METHOD FOR PRODUCING OLIVETOLIC ACID IN AN AMOEBOZOA HOST SPECIES
20230272439 · 2023-08-31
Inventors
- FALK HILLMANN (JENA, DE)
- CHRISTIN REIMER (JENA, DE)
- JOHANN ELIAS KUFS (JENA, DE)
- VITO VALIANTE (JENA, DE)
Cpc classification
C12N9/1029
CHEMISTRY; METALLURGY
C12Y203/01074
CHEMISTRY; METALLURGY
C12Y203/01206
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to a method for the recombinant production of olivetolic acid (OA) in a host species selected from amoebozoa, based on a hybrid-gene or enzyme of polyketide synthase 37 (PKS37) in which the C-terminal type III PKS domain from an amoeba is replaced by an olivetol synthase (OLS) from a plant, and is expressed together with an olivetolic acid cyclase from a multi-gene expression vector. Further provided is a recombinant amoebozoa host species, and an improved method for producing Δ.sup.9-tetrahydrocannabinol (THC) or other cannabinoids.
Claims
1. A hybrid or protein comprising a polyketide synthase 37 (PKS37) in which the C-terminal type III PKS domain or a functional fragment thereof is replaced by a plant olivetol synthase (OLS) or a functional fragment thereof.
2. The hybrid gene or protein according to claim 1, wherein said hybrid gene or protein comprises amino acids 1 to 2618 of PKS37 from Dictyostelium discoideum, and amino acids 2 to 385 of OLS from Cannabis sativa.
3. A multi-gene expression vector for the recombinant production of olivetolic acid (OA) in a host species selected from amoebozoa, comprising the genes for medium-chain fatty acid CoA ligase (FadK), plant olivetol synthase (OLS), plant olivetolic acid cyclase (OAC), and Tobacco etch virus (TEV) peptidase or homologous genes thereof or functional fragments thereof, wherein said genes are transcribed as a single polycistronic mRNA spaced apart by viral 2A sequences.
4. The multi-gene expression vector according to claim 3, wherein said medium-chain fatty acid CoA ligase FadK is selected from FadK of E. coli, Eggerthella lenta, Kosakonia radicincitans, Enterobacteriaceae bacterium, Dickeya zeae EC1, Sutterella wadsworthensis, Clostridium clostridioforme, Citrobacter amalonaticus, Citrobacter braakii, Citrobacter werkmanii, Escherichia albertii, Actinobacteria bacterium, Citrobacter portucalensis, Yersinia massiliensis, and Serratia fonticola.
5. A multi-gene expression vector for the recombinant production of olivetolic acid (OA) in a host species selected from amoebozoa, comprising and expressing genes that encode the hybrid protein according to claim 1, a plant olivetolic acid cyclase (OAC), and Tobacco etch virus (TEV) peptidase or homologous genes thereof or functional fragments thereof, wherein said genes are transcribed as a single polycistronic mRNA spaced apart by viral 2A sequences.
6. The multi-gene expression vector according to claim 3, wherein said plant olivetol synthase (OLS) and/or plant olivetolic acid cyclase (OAC) is derived from C. sativa.
7. The multi-gene expression vector according to claim 3, wherein said genes are codon optimized for expression in said amoebozoa host species.
8. The multi-gene expression vector according to claim 3, wherein said vector further comprises a suitable selection marker.
9. A recombinant amoebozoa host species, comprising and expressing the multi-gene expression vector according to claim 3, wherein said species is preferably selected from Dictyostelium discoideum.
10. A method for the recombinant production of olivetolic acid (OA) in a species selected from amoebozoa, comprising the steps of a) culturing a recombinant amoebozoa species comprising and expressing the multi-gene expression vector according to claim 3 in a suitable culture medium, wherein said culture medium is supplemented with hexanoic acid as precursor, and b) isolating said OA from said culture of step a).
11. A method for the recombinant production of olivetolic acid (OA) in a species selected from amoebozoa, comprising the steps of a) culturing a recombinant amoebozoa species comprising and expressing the multi-gene expression vector according to claim 5 in a suitable culture medium without the need of hexanoic acid supplementation, and b) isolating said OA from said culture of step a).
12. The method according to claim 10, further comprising induction of the expression by tetracycline or doxycycline, and/or comprising G418 selection.
13. A method for producing Δ.sup.9-tetrahydrocannabinol or another cannabinoid, comprising the steps of performing the method according to claim 10, and chemically and/or enzymatically converting the olivetolic acid (OA) as produced into Δ.sup.9-tetrahydrocannabinol or another cannabinoid.
14. (canceled)
15. The hybrid protein according to claim 1, wherein said pks37 or functional fragment thereof is derived from Dictyostelium discoideum.
16. The multi-gene expression vector according to claim 5, wherein said plant olivetol synthase (OLS) and/or plant olivetolic acid cyclase (OAC) is derived from C. sativa.
