Production of D-lysergic acid

Abstract

The present invention relates to modified cells suitable for use in the production of ergot alkaloids. More specifically, the present invention provides engineered recombinant cells that contain one or more genes encoding one or more enzymes in the biosynthetic pathway from tryptophan to D-lysergic acid (DLA). The present invention also provides methods for culturing the engineered recombinant cells to produce DLA and other ergot alkaloids.

Description

[Technical field]

[0001] The present invention relates to modified cells suitable for use in the production of ergot alkaloids. More specifically, the present invention provides engineered recombinant cells that contain one or more genes encoding one or more enzymes in the biosynthetic pathway from tryptophan to D-lysergic acid (DLA). The present invention also provides methods for culturing the engineered recombinant cells to produce DLA and other ergot alkaloids. [Background technology]

[0002] Ergot alkaloids are a class of natural products that have been widely used as therapeutic agents throughout history. In modern medicine, these compounds and their semi-synthetic derivatives are used to treat several neurological disorders, especially Parkinson's disease, dementia, and hypertension (Lieberman, A et al. 295, 1400-1404 (1976); Winblad, B et al., Clin. 28, 533-552 (2008); Tandowsky, R., M., Circulation 9, 48-56 (1954)). Ergot alkaloids are broadly divided into three groups: clavines, ergoamides, and ergopeptines, all of which are distinguished by different modifications to the ergoline skeleton. These compounds are produced by several filamentous fungi of the Ascomycota phylum, but are particularly produced by the parasitic fungus Claviceps purpurea, more commonly known as the ergot fungus from which it takes its name (de Groot, AN et al., Drugs 56, 523-535 (1998)).

[0003] The pharmacological actions of ergot alkaloids are due to the molecular similarity of the ergoline skeleton to monoamine neurotransmitters such as adrenaline, dopamine, and serotonin (Pertz, H. & Eich, E., Amsterdam: Harwood Academic Publishers, 411-440 (1999); Mantegani, S., et al., Il Farm. 54, 288-296 (1999)). Therefore, the ergoline pharmacophore provides an important scaffold for the discovery and development of potential therapeutics, especially in the treatment of neurological and psychiatric disorders. As an illustration, D-lysergic acid diethylamide (LSD), a chemically derived ergot alkaloid, inhibits 5-HT 2A It is one of the most potent agonists for serotonergic receptors (K d =0.33 nM) (Wacker, D. et al., Cell 168, 377-389, e312 (2017)). The primary active pharmaceutical ingredient (API) of these ergoline derivatives is derived from D-lysergic acid (DLA).

[0004] To produce therapeutically relevant ergoline-derived medicines, the recovered ergopeptines usually need to be hydrolyzed to DLA followed by further semi-synthetic derivatization. To meet the global demand for DLA, 8 tons of ergopeptines and up to 10-15 tons of DLA are produced annually. About 60% of these are produced by submerged fermentation of specially developed strains of Claviceps purpurea, the rest by field cultivation (Cvak, L., 373 (CRC Press, 1999)). However, the main limitations of these existing production methods are, first, the high variability of the produced ergot alkaloids, which complicates the downstream extraction workflow and increases production costs (Chen, J.-J. et al., RSC Adv. 7, 27384-27396 (2017)), and, second, the tendency of these strains to degenerate during the cultivation and storage process (Cvak, L., 373 (CRC Press, 1999)).

[0005] A number of chemical total synthesis routes to DLA have also been reported (Liu, H. & Jia, Y., Nat. Prod. Rep. 34, 411-432 (2017)). However, such routes are highly complex, requiring 8–19 chemical transformation steps and harsh reaction conditions. Moreover, the yields are low and the products are often not enantiomerically pure. For example, the highest yielding process requires 19 steps and a reported yield of 12% (Umezaki, S. et al. Lett. 15, 4230-4233 (2013)). In contrast, the simplest method is an 8-step process with a reported yield of 10.6%, but is not enantioselective (Hendrickson, JB & Wang, J., Org. Lett. 6, 3-5 (2004)). These issues have significantly hindered the use of chemical synthesis to meet the commercial demand for DLA, as evidenced by its lack of use in industry. Thus, the synthesis of these compounds by chemical and biological routes, although of high industrial relevance, still suffers from several challenges.

[0006] Therefore, there is a great need to provide improved microbial production systems and methods for producing DLA that overcome, or at least ameliorate, one or more of the above-mentioned disadvantages. Summary of the Invention

[0007] The present invention provides an isolated recombinant cell comprising one or more genes, where each gene encodes an enzyme in the biosynthetic pathway from tryptophan to DLA.

[0008] For example, the one or more genes may be dmaW, easF, easC, easE, easD, easA イソメラーゼ and cloA. It will be understood that the isolated recombinant cell may contain one or more orthologs of each gene from the group.

[0009] The isolated recombinant cell contains at least one dmaW, at least one easF, at least one easE, at least one easD, at least one easA イソメラーゼ , and at least one cloA gene.

[0010] Alternatively, the isolated recombinant cell comprises at least one dmaW, at least one easF, at least one easE, at least one easC, at least one easD, at least one easA, イソメラーゼ , at least one easG, and at least one cloA gene.

[0011] The invention includes a method of culturing the recombinant cells described herein in a suitable medium, as will be understood to be for the production of 4-dimethylallyl-L-tryptophan (DMAT), 4-dimethylallyl-L-abrin (4DMA), chanoclavine I, chanoclavine I-aldehyde, agroclavine, and / or DLA.

[0012] In particular, the method is for preparing DLA.

[0013] Advantageously, the engineered recombinant cells of the present disclosure can directly produce DLA and are stable in high density liquid culture. Further advantageously, the engineered recombinant cells of the present disclosure eliminate the need to hydrolyze ergopeptines and provide a means to directly produce DLA (the major ergoline derivative API), thereby minimizing purification and downstream processing complications. These and other advantages of the present disclosure will become readily apparent to those skilled in the art from the detailed description that follows. [Brief description of the drawings]

[0014] The accompanying drawings illustrate disclosed embodiments and serve to explain the principles of the disclosed embodiments, but it is to be understood that the drawings are for purposes of illustration only and are not intended to define the limits of the invention.

[0015] [Figure 1] A schematic diagram of the complete biosynthetic pathway of ergopeptides is shown. It starts with tryptophan and goes through the key intermediate DLA. Alternative branches of the ergot pathway are indicated by faded regions. Colored arrows indicate steps of the pathway. Green: early pathway common to all ergot producing species, leading to chanoclavine I-aldehyde, the first major branch of the pathway. The early pathway consists of four steps and requires five enzymes (DmaW, EasF, EasC, EasE, and EasD). Blue: intermediate and late pathways that explain the diversification of ergoline products found in different lineages of ergot producing species. This stage of the pathway consists of two steps and requires at least three enzymes (EasA, EasG, and CloA). Orange: alternative branches of the pathway leading to other ergoline derivatives.

[0016] [Diagram 2] The results of screening for EasE orthologues using a yeast screening strain (YMC17) in which the dmaW, easF, and easC genes were stably integrated into the yeast genome are shown. Introduction of episomal plasmids expressing various identified EasE orthologues into the strain enables a convenient screening platform. Figure 2A Biosynthetic reaction producing chanoclavine I from 4DMA. Figure 2B LC-MS / MS extracted ion chromatograms of the screened EasE orthologues show an ion transition from 257 to 226 m / z, indicating the production of chanoclavine I. The highlighted area of ​​the peak eluting at 121 s indicates the production of chanoclavine I. Chromatogram order (bottom to top): GFP control, easE_Ec, easE_Aj, easE_Al, easE_Cf, easE_Pi, easE_Nl, easE_Ei, easE_Ee, easE_Ef. Figure 2C. The relative amounts of chanoclavine I produced by EasE_Aj and EasE_Ec were estimated by the peak area of ​​the transition from 257 to 226 m / z in MS / MS. Data are shown as mean ± standard deviation. Error bars represent standard deviation calculated from three biological replicates.

[0017] [Diagram 3] The results of screening for EasA orthologues are shown. Figure 3A The reaction forming the ergoline D-ring from chanoclavine I. Different combinations of enzymes and isoforms (EasA, EasG, and EasH) control the branching of the tetracyclic ergoclavine product formed at this branch point. Orthologues of EasA were screened using episomal plasmids of strains containing genes for early pathway enzymes dmaW, easF, easC, and easE integrated on the yeast genome and controlled by the PTEF2, PGPM1, PGAL10, and PGAL1 promoters, respectively. Figure 3B LC-MS chromatograms of the screened EasA orthologues. All selected orthologues were found to produce compounds that co-elute with the commercially available agroclavine standard. The chromatograms are displayed in order (from bottom to top): YOCE, pCKU-RFP, easA_Pi, easA_Ec, easA_Cpur, easA_Nl, and the agroclavine standard.

[0018] [Figure 4]The results of screening for cloA orthologues are shown. Figure 4A Putative oxidation and isomerization reactions catalyzed by cloA. Schematic of the assay design for screening of cloA product profiles. The cloA-expressing strain was fed commercial agroclavine and analyzed for DLA production. Figure 4B LC-MS / MS chromatograms of products showing the 269 → 223 m / z ion transition produced by cloA orthologues. Chromatograms are displayed in the following order (bottom to top): pYES2-CT, DLA standard, C. glo (X90), M. rob (392), E. coe (253), C. fus, C. pur (20.1), B. cin, M. acr (SJ7), C. glo (ET3), P. ipo, M. acr (AT5), C. pas, C. pur, N. lol, E. coe (XN6). Figure 4C: Relative amounts of agroclavine consumed by the screened cloA orthologues. Figure 4D: Relative amounts of DLA produced by the various cloA orthologues. Data are shown as mean + / - standard deviation. Error bars represent standard deviation calculated from three biological replicates.

[0019] [Diagram 5] Figure 5A shows the assembly of functional parts into a DLA-producing yeast strain. Figure 5A A stepwise expansion approach to pathway construction in engineered strains that allows each predecessor strain to be used as a control for the subsequent strain. Figure 5B LC-MS / MS extracted ion chromatogram monitoring observed ion transition of [M+H]+=257→226 m / z, indicating the production of chanoclavine I from both AgcM1B and AgcM2B. Figure 5C LC-MS / MS extracted ion chromatogram of [M+H]+=239→208 m / z, indicating the production of agroclavine from AgcM33B. Figure 5D LC-MS / MS extracted ion chromatogram showing detected ion transition of [M+H]+=269→223 m / z, indicating the production of DLA from DLAM33B. Peaks eluted in the time segment (highlighted) correspond to the target compounds.

