METHOD FOR PRODUCING A COMPOUND OF THE STEROL BIOSYNTHESIS PATHWAY IN A EUKARYOTIC ORGANISM
20170253901 · 2017-09-07
Inventors
- PIERRE BERTHOMIEU (MONTPELLIER, FR)
- ORIANE MITH (MONTPELLIER, FR)
- LAURENCE MARQUES-BASTIDE (MONTPELLIER, FR)
- VÉRONIQUE PERRIER (VENDRES, FR)
- ERIC DUBREUCQ (MONTPELLIER, FR)
Cpc classification
C12N1/38
CHEMISTRY; METALLURGY
International classification
Abstract
The invention concerns a method for producing a compound of interest of the sterol biosynthesis pathway in a eukaryotic organism or a derivative of a compound of the sterol biosynthesis pathway in said eukaryotic organism, in particular a sterol. This method comprises a step for the in vitro culturing of cells of said eukaryotic organism modified to express or overexpress a defensin, in a culture medium suitable for the development of said cells and containing at least one element chosen from the transition metals, lead and selenium, at a concentration greater than or equal to the concentration of said element that is necessary for inducing the overproduction of said compound of interest by said cells.
Claims
1. A method for producing a compound of interest of a sterol biosynthesis pathway in a eukaryotic organism or derived from a compound of the sterol biosynthesis pathway in said eukaryotic organism, comprising a step of in vitro culture of cells of said eukaryotic organism, modified to express or overexpress a defensin, in an enriched culture medium configured to grow said cells and containing at least one element chosen from a group consisting of transition metals, lead and selenium, at a concentration greater than or equal to a concentration of said at least one element for inducing production by said cells of an amount of said compound of interest at least 2 times greater than an amount produced by said cells under identical culture conditions in a culture medium containing said at least one element at a minimum concentration for an optimal growth of said cells.
2. The method as claimed in claim 1, wherein the defensin is a defensing of a plant origin encoded by a gene of a Plant Defensin type 1 family having at least one of a three-dimensional structure comprising a CSαβ type motif, a gamma-core loop, and at least 30% amino acid identity with a defensin AhPDF1.1b of Arabidopsis halleri of sequence SEQ ID NO:2.
3. The method as claimed in claim 1, wherein said at least one element is zinc.
4. The method as claimed in claim 1, wherein said at least one element is present in said enriched culture medium at a concentration greater than or equal to the concentration of said at least one element for inducing the production by said cells of an amount of said compound of interest at least five times greater than the amount produced by said cells under identical culture conditions in said culture medium containing said at least one element at the minimum concentration for the optimal growth of said cells.
5. The method as claimed in claim 1, wherein said eukaryotic organism is chosen from a group consisting of yeasts, fungi, algae, microalgae, plants and animals.
6. The method as claimed in claim 1, wherein the cells of said eukaryotic organism are recombinant cells into which a nucleotide sequence coding for said defensin has been introduced.
7. The method as claimed in claim 1, further comprising a subsequent step of collecting the cells of said eukaryotic organism having accumulated said compound of interest.
8. The method as claimed in claim 1, wherein the compound of interest is a final compound in the sterol biosynthesis pathway.
9. The method as claimed in claim 1, wherein the compound of interest is an intermediate compound in the sterol biosynthesis pathway and said cells of said eukaryotic organism are recombinant cells modified to inhibit an enzyme activity involved in a biotransformation of said intermediate compound in the sterol biosynthesis pathway.
10. The method as claimed in claim 1, wherein the compound of interest is a compound derived from a compound of the sterol biosynthesis pathway, and said cells of said eukaryotic organism are recombinant cells modified to activate an enzyme activity involved in a biotransformation of said compound of the sterol biosynthesis pathway into said compound of interest.
11. A recombinant eukaryotic cell modified to express or overexpress a defensin and modified to inhibit an enzyme activity involved in a biotransformation of an intermediate compound in a sterol biosynthesis pathway in said recombinant eukaryotic cell.
12. The recombinant eukaryotic cell as claimed in claim 11, modified to activate an enzyme activity involved in the biotransformation of said intermediate compound into a compound of interest.
13. A method of using a recombinant yeast strain modified to express or overexpress a defensing for production of a compound of interest of a sterol biosynthesis pathway or derived from a compound of the sterol biosynthesis pathway in said recombinant yeast strain.
