Formation of oxygenated diterpenoid compounds

Abstract

Disclosed are methods for the production of oxygenated diterpenoid compounds, such as tryptophenolide, triptonide, and triptolide, by inserting and expressing a gene encoding a specific cytochrome P450 enzyme in a host cell selected for the synthesis of the oxygenated diterpenoid compound. Also disclosed are specific cytochrome P450 enzymes suitable for this synthesis.

Description

[Technical Field]

[0001] Sequence Listing Reference This application contains a Sequence Listing in computer readable form, which is incorporated by reference.

[0002] FIELD OF THE INVENTION The present invention relates to the production of oxygenated diterpenoid compounds in recombinant cells, such as yeast cells. The oxygenated diterpenoid compounds are useful as intermediates or final compounds in the synthesis of useful biologically active compounds for use in the pharmaceutical treatment of diseases such as cancer. The invention further relates to genes, enzymes, and cells, such as yeast cells, particularly suitable for the production of such compounds. [Background technology]

[0003] Background of the Invention Terpenes are a diverse group of compounds made from the basic 5-carbon structure isoprene (2-methyl-1,3-butadiene). Diterpenes are compounds with 20 carbon structures made by the action of the enzyme diterpene synthase, which converts the compound geranylgeranyl diphosphate (GGPP) into a diterpene structure, which can be further modified to form a wide range of diterpene or diterpenoid compounds. Diterpenes, diterpenoids, and their derivatives are widely used, for example, as pharmaceuticals, cosmetics, nutrients, flavorings, fragrances, and insecticides. There are many methods in the art for increasing the production of these compounds in natural or engineered cells.

[0004] Tripterygium wilfordii, a traditional Chinese medicine plant, is known to produce multiple sesquiterpenoids, diterpenoids, and triterpenoids with potential pharmacological properties, including the diterpenoid compounds triptonide and triptolide. Triptolide, an oxygenated diterpenoid compound, and its derivatives have been identified as potentially valuable pharmacological compounds and are currently being investigated as immunosuppressants and for the treatment of cancer. Triptolide may also be used in the treatment of COVID-19. Triptonide may be useful as a male contraceptive.

[0005] The use of engineered microorganisms to produce valuable molecules from renewable feedstocks is a desirable alternative to traditional production means, but achieving economically viable yields, titers, and production capacity remains a significant barrier to industrialization.

[0006] N.L. Hansen et al. (2017) described a diterpene synthase capable of converting GGPP to the diterpene miltiradiene, a precursor of triptolide, in The Plant Journal 2017, 89, 429-441. This finding was confirmed by P. Su et al. (2018) in The Plant Journal 2018, 50-65, and by J. Guo et al. in PNAS 2013, 110, 12108-12113.

[0007] The complete pathway for the conversion of miltiradiene to other diterpenoid compounds such as triptolide remains to be elucidated.

[0008] Cytochrome P450 enzymes (CYPs) are involved in the biosynthesis of terpenoids, and for many cytochrome P450 enzymes, nothing is known about the substrates they act on, which compounds they make, or their role in the biosynthesis of specific compounds.

[0009] U.S. Patent Application Publication No. 20190270971 discloses methods for increasing the productivity of microbial host cells that functionally express P450 enzymes. The document describes how P450 genes can be modified to improve performance in microorganisms such as yeast, and mentions that co-expression with cytochrome P450 reductase can be beneficial for improving yield, but the document does not provide any link between triptolide and any specific P450 enzymes or cytochromes.

[0010] Chinese Patent No. 108395997 describes a yeast with increased GGPP production. The yeast is transformed with different diterpene synthases and P450 enzymes to synthesize diterpenoid compounds. The team of scientists behind this patent also supports further patents and patent applications, such as Chinese Patent No. 108866029 (Friedelin), Chinese Patent No. 107058419 (Kauren-type), and WO2020029564 (Fridelin and Amyrins), which disclose the synthesis of different di- and triterpenoid compounds using appropriate terpene synthases and P450 enzymes.

[0011] Chinese Patent No. 110747178 describes the P450 gene TwCYP728B70 as encoding a cytochrome P450 enzyme that has a role in triptolide synthesis. Summary of the Invention [Problem to be solved by the invention]

[0012] Short description of the invention The present inventors have solved the problem of providing improved methods for producing oxygenated diterpenoid compounds such as triptophenolide, triptonide and triptolide. [Means for solving the problem]

[0013] In a first aspect, the present invention relates to a method for producing oxygenated diterpenoid compounds, the method comprising: a. providing a host cell capable of producing miltiradiene and / or dehydroabietadiene; b. transforming a host cell with a first gene encoding an enzyme having cytochrome P450 activity; c. growing the transformed cells under conditions conducive to expression of the transformed genes, thereby forming oxygenated diterpenoid compounds; where: The first gene encoding an enzyme having cytochrome P450 activity encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity to SEQ ID NO:1 (TwCYP82D274v1), SEQ ID NO:2 (TwCYP82D274v2), SEQ ID NO:74 (TwCYP82D274v3) or SEQ ID NO:75 (TwCYP82D274v4), or a mature polypeptide thereof.

[0014] In a second aspect, the present invention relates to a method for producing oxygenated diterpenoid compounds, comprising transforming a host cell with a first gene encoding an enzyme having cytochrome P450 activity, further with a second gene encoding a second enzyme having cytochrome P450 activity, and with a third gene encoding a third enzyme having cytochrome P450 activity; where: the second gene encoding an enzyme having cytochrome P450 activity encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity to SEQ ID NO: 3 (TwCYP71BE85), or a mature polypeptide thereof; The third gene encoding an enzyme having cytochrome P450 activity encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity to SEQ ID NO:4 (TwCYP71BE85), or a mature polypeptide thereof.

[0015] In a third aspect, the present invention relates to a method for producing oxygenated diterpenoid compounds, said method comprising transforming a host cell with first, second and third genes encoding enzymes having cytochrome P450 activity, and further transformed with a fourth gene encoding a fourth enzyme having cytochrome P450 activity, wherein: The fourth gene encoding the fourth enzyme having cytochrome P450 activity encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity to SEQ ID NO:5 (TwCYP82D213v1) or SEQ ID NO:76 (TwCYP82D213v2), or a mature polypeptide thereof.

[0016] The present invention may provide useful oxygenated diterpenoid compounds, triptophenolide, triptonide, or triptolide.

[0017] The present invention further relates to polypeptides, polynucleotides, plasmids and expression constructs, and recombinant host cells that are useful in the methods of the present invention. [Brief explanation of the drawings]

[0018] [Figure 1]LCMS profiles of extracts from N. benthamiana leaves expressing miltiradiene biosynthetic genes and selected T. wilfordii CYPs are shown. TwCYPs were coexpressed with CfDXS, CfGGPPS, CfTPS1, and CfTPS3. "3xSTD" represents an LCMS run of a sample containing three mixed authentic standards: triptolide, triptonide, and triptophenolide. The solid line represents the extracted ion chromatogram in the m / z range of 280-380. The dashed line (- - -) represents the extracted ion chromatogram at m / z 313.1800 ± 0.015, corresponding to the parent ion [M+H] of triptophenolide. The dashed line (- - -) represents the extracted ion chromatogram at m / z 3593.1490 ± 0.015, corresponding to the parent ion [M+H] of triptophenolide. LC Protocol 1 was used. For more details, see Example 1. [Figure 2] LCMS profiles of extracts from genetically engineered Saccharomyces cerevisiae (S. cerevisiae) strains are shown. In the Bakkergrund strain (-), the diterpene biosynthetic enzyme-encoding genes SPGGPPS7, CftTPS1, CftTPS3, and TwCPR1 were integrated into the wild-type S. cerevisiae genome. In the TwCYP82D274v1 strain, the diterpene biosynthetic enzymes were expressed in TwCYP82D274v1, resulting in the formation of compound 3, identified as 14-OH-dehydroabietadiene (shown in gray). LC Method 1 was used for analysis. For more details, see Example 3. [Figure 3]

[0023] Figure 1 shows the H NMR spectrum of 14-OH-dehydroabietadiene in CDCl3 at 599.85 MHz. For more details, see Example 4. [Figure 4]

[0023] Figure 1 shows the C NMR spectrum of 14-OH-dehydroabietadiene in CDCl at 150.83 MHz. For more details, see Example 4. [Figure 5]Figure 1 shows LCMS profiles of extracts of yeast with the indicated integrated gene combination genomes. All yeast strains have genomes integrated with spGGPPs7, CftTPS1, CftTPS3, and TwCPR1. "0.5 ppm 3xSTD" represents an LCMS run of a sample of three mixed authentic standards: triptolide, triptonide, and triptophenolide. Non-dashed lines represent the ion chromatogram in the m / z range of 280-380. The dashed line represents the extracted ion chromatogram at m / z 359.1490 ± 0.015, corresponding to the parent ion of triptonide ([M+H]+). LC protocol 2 was used. For more details, see Example 5. [Figure 6] Co-expression of TwCYPs with different variants of the B5 protein isolated from Tripterygium wilfordii is shown. Levels of tryptophenolide, triptonide, and 14-OH-dehydroabietadiene were quantified from cultures of engineered S. cerevisiae strains. Each column represents an engineered yeast strain and its production of selected compounds. The genes integrated into each individual strain are shown in the bottom panel. Quantification was based on the area of ​​the peak representing each compound of interest. Individual scales apply to each compound. For more details, see Example 5. [Figure 7]The relative abundances (bars) of key intermediates in the proposed biosynthetic pathway of tryptonides, established in vivo via heterologous gene expression in N. benthamiana (panels A and D) and S. cerevisiae (panels B and E, strains listed in Table 3), are shown. Gene expression is indicated by the black square on the left, and relative abundance is indicated by the bar (average of 3–4 biological replicates; black diamond). White and gray fills distinguish between expressing and non-expressing Twb5#1, respectively. Error bars represent standard deviations. "DiTPSs" refers to CftTPS1 and Cft-TPS3. For peak area quantification, the mass tolerance of indicators was ±0.1 m / z by GCMS (miltiradiene and 14-OH-dehydroabietadiene) and ±0.005 m / z by LCMS (all other compounds). Panel C: Hypothesized biosynthetic pathway from myrtildienes to triptonides in vivo in N. baenthamiana and S. cerevisiae, including a Wagner-Meerwein rearrangement reaction describing a methyl shift from C-19 or C-18 to C3 within the abietane carbon skeleton. [Figure 8] Accumulation of tryptophenolide and tryptonide produced by yeast strain NVJ8.15 over 7 days when grown in a bioreactor. Triptonide (solid black line) and tryptophenolide (dotted black line) levels represent absolute amounts (ppm, w / v) in culture samples taken each day. Biomass was quantified by absorbance at 600 nm (dotted gray line). [Figure 9]Yeast strains expressing genes required for tryptonide biosynthesis but with gene mutations substituting TwCYP82D274v1 or TwCYP82D213v1 are shown to be related to their ability to produce tryptophenolide (Panel A) and tryptonide (Panel B) and to yield similar LCMS profiles (Panel C). Genes present in the engineered strains are represented by black squares. Panels A and B: Bars represent the average relative abundance (2-3 biological replicates, crosses), and error bars indicate standard error. From left to right, bars represent yeast strains NVJ10-1, NVJ10-3, NVJ10-6, and NVJ10-8 (see Table 3). Panel C: EICs (m / z 280-360) of yeast cultures analyzed by LCMS. From top to bottom, the pairs of chromatograms represent yeast strains NVJ10-1, NVJ10-3, NVJ10-6, and NVJ10-8. [Figure 10] NMR spectrum of the compound produced. [Figure 11] NMR spectrum of the compound produced. [Figure 12] NMR spectrum of the compound produced. [Figure 13] NMR spectrum of the compound produced. [Figure 14] NMR spectrum of the compound produced. [Figure 15] NMR spectrum of the compound produced. [Figure 16] NMR spectrum of the compound produced. [Figure 17] NMR spectrum of the compound produced. [Figure 18] NMR spectrum of the compound produced. [Figure 19] NMR spectrum of the compound produced. [Figure 20] NMR spectrum of the compound produced. [Figure 21] NMR spectrum of the compound produced. [Figure 22] NMR spectrum of the compound produced. [Figure 23] NMR spectrum of the compound produced. [Figure 24] NMR spectrum of the compound produced. [Figure 25] NMR spectrum of the compound produced. [Figure 26] NMR spectrum of the compound produced. DETAILED DESCRIPTION OF THE INVENTION

[0019] A short description of the sequence SEQ ID NO:1 shows the amino acid sequence of a cytochrome P450 enzyme from T. wilfordii, also known as TwCYP82D274v1.