[0020] [Figure 6]Production of DLA from DLAM33B in 1L and 4L scale fermentations is shown. The fermentation process modeled the induction protocol used in the shake flask experiments, supplemented with additional galactose and 10X SC-URA medium in fed-batch mode with defined feeding phases (I, II, III). 50 mM ammonium succinate was added to the medium from the beginning to maintain the pH at 5.8. The maximum wet cell weight was approximately 26 g L-1 and the maximum DLA titers were 2.0 mg L-1 and 1.6 mg L-1 for both 1L and 4L fermentations. Feeding phase I: Initial induction phase mimicking galactose supplementation for induction in shake flask experiments. The 10X feed solution was replenished to the vessel to a final concentration of 1X at a flow rate of 3.5 mL min-1. Feeding phase II: Sustained feeding phase, a second 10X concentration of feed solution was replenished to the culture over 52 h to a final concentration of 1X. Feeding phase III: Starvation phase, no additional carbon or nitrogen sources were added. Data are presented as mean values ​​+ / - standard deviation. Error bars are calculated from three biological replicates.

[0021] [Figure 7] The modifications made to the yeast Fab assembly system are shown. Two sets of plasmids were created for the refinement of the entire workflow: the first is a set of pathway acceptor vectors (level 2) that allow the screening of pathway modules created in E. coli and rapidly tested as episomal plasmids in yeast. The second is a set of yeast genome integration vectors to broaden the repertoire of available integration sites. The URA3 marker on the integration fragment is designed to be flanked by the homologous sequence URR1, which allows the marker to be removed by homologous recombination upon counterselection with 5-fluoroorotic acid.

[0022] [Figure 8]Promoter strength measurements at 12 and 18 hours of growth are shown in descending order of strength at 12 hours. Figure 8A Schematic of the promoter reporter plasmid pGLO3. The promoter released from HCKan_P replaces the RFP cassette in a Golden Gate reaction with Esp3I. When transformed into yeast, the promoter drives expression of mKOK (orange fluorescent protein). The PCYC1-yeGFP-TCYC1 cassette serves to distinguish cells that carry the plasmid from those that do not. Figure 8B Weak promoter - relative strength to PMDN1 below 1 at 12 hours of growth. Figure 8C Medium strength promoter - relative strength between 1 and 6. Figure 8D Strong promoter - defined by having a strength 6-fold or greater than PMDN1.

[0023] [Figure 9] Testing of pathway acceptor plasmids and validation of promoter strength data by production of DMAT are shown. Figure 9A Illustrated assembly process to generate the pMKU-dmaW series of plasmids. Figure 9B The first reaction of the ergot alkaloid pathway catalyzed by dmaW producing DMAT. Figure 9C Overlay of LC-MS chromatograms at 273 m / z of analyzed samples showing peaks of various sizes corresponding to the amount of DMAT produced. Chromatograms are displayed in order (bottom to top): PDC1, PGI1, PYK1, SSA1, TDH2, RPL8, RPL15, PGK1, ENO2, PDA1, GPM1, TPI1, TEF2, and TDH3. Figure 9D Comparison of the relative amounts of DMAT produced as estimated from the peak area responses of extracted ion chromatograms. Error bars were calculated from three biological replicates.

[0024] [Figure 10]The results of verifying the production of DMAT produced from the pMKU-dmaW series of plasmids are shown. Comparison of retention time and mass spectrum of DMAT produced in vivo (Figure 10A) and in vitro (Figure 10B) from the purified enzyme supplemented with DMAPP and tryptophan. Structure of the simplest fragment ion observed. [Figure 11] The results of testing the genome integration vector by producing 4DMA are shown. Fig. 11A Schematic diagram of the strain (YMWF) containing the expression cassettes: PTEF2-dmaW-TENT2 and PGPM1-easF-TPRX1 integrated at the YMRWΔ15 transposon site. Fig. 11B Reaction catalyzed by dmaW and easF to produce 4DMA from tryptophan and DMAPP. Comparison of retention time and mass spectrum of 4DMA produced in vivo (Fig. 11C) and in vitro (Fig. 11D) from purified enzyme supplemented with DMAPP, tryptophan, and S-adenosylmethionine (SAM). Structure prediction of the simplest fragment ion observed.

[0025] [Figure 12]Sequence similarity networks (SSNs) generated from known gene targets of the Ergo pathway using the EFI-EST web tool (Gerlt, JA et al., Biochimica Et Biophysica Acta (BBA)-Proteins and Proteomics 1854, 1019-1037 (2015); Zallot, R. et al., Biochemistry 58, 4169-4182 (2019)). In the representation of the SSN, each investigated sequence is shown as a node and linked to other related nodes by edges. Each edge represents a predefined degree of similarity. By defining appropriate alignment scores as edges, the nodes can be partitioned into isofunctional clusters that group related sequences in a similar way that multiple sequence alignments draw consensus sequences. Enzyme sequences within isofunctional clusters are expected to be able to catalyze similar reactions. Uncharacterized enzymes closely related to known targets can be identified and tested for properties of interest, in this case functional expression in yeast. Expanding the hypothetical isofunctional cluster of easE, easA, and cloA allows us to further define more closely related sequences and better predict their specific activities. Figure 12A Expanded SSN of the easE isofunctional cluster. Nodes are color-coded by the genus from which the sequences originated; purple: Epichloe, orange: Claviceps, green: Aspergillus, blue: mainly Penicillium, Pseudogymnoascus, and Trichophyton. Figure 12B Expanded SSN of the easA isofunctional cluster. The reductase variants of easA show more sequence divergence and move away from the main cluster. The color of the nodes indicates the genus of the source organism; purple: Epichloe, orange: Claviceps, green: Aspergillus, red: Penicillium, brown: Claviceps gigantea and africana, blue: others.FIG 12C Expanded SSN of the cluster containing known ergoline-C17 oxidases. Subclusters are grouped according to known product profiles from closely related source organisms.

[0026] [Figure 13] The results of the MS / MS fragmentation spectra of easE_Aj and easE_Ec are shown. Figure 13A MS / MS fragmentation spectrum of the peak eluting at 20.6 seconds from the screen of easE orthologues showing that the same product eluted from both easE_Aj and easE_Ec. Figure 13B Predicted structures of the two simplest fragment ions, 226 and 208 m / z.

[0027] [Figure 14] MS / MS fragmentation spectrum of the peak eluting at 149.6 seconds from the easA orthologues screen is shown. Comparison of the fragmentation pattern with a commercially available agroclavine standard indicates that agroclavine is produced in strains carrying the easA_Ec, easA_Nl, and easA_Cp orthologues (at a collision energy of 10 eV).

[0028] [Figure 15] Quantification of agroclavine produced from easA screening strains is shown. FIG. 15A Standard curve of agroclavine established with negative control (YOCE). The curve was generated by plotting the peak area response for the ion transition of 239→208 m / z against the spiked agroclavine concentration. FIG. 15B Agroclavine titer of screening strains calculated from the standard curve measured by the peak area response of the ion transition of 239→208 m / z. Error bars were calculated from three biological replicates.

[0029] [Figure 16]MS / MS fragmentation spectrum of the peak eluting at 126.3 seconds from a screen of cloA orthologues is shown. Comparison of fragmentation patterns against a commercial DLA standard for compounds produced from various cloA orthologues showed the production of similar fragmentation ions (at a collision energy of 10 eV).

[0030] [Figure 17] Evaluation of the performance of cloA orthologues on the agroclavine-producing yeast chassis is shown. Figure 17A: Schematic of the performed experiment. Figure 17B: LC-MS / MS chromatograms of the products showing the 269→223 m / z ion transition. The chromatograms are displayed in the following order (bottom to top): pYES2-CT, cloA_Pi, cloA_Ec, cloA_Cp, and DLA. Figure 17C: Standard curve of empty vector control samples spiked with DLA. The curve was obtained by plotting the peak area response for the 269→223 m / z ion transition against the spiked DLA concentration. Figure 17D: Quantification of DLA produced from strain AgcM33B expressing the cloA orthologue from an episomal plasmid. Error bars were calculated from three biological replicates.

[0031] [Figure 18] Schematic diagram of modular introduction of four segments to sequentially reconstitute the pathway to D-lysergic acid. Following the modified yeast Fab workflow, genetic components were packaged as transcription units on POT plasmids and then assembled as linked transcription units on pathway acceptor vectors (p[C / M]K[U / L / H]). Validated pathway segments were transferred to genome integration vectors (pGAU series). Integrated constructs were verified and added with a URA3 selection marker for subsequent integration.

[0032] [Figure 19]MS / MS fragmentation spectra of a 250 nM agroclavine standard spiked into AgcM2B (top) and the peak eluted from AgcM33B (bottom) are shown, demonstrating the production of agroclavine in the reconstituted strain. Fragmentation spectra obtained at a collision energy of 20 eV.

[0033] [Figure 20] MS / MS fragmentation spectra of 250 nM DLA standard spiked into AgcM33B (top) and peaks eluted from DLAM33B (bottom) are shown, demonstrating production of DLA in the reconstituted strain. Fragmentation spectra obtained at a collision energy of 20 eV.

[0034] [Figure 21] Figure 21A shows confirmation of DLA production with 13C-tryptophan feedstock for DLAM33B. Figure 21A Schematic of 13C-W incorporation and production of 13C-DLA by DLAM33B. Figure 21B LC-MS chromatogram shows [M+H]+ shift of peaks corresponding to elution of DLA and 13C-DLA. Figure 21C (Top panel) Overlay of MS / MS spectra obtained from samples fed with tryptophan (black) and 13C-W (red). (Bottom panel) MS / MS difference spectrum between samples spiked with tryptophan and 13C-W. The +1 m / z shift of [M+H]+ expected for DLA and its fragmentation ions is highlighted.

[0035] [Figure 22]LC-MS chromatograms showing the incorporation of 13C-labeled tryptophan in all intermediates along the ergot alkaloid biosynthetic pathway are shown: Figure 22A agroclavine (239 m / z) and 13C-agroclavine (240 m / z); Figure 22B chanoclavine I (257 m / z) and 13C-chanoclavine I (258 m / z); Figure 22C DMAT (273 m / z), 4DMA (287 m / z), 13C-DMAT (274 m / z), and 13C-4DMA (288 m / z). Figures 22D-22F Overlay of the mass spectra of samples fed with 13C-tryptophan (red spectrum) and tryptophan (black spectrum) highlights the +1 m / z shift of all precursor ions and their corresponding fragment ions.

[0036] [Figure 23] Quantification of DLA production titer from strain DLAM33B by standard addition. Calibration curves were obtained by plotting the area under the curve of the peak response of the MS / MS transition 269→223 m / z against the final concentration of DLA added to aliquots of DLAM33B samples. Error bars were calculated from three biological replicates per calibration curve.

[0037] [Figure 24] 1 provides a list of promoter sequences featured in embodiments of the present invention.

[0038] [Diagram 25] 1 shows a list of terminator sequences disclosed in an embodiment of the present invention.

[0039] [Figure 26] 1 shows a list of UniProt IDs of ORFs used in embodiments of the present invention.

[0040] [Figure 27]A table summarizing the accurate mass and retention time of chanoclavine I detected from screening for easE orthologs using an electrospray ion source (ESI) in positive mode is shown.

