Process for producing chimeric cyclooligodepsipeptides in filamentous fungi

Abstract

A method for obtaining at least one microbial secondary metabolite or a derivative thereof, the method includes the step of heterologous expression of at least one synthetase of the secondary metabolite in at least one filamentous fungus. Also disclosed is an expression cassette, a plasmid vector including the expression cassette, an expression host, cyclodepsipeptides and a chimeric cyclodepsipeptide synthetase.

Claims

1. A method for obtaining at least one non-ribosomal peptide, the method comprising: the step of heterologous expression of at least one non-ribosomal peptide synthetase (NRPS) of at least one non-ribosomal peptide in filamentous fungus Aspergillus niger, wherein the at least one NRPS is a cyclodepsipeptide synthetase selected from the group containing Enniatin, PF1022, Beauvericin and Bassianolide synthetase, wherein an inducible expression system integrated into the genome of the filamentous fungus Aspergillus niger is used for heterologous expression of the at least one NRPS, wherein the inducible expression system comprises at least one expression cassette, the at east one expression cassette comprising a first module for constitutive expression of a tetracycline dependent transactivator rtTA2, a second module harboring the rtTA2-dependent promoter for inducible expression of the at least one NRPS and a polynucleotide encoding the at least one non-ribosomal peptide synthetase and a third module for integrating the at least one expression cassette into the fungal genome by homologous or heterologous recombination using appropriate selection markers.

2. The method according to claim 1, wherein the at least one NRPS comprises modules and/or domains of at least two different cyclodepsipeptide synthetases.

3. The method according to claim 2, wherein the at least one NRPS comprises module 1 of the cyclodepsipeptide synthetase and module 2, PCP domain and C-domain of another cyclodepsipeptide synthetase.

4. The method according to claim 1, wherein the expression cassette further comprises genes encoding for biosynthetic enzymes of metabolic precursors or metabolic intermediates.

5. The method according to claim 4, wherein the genes encode dehydrogenases capable of transforming amino acids to hydroxycarboxylic acids.

6. The method according to claim 1, wherein the culture media used for heterologous expression of the at least one NRPS comprises talcum, titanate, silica, or aluminum oxide particles.

7. The method according to claim 1, wherein the culture media used for heterologous expression of the at least one NRPS comprises 5-20 mM, of at least one hydroxycarboxylic acid and 10-30 mM of at least one amino acid.

8. The method according to claim 1, that the culture media used for heterologous expression of the at least one NRPS comprises at least on D- or L-hydroxycarboxylic add of the general formula RCHOHCO.sub.2H wherein R is selected from a group comprising: substituted and non-substituted C.sub.1-C.sub.50-alkyl, substituted and non-substituted C.sub.2-C.sub.50-alkenyl, substituted and non-substituted C.sub.2-C.sub.50-alkinyl substituted and non-substituted C.sub.3-C.sub.10-cycloalkyl, substituted and non-substituted C.sub.5-C.sub.7-cycloalkenyl, which in each case can be interrupted by one or more oxygen atoms, sulfur atoms, substituted or mono-substituted nitrogen atoms, double bonds and/or by one or more groups of the type C(O)O, OC(O), C(O), NHC(O)O, OC(O)NH and/or OC(O)O-aryl, heteroaryl, CH.sub.2-aryl or CH.sub.2-heteroaryl, wherein aryl and heteroaryl are substituted or non-substituted.

9. The method according in to claim 1, wherein the culture media used for heterologous expression of the at least one NRPS comprises at least one D- or L-amino add of the general formulate R.sup.2CHNH.sub.2CO.sub.2H, where in R.sup.2 is selected from the group comprising: substituted and non-substituted C.sub.1-C.sub.50-alkyl, substituted and non-substituted C.sub.2-C.sub.50-alkenyl, substituted and non-substituted C.sub.2-C.sub.50-alkinyl, substituted and non-substituted C.sub.3-C.sub.10-cycloalkyl, substituted and non-substituted C.sub.5-C.sub.7-cycloalkenyl, which in each case can be interrupted by one or more oxygen atoms, sulfur atoms, substituted or mono-substituted nitrogen atoms, double bonds and/or by one or more groups of the type C(O)O, OC(O), C(O), NHC(O)O, OC(O)NH and/or OC(O)O.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) The invention is further explained by the following examples with reference to the Figures. It shows:

(2) FIG. 1 a schematic view of the module arrangement of the cyclohexadepsipeptide synthetase (here enniatin synthetase);

(3) FIG. 2 Schematic overview of the integrated and inducible expression system in filamentous fungi;