17. The multi-gene expression vector according to claim 5, wherein said genes are codon optimized for expression in said amoebozoa host species.
18. A recombinant amoebozoa host species, comprising and expressing the multi-gene expression vector according to claim 5.
19. A method for producing Δ9-tetrahydrocannabinol or another cannabinoid, comprising the steps of performing the method according to claim 11, and chemically and/or enzymatically converting the olivetolic acid (OA) as produced into Δ.sup.9-tetrahydrocannabinol or another cannabinoid.
20. A polynucleotide sequence that encodes the hybrid protein, or fragment thereof, of claim 1.
21. The polynucleotide sequence according to claim 20, wherein said hybrid protein comprises amino acids 1 to 2618 of PKS37 from Dictyostelium discoideum, and amino acids 2 to 385 of OLS from Cannabis sativa.
Description
[0056] The present invention will now be described further in the following examples with reference to the accompanying Figures, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties.
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EXAMPLES
[0065] Materials and Methods
[0066] Dictyostelium strains and cultivation. Dictyostelium discoideum AX2 cells were grown in HL5 complex medium (Formedium) supplemented with 1% [w/v] glucose at 22° C. in cell culture dishes or shake flasks at 140 rpm. Selection of recombinant amoebae was performed by adding 10 μg/mL G418 (InvivoGen) unless otherwise stated. For induction of the Tet.sup.ON polycistronic genes, amoeba cultures were supplied with 20 μg/mL doxycycline (AppliChem). For exogenous supply of precursors, cultures were supplemented with 1 mM p-coumaric acid (Merck) or 1 mM hexanoic acid (Merck) as indicated.
[0067] Construction of single-gene overexpression plasmids. Since the complete amplification of the full-length pks37 gene (Gene ID: 8627855) from genomic DNA did not yield in any product, the gene was amplified in two overlapping fragments and assembled into the pSK-GFP-srbA (30) as a cloning vector using seamless cloning. Oligonucleotides were obtained from Eurofins Genomics and are listed in Table 1. PCR amplifications were performed using the Phusion Flash High-Fidelity PCR Master Mix (Thermo Fisher). For expression analysis of pks37 in amoeba, the full-length gene was amplified from the cloning vector using primers [1]/[2] and introduced into the pTX vector in frame with a C-terminal GFP-tag (31, 32), under control of a constitutive actin15 promoter. For this purpose, the pTX-GFP vector was amplified by PCR using primer pair [3]/[4]. After DpnI digest of the template plasmid, PCR fragments were purified by gel extraction (GeneJET Gel Extraction Kit, Thermo Fisher) and assembled using the GeneArt Seamless Cloning & Assembly Kit (Thermo Fisher) according to the manufacturer's instructions. Recombinant plasmids were isolated from E. coli DH5α (GeneJET Plasmid Miniprep Kit, Thermo Fisher) and correct vector assemblies were verified by PCR and Sanger sequencing (LGC Genomics). A tag-free construct was generated as described above using primer pair [1]/[5] for amplification of pks37 and pair [6]/[7] for pTX vector linearization. In addition, the pTX vector was amplified using primers [8]/[4] and combined with pks1 (Gene ID: 8616850), pks5 (Gene ID: 8618014) and pks13 (Gene ID: 8618586) genes that were PCR-amplified using primer pair [9]/[10], [11]/[12] and [13]/[14], respectively. For resveratrol production, the pTX vector was linearized by PCR using primers [15]/[16] and assembled with the V. vinifera STS gene (Gene ID: 100256566) that was amplified using primer pair [17]/[18].
[0068] Generation of polycistronic multi-gene expression plasmids. For olivetolic acid production in amoeba, the genes of interest were provided with homologous tail regions for direct integration into the EcoRV-linearized pV2A-T vector (22). The fadK gene (Gene ID: 946213) was amplified from genomic DNA of E. coli K12 MG1655 using primers [19] and [20]. The genes for OLS (GenBank: AB164375.1) and OAC (JN679224.1) from C. sativa were synthesized by Eurofins Genomics and amplified using primer pair [21]/[22] and [23]/[24], respectively. The generated fragments were gel-purified and separately assembled into the pV2A-T vector by seamless cloning. According to the selected order, each plasmid was restricted either with SwaI (acceptor plasmid) or with PmeI (donor plasmid) for a stepwise combination of genes as reported (22). The final pV2A-T vector containing fadK, OLS, OAC and TEV (named as pV2A-T_OA) served as donor of the desired genes and was combined with a modified pDM310 vector that contains an inducible Tet.sup.ON promoter for expression in amoeba (33). For this purpose, the Venus-TEV cassette was amplified from the pV2A-T_TEV plasmid using primers [25]/[26] that contain an integrated Kozak-sequence and homologous regions for direct insertion into the Bg/II-linearized pDM310 vector (33). The resulting pDM310_Venus-TEV plasmid served as acceptor, which was subsequently digested with ZraI/AleI and combined with the PacI/SwaI-restricted pV2A-T_OA donor vector to create the final expression vector pChR2. Correct assembly was verified by sequencing. To generate the pChR1 control vector, the pDM310 Venus-TEV plasmid was linearized by PCR using primer pair [27]/[28] and assembled with the PacI/SwaI-restricted pV2A-T_fadK-OLS-OAC plasmid.
