ ID NO: 35), ZmMSBP (SEQ ID NO: 36), AtMSBPI (SEQ ID NO: 37), AtMSBP2 (SEQ ID NO: 38), HsPGRMCI (SEQ ID NO: 39), PinfMSBPI (SEQ ID NO: 42), or ScDAPI (SEQ ID NO: 40), or a functional homologue thereof sharing at least 70% sequence identity, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity or 100% sequence identity with any of the aforementioned. The host cell according to any one of the preceding items, wherein the truncated MSBP shares at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity or 100% sequence identity with the corresponding N- terminal part of the corresponding wild-type MSBP, preferably wherein said MSBP is SbMSBP as set forth in SEQ ID NO: 5, or a functional homologue thereof sharing at least 70% sequence identity thereto. 19. The host cell according to any one of the preceding items, wherein the truncated MSBP consists of amino acids 1 to n of SEQ ID NO: 5, or a functional homologue thereof sharing at least 70%, for example at least 80%, such as at least 90%, for example at least 95% sequence identity therewith, 167 to 222, such as in the range of 169 to 215, for example in the range of 170 to 200, such as in the range of 170 to 190, for example in the range of 170 to 180, for example n is 174.

[0230] 20. The host cell according to any one of the preceding items, wherein the truncated MSBP is SbMSBPAC as set forth in SEQ ID NO: 6, or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity thereto.

[0231] 21. The host cell according to any one of the preceding items, wherein said truncated MSBP is SbMSBPACIO as set forth in SEQ ID NO: 92, or SbMSBPAC20 as set forth in SEQ ID NO: 93, or SbMSBPAC30 as set forth in SEQ ID NO: 94, or SbMSBPAC40 as set forth in SEQ ID NO: 95, or SbMSBPAC50 as set forth in SEQ ID NO: 96, or a functional homologue thereof sharing at least 70%, for example at least 80%, such as at least 90%, for example at least 95% sequence identity thereto.

[0232] 22. The host cell according to any one of the preceding items, wherein said nucleic acid encoding a CYP450 enzyme is a heterologous nucleic acid, optionally said CYP450 enzyme is a heterologous CYP450 enzyme, for example said CYP450 enzyme is native to a plant, a yeast, a fungus, or a mammal.

[0233] 23. The host cell according to any one of the preceding items, wherein said CYP450 enzyme is a Sorghum CYP450 enzyme, such as a Sorghum CYP450 enzyme, for example SbCYP79A1 as set forth in SEQ ID NO: 1 and / or SbCYP71E1 as set forth in SEQ ID NO: 2, or functional homologues thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity to any of the aforementioned. The host cell according to any one of the preceding items, wherein the host cell further comprises a nucleic acid encoding a UDP-glucuronosyltransferase (UGT), such as a Sorghum UGT, for example a Sorghum bicolor UGT, such as SbllGT85B1 as set forth in SEQ ID NO: 3, or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity thereto. The host cell according to any one of the preceding items, wherein the host cell further comprises a nucleic acid encoding a cytochrome P450 oxidoreductase (POR). The host cell according to item 25, wherein said POR is a Sorghum POR, such as a Sorghum bicolor POR, for example said POR is SbPOR2a as set forth in SEQ ID NO: 4, or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity thereto. The host cell according to any one of the preceding items, wherein said host cell further comprises a nucleic acid encoding a cytochrome b5 (CYB5). The host cell according to any one of the preceding items, wherein said host cell further comprises a nucleic acid encoding a glycosyltransferase. The host cell according to any one of the preceding items, wherein the host cell further comprises at least one nucleic acid encoding one or more of polypeptides of said biosynthetic pathway comprising a CYP450 enzyme, optionally said at least one nucleic acid is codon-optimised for said host cell. The host cell according to any one of the preceding items, wherein the host cell is a bacterial cell, a microalgae cell, a fungal cell or a yeast cell. The host cell according to item 30, wherein the microalgae cell is a cell belonging to the genus of Chlamydomonas, such as Chlamydomonas reinhardtii, Dunaliella, such as Dunaliella salina, Chlorella, such as Chlorella vulgaris, or to the genus of Haematococcus, such as Haematococcus pluvialis. 32. The host cell according to item 30, wherein the bacterial cell is a cell belonging to the genus of Escherichia, such as E. coli, Pseudomonas, such as Pseudomonas putida, Corynebacterium, such as Corynebacterium glutamicum, Streptomyces, such as Streptomyces coelicolor, or to the genus of Bacillus, such as Bacillus subtilis.