[0020] SEQ ID NO:2 shows the amino acid sequence of a cytochrome P450 enzyme from T. wilfordii. This enzyme is also known as TwCYP82D274v2. SEQ ID NO:2 differs from SEQ ID NO:1 at only three positions; therefore, SEQ ID NO:1 and SEQ ID NO:2 are predicted to represent different alleles of the same gene.

[0021] SEQ ID NO:3 shows the amino acid sequence of a cytochrome P450 enzyme from T. wilfordii, also known as TwCYP71BE85.

[0022] SEQ ID NO:4 shows the amino acid sequence of a cytochrome P450 enzyme from T. wilfordii, also known as TwCYP71BE86.

[0023] SEQ ID NO:5 shows the amino acid sequence of a cytochrome P450 enzyme from T. wilfordii, also known as TwCYP82D213v1.

[0024] SEQ ID NO:6 shows the amino acid sequence of a cytochrome P450 enzyme from T. wilfordii, also known as TwCYP82D217.

[0025] SEQ ID NO:7 shows the amino acid sequence of a cytochrome P450 enzyme from T. wilfordii, also known as TwCYP82D275.

[0026] SEQ ID NO:8 shows the amino acid sequence of the cytochrome B5 enzyme from T. wilfordii, also known as TwB5#1.

[0027] SEQ ID NO:9 shows the amino acid sequence of a cytochrome P450 enzyme from T. wilfordii, also known as TwCPR1.

[0028] SEQ ID NOs: 10-66 show PCR primers as further described in Example 2.

[0029] SEQ ID NO:67 shows the amino acid sequence of diterpene synthase TPS1 from Plectranthus barbatus. This enzyme is also known as CfTPS1.

[0030] SEQ ID NO:68 shows the amino acid sequence of diterpene synthase TPS3 from Plectranthus barbatus. This enzyme is also known as CfTPS3.

[0031] SEQ ID NO:69 shows the amino acid sequence of the terpene synthase TPS9 from T. wilfordii. This enzyme is also known as TwTPS9.

[0032] SEQ ID NO:70 shows the amino acid sequence of a terpene synthase from T. wilfordii. This enzyme is also known as TwTPS27.

[0033] SEQ ID NO:71 shows the amino acid sequence of copalyl diphosphate synthase CPS1 from Salvia miltiorrhiza. This enzyme is also known as SmCPS.

[0034] SEQ ID NO:72 shows the amino acid sequence of miltiradiene synthase KSL1 from Salvia miltiorrhiza. This enzyme is also known as SmKSL.

[0035] SEQ ID NO:73 shows the amino acid sequence of generanylgeneranyl diphosphate synthase from Synechococcus sp. This enzyme is also known as SpGGPPs7v1.

[0036] SEQ ID NO:74 shows the amino acid sequence of a cytochrome P450 enzyme from T. wilfordii, also known as TwCYP82D274v3.

[0037] SEQ ID NO:75 shows the amino acid sequence of a cytochrome P450 enzyme from T. wilfordii, also known as TwCYP82D274v4.

[0038] SEQ ID NO:76 shows the amino acid sequence of a cytochrome P450 enzyme from T. wilfordii, also known as TwCYP82D213v2.

[0039] SEQ ID NO:77 shows the truncated amino acid sequence of diterpene synthase TPS1 from Plectranthus barbatus. The amino acid sequence was truncated to remove the transit peptide. This enzyme is also known as CftTPS1.

[0040] SEQ ID NO:78 shows the truncated amino acid sequence of diterpene synthase TPS3 from Plectranthus barbatus. The amino acid sequence was truncated to remove the transit peptide. This enzyme is also known as CfTPS3.

[0041] SEQ ID NO:79 shows the amino acid sequence of the DXS enzyme from Plectranthus barbatus. This enzyme is also known as CfDXS.

[0042] SEQ ID NO:80 shows the amino acid sequence of the truncated HMGR enzyme from S. cerevisiae, which is also known as SctHMGR.

[0043] SEQ ID NO:81 shows the amino acid sequence of geranylgeranyl diphosphate synthase from Synechococcus sp. This enzyme is also known as SpGGPPs7v2.

[0044] Detailed Description of the Invention In accordance with a first aspect of the present invention, a method for producing an oxygenated diterpenoid compound is disclosed, the method comprising: a. providing a host cell capable of producing miltiradiene and / or dehydroabietadiene; b. transforming a host cell with a first gene encoding an enzyme having cytochrome P450 activity; c. growing the transformed cells under conditions that result in expression of the transformed genes, thereby forming oxygenated diterpenoid compounds; where: The first gene encoding an enzyme having cytochrome P450 activity encodes a polypeptide comprising or consisting of an amino acid sequence having at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity to SEQ ID NO:1, or SEQ ID NO:1 (TwCYP82D274V1), or a mature polypeptide thereof.

[0045] The polypeptide having SEQ ID NO:1 is a preferred example of a first gene having cytochrome P450 activity, and the polypeptides having SEQ ID NO:2, SEQ ID NO:74 and SEQ ID NO:75 are other examples of such polypeptides.

[0046] Thus, the enzyme encoded by the first gene has the ability to convert miltiradiene and / or dehydroabietadienes to 14-OH-dehydroabietadienes by inserting an OH group at the 14-position of the diterpene skeleton of miltiradiene.

[0047] In some embodiments, the synthesis of 14-OH-dehydroabietadiene is carried out via the compound 14-OH-miltiradiene, which is subsequently converted to 14-OH-dehydroabietadiene. However, the present invention is not limited to any particular mechanism for converting miltiradiene to 14-OH-dehydroabietadiene.

[0048] Use of the method according to the first aspect of the present invention results in the formation of the oxygenated diterpenoid compound 14-OH-dehydroabietadiene, which is a useful intermediate in the synthesis of oxygenated diterpenoid compounds of pharmaceutical use, including well-known compounds such as triptophenolide, triptonide and triptolide. [ka]

[0049] In a second embodiment of the invention, method step b comprises transforming a host cell with a first gene encoding an enzyme having cytochrome P450 activity, further with a second gene encoding a second enzyme having cytochrome P450 activity, and a third gene encoding a third enzyme having cytochrome P450 activity, wherein the second gene encoding an enzyme having cytochrome P450 activity encodes a polypeptide comprising or consisting of an amino acid sequence having at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity to SEQ ID NO: 4, or SEQ ID NO: 4 (TwCYP71BE86), or a mature polypeptide thereof; The third gene encoding an enzyme having cytochrome P450 activity encodes a polypeptide comprising or consisting of an amino acid sequence having at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity to SEQ ID NO:3, or SEQ ID NO:3 (TwCYP71BE85), or a mature polypeptide thereof.

[0050] In this second embodiment, the host cell preferably further produces the oxygenated diterpenoid compound tryptophenolide: (3bR,9bS)-6-hydroxy-9b-methyl-7-propan-2-yl-3,3b,4,5,10,11-hexahydronaphtho[2,1-e]isobenzofuran-1-one. [ka]

[0051] Tryptophenolide is a valuable compound identified as an antiandrogen. In addition, it may be useful as a starting point for further modifications leading to additional biologically active compounds.

[0052] In a preferred embodiment of the second aspect of the present invention, the host cell is further transformed with a first gene encoding a polypeptide having cytochrome B5 activity and comprising or consisting of an amino acid sequence having at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity to SEQ ID NO:8, or SEQ ID NO:8 (TwB5#1), or a mature polypeptide thereof. Surprisingly, it has been found that expressing a polypeptide having cytochrome B5 activity in the same cell expressing the first, second, and third genes encoding enzymes with cytochrome P450 activity results in significantly higher production of oxygenated diterpenoid compounds. Production is increased by at least 50%, preferably at least 100%, preferably at least 200%, or more, compared to production in a similar cell not containing a polypeptide having cytochrome B5 activity.

[0053] In a third aspect of the present invention, a host cell is transformed with first, second and third genes encoding enzymes having cytochrome P450 activity, and further transformed with a fourth gene encoding a fourth enzyme having cytochrome P450 activity, wherein The fourth gene encoding the fourth enzyme having cytochrome P450 activity encodes a polypeptide comprising or consisting of an amino acid sequence having at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity to SEQ ID NO:5, or SEQ ID NO:5 (TwCYP82D213v1), or a mature polypeptide thereof. The polypeptide having SEQ ID NO:5 is a preferred example of a fourth gene having cytochrome P450 activity, and the polypeptide having SEQ ID NO:76 is another example of such a polypeptide.

[0054] In the third aspect of the invention, the transformed eukaryotic cells preferably produce triptonides, which are oxygenated diterpenoid compounds. [ka]

[0055] The compound triptonide has been reported to have potent inhibitory activity in cancer (Fulu Dong et al. 2019, The Prostate, Volume 19, Issue 11, pages 1284-1293). This compound is also useful as a male contraceptive. Furthermore, this compound is useful as a starting point for further modification to yield additional bioactive compounds.

[0056] In a preferred embodiment of the third aspect of the present invention, the host cell is further transformed with a fifth gene encoding a polypeptide having cytochrome B5 activity and comprising or consisting of an amino acid sequence having at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity to SEQ ID NO:8, or SEQ ID NO:8 (TwB5#1), or a mature polypeptide thereof. Surprisingly, it has been found that expressing a polypeptide having cytochrome B5 activity in the same cell expressing the first, second, third, and fourth genes encoding enzymes with cytochrome P450 activity results in significantly higher production of oxygenated diterpenoid compounds. Production is increased by at least 50%, preferably at least 100%, preferably at least 200%, or more, compared to production in a similar cell not containing a polypeptide having cytochrome B5 activity.

[0057] In a further preferred embodiment of the third aspect of the present invention, the host cell is further transformed with a sixth gene encoding a fifth enzyme having cytochrome P450 activity and / or a seventh gene encoding a sixth enzyme having cytochrome P450 activity, wherein the sixth gene encoding the fifth enzyme having cytochrome P450 activity encodes a polypeptide comprising or consisting of an amino acid sequence having at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity to SEQ ID NO:6, or SEQ ID NO:6 (TwCYP82D217), or a mature polypeptide thereof; The seventh gene encoding a sixth enzyme having cytochrome P450 activity encodes a polypeptide comprising or consisting of an amino acid sequence having at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity to SEQ ID NO:7, or SEQ ID NO:7 (TwCYP82D275), or a mature polypeptide thereof. Surprisingly, it has been found that expressing the sixth and / or seventh genes encoding enzymes having cytochrome P450 activity results in significantly higher production of oxygenated diterpenoid compounds. Preferably, production is increased by at least 10%, preferably at least 20%, more preferably at least 50%, or more compared to production in a similar cell that does not contain the sixth and / or seventh genes encoding enzymes having cytochrome P450 activity.