[0069] To create the domain swapping constructs, the pChR2 vector backbone was amplified by PCR using primers [29] and [30] with an integrated XbaI recognition site between the 2A-tag and fadK to remove the generated linker region. After DpnI/XbaI digest, the vector backbone was gel-purified and combined with three different versions of the PKS37/FAS (A: 1-2618 aa, B: 1-2618 aa, C: 1-2605 aa) and OLS (A: 25-385 aa, B: 2-385 aa, C: 14-385 aa) fragments that were amplified using primer pair [31]/[32], [31]/[33], [31]/[34], [35]/[38], [36]/[38] and [37]/[38], respectively. The obtained plasmids (version A,B,C) are referred to as pChR3, pChR5 and pChR4, respectively. To generate the loss-of-function plasmid pChR6, a S2458A substitution was introduced into the PP-binding site of PKS37/FAS by pChR5 vector amplification using overlapping primers [39]/[40] that contain the corresponding nucleotide exchange. A new NsiI recognition site was introduced to screen for positive mutants and the correct mutagenesis was verified by sequencing. The functional pChR5 hybrid vector was further used as template for replacing the OLS and OAC genes with their codon-optimized version for D. discoideum. Codon optimization was performed based on the Kazusa Codon Usage Database using the GENEius software designed by Eurofins Genomics. The OLS-DD and OAC-DD optimized genes were synthesized from GenScript Biotech and Eurofins Genomics, respectively. The pChR5 vector backbone, and the synthetic OLS-DD and OAC-DD genes were amplified using primer pair [41]/[42], [43]/[44] and [45]/[46], respectively, and combined by seamless cloning to yield plasmid pChR7. In addition, the OLS-DD gene was PCR-amplified using primers [43]/[47] and used to replace the PKS III sequence of the pTX_pks37 plasmid that was amplified using primers [48]/[42], resulting in the pChR8 plasmid.
[0070] Transfection of amoeba by electroporation. Transfection of D. discoideum was performed as previously described (34) with some modifications. Briefly, Dictyostelium cells were harvested from mid-log phase by centrifugation for 5 min at 500×g and 4° C. The cells were washed in ice-cold electroporation buffer and re-suspended at a concentration of 4×10.sup.7 cells/mL, determined using the CASY cell counter (Roche). Then, 1-5 μg of plasmid DNA were added to 100 μL of cell suspension in a pre-chilled 1 mm gap electroporation cuvette (BTX) and incubated for 5 min on ice. Electroporation was performed at 850 V, 25 μF and 25Ω using the ECM 630 cell electroporator (BTX). After pulsing, the cell suspension was mixed with 500 μL of HL5 medium containing 1% glucose and incubated for 4 h at 22° C. and 400 rpm. Then, the cells were dispensed on tissue culture plates containing HL5 medium supplemented with 1% glucose and 10 μg/mL G418 for selection of drug-resistant amoeba. Recombinant amoeba cells were sub-cultured at least two times before preparing permanent frozen stocks.
[0071] Western blot analysis. For Western blotting of the wild-type (as negative control) and the pks37-GFP strain, 1×10.sup.7 cells of overnight cultures were pelleted by centrifugation for 5 min at 800×g and 4° C., and re-suspended into 150 μL lysis buffer (100 mM Tris/HCl, 1% SDS, 8 M Urea). The cell lysates were mixed with 6×SDS sample buffer and denatured for 10 min at 98° C. and 700 rpm. The extracted proteins were separated on Bolt™ 4-12% Bis-Tris Plus Gels (Thermo Fisher) and the corresponding 1× Bolt™ MOPS SDS Running Buffer (Thermo Fisher) for 40 min at 140 V. After separation by SDS-PAGE, the proteins were transferred to a 0.2 μm nitrocellulose membrane for 1 h at 25 V using the Mini Gel Electrophoresis Tank (Thermo Fisher). The membrane was blocked with TBS-T (20 mM Tris, 150 mM NaCl, 0.1% Tween 20) containing 3% BSA, and incubated overnight at 4° C. with mild shaking. For detection, an anti-GFP antibody (GF28R, Thermo Fisher) was used in combination with a goat anti-mouse IgG-AP antibody (Santa Cruz). The membrane was washed in assay buffer (20 mM Tris, 1 mM MgCl.sub.2, pH 9.8), developed by adding CDP-Star solution (Applied Biosystems) and imaged using the FUSION FX imaging system (Vilber Lourmat). For Western blot analysis of the 2A-tagged proteins, the D. discoideum pChR1 strain was grown under non-inducing conditions (as negative control) or in the presence of 20 μg/mL doxycycline. Cell harvesting and sample preparation was performed as described above. The 2A-tagged proteins were separated on Novex™ 8-16% Tris-Glycine Gels (Thermo Fisher) and blotted onto a PVDF membrane. For detection, an anti-2A antibody (ABS31, Merck) was used in combination with a goat anti-rabbit IgG-AP antibody (A3687, Merck).