[0041] [Figure 28] A table summarizing the accurate masses and retention times of agroclavines detected from screening for easA orthologs using an electrospray ionization source (ESI) in positive mode is shown.

[0042] [Figure 29] A table summarizing the accurate masses and retention times of DLA detected from screening for cloA orthologs using an electrospray ionization source (ESI) in positive mode is shown.

[0043] [Diagram 30] FIG. 1 shows a table summarizing the measured accurate masses of detectable intermediates in the ergot alkaloid pathway with and without incorporation of 13C-2-indole-L-tryptophan in positive mode using an electrospray ionization source (ESI).

[0044] [Diagram 31] 1 shows a table summarizing values ​​for DLA titers calculated from strain DLAM33B in shake flasks.

[0045] [Diagram 32] The following shows a list of yeast strains disclosed in the present invention. All strains were derived from the base strain S. klevisiae BY4741.

[0046] [Diagram 33] FIG. 1 is a table showing the composition of 10× PBS solution used for preparation of 1× PBS (pH 7.4) for screening of cloA orthologs.

[0047] [Diagram 34]1H-NMR assignments for D-lysergic acid in DO are shown.

[0048] [Diagram 35] A table summarizing endpoint DLA titers from 4 L and 1 L fermentations of DLAM33B is shown.

[0049] [Diagram 36] FIG. 1 shows a table providing the DNA sequences of orthologs of FAD1, PDI1, and dmaW, easF, easC, easE, easD, easA, easG, and cloA genes described in embodiments of the present invention.

[0050] [Figure 37] FIG. 1 shows a table providing the amino acid sequences corresponding to orthologs of FAD1, PDI1, and dmaW, easF, easC, easE, easD, easA, easG, and cloA genes described in embodiments of the present invention.

[0051] definition Generally, technical, scientific and medical terms used herein have the same meaning as understood by those skilled in the art to which the present invention belongs. In addition, the following technical comments and definitions are provided. These definitions should in no way limit the scope of the present invention to these terms alone, but are presented to better understand the following description.

[0052] As used herein, "a" or "an" may mean one or more, unless indicated to the contrary or clear from the context.

[0053] As used herein, the term "comprising" or "including" is to be construed as specifying the presence of the stated features, integers, steps, or components as referred to, but does not exclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. However, in the context of this disclosure, the term "comprising" or "including" also includes "consisting of." Variations of the word "comprising," such as "comprise" and "comprises," and "including," such as "include" and "includes," have corresponding different meanings.

[0054] As used herein, "enzyme" has its typical meaning in the art and refers to a polypeptide, protein, or in some cases, an RNA molecule that acts as a biological catalyst to effect a specific biochemical reaction. Thus, the term "functional enzyme" refers to an enzyme that retains some or all of its intended activity or function (e.g., a biological activity or function, such as an enzymatic activity).

[0055] The term "functional fragment" refers to a portion of a protein that retains some or all of the activity or function (e.g., a biological activity or function, such as an enzymatic activity) of the full-length protein, such as the ability to bind and / or interact with or modulate another protein or nucleic acid. Functional fragments can be of any size, so long as they retain the ability to bind and interact with, for example, another protein or nucleic acid.

[0056] As used herein, "gene product" has its typical meaning in the art and refers to a biochemical substance, either a protein or an RNA molecule, produced from the expression of a gene.

[0057] As used herein, the term "recombinant cell" means that the cell contains at least one nucleic acid sequence that is not naturally occurring in the cell, or that is naturally occurring but associated with a sequence that is not naturally associated in the cell, such as a promoter with which the nucleic acid sequence encoding the protein is not naturally associated. For example, in the context of the present invention, recombinant cells differ from naturally occurring cells in that they contain at least one expression cassette that is not present in naturally occurring cells.

[0058] As used herein, "dmaW" gene refers to a gene sequence that encodes a polypeptide, enzyme, or gene product having tryptophan dimethylallyltransferase activity and functioning to catalyze the conversion of L-tryptophan to 4-dimethylallyl-L-tryptophan (DMAT).

[0059] As used herein, the "easF" gene refers to a gene sequence that encodes a polypeptide, enzyme, or gene product that has N-methyltransferase activity and functions to catalyze DMAT to 4-dimethylallyl-L-abrin (4DMA).

[0060] As used herein, the "easC" gene refers to a gene sequence that encodes a polypeptide, enzyme, or gene product that has catalase activity and, in the presence of EasE activity, functions to catalyze 4DMA to chanoclavine I.

[0061] As used herein, "easE" gene refers to a gene sequence that encodes a polypeptide, enzyme, or gene product that has flavin adenine dinucleotide (FAD)-dependent oxidoreductase activity and, in the presence of easC activity, functions to catalyze 4DMA to chanoclavine I.

[0062] As used herein, the "easD" gene refers to a gene sequence that encodes a polypeptide, enzyme, or gene product that has oxidoreductase activity and functions to catalyze chanoclavine I to chanoclavine I-aldehyde.

[0063] As used herein, the "easA" gene refers to a gene that, in the presence of EasG activity, exhibits reductase activity (easA レダクターゼ ) or isomerase activity (easA イソメラーゼ ) or both, and encodes a polypeptide, enzyme or gene product having the function of catalyzing chanoclavine I-aldehyde to fesclavine and / or agroclavine.

[0064] As used herein, "easA レダクターゼ " Gene " refers to a genetic sequence that encodes a polypeptide, enzyme, or gene product having reductase activity and the function of catalyzing the conversion of chanoclavine I-aldehyde to fesclavine in the presence of easG activity.

[0065] As used herein, "easA イソメラーゼ " gene " refers to a genetic sequence that encodes a polypeptide, enzyme, or gene product that has isomerase activity and functions to catalyze the conversion of chanoclavine I-aldehyde to agroclavine in the presence of easG activity.

[0066] As used herein, the "easG" gene refers to a gene sequence that encodes a polypeptide, enzyme, or gene product having oxidoreductase activity and the function of catalyzing the conversion of chanoclavine I-aldehyde to either fesclavine and / or agroclavine in the presence of easA activity. レダクターゼ In the presence of easA activity, easG catalyzes the conversion of chanoclavine I-aldehyde to fesclavine, and easB catalyzes the conversion of chanoclavine I-aldehyde to fesclavine. イソメラーゼ In the presence of this activity, it catalyzes the conversion of chanoclavine I-aldehyde to agroclavine.

[0067] As used herein, "cloA" gene refers to a genetic sequence that encodes a polypeptide, enzyme, or gene product that functions to catalyze the conversion of agroclavine to oxidized agroclavine products, such as, but not limited to, D-lysergic acid (DLA), paspalic acid, lysergol, and / or elimoclavine.

[0068] As used herein, "FAD1" refers to a genetic sequence that encodes a polypeptide, enzyme, or gene product having FAD synthase activity and the function of catalyzing the adenylation of flavin mononucleotide to form the FAD coenzyme.

[0069] As used herein, "PDI1" refers to a genetic sequence that encodes a polypeptide, enzyme, or gene product that has protein disulfide isomerase activity.

[0070] As used herein, "polypeptide" and "protein" are used interchangeably to refer to a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). The term "protein" encompasses naturally occurring, artificially produced (e.g., engineered or mutant), full-length proteins, and functional fragments of proteins.

[0071] As used herein, the terms "orthologue" or "ortholog" are used interchangeably to refer to homologous genes in different species that have evolved from a common ancestral gene by speciation. Orthologous genes typically have significant sequence similarity and share functional domains inherited from a common ancestor. Orthologous genes may share at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. As will be appreciated by those skilled in the art, orthologous genes may be identified using bioinformatics approaches such as BLAST (Basic Local Alignment Search Tool), MSA (multiple sequence alignment), and EFI-EST (Enzyme Function Initiative - Enzyme Similarity Tool) for enzyme prospecting, among others. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0072] The following is an exemplary description of non-limiting embodiments of the invention.

[0073] Disclosed herein are isolated recombinant cells that contain one or more genes, each encoding an enzyme in the biosynthetic pathway from tryptophan to D-lysergic acid (DLA). Advantageously, the engineered recombinant cells of the present disclosure function as effective host strains for the production of DLA and other ergot alkaloids directly from central metabolism.

[0074] The biosynthetic pathway of ergot alkaloids is shown in Figure 1. All ergot alkaloids are derived from L-tryptophan and share a series of early biosynthetic steps that form the ergoline C ring. The presence of various isoforms of enzymes from different species of ergot-producing organisms, involved in the middle and late steps of the biosynthesis, determines the product profile produced by each of these organisms (Cheng, JZ, et al., Journal of the American Chemical Society 132, 1776-1777 (2010)).

[0075] Disclosed herein is a recombinant cell suitable for producing D-lysergic acid (DLA) and other ergot alkaloids. The recombinant cell of the disclosure is engineered to contain at least one gene encoding an enzyme in the DLA biosynthetic pathway starting from tryptophan. Also disclosed is a method for culturing the recombinant cell to produce DLA and other ergot alkaloids.

[0076] The present invention provides an isolated recombinant cell comprising one or more genes, where each gene encodes an enzyme in the biosynthetic pathway from tryptophan to DLA.

[0077] In particular, one or more of the genes are dmaW, easF, easC, easE, easD, easA イソメラーゼ and cloA. It will be understood that the isolated recombinant cell may contain one or more orthologs of each gene from the group.

[0078] The isolated recombinant cell contains at least one dmaW, at least one easF, at least one easE, at least one easD, at least one easA イソメラーゼ , and at least one CloA gene.

[0079] Examples of easA orthologs include, but are not limited to, easA from Claviceps purpurea (easA_Cp), easA from Periglandula ipomoeae (easA_Pi), easA from Neotyphodium lolii (easA_Nl), and easA from Epichloe coenophiala (easA_Ec). イソメラーゼ An example of an ortholog is EasA from Claviceps purpurea. イソメラーゼ (easA_Cp), EasA from Periglandula ipomoea イソメラーゼ (easA_Pi), EasA from Neotyphodium roryi イソメラーゼ (easA_Nl), EasA from Epichloe coenophiala イソメラーゼ (easA_Ec), but is not limited to.

[0080] Examples of cloA orthologues include cloA from Claviceps paspali (cloA_Cpas), cloA from Neotyphodium lolii (cloA_Nlol), cloA from Periglandula ipomoea (cloA_Pipo), cloA from Epichloe coenophiala (cloA_XN6, cloA_253), cloA from Claviceps purpurea (cloA_Cpur), cloA from Claviceps fusiformis (cloA_Cfus), cloA from Botrytis cinerea (cloA_Bcin), and cloA from Metarhizium acridum (cloA_Bcin). These include, but are not limited to, cloA from Bacillus acridum (cloA_AT5, cloA_SJ7), cloA from Claviceps purpurea 20.1 (cloA_Cpur), cloA from Metarhizium robertsii (cloA_0X7, cloA_392), and cloA from Colletotrichum gloesporioides (cloA_ET3, cloA_X90).