[0234] 33. The host cell according to item 30, wherein the fungal cell is a cell belonging to the genus of Aspergillus, such as Aspergillus nidulans, Aspergillus niger, or Aspergillus pseudoterreus, or to the genus of Penicillium, such as Penicillium chrysogenum.

[0235] 34. The host cell according to item 30, wherein the yeast cell is a cell belonging to the genus of Saccharomyces, such as S. cerevisiae, Pichia, such as Pichia pastoris, Yarrowia, such as Yarrowia lipolytica, Kluyveromyces, such as Kluyveromyces lactis, or to the genus of Candida, such as Candida tropicalis.

[0236] 35. The host cell according to any one or the preceding items, wherein the cell comprises a deletion of a gene encoding a glucan 1,3-beta-glucosidase, such as EXG1 as set forth in SEQ ID NO: 43, or a functional homologue thereof sharing at least 70% sequence identity thereto, optionally wherein the gene encoding said glucan 1,3-beta-glucosidase is EXG1 as set forth in SEQ ID NO: 41.

[0237] 36. The host cell according to any one of the preceding items, wherein the compound is a product of a biosynthetic pathway comprising said CYP450 enzyme.

[0238] 37. The host cell according to any one of the preceding items, wherein the biosynthetic pathway is a cyanogenic glucoside pathway, a anthocyanin pathway, a flavonoid pathway, an isoflavonoid pathway, a cannabinoid pathway, an alkaloid pathway, a betalain pathway, a betaxanthin pathway, or a terpenoid pathway. The host cell according to any one of the preceding items, wherein the compound is a plant compound selected from the group consisting of cyanogenic glucosides, anthocyanins, flavonoids, betalains, betaxanthins, isoflavonoids, cannabinoids, alkaloids, and terpenoids. The host cell according to any one of the preceding items, wherein the compound is dhurrin and / or betanin. The host cell according to item 39, wherein said host cell comprises: i. a nucleic acid encoding SbMSBPAC as set forth in SEQ ID NO: 6, such as SEQ ID NO: 12; ii. a nucleic acid encoding SbCYP79A1 as set forth in SEQ ID NO: 1, such as SEQ ID NO: 7, and SbCYP71E1 as set forth in SEQ ID NO: 2, such as SEQ ID NO: 8; iii. a nucleic acid encoding SbPOR2a as set forth in SEQ ID NO: 4, such as SEQ ID NO: 10; and iv. a nucleic acid encoding SbllGT85B1 as set forth in SEQ ID NO: 3, such as SEQ ID NO: 9, or functional homologues thereof sharing at least 70% sequence identity to any of the aforementioned. The host cell according to any one of items 39 to 40, wherein the dhurrin is produced with a titer of at least 120 mg / L, such as at least 130 mg / L, such as at least 140 mg / L, such as at least 150 mg / L, such as at least 160 mg / L, such as at least 165 mg / L, such as at least 170 mg / L, such as at least 175 mg / L, such as at least 180 mg / L, such as at least 190 mg / L, such as at least 200 mg / L, or more. The host cell according to item 39, wherein said host cell comprises: i. a nucleic acid encoding a truncated MSBP, for example SbMSBPAC as set forth in SEQ ID NO: 6, such as SEQ ID NO: 12; ii. a nucleic acid encoding a CYP450 enzyme, for example BvCYP45076AD1 as set forth in SEQ ID NO: 44, such as SEQ ID NO: 48; and further comprises one or more of the following, preferably all of the following: iii. a nucleic acid encoding a cytochrome P450 reductase, for example AtATRI as set forth in SEQ ID NO: 47, such as SEQ ID NO: 51; iv. a nucleic acid encoding a dioxygenase, for example BvDODA as set forth in SEQ ID NO: 45, such as SEQ ID NO: 49; and / or v. a nucleic acid encoding a glycosyltransferase, for example MjcDOPA5GT as set forth in SEQ ID NO: 46, such as SEQ ID NO: 50, or functional homologues thereof sharing at least 70% sequence identity to any of the aforementioned.