14. The method as claimed in claim 13, wherein cells of said recombinant yeast strain are subjected to a step of in vitro culture in an enriched culture medium configured to grow said cells and containing at least one element chosen from a group consisting of transition metals, lead and selenium, at a concentration greater than or equal to a concentration of said at least one element for inducing the production by said cells of an amount of said compound of interest at least 2 times greater than an amount produced by said cells under identical culture conditions in a culture medium containing said at least one element at a minimum concentration for an optimal growth of said cells.
15. The method as claimed in claim 13, wherein the defensin is a defensing of a plant origin encoded by a gene of a Plant Defensin type 1 family having at least one of a three-dimensional structure comprising a CSαβ type motif, a gamma-core loop, and at least 30% amino acid identity with a defensin AhPDF1.1b of Arabidopsis halleri of sequence SEQ ID NO:2.
16. The method as claimed in claim 1, comprising subsequent steps of collecting the cells of said eukaryotic organism having accumulated said compound of interest; and extracting said compound of interest.
17. The method as claimed in claim 10, wherein said cells of said eukaryotic organism are said recombinant cells modified to inhibit the enzyme activity involved in the biotransformation of an intermediate compound in the sterol biosynthesis pathway.
18. The method as claimed in claim 4, wherein said at least one element is present in said enriched culture medium at a concentration greater than or equal to the concentration of said at least one element for inducing the production by said cells of an amount of said compound of interest at least 10 times greater than the amount produced by said cells under identical culture conditions in said culture medium containing said at least one element at the minimum concentration for the optimal growth of said cells.
Description
[0080] The features and advantages of the invention will become more clearly apparent in light of the exemplary embodiment below, provided simply by way of nonlimiting illustration of the invention, with the support of
[0081]
[0082]
[0083]
[0084]
[0085]
[0086]
EXAMPLE 1—DEFENSIN AhPDF1.1b AND EXCESS OF ZINC
[0087] A strain of Saccharomyces cerevisiae was modified to express the plant defensin of Arabidopsis halleri AhPDF1.1b, then cultured in the presence of an excess of zinc in accordance with the present invention, in the following way.
[0088] The defensin AhPDF1.1b of Arabidopsis halleri (Genbank accession number: HF545648, nucleotide sequence SEQ ID NO:1, amino acid sequence SEQ ID NO:2) was firstly the subject of a molecular modeling study (Swiss Model, Geno3D or PyMol) and its structure was superimposed onto that of radish defensin RsAFP1 (amino acid sequence: GenBank accession number AAA69541; nucleotide sequence: GenBank accession number U18557) which has been widely described in the literature (Terras et al., 1995) as having a structure with a CSαβ type motif and a gamma-core loop. The representation obtained is shown in
[0089] The defensin AhPDF1.1b coding sequence was amplified by polymerase chain reaction (PCR) and cloned into the vector pYX212 (sold by Ingenenius), the map of which is shown in
[0090] The coding sequence is under the control of the yeast triose phosphate isomerase (TPI) constitutive promoter and upstream of the yeast heat shock protein HSF1 terminator (YGL073w).
[0091] The recombinant plasmid obtained, and also the empty plasmid pFL38H carrying the gene HIS3 which confers auxotrophy to histidine, described in the publication by Talke et al., 2006, were introduced into the yeast strain BY4741 (Mat a, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0) using the method for transformation using PEG and lithium chloride (Gietz & Woods, 2002). The strain BY4741 was transformed in parallel with the empty plasmids pYX212 and pFL38H and serves as negative control.
[0092] The recombinant strains were cultured at 30° C. at the density of approximately 50 CFU/cm.sup.2 on solid medium (1.43 g/l of Yeast Nitrogen Base without amino acids or ammonium (Ref. 233520, Difco), 20 g/l glucose, 6.4 g/l NH.sub.4NO.sub.3, 50 mM succinic acid-KOH pH 4.5, methionine 20 mg/l, leucine 60 mg/l, 20 g/l agarose (ref. D5, Euromedex)), supplemented: [0093] either by zinc sulfate (ZnSO.sub.4) at the final concentration of 2.5 μM, which corresponds to the minimum concentration necessary for the optimal growth of the cells, or [0094] by zinc sulfate (ZnSO.sub.4) at the final concentration of 20 mM, that is to say in excess in accordance with the present invention.
