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Definitions

• the invention relates to a genetically modified fungus Claviceps purpurea.
• This fungus species is widely used in the pharmaceutical industry as a production microorganism for its ability to produce ergot alkaloids.
• the invention further relates to a method of genetic modification of said fungus.
• Claviceps purpurea a sac fungus from the order Hypocreales, is one of the best known producers of ergot alkaloids.
• These secondary metabolites, derivatives of D-lysergic acid, are widely used in the pharmaceutical industry.
• the utility of these compounds in medicine is due to their structural similarity to the neurotransmitters adrenaline, noradrenaline and serotonin, and the interaction of ergot alkaloids with receptors for these neurotransmitters.
• ergot alkaloids are mainly used in obstetrics, and to treat headache and prolactin-related diseases.
• ergot alkaloids are used in the treatment of Parkinson's and Alzheimer's disease.
• C. purpurea strain PI derived from strain 1029 was also obtained in this way (Keller U. (1983), Applied and Environmental Microbiology, 46(3), 580-584). Furthermore, a cluster of ergot alkaloid biosynthetic genes (EAS cluster) was identified for the first time in this C. purpurea strain (Tudzynski P, Fldlter K, Correia T, Arntz C, Grammel N, Keller U. (1999), Molecular and General Genetics, 261(1), 133-141).
• the EAS cluster contains a total of 13 genes and one pseudogene, wherein the dmaW gene encodes dimethylallyltryptophan synthetase (DMATS, EC: 2.5.1.34), which catalyzes the first pathway specific step leading to ergot alkaloids, the prenylation of L-tryptophan withdimethylallyl diphosphate (DMAPP)producing 4-g,g-dimethyl allyl tryptophan (DMAT).
• DMATS dimethylallyltryptophan synthetase
• DMAPP L-tryptophan withdimethylallyl diphosphate
• DMAT 4-g,g-dimethyl allyl tryptophan
• the amino acid tryptophan also inhibits (in a feedback loop manner) anthranilate synthase (EC: 4.1.3.27), a key enzyme involved in tryptophan biosynthesis, which catalyses the conversion of chorismic acid to anthranilic acid (Krupinski VM., Robbers JE, Floss HG. (1976), Journal of Bacteriology, 725(1), 158-165).
• the second step of ergot alkaloid biosynthesis is methylation of DMAT to produce N-methyl-di methyl al I ytryptophan (MeDMAT).
• This step is catalyzed by 4-dimethylallyltryptophan N-methy I transferase encoded by EasF gene.
• chanoclavin-I occurs, the reaction being catalyzed by two enzymes, chanoclavin-I synthase encoded by EasE gene and catalase encoded by EasC gene.
• Chanoclavin-I is further oxidized to chanoclavin-1 -aldehyde by chanoclavin-1 -dehydrogenase encoded by EasD gene.
• chanoclavine-I-aldehyde is isomerised and reduced to agroclavine by orthologs of the flavin-dependent oxidoreductase encoded by easA gene and the reductase encoded by easG, respectively. This is followed by oxidation of agroclavin to elymoclavin and its subsequent oxidation to D-lysergic acid. These three rounds of 2-electron oxidations are catalyzed by NADPH-dependent cytochrome P450 monooxygenase encoded by cloA gene. Finally, ergopeptines are formed in C. purpurea.
• IpsAl , lpsA2, IpsB and IpsC genes encoding four different non-ribosomal peptide lysergyl synthetases have been found in the genome of C. purpurea 20.1.
• the IpsAl gene encodes trimodular lysergyl peptide synthetase LPS 1 , whichhas a similar function to the enzyme LPS4 encoded by the lpsA2 gene. The difference between these two enzymes is probably only in their ability to bind various amino acids that are part of the ergopeptines.
• the IpsB gene encodes monomodular enzyme LPS2, which activates the D-lysergic acid chain.
• LPS1 or LPS4 Upon activation of D-lysergic acid by LPS2, the acid is transferred to LPS1 or LPS4, both containing three domains for binding three amino acids. After their binding, a peptide lactam is formed, the subsequent cyclisation of which is catalyzed by a dioxygenase encoded by the easH gene.
• the IpsC gene encodes monomodular synthetase LPS3, which, together with LPS2, catalyzes ergometrine biosynthesis (Florea S., Panaccione D. G., Schardl C. L. (2017), Phytopathology, 107(5), 504-518).
• vectors containing trpC promoter from A. nidulans and an ogfp gene encoding green fluorescent protein were prepared using yeast recombinational cloning method (Schumacher J. (2012), Fungal Genetics and Biology, 49, 483-497). These vectors were further used to transform C.