[0239] 43. The host cell according to item 39, said host cell comprises: i. a nucleic acid encoding a truncated MSBP, for example SbMSBPAC as set forth in SEQ ID NO: 6, such as SEQ ID NO: 12; ii. a nucleic acid encoding a CYP450 enzyme, for example PhF3’H as set forth in SEQ ID NO: 70, such as SEQ ID NO: 74; and further comprises one or more of the following, preferably all of the following: iii. a nucleic acid encoding a POR, for example PinfPORa as set forth in SEQ ID NO: 69, such as SEQ ID NO: 73; iv. a nucleic acid encoding a flavanone 3-hydroxylase, for example PinfF3H as set forth in SEQ ID NO: 68, such as SEQ ID NO: 72; and / or v. a nucleic acid encoding a flavonol synthase, for example PinfFLS as set forth in SEQ ID NO: 71, such as SEQ ID NO: 75, or functional homologues thereof sharing at least 70% sequence identity to any of the aforementioned.

[0240] 44. A method for producing a compound of a biosynthetic pathway comprising a cytochrome P450 (CYP450) enzyme, said method comprising the steps of: i. providing a host cell according to any one of the preceding items; and ii. cultivating said host cell in a medium, whereby said compound is produced. A method for increasing the titer of a compound of a biosynthetic pathway comprising a cytochrome P450 (CYP450) enzyme in a host cell capable of producing said compound, said method comprising the steps of: iii. expressing a heterologous nucleic acid encoding a truncated membrane-steroid-binding protein (MSBP)(truncated MSBP) lacking at least 10% of the amino acids of the C-terminus of the corresponding wild-type MSBP in said host cell; iv. cultivating said host cell in a medium, whereby the titer of said compound is increased, wherein said increase is compared to the titer produced by a host cell not expressing said nucleic acid encoding a truncated MSBP, when cultivated in the same conditions. A method for increasing the purity of a compound of a biosynthetic pathway comprising a cytochrome P450 (CYP450) enzyme in a host cell capable of producing said compound, said method comprising the steps of: i. expressing a heterologous nucleic acid encoding a truncated membrane-steroid-binding protein (MSBP)(truncated MSBP) lacking at least 10% of the amino acids of the C-terminus of the corresponding wild-type MSBP in said host cell; ii. cultivating said host cell in a medium, whereby the purity of said compound is increased, wherein said increase is compared to the purity of said compound obtained from a host cell not expressing said nucleic acid encoding a truncated MSBP, when cultivated in the same conditions. The method according to any one of items 44 to 46, wherein said host cell is a host cell according to any one of items 1 to 41 or 56, and / or wherein the truncated MSBP and / or the corresponding wild-type MSBP is as defined in any one of items 3 to 20. The method according to any one of items 44 to 47, further comprising a step of isolating said compound. 49. The method according to item 48, wherein the isolation comprises liquidchromatography (LC), and / or two-phase partitioning during cultivation of said host cell.

[0241] 50. The method according to any one of items 44 to 49, further comprising a step of converting said compound into a derivative thereof.

[0242] 51. A system of nucleic acids for expression in a host cell, comprising: i. a heterologous nucleic acid encoding a truncated membrane-steroid- binding protein (MSBP)(truncated MSBP) lacking at least 10% of the amino acids of the C-terminus of the corresponding wild-type MSBP; and ii. a nucleic acid encoding said CYP450 enzyme.

[0243] 52. The nucleic acid construct according to item 51 , wherein the truncated MSBP and / or the wild-type MSBP is as defined in any one of items 3 to 20.