[0081] It will be understood that the recombinant cell may contain one or more orthologs of each gene in the biosynthetic pathway from tryptophan to DLA. For example, the isolated recombinant cell may contain at least one ortholog of a gene. In particular, the isolated recombinant cell may contain at least one easE.

[0082] Examples of easE orthologs include easE from Epichloe coenophiala (easE_Ec), easE from Aspergillus japonicas (easE_Aj), easE from Aspergillus lentulus (easE_Al), easE from Claviceps fusiformis (easE_Cf), easE from Periglandula ipomoea (easE_Pi), easE from Neotyphodium roryi (easE_Nl), easE from Epichloe inebrians (easE_Ei), easE from Epichloe elymi (easE_Ee), and easE from Epichloe fungii (easE_F). funkii (easE_Ef), and easE from Aspergillus indologenus.

[0083] For example, easE includes easE from Epichloe coenophiala (easE_Ec) and / or easE from Aspergillus japonicus (easE_Aj) and / or easE from Aspergillus indologenus. By way of further example, easE can include a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs:6-14.

[0084] In particular, easE from Epichloe coenophiala (easE_Ec) may comprise a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:14, and / or easE from Aspergillus japonicus (easE_Aj) or easE from Aspergillus indologenus may comprise a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:6.

[0085] More particularly, easE comprises easE from Aspergillus japonicus (easE_Aj) or easE from Aspergillus indologenus. More particularly, easE comprises a sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:6.

[0086] It will be appreciated that easE in recombinant cells expresses an enzyme that, in particular, catalyzes the conversion of 4-dimethylallyl-L-abrin (4DMA) to chanoclavine I.

[0087] Additionally, the isolated recombinant cell comprises at least one easA イソメラーゼ For example, easA イソメラーゼ easA from Neotyphodium roryi イソメラーゼ (easA_Nl), easA from Periglandula ipomoea イソメラーゼ (easA_Pi), easA from Claviceps purpurea イソメラーゼ (easA_Cp), and / or easA from Epichloë coenophiala イソメラーゼ (easA_Ec). As a further example, easA イソメラーゼ may comprise a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 15-18.

[0088] In particular, easA イソメラーゼ easA from Neotyphodium roryi イソメラーゼ (easA_Nl), easA from Claviceps purpurea イソメラーゼ (easA_Cp), and / or easA from Epichloë coenophiala イソメラーゼ (easA_Ec). In some embodiments, the easA from Neotyphodium roryi イソメラーゼ (easA_Nl) is an easA from Claviceps purpurea that contains a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 15. イソメラーゼ(easA_Cp) comprises a sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO: 18, and easA from Epichloe coenophiala イソメラーゼ (easA_Ec) comprises a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:17.

[0089] More specifically, easA イソメラーゼ easA from Epichloe coenophiala イソメラーゼ (easA_Ec). In some embodiments, easA イソメラーゼ comprises a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:17.

[0090] easA イソメラーゼ It will be appreciated that the present invention relates to a method for the preparation of agroclavine comprising the steps of: (a) expressing an enzyme that catalyzes the conversion of chanoclavine I-aldehyde to agroclavine;

[0091] Further, the isolated recombinant cell comprises cloA. For example, the isolated recombinant cell comprises cloA from Claviceps paspalii (CloA_Cpas), cloA from N. lolii (CloA_Nlol), cloA from P. ipomoeae (CloA_Pipo), cloA from E. coenophiala (CloA_XN6), and / or cloA from C. purpurea (CloA_Cpur). In some embodiments, cloA comprises cloA from E. coenophiala (CloA_XN6) and / or cloA from C. purpurea (CloA_Cpur).

[0092] As a further example, cloA from E. coenophiala (CloA_XN6) may comprise a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 27 or 28, and cloA from C. purpurea (CloA_Cpur) comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 22, 23 or 24.

[0093] In particular, cloA includes cloA from C. purpurea (CloA_Cpur). More particularly, cloA from C. purpurea (CloA_Cpur) includes a sequence having at least 80%, at least 85%, at least 90%, at least 95% identity to SEQ ID NO:23.

[0094] It will be appreciated that cloA expresses an enzyme in recombinant cells, which in particular catalyzes the conversion of agroclavine to D-lysergic acid (DLA).

[0095] The recombinant cell can contain two or more genes encoding enzymes in the biosynthetic pathway from tryptophan to D-lysergic acid (DLA).

[0096] For example, the recombinant cell may contain at least one easE, at least one easA イソメラーゼ , and at least one cloA.

[0097] In this example, easE may include easE from Aspergillus japonicus (easE_Aj) or easE from Aspergillus indologenus, and easA イソメラーゼ easA from Epichloe coenophiala イソメラーゼ(easA_Ec), and cloA may comprise cloA from C. purpurea (CloA_Cpur). The recombinant cell may further comprise at least one dmaW, at least one easF, and at least one easD. In particular, dmaW may comprise a sequence at least 80%, at least 85%, at least 90%, at least 95% identical to SEQ ID NO:1, easF may comprise a sequence at least 80%, at least 85%, at least 90%, at least 95% identical to SEQ ID NO:2; and easD may comprise a sequence at least 80%, at least 85%, at least 90%, at least 95% identical to SEQ ID NO:4.

[0098] It will be appreciated that dmaW, easF, and easD each express their respective enzymes, particularly, each enzyme is functional.

[0099] At least one easE, at least one easA イソメラーゼ In addition to containing at least one cloA, the recombinant cell further contains at least one easC and / or at least one easG. In particular, easC comprises a sequence at least 80%, at least 85%, at least 90%, at least 95% identical to SEQ ID NO:3, and easG comprises a sequence at least 80%, at least 85%, at least 90%, at least 95% identical to SEQ ID NO:5.

[0100] In a specific embodiment, at least one dmaW, at least one easF, at least one easE or easC, at least one easD, at least one easA イソメラーゼ Alternatively, an isolated recombinant cell is provided that contains at least one easG, easG, and at least one cloA gene.

[0101] As a second specific embodiment, at least one dmaW, at least one easF, at least one easE, at least one easD, at least one easA イソメラーゼand at least one cloA gene.

[0102] As a third specific embodiment, at least one dmaW, at least one easF, at least one easC, at least one easD, at least one easA イソメラーゼ An isolated recombinant cell is provided that contains at least one easG and at least one cloA gene.

[0103] dmaW comprises a sequence at least 80%, at least 85%, at least 90%, at least 95% identical to SEQ ID NO:1; easF comprises a sequence at least 80%, at least 85%, at least 90%, at least 95% identical to SEQ ID NO:2; easE comprises a sequence at least 80%, at least 85%, at least 90%, at least 95% identical to SEQ ID NO:6; easC comprises a sequence at least 80%, at least 85%, at least 90%, at least 95% identical to SEQ ID NO:3; easD comprises a sequence at least 80%, at least 85%, at least 90%, at least 95% identical to SEQ ID NO:4; easA イソメラーゼ It will be understood that: comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95% identical to SEQ ID NO:17; easG comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95% identical to SEQ ID NO:5; and cloA comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95% identical to SEQ ID NO:23.

[0104] The recombinant cell of the present disclosure can be further designed to incorporate genes that enhance the production of FAD. In some embodiments, the isolated recombinant cell described herein further comprises FAD1 gene and PDI1 gene. In some embodiments, the isolated recombinant cell further comprises multiple copies of FAD1 gene and PDI1 gene. In some embodiments, FAD1 comprises a sequence with at least 80%, at least 85%, at least 90%, at least 95% identity to SEQ ID NO:35, and PDI1 comprises a sequence with at least 80%, at least 85%, at least 90%, at least 95% identity to SEQ ID NO:36.

[0105] It will be understood that an isolated recombinant cell can express one or more genes. Expression of one or more genes produces a respective gene product. In particular, each gene product is functional. More particularly, each gene product is a respective enzyme.

[0106] It will be understood that any suitable cell may be used for the recombinant cell. The recombinant cell may be any eukaryotic recombinant cell. For example, the recombinant cell may be a recombinant yeast cell. In particular, the recombinant cell may be from Saccharomyces sp. More particularly, the recombinant cell may be a recombinant Saccharomyces cerevisiae.

[0107] In one particular embodiment, the isolated recombinant yeast cell is strain DLAM33B.

[0108] Also described herein is a method for culturing a recombinant cell of the present disclosure. In one aspect, a method for culturing a recombinant cell of any one of the previous aspects in a suitable medium is provided. In particular, the method comprises culturing the recombinant in a suitable medium.

[0109] It will be understood by those skilled in the art that the recombinant cells of the present disclosure can be cultured in media such as, but not limited to, SC medium, SM medium, YPD medium, YPG medium, and YPAD medium. In some embodiments, the medium is SC medium.

[0110] In some embodiments, the method is suitable for producing 4-dimethylallyl-L-tryptophan (DMAT), 4-dimethylallyl-L-abrine (4DMA), chanoclavine I, chanoclavine I-aldehyde, agroclavine, and / or D-lysergic acid.

[0111] In some embodiments, the method is suitable for preparing D-lysergic acid.

[0112] Unless otherwise indicated or clear from the context or understanding of one of ordinary skill in the art, values ​​expressed as ranges can assume any particular value or subrange within the stated range in various embodiments, to the tenth of the unit of the lower limit of the range, unless otherwise clearly indicated from the context. "About" in relation to a numerical value generally refers to a range of values ​​within ±10%, in some embodiments ±5%, in some embodiments ±1%, and in some embodiments ±0.5% of the numerical value, unless otherwise specified or clear from the context. In embodiments where a numerical value is preceded by "about", an embodiment is provided in which the exact numerical value is described. When an embodiment is provided in which a numerical value is not preceded by "about", an embodiment is also provided in which the numerical value is preceded by "about". When a range is preceded by "about", an embodiment is provided in which "about" applies to either the lower limit and upper limit, or the lower limit and upper limit, of the range, unless otherwise clearly indicated from the context. When a phrase such as "at least," "up to," or "no more than" precedes a series of numerical values, it is to be understood that the phrase applies to each numerical value in the list in various embodiments unless the context clearly dictates otherwise (with the understanding that the context may provide an upper limit of 100% of the value, e.g., a value expressed as a percentage). For example, "at least 1, 2, or 3" is to be understood to mean "at least 1, at least 2, or at least 3" in various embodiments. It is also to be understood that all reasonable lower and upper limits are expressly contemplated.

[0113] Having now generally described the invention, the same will be more readily understood by reference to the following examples, which are provided for purposes of illustration and are not intended to limit the invention. EXAMPLES

[0114] Standard molecular biology techniques known in the art, unless otherwise specified, were generally followed as described in Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2001).