• the PI strain is not the only C. purpurea strain that produces ergot alkaloids (namely ergotamine a- ergocryptine and their stereoisomers ergotaminine and a-ergocryptinine) in submerged culture.
• This characteristic is possessed also by, for example, ergotamine producing strain 275 FI (Arcamone F., Cassinenelli G., Femifni G., Penco S., Pennella P., Pol C. (1970), Canadian Journal of Microbiology, 16(10), 923-931) or strain 59 (Kfen V., Pazoutova S., Sedmera P., Rylko V., Rehacek Z.
• the present invention provides genetically engineered transformants of Claviceps purpurea with increased ergot alkaloid production.
• the transformants comprise in their genome an inserted nucleotide sequence comprising at least one gene selected from TrpE gene encoding a-subunit of anthranilate synthase having S76F mutation, and dmaW gene encoding dimethylallyltryptophan synthetase, wherein the inserted gene is under the control of a constitutive promoter.
• insertion of the gene encoding a-subunit of the key enzyme for the production oftryptophan, i.e., a-subunit of anthranilate synthase with the S76F mutation, under the control of a constitutive promoter unblocks the feedback loop inhibition of tryptophan biosynthesis, thereby enhancing the biosynthetic pathway leading to ergot alkaloid production.
• the ergot alkaloid biosynthesis is also enhanced by inserting the gene for transformation of tryptophan to dimethylallyltryptophan, a key intermediate in ergot alkaloid biosynthesis, under the control of a constitutive promoter.
• Such genetically modified microorganisms are suitable for use in the manufacture of ergot alkaloids for the pharmaceutical industry.
• the gene encoding a-subunit of anthranilate synthase (TrpE) with the S76L mutation i.e. the gene carrying the mutation of the codon for serine (codon AGC) at position 76 to the codon for leucine (CTC codon), has a sequence with at least 90% identity to SEQ ID NO. 1, wherein a mutation of the serine codon at position 76 to the leucine codon (S76L) must be present. More preferably, the gene encoding a-subunit of anthranilate synthase having the S76L mutation has the sequence SEQ ID NO. 1.
• the gene encoding dimethylallytryptophan synthetase has a sequence with at least 90% identity to SEQ ID NO. 2. More preferably, the gene encoding dimethylallytryptophan synthetase has the sequence SEQ ID NO. 2.
• Both above discussed genes are genes derived from Claviceps purpurea, optionally modified by introduction of a mutation, and inserted into the genome of Claviceps purpurea under the control of a constitutive promoter.
• the resulting microorganism carries its original TrpE and dmaW genes, and also carries the additional at least one gene ( TrpE S76L and/or dmaW) inserted through said genetic transformation under the control of a constitutive promoter.
• the inserted nucleotide sequence further comprises a terminator (e.g., a Glue terminator) and may contain an ogfp gene encoding a green fluorescent protein codon-optimized for expression in Botrytis cinerea.
• a terminator e.g., a Glue terminator
• Reducing the sequence identity to at least 90% may be due to replacing nucleotides with other nucleotides, especially nucleotides resulting in a codon encoding the same amino acid (synonymous mutations), natural differences and mutations in Claviceps purpurea genes, and replacing nucleotides with other nucleotides in non-coding portions of the gene (introns).
• the sequence identity is at least 95%.
• the amount of ergot alkaloids in submerged culture, mycelium and medium of transformed Claviceps purpurea according to the present invention is significantly higher than the amount of ergot alkaloids in the unmodified Claviceps purpurea strain.
• the inserted nucleotide sequences are under the control of a constitutive promoter.
• constitutive promoters are an OliC promoter of the nuclear gene encoding mitochondrial ATP synthetase subunit 9 or a gpdA promoter of the gene encoding glyceraldehyde-3-phosphate dehydrogenase, both promoters are derived from Aspergillus nidulans.
• the genetically engineered transformants of Claviceps purpurea with increased ergot alkaloid production according to the present invention are selected from the group comprising strains designated in the examples as OE TrpE S76L 4, OE ogfp_dmaW 8 and deposited with the Czech Collection of Microorganisms (CCM) on 25.06.2019 as CCM 8984 ( Claviceps purpurea PN with TrpE gene inserted with S76L mutation (Cp TrpE SL)) and CCM 8983 ( Claviceps purpurea PN with inserted gene ogfp-dmaW (Cp ogfp-dmaW)).
• CCM 8984 Claviceps purpurea PN with TrpE gene inserted with S76L mutation (Cp TrpE SL)
• CCM 8983 Claviceps purpurea PN with inserted gene ogfp-dmaW (Cp ogf
• the present invention also provides a method for producing genetically engineered transformants of Claviceps purpurea comprising preparing an expression cassette comprising a nucleotide sequence comprising at least one gene selected from a gene encoding a-subunit of anthranilate synthase having the S76L mutation and a gene dmaW encoding dimethylallyltryptophan synthetase, said gene being under the control of a constitutive promoter, and a terminator, said expression cassette optionally further comprising an ogfp gene encoding a green fluorescent protein codon-optimized for expression in Botrytis cinerea, and this expression cassette is inserted into the genome of Claviceps purpurea.