[0244] 53. The nucleic acid construct according to any one of items 51 to 52, comprising: i. a nucleic acid encoding a truncated MSBP, for example SbMSBPAC as set forth in SEQ ID NO: 6, such as SEQ ID NO: 12; ii. a nucleic acid encoding a CYP450 enzyme, for example SbCYP79A1 as set forth in SEQ ID NO: 1, such as SEQ ID NO: 7, and / or SbCYP71 E1 as set forth in SEQ ID NO: 2, such as SEQ ID NO: 8; and further comprising: iii. a nucleic acid encoding a POR, for example SbPOR2a as set forth in SEQ ID NO: 4, such as SEQ ID NO: 10; and / or iv. a nucleic acid encoding a UDP-glucuronosyltransferase, for example SbUGT85B1 as set forth in SEQ ID NO: 3, such as SEQ ID NO: 9, or functional homologues thereof sharing at least 70% sequence identity to any of the aforementioned.

[0245] 54. The nucleic acid construct according to any one of items 51 to 52, comprising: i. a nucleic acid encoding SbMSBPAC as set forth in SEQ ID NO: 6, such as SEQ ID NO: 12; ii. a nucleic acid encoding BvCYP45076AD1 as set forth in SEQ ID NO:

[0246] 44, such as SEQ ID NO: 48; and further comprising: iii. a nucleic acid encoding AtATRI as set forth in SEQ ID NO: 47, such as SEQ ID NO: 51 ; iv. a nucleic acid encoding BvDODA as set forth in SEQ ID NO: 45, such as SEQ ID NO: 49; and v. a nucleic acid encoding MjcDOPA5GT as set forth in SEQ ID NO: 46, such as SEQ ID NO: 50, or functional homologues thereof sharing at least 70% sequence identity to any of the aforementioned.

[0247] 55. The nucleic acid construct according to any one of items 51 to 52, comprising: i. a nucleic acid encoding SbMSBPAC as set forth in SEQ ID NO: 6, such as SEQ ID NO: 12; ii. a nucleic acid encoding PhF3’H as set forth in SEQ ID NO: 70, such as SEQ ID NO: 74; and further comprising: iii. a nucleic acid encoding PinfPORa as set forth in SEQ ID NO: 69, such as SEQ ID NO: 73; iv. a nucleic acid encoding PinfF3H as set forth in SEQ ID NO: 68, such as SEQ ID NO: 72; and v. a nucleic acid encoding PinfFLS as set forth in SEQ ID NO: 71 , such as SEQ ID NO: 75, or functional homologues thereof sharing at least 70% sequence identity to any of the aforementioned.

[0248] 56. The host cell according to any one of items 1 to 41 , comprising the nucleic acid construct according to any one of items 51 to 55.

[0249] 57. Use of a truncated membrane-steroid-binding protein (MSBP) lacking at least a part of the C-terminus compared to a corresponding wild-type MSBP in a method for producing a compound of a biosynthetic pathway comprising a cytochrome P450 (CYP450) enzyme, and / or for increasing the production of a compound of a biosynthetic pathway comprising a cytochrome P450 (CYP450) enzyme, such as for increasing the titer of said compound, and / or for increasing the purity of said compound, preferably said compound is a plant compound. The use according to item 57, wherein the truncated MSBP and / or the wild-type MSBP is a truncated MSBP and / or a wild-type MSBP, respectively, as defined in any one of items 3 to 20. The use according to any one of items 57 to 58, wherein the compound is a compound as defined in any one of items 36 to 39. The host cell, the method and / or the use according to any one of the preceding items, wherein the titer of said compound is increased compared to the titer of said compound produced in the absence of the truncated MSBP, when the production is performed under the same conditions, and the titer is measured under the same conditions. The host cell, the method and / or the use according any one of the preceding items, wherein the increased titer of said compound is compared to the titer of said compound produced in the presence of the wild-type MSBP. The host cell, the method and / or the use according to any one of the preceding items, wherein the titer of said compound is increased at least 1-fold, for example at least 1.1 -fold, such as at least 1.2-fold, for example at least 1.3-fold, such as at least 1.5-fold, for example at least 1.7-fold, such as at least 2-fold, for example at least 2.3-fold, such as at least 2.5-fold, for example at least 3- fold, or more. The host cell, the method and / or the use according to any one of the preceding items, wherein the titer of said compound is defined as the total titer of said compound, precursors, pathway intermediates, and / or derivatives thereof. The host cell, the method and / or the use according to any one of the preceding items, wherein the purity of said compound is increased compared to the purity of said compound obtained in the absence of the truncated MSBP, when the production is performed under the same conditions, and the purity is measured under the same conditions. The host cell, the method and / or the use according any one of the preceding items, wherein the increased purity of said compound is compared to the purity of said compound produced in the presence of the wild-type MSBP. The host cell, the method and / or the use according to any one of the preceding items, wherein the purity of said compound is defined as the total purity of said compound, precursors, pathway intermediates, and / or derivatives thereof. The host cell, the method and / or the use according to any one of the preceding items, wherein the purity of said compound is increased by at least 1% compared to the purity of the same compound obtained from a host cell not expressing said nucleic acid encoding a truncated MSBP, such as at least 5%, for example at least 10%, such as at least 15%, for example at least 20%, such as at least 20%, for example at least 25%, such as at least 30%, for example at least 35%, such as at least 40%, for example at least 45%, such as at least 50%, for example at least 55%, such as at least 60%, for example at least 65%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 100%, such as at least 125%, for example at least 150%, or more.