[0058] The first, second, third, fourth, fifth, sixth, and seventh genes may be contained in one or more nucleic acid molecules, such as one or more heterologous nucleic acids. A heterologous nucleic acid encoding a first enzyme having cytochrome P450 activity may be referred to herein as a "first heterologous nucleic acid." A heterologous nucleic acid encoding a second enzyme having cytochrome P450 activity may be referred to herein as a "second heterologous nucleic acid." A heterologous nucleic acid encoding a third enzyme having cytochrome P450 activity may be referred to herein as a "third heterologous nucleic acid." A heterologous nucleic acid encoding a fourth enzyme having cytochrome P450 activity may be referred to herein as a "fourth heterologous nucleic acid." A heterologous nucleic acid encoding a fifth enzyme having cytochrome P450 activity may be referred to herein as a "fifth heterologous nucleic acid." A heterologous nucleic acid encoding a sixth enzyme having cytochrome P450 activity may be referred to herein as a "sixth heterologous nucleic acid." A heterologous nucleic acid encoding an enzyme having cytochrome B5 activity may be referred to herein as a "seventh heterologous nucleic acid." This does not imply that a recombinant host cell must contain a total of seven heterologous nucleic acids; in some embodiments, the cell contains only one or more of the first, second, third, fourth, fifth, sixth, and seventh heterologous nucleic acids.

[0059] The oxygenated diterpenoid compounds produced by the methods of the invention may be further modified by biosynthesis or chemical synthesis. In this context, biosynthesis is understood as a method in which the host cell containing the genes of the invention is further provided with one or more additional genes encoding additional enzymes capable of modifying the oxygenated diterpenoid compounds produced by the methods of the invention.

[0060] Chemical modification of the oxygenated diterpenoid compounds produced by the methods of the present invention may be performed directly on the culture broth prior to recovery of the oxygenated diterpenoid compounds, or it may be performed on the recovered oxygenated diterpenoid compounds.

[0061] The reduction of triptonide to triptolide can be achieved by organic synthesis. An example of such a synthesis is the reduction by nucleophilic attack by a hydride on a C-14 ketone. For this reason, sodium borohydride is a suitable agent to catalyze this reaction at neutral pH in a suitable solvent, such as water or MetOH.

[0062] In one preferred embodiment, triptonide produced by the methods of the present invention is converted to the compound triptolide, which is reported to be an immunosuppressant and is currently being investigated for use in cancer treatment. [ka]

[0063] host cell The host cell capable of producing miltiradiene and / or dehydroabietadienes can in principle be any such cell. The cell may be one that naturally produces miltiradiene and / or dehydroabietadienes, or it may be a cell that has been engineered to produce one or both of these compounds.

[0064] It is believed that miltiradiene may be naturally converted to dehydroabietadiene under at least some circumstances, and therefore the invention may be practiced using cells that produce miltiradiene that is naturally converted to dehydroabietadiene, or it may be practiced in cells that contain enzymes that facilitate the conversion of miltiradiene to dehydroabietadiene (see J. Zi, et al., Organic & Biomolecular Chemistry 2013, 11, 7650-7652).

[0065] The synthesis of miltiradienes generally begins with the formation of GGPP, which may be synthesized by the condensation of one dimethylallyl pyrophosphate (DMAP) molecule with three isopentenyl pyrophosphate (IPP) molecules and is typically catalyzed by geranylgeranyl diphosphate synthase, such as the SpGGPPs7 enzyme from Synechococcus sp., having the amino acid sequence set forth in SEQ ID NO:73 or SEQ ID NO:81.

[0066] GGPP is converted to mirtiradiene by the action of a diterpene synthase or by the combined action of two or more diterpene synthases, such as the combination of two diterpene synthases CfTPS1 and CfTPS3 from Plectranthus barbatus having the amino acid sequences of SEQ ID NOs: 67 and 68, or CftTPS1 represented by SEQ ID NOs: 77 and CftTPS3 represented by SEQ ID NOs: 78; the combination of two diterpene synthases TwTPS9 and TwTPS27 from T. wilfordii having the amino acid sequences of SEQ ID NOs: 69 and 70; or the combination of copalyl diphosphate synthase SmCPS from Salvia miltiorrhiza having the amino acid sequence of SEQ ID NO: 71 and mirtiradiene synthase SmKSL from Salvia miltiorrhiza having the amino acid sequence of SEQ ID NO: 72. [ka]

[0067] One preferred method for providing a host cell that produces miltiradiene and / or dehydroabietadiene is to select a host cell that produces GGPP and transform the cell with a diterpene synthase that catalyzes the transformation of GGPP to miltiradiene. Alternatively, a host cell that has been genetically engineered to produce GGPP may be used as a starting point.

[0068] Techniques for transforming host cells with diterpene synthases that catalyze the transformation of GGPP to miltiradiene are described in the prior art, for example, N.L. Hansen et al. (2017) The Plant Journal 2017, 89, 429-441 (incorporated herein by reference), P. Su et al. (2018) The Plant Journal 2018, 50-65, and J. Guo, et al., Proceedings of the National Academy of Sciences 2013, 110, 12108-12113, and the procedures and methods disclosed in these publications are also useful for providing host cells for use in the present invention.

[0069] The host cell may be a prokaryotic cell, such as a eubacterium or archaebacterium; or a eukaryotic cell, such as a plant cell, animal cell, insect cell, fungus cell or yeast cell.

[0070] Although virtually all eukaryotic cells produce GGPP due to their biosynthesis, in some embodiments, eukaryotic cells produce increased amounts of GGPP, which can increase the production of miltiradiene compared to similar eukaryotic organisms that do not produce increased amounts of GGPP. Methods for increasing GGPP in eukaryotic cells have also been described in the prior art.

[0071] The host cell may be a unicellular organism, or it may be contained within a multicellular organism, such as a plant. Examples of plants or plant cells suitable for use as host cells in accordance with the present invention include maize (Zea mays), rapeseed (Brassica napus, Brassica rapa subsp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cerer), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annuus), wheat (Triticum aestivum and other species), triticale, and rye (Secale Le), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea batatas), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), sis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifer indica), olive (Olea ouropaea), papaya (Carica papaya), cashew (Acer occidentale), macadamia (Macadamia intergrifolia), almond (Primus amygdalus), apple (Malus spp.), pear (Pyrus spp.), plum and sa These include crumbo trees (Prunus spp.), gooseberries (Cranthus et al.), grapes, Jerusalem artichokes (Cassius spp.), non-cereal grasses (Poaceae), sugar and fodder beets (Beta vulgaris), chicory, oats, barley, vegetable or ornamental plants, crop plants (e.g., cereals and pulses, maize, wheat, potato, tapioca, rice, sorghum, millet, cassava, barley, peas, sugar beet, sugarcane, soybean, rapeseed, sunflower and other root, tuber or seed crops).Other important plants may be fruit trees, crop trees, forest trees, or plants grown for use as spices or medicinal products (Mentha, Clove, Artemisia, Thymus, Lavendula, Allium, Hypericum, Catharanthus roseus, Vinca, Papaver spp., Digitalis, Ophiopogon, Vanilla, Petrusilium, Eucalyptus, Tea tree, Picea, Pinus, Abies, Juniperus). Horticultural plants that may be used in the present invention include lettuce, endive, and cruciferous vegetables including cabbage, broccoli, and cauliflower, carrots, and carnations and geraniums.

[0072] The plant may also be tobacco, cucurbit, carrot, strawberry, sunflower, tomato, pepper and chrysanthemum.

[0073] Further examples of plants include cereal plants, such as oilseed plants or legumes. Seeds of interest include cereal seeds such as corn, wheat, barley, sorghum, and rye. Oilseed plants include cotton, soybeans, safflowers, sunflowers, rapeseed, corn, alfalfa, palms, coconuts, and the like. Legumes include kidney beans and peas. Kidney beans include guar, locust beans, fenugreek seeds, soybeans, garden beans, cowpeas, mung beans, lima beans, fava beans, lentils, and chickpeas.

[0074] Particularly preferred plant species include Physcomitrella species such as Physcomitrella patens; Arabidopsis species such as Arabidopsis thaliana; Nicotiana species such as Nicotiana benthamiana; Chlamydomonas species such as Chlamydomonas reinhardtii; and Nannochloropsis species such as Nannochloropsis oceanica.

[0075] Examples of suitable eukaryotic cells for use with the present invention include fungal cells such as Agaricus, Aspergillus, Candida, Eremothecium, Fusarium / Gibberella, Kluyveromyces, Recipiporus, Lentinus, Phaffia, Phanerochaete, Pichia, Physcomitrella, Rhodotorula, Saccharomyces, Schizosaccharomyces, Sphaceloma, Xanthophyllomyces, or Yarrowia. Exemplary species from such genera include Lentinus tigrinus, Reciporus sulphureus, Phanerochaete chrysosporium, Pichia pastoris, Sibelindonella jadinii, Physcomitrella patens, Rhodotorula glutinus, Rhodotorula mukiraginosa, Phaffia rhodozyma, Xanthophyllomyces dendrorhous, Fusarium fujikuroi / Giberella fujikuroi, Candida utilis, Candida glabrata, Candida albicans, and Yarrowia lipolytica.

[0076] In some embodiments, the host cell can be an ascomycete such as Gibberella fujikuroi, Kluyveromyces lactis, Schizosaccharomyces pombe, Aspergillus niger, Yarrowia lipolytica, Ashbya gossypii, or S. cerevisiae.

[0077] In some embodiments, the host cell can be an algal cell such as Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp., Undaria pinnatifida, Sargassum, Laminaria japonica, Scenedesmus armeriensis, and the like.

[0078] In some embodiments, the host cell can be a prokaryote, such as a Bacillus cell, e.g., Bacillus subtilis; an Escherichia cell, e.g., an Escherichia coli cell; a Lactobacillus cell; a Lactococcus cell; a Streptomyces cell, a Streptococcus cell, a Corynebacterium cell; an Acetobacter cell; an Acinetobacter cell; or a Pseudomonas cell.

[0079] In some embodiments, the host cell can be a cyanobacterial cell, such as a Synechocystis or Synechococcus cell.

[0080] In one embodiment, host cells are selected that are suitable for growth in fermentors. Growing recombinant host cells according to the present invention is a convenient method of growing host cells for the production of oxygenated diterpenoid compounds of the present invention.

[0081] In another embodiment, the host cells are phototropic cells and the cells are cultured in a greenhouse or photobioreactor.

[0082] Genes and Enzymes The recombinant host cells of the invention are capable of producing miltiradiene and / or dehydroabietadiene. Miltiradiene may be converted to dehydroabietadiene naturally or it may be converted by an enzyme that facilitates the conversion of miltiradiene to dehydroabietadiene.

[0083] As described earlier herein, the synthesis of miltiradienes typically begins with the condensation of one dimethylallyl pyrophosphate (DMAP) molecule with three isopentenyl pyrophosphate (IPP) molecules by geranylgeranyl diphosphate synthase to form GGPP.

[0084] Recombinant host cells and heterologous nucleic acids encoding enzymes that catalyze the synthesis of GGPP in recombinant host cells are generally known in the art; see, e.g., WO 2015 / 113570. In addition, many host organisms can naturally produce GGPP, and heterologous nucleic acids may therefore not be necessary for the production of GGPP.