[0115] Example 1 Materials and Methods Culture, medium, and strains E. coli XL1Blue (Stratagene), E. coli NEB Stable (New England Biolabs), and S. cerevisiae strain BY4741 were used as base strains in this experiment. E. coli constructs were grown in lysogeny broth (LB) at 37°C with the appropriate antibiotics. Competent E. coli cells were prepared and transformed according to the Inoue protocol, with the modification that cells were grown to mid-log phase at 30°C before further processing (Inoue, H., et al.; Gene 96, 23-28 (1990)). Yeast strains were grown in yeast extract-peptone-dextrose (YPD) medium or synthetic complete (SC) medium minus the appropriate nutrients for selection (Treco, DA & Lundblad, V.; Curr. Protoc. Mol. Biol. 23, 13.11.11-13.11.17 (1993)). Transformation of plasmids and DNA fragments for chromosomal integration in S. cerevisiae was carried out using a lithium acetate / PEG-3350 / single-stranded carrier DNA protocol (Gietz, RD & Schiestl, RH; Nat. Protoc. 2, 1 (2007)).

[0116] Assembly of modified yeast Fab plasmids The pathway acceptor plasmid and genome integration plasmid were constructed by Gibson assembly (Gibson, DG et al.; Nature methods, 6(5), 343-345 (2009)). The pathway acceptor plasmid was assembled from four fragments, each of which contained several core elements (Kan R The genome integration plasmid contains five fragments: a URA3 selection marker flanked by URR sites, an RFP expression cassette with an insertion site, an Amp R and ColE1 origin, upstream and downstream genome integration homology regions. All fragments were PCR amplified (Takara PrimeSTAR (商標) The plasmids were verified by restriction enzyme digestion and Sanger sequencing.

[0117] Golden Gate Assembly of Parts and Paths The Golden Gate assembly used in this study largely follows existing published protocols, except for some modifications (Engler, C. et al., PloS one 4, e5553 (2009); Tan, YQ et al., Biomacromolecules (2021)). Reactions were prepared as 10 μL pots, each containing 1 μL of 10X T4 ligase buffer (New England Biolabs), 1 μL of 100X bovine serum albumin (New England Biolabs), 5 U of restriction enzyme (BsaI-HFv2 or Esp3I, New England Biolabs), 10 U T4 DNA ligase (New England Biolabs), 15 ng of the desired plasmid, 1 μL per insert, and made up to 10 μL with sterile deionized distilled water (ddH2O). The assembly reaction was subjected to 25 cycles of 5 min at 37 °C and 10 min at 25 °C, followed by 20 min at 55 °C and an additional 20 min at 80 °C. The reaction mixture was then used directly for transformation of chemically competent XL1Blue (level 0 / 1 constructs) or NEB Stable (level 2 and above).

[0118] Yeast promoter and terminator parts were PCR amplified from S. cerevisiae S288C genomic DNA and cloned into HcKan_P and HcKan_T, respectively. For simplicity, promoter and terminator sequences were defined as 500 base pairs (bp) upstream or downstream of the ORF used to name the genetic element. Genes encoding pathway enzymes were codon-optimized for yeast expression, synthesized, and cloned into HcKan_O. FAD1 and PDI1 genes were PCR amplified from S. cerevisiae S288C genomic DNA (Takara PrimeSTAR (商標) ) and cloned into HcKan_O. All assembled Level 0 and Level 1 constructs were verified by colony PCR using Taq DNA polymerase (New England Biolabs) and Sanger sequencing, and Level 2 and genomic integration constructs were verified by colony PCR.

[0119] Small scale yeast cultures for the production of ergot alkaloids. For each assay construct, triplicate freshly isolated transformed or streaked colonies were used to inoculate 10 mL precultures of the appropriate growth medium in 50 mL tubes and grown for at least 18 h at 30°C in a shaking incubator at 210 rpm. These cultures were then cultured in 10 mL of the appropriate SC medium supplemented with 0.1% (w / v) D-glucose until a final OD 600 The OD was then adjusted to 0.0125. 600 After incubation until the β-amyloid ratio reached 0.8, filter-sterilized 20% (w / v) stock solution of galactose was added to a final concentration of 2% (w / v). The caps of the culture tubes were then replaced with autoclaved aluminum foil caps and incubated at 24°C for 120 h. Cultures were then pelleted by centrifugation at 4000 rpm for 10 min. A 1 mL aliquot of the supernatant from each sample was filtered through a 0.2 um PTFE syringe filter and analyzed using liquid chromatography-tandem mass spectrometry (LC491 MS / MS).

[0120] Fed-batch fermentation production of DLA from engineered yeast The media used in the 4L and 1L fermentations consisted primarily of SC-URA supplemented with 0.1% (w / v) glucose and 50 mM ammonium succinate, pH 5.8 (SCUS). Feed 1 consisted of 10X SC-URA amino acid mix and 10X yeast nitrogen base, and Feed 2 consisted of 20% (w / v) galactose and 100 mM ammonium succinate, pH 5.8.

[0121] Seed cultures were prepared by culturing a single colony of DLAM33B from a freshly streaked plate in 10 mL of SC-URA medium supplemented with 2% (w / v) glucose overnight at 30°C. Fermentations were performed in fermentation vessels (INFORS HT Minifors 2, Bottmingen, Basel, Switzerland) filled with 2 L or 500 mL SCUS until a final OD 600 The incubation period was started by inoculating the seed bacteria so that the β-

[0122] The fermentation process was carried out at 30 °C, with stirring at 1000 rpm and compressed air (Ekom DK40 2V, Singapore) at 1 volumetric airflow per minute (4 L min−1). -1 Or 1L -1 The initial growth phase (phase 0) was started by supplying either 0 or 100 mL of ethanol, and the dissolved oxygen (DO) level was adjusted by increasing the agitation speed to 1500 rpm and the airflow to 2 volumes per minute (8 L min−1). -1 Or 2L -1 The fermentation was maintained above 90% saturation by an automated cascade of increasing the concentration of glucose to 3.5 mL min−1. This stage starved the presence of glucose and allowed the culture to grow to a cell density suitable for induction. After 24 hours, the inflow of each feed was increased from 1 part to 8 parts of starting culture volume by 3.5 mL min−1. -1 Both feeds 1 and 2 were flowed into the vessel at 0°C to initiate the induction phase (Phase I). The temperature was also reduced to 24°C for pathway induction. Phases 0 and I directly mimic the conditions used for pathway induction at shake flask scale. Phase II was preprogrammed to start 20 hours after Phase I, when the galactose supplemented in Phase I is expected to begin to exponentially decrease (Sanchez, RG et al., Microbial Cell Factories 9, 1-8 (2010)). During this phase, both Feed 1 and Feed 2 were flowed at 0.05 mL min−1. -1 (1L) or 0.18mL -1A steady low-level flow rate of (4 L) was maintained for 28 h to maintain sufficient nutrients in the culture and ensure sufficient expression of pathway genes. In phase III or starvation stage, no additional feed was supplemented and fermentation was continued for an additional 2 days. Fermentation was monitored by taking 10 mL samples daily to assess wet cell mass (WCM) and DLA production titers.

[0123] Screening for cloA orthologues Genes encoding various cloA orthologues were synthesized (BioBasic) and cloned into the pYES2 / CT vector (Invitrogen). Cells were cultured and expression was induced as described previously, except that a 1 mL aliquot of induced cells was removed from each tube and transferred to a 1.5 mL microcentrifuge tube. Cells were then pelleted by centrifugation at 21000 g for 1 min. The pellet was resuspended in 1 mL of phosphate-buffered saline (PBS, pH 7.4). Agroclavine was added to each tube to a final concentration of 5 μM and incubated overnight at 30°C. The PBS incubations were then pelleted by centrifugation, and the supernatants from each sample were filter-sterilized through 0.2 um PTFE syringe filters and similarly analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0124] 13 C-2-indole-L-tryptophan 13 C-labeling experiments 13 A stock solution of C-2-indole-L-tryptophan (Sigma) was prepared by dissolving the powder in 20% (w / v) galactose to a final concentration of 10 mM and filter sterilizing. 13 C labeling was performed using the same protocol as described for the production of ergot alkaloids, but with 1H-galactose instead of 1H-galactose alone. 13 Use a stock solution of C-2-indole-L-tryptophan to a final concentration of 1 mM 13The negative control was derived from C-2-indole-L-tryptophan and 2% (w / v) galactose. 13 A similar preparation was used using L-tryptophan (Sigma) instead of C-2-indole-L-tryptophan.

[0125] Analysis of ergot alkaloids by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) All samples were analyzed using an Agilent 1290 Infinity LC System coupled with an Agilent 6550 iFunnel QTOF equipped with an electrospray ionization source. Samples were separated using an Agilent InfinityLab Poroshell 120 EC-C18 column with dimensions 2.1X100mm and particle size 1.9μm. The mobile phases used were as follows: A, water with 0.1% formic acid; B, acetonitrile with 0.1% formic acid. Chromatography was performed with a constant flow rate of 0.5mL / min, injection volume of 1μL, and the following step gradient: 95%A / 5%B for 0.6min, 65%A / 35%B for 2.6min, 1%A / 99%B for 4.6min. The column was washed with 100%B for 2min, followed by re-equilibration with 95%A / 5%B for 1min.

[0126] For mass data acquisition, the eluent polarity (positive) was fixed and the run was set to target MS / MS mode from 1 min to 4.5 min. The instrument parameters were set to run as follows; source gas temperature 200 °C with flow rate 10 L / min, sheath gas temperature 350 °C with flow rate 10 L / min, and nebulizer pressure 50 psig. The capillary voltage was set to 4000 V and nozzle voltage to 0 V. MS1 was set to a mass range of 40-1000 m / z with a scan rate of 3 spectra / s. MS2 was set to a mass range of 40-1000 m / z with a fixed collision energy of either 10 eV for screening experiments or 20 eV for analysis of reconstituted strains with a scan rate of 6 spectra / s.

[0127] The target mass for screening easE orthologues to produce chanoclavine I was set at 257.1648 m / z with a narrow isolation bandwidth (1.3 amu). For screening easA isomerase mutants, the target mass was set at 239.1543 m / z with a narrow isolation bandwidth (1.3 amu). For screening cloA orthologues, the target masses were set to: 1) 239.1543 m / z, narrow isolation bandwidth (1.3 amu); 2) 269.1285 m / z, narrow isolation bandwidth (1.3 amu). For the ergot alkaloid pathway reconstitution assay, the target masses were set as follows: 1) 287.1754 m / z, narrow isolation width (1.3 amu); 2) 257.1648 m / z, narrow isolation width (1.3 amu); 3) 239.1543 m / z, narrow isolation width; 4) 269.1285 m / z, narrow isolation width (1.3 amu). 13 To detect incorporation of C-labeled tryptophan, the target masses were set as follows: 1) 269.1285 m / z, narrow isolation width (1.3 amu), 2) 270.1318 m / z, narrow isolation width. All data analysis and instrument control were performed using the Mass Hunter software suite (Agilent).