Claims

Claims1. A host cell capable of producing a compound of a biosynthetic pathway comprising a cytochrome P450 (CYP450) enzyme, said host cell comprising: i. a heterologous nucleic acid encoding a truncated membrane-steroid- binding protein (MSBP)(truncated MSBP) lacking a part of the C- terminus of the corresponding wild-type MSBP, such as at least 10% of the amino acids of the C-terminus of the corresponding wild-type MSBP; and ii. a nucleic acid encoding said CYP450 enzyme.

2. A host cell capable of producing a compound, which is a product of a biosynthetic pathway comprising a cytochrome P450 (CYP450) enzyme, said host cell comprising: i. a heterologous nucleic acid encoding a truncated membrane-steroid- binding protein (MSBP)(truncated MSBP) lacking at least 10% of the amino acids of the C-terminus of the corresponding wild-type MSBP; and ii. a nucleic acid encoding said CYP450 enzyme, wherein the C-terminus of the wild-type MSBP is defined as the residues downstream of the CYB5-like domain, and wherein the compound is selected from the group consisting of cyanogenic glucosides, anthocyanins, flavonoids, betalains, betaxanthins, isoflavonoids, cannabinoids, alkaloids, and terpenoids.

3. The host cell according to claim 1 or 2, wherein the C-terminus of the wild-type MSBP is defined as the residues downstream of the CYB5-like domain and / or wherein the truncated MSBP is N-terminus-membrane-anchored, optionally wherein the C-terminus of the wild-type MSBP is defined as the residues downstream the residues corresponding to amino acids 70 to 167 of SbMSBP as set forth in SEQ ID NO: 5.

4. The host cell according to any one of the preceding claims, wherein said truncated MSBP lacks at least 15% of the amino acids of the C-terminus of the corresponding wild-type MSBP, such as at least 20%, for example at least 25%, such as at least 30%, for example at least 35%, such as at least 40%, forRECTIFIED SHEET (RULE 91 ) ISA / EPexample at least 45%, such as at least 50%, for example at least 55%, such as at least 60%, for example at least 65%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% of the amino acids of the C-terminus of the corresponding wild-type MSBP, or more.

5. The host cell according to any one of the preceding claims, wherein the wildtype MSBP is native to a plant, a mammal, such as Homo sapiens (human), or a yeast, optionally wherein the wild-type MSBP is derived from Sorghum, such as Sorghum bicolor, Arabidopsis, such as Arabidopsis thaliana, Zea, such as Zea mays, Oryza, such as Oryza sativa spp. japonica, or Petunia, such as P. inflata, preferably wherein the wild-type MSBP is a Sorghum bicolor MSBP, such as SbMSBP as set forth in SEQ ID NO: 5, or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity thereto.