[0085] In some embodiments, the recombinant host cell comprises a heterologous nucleic acid encoding a geranylgeranyl diphosphate synthase such as the geranylgeranyl diphosphate synthase SpGGPPs7 represented by SEQ ID NO:73 or SEQ ID NO:81, or a functional homolog thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto, or a mature polypeptide thereof.

[0086] GGPP may then be converted to miltiradiene by the action of one or more diterpene synthases, copalyl diphosphate synthase and / or miltiradiene synthase.

[0087] In some embodiments, the recombinant host cell comprises one or more heterologous nucleic acids encoding one or more diterpene synthases, such as the diterpene synthases CfTPS1 (SEQ ID NO:67) and CfTPS3 (SEQ ID NO:68), or CftTPS1 (SEQ ID NO:77) and CftTPS3 (SEQ ID NO:78), or respective functional homologs having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto, or a mature polypeptide thereof.

[0088] In some embodiments, the recombinant host cell comprises one or more heterologous nucleic acids encoding one or more diterpene synthases, such as diterpene synthases TwTPS9 (SEQ ID NO:69) and TwTPS27 (SEQ ID NO:70), or respective functional homologs thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto, or a mature polypeptide thereof.

[0089] In some embodiments, the recombinant host cell comprises a combination of one or more copalyl diphosphate synthases and one or more miltiradiene synthases, such as a combination of copalyl diphosphate synthase SmCPS (SEQ ID NO:71) and miltiradiene synthase SmKSL (SEQ ID NO:72), or respective functional homologs thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto, or mature polypeptides thereof.

[0090] In a further aspect, the present invention relates to a polypeptide having the enzymatic activity of a cytochrome P450 and comprising an amino acid sequence having at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, preferably at least 98% sequence identity or 100% sequence identity to one of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, or a mature polypeptide thereof.

[0091] In a further aspect, the present invention relates to a polypeptide having cytochrome B5 activity and comprising an amino acid sequence having at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, preferably at least 98% sequence identity or 100% sequence identity to SEQ ID NO:8, or a mature polypeptide thereof.

[0092] The present invention also relates to a polynucleotide sequence or gene encoding a polypeptide having cytochrome P450 enzymatic activity and comprising an amino acid sequence having at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, preferably at least 98% sequence identity or 100% sequence identity to one of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, or a mature polypeptide thereof, or a polypeptide having cytochrome B5 activity and comprising an amino acid sequence having at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, preferably at least 98% sequence identity or 100% sequence identity to SEQ ID NO:8, or a mature polypeptide thereof.

[0093] In a preferred embodiment, one or more of the first, second, third, fourth, fifth and sixth enzymes having cytochrome P450 activity are selected from the group consisting of SEQ ID NO:1 (TwCYP82D274v1), SEQ ID NO:2 (TwCYP82D274v2), SEQ ID NO:74 (TwCYP82D274v3), SEQ ID NO:75 (TwCYP82D274v4), SEQ ID NO:3 (TwCYP71BE85), SEQ ID NO:4 (TwCYP71BE86), SEQ ID NO:5 (TwCYP82D213v1) and SEQ ID NO:6 (TwCYP82D213v2). It comprises or consists of an amino acid sequence according to any one of NO:76 (TwCYP82D213v2), or each functional homolog thereof having at least 80% sequence identity thereto, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, preferably at least 98% sequence identity, preferably at least 98% sequence identity, or a mature polypeptide thereof.

[0094] In some embodiments, the first heterologous nucleic acid encoding a first enzyme having cytochrome P450 activity is selected from the group consisting of SEQ ID NO:1 (TwCYP82D274v1), SEQ ID NO:2 (TwCYP82D274v2), SEQ ID NO:74 (TwCYP82D274v3), SEQ ID NO: Encodes TwCYP82D274, represented by NO:75 (TwCYP82D274v4), or a functional homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto.

[0095] In some embodiments, the second heterologous nucleic acid encoding a second enzyme having cytochrome P450 activity encodes the cytochrome P450 enzyme TwCYP71BE86 set forth in SEQ ID NO:4, or a functional homolog having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto.

[0096] In some embodiments, the third heterologous nucleic acid encoding a third enzyme having cytochrome P450 activity encodes the cytochrome P450 enzyme TwCYP71BE85 set forth in SEQ ID NO:3, or a functional homolog having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto.

[0097] In some embodiments, the fourth heterologous nucleic acid encoding a fourth enzyme having cytochrome P450 activity encodes the cytochrome P450 enzyme TwCYP82D213v1 represented by SEQ ID NO:5 or TwCYP82D213v2 represented by SEQ ID NO:76 (TwCYP82D213v2), or a functional homolog having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto.

[0098] In some embodiments, the fifth heterologous nucleic acid encoding a fifth enzyme having cytochrome P450 activity encodes the cytochrome P450 enzyme TwCYP82D217 set forth in SEQ ID NO:6, or a functional homolog having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto.

[0099] In some embodiments, the sixth heterologous nucleic acid encoding a sixth enzyme having cytochrome P450 activity encodes the cytochrome P450 enzyme TwCYP82D275 set forth in SEQ ID NO:7, or a functional homolog having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto.

[0100] In some embodiments, the seventh heterologous nucleic acid encoding an enzyme having cytochrome B5 activity encodes the cytochrome B5 enzyme TwB5#1 set forth in SEQ ID NO:8, or a functional homolog having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto.

[0101] In some embodiments, a recombinant host cell is provided, i. wherein the host cell is capable of producing miltiradiene and / or dehydroabietadiene; ii. comprises a heterologous nucleic acid encoding TwCYP82D274 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:74 or SEQ ID NO:75, or a functional homolog having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto; The cells are now capable of producing 14-hydroxydehydroabietadiene.

[0102] In some embodiments, a recombinant host cell is provided, i. wherein the host cell is capable of producing miltiradiene and / or dehydroabietadiene; ii. comprises a heterologous nucleic acid encoding TwCYP82D274 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:74 or SEQ ID NO:75, or a functional homolog having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto; iii. TwCYP71BE86 of SEQ ID NO:4, or a functional homologue having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto, or a heterologous nucleic acid encoding a mature polypeptide thereof; wherein the cells are capable of producing 14-hydroxydehydroabietadiene, 3,14-dihydroxydehydroabietadiene, 3,14-dihydroxyabeodyene and / or 14-hydroxy-18-aldo-abeodyene.

[0103] In some embodiments, a recombinant host cell is provided, i. wherein the host cell is capable of producing miltiradiene and / or dehydroabietadiene; ii. comprises a heterologous nucleic acid encoding TwCYP82D274 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:74 or SEQ ID NO:75, or a functional homolog having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto; iii. TwCYP71BE86 of SEQ ID NO:4, or a functional homologue having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto, or a heterologous nucleic acid encoding a mature polypeptide thereof; iv. TwCYP71BE85 of SEQ ID NO:3, or a functional homologue having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto, or a heterologous nucleic acid encoding a mature polypeptide thereof; wherein the cells are capable of producing 14-hydroxydehydroabietadiene, 3,14-dihydroxydehydroabietadiene, 3,14-dihydroxyabeodyene, 14-hydroxy-18-aldo-abeodyene and / or tryptophenolide.

[0104] In some embodiments, a recombinant host cell is provided, i. wherein the host cell is capable of producing miltiradiene and / or dehydroabietadiene; ii. comprises a heterologous nucleic acid encoding TwCYP82D274 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:74 or SEQ ID NO:75, or a functional homolog having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto; iii. TwCYP71BE86 of SEQ ID NO:4, or a functional homologue having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto, or a heterologous nucleic acid encoding a mature polypeptide thereof; iv. TwCYP71BE85 of SEQ ID NO:3, or a functional homologue having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto, or a heterologous nucleic acid encoding a mature polypeptide thereof; V. TwCYP82D213 of SEQ ID NO:75 or SEQ ID NO:76, or a functional homologue having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto, or a heterologous nucleic acid encoding a mature polypeptide thereof; wherein the cells are capable of producing 14-hydroxydehydroabietadiene, 3,14-dihydroxydehydroabietadiene, 3,14-dihydroxyabeodyene, 14-hydroxy-18-aldo-abeodyene, tryptophenolide and / or triptonide.

[0105] In some embodiments, a recombinant host cell is provided, i. wherein the host cell is capable of producing miltiradiene and / or dehydroabietadiene; ii. comprises a heterologous nucleic acid encoding TwCYP82D274 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:74 or SEQ ID NO:75, or a functional homolog having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto; iii. TwCYP71BE86 of SEQ ID NO:4, or a functional homologue having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto, or a heterologous nucleic acid encoding a mature polypeptide thereof; iv. TwCYP71BE85 of SEQ ID NO:3, or a functional homologue having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto, or a heterologous nucleic acid encoding a mature polypeptide thereof; V. TwCYP82D213 of SEQ ID NO:75 or SEQ ID NO:76, or a functional homologue having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto, or a heterologous nucleic acid encoding a mature polypeptide thereof; vi. TwB5#1 of SEQ ID NO:8, or a functional homologue having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto, or a heterologous nucleic acid encoding a mature polypeptide thereof; Here, the cells are capable of producing tryptonides with a titer that is at least twice, at least three times, at least four times, at least five times, etc., that of identical yeast cells, except that the yeast does not express TwB5#1 or its functional homologue.

[0106] In some embodiments, a recombinant host cell is provided, i. wherein the host cell is capable of producing miltiradiene and / or dehydroabietadiene; ii. comprises a heterologous nucleic acid encoding TwCYP82D274 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:74 or SEQ ID NO:75, or a functional homolog having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto; iii. TwCYP71BE86 of SEQ ID NO:4, or a functional homologue having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto, or a heterologous nucleic acid encoding a mature polypeptide thereof; iv. TwCYP71BE85 of SEQ ID NO:3, or a functional homologue having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto, or a heterologous nucleic acid encoding a mature polypeptide thereof; V. TwCYP82D213 of SEQ ID NO:75 or SEQ ID NO:76, or a functional homologue having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto, or a heterologous nucleic acid encoding a mature polypeptide thereof; vi. TwB5#1 of SEQ ID NO:8, or a functional homologue having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or at least 99% sequence identity thereto, or a heterologous nucleic acid encoding a mature polypeptide thereof; wherein the cells are grown in a fermentation medium, and after 7 days of fermentation, the fermentation medium contains: - at least 3 ppm triptonide and / or - At least 1 ppm of tryptophenolide Includes:

[0107] The recombinant host cells described above may be capable of producing miltiradiene and / or dehydroabietadienes for a number of different reasons. For example, the host cells may be capable of endogenously producing miltiradienes. Alternatively, the recombinant host cell may comprise one or more heterologous nucleic acid sequences encoding one or more enzymes involved in the production of miltiradiene, such as the diterpene biosynthetic enzymes SPGGPPS7 of SEQ ID NO:73 or SEQ ID NO:81, CfTPS1 of SEQ ID NO:67, CftTPS1 of SEQ ID NO:77, CfTPS3 of SEQ ID NO:68, CftTPS3 of SEQ ID NO:78 and / or TwCPR1 of SEQ ID NO:9, or their respective functional homologs having at least 80% sequence identity thereto, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, preferably at least 98% sequence identity, preferably at least 98% sequence identity, or mature polypeptides thereof.

[0108] Functional homologs of the first (e.g., TwCYP82D274), second (e.g., TwCYP71BE86), third (e.g., TwCYP71BE85), fourth (e.g., TwCYP82D213), fifth (e.g., TwCYP82D217), and sixth (e.g., TwCYP82D275) enzymes with cytochrome P450 activity and enzymes with cytochrome B5 activity (e.g., TwB5#1) may be verified by expressing the relevant proteins in yeast cells and assessing whether they can produce the specific compounds described herein below.