• the expression cassette can be prepared, for example, by yeast recombinational cloning method.
• the expression cassette is inserted using a protoplast transformation method.
• the invention further provides an expression cassette comprising a constitutive promoter, at least one gene selected from a gene encoding a-subunit of anthranilate synthase having the S76L mutation, and a gene dmaW encoding dimethylallyltryptophan synthetase, and a terminator.
• the promoter is preferably the OliC promoter of the nuclear gene encoding the mitochondrial ATP subunit 9 of Aspergillus nidulans or the gpdA promoter of the gene encoding glyceraldehyde-3-phosphate dehydrogenase from Aspergillus nidulans.
• the terminator is preferably a Glue terminator.
• the expression cassette may optionally further comprise an ogfp gene encoding a green fluorescent protein, codon-optimized for expression in Botrytis cinerea, which serves as the N-terminal fusion domain of the target enzyme.
• the expression cassette comprises: OliC promoter ::TrpE S76L ::Gluc terminator and has a sequence with at least 90% identity, more preferably with at least 95% identity, to sequence SEQ ID NO. 3.
• the expression cassette comprises: OliC promotor: :ogfp- dmaWv.Gluc terminator and has a sequence with at least 90% identity, more preferably with at least 95% identity, to sequence SEQ ID NO. 4.
• Another object of the present invention is the use of the said expression cassettes for the preparation of the fungus Claviceps purpurea as a production microorganism for producing ergot alkaloids.
• ATAG Nucleotide sequence of the dmaW gene encoding dimethylallyltryptophan synthetase (1466 bp, SEQ ID NO. 2):
• Fig 1 Scheme of preparation of expression vectors by yeast recombinational cloning: pNDH-OGG vector containing ogfp (A), cloning of mutated TrpE (amplification of the TrpE gene from genomic DNA in two parts, the position of the mutation is shown by an asterisk) into pNDH-OGG with cleaved ogfp (B), cloning of dmaW (N-terminal fusion of DMATS with OGFP) into pNDH-OGG (C).
• 3 3 "flank Botrytis cinerea reductase nitrate
• 5' 5 "flank Botrytis cinerea reductase nitrate.
• Fig. 2 PCR detection of transgenes in the genetically modified C. purpurea (strain PI) fungi expressing a mutated version of TrpE ( TrpE S76L ) under the control of the constitutive promoter OliC ( PoliC ).
• the detected transgenic DNA region is represented by arrows. Black arrows show the primers annealing to the OliC promoter and the GluC terminator, flanking the inserted TrpE gene with a mutation.
• Gray arrows indicate primers annealing to the hph gene reading frame, 3": 3 "flank Botrytis cinerea reductase nitrate, 5': 5 "flank Botrytis cinerea reductase nitrate (A). Detection of part of the Olic promoter, the gene and part of the GluC terminator. Amplicon size 2353 bp (B). Detection of hph gene. Amplicon size 366 bp (C). M: 1 kb Plus DNA marker, 4, 12: independent C. purpurea transformants with the integrated transgene, PC positive control (plasmid DNA carrying the transgene), NC: negative control (genomic DNA of a wild type fungus).
• Fig. 3 PCR detection of a transgene in the genetically modified C. purpurea (strain PI) fungus expressing ogfp under the control of the constitutive promoter OliC ⁇ PoliC ).
• the detected transgenic DNA region is represented by arrows.
• Black arrows show the primers annealing to the OliC promoter and the GluC terminator flanking the inserted ogfp gene.
• Gray arrows indicate primers annealing to the hph gene reading frame, 3": 3 "flank Botrytis cinerea reductase nitrate, 5': 5 "flank Botrytis cinerea reductase nitrate (A).
• Fig. 4 PCR detection of transgenes in genetically modified C. purpurea (strain PI) fungi expressing dmaW fused to ogfp ⁇ ogfp-dmaW) under the control of the constitutive promoter OliC ⁇ PoliC).
• the detected transgenic DNA region is represented by arrows. Black arrows show the primers annealing to the OliC promoter and the GluC terminator with the inserted ogfp and dmaW genes.