6. The host cell according to any one of the preceding claims, wherein said truncated MSBP lacks between 10 to 56 amino acids of the most C-terminal amino acids of the corresponding wild-type MSBP, such as between 20 and 53 amino acids, for example between 30 and 53 amino acids, such as between 40 and 50 amino acids, for example between 45 and 49 amino acids of the most C-terminal amino acids of the corresponding wild-type MSBP, preferably wherein the corresponding wild-type MSBP is SbMSBP as set forth in SEQ ID NO: 5, or a functional homologue thereof sharing at least 70% sequence identity thereto, and / or wherein the truncated MSBP shares at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity or 100% sequence identity with the N- terminal part of the corresponding wild-type MSBP, preferably wherein said MSBP is SbMSBP as set forth in SEQ ID NO: 5, or a functional homologue thereof sharing at least 70% sequence identity thereto.

7. The host cell according to any one of the preceding claims, wherein said truncated MSBP lacks between 10 to 56 amino acids of the most C-terminalRECTIFIED SHEET (RULE 91 ) ISA / EPamino acids of the corresponding wild-type MSBP, wherein the corresponding wild-type MSBP is SbMSBP as set forth in SEQ ID NO: 5.

8. The host cell according to any one of the preceding claims, wherein the truncated MSBP consists of amino acids 1 to n of a wild-type MSBP, wherein n is an integer in the range of 100 to 264, for example in the range 110 to 245, such as in the range of 120 to 230, for example in the range of 130 to 210, such as in the range of 140 to 200, for example in the range of 150 to 190, such as in the range of 160 to 180, for example in the range of 160 to 175, and wherein the wild-type MSBP may be SbMSBP (SEQ ID NO: 5), OsMSBPI (SEQ ID NO: 34), OsMSBP2 (SEQ ID NO: 35), ZmMSBP (SEQ ID NO: 36), AtMSBPI (SEQ ID NO: 37), AtMSBP2 (SEQ ID NO: 38), HsPGRMCI (SEQ ID NO: 39), PinfMSBPI (SEQ ID NO: 42), or ScDAPI (SEQ ID NO: 40), or a functional homologue thereof sharing at least 70% sequence identity, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity or 100% sequence identity with any of the aforementioned.

9. The host cell according to any one of the preceding claims, wherein the truncated MSBP consists of amino acids 1 to n of SEQ ID NO: 5, or a functional homologue thereof sharing at least 70%, for example at least 80%, such as at least 90%, for example at least 95% sequence identity therewith, wherein n is a integer in the range of 167 to 222, such as in the range of 169 to 215, for example in the range of 170 to 200, such as in the range of 170 to 190, for example in the range of 170 to 180, for example n is 174.

10. The host cell according to any one of the preceding claims, wherein the truncated MSBP consists of amino acids 1 to n of SEQ ID NO: 5, wherein n is a integer in the range of 167 to 222, for example n is 174.11 . The host cell according to any one of the preceding claims, wherein the truncated MSBP consists of amino acids 1 to 174 of SEQ ID NO: 5.RECTIFIED SHEET (RULE 91 ) ISA / EP12. The host cell according to any one of the preceding claims, wherein the truncated MSBP is SbMSBPAC as set forth in SEQ ID NO: 6, or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity thereto.

13. The host cell according to any one of the preceding claims, wherein the compound is a plant compound selected from the group consisting of cyanogenic glucosides, anthocyanins, flavonoids, betalains, betaxanthins, isoflavonoids, cannabinoids, alkaloids, and terpenoids.

14. A method for producing a compound of a biosynthetic pathway comprising a cytochrome P450 (CYP450) enzyme, said method comprising the steps of: i. providing a host cell according to any one of the preceding claims; and ii. cultivating said host cell in a medium, whereby said compound is produced, optionally wherein the compound is a plant compound as defined in claim 13.