[0109] expressing a functional homolog of TwCYP82D274 (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:74 or SEQ ID NO:75); and i. diterpene biosynthetic enzymes SPGGPPS7v2 (SEQ ID NO: 81), CftTPS1 (SEQ ID NO: 77), CftTPS3 (SEQ ID NO: 78) and TwCPR1 (SEQ ID NO: 9); The yeast cell further expressing the 14-hydroxydehydroabietadiene is preferably capable of producing 14-hydroxydehydroabietadiene.

[0110] expressing a functional homolog of TwCYP71BE86 (SEQ ID NO:4); and i. the diterpene biosynthetic enzymes SPGGPPS7v2 (SEQ ID NO: 81), CftTPS1 (SEQ ID NO: 77), CftTPS3 (SEQ ID NO: 78), and TwCPR1 (SEQ ID NO: 9); and ii. TwCYP82D274 (SEQ ID NO: 1 or SEQ ID NO: 2), The yeast cell further expressing is preferably capable of producing 14-hydroxydehydroabietadiene, 3,14-dihydroxydehydroabietadiene, 3,14-dihydroxyabeodyene and 14-hydroxy-18-aldo-abeodyene.

[0111] expressing a functional homolog of TwCYP71BE85 (SEQ ID NO:3); and i. diterpene biosynthetic enzymes SPGGPPS7v2 (SEQ ID NO: 81), CftTPS1 (SEQ ID NO: 77), CftTPS3 (SEQ ID NO: 78) and TwCPR1 (SEQ ID NO: 9); ii. TwCYP82D274 (SEQ ID NO: 1 or SEQ ID NO: 2); and iii.TwCYP71BE86(SEQ ID NO:4), Yeast cells further expressing the 14-hydroxydehydroabietadiene, 3,14-dihydroxydehydroabietadiene, 3,14-dihydroxyabeodyene, 14-hydroxy-18-aldo-abeodyene, and tryptophenolide are preferably capable of producing 14-hydroxydehydroabietadiene, 3,14-dihydroxydehydroabietadiene, 3,14-dihydroxyabeodyene, 14-hydroxy-18-aldo-abeodyene, and tryptophenolide.

[0112] expressing a functional homolog of TwCYP82D213 (SEQ ID NO:5 or SEQ ID NO:76); and i. diterpene biosynthetic enzymes SPGGPPS7v2 (SEQ ID NO: 81), CftTPS1 (SEQ ID NO: 77), CftTPS3 (SEQ ID NO: 78) and TwCPR1 (SEQ ID NO: 9); ii. TwCYP82D274 (SEQ ID NO: 1 or SEQ ID NO: 2); iii. TwCYP71BE86 (SEQ ID NO: 4); and iv.TwCYP71BE85(SEQ ID NO:3), The yeast cell further expressing preferably is capable of producing 14-hydroxydehydroabietadiene, 3,14-dihydroxydehydroabietadiene, 3,14-dihydroxyabeodyene, 14-hydroxy-18-aldo-abeodyene, tryptophenolide and triptonide.

[0113] expressing a functional homolog of TwB5#1 (SEQ ID NO:8); and i. diterpene biosynthetic enzymes SPGGPPS7v2 (SEQ ID NO: 81), CftTPS1 (SEQ ID NO: 77), CftTPS3 (SEQ ID NO: 78) and TwCPR1 (SEQ ID NO: 9); ii. TwCYP82D274 (SEQ ID NO: 1 or SEQ ID NO: 2); iii.TwCYP71BE86 (SEQ ID NO: 4); iv. TwCYP71BE85 (SEQ ID NO: 3); and v.TwCYP82D213 (SEQ ID NO: 5 or SEQ ID NO: 76), Preferably, the yeast cell further expressing TwB5#1 is capable of producing tryptonides with a titer that is at least 2-fold, such as at least 3-fold, such as at least 4-fold, such as at least 5-fold higher than the same yeast cell, except that the yeast does not express the functional homolog of TwB5#1.

[0114] In a preferred embodiment, the enzyme having cytochrome B5 activity comprises or consists of an amino acid sequence according to SEQ ID NO:8 (TwB5#1), or a functional homologue thereof having at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, preferably at least 98% sequence identity, preferably at least 98% sequence identity, or a mature polypeptide thereof.

[0115] The polynucleotide of the present invention may be provided by cloning from an organism that naturally produces the polypeptide, such as the plant T. wilfordii or a closely related plant, or it may be produced by chemical synthesis of a polynucleotide sequence according to techniques known in the art. The polynucleotide may have a sequence identical to a sequence found in nature, or it may have a sequence that is not found in nature, for example, the sequence may be codon-optimized for a particular selected host cell.

[0116] The polypeptides of the present invention may be provided from organisms that naturally produce the polypeptides, such as the plant T. wilfordii or related organisms, or they may be provided by inserting and expressing a polynucleotide encoding the polypeptide into a suitable host cell and recovering the polypeptide from a culture broth containing host cells transformed with the respective gene. It is preferred to provide the polypeptides of the present invention from an appropriate selected recombinant host cell.

[0117] To transform and express a gene in a suitable host cell, the gene is usually operably linked to suitable regulatory elements and inserted into an expression vector that is appropriate for the specific selected host cell. Selecting appropriate regulatory elements, constructing a suitable expression vector, and transforming the selected host cell is within the skill of the average practitioner, and the present invention is not limited to any particular choice of such elements.

[0118] The engineered host cells containing the genes of the invention are suitably grown in containers, such as fermentors or shake flasks, under conditions whereby the genes are expressed and oxygenated diterpenoid compounds are formed. Once growth has ceased, or a sufficiently large amount of oxygenated diterpenoid compound has accumulated in the culture broth, the oxygenated diterpenoid compound may be further modified and recovered from the culture broth.

[0119] Sequence identity is understood as a measure of similarity between two amino acid or nucleotide sequences. Sequence identity is calculated by first aligning the two sequences, counting the number of positions where the two sequences contain the same amino acid residue or nucleotide, and calculating the percentage identity as the number of positions with the same amino acid residue or nucleotide over the entire length of the alignment.

[0120] Several algorithms have been developed and are available to those skilled in the art.

[0121] In this specification and claims, sequence identity for amino acid sequences is calculated using the NCBI BLAST+ pairwise alignment algorithm with default parameters (BLOSUM 62 matrix, gap open penalty 11; gap extend penalty 1, exp. Thr 10), and sequence identity for nucleotide sequences is calculated using the NCBI BLAST+ pairwise alignment algorithm with default parameters (match / mismatch score 1, -3; gap open penalty 5; gap extend penalty 2; exp. Thr 10). The NCBI BLAST+ program is further described in Madeira F el at (2019) NAR 47: W636-W641.

[0122] Example Materials and Methods Genetic manipulation of Nicotiana benthamiana Tripterygium wilfordii CYP genes were cloned from plant material using constructs and methods previously described (1-4) and coexpressed in Nicotiana benthamiana with the diterpene biosynthetic genes CfDXS (SEQ ID NO:79) or SctHMGR (SEQ ID NO:80), CfGGPPS or SpGGPPS7 (SEQ ID NO:81), CfTPS1 (SEQ ID NO:67) or CftTPS1 (SEQ ID NO:77), and CfTPS3 (SEQ ID NO:68) or CftTPS3 (SEQ ID NO:78). The gene silencing suppressor p19 was also coexpressed. Briefly, binary vectors, each containing an individual diterpene biosynthetic gene or a Tripterygium wilfordii CYP (TwCYP), were transformed into Agrobacterium. Liquid cultures of Agrobacterium, each containing a specific plasmid, were mixed for co-expression of specific combinations of TwCYPs.

[0123] Genetic manipulation of Saccharomyces cerevisiae and growth conditions for engineered S. cerevisiae Culture medium YPD medium: 20 g / L Bacto™ peptone, 10 g / L Bacto™ yeast extract, 20 g / L glucose. Uracil-free synthetic complete (SC) medium: 1.92 g / L uracil-free yeast synthetic dropout medium supplement (Sigma-Aldrich Co. LLC, catalog number Y1501), 6.7 g / L amino acid-free nitrogen base for yeast culture (Sigma-Aldrich Co. LLC, catalog number Y0626), 20 g / L glucose. Feed-in-time (FIT) plates were prepared using the EnPump200 (Enpresso GmbH) according to the enclosed protocol. Agar plates: SC medium containing agar (15 g / L).

[0124] Uracil auxotrophy in the parent strain was introduced by selecting for the absence of URA3 function on agar plates of uracil-free SC medium containing 5-fluoroorotic acid (5-FOA, 0.74 g / L) and uracil (30 mg / L).

[0125] Yeast transformants were isolated on uracil-free SC agar plates.

[0126] Fed-batch fermentation of engineered S. cerevisiae strains for the isolation of miltiradiene-derived diterpenoids All engineered S. cerevisiae strains were cultured in 96-deep-well plates using a feed-in-time (FIT; m2p-labs) approach similar to that previously described (see insert reference Forman et al., 2018). For isolation and purification of key intermediates in the tryptonide pathway, selected engineered S. cerevisiae strains were cultured in fed-batch fermenters using 2L Biostat® A bioreactors (Sartorius AG). Fed-batch fermentation was initiated by adding 100 mL of starter culture to a pre-autoclaved reactor tank (with impeller) containing 200 mL of batch glucose and 300 mL of batch salt mix. 5 mL of vitamin mix, 5 mL of microelements, and 0.5 mL of trace elements were also added. Bioreactor cultivation was initiated under the following conditions (monitored and automatically controlled): pH = 5, temperature = 30 °C, and dissolved oxygen (DO) = 20%. The pH was controlled by feeding ammonium hydroxide (32%) and sulfuric acid (10%), while the dissolved oxygen was controlled by aeration with stirring. The foaming level was also adjusted by adding antifoam emulsion (35119, Serva Electrophoresis GmbH). After 18 hours of initial cultivation in the bioreactor, feeding of the feed solution at a rate of 1.3% was started. The fermentation process continued for 7 days, and the culture was harvested daily.

[0127] Extraction of engineered S. cerevisiae for LC-MS analysis The engineered S. cerevisiae strain was transferred into 0.5 mL of medium in a 96-well plate and grown at 30°C for 3 days with orbital shaking at 350 rpm. For extraction, 0.1 mL of S. cerevisiae culture was transferred to a 1.5 mL glass vial. 0.4 mL of uHPLC-grade MeOH was added. The S. cerevisiae extract was filtered using a 0.22 μm 96-well filter plate (Merck Millipore, Darmstadt, Germany) and stored at 4°C before LC-MS analysis.

[0128] Extraction of diterpenoid metabolites for LC-MS analysis Yeast culture samples for LCMS analysis were prepared in 1.5 mL glass vials by mixing the yeast culture with methanol supplemented with 5 ppm andrographolide at a 1:19 ratio (v / v) for daily bioreactor samples and a 1:4 ratio (v / v) for 96-deep-well cultures. Mixing proceeded for 30 minutes with shaking at room temperature. For tobacco samples, two leaf disks (diameter = 3 cm) placed in 1.5 mL glass vials were extracted with 1 mL of methanol extraction solution at room temperature with shaking for 1 hour. Prior to LCMS analysis, samples were passed through a 0.22 μM 96-well plate filter (Merck Millipore, Darmstadt, Germany) and stored at 5°C.