• Gray arrows indicate primers annealing to the hph gene reading frame, 3": 3 "flank Botrytis cinerea reductase nitrate, 5': 5 "flank Botrytis cinerea reductase nitrate (A). Detection of part of the Olic promoter, the gene and part of the GluC terminator. Amplicon size 3098 bp (B). Detection of hph gene. Amplicon size 366 bp (C). M:1 kb Plus DNA marker, 8, 13: independent C. purpurea transformants with the integrated transgene, PC: positive control (plasmid DNA carrying the transgene), NC: negative control (genomic DNA of a wild type fungus).
• Fig. 5 RT-PCR detection of transgenes in genetically modified C.
• purpurea strain PI
• fungi expressing a mutated version of TrpE ⁇ TrpE S76L ) or dmaW fused to ogfp ⁇ ogfp-dmaW), and free ogfp under the control of the constitutive promoter OliC ⁇ PoliC).
• Detection of hph gene Amplicon size 366 bp (A). Detection of g-actin.
• M 1 kb Plus DNA marker
• NC negative control (genomic DNA of a wild type fungus)
• 4, 12 independent C.
• purpurea transformants expressing the mutated TrpE S76L 1: ogfp-ex pressing C. purpurea transformant; 8, 13: independent C. purpurea PI transformants expressing ogfp-dmaW.
• Fig. 6 PCR detection of transgenes in my celia of wild type and genetically modified C. purpurea (strain PI) fungi expressing the mutated version of TrpE ⁇ TrpE S76L ) under the control of the constitutive promoter OliC ⁇ PoliC) after 10 days of incubation in T25N induction medium.
• the detected transgenic DNA region is represented by arrows.
• Black arrows show the primers annealing to the OUC promoter and the GluC terminator, flanking the inserted TrpE gene wit a mutation, 3': 3 'flank Botrytis cinerea reductase nitrate, 5': 5 'flank Botrytis cinerea reductase nitrate (A). Detection of part of the Olic promoter, the gene and part of the GluC terminator. Amplicon size 2353 bp (B). M:1 kb Plus DNA marker, 4, 12: independent C.
• Fig. 7 PCR detection of transgenes in wild type my celia and in genetically modified C. purpurea (strain PI) fungi expressing a mutated version of TrpE ⁇ TrpE S76L ) under the control of the constitutive promoter OUC ⁇ PoliC) after 10 days of incubation in T25N induction medium.
• the detected transgenic DNA region is represented by arrows. Gray arrows indicate primers annealing to the hph gene reading frame, 3': 3 'flank Botrytis cinerea reductase nitrate, 5': 5 'flank Botrytis cinerea reductase nitrate (A). Detection of hph gene.
• M 1 kb Plus DNA marker; 4, 12: independent
• C purpurea transformants expressing a mutated version of TrpE S76L
• PC positive control
• NC negative control (genomic DNA of a control wild type fungus)
• A-F 6 biological replicates.
• Fig. 8 PCR detection of transgenes in wild type mycelia and in genetically modified C. purpurea (PI strain) expressing ogfp or dmaW fused to ogfp ⁇ ogfp-dmaW) under the control of the constitutive promoter OUC ⁇ PoliC) after 14 days of incubation in T25N induction medium.
• the detected transgenic DNA region is represented by arrows.
• Black arrows show the primers annealing to the OUC promoter and the GluC terminator, with the inserted ogfp and dmaW genes, 3': 3 'flank Botrytis cinerea reductase nitrate, 5': 5 'flank Botrytis cinerea reductase nitrate (A). Detection of part of the Olic promoter, the gene and part of the GluC terminator. Amplicon size of 1292 bp or 3098 bp (B). M: 1 kb Plus DNA marker, 1: ogfp-expressing C. purpurea transformant; 8, 13: independent C.
• PCI positive control 1 (ogfp-carrying plasmid DNA)
• PC2 positive control 2 (ogfp-dma VF-carrying plasmid DNA)
• NC negative control (genomic DNA of a wild type fungus control)
• A-F 6 biological replicates.
• Fig. 9 PCR detection of transgenes in wild type mycelia and in genetically modified C. purpurea (PI strain) expressing ogfp or dmaW fused to ogfp ⁇ ogfp-dmaW) under the control of the constitutive promoter OUC ⁇ PoliC) after 14 days of incubation in T25N induction medium.
• the detected transgenic DNA region is represented by arrows. Gray arrows indicate primers annealing to the hph gene reading frame, 3': 3 'flank Botrytis cinerea reductase nitrate, 5': 5 'flank Botrytis cinerea reductase nitrate (A).
• Example 1 Preparation of genetically modified fungi 1. Preparation of expression vectors
• Two expression vectors were prepared (SEQ ID NO. 4, SEQ ID NO. 3): the first vector contained the dmaW gene, which encodes dimethylallyltryptophan synthetase, DMATS ( EnsemblFungi : CPUR_04076, SEQ ID NO. 2), the other vector contained a mutated version of the TrpE gene ( TrpE S76L ) for a-subunit of anthranilate synthase ( EnsemblFungi : CPUR_05013, SEQ ID NO. 1).