[0128] Compound identities were determined by comparison of retention times and MS / MS product ion spectra with commercially available standards (D-lysergic acid; Chiron) (agroclavine; Chiron / Toronto Research Chemicals). Quantification of 4DMAT, 4DMA, and chanoclavine I was performed by comparison of retention times and MS / MS product ion spectra to their in vitro biosynthetic products in cell extracts expressing purified dmaW, easF, easC, and easE_Aj. Quantification of produced agroclavine and DLA was performed by standard curves constructed from commercially available standards or by standard addition. Agroclavine was quantified by monitoring the transition from the precursor ion at 239 m / z to 208 m / z, and DLA was quantified by monitoring the transition from the precursor ion at 269 m / z to 223 m / z. Linear regression analysis of standard curves was performed using Graphpad Prism version 7.00 for Windows (Graphpad Software, San Diego, CA).

[0129] Bioinformatics Analysis All SSNs used in this experiment were generated using a protein sequence query in an initial BLAST search (option A) via the EFI-EST web tool (Zallot, R. et al., Current opinion in chemical biology 47, 77-85 (2018)), with parameters set to search up to 9000 sequences with a minimum alignment E-value of 5. An initial network was calculated by defining edges representing relationships with an alignment score equivalent to 40% or higher sequence identity, and each SSN was then individually refined by increasing edge scores until the "hair ball" was fragmented into smaller hypothetical iso-functional clusters. Manipulation and visualization of SSNs was performed using Cytoscape software (Shannon, P. et al. Genome research 13, 2498-2504 (2003)). Selected sequences were retrieved from the Uniprot database using the Uniprot numbers associated with the SSNs.

[0130] Flow cytometric analysis of yeast promoter libraries The promoter reporter plasmid pGlo3 containing the promoter library insert was transformed into yeast BY4741 cells. Three individual colonies were picked from the transformants and grown in liquid SC-URA at 25 °C and 220 rpm for 30 h until saturation (OD ≈ 3). Fresh medium was then inoculated with a 1:200 dilution of the saturated cell culture and grown at 25 °C for 12 h. At this point, the optical density at 600 nm (OD 600 ) reached approximately 0.9–1.2, corresponding to the exponential growth phase. 20 μL of cell culture was added to 180 μL of ice-cold PBS, and the 96-well plate containing the samples was kept at 4 °C until flow cytometry analysis within 4 h. The remaining cell cultures were incubated at OD 600 The cells were incubated at 25 °C for an additional 6 h until the β-actin ratio reached approximately 2.0–2.5, which corresponds to the early stationary phase. Similarly, 20 μL of the culture was diluted with 180 μL of ice-cold PBS and kept cold until analysis.

[0131] The yeGFP and mKOκ fluorescence of each cell was analyzed by BD Accurio. (商標) The measurements were performed using a flow cytometer. For each sample, 20,000 cells were measured, and the flow rate was approximately 2,000 cells s -1 For each batch of samples, P PGK1 A strain expressing a driven yeGFP and another strain expressing mKOκ with a high-copy 2μ plasmid were included as fluorescence compensation controls, while a strain containing the plasmid pCKU (which does not express a fluorescent protein) was included to account for background fluorescence. Results were analyzed using FlowJo (version 10) software. Fluorescence bleed-through between the green and orange emission channels was first compensated for using a 2μ plasmid control. The signal readout from promoter activity was then calculated as the geometric mean of the orange emission of the plasmid-carrying strains (identified by yeGFP emission) minus the background orange emission measured using the pCKU strain.

[0132] Expression and purification of dmaW and easF from E. coli for in vitro assays The genes encoding these two proteins were cloned into the pET15B vector and recombinantly expressed in E. coli BL21(DE3) cells. Expression was observed at OD 200 / mL in 2YT medium at 37°C. 600 Cells were grown to a pH of 0.7 and then induced with IPTG (2 mM) for 20 h at 20 °C. Cells were then pelleted by centrifugation at 5000 rpm for 10 min at 4 °C. Pelleted cells were resuspended in binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) at 4 °C and lysed by sonication. The cell debris was then clarified by centrifugation at 15000 rpm for 20 min at 4 °C. All subsequent purification steps were performed at 4 °C. The clarified cell supernatant was transferred to 200 μL of NiCl equilibrated in binding buffer. 2+-NTA chelating Sepharose resin. This was incubated for 30 min with shaking at 140 rpm. The resin was washed three times with 2 mL of wash buffer (60 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) for 5 min for each wash step with shaking at 90 rpm. Bound proteins were eluted from the resin twice with 500 μL of His-elution buffer (100 mM L-histidine, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) for 10 min with shaking at 70 rpm. Fractions were analyzed by SDS-PAGE and fractions containing protein were concentrated using a 3 kDa molecular weight cut-off (MWCO) Ultra-0.5 spin filter (Amicon). The purified solution was dialyzed against a storage buffer containing 50 mM Tris-HCl, pH 7.5, 5 mM CaCl2, and 50% glycerol, and stored at -20°C.

[0133] In vitro biosynthesis of DMAT was prepared in a 60 μL reaction mixture containing 50 mM Tris-HCl, pH 7.5, 5 mM CaCl2, 1 mM L-tryptophan, 1 mM DMAPP, and 10 μL of purified dmaW. The reaction was incubated at 30 °C for 18 h. The reaction was stopped by filtration of the enzyme using a 3 kDa MWC7O Ultra-0.5 spin filter (Amicon). Samples were stored at -20 °C or immediately analyzed by LC-MS. In vitro biosynthesis of 4DMA was similarly prepared, but with the addition of 1 mM SAM and 10 μL of purified easF.

[0134] Nuclear magnetic resonance (NMR) analysis of biosynthesized DLA Cell cultures to produce DLA for NMR analysis were performed as previously described. DLA was purified from culture medium (8 L) by first lyophilizing the harvested clarified medium. The dried culture medium was reconstituted with 300 mL of ddH2O and purified by liquid chromatography using an AKTA Pure 25M (Cytiva) equipped with a C18 preparative column (Agilent Zorbax Eclipse XDB-C18, semi-preparative; 9.4 x 646 250 mm, 5 μm particle size). The mobile phase consisted of: A, water with 0.1% trifluoroacetic acid; B, acetonitrile with 0.1% trifluoroacetic acid. Semi-preparative chromatography was performed with a constant flow rate of 2 mL / min, injecting 2 mL, with a stepwise gradient as follows: 90% A / 10% B for 10 min, 90% A / 10% B to 80% A / 20% B for 50 min. Between runs, the column was washed with 4 column volumes (80 mL) of 100% B at a flow rate of 10 mL / min, followed by 4 CV re-equilibration to 90% A / 10% B at a flow rate of 2 mL / min. Elution of DLA was monitored by absorbance at 310 nm, and fractions corresponding to the peak at 310 nm were collected and pooled. The pooled fractions were concentrated by lyophilization, and a 150 μL aliquot was taken for LC-MS / MS analysis of purity and confirmation of the presence of DLA. The remaining pooled fractions were lyophilized and stored at -20°C.

[0135] Samples for NMR analysis were prepared by adding 2 mL of DO (Sigma) to the dried fraction and removing insoluble material by centrifugation at 4000 rpm for 20 min. Then, 1 mL of DO saturated with the sample was used for analysis of 1H-NMR spectra using a Bruker AVANCE 500 MHz NMR spectrometer at the Department of Chemistry, National University of Singapore.

[0136] result Biosynthetic resolution of the ergot alkaloid pathway. The complete biosynthesis of DLA from L-tryptophan requires eight enzymes encoded by the following genes - DmaW, EasF, EasC, EasE, EasD, EasA, EasG, and CloA. (Chen, J.-J. et al., RSC Advances 7, 27384-27396 (2017)). The conversion of DmaW to EasD has been biochemically characterized (Figure 1) (Chen, J.-J. et al., RSC Advances 7, 27384-27396 (2017); Metzger, U. et al., Proceedings of the National Academy of Sciences 106, 14309-14314 (2009); Rigbers, O. & Li, S.-. M., Journal of Biological Chemistry 283, 26859-26868 (2008); Nielsen, CA et al., Microbial cell factories 13, 1 (2014); Wallwey, C. et al., Archives of microbiology 192, 127-134 (2010)). All ergot alkaloids are derived from L-tryptophan and share a series of initial biosynthetic steps that form the ergoline C ring. The presence of various isoforms of enzymes involved in the middle and late biosynthetic steps from different species of ergot-producing organisms determines the product profile produced by each of these organisms (Cheng, JZ et al., Journal of the American Chemical Society 132, 1776-1777 (2010)). However, it has been reported that some enzymes are not amenable to heterologous expression (Nielsen, CA et al., Microbial cell factories 13, 1 (2014); Cheng, JZ et al., Journal of the American Chemical Society 132, 1776-1777 (2010)).Therefore, we searched for alternative orthologs of these enzymes (encoded by the genes easE, easA, and cloA) to reconstruct the pathway using EFI-ESTs (Gerlt, JA et al., Biochimica Et Biophysica Acta (BBA)-Proteins and Proteomics 1854, 1019-1037 (2015); Zallot, R. et al., Biochemistry 58, 4169-4182 (2019)) (Figure 12).

[0137] Example 2: Screening for functional expression of easE in yeast The enzymes EasC and EasE have been reported in previous publications from several groups to be essential for the conversion of 4DMA to chanoclavine I (Nielsen, CA et al., Microbial cell factories 13, 1 (2014); Lorenz, N. et al., Appl. Microbiol. 76, 1822-1830 (2010); Goetz, KE et al., Current genetics 57, 201 (2011); Kozikowski, AP et al., Journal of the American Chemical Society 115, 2482-2488 (1993)). However, EasE from most ergot-producing fungi has been shown to have suboptimal activity in heterologous yeast systems (Nielsen, CA et al., Microbial cell factories 13, 1 (2014)). To date, only the EasE orthologue from Aspergillus japonicus (EasE_Aj), reported by Nielsen, CA et al. (2014), has been functionally expressed in S. cerevisiae.

[0138] Therefore, to identify further active EasE orthologues, we used this orthologue to generate a Sequence Similarity Network (SSN). We then identified a putative isofunctional cluster around EasE_Aj and selected eight sequences to screen for expression and enzymatic function. To facilitate this screening, we generated a modified strain (YMC17; Supplementary Table 9) in which the genes for dmaW, easF, and easC were stably integrated into the genome at the YMRWδ15 site. We then transformed the eight EasE orthologues on episomal vectors into YMC17. Of the eight orthologues, only the orthologue from Epichloe coenophiala (EasE_Ec) and EasE_Aj showed detectable production of chanoclavine I (Figure 2B, Figure S13). The relative amount of chanoclavine I produced by EasE_Ec was 10-fold lower than that produced by easE_Aj (Figure 2C). Nevertheless, both sequences could be used to complete the ergot alkaloid pathway in yeast.