(4) FIG. 3 Vector map of the expression plasmid with enniatin synthetase;

(5) FIG. 4 Overview of single and multiple integration events in the pyrG locus;

(6) FIG. 5 1H-NMR spectra of enniatin B obtained by heterologous expression of the corresponding synthase in A. niger;

(7) FIG. 6 MS-spectra of enniatin B obtained by heterologous expression of the corresponding synthase in A. niger;

(8) FIG. 7 MS/MS-spectra of enniatin B obtained by heterologous expression of the corresponding synthase in A. niger;

(9) FIG. 8 13C-NMR spectra of enniatin B obtained by heterologous expression of the corresponding synthase in A. niger;

(10) FIG. 9 X-Ray crystal structure of enniatin B;

(11) FIG. 10 preferred enniatin derivatives;

(12) FIG. 11 Analytical data of chloroenniatin;

(13) FIG. 11a Analytical data for Beauvericin obtained from A. niger;

(14) FIG. 12 Strategy for swapping whole modules between two fungal NRPS encoding sequences;

(15) FIG. 13 Selected homologous region for swapping of hydroxy carboxylic acid activating module between PF1022- and enniatin/beauvericin synthetase;

(16) FIG. 14 Overview of generating hybrid fungal NRPS: Sketch of a hybrid PFSYN/ESYN containing module 1 from PFSYN and module 2 from ESYN. The hydroxy carboxylic acid specificity comes from PF1022 (D-PheLac/D-Lac) the amino acid specificity from ESYN (L-Leu) to generate new cyclohexadepsipeptides;

(17) FIG. 15 HPLC-ESI-MS of chimeric [PheLac].sub.0-3-enniatin derivatives. Solvent A: water with 0.1% HCOOH, solvent B: acetonitrile with ACN mit 0.1% HCOOH. Flow rate: 0.2 mL/min. Measurements were performed with ESI-Orbitrap-MS, Exactive, Thermo Fisher Scientific, HPLC 1200 Series (Agilent Technologies) and a gradient from 5% to 100% from 1 to 8 min and subsequently 100% B for 5 more minutes. Column: Grace Grom-Sil 120 ODS-4 HE, 250 mm, 3 m. TCC=20 C.;

(18) FIG. 16 Determination of identity of chimeric [PheLac].sub.0-3-enniatin cyclodepsipeptides by tandem MS measurements. m/z of fragments (cleavage at peptide or ester bonds) corresponds to loss of one or several amino or hydroxy acids as indicated. Solvent A: water, solvent B: isopropanol. Flow rate: 0.4 mL/min. Agilent Technologies ESI-Triple-Quadrupol-MS, 6460 Series, UHPLC 1290 Infinity-Series (Agilent Technologies). Column: Agilent Poroshell 120 EC-C18 3.050 mm. TCC=45 C.

(19) FIG. 17 Structure formula of [PheLac].sub.3-Enniatin and HR-ESI-Orbitrap MS for determination of the molecular formula;

(20) FIG. 18 Changing hydroxy carboxylic acid specification from beauvericin (D-Hiv) into PF1022 (D-PheLac/D-Lac);

(21) FIG. 19 HPLC-ESI-MS of chimeric [PheLac].sub.2-3-beauvericin derivatives. Solvent A: water with 0.1% HCOOH, solvent B: acetonitrile with ACN mit 0.1% HCOOH. Flow rate: 0.2 mL/min. Measurements were performed with ESI-Orbitrap-MS, Exactive, Thermo Fisher Scientific, HPLC 1200 Series (Agilent Technologies) and a gradient from 5% to 100% from 1 to 8 min and subsequently 100% B for 5 more minutes. Column: Grace Grom-Sil 120 ODS-4 HE, 250 mm, 3 m. TCC=20 C.;

(22) FIG. 20 Determination of identity of chimeric [PheLac].sub.2-3-beauvericin cyclodepsipeptides by tandem MS measurements. m/z of fragments (cleavage at peptide or ester bonds) corresponds to loss of one or several amino or hydroxy acids as indicated. Solvent A: water, solvent B: isopropanol. Flow rate: 0.4 mL/min. Agilent Technologies ESI-Triple-Quadrupol-MS, 6460 Series, UHPLC 1290 Infinity-Series (Agilent Technologies). Column: Agilent Poroshell 120 EC-C18 3.050 mm. TCC=45 C.;

(23) FIG. 21 Extracted ion chromatogramm of d18 enniatin B obtained from the feeding dependent A. niger strain. This substance can be used as an internal standard for the determination of enniatin in crops. A deuterated standard has never been reported yet. The system allows the synthesis of deuterated enniatin variants as well as beauvericin variants.