• TrpE S76L a mutated version of the TrpE gene for a-subunit of anthranilate synthase
• Each of the above genes was placed under the control of the constitutive OliC promoter of the Aspergillus nidulans nuclear gene encoding mitochondrial ATP synthetase subunit 9.
• ogfp i.e. codon-optimized egfp for expression in Botrytis cinerea
• DMATS Leroch M, Mernke D, Koppenhoefer D, Schneider P, Mosbach A, Doehlmann G, Hahn M. (2011), Applied and Environmental Microbiology 77(9), 2887-2897.
• dmaW and TrpE gene sequences were amplified from the genomic DNA of Claviceps purpurea strain 20.1, using Phusion High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA). In the case of the TrpE gene with the S76L mutation, the sequence of this gene was amplified in two steps, the desired mutation being introduced into the sequence of the primers used.
• the pNDH-OGG vector was digested with restriction endonucleases Notl and Ncol (New England Biolabs), and for the cloning of the dmaW gene, the vector pNDH-OGG was digested with restriction endonuclease Notl (New England Biolabs).
• the obtained DNA fragments were transformed into uracil-auxotrophic strain of Saccharomyces cerevisiae (strain FGSC 9721, Fungal Genetics Stock Center, Manhattan, USA). The transformation was performed according to Winston F, Dollard C, Ricupero-Hovasse SF. (1995), Yeast, 11(1), 53-55. In a microtube, 50% PEG 3350, 1M lithium acetate, the digested pNDH-OGG vector, and a PCR fragment amplified from C. purpurea 20.1 genomic DNA were mixed. S.
• cerevisiae FGSC 9721 cells pre-washed with distilled water and 100 mM lithium acetate, were added to this mixture, and resuspended in 100 mM lithium acetate.
• a microtube with undigested vector pNDH-OGG served as a positive control, and the negative control microtube contained only the digested vector without the DNA amplicon.
• Denaturated salmon sperm DNA (Sigma-Aldrich) was added to the microtubes.
• the mixture was incubated at 30 °C, then at 42 °C and then plated on uracil-free SD agar plates (20 g D- glucose monohydrate, 6.7 g yeast nitrogen base without amino acids, 0.77 g amino acid mixture and nutrients without uracil, made up to 1 liter with distilled water, pH adjusted to 5.8, autoclaved for 20 min at 120 °C). After three days of incubation at 30 °C, the grown colonies of S.
• plasmid DNA was isolated using the QIAprep Spin Miniprep kit (Qiagen, Hilden, Germany) and subsequently used to transform Escherichia coli (strain TOP 10) (New England Biolabs). Plasmid DNA was pipetted into a microtube with E. coli TOP 10. After incubation on ice, the cells were heat shocked at 42 °C and the mixture was again transferred to ice.
• SOC medium (20 g of tryptone, 5 g of yeast extract and 0.5 g of sodium chloride dissolved in 950 ml of distilled water, followed by addition of 10 ml of 250 mM potassium chloride, the mixture was adjusted to pH 7.0, and made up to 1 liter with distilled water; after autoclaving for 20 min at 120 °C, 20 ml of 1 M D-glucose and 5 ml of 2 M magnesium chloride were added to the solution) was added to the mixture, and after 1 h incubation at 37 °C, E.
• coli TOP10 cells were plated on FB agar plates (9.5 g sodium chloride, 15.5 g FB Broth, 15 g agar, made up to 1 liter with distilled water, pH adjusted to 7.2, autoclaved for 20 min at 120 °C) with ampicillin (Sigma-Aldrich) of the final concentration 100 mg/ml. The plates were incubated until the next day at 37 °C. Plasmid DNA was then isolated from the grown E. coli TOP10 colonies using the QIAprep Spin Miniprep kit (Qiagen). The resulting constructs were verified by restriction analysis and commercial sequencing (SEQme, Dobns, Czech Republic).
• the plasmid DNA and also vector pNDH-OGG containing free ogfp were used for the transformation of C. purpurea (strain PI) protoplasts (Tudzynski P, Hdlter K, Correia T, Arntz C, Grammel N, Keller U. (1999), Molecular and General Genetics MGG, 261(1), 133-141).
• the vectors Prior to transformation, the vectors were linearized with Pstl restriction endonuclease (New England Biolabs), and the restriction mixtures were purified using a NucleoSpin Gel and PCR Clean-up kit (MACHEREY-NAGEL GmbH and Co. KG, Berlin, Germany). Purified linearized vectors were then transformed into C.