[0072] Northern Hybridizations. RNA was isolated from 5×10.sup.7 amoeba cells using the RNeasy Mini Kit (QIAGEN). For each sample, 20 μg of total RNA were separated on a 1.2% denaturating agarose gel containing 2% formaldehyde in MOPS buffer. The gel was washed in 0.25 M HCl solution, denaturation solution (10 g NaOH, 44 g NaCl, 500 mL) and 10×SSC solution (87.7 g NaCl, 44.1 g sodium citrate, 500 mL), and blotted overnight on a Nylon membrane. After UV-crosslinking, the membrane was pre-hybridized at 48° C. for 5 h in hybridization buffer (5×SSC, 1% SDS, 0.1% N-laurylsarcosyl, 5 M Urea, 50 mM sodium phosphate, pH 7.0) containing 5% Roche blocking solution. Hybridization was performed overnight at 60° C. using a pks37-specific Digoxigenin (DIG)-labeled DNA probe that was constructed by amplification from genomic DNA using the DreamTaq Green PCR Master Mix (Thermo Fisher), DIG-ddUTPs (Jena Bioscience) and primers [49]/[50]. The membrane was washed, blocked with maleic acid buffer (0.1 M maleic acid, 0.1 M NaCl, pH 7.5) containing 10% Roche blocking reagent and incubated with an Anti-DIG AP-conjugated antibody (Roche). The blot was equilibrated in detection buffer and developed as described above.
[0073] Chemical extraction and HPLC-HRMS analysis. For chemical extractions, amoeba cells at a start density of 3×10.sup.5 cell/mL were grown in liquid cultures for 3 days at 22° C. under shaking conditions. Cultures were extracted twice with equal volume of ethyl acetate. The organic phases were dried with sodium sulfate and concentrated under reduced pressure using a rotary evaporator (Buechi). Crude extracts were dissolved in methanol and filtered through a 0.2 μm PTFE filter (Carl Roth). For detection of secondary metabolites, high-performance liquid chromatography (HPLC) analyses were performed using a Shimadzu LC-20AD instrument, equipped with a reverse-phase Kinetex C18 column (4.6×250 mm, 5 μm, 100 Å, Phenomenex) maintained at 30° C. A volume of 10 μL was injected using an autosampler and separated by applying a linear gradient of solvents A (water, 0.1% formic acid) and B (acetonitrile) at a flow rate of 0.8 mL/min: 10% B for 1 min, a linear increase to 95% B for 23 min, followed by column rinsing with 95% B for 10 min and re-equilibration with 10% B for 10 min. Organic compounds were detected by monitoring the absorbance at 280 and 400 nm using a PDA detector (Shimadzu). High-resolution mass spectrometry (HRMS) was performed on a Q-Exactive Plus Hybrid Quadrupole Orbitrap mass spectrometer using electrospray ionization and an UltiMate 3000 UHPLC system (Thermo Fisher) equipped with a Kinetex C18 column (2.1×150 mm, 2.5 μm, 100 Å, Phenomenex). UHPLC was performed with an injection volume of 3 μL and a gradient elution of solvents A (water, 0.1% formic acid) and B (acetonitrile) at a flow rate of 0.3 mL/min: 5% B for 0.5 min, a linear gradient to 97% B for 11.5 min, then 97% B for 3 min and 5% B for 3 min. To quantify OA titers in culture extracts, the areas under the curve (AUCs) from extracted ion chromatograms were determined. The concentration was calculated by using a standard curve recorded with the pure reference compound.
[0074] Metabolic analysis of fruiting bodies. For inducing the development of amoeba, the protocol was performed as previously reported (16). Briefly, amoeba cells were pelleted from mid-log phase (2-5×10.sup.6 cells/mL) by centrifugation for 5 min at 500×g and 4° C. The cells were washed twice in ice-cold development buffer (5 mM Na.sub.2HPO.sub.4, 5 mM KH.sub.2PO.sub.4, 1 mM CaCl.sub.2, 2 mM MgCl.sub.2, pH 6.5) and re-suspended at a concentration of 2-3×10.sup.8 cells/mL. For metabolic analysis of mature fruiting bodies, 8×10.sup.8 cells were spread on KK2 plates and incubated at 22° C. for 26 h. The cell aggregates were scraped off, re-suspended in development buffer, freeze-dried and extracted with methanol. The liquid phase was separated by centrifugation and concentrated under reduced pressure. Crude extracts were analyzed by HR-MS as already described.