(24) FIG. 22 Total Ion Chromatogram of [3-Br-Lac].sub.1 [Lac].sub.2 Enniatin B and Tandem MS of [3-Br-Lac].sub.1 [Lac].sub.2 Enniatin B;

(25) FIG. 23 Tandem MS of [3-F-Lac].sub.3 Enniatin B;

(26) FIG. 24 Tandem MS [3-N.sub.3-Lac].sub.3 enniatin B. The azido group allows 1,3 dipolar cyclo addition for further chemical modification of the enniatin variant; and

(27) FIG. 25 Tandem MS of [3-I-Lac].sub.3 enniatin B obtained by precursor directed biosynthesis in A. niger. Iodine containing enniatin variants undergo Elimination and substitution reactions.

(28) FIG. 26 Polynucleotide sequence (SEQ ID NO: 1) of PF/Enniatin Chimeric Synthetase

(29) FIG. 27 Polynucleotide sequence (SEQ. ID NO: 2) of PF/Beauvericin Chimeric Synthetase

A. FILAMENTOUS FUNGI AS EXPRESSION HOST

1. Terms and Definitions

(30) Host: A filamentous fungus belonging to the genera Aspergillus, Trichoderma, Penicillium, Fusarium, Rhizopus Dependent Host: A host that is able to synthesize cyclodepsipeptides only if precursors (hydroxy acids) are added to the medium (example: A. niger strain DS3.1) Independent Host: A host that is able to synthesize cyclodepsipeptides without addition of the precursors (example: A. niger strain V3.4)

2. Protocol: PEG-Mediated Transformation of A. niger

(31) The esyn1 gene of F. oxysporum was integrated in plasmid pVG2.2 to give plasmid pDS4.2 (FIG. 3) which comprises all three components of the Tet-on system:PgpdA::rtTA2S-M2 for constitutive expression of the transactivator rtTA, tetO7::Pmin::esyn1, which mediates esyn1 expression in a Dox-dependent manner and the pyrG* cassette, necessary for selection and targeting of the system to the pyrG locus of A. niger.

(32) The performed transformation of A. niger is based on the method described by Punt et al. The used recipient strains were protease-negative (prtT) and uracil-auxotroph (pyrG.sup.). The used constructs carry a mutated pyrG gene (pyrG*), which allows the transformants to grow on medium lacking uridine only after uptake of the foreign DNA and after homologous recombination of pyrG* with a mutated pyrG gene version of A. niger (both mutations are at different locations). The enniatin synthetase was expressed under control of the Tet-On expression system.

3. Materials and Solutions

(33) SMC: 1.33 M sorbitol 50 mM CaCl.sub.2 20 mM MES Buffer pH 5.8 TC: 50 mM CaCl.sub.2 10 mM Tris/HCl pH 7.5 STC: 1.33 M sorbitol in TC PEG buffer: 7.5 g PEG-6000 TC up to 30 mL ASP+N (50): 350 mM KCl 550 mM KH.sub.2PO.sub.4 3.5 M NaNO.sub.3 pH 5.5 Vishniac: 76 mM ZnSO4 178 mM H.sub.3BO.sub.3 25 mM MnCl.sub.2 18 mM FeSO.sub.4 7.1 mM CoCl.sub.2 6.4 mM CuSO.sub.4 6.2 mM NaMoO.sub.4 174 mM EDTA protoplastation solution: 250 mg lysing enzyme from Trichoderma harzianum (Sigma) SMC up to 10 mL pH 5.6 MM: 20 mL ASP+N (50) 20 mL glucose (50%) 2 mL MgSO.sub.4 1 mL Vishniac H.sub.2O to 1 L (addition of 2% agar for solid medium) CM: MM, supplemented with: 10 mL casamino acids (10%) 50 mL yeast extract (10%) H.sub.2O to 1 L (addition of 2% agar for solid medium) Tranformation plates: 325.19 g sucrose 20 mL ASP+N 2 mL MgSO.sub.4 1 mL Vishniac 12 g agar Top agar: 325.19 g sucrose 20 mL ASP+N 2 mL MgSO.sub.4 1 mL Vishniac 6 g agar