• C. purpurea (strain PI), which produces ergotamine, ergotaminine, a-ergocryptine and a-ergocryptinine in submerged culture (Haarmann T, Lorenz N, Tudzynski P. (2008), Fungal Genetics and Biology, 45(1), 35-4). Transformation of C. purpurea (strain PI) protoplasts was performed as follows. After 3 days, the culture of C.
• purpurea PI in BII cultivation medium 100 g sucrose, 5 g peptone, 5 g L- asparagine monohydrate, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulphate heptahydrate, 0.01 g ferrous sulphate heptahydrate, made up to 1 liter with distilled water, pH adjusted to 5.8, autoclaved for 20 min at 120 °C) was centrifuged and then washed with SmaC buffer (123.88 g sorbitol, 5.88 calcium chloride, made up to 800 ml with 0.2 M potassium malate, autoclaved for 20 min at 120 °C).
• SmaC buffer 123.88 g sorbitol, 5.88 calcium chloride, made up to 800 ml with 0.2 M potassium malate, autoclaved for 20 min at 120 °C).
• a protoplastization solution (100 mg of Trichoderma harzianum lysing enzyme in 20 ml of SmaC buffer, sterilized through a 0.22 pm pore size filter) was added to the mycelium. After 2 h incubation at 28 °C, the mixture was filtered through a Nytex membrane (Fluka, Germany). The filtrate was centrifuged and washed twice with STC buffer (154.84 g sorbitol, 1.22 g Tris, 7.36 g calcium chloride, made up to 1 liter with distilled water, pH adjusted to 7.5, autoclaved for 20 min at 120 °C), in which the pellet was subsequently resuspended. Purified linearized plasmid DNA, STC buffer and C.
• purpurea PI protoplasts were mixed in a microtube and after 20 min of incubation, PEG 6000 was added to the tube. After another 5 min of incubation, STC buffer was added.
• 20 ml of BII transformation agar 200 g sucrose, 5 g peptone, 5 g L- asparagine monohydrate, 1 g potassium hydrogen phosphate, 0.5 g magnesium sulphate heptahydrate, made up to 1 liter with distilled water, pH adjusted to 8.0, autoclaved for 20 min at 120 °C was poured into a first petri dish together with C. purpurea PI protoplasts).
• transformation BII agar 20 ml of transformation BII agar were together with a transformation mixture containing DNA and protoplasts poured into a second petri dish.
• 160 ml of transformation BII agar were together with a transformation mixture containing DNA and protoplasts were poured into eight petri dishes which were after 24 h overlaid with BII transformation agar containing Hygromycin gold (InvivoGen, San Diego, CA, USA) after 24 h so that the final antibiotic concentration in the medium was 200 mg/ml.
• the hygromycin resistance gene (hph) which is present in the pNDH-OGG vector, was used as a marker for selection of transgenic fungi. Fungi grown after transformation were transferred to new BII cultivation medium containing Hygromycin gold at a final concentration of 200 mg/ml . The fungi were cultivated in the dark at 26 °C.
• Genomic DNA was isolated from the mycelia of C. purpurea (strain PI) transformants (Cenis JL. (1992), Nucleic Acids Res 20:2380).
• Lysis buffer (4.84 g Tris, 2.92 g sodium chloride, 1.86 g EDTA, 1 g SDS, made up to 200 ml with distilled water, pH adjusted to 8,5) was added to the lyophilized mycelia of C. purpurea PI transformants in a microtube. After 15 min of incubation at room temperature, 5 M potassium acetate was added to the mixture. After 20 min incubation at -20 °C, the samples were centrifuged, and the supernatant was mixed with isopropanol. After 45 min incubation at -20 °C, the samples were centrifuged and the pellet was washed with 70% ethanol (-20 °C). The pellet was dried and resuspended in nuclease-free water.
• Transgene integration was verified at the genomic level by PCR using GoTaq G2 Flexi DNA polymerase (Promega corporation, Madison, Wisconsin, USA) and two sets of primers.
• the presence of the hph gene, which is part of the pNDH-OGG vector (5 '-GA ATTC AGCGAGAGCCTGAC-3 ' (SEQ ID NO. 11) and 5 "-ACATTGTTGGAGCCGAAATC-3 ' (SEQ ID NO. 12)
• the transgene 5 '-CCCGG A A ACTC ACT CTCCTT-3 ' (SEQ ID NO. 13) and
• Genomic DNA from wild type C. purpurea (strain PI) was used as a negative control, and the plasmid DNAs with individual genes were used as positive controls (Fig. 2-4).