[0095] For each experiment, when the diameter of the colonies reached 0.48±0.02 mm on average, which corresponds to an exponential growth phase, the yeasts were harvested, rinsed twice in pure water and then lyophilized.
[0096] The neutral lipids were extracted with hexane and the remaining (polar) lipids were extracted with a mixture of chloroform/methanol (2V/1V) according to the protocol described by Lomascolo et al., 1994.
[0097] Each of the lipid fractions was then saponified according to standard AFNOR NFT 60-205 and samples of the unsaponifiable fractions containing between 50 and 100 μg of lipids were taken off.
[0098] The samples were analyzed by thin-layer chromatography (TLC) on silica gel TLC plate (ref. 60 F 254 from E. Merck KGaA).
[0099] As controls, different amounts (respectively five, 10, 25 and 50 μg) of pure ergosterol (Ref. 45480, Sigma) were deposited in parallel. Migration was carried out in a buffer hexane:ether:formic acid in the proportions 70:30:1. At the end of migration, the plate was air-dried then sprayed uniformly with a 50:50 solution of pure orthophosphoric acid and saturated copper sulfate. After drying, the plate was heated at 180° C. for 8 to 10 min. The amounts of sterols were determined by comparison with the samples constituting the calibration range, by means of a densitometer (CAMAG TLC Scanner 3 model “Scanner3_160813” S/N 160813 (1.14.28), E. Merck KGaA) which analyzes the plate at the wavelength of 325 nm.
[0100] The results obtained are shown in
##STR00001##
[0101] Ergosterol is considerably predominant in this fraction. It can be established that it represents more than 80% thereof.
[0102] The confirmation that this particular compound is indeed ergosterol was established by gas chromatography analysis coupled with mass spectrometry (GC/MS) in the following way.
[0103] A preparative TLC was carried out starting from 100 μL of the neutral unsaponifiable lipid fraction obtained for the culture condition expressing the plant defensin AhPDF1.1b, in the presence of 20 mM of zinc in the culture medium. After migration, the plate was scraped at the mark identified as corresponding to ergosterol. The product was extracted from the silica with chloroform. After filtration to totally eliminate the silica particles, the chloroform was evaporated and the extract was redissolved in hexane before being injected into gas chromatography coupled with a mass spectrometer.
[0104] The chromatogram obtained is shown in
[0105] Moreover, the mass spectrum determined for this peak indicates unambiguously that it is ergosterol, as shown in
[0106] The production yields of ergosterol by the yeasts, expressed in mg per g of dry weight of yeasts (mg/gDW) were moreover determined for each of the culture conditions. The results are indicated in table 1 below.
TABLE-US-00001 TABLE 1 production yields of ergosterol by yeasts as a function of the transformation/culture conditions Zinc concentration Defensin AhPDF1.1b Yield (mg/gDW) 2.5 μM No expression 4 2.5 μM Expression 4 20 mM No expression 5 20 mM Expression >120
[0107] For the combination “20 mM of zinc—expression of defensin”, the ergosterol production yield is considerably greater than that obtained for the other conditions.
[0108] The sterols represent more than 12% by weight relative to the total dry weight of yeasts.
[0109] Which genes of the sterol biosynthesis pathway are affected by the activation process according to the invention was also studied.
[0110] To this end, a transcriptomic approach was used.
[0111] The same yeast strains as those described above were cultured in the same conditions as described above. When the diameter of the colonies reached an average of 0.48±0.02 mm, the yeasts were harvested and immediately frozen in liquid nitrogen.
[0112] The RNA was then extracted using the method with TRIZOL®, described in the publication by Chomczynski and Sacchi, 1987. Briefly, the cells were milled by intense agitation in the presence of 0.3 mm glass beads in Trizol at 4° C. for 15 min. Chloroform was added at an amount of 1/5 of the volume. After centrifugation for 15 min at 9000×g, the supernatant was recovered and isopropyl alcohol was added at an amount of 50% of the volume of supernatant. After 10 min of incubation at −20° C. then 10 min of centrifugation at 9000×g, the pellet was recovered and rinsed twice in 75% ethanol then taken up again in 150 μl of water.
[0113] 100 μg of total RNA were then purified using the RNeasy kit (Qiagen). The DNA was eliminated by using a DNase directly on the kit column. The RNA was eluted in 30 μl of water.