[0075] Isotope labeling studies. For the feeding experiments, 6 mM sodium [1,2-.sup.13C.sub.2]acetate (Merck) was added to the appropriate overexpression strains as indicated. Separate growth experiments were performed without the addition of acetate as precursor. After 3 days of growth, the cultures were extracted and analyzed for their metabolic profile by HR-MS as described.
[0076] PCP isolation, purification and NMR analysis. For NMR analysis, a 2 L large-scale culture was inoculated with 3×10.sup.5 cells/mL of the pks37 overexpression mutant and extracted as described above. For pre-purification, the dried extract was dissolved in 6 mL of 50% methanol and loaded onto a Chromabond C18 SPE column (Macherey-Nagel) that was pre-equilibrated with 2 column volumes (CVs) of methanol followed by 1 CV water. After washing the column with 1 CV of water and 1.5 CVs of 30% methanol, the sample was eluted using 1 CV of 100% methanol. The collected methanol fraction was concentrated under reduced pressure to 1.5 mL and separated by semi-preparative HPLC using a Shimadzu LC-20AD instrument, equipped with a reverse-phase Kinetex C18 column (10×250 mm, 5 μm, 100 Å, Phenomenex) maintained at 30° C. Extracts were fractionated by using a linear gradient of solvents A (water, 0.1% formic acid) and B (acetonitrile) at a flow rate of 2 mL/min under the following conditions: 10% B for 1 min, a linear increase to 60% B for 14 min, held at 60% B for 15 min, increase to 97% for 1 min, followed by column rinsing with 97% B for 10 min, decrease to 10% in 2 min and re-equilibration with 10% B for 10 min. Organic compounds were detected by monitoring the absorbance at 254 and 280 nm using a PDA detector (Shimadzu). Collected fractions were evaporated using the Genevac EZ-2 sample concentrator (SP Scientific). Nuclear magnetic resonance (NMR) measurements (.sup.1H, .sup.13C, DEPT) were performed in deuterated methanol (VWR) on a Bruker Avance III 500 MHz spectrometer. The chemical shifts are reported in ppm relative to the solvent residual peak (δ (CD.sub.3OD)=3.31 (49.05) ppm). The purified compound was used as a reference for further metabolic analyses.
[0077] Lab-scale fermentation of the pks5-oex strain. For up-scaling the polyketide production, 5 L of HL5 medium supplemented with 10 g/L glucose and 10 μg/mL G418 were inoculated with 1×10.sup.6 cells/mL of the pks5-oex strain and grown in a 7 L-stirred tank reactor (Sartorius) as a batch culture. To ensure fully aerated and oxygen unlimited conditions at all time, the level of dissolved oxygen tension (DOT) was controlled in cascade mode at a value of 20% by increasing the gas flow rate to a maximum value of 4 L/min followed by an increase of the stirring rate to a maximum value of 307 rpm. This aeration strategy was conducted to keep the hydro-mechanically stress on the cells to a minimum level. The pH was adjusted to 6.5 by addition of sulfuric acid if necessary. The batch culture was grown for 4 days at a constant temperature of 22° C.
[0078] Purification and NMR analysis of PKS5-derived metabolites. After fermentation of the pks5-oex strain, the culture was centrifuged for 20 min at 1,000×g and the supernatant was extracted twice with an equal volume of ethyl acetate. The organic phase was dried with sodium sulfate and concentrated to dryness under reduced pressure. The pellet was lyophilized and extracted with 800 mL methanol for 1 h. Then, the methanol extract was filtered and concentrated under reduced pressure. The crude extracts were dissolved in methanol and the compounds were isolated by preparative HPLC (Jasco), equipped with a reverse-phase Eurospher E C18 column (32×240 mm, 10 μm, 100 Å, Knauer). The extract was fractionated by using a linear gradient of solvents A (water, 0.1% formic acid) and B (acetonitrile) at a flow rate of 20 mL/min under the following conditions: 10% B for 1 min, a linear increase to 95% B for 24 min, held at 95% B for 10 min and re-equilibration with 10% B. Organic compounds were detected by monitoring the absorbance at 400 nm using a PDA detector (Jasco). Fractions showing an absorption maximum at 400 nm were combined, evaporated, dissolved in methanol and further purified by semi-preparative HPLC as described above, except the following changes in the gradient: 10% B for 1.5 min, a linear increase to 95% B for 38.5 min, followed by column rinsing with 95% B for 12 min, decrease to 10% in 3 min and re-equilibration with 10% B for 12 min. Collected fractions were evaporated and dissolved in deuterated DMSO (VWR). NMR spectra (.sup.1H, .sup.13C, DEPT, COSY, HSQC, HMBC) were recorded on a Bruker Avance III 600 MHz spectrometer. Chemical shifts are reported in ppm relative to the solvent residual peak (δ (DMSO-d.sub.6)=2.50 (39.52) ppm).