• C. purpurea strain PI
• strain PI C. purpurea
• the fungi were grown for 1 week on Mantle agar (100 g sucrose, 10 g L-asparagine monohydrate, 1 g calcium nitrate tetrahydrate, 0.25 g potassium dihydrogen phosphate, 0.25 g magnesium sulphate heptahydrate, 0.033 g ferrous sulphate heptahydrate, 0.27 g ferrous sulphate heptahydrate, 0.01 g L- cysteine, 0.1 g yeast extract, 20 g agar, made up to 1 liter with distilled water, pH adjusted to 5.2, autoclaved for 20 min at 112 °C), and then hyphae tips were cut under a stereomicroscope behind dichotomous branching. The hyphae tips were transferred to new Mantle agar. This procedure was repeated three times.
• RNA from transformants and wild type C was isolated using an RN Aqueous kit (Thermo Fischer Scientific, Waltham, MA, USA). The obtained RNA was treated with DNAse (Thermo Fischer Scientific) and then precipitated with LiCl (Thermo Fischer Scientific). Subsequently, transcription into cDNA was performed using RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fischer Scientific). RT-PCR was performed using GoTaq G2 Flexi DNA polymerase (Promega corporation), two sets of primers were used. One set of primers was used to amplify the hph gene (5 '-G A ATTC AGCG AG AGCCTG AC-3 ' (SEQ ID NO.
• Example 3 Determination of the content of ergot alkaloids in submerged cultures of C. purpurea PI
• C. purpurea (strain PI) transformants were cultivated in parallel with wild type C. purpurea (strain PI) for 3 weeks on Mantle agar. Then, each fungus was inoculated into six 250 ml Erlenmeyer flasks with 50 ml of liquid preinoculation InoCN medium (100 g sucrose, 10 g citric acid monohydrate, 0.12 g potassium chloride, 0.5 g potassium dihydrogen phosphate, 0.5 g magnesium sulphate heptahydrate, 1 g calcium nitrate tetrahydrate, 0.075 g nicotinic acid amide, 0.006 g zinc sulphate heptahydrate, 0.007 g ferrous sulphate heptahydrate, pFl adjusted to 5.2 with 10% NH 4 OH, made up to 1 liter with distilled water, autoclaved for 15 min at 105 °C).
• InoCN medium 100 g sucrose, 10
• the culture was homogenized and 2 ml of the obtained biomass were pipetted into a 250 ml Erlenmeyer flask containing 50 ml of T25N induction medium (300 g sucrose, 15 g citric acid monohydrate, 0.12 g potassium chloride, 0.5 g potassium dihydrogen phosphate, 0.5 g magnesium sulphate heptahydrate, 1 g calcium nitrate tetrahydrate, 0.075 g nicotinic acid amide, 0.006 g zinc sulphate heptahydrate, 0.007 g ferrous sulphate heptahydrate, pFl adjusted to 5.2 with 10% NH 4 OH, made up to 1 liter with distilled water, autoclaved for 20 min at 115 °C).
• T25N induction medium 300 g sucrose, 15 g citric acid monohydrate, 0.12 g potassium chloride, 0.5 g potassium dihydrogen phosphate, 0.5 g magnesium sulphate
• the cultures were filtered through a Miracloth membrane (Merck, Darmstadt, Germany), and the media and mycelia were used for further analyzes.
• the volume of the media was measured, the mycelia were lyophilized in a SCANVAC lyophilizer (Trigon-plus, suprany u Prahy, Czech Republic).
• Genomic DNA was again isolated from all obtained lyophilized mycelia, and the presence of the transgene and the hph gene in all samples was verified by PCR (see Example 1, item 3, detection of the transgene by PCR analysis) (Fig. 6-9).
• the eluate was evaporated under reduced pressure at 37 °C in an SPD SpeedVac instrument (Thermo electron, Waltham, MA, USA), the obtained residue was resuspended in 1 ml of 80% methanol. Subsequently, the sample was filtered through Costar SpinX 22 pm columns (Sigma-Aldrich, St. Louis, Missouri, USA) and 5 pi of this mixture were analyzed on UPLC.
• the prepared samples were analyzed by UPLC (ultra high performance liquid chromatography) using a Nexera system (Shimadzu, Prague, Czech Republic) equipped with a Cl 8 reversed phase column (Zorbax RRHD Eclipse Plus 1.8 pm, 2.1 mm ID x 150 mm, Agilent, Santa Clara, CA, USA).
• mobile phase B contained acetonitrile and water in a ratio of 4:1.
• ergopeptines were determined in the samples: ergotamine, ergotaminine, a-ergocryptine and a-ergocryptinine (methodology based on: Cudejkova MM, Vojta P, Valfk J, Galuszka P. (2016), New Biotechnology, 33(5), 743-754, 2016).