4. Experimental Procedure

(34) The recipient strains (e.g. MA169.4 or AB1.13) were cultured in 100 mL of CM (+10 mM uridine) at 30 C. and 120 rpm for 10-16 h. The mycelium was collected over a myracloth filter and washed once with SMC. Afterwards, the collected mycelium was added to the protoplastation solution (in 50 mL test tube) and incubated for 1-1.5 h at 37 C. and 80 rpm. The protoplastation was confirmed by microscopy. Then, the protoplasts were collected through a myracloth filter and washed once with STC. The suspension was centrifuged for 10 min at 10 C. and 2000 rpm. The supernatant was decanted and the pellet gently resuspended in 1 mL of STC. The suspension was transferred to an Eppendorf tube and centrifuged for 5 min at 10 C. and 6000 rpm. Again, the supernatant was decanted and the protoplasts resuspended in 1 mL of STC. The wash step was repeated twice. For each transformation, 100 L of protoplasts, 10 g plasmid pDS4.2 (in 10 L H.sub.2O) and 25 L PEG buffer were added to a 50 mL test tube and mixed gently. 1 mL of PEG buffer was added and the tube mixed gently. After 5 min, 2 mL STC were added and mixed. 20-25 mL of top agar (cooled to 40 C.) were added to the mixture and poured onto the prepared transformation plates ( 15 cm). The plates were incubated at 30 C. for 3-4 days. Afterwards, the transformants were purified twice by streaking the spores on MM plates for colony isolation. The purified strains were analyzed by PCR and Southern Blot to confirm proper uptake and insertion of plasmid pDS4.2 into the genome of the recipient strains (see FIG. 3).

5. Analytical Data for the Dependent Host Mutant DS3.1 Synthesizing Enniatin and Chloroenniatin (FIGS. 9 5 to 11)

(35) Cultivation Conditions:

(36) In order to identify the optimum condition for high yield production of enniatin a design-of-experiment approach was followed using the statistical software program MODE. The following parameters were varied in shake flask cultures of esyn1-expressing strain DS3.1: medium composition (minimal medium, complete medium, Fusarium defined medium), amino acid (in particular L-valine, L-leucine, L-isoleucine) supplementation (0-20 mM), hydroxyacid (in particular D-Hiv) supplementation, 0-50 mM), glucose concentration (1-5%), temperature, cultivation time (1-92 h) and Dox-concentration (0-20 g/ml). The parameters which mainly affected the enniatin yields were Dox and D-Hiv. The best cultivation medium identified contained 20 mM D-Hiv, 20 mM of one of the amino acids and 10 g/ml Dox. This medium composition improved the enniatin yield by a factor of 200. The enniatin yield was further increased about 4.75 fold by increasing the glucose concentration to 5% and by adding talcum. Enniatin Production Medium (EM): 20 mL ASP+N (50) 100 mL glucose (50%) 2 mL MgSO.sub.4 1 mL Vishniac 10 mL casamino acids (10%) 50 mL yeast extract (10%) 100 mL talc (10% in 50 mM Na-acetate buffer, pH 6.5) H.sub.2O to 1 L

(37) Strain DS3.1 (inoculation with 510.sup.6 spores/mL) was cultivated in EM at 26 C. and 250 rpm. After 16 h, 10 mM or 20 mM D-Hiv, 20 mM I-Val and 10 g/mL Dox were added. The biomass was harvested after 92 h, lyophilized and enniatin B extracted with ethyl acetate.

(38) Analytical Methods (See FIGS. 5-9, 11):

(39) .sup.1H-NMR and .sup.13C-NMR spectra of enniatin B were recorded on a Bruker Avance 400 NMR-spectrometer. Chemical shifts are given in -units (ppm) relative to the solvent signal. IR spectra were recorded on a Jasco FT-IR 4100 spectrometer. HRMS using ESI-technique was performed on a LTQ Orbitrap XL apparatus. The enniatin samples were directly infused into the mass spectrometer.

(40) Data for single-crystal structure determination of enniatin B were collected on an Oxford-Diffraction Xcalibur diffractometer, equipped with a CCD area detector Sapphire S and a graphite monochromator utilizing MoK.sub. radiation (=0.71073 ). Suitable crystals were attached to glass fibers using fluoropolyalkylether oil (ABCR) and transferred to a goniostat where they were cooled to 150 K for data collection. Software packages used: CrysAlis CCD for data collection, CrysAlis Pro for cell refinement and data reduction.

(41) Results:

(42) Enniatin B:

(43) A production of enniatin B up to 1000 mg/L in strain DS3.1 could be obtained and verified by MRM-analysis.

(44) 393 mg of purified enniatin B could be isolated from the biomass (27.5 mg) and the broth of a 1-L cultivation of transformant DS3.1 with supplementation of 10 mM d-Hiv and 20 mM I-Val to the medium.