[0079] Growth and metabolic activity monitoring. For determining the respiration activity, the D. discoideum pChR2, pChR5 and pChR7 strains were grown at an initial density of 1×10.sup.6 cells/mL of HL5 medium supplemented with 1% [w/v] glucose and 20 μg/mL G418 in the presence or absence of 20 μg/mL doxycycline and 1 mM hexanoic acid as indicated. The respiration activity was measured by using the Kuhner TOM (transfer-rate online measurement) system equipped with oxygen partial pressure sensors and infrared sensors to calculate the oxygen transfer rate (OTR) and the carbon dioxide transfer rate (CTR). The cultures were grown in 250 mL TOM flasks for 144 h at 140 rpm and 22° C. without interruption to allow a constant OTR assessment. A filling volume of 52 mL was calculated in order to allow a maximum OTR of 8.5 mmol/L/h43, similar to culture conditions of previous experiments using 25 mL per 100 mL Erlenmeyer shake flasks.
[0080] For offline analysis, the strains were grown in parallel in 100 mL shake flasks filled with 25 mL liquid cultures, inoculated from the same master mix. At the indicated time points, 20 mL of each culture flask were harvested for chemical extraction and OA quantification by HR-MS as already described.
TABLE-US-00001 TABLE 1 Oligonucleotides as used. No Primer Name DNA Sequence (5′ .fwdarw. 3′) 1 pTX-37_F AAAAATAAAAATCAGATCCAAGCTTAAAAAATGA ACAACAACAAAAGTATAAAC (SEQ ID NO: 1) 2 pTX-37_R AACTCCAGTGAAAAGTTCTTCTCCTTTACTTTTAA TTAATTTAAATAAAATTGC (SEQ ID NO: 2) 3 pTX-GFP-lin_F AGTAAAGGAGAAGAACTTTTCACTGGA (SEQ ID NO: 3) 4 pTX-lin_R TTTTTAAGCTTGGATCTGATTTTTATTTTT (SEQ ID NO: 4) 5 pTX-37_R2 GAATTCCTGCAGCCCGGGGGATCGTCTAGATTATT TAATTAATTTAAATAAAAT (SEQ ID NO: 5) 6 37/pTX_F AAATTAATTAAATAATCTAGACGATCCCCCGGGC TG (SEQ ID NO: 6) 7 37/pTX_R TTTGTTGTTGTTCATTTTTTAAGCTTGGATCTGAT TTTTATTTTT (SEQ ID NO: 7) 8 pTX-lin_F TCTAGACGATCCCCCGGG (SEQ ID NO: 8) 9 pTX-1_F AAAAATAAAAATCAGATCCAAGCTTAAAAAATGA ATAAAAATTCAAAAATCCAATCA (SEQ ID NO: 9) 10 pTX-1_R GAATTCCTGCAGCCCGGGGGATCGTCTAGATTAG ACAACATTTTTTAAGAAACAACC (SEQ ID NO: 10) 11 pTX-5_F AAAAATAAAAATCAGATCCAAGCTTAAAAAATGG ATATGAAATTAAATGATATTGAA (SEQ ID NO: 11) 12 pTX-5_R GAATTCCTGCAGCCCGGGGGATCGTCTAGATTATT TATTTATCTTTTTCAAAAAATTTAA (SEQ ID NO: 12) 13 pTX-13_F AAAAATAAAAATCAGATCCAAGCTTAAAAAATGG AAAACTTTAAATATAGAAATAATG (SEQ ID NO: 13) 14 pTX-13_R GAATTCCTGCAGCCCGGGGGATCGTCTAGATTATT CTTGGCTTTGAACTT (SEQ ID NO: 14) 15 VINST1/pTX_F ACGGTTACAAATTAATCTAGACGATCCCCCGGGC TG (SEQ ID NO: 15) 16 VINST1/pTX_R CTCAACTGAAGCCATTTTTTAAGCTTGGATCTGAT TTTTATTTTT (SEQ ID NO: 16) 17 pTX-VINST1_F AAAAATAAAAATCAGATCCAAGCTTAAAAAATGG CTTCAGTTGAGGAATTTAGA (SEQ ID NO: 17) 18 pTX-VINST1_R GAATTCCTGCAGCCCGGGGGATCGTCTAGATTAA TTTGTAACCGTAGGAACGCT (SEQ ID NO: 18) 19 pV2AT-fadK_F GGCGACGTTGAAGAAAACCCTGGCCCTGATATGC ATCCCACAGGCCCGCAT (SEQ ID NO: 19) 20 pV2AT-fadK_R CATCGCACCTTGAAAATAAAGATTTTCGATTTCAA TCTCTTCACAGACATCCTGCGTTAAACG (SEQ ID NO: 20) 21 pV2AT-OLS_F GGCGACGTTGAAGAAAACCCTGGCCCTGATATGA ATCATCTTCGTGCTGAGGGT (SEQ ID NO: 21) 22 pV2AT-OLS_R CATCGCACCTTGAAAATAAAGATTTTCGATATATT TGATGGGAACACTACGCAC (SEQ ID NO: 22) 23 pV2AT-OAC_F2 GGCGACGTTGAAGAAAACCCTGGCCCTGATATGG CAGTGAAGCATTTGATTGTATTGAAG (SEQ ID NO: 23) 24 pV2AT-OAC_R2 CATCGCACCTTGAAAATAAAGATTTTCGATCTTTC GTGGTGTGTAGTCAAAAATGAGAAG (SEQ ID NO: 24) 25 TRE-VenusN_F TAAATTAAATTAAATAAAAAATAAAAATCAAAAA AATGGATAAAGCGGAATTAATTCCC (SEQ ID NO: 25) 26 Tact8- TTAAATAATTTATTTATTTAACTAGTACTACTACT VenusC_R TGTACAGCTCGTCCA (SEQ ID NO: 26) 27 pDM-TEVsite- ATCGAAAATCTTTATTTTCAAGGTGCGATGG lin_F (SEQ ID NO: 27) 28 pDM- GTCGCCGTCAAGCTCGAC (SEQ ID NO: 28) ZraI-lin_R 29 OLS-seq_F GTGGAGCACGAGATGCAAACT (SEQ ID NO: 29) 30 