• Ergopeptines were eluted with a mobile phase A (0.15% acetic acid pH 4.4 and acetonitrile, 4:1, v/v) and B (water and acetonitrile, 1:4, v/v) with the following gradient: 0-5 min, 7- 9% B; 5-7 min, 9-13% B; 7-9 min, 13-29% B; 9-11 min, 29-44% B; 11-13 min, 44% B; 13-15 min, 44-59% B; 15-18 min, 59% B; 18-19 min, 59-7% B; and 19-25 min, 7% B.
• the column temperature was set at 15 °C.
• the monitoring was performed at 317 nm. Alkaloid concentrations were read from the peak area using LabSolution software (Shimadzu). The concentrations of the individual alkaloids were calculated to 1 mg of lyophilized mycelium and 1 ml of T25N induction medium.
• Table 1 summarizes the average amounts of ergopeptines in 1 mg of lyofilized mycelium (WT, OE ogfp, OE ogfp_dmaW 8, OE ogfp_dmaW 13).
• the table clearly shows that the transformants expressing only ogfp did not show any difference in comparison to C. purpurea PI WT (wild type).
• the transformants expressing dmaW fused with ogfp show increased levels of ergotamine and ergotaminine.
• Transformant No. 13 has shown also a significant increase of a- ergocryptinine.
• Table 2 summarizes the average amounts of ergopeptines in 1 ml of T25N induction medium (WT, OE gfp, OE gfp_dmaW 8, OE gfp_dmaW 13).
• the table shows that the transformants expressing only ogfp did not show any difference in comparison to wild type of C. purpurea PI.
• the transformants expressing dmaW fused with ogfp show increased levels of all examined ergopeptines, i.e., ergotamine, ergotaminine, a-ergocryptine and a-ergocryptinine.
• Table 3 summarizes the average amounts of ergopeptines in 1 mg of lyofilized mycelium (WT, OE TrpE S76L 4, OE TrpE S76L 12). Significant increase of all examined ergopeptines (ergotamine, ergotaminine, a-ergocryptine and a-ergocryptinine) was observed only for transformant No. 4.
• Table 4 summarizes the average amounts of ergopeptinesin 1 ml of T25N induction medium (WT, OE TrpE S76L 4, OE TrpE S76L 12). The table clearly shows that in both transformants, a significant increase of ergotamine and ergotaminine levels was observed. Transformant No. 4 showed also an increase in the levels of a-ergocryptine and a-ergocryptinine.
• Table 1 Average content of ergopeptines ergotamine, ergotaminine, a-ergocryptine, a-ergocryptinine) in mycelia of wild type (WT) and transformants of C. purpurea (strain PI) expressing ogfp (OE ogfp) and dmaW fused with ogfp (OE ogfp-dmaW 8, OE ogfp-dmaW 13).
• the amount of alkaloids was calculated to 1 mg of lyofilized mycelium in the culture after 14 days of cultivation ⁇ standard error. The values were obtained from 6 biological replicates. The data were analyzed by non-parametric Kruskal-Wallis ANOVA test: * p ⁇ 0.05, ** p ⁇ 0.01).
• Table 2 Average content of ergopeptines ergotamine, ergotaminine, a-ergocryptine, a-ergocryptinine in media of wild type (WT) and transformants of C. purpurea (strain PI) expressing ogfp ( OE ogfp ) and dmaW fused with ogfp (OE ogfp-dmaW 8, OE ogfp-dmaW 13).
• the amount of alkaloids was calculated to 1 mg of medium in the culture after 14 days of cultivation ⁇ standard error. The values were obtained from 6 biological replicates. The data were analyzed by non-parametric Kruskal -Wallis ANOVA test: * p ⁇ 0.05, ** p ⁇ 0.01).
• Table 3 Average content of ergopeptines ergotamine, ergotaminine, a-ergocryptine, a-ergocryptinine in media of wild type (WT) and transformants of C. purpurea (strain PI) expressing the mutated version of TrpE (OE TrpE S76L 4, OE TrpE S76L 12).
• the amount of alkaloids was calculated to 1 mg of lyofilized mycelium in the culture after 10 days of cultivation ⁇ standard error. The values were obtained from 6 biological replicates. The data were analyzed by non-parametric Kruskal -Wallis ANOVA test: * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.01).
• Table 4 Average content of ergopeptines ergotamine, ergotaminine, a-ergocryptine, a-ergocryptinine in media of wild type (WT) and transformants of C. purpurea (strain PI) expressing the mutated version of TrpE (OE TrpE S76L 4, OE TrpE S76L 12).
• the amount of alkaloids was calculated to 1 mg of medium in the culture after 10 days of cultivation ⁇ standard error. The values were obtained from 6 biological replicates. The data were analyzed by non-parametric Kruskal-Wallis ANOVA test: * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.01).

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