[0139] Example 3: Identification of easA isomerase mutants The next biosynthetic step in the construction of the DLA pathway involves a branch point linking tricyclic clavines to tetracyclic clavines: isoforms of EasA from different lineages of ergot alkaloid-producing organisms catalyze either the reduction or the cis-trans isomerization of the C8-C9 double bond of the ergoline moiety, while EasG combines the aldehyde group with a methylamino group to form the ergoline D ring (Floss, HG et al., Journal of the American Chemical Society 90, 6500-6507 (1968)). Reduction or maintenance of the C8-C9 double bond at the end of this process depends on the EasA isoform, with the pathway branching towards either agroclavine, festuclavine, pyroclavine, or, if certain orthologues of EasH are present, cycloclavine (Figure 3A) (Cheng, JZ, et al., Journal of the American Chemical Society 132, 1776-1777 (2010); Floss, HG, et al., Journal of the American Chemical Society 90, 6500-6507 (1968); Cheng, JZ, et al., Journal of the American Chemical Society 132, 12835-12837 (2010)).

[0140] The EasA isomerase mutant is required to redirect metabolic flux into the agroclavine branch and then into DLA. As in the previous study, we created an EasA SSN using sequences from C. purpurea and identified an isofunctional cluster of isomerases. An earlier paper by Cheng, JZ et al. (2010) reported the importance of the F176 residue, and four sequences with structurally corresponding F176 residues from this cluster were selected and synthesized. These orthologs were screened by co-expressing easD and easG in a strain in which dmaW, easF, easC, and easE_Aj were integrated into the yeast genome (YOCE; Figure 32).

[0141] All easA orthologs have an [M+H] of 239 m / z, which corresponds to the agroclavine standard. + The strains expressing the different EasA orthologs produced a compound with the same structure (Figure 3B). Further comparison of the MS / MS fragmentation spectra confirmed that the eluted compound was agroclavine (Figure 14). The amount of agroclavine produced by strains expressing different EasA orthologs, EasA_Ec, EasA_Cp, and EasA_Nl, was approximately 2.8-3.1 μg / L, whereas the strain containing the EasA_Pi ortholog produced approximately 7-fold less agroclavine (Figure 15), and no production of alternative products was observed for any of the selected EasA orthologs. From these results, three equally suitable EasA candidates that could be used to construct the DLA pathway were identified; to simplify future steps, EasA_Ec was selected for further pathway construction.

[0142] Example 4: Screening of functional agroclavine oxidase for DLA production To complete the biosynthetic pathway of DLA, we addressed the oxidation of agroclavine to DLA. This two-step oxidation was proposed to be catalyzed by the cytochrome P450 (CYP450) monooxygenase clavine oxidase (CloA) (Haarmann, T. et al., ChemBioChem 7, 645-652 (2006)), but the reaction mechanism has not yet been biochemically elucidated. A diverse set of oxidized agroclavine products has been isolated from numerous ergot alkaloid-producing fungi. These products are characterized by a single oxidation (erimoclavine / lysergol: [M+H]) with the possibility of isomerization from the C8-C9 double bond (erimoclavine / pasparic acid) to the C9-C10 position (lysergol / DLA) (Figure 4A). + = 255 m / z) or double oxidation (pasparic acid / DLA: [M+H] + = 269 m / z).

[0143] We identified orthologs that express in yeast and specifically produce DLA. Using SSNs generated from the predicted cloA sequence from C. purpurea, we identified 15 orthologs from a cluster of sequences from organisms in which DLA has been isolated. These 15 orthologs were screened for functional expression in yeast (Figure 4B). Upon feeding with agroclavine substrate, 5 of the 15 orthologs shared the same retention time [M+H] with the DLA standard. + = 269 m / z (Figure 4C), which also showed an MS / MS fragmentation spectrum identical to that of the DLA standard, confirming that this molecule was DLA (Figure 16).

[0144] This screen identified five orthologs of cloA (C.pur., ​​C.pas., N.lol., E.coe, P.ipo.) that could be used for pathway construction. The top two producers (C.pur., ​​E.coe.) and the lowest producer (P.ipo.) from this screen (Figure 4) were then further tested for DLA production in the context of an agroclavine-producing host (Figures 17A, 17B). The C.purpurea and E.coenophila orthologs were found to produce similar levels of DLA for this test (Figure 17D), and the C.purpurea ortholog was selected for incorporation into the engineered strain due to its lower variability in DLA titers.

[0145] Example 5: Assembly of components to complete the DLA biosynthetic pathway in yeast We sought to construct a prototype DLA biosynthetic strain equipped with a set of functional enzymatic and genetic components for the reconstitution of the DLA biosynthetic pathway in yeast. The first prototype produced 529 mg L -1The prototype design was modeled on a strain developed for cycloclavine production that reportedly achieved impressive yields of 1000 kDa (Jakubczyk, D et al., Angewandte Chemie International Edition 54, 5117-5121 (2015)). The prototype design attempted to reduce the metabolic burden by using stronger promoters for less functional enzymes such as easE and multiple copies driven by weaker promoters for other pathway enzymes. It also contains additional copies of the yeast-derived FAD1 and PDI1 genes, which have been shown to aid in protein folding and promote the production of flavin adenine dinucleotide (FAD), a key cofactor for EasE activity (Nielsen, CA et al., Microbial cell factories 13, 1 (2014)).

[0146] Following the modified yeast Fab pipeline (Figure 7), prototype strains were sequentially constructed and expanded as four segments (Figure 18). Each segment was designed to produce easily detectable intermediate products along the biosynthetic pathway of DLA, namely, chanoclavine I, agroclavine, and DLA. This approach allowed for easy troubleshooting and allowed each intermediate strain to serve as a negative control for subsequent strains. Introduction of the first two segments (AgcM1B, AgcM2B; Figure 32), which contain genes of the early ergot pathway, resulted in the production of chanoclavine I (Figure 5A). Increasing the copy number of easC and dmaW, as well as the complementary expression of FAD1 and PDI1, improved the yield of chanoclavine I, as previously reported (Nielsen, CA et al., Microbial cell factories 13, 1 (2014)). In the third segment, additional copies of easG, easA_Ec, as well as easF, easC, and easD were introduced. This strain, AgcM33B, which has three segments integrated into the yeast genome, coelutes with the agroclavine standard [M+H]. += 239 m / z (Figure 5B), which was confirmed to be agroclavine by MS / MS fragmentation (Figure 19). The final reaction to DLA was incorporated by incorporation of cloA_Cpur as the last segment into the ARS208 site. LC-MS analysis of post-culture medium from this strain (DLAM33B; Figure 32) revealed a compound [M+H] co-eluting with the DLA standard. + = 269 m / z (Figure 5C), which was further confirmed by MS / MS fragmentation (Figure 20).

[0147] Next, the DLAM33B strain was used as the raw material. 13 Reconstitution of the pathway to produce DLA was verified by feeding C-2-indole-L-tryptophan (13C-W) and following the progression of tryptophan through the reconstituted pathway (Figure 21A). In these experiments, a 1 m / z shift was detected in the chromatogram peak corresponding to DLA, indicating that the naturally occurring 13 C-DLA was more abundant than expected (Figure 21B). The same mass shift was observed for all intermediates in the DLA biosynthetic pathway (Figure 22). These data indicate that 13 Incorporate CW into the route and then 13 C-DLA and its 13 The predicted [ 13 C-M+H] + The values ​​obtained were demonstrated. 13Comparison of the mass spectral patterns of C-DLA further showed this +1 / z shift across all major fragmentation peaks (Figure 22C, top), indicating that tryptophan is utilized to produce DLA in the introduced pathway. Considering that the base strain of S. cerevisiae used in this experiment is not tryptophan autotrophic, this experiment shows that the engineered strain can produce DLA from tryptophan derived from central metabolism or medium. The next step to verify the production of DLA is NMR analysis. To achieve this, a scale-up of the shake flask process was performed to obtain the required material. The chemical shifts of the concentrated extract were assigned based on the predicted values ​​and compared to the DLA standard (Figure 34).

[0148] The end-point production titer of the DLAM33B strain under small-scale shake flask conditions was 71.5 μg L -1 was measured (Figure 23). To demonstrate the scalable application of the engineered strain, 1 L and 4 L scale bioreactor fermentations were further attempted. In these fermentation experiments, improved control of the culture oxygen supply, carbon source, and inducer (galactose was used as both the carbon source and expression inducer), as well as pH control, resulted in approximately 25–26 g L -1 Cell growth was sustained and production titers improved (Figure 6), reaching a maximum cell density of 2.0 mg L at the end of 1 L and 4 L fermentations, respectively. -1 and 1.6 mg L -1 (Figure 35). Therefore, further bioprocess and strain optimization could achieve commercial titers.

[0149] Consideration In this work, we describe the identification of alternative orthologues of fastidious enzymes along the ergot alkaloid pathway using SSNs generated from the EFI-EST web tool, along with the development of customized strains for their systematic screening. Through this approach, which is central to the tenets of synthetic biology, we successfully identified candidates for the reconstruction of the DLA pathway in S. cerevisiae. These candidates were then used to generate engineered yeasts capable of producing DLA from central metabolism. To the best of our current knowledge, this is the first demonstration of heterologous total biosynthesis of DLA from simple sugars. Advantageously, the production costs may be reduced by providing an engineered microorganism capable of producing lysergic acid directly from simple sugars and suitable for commercial-scale fermentation. Further optimization of the strain aimed at identifying and eliminating bottlenecks in the pathway, as well as improved optimization of the bioprocess, will undoubtedly boost the production titer to commercially required levels.

[0150] This effort builds on a growing body of work demonstrating the use of industrially tractable microorganisms for the production of complex natural products using economical and renewable feedstocks, as has been done for opioid biosynthesis (Galanie, S. et al., Science 349, 1095-1100 (2015)). The engineered strains provide an excellent platform to drive the discovery of semisynthetic therapeutic leads, as well as for the development of pilot strains to produce important naturally derived therapeutics.

[0151] Finally, given the recent resurgence of research into the use of hallucinogenic compounds for antidepressant and anxiolytic applications (De Gregorio, D. et al., Elsevier Vol. 242 69-96 (2018)), the recombinant strain as described could be used to aid efforts in exploring ergoline-based therapeutics in natural and semi-synthetic chemical space and to identify lead compounds with improved therapeutic potential and fewer side effects.

[0152] The teachings of all patents, publications, and references cited herein are incorporated by reference in their entirety.

[0153] While the present invention has been particularly shown and described with reference to illustrative embodiments thereof, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as encompassed by the appended claims.