fadK- CGGGCCTGTGGGATGCATTCTAGATCAGGGCCA XbaI-2A_R (SEQ ID NO: 30) 31 pV2AT-37_F GGCGACGTTGAAGAAAACCCTGGCCCTGATATGA ACAACAACAAAAGTATAAACGATTTAAG (SEQ ID NO: 31) 32 37-A_R AAACTCATCTTGTAAAATTGGTGAAATTTCATAAA TTACTGTTGC (SEQ ID NO: 32) 33 37-B_R AGCACGAAGATGATTAATTGGTGAAATTTCATAA ATTACTGTTGC (SEQ ID NO: 33) 34 37-C_R GGCGGTGCCAATGGCATCGTTATCACTATTATTAT CTTCTGATATTATTGG (SEQ ID NO: 34) 35 OLS-A_F GAAATTTCACCAATTTTACAAGATGAGTTTCCTGA CTACT (SEQ ID NO: 35) 36 OLS-B_F GAAATTTCACCAATTAATCATCTTCGTGCTGAGGG T (SEQ ID NO: 36) 37 OLS-C_F AATAGTGATAACGATGCCATTGGCACCGCCAAT (SEQ ID NO: 37) 38 OLS-seq_R GATGGCCTTTGCACAAGCATC (SEQ ID NO: 38) 39 37PP- GACTATGGTTTAGATGCATTACTATCAAGTGAATT S2458A_F ATCA (SEQ ID NO: 39) 40 37PP- CACTTGATAGTAATGCATCTAAACCATAGTCACTA S2458A_R AATG (SEQ ID NO: 40) 41 pDM-B_DD- GGAGAAAGCTTGTTTAAGGGGCC (SEQ ID lin_F NO: 41) 42 pDM-B_DD- AGCTCTGAGATGGTTAATTGGTGAAATTTCATAA lin_R ATTACTGTTGCCA (SEQ ID NO: 42) 43 OLS-DD_F GAAATTTCACCAATTAACCATCTCAGAGCTGAAG GAC (SEQ ID NO: 43) 44 OLS-DD_R ATATTTAATTGGAACTGATCTAACAACAACTC (SEQ ID NO: 44) 45 OAC-DD_F GAGTTGTTGTTAGATCAGTTCCAATTAAATAT (SEQ ID NO: 45) 46 OAC-DD_R ATCACGCGGCCCCTTAAAC (SEQ ID NO: 46) 47 OLS-DD_R2 GGATCGTCTAGATTAATATTTAATTGGAACTGATC TAACAACAACTC (SEQ ID NO: 47) 48 OLS-DD/pTX_F GTTCCAATTAAATATTAATCTAGACGATCCCCCGG G (SEQ ID NO: 48) 49 pks37_NB_F1 GCCATTAGATTCATGTCATCCGGTAATC (SEQ ID NO: 49) 50 pks37_GenR2 GCCATACCAACATCAGGTATAGAAGC (SEQ ID NO: 50)
[0081] PCP Production by Overexpression of Pks37 in D. discoideum
[0082] As a proof-of-concept study, the inventors first overexpressed the PKS37 encoding gene (pks37 or also named stlB) in D. discoideum. Gene activation was achieved by expressing pks37 under the control of a constitutive actin15 promoter, either alone or as a pks37-GFP fusion construct, of which the latter was used solely for expression analysis (
[0083] Since PCP is the precursor for the DIF-1 biosynthesis (19) (
[0084] Secondary Metabolite Production by Amoeba PKSs
[0085] Since overexpression of pks37 resulted in a high-level polyketide production in vivo, the inventors decided to further investigate the natural product potential of D. discoideum by expression of still uncharacterized PKS genes. Under vegetative growth conditions, the newly generated overexpression mutants were cultured for three days, extracted and analyzed by HPLC and HR-MS. The extracted ion chromatogram of the pks5-oex mutant revealed multiple peaks with the same mass in the positive mode suggesting the presence of different structural isomers. In order to obtain the PKS5-derived metabolites in a sufficient amount for structure elucidation, the inventors performed a lab-scale fermentation and compound purification using preparative HPLC. The NMR-guided structure elucidation showed the presence of an unsaturated long-chain fatty acid (C18) containing a secondary hydroxyl group at carbon 17, and a gamma or alpha methyl group. The newly identified molecules were named Dictyodene B and A, with the m/z of 301.1792 [M+H].sup.+ (5) and 301.1787 [M+H].sup.+ (6), respectively (
[0086] For the pks13-oex strain, the HPLC chromatogram showed two peaks at the retention time of 21.7 and 23.2 min, whereby subsequent HR-MS analysis identified ions with the m/z of 283.1340 [M−H].sup.+ (7) and 311.1641 [M+H].sup.+ (8), respectively (
[0087] These data give further evidence that the here developed homologous expression system makes D. discoideum an optimal platform for the functional analysis of PKS genes. It enables the inventors to confirm the production of suspected polyketides in vivo, and even more relevant, to elucidate novel natural products of polyketide origin.