[0129] LC-MS analysis: The methanol (MeOH) extract was analyzed using an Ultimate 3000 UHPLC+ Focused system (Dionex Corporation, Sunnyvale, CA) coupled to a Bruker Compact ESI-QTOF-MS (Bruker) system. Samples were separated on a Kinetex XB-C18 column (100 × 2.1 mm ID, 1:7 μm particle size, 100 Å pore size; Phenomenex Inc., Torrance, CA) maintained at 40 °C with a flow rate of 0.3 mL / min and a mobile phase consisting of 0.05% (v / v) formic acid in water (solvent A) and 0.05% (v / v) formic acid in acetonitrile (solvent B).

[0130] Two LC protocols were used: LC method 1: 0–0.5 min, 10% B; 0.5–21 min, linear increase from 10% B to 80% B; 21–31 min, to 90% B; 31–34 min, to 100% B; 34–39 min, 100% B; 39–40 min, linear decrease from 100% B to 10% B.

[0131] LC method 2: 0–0.5 min, 20% B; 0.5–11 min, linear increase from 20% B to 80% B; 11–20 min, to 90% B; 20–22 min, to 100% B; 22–27 min, 100% B; 27–28 min, linear decrease from 100% B to 20% B.

[0132] LC method 3: 0–0.5 min, 20% B; 0.5–9 min, linear increase from 20% B to 100% B; 9–11 min, 100% B; 11–11.5 min, linear decrease from 100% B to 20% B; 11.5–15 min, 20% B.

[0133] Extraction of diterpenoid metabolites for GC-MS analysis Yeast culture samples for GCMS analysis were prepared in 1.5 mL glass vials by mixing yeast culture with pure methanol in a 1:4 ratio (v / v). After brief mixing, nonpolar components were liquid-liquid extracted into hexane, to which 10 ppm of 1-eicosene was added, by mixing in a 1:1 ratio (v / v) and shaking for 1 h. For tobacco samples, two leaf disks (diameter = 3 cm) placed in a 1.5 mL glass vial were extracted with 1 mL of the same hexane solution by shaking for 1 h. Prior to GCMS analysis, the hexane layer was transferred to a new vial.

[0134] Gas chromatography-mass spectrometry (GC-MS) analysis GC-MS analysis was performed on a Shimadzu GCMS-QP2010 Ultra (Shimadzu Corp.) with an Agilent HP-5MS column (Agilent Technologies) 20 m × 0.18 mm i.d., 0.18 μm film thickness. Hydrogen was used as the carrier gas at a constant linear velocity of 50 cm / s, and the injection volume was 1 μL at 250 °C (splitless mode). The oven program was 80 °C for 2 min, ramped to 180 °C at 20 °C / min, ramped to 300 °C at 10 °C / min, ramped to 310 °C at 20 °C / min, and held for 3 min. Data were saved in CDF format and processed using MZmine2.

[0135] Relative quantification of miltiradiene-derived diterpenoids Relative compound amounts in yeast cultures were based on normalized peak areas of characteristic ions (data obtained using targeted feature detection with MZmine2 software). The following ion signals were quantified: 1: miltiradiene m / z 91.1, 2: 14-hydroxyabietadiene m / z 189.1, 3: F15P1 m / z 303.2318, 4: F20P2 m / z 283.2059, 5: F15P2 m / z 299.2002, 6: tryptophenolide m / z 313.1794, and 8: triptonide m / z 359.1481. Mass deviations of 5 ppm and 100 ppm were allowed for LCMS and GCMS data, respectively.

[0136] The peak area of ​​the base peak ion of the internal standard andrographolide (m / z 315.1947) was used for normalization.

[0137] Absolute quantification Absolute quantification of tryptophenolide (FT65732, CarboSynth) and triptonide (FT65197, CarboSynth) was performed by simultaneous analysis of authentic standards prepared in methanol and an internal standard (andrographolide) at a final concentration of 5 ppm. Quantitation was based on normalized peak areas and calculated from the slope of linear extrapolation of the standard response curves (tryptophenolide at 0.05, 0.5, 1, and 2 ppm; triptonide at 0.5, 1, 2, 10, and 20 ppm).

[0138] Isolation and purification of miltiradiene-derived compounds from engineered S. cerevisiae strains for NMR analysis The compounds of the present invention were isolated from bioreactor-grown yeast strains NVJ8.15 and NVJ3.10 and structurally elucidated by NMR. The combined ethyl acetate extracts of broth and methanol-lysed cells (cells:methanol = 1:4 v / v) were first dried via rotary evaporation in the presence of Celite S® (06858, Sigma-Aldrich). The compounds were then isolated by sequential fractionation using a puriFlash® 5.250 (Interchim, Montluçon, France) system with UV absorption and evaporative light scattering detection (ELSD). This was equipped with either a (C1) PF-15SIHP-F0025 (OV002A, Interchim) or a (C2) US5C18HQ-100 / 300 (SSP750, Interchim) column for normal-phase and reverse-phase separation, respectively.

[0139] Initial pre-fractionation of the Celite S® / crude extract dry mix was achieved using a column loaded from a manually packed dry-loading column (Reference 9). Separation was achieved using mobile phases hexane (A) and ethyl acetate (B) at a constant flow rate of 15 mL / min, followed by a final wash step with 100% methanol. The target compounds were detected by UV and ELSD and collected. Collected fractions were subsequently evaluated by LCMS and TLC analysis using LC-MS Method 3 prior to further fractionation or NMR testing. Further purification of the target compounds from fractions containing multiple compounds was performed by additional normal-phase fractionation using C1 or reverse-phase column fractionation using C2.

[0140] For reverse-phase purification on C2, the sample was evaporated using rotary evaporation and resuspended in 2 mL of methanol. The sample was injected directly onto the pre-conditioned column C2. The mobile phase for C2 consisted of solvent C: deionized water and solvent D: acetonitrile, each acidified with 0.05% (v / v) formic acid. A constant flow rate of 32 mL / min was used with a linear gradient of increasing concentrations of solvent D. The target compounds were detected and collected by ELSD and UV.

[0141] Further reverse-phase purification was performed by multiple injections of 100 μL onto a semi-preparative Phenomenex Luna 5 μm C18(2) 100 Å 250 x 10 mm (fully porous) (Phenomenex, Inc., Torrance, CA, USA) column on a Shimadzu HPLC (SPD-M20A diode array detector, FRC-10A fraction collector, DGU-20A5 degasser, LC-20AT pump, CBM-20A system controller, CTO-10AS VP column oven, SIL-10AP autosampler). The mobile phase was a linear gradient of C and D, increasing the amount of D from 50% to 100%. The target compounds were detected by UV absorption at 210 nm and collected.

[0142] Mass spectrum Mass spectra were acquired in positive ion mode with a scan range of m / z 50-1200 with the following ESI and MS settings: capillary voltage, 4000 V; end plate offset, 500 V; dry gas temperature, 220 °C; dry gas flow rate, 8 L / min; nebulizer pressure, 2 × 10 5Pa (2 bar); in-source CID energy, 0 eV; hexapole RF, 50 Vpp; quadrupole ion energy, 4 eV; collision cell energy, 7 eV. Raw chromatogram data were calibrated using sodium formate internal standard and then exported as mzML format using Bruker's DataAnalysis 4.3 (Build 110.102.1532) (64-bit). MZmine version 2.53 was used to visualize LC-MS chromatograms.

[0143] Media recipes for bioreactor starter and feed media Batch glucose: Glucose monohydrate 55g / L

[0144] Batch Salt Mix: Ammonium sulfate 25g / L Monobasic potassium phosphate 5g / L Magnesium sulfate pentahydrate 1.7g / L

[0145] Feed glucose: Glucose monohydrate 880g / L

[0146] Feed Salt Mix: Monobasic potassium phosphate 21.6g / L Magnesium sulfate pentahydrate 24.24g / L Potassium sulfate 8.4g / L Sodium sulfate 0.672g / L

[0147] Preparation notes: Batch and feed salt mix and batch and feed glucose were prepared in separate BlueCap bottles by dissolving the ingredients in Milli-Q water and sterilizing by autoclaving.

[0148] Feeding solution The feeding solution was made by mixing 500 mL of feed glucose with 500 mL of feed salt mix, 10 mL of vitamin mix, 10 mL of microelements and 1 mL of trace elements.

[0149] Example 1: Expression in Nicotiana benthamiana Leaf material from N. benthamiana plants co-expressing specific combinations of genes of interest (GOIs) was harvested 7 days after Agrobacterium infiltration. 1 mL of methanol (MeOH) was added to two leaf disks (diameter = 2 cm). Extraction was performed at room temperature under orbital shaking at 200 rpm. 200 μL of extract was filtered using a 0.22 μm 96-well filter plate (Merck Millipore, Darmstadt, Germany) and stored at 4 °C prior to LC-MS analysis. Figure 1 shows the resulting LC-MS profile. The results show that N. benthamiana cells transformed with CYP82D274V1, which encodes the enzyme having SEQ ID NO:1, result in the production of 14-OH-dehydroabietadiene; when the cells are further transformed with CYP71BE85 and CYP71BE86, which encode the enzymes having the amino acid sequences SEQ ID NO:3 and SEQ ID NO:4, respectively, tryptophenolide is formed; and when the cells are further transformed with CYP82D213, which encodes the enzyme having the amino acid sequence of SEQ ID NO:5, tryptonide is formed. Furthermore, it can be seen that the enzyme having the sequence of SEQ ID NO:6 and encoded by the gene CYP82D217 increases the production of tryptophenolide and tryptonide.

[0150] Example 2: Construction of S. cerevisiae strains Strain construction The parent yeast strain was S. cerevisiae S288C (NCYC 3608; National Collection of Yeast Cultures, Norwich, UK).

[0151] The genotypes and sources of the strains are listed in Table 3.

[0152] The constructed yeast strains were made using the lithium acetate transformation method (8). The parent strain lacking a functional URA3 gene was made competent by the following procedure: inoculating 5 ml of YPD medium from a glycerol stock and growing overnight at 30°C. Then, 3 ml of the overnight culture was transferred to 50 ml of YPD medium and continued to grow for 4–5 h, followed by centrifugation at 4000 RPM for 10 min and discarding the supernatant. The cells were then ready for transformation after two washes with sterile water (first with 25 ml and then with 1 ml) and resuspension in 0.4 ml of sterile water.

[0153] Transformation of competent yeast cells was carried out as follows: a mixture of the designated NotI-digested plasmids (2 μL each) was added to 10 μL of competent yeast cells and mixed with 60 μL of PEG 3350 (50% w / v), 9 μL of LiAc (1 M), and 12.5 μL of boiled salmon sperm DNA (10 mg / ml). The resulting mixture was then incubated at 42°C for 40 minutes, after which the cells were harvested by centrifugation (3000 RPM for 5 minutes) and removal of the supernatant. The cells were then resuspended in 100 μL of sterile water and plated on uracil-free SC agar plates. Isolated transformants appeared as single colonies after 2 days of incubation at 30°C. Insertion of the gene constructs was confirmed by colony PCR using primers specific to the genes and constructs found in Table 1. For colony PCR, yeast colonies were resuspended in 50 μL of 20 mM NaOH and incubated for 15 minutes at 99° C. 1 μL of the colony suspension was used for PCR. [Table 1] TIFF0007804353000007.tif242160TIFF0007804353000008.tif109162

[0154] Assembly of gene constructs for S. cerevisiae genome engineering Plasmid names and encoded gene constructs are listed in Table 2. All plasmids were generated by USER cloning as previously described (5). Parent vectors designated Assembler-1, -2, and -3, for simultaneous genomic integration of up to six gene constructs and containing an AsiSI / Nb.BsmI USER cassette, were also prepared for USER cloning as previously described (6). Primers used for PCR amplification with USER-compatible PfuX7 polymerase (7) are listed in Table 1. Vectors used and generated in this work are listed in Table 3.