15. A method for increasing the titer of a compound of a biosynthetic pathway comprising a cytochrome P450 (CYP450) enzyme in a host cell capable of producing said compound, said method comprising the steps of: i. expressing a heterologous nucleic acid encoding a truncated membrane-steroid-binding protein (MSBP)(truncated MSBP) lacking at least 10% of the amino acids of the C-terminus of the corresponding wild-type MSBP in said host cell; ii. cultivating said host cell in a medium, whereby the titer of said compound is increased, wherein said increase is compared to the titer produced by a host cell not expressing said nucleic acid encoding a truncated MSBP, when cultivated in the same conditions, optionally wherein said host cell is a host cell according to any one of claims 1 to 13, and / or wherein the truncated MSBP and / or the corresponding wild-type MSBP is as defined in any one of claims 2 to 12, further optionally wherein the compound is a plant compound as defined in claim 13.RECTIFIED SHEET (RULE 91 ) ISA / EP16. A method for increasing the purity of a compound of a biosynthetic pathway comprising a cytochrome P450 (CYP450) enzyme in a host cell capable of producing said compound, said method comprising the steps of: i. expressing a heterologous nucleic acid encoding a truncated membrane-steroid-binding protein (MSBP)(truncated MSBP) lacking at least 10% of the amino acids of the C-terminus of the corresponding wild-type MSBP in said host cell; ii. cultivating said host cell in a medium, whereby the purity of said compound is increased, wherein said increase is compared to the purity of said compound obtained from a host cell not expressing said nucleic acid encoding a truncated MSBP, when cultivated in the same conditions, optionally wherein said host cell is a host cell according to any one of claims 1 to 13, and / or wherein the truncated MSBP and / or the corresponding wild-type MSBP is as defined in any one of claims 2 to 12, further optionally wherein the compound is a plant compound as defined in claim 13.

17. Use of a truncated membrane-steroid-binding protein (MSBP) lacking at least a part of the C-terminus compared to a corresponding wild-type MSBP in a method for producing a compound of a biosynthetic pathway comprising a cytochrome P450 (CYP450) enzyme, and / or for increasing the production of a compound of a biosynthetic pathway comprising a cytochrome P450 (CYP450) enzyme, such as for increasing the titer of said compound, and / or for increasing the purity of said compound, preferably said compound is a plant compound, such as a plant compound as defined in claim 13, optionally wherein said host cell is a host cell according to any one of claims 1 to 13, and / or wherein the truncated MSBP and / or the corresponding wild-type MSBP is as defined in any one of claims 2 to 12, further optionally wherein the compound is a plant compound as defined in claim 13.

18. The host cell, the method and / or the use according to any one of the preceding claims, wherein the titer of said compound is increased compared to the titer of said compound produced in the absence of the truncated MSBP,RECTIFIED SHEET (RULE 91 ) ISA / EPwhen the production is performed under the same conditions, and the titer is measured under the same conditions, preferably wherein the increased titer of said compound is compared to the titer of said compound produced in the presence of the wild-type MSBP, optionally wherein the titer of said compound is increased at least 1-fold, for example at least 1.1 -fold, such as at least 1.2-fold, for example at least 1.3-fold, such as at least 1.5-fold, for example at least 1.7-fold, such as at least 2-fold, for example at least 2.3-fold, such as at least 2.5-fold, for example at least 3- fold, or more, further optionally wherein the titer of said compound is defined as the total titer of said compound, precursors, pathway intermediates, and / or derivatives thereof.

19. The host cell, the method and / or the use according to any one of the preceding claims, wherein the purity of said compound is increased compared to the purity of said compound obtained in the absence of the truncated MSBP, when the production is performed under the same conditions, and the purity is measured under the same conditions, preferably wherein the increased purity of said compound is compared to the purity of said compound produced in the presence of the wild-type MSBP, optionally wherein the purity of said compound is defined as the total purity of said compound, precursors, pathway intermediates, and / or derivatives thereof, further optionally wherein the purity of said compound is increased by at least 1% compared to the purity of the same compound obtained from a host cell not expressing said nucleic acid encoding a truncated MSBP, such as at least 5%, for example at least 10%, such as at least 15%, for example at least 20%, such as at least 20%, for example at least 25%, such as at least 30%, for example at least 35%, such as at least 40%, for example at least 45%, such as at least 50%, for example at least 55%, such as at least 60%, for example at least 65%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 100%, such as at least 125%, for example at least 150%, or more.RECTIFIED SHEET (RULE 91 ) ISA / EP

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