[0088] Functional Expression of a Plant Type III PKS in D. discoideum
[0089] Since PKS37 contains a C-terminal type III PKS domain, the inventors investigated the potential of amoebae to functionally express a stand-alone plant type III PKS (
[0090] The inventors first aimed to produce resveratrol, a stilbene that is mainly present in grapes, peanuts and a variety of berries, and known for its antioxidant, anti-tumorigenic and anti-inflammatory properties (20). The inventors heterologously expressed the stilbene synthase (STS) encoding gene from the grapevine Vitis vinifera under control of the constitutively active actin15 promoter. The STS overexpression strain was grown as a liquid culture supplemented with p-coumaric acid for three days and analyzed for metabolite production by HR-MS. The inventors confirmed the heterologous production of cis-resveratrol (11) in D. discoideum (
[0091] Heterologous Production of Olivetolic Acid in Amoeba
[0092] The heterologous production of resveratrol suggested that the amoeba can potentially be used as a chassis for the production of plant metabolites. To further explore this, the inventors decided to produce an additional polyketide, the olivetolic acid (OA), which is a major intermediate in the cannabinoid biosynthetic pathway of Cannabis sativa. Remarkably, the PCP of D. discoideum and OA from C. sativa are both synthesized from hexanoyl starter molecules and also share the malonyl-CoA derived aromatic ring.
[0093] The inventors designed a biosynthetic pathway for the production of OA built on the medium-chain fatty acid CoA ligase FadK of E. coli (21), and the C. sativa enzymes olivetol synthase (OLS) and olivetolic acid cyclase (OAC,
[0094] Construction of an Inter-Kingdom PKS Hybrid for OA Biosynthesis
[0095] To circumvent the need for external supplements and directly produce OA in D. discoideum from primary metabolic precursors, the inventors modified the biosynthetic pathway by taking advantage of PKS37 as a hybrid of a FAS and a PKS III domain. Here, the FAS domain synthesizes a hexanoyl moiety, which is then transferred to the PKS III domain for PCP biosynthesis. By swapping the PKS III domain with the OLS of C. sativa, the inventors expected that the amoeba FAS domain could directly provide the hexanoyl-intermediate to OLS leading to the formation of the tetraketide intermediate (
[0096] To exclude another intracellular source of the starter unit and proof that the hexanoyl-intermediate was solely provided via the PKS37/FAS domain, the inventors additionally created a loss-of-function mutant of the pChR5 hybrid strain by introducing a S2458A substitution in the phosphopantetheine-binding site of the FAS-ACP domain (pChR6). Similar transcription levels of the functional pChR5 hybrid and its loss-of-function mutant pChR6 were verified by northern hybridization (
[0097] For further evidence of the functional involvement of PKS37/FAS in OA biosynthesis, the optimized pChR7 hybrid and the fadK containing pChR2 strain were grown in medium enriched with [1,2-.sup.13C.sub.2]acetate and analyzed for their metabolic profile by HR-MS (
[0098] Taken together, the inventors report here the first synthetic inter-kingdom hybrid enzyme that enables OA biosynthesis exclusively from acetyl- and malonyl-CoA.
[0099] Growth and Metabolic Activity of OA Production Strains
[0100] We compared the growth and metabolic activities of the pChR5 hybrid, the codon-optimized pChR7 hybrid, and the fadK expressing pChR2 strain requiring hexanoic acid supplementation in real-time (
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