[0155] Codon-optimized genes for S. cerevisiae were obtained from TWIST Biosciences, San Francisco, USA. All genes designated with the prefix "CO_" in the table below were codon-optimized. The codon-optimized genes were amplified using primers identical to those listed in Table 1 above, except that the primers were modified to accommodate hybridization to any nucleotide change in the codon-optimized genes. Primers for amplification of codon-optimized genes are also disclosed in J. Andersen-Ranberg et al., Expanding the Landscape of Diterpene Structural Diversity through Stereochemically Controlled Combinatorial Biosynthesis. Angewandte Chemie International Edition, n / a (2016). [Table 2] JPEG0007804353000010.jpg175162 [Table 3] TIFF0007804353000012.tif242160TIFF0007804353000013.tif64160

[0156] Example 3: Expression in the yeast Saccharomyces cerevisiae Extraction and analysis of metabolites The engineered S. cerevisiae strains were transferred to 0.5 mL of medium in a 96-well plate and grown at 30°C for 3 days with orbital shaking at 350 rpm. For extraction, 0.1 mL of S. cerevisiae culture was transferred to a 1.5 mL glass vial. 0.4 mL of uHPLC-grade MeOH was added. The S. cerevisiae extract was filtered using a 0.22 μm 96-well filter plate (Merck Millipore, Darmstadt, Germany) and stored at 4°C before LC-MS analysis.

[0157] The LCMS profile of the extract can be seen in Figure 2, where it can be observed that transforming the background strain with TwCYP82D274V1 encoding the enzyme having the amino acid sequence of SEQ ID NO: 1 results in the formation of 14-OH-dehydroabietadiene.

[0158] Example 4: Detection of 14-OH-dehydroabietadiene by NMR analysis The compound identified in Example 3 as 14-OH-dehydroabietadiene was analyzed by NMR to confirm its identity.

[0159] Purification for NMR Purification of triptolide intermediates from engineered yeast Engineered yeast producing the desired compound of interest was inoculated from SC agar in 10 mL of YDP and grown overnight at 30°C. 5 mL of the overnight culture was inoculated into 500 mL of FIT medium and grown at 30°C for 5 days. The compound of interest was extracted from the culture with 500 mL of EtAc. The solvent was removed by rotary evaporation, and the analyte was resuspended in hexane. The extraction was repeated three times. The hexane extract was applied to a Supelclean™ Florisil® / Na2SO4 SPE Tube (Sigma-Aldrich), and the analyte was eluted from the column using a stepwise gradient of EtAC:hexane from 1:99 to 5:95. Each fraction was analyzed by either LC-MS or GC-MS, and fractions containing the compound of interest were selected for NMR analysis.

[0160] NMR analysis NMR data were acquired on a Bruker Avance III HD 600 MHz NMR spectrometer (H operating frequency 599.85 MHz) (Bruker Biospin, Karlsruhe, Germany) equipped with a 5 mm cryogenically cooled DC probe optimized for C and H. NMR data were recorded in 5 mm tubes in CDCl (Euriso-top, 99.8 atomic % D) equilibrated to 300 K. Lock parameter optimization, gradient shimming, and receiver gain settings were all automatically controlled by Topspin ver. 3.2 and IconNMR ver. 4.7.5 (Bruker Biospin, Karlsruhe, Germany). H and C chemical shifts were referenced to the residual solvent signals at pH 7.26 ppm and pH 77.16 ppm, respectively. 1D 1H and 13C NMR spectra were acquired with a 30° pulse and 64k data points, zero-padded to 256k data points. 1H spectra were acquired with a 12 kHz spectral width, a 1-second delay, and a 2.7-second data acquisition time. 13C spectra were H-decoupled using the Waltz16 composite pulse decoupling scheme. 2D homonuclear and heteronuclear experiments were acquired with 4096 (HMBC), 2048 (DQF-COSY and ROESY), or 1024 (multiplicity-edited HSQC) data points in the direct dimension, and 256 (DQF-COSY, HMBC, and ROESY) or 128 (multiplicity-edited HSQC) data points in the indirect dimension. 2D NMR data were zero-padded to 1k in F1 and to twice the number of points in F2, and followed forward linear prediction in F1 (LPBIN = 0). Processing of NMR data was performed using Topspin ver. 4.0.9 (Bruker Biospin, Karlsruhe, Germany).

[0161] The NMR spectroscopic data for 14-OH-dehydroabietadiene is shown in Table 4. [Table 4]

[0162] of 14-OH-dehydroabietadiene in CDCl3 at 599.85 MHz 1 The H NMR spectrum of 14-OH-dehydroabietadiene in CDCl3 at 150.83 MHz is shown in Figure 3. 13 The C NMR spectrum is shown in Figure 4 and confirms the identity of the compound.

[0163] Example 5: Expression in S. cerevisiae of genes leading to the production of tryptophenolide and triptonide This was a preliminary study to assess the effect of gene expression on the production of tryptophenolide and triptonide in S. cerevisiae.

[0164] The background yeast strains generated in Example 2 were further transformed with vectors each containing an individual diterpene biosynthetic gene or a Tripterygium wilfordii CYP (TwCYP).

[0165] The LCMS profiles of the extracts can be seen in Figure 5, where it can be seen that transformation of the background strain with TwCYP82D274V1, TwCYP71BE85 and TwCYP71BE86, which encode enzymes having the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:4, respectively, results in the formation of tryptophenolide, and further transformation with CYP82D213, which encodes an enzyme having the amino acid sequence of SEQ ID NO:5, results in the formation of tryptonide.

[0166] FIG. 6 shows a summary of the content of oxygenated diterpenoid compounds detected in extracts of the generated transformants.

[0167] The left panel shows the contents of tryptophenolide and triptonide, and the right panel shows the content of 14-OH-dehydroabietadiene. Expression of the gene TwB5#1 encoding the enzyme having the amino acid sequence of SEQ ID NO:8 resulted in significantly higher production of tryptophenolide and triptonide.

[0168] Genes TwB5#2-6 are other T. wilfordii cytochrome B5 genes that do not increase tryptophenolide or tryptonide production (sequences not provided).

[0169] Example 6: Production of oxygenated diterpenoid compounds in S. cerevisiae and N. benthamiana All engineered S. cerevisiae strains and N. benthamiana were cultured as described in the "Materials and Methods" section hereinabove. Similarly, terpenoid metabolites were extracted, analyzed by LC-MS, GC-MS, and NMR, and quantified as described hereinabove.

[0170] It is preferred that the experimental organism be yeast, and that the heterologous gene be stably transfected into the organism, as this gives the most accurate and reproducible results.

[0171] The results are shown in Figures 7-9. As can be clearly seen from the figures, organisms as different as yeast cells and tobacco plants are both capable of producing high titers of the claimed key intermediates in the proposed tryptonide biosynthetic pathway according to the methods of the present invention.

[0172] NMR spectra of other important compounds produced are shown in Figures 10 to 26. NMR spectroscopic data of the compounds produced are shown in Tables 5 to 21 below. [Table 5] [Table 6] [Table 7] Table 8 Table 9 Table 10 Table 11 Table 12 Table 13 Table 14 Table 15 Table 16 Table 17 Table 18 Table 19 Table 20 Table 21 References 1. J. Andersen-Ranberg et al., Expanding the Landscape of Diterpene Structural Diversity through Stereochemically Controlled Combinatorial Biosynthesis. Angewandte Chemie International Edition, n / a (2016). 2. I. Pateraki et al., Total biosynthesis of the cyclic AMP booster forskolin from Coleus forskohlii. Elife 6, e23001 (2017). 3. I. Pateraki et al., Manoyl Oxide (13R)、 the Biosynthetic Precursor of Forskolin, Is Synthesized in Specialized Root Cork Cells in Coleus forskohlii. Plant Physiology 164, 1222-1236 (2014). 4. N. L. Hansen et al., The terpene synthase gene family in Tripterygium wilfordii harbors a labdane-type diterpene synthase among the monoterpene synthase TPS-b subfamily. The Plant Journal 89, 429-441 (2017). 5. H. H. Nour-Eldin, B. G. Hansen, M. H. H. Norholm, J. K. Jensen, b. A. Halkier, Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments. Nucleic Acids Research 34, e122 (2006). 6. N. B. Jensen et al., EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae. FEMS Yeast Research 14, 238-248 (2014). 7. M. H. H. Norholm, A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC Biotechnology 10, 21 (2010). 8. R. D. Gietz, R. H. Schiestl, High-efficiency yeast transformation using the LiAc / SS carrier DNA / PEG method. Nature Protocols 2, 31-34 (2007). 9. Hansen, N.L., et al., Integrating pathway elucidation with yeast engineering to produce polpunonic acid the precursor of the anti-oDesity agent celastrol. MicroD Cell Fact, 2020. 19(1): p. 15. 10. Voinnet O, Rivas S, Mestre P, Baulcombe D. An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus (retracted in: Plant J. 2015 Nov;84(4):846). Plant J. 2003;33(5):949-956. doi:10.1046 / j.1365-313x。2003.01676.x item 1. A recombinant host cell capable of producing oxygenated diterpenoid compounds, wherein the recombinant host cell is capable of producing miltiradiene and / or dehydroabietadienes and has been transformed with a first gene encoding an enzyme having cytochrome P450 activity, wherein the enzyme is capable of converting miltiradiene and / or dehydroabietadienes to 14-OH-dehydroabietadienes. 2. The recombinant host cell of item 1, wherein the first gene encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity to SEQ ID NO: 1 (TwCYP82D274V1), or a mature polypeptide thereof. 3.Furthermore, a second gene encoding a second enzyme having cytochrome P450 activity; and a third gene encoding a third enzyme having cytochrome P450 activity. where the second gene encoding an enzyme having cytochrome P450 activity encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity to SEQ ID NO: 4 (TwCYP71BE86), or a mature polypeptide thereof; the third gene encoding an enzyme having cytochrome P450 activity encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity to SEQ ID NO: 3 (TwCYP71BE85), or a mature polypeptide thereof; 3. The recombinant host cell of item 1 or 2. 4. The recombinant host cell of item 3, further comprising a gene encoding a polypeptide having cytochrome B5 activity and comprising an amino acid sequence having at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity to SEQ ID NO: 8 (TwB5#1), or a mature polypeptide thereof. 5. Further comprising a fourth gene encoding a fourth enzyme having cytochrome P450 activity, the fourth gene encoding a fourth enzyme having cytochrome P450 activity encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity to SEQ ID NO: 5 (TwCYP82D213), or a mature polypeptide thereof; 5. The recombinant host cell of item 3 or 4. 6. Further comprising a fifth gene encoding a fifth enzyme having cytochrome P450 activity and / or a sixth gene encoding a sixth enzyme having cytochrome P450 activity, the fifth gene encoding a fifth enzyme having cytochrome P450 activity encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity to SEQ ID NO:6 (TwCYP82D217), or a mature polypeptide thereof; the sixth gene encoding the sixth enzyme having cytochrome P450 activity encodes a polypeptide comprising an amino acid sequence having at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity to SEQ ID NO: 7 (TwCYP82D275), or a mature polypeptide thereof; The recombinant host cell of item 5. 7. A host cell capable of producing miltiradiene and / or dehydroabietadiene, a. Geranylgeranyl diphosphate synthase; b. Diterpene synthase capable of converting GGPP to miltiradiene; c. a combination of two or more diterpene synthases that in combination can convert GGPP to miltiradiene; or d. copalyl diphosphate synthase and miltiradiene synthase; A recombinant host cell according to any of the preceding items, which is a recombinant cell transformed with one or more genes encoding: 8. The recombinant host cell of item 7, wherein the geranylgeranyl diphosphate synthase is a polypeptide comprising SEQ ID NO:73 or SEQ ID NO:81. 9. The recombinant host cell of item 7, wherein the combination of two or more diterpene synthases capable of converting GGPP to miltiradiene is a combination of a polypeptide comprising the amino acid sequence of SEQ ID NO:67 and a polypeptide comprising the amino acid sequence of SEQ ID NO:68, or a combination of a polypeptide comprising the amino acid sequence of SEQ ID NO:69 and a polypeptide comprising the amino acid sequence of SEQ ID NO:70. 10. The recombinant host cell of item 7, wherein the combination of copalyl diphosphate synthase and miltiradiene synthase is a combination of a polypeptide comprising the amino acid sequence of SEQ ID NO:71 and a polypeptide comprising the polypeptide of SEQ ID NO:72. 11. A recombinant host cell according to any of the above items, selected from a prokaryotic cell and a eukaryotic cell. 12. The recombinant host cell according to item 11, which is a prokaryotic cell selected from the species Escherichia, Bacillus, Lactobacillus and Corynebacterium. 13. The recombinant host cell of item 11, which is a eukaryotic cell selected from the species Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia, Candida, and Yarrowia. 14. The recombinant host cell of item 11, which is an S. cerevisiae cell. 15. Use of a recombinant host cell according to any of the preceding items for the production of oxygenated diterpenoid compounds. 16. Use of item 15, wherein the oxygenated diterpenoid compound is selected from 14-OH-dehydroabietadienes, tryptophenolides and triptonides. 17. Use according to item 16, wherein the oxygenated diterpenoid compound is triptonide, which is further converted to triptolide. 18. Use according to one of items 15 to 17, wherein the oxygenated diterpenoid compounds are recovered using one or more separation and / or chromatography steps. 19. A polypeptide having cytochrome P450 enzyme activity and comprising an amino acid sequence having at least 80% sequence identity, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, preferably at least 98% sequence identity, or 100% sequence identity to one of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or a mature polypeptide thereof. 20. A polynucleotide encoding the polypeptide of item 19. 21. A plasmid, expression vector, expression construct or recombinant host cell comprising the polynucleotide of item 20. 22. The compound 14-OH-dehydroabietadiene.