[0154] References The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the art or is common general knowledge to those skilled in the art. Chen, J.-J., Han, M.-Y., Gong, T., Yang, J.-L. & Zhu, P. Recent progress in ergot alkaloid research. RSC Adv. 7, 27384-27396 (2017). Cheng, JZ, Coyle, CM, Panaccione, DG & O'Connor, SE A role for old yellow enzyme in ergot alkaloid biosynthesis. J. Am. Chem. Soc. 132, 1776-1777 (2010). Cheng, JZ, Coyle, CM, Panaccione, DG & O'Connor, SE Controlling a structural branch point in ergot alkaloid biosynthesis. J. Am. Chem. Soc. 132, 12835-12837 (2010). Cvak, L. In Ergot: the genus Claviceps 373 (CRC Press, 1999). De Gregorio, D., Enns, J. P., Nunez, N. A., Posa, L. & Gobbi, G. In Progress in brain research, 242 69-96 (Elsevier, 2018). de Groot, A. N., van Dongen, P. W., Vree, T. B., Hekster, Y. A. & van Roosmalen, J. Ergot alkaloids. Drugs 56, 523-535 (1998). Engler, C., Gruetzner, R., Kandzia, R. & Marillonnet, S. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PloS one 4, e5553 (2009). Floss, H. G. et al. Biosynthesis of ergot alkaloids. Evidence for two isomerizations in the isoprenoid moiety during the formation of tetracyclic ergolines. J. Am. Chem. Soc. 90, 6500-6507 (1968). Galanie, S., Thodey, K., Trenchard, I. J., Interrante, M. F. & Smolke, C. D. Complete biosynthesis of opioids in yeast. Science 349, 1095-1100 (2015). Gerlt, J. A. et al. Enzyme function initiative-enzyme similarity tool (EFI-EST): A web tool for generating protein sequence similarity networks. Biochimica Et. Biophysica Acta (BBA)-Proteins Proteom. 1854, 1019-1037 (2015). Gibson, D.G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature methods 6, 343-345 (2009). Gietz, R. D. & Schiestl, R. H. Frozen competent yeast cells that can be transformed with high efficiency using the LiAc / SS carrier DNA / PEG method. Nat. Protoc. 2, 1 (2007). Goetz, K. E., Coyle, C. M., Cheng, J. Z., O’Connor, S. E. & Panaccione, D. G. Ergot cluster-encoded catalase is required for synthesis of chanoclavine-I in Aspergillus fumigatus. Curr. Genet. 57, 201 (2011). Haarmann, T., Ortel, I., Tudzynski, P. & Keller, U. Identification of the cytochrome P450 monooxygenase that bridges the clavine and ergoline alkaloid pathways. ChemBioChem 7, 645-652 (2006). Hendrickson, J. B. & Wang, J. A new synthesis of lysergic acid. Org. Lett. 6, 3-5 (2004). Inoue, H., Nojima, H. & Okayama, H. High efficiency transformation of Escherichia coli with plasmids. Gene 96, 23-28 (1990). Jakubczyk, D. et al. Discovery and reconstitution of the cycloclavine biosynthetic pathway-enzymatic formation of a cyclopropyl group. Angew. Chem. Int. Ed. 54, 5117-5121 (2015). Kozikowski, A. P. et al. Probing ergot alkaloid biosynthesis: Intermediates in the formation of ring C. J. Am. Chem. Soc. 115, 2482-2488 (1993). Lieberman, A., Kupersmith, M., Estey, E. & Goldstein, M. Treatment of Parkinson’s disease with bromocriptine. N. Engl. J. Med. 295, 1400-1404 (1976). Liu, H. & Jia, Y. Ergot alkaloids: synthetic approaches to lysergic acid and clavine alkaloids. Nat. Prod. Rep. 34, 411-432 (2017). Lorenz, N., Olsovska, J., Sulc,M.& Tudzynski, P. Alkaloid cluster gene ccsA of the ergot fungus Claviceps purpurea encodes chanoclavine I synthase, a flavin adenine dinucleotide-containing oxidoreductase mediating the transformation of Nmethyl-dimethylallyltryptophan to chanoclavine I. Appl. Environ. Microbiol. 76, 1822-1830 (2010). Mantegani, S., Brambilla, E. & Varasi, M. Ergoline derivatives: receptor affinity and selectivity. Il Farm. 54, 288-296 (1999). Metzger, U. et al. The structure of dimethylallyl tryptophan synthase reveals a common architecture of aromatic prenyltransferases in fungi and bacteria. Proc. Natl Acad. Sci. 106, 14309-14314 (2009). Nielsen, C. A. et al. The important ergot alkaloid intermediate chanoclavine-I produced in the yeast Saccharomyces cerevisiae by the combined action of EasC and EasE from Aspergillus japonicus. Microb. cell factories 13, 1 (2014). Pertz, H. & Eich, E. Ergot alkaloids and their derivatives as ligands for serotoninergic, dopaminergic, and adrenergic receptors. Ergot: The Genus Claviceps. Amsterdam: Harwood Academic Publishers, 411-440 (1999). Rigbers, O. & Li, S.-M. Ergot alkaloid biosynthesis in Aspergillus fumigatus overproduction and biochemical characterization of a 4-dimethylallyltryptophan N-methyltransferase. J. Biol. Chem. 283, 26859-26868 (2008). Sanchez, R. G., Hahn-Hagerdal, B. & Gorwa-Grauslund, M. F. PGM2 overexpression improves anaerobic galactose fermentation in Saccharomyces cerevisiae. Microb. Cell Factories 9, 1-8 (2010). Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome research 13, 2498-2504 (2003). Tan, Y. Q. et al. Structure of a Minimal α-Carboxysome-Derived Shell and Its Utility in Enzyme Stabilization. Biomacromolecules (2021). Tandowsky, R. M. Clinical Evaluation of Combined Hydrogenated Ergot Alkaloids (Hydergine) in Arterial Hypertension: With Special Reference to Their Action in Central Manifestations. Circulation 9, 48-56 (1954). Treco, D. A. & Lundblad, V. Preparation of Yeast Media. Curr. Protoc. Mol. Biol. 23, 13.11.11-13.11.17 (1993). Umezaki, S., Yokoshima, S. & Fukuyama, T. Total synthesis of lysergic acid. Org. Lett. 15, 4230-4233 (2013). Wacker, D. et al. Crystal structure of an LSD-bound human serotonin receptor. Cell 168, 377-389. e312 (2017). Wallwey, C., Matuschek, M. & Li, S.-M. Ergot alkaloid biosynthesis in Aspergillus fumigatus: conversion of chanoclavine-I to chanoclavine-I aldehyde catalyzed by a short-chain alcohol dehydrogenase FgaDH. Arch. Microbiol. 192, 127-134 (2010). Winblad, B. et al. Therapeutic use of nicergoline. Clin. drug Investig. 28, 533-552 (2008). Zallot, R., Oberg, N.O. & Gerlt, J.A. ‘Democratized’genomic enzymology web tools for functional assignment. Current opinion in chemical biology 47, 77-85 (2018). Zallot, R., Oberg, N. & Gerlt, J. A. The EFI Web Resource for Genomic Enzymology Tools: Leveraging Protein, Genome, and Metagenome Databases to Discover Novel Enzymes and Metabolic Pathways. Biochemistry 58, 4169-4182 (2019).

Claims

1. dmaW, easF, easC, easE, easD, easA イソメラーゼ , easG, and cloA, each gene encoding an enzyme in a biosynthetic pathway from tryptophan to D-lysergic acid (DLA).

2. At least one easE, at least one easA イソメラーゼ 10. The isolated recombinant cell of claim 1, comprising at least one cloA gene.

3. easE comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 6-14; easA イソメラーゼ comprises a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 15-18; and cloA comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 22, 23, 24, 27, or 28; The isolated recombinant cell of claim 2.

4. easE comprises easE from Epichloe coenophila (easE_Ec), easE from Aspergillus japonicus (easE_Aj), and / or easE from Aspergillus indologenus; easA イソメラーゼ However, easA derived from Neotyphodium lolii イソメラーゼ (easA_Nl), easA from Periglandula ipomoeae イソメラーゼ (easA_Pi), easA from Claviceps purpurea イソメラーゼ (easA_Cp), and / or easA from Epichloe coenophila イソメラーゼ (easA_Ec), and cloA comprises cloA from Claviceps paspali (CloA_Cpas), cloA from N. lolii (CloA_Nlol), cloA from P. ipomoeae (CloA_Pipo), cloA from E. coenophiala (CloA_XN6), and / or cloA from C. purpurea (CloA_Cpur); The isolated recombinant cell of claim 2.

5. easE from Epichloe coenophiala (easE_Ec) comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 14; easE from Aspergillus japonicus (easE_Aj) or easE from Aspergillus indologenus comprises a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:6; easA from Neotyphodium lolii イソメラーゼ (easA_Nl) comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 15; easA from Claviceps purpurea イソメラーゼ (easA_Cp) comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 18; easA from Epichloe coenophiala イソメラーゼ (easA_Ec) comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 17; cloA from E. coenophiala (CloA_XN6) comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 27 or 28; and cloA from C. purpurea (CloA_Cpur) comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 22, 23, or 24; The isolated recombinant cell of claim 4.

6. easE comprises easE from Aspergillus japonicus (easE_Aj) or easE from Aspergillus indologenus; easA イソメラーゼ However, easA from Epichloe coenophiala イソメラーゼ (easA_Ec), and cloA comprises cloA from C. purpurea (CloA_Cpur); The isolated recombinant cell of claim 2.

7. easE comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:6; easA イソメラーゼ comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 17, and cloA comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:23; The isolated recombinant cell of claim 6.

8. easE expresses an enzyme that catalyzes the conversion of 4-dimethylallyl-L-abrin to chanoclavine I; easA イソメラーゼ expresses an enzyme that catalyzes the conversion of chanoclavine I-aldehyde to agroclavine, and cloA expresses an enzyme that catalyzes the conversion of agroclavine to D-lysergic acid; The isolated recombinant cell of claim 7.

9. 9. The isolated recombinant cell of claim 8, wherein the recombinant yeast cell further comprises at least one dmaW, at least one easF, and at least one easD.

10. dmaW comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:1; easF comprises a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:2, and easD comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:4; The isolated recombinant cell of claim 9.

11. 11. The isolated recombinant cell of claim 10, wherein each of dmaW, easF, and easD expresses a respective enzyme, and each enzyme is functional.

12. 12. The isolated recombinant cell of claim 11, wherein the recombinant yeast cell further comprises at least one easC and / or at least one easG.

13. easC comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:3, and easG comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:5; The isolated recombinant cell of claim 12.

14. At least one dmaW, at least one easF, at least one easE or easC, at least one easD, at least one easA イソメラーゼ or an isolated recombinant cell comprising easG, and at least one cloA gene.

15. At least one dmaW, at least one easF, at least one easE, at least one easD, at least one easA イソメラーゼ and an isolated recombinant cell comprising at least one cloA gene.

16. At least one dmaW, at least one easF, at least one easC, at least one easD, at least one easA イソメラーゼ , at least one easG, and at least one cloA gene.

17. dmaW comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:1; easF comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:2; easE comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:6; easC comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:3; easD comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:4; easA イソメラーゼ comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 17; easG comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:5; and cloA comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:23; 15. The isolated recombinant cell of claim 14.

18. further comprising multiple copies of the FAD1 gene and the PDI1 gene; FAD1 comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 35, and PDI1 comprises a sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 36; 18. The isolated recombinant cell of claim 17.

19. capable of expressing one or more genes, Expression of one or more genes produces respective gene products, Each gene product is an enzyme, The isolated recombinant cell of claim 1.

20. 20. A method for preparing D-lysergic acid, comprising culturing the recombinant cell of any one of claims 1 to 19 in a suitable medium.

Images