(45) The isolated enniatin B was analyzed my MS, MS/MS, IR, NMR and X-Ray crystallography (see FIGS. 5-10):

(46) .sup.1H-NMR (400.1 MHz, CDCl.sub.3) =5.11 (d, .sup.3J.sub.H,H=8.7 Hz, 3H), 4.49 (d, .sup.3J.sub.H,H=9.7 Hz, 3H), 3.11 (s, 9H), 2.32-2.21 (m, 6H), 1.05-0.86 ppm (m, 36H); .sup.13C-NMR (100.6 MHz, CDCl.sub.3) =170.25, 169.31, 75.67, 63.20, 33.26, 29.91, 27.92, 20.42, 19.34, 18.72, 18.50 ppm; IR (Neat): v=2963.6-2873.4 (CH, CH.sub.3 and CH), 1736.1 (CO, ester), 1660.9 (CO, amide), 1183.6 (CH, isopropyl) 1011.0 (CO, -hydroxycarboxylic acid); ESI-HRMS: m/z calcd for [C.sub.33H.sub.57N.sub.3O.sub.9+Na].sup.+: 662.39870; found: 662.39859.

(47) Generation of Enniatin Analogs:

(48) Protocol for Feeding Experiments:

(49) Strain DS3.1 was cultivated under the same conditions as described above. Instead of D-Hiv, the corresponding hydroxy acid chlorolactate were added (10 mM final concentration). Feeding can be performed once or repeatedly at different time intervals during batch or fed batch cultivation.

(50) An equivalent approach provided Beauvericin (see FIG. 11a).

B) METHODS AND ANALYTICAL DATA FOR GENERATION OF HYBRID SYNTHETASE

1. Cloning Strategy for Generating Chimeric Synthetases (See FIGS. 12-14, 18)

(51) Expression plasmids using T7 promoter for heterologous expression in E. coli were purchased from Merck Millipore. Protein expression of all hybrid genes coding for NRPS were cloned by insertion via restrictions sites into the high copy pRSF-Duet1 plasmid. Combinatorial biosynthesis was performed by -recombination mediated system. Sequences of desired domain or module were amplified via PCR (Q5-polymerase, High-Fidelity, New England Biolabs) and sub cloned into pRSF-Duet1. A selection marker (coding sequence for streptomycin resistance) which is needed after recombination was integrated at a single restriction site into the subcloned fragment. Positive clones were screened for streptomycin resistance and subsequently removed resistance by restriction and self-ligation of the flanking single cutter site. Electro competent E. coli BW25113 cells were transformed with the desired vector with the original NRPS gene cultivated at 30 C. to save the heat labile plasmid pIJ790 harbouring -recombinase RED (gam, bet, exo) and single DNA protecting proteins. Recombinase expression was induced by addition of arabinose before E. coli BW25113 cells were transformed with the module containing plasmid up to 70 base pairs were chosen as overlapping homologous region (modified after Gust et al./Zhang et al. 1998).

2. Production of Chimeric Enniatins and Beauvericins with Chimeric Synthetases Expressed in E. coli

(52) Culture Conditions and Extraction

(53) E. coli BI21gold cells were pre cultured over night at 37 C. in 20 ml of LB-medium at 200 rpm. Inoculation of main culture (50 ml LB-medium) with pre culture in the ration 1:100 was incubated to an OD.sub.600 of 0.6 at 37 C. and induced with IPTG at a final concentration of 0.25 mM (Carl Roth) and further incubated for protein expression and peptide production at 18 C. for 24-48 h 200 rpm. Cells were harvested and cell pellet was extracted with 5 ml of MeOH (technical grade). The suspension was sonified for 5 minutes and supernatant was evaporated.

(54) Analysis with HPLC-ESI-MS, HPLC-ESI-MS and Tandem MS (See FIGS. 15-17, 19-25)

(55) For complex analytics crude extracts were resolved in 200-500 l MeOH (HPLC grade) and suspended solids were centrifuged at 14 000g. HPLC-ESI-MS was carried out for scanning new derivatives. For all measurements we used Agilent technologies regarding columns and HPLC/MS equipment (Eclipse Plus C18, 2.150 mm; UHPLC 1290 Infinity-Series, ESI-Triple-Quadrupol-MS, 6460 Series). By using a mobile phase system we added of 0.1% of HCOOH in H.sub.2O (A) and ACN (B) with a gradient from 5% to 100% over 2.5 min and subsequently 100% B for 5.5 more minutes.