Claims

1. 1. A recombinant host cell that produces oxygenated diterpenoid compounds, comprising: i. producing miltiradiene and / or dehydroabietadienes; and ii. A recombinant host cell comprising a first heterologous nucleic acid encoding a first enzyme having cytochrome P450 activity, wherein said first enzyme having cytochrome P450 activity is the cytochrome P450 enzyme TwCYP82D274 set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 74 or SEQ ID NO: 75, or a functional homolog having at least 90% sequence identity thereto, or a mature polypeptide thereof, thereby converting miltiradiene and / or dehydroabietadiene to 14-hydroxydehydroabietadiene.

2. 2. The recombinant host cell of claim 1, further comprising a second heterologous nucleic acid encoding a second enzyme having cytochrome P450 activity, wherein the second enzyme having cytochrome P450 activity is the cytochrome P450 enzyme TwCYP71BE86 represented by SEQ ID NO: 4, or a functional homolog having at least 90% sequence identity thereto, or a mature polypeptide thereof.

3. 3. The recombinant host cell of claim 2, wherein the recombinant host cell contains and expresses the first heterologous nucleic acid and the second heterologous nucleic acid, thereby producing 14-hydroxydehydroabietadiene, 3,14-dihydroxydehydroabietadiene, 3,14-dihydroxyabeodienes, and 14-hydroxy-18-aldo-abeodienes.

4. 4. The recombinant host cell of any one of claims 1 to 3, further comprising a third heterologous nucleic acid encoding a third enzyme having cytochrome P450 activity, wherein the third enzyme having cytochrome P450 activity is the cytochrome P450 enzyme TwCYP71BE85 represented by SEQ ID NO: 3, or a functional homolog having at least 90% sequence identity thereto, or a mature polypeptide thereof.

5. 5. The recombinant host cell of claim 4, wherein the recombinant host cell contains and expresses the first heterologous nucleic acid and the third heterologous nucleic acid, thereby producing 14-hydroxydehydroabietadiene.

6. 5. The recombinant host cell of claim 4, wherein the recombinant host cell contains and expresses the first heterologous nucleic acid, the second heterologous nucleic acid, and the third heterologous nucleic acid, thereby producing 14-hydroxydehydroabietadiene, 3,14-dihydroxydehydroabietadiene, 3,14-dihydroxyabeodienes, 14-hydroxy-18-aldo-abeodienes, and tryptophenolides.

7. 7. The recombinant host cell of any one of claims 1 to 6, further comprising a fourth heterologous nucleic acid encoding a fourth enzyme having cytochrome P450 activity, wherein the fourth enzyme having cytochrome P450 activity is the cytochrome P450 enzyme TwCYP82D213 represented by SEQ ID NO: 5 or SEQ ID NO: 76, or a functional homolog having at least 90% sequence identity thereto, or a mature polypeptide thereof.

8. 8. The recombinant host cell of claim 7, wherein the recombinant host cell contains and expresses the first heterologous nucleic acid, the second heterologous nucleic acid, the third heterologous nucleic acid, and the fourth heterologous nucleic acid, thereby producing 14-hydroxydehydroabietadienes, 3,14-dihydroxydehydroabietadienes, 3,14-dihydroxyabeodiene, 14-hydroxy-18-aldo-abeodiene, tryptophenolides, and triptonides.

9. 9. The recombinant host cell of any one of claims 1 to 8, further comprising a fifth heterologous nucleic acid encoding a fifth enzyme having cytochrome P450 activity, wherein the fifth enzyme having cytochrome P450 activity is the cytochrome P450 enzyme TwCYP82D217 represented by SEQ ID NO: 6, or a functional homolog having at least 90% sequence identity thereto, or a mature polypeptide thereof.

10. 10. The recombinant host cell of any one of claims 1 to 9, further comprising a sixth heterologous nucleic acid encoding a sixth enzyme having cytochrome P450 activity, wherein the sixth enzyme having cytochrome P450 activity is the cytochrome P450 enzyme TwCYP82D275 set forth in SEQ ID NO: 7, or a functional homolog having at least 90% sequence identity thereto, or a mature polypeptide thereof.

11. 11. The recombinant host cell of any one of claims 1 to 10, further comprising a seventh heterologous nucleic acid encoding an enzyme having cytochrome B5 activity, wherein the enzyme having cytochrome B5 activity is cytochrome B5 enzyme TwB5#1 represented by SEQ ID NO: 8, or a functional homolog having at least 90% sequence identity thereto, or a mature polypeptide thereof.

12. i. geranylgeranyl diphosphate synthase; ii. a diterpene synthase that converts geranylgeranyl diphosphate (GGPP) to miltiradiene; iii. A combination of two or more diterpene synthases that in combination convert GGPP to miltiradiene; or iv. copalyl diphosphate synthase and miltiradiene synthase; and expressing one or more of:

12. The recombinant host cell according to any one of claims 1 to 11, thereby producing miltiradiene and / or dehydroabietadienes.

13. 13. The recombinant host cell of claim 12, wherein the geranylgeranyl diphosphate synthase is a polypeptide comprising the amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 81, or a functional homolog thereof having at least 90% sequence identity thereto.

14. 14. The recombinant host cell of claim 12 or 13, wherein the combination of two or more diterpene synthases that convert GGPP to miltiradiene is a combination of CfTPS1 represented by SEQ ID NO: 67 and CfTPS3 represented by SEQ ID NO: 68, or a combination of CftTPS1 represented by SEQ ID NO: 77 and CftTPS3 represented by SEQ ID NO: 78, or a combination of their respective functional homologs that have at least 90% sequence identity thereto; or a combination of TwTPS9 represented by SEQ ID NO: 69 and TwTPS27 represented by SEQ ID NO: 70, or a combination of their respective functional homologs that have at least 90% sequence identity thereto.

15. The recombinant host cell according to any one of claims 12 to 14, wherein the combination of copalyl diphosphate synthase and miltiradiene synthase is a combination of SmCPS represented by SEQ ID NO: 71 and SmKSL represented by SEQ ID NO: 72, or a combination of their respective functional homologs having at least 90% sequence identity thereto.

16. The recombinant host cell of any one of claims 1 to 15, which is a prokaryotic or eukaryotic cell.

17. 17. The recombinant host cell of any one of claims 1 to 16, which is a eukaryotic cell of a species selected from the group consisting of Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia, Candida and Yarrowia.

18. 18. The recombinant host cell of any one of claims 1 to 17, which is S. cerevisiae.

19. 17. The recombinant host cell according to any one of claims 1 to 16, which is a prokaryotic cell of a species selected from the list consisting of Escherichia, Bacillus, Lactobacillus and Corynebacterium.

20. The recombinant host cell according to any one of claims 1 to 16, which is a plant cell and is a cell from a multicellular host.

21. 1. A method for the production of oxygenated diterpenoid compounds, such as triptonides, comprising: i. providing a recombinant host cell according to any one of claims 1 to 20; ii. Culturing the recombinant host cell under conditions suitable for the production of the oxygenated diterpenoid compound; A method comprising:

22. 22. The method of claim 21, wherein the oxygenated diterpenoid compound is selected from the list consisting of 14-OH-dehydroabietadiene, tryptophenolide, and triptonide.

23. 23. The method of any one of claims 21 to 22, wherein the oxygenated diterpenoid compound is a triptonide.

24. 24. The method of any one of claims 22 to 23, further comprising recovering and optionally purifying the triptonide.

25. 1. A method for the production of triptolide, comprising: i. Producing triptonide according to the method of any one of claims 22 to 24, and ii. Converting the triptonide to triptolide; and iii. Optionally, recovering and / or purifying the triptolide; A method comprising:

26. Use of a recombinant host cell according to any one of claims 1 to 20 for the production of oxygenated diterpenoid compounds.

27. 27. The use according to claim 26, wherein the oxygenated diterpenoid compound is selected from the list consisting of 14-OH-dehydroabietadiene, tryptophenolide and triptonide.

28. 27. The use of claim 26, wherein the oxygenated diterpenoid compound is triptonide, and the triptonide is further converted to triptolide.

29. 29. The use according to any one of claims 26 to 28, wherein the oxygenated diterpenoid compounds are recovered using one or more separation and / or chromatography steps.

30. A polypeptide having cytochrome P450 enzyme activity and comprising an amino acid sequence having at least 90% sequence identity to one of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or a mature polypeptide thereof; or a polypeptide having cytochrome B5 enzyme activity and comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

8.

31. A polynucleotide encoding the polypeptide of claim 30.

32. 32. A plasmid, expression vector, expression construct, or recombinant host cell comprising the polynucleotide of claim 31.

33. 21. The recombinant host cell of any one of claims 1 to 20, comprising the compound 14-OH-dehydroabietadiene.

34. The following formulas (1) to (17): 【Chemistry 1】 【change】 【change】 【change】 【change】 21. The recombinant host cell of any one of claims 1 to 20, comprising a compound selected from the group consisting of:

35. The recombinant host cell of claim 34, wherein the compound is a compound according to formula (6) (F20P2).

36. The recombinant host cell of claim 34, wherein the compound is a compound according to formula (10) (F15P1).

37. The recombinant host cell of claim 34, wherein the compound is a compound according to formula (15) (F15P2).

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