[0114] 100 ng of purified RNA were labelled with cyanin3-dCTP using the “Quick Amp Labeling one-color kit” from Agilent Technologies. The labeled RNAs were hybridized on a yeast DNA chip “8-15 15 karray Agilent standard Yeast V2 g-Gene Expression Microarray” (Agilent Technologies) for 17 h at 65° C. After rinsing, the slides were read on a GENEPIX® 4000B scanner.
[0115] From the datasheet giving the hybridization intensities for all the genes under the four conditions tested (control yeasts (“Def−”) and yeasts expressing defensin (“Def+”) cultured in the presence of zinc at the final concentration of 2.5 μM (“Zn−”) or of zinc at the final concentration of 20 mM (“Zn+”)), the results obtained relating to the expression of all the genes involved in the ergosterol biosynthesis pathway are given in table 2 below.
TABLE-US-00002 TABLE 2 Expression of the genes involved in the ergosterol biosynthesis pathway as a function of the transformation/culture conditions (presence (“Def+”) or absence (“Def−”) of defensin expression, in the presence of zinc at 20 mM (“Zn+”) or zinc at 2.5 μM (“Zn−”) in the culture medium) Gene Def−/Zn− Def+/Zn− Def−/Zn+ Def+/Zn+ ERG1 10.063 10.011 8.8487 9.5168 ERG2 10.666 10.483 9.9281 10.286 ERG3 11.533 11.444 11.790 12.830 ERG4 10.527 10.377 10.816 11.373 ERG5 9.0856 9.1176 9.2295 9.4736 ERG6 9.1971 8.7485 8.9209 9.1201 ERG7 9.9551 10.065 9.4971 10.002 ERG8 10.597 10.686 11.022 10.367 ERG9 10.373 10.337 10.245 10.733 ERG10 10.835 10.742 9.7926 10.790 ERG11 11.167 11.170 10.396 11.028 ERG12 8.0901 7.8844 8.0822 8.0168 ERG13 9.5652 9.4018 8.9288 9.4047 ERG20 11.760 11.716 10.034 11.120 ERG24 10.013 9.9343 9.1541 9.8254 ERG25 11.768 11.733 11.603 12.798 ERG26 9.8690 9.7937 9.4158 9.9413 ERG27 10.072 10.279 9.6617 10.612 ERG28 9.4582 9.4305 8.6241 9.4682 HMG1 8.9564 8.9347 8.8616 9.0901 HMG2 10.308 10.321 9.8840 10.198 IDI1 10.804 10.842 10.539 10.394 MVD1 8.2992 8.3027 8.0140 8.6319
[0116] It is observed in particular that the expression of the genes ERG1, ERG3, ERG4, ERG7, ERG9, ERG10, ERG11, ERG13, ERG20, ERG24, ERG25, ERG26, ERG27, ERG28, MVD1 is increased, sometimes greatly, for the combination “expression of defensin+addition of zinc in excess (20 mM) in the culture medium” compared to the condition “normal yeast+addition of zinc in excess in the culture medium”; and as regards the genes ERG3, ERG4, ERG9, ERG25, ERG27 and MVD1, compared to all the conditions tested.
[0117] The results obtained above are projected onto the ergosterol biosynthesis pathway in yeast, comparing the condition “expression of defensin/presence of zinc in excess in the culture medium” (“Def+/Zn+”) with the condition “absence of defensin/presence of zinc in excess in the culture medium” (“Def−/Zn+”). The result is shown in
EXAMPLE 2—DEFENSIN AhPDF1.4 AND EXCESS OF ZINC
[0118] A strain of Saccharomyces cerevisiae was modified to express the plant defensin of Arabidopsis halleri AhPDF1.4 (nucleotide sequence SEQ ID NO:3, amino acid sequence SEQ ID NO:4; this defensin has a CSαβ type motif and a gamma-core loop, and has 38% amino acid identity with AhPDF1.1b), then cultured in the presence of an excess of zinc (20 mM), in accordance with the present invention, following the same protocol as that described in example 1 above. By way of comparison, a culture in the same culture medium containing zinc at a much lower concentration (2.5 μM), which is not considered as an excess within the context of the present invention, was also carried out.
[0119] The yields of production of ergosterol by yeasts, expressed in mg per g of dry weight of yeasts (mg/gDW) were determined for each of the culture conditions. The results are indicated in table 3 below.
TABLE-US-00003 TABLE 3 Yields of production of ergosterol by yeasts as a function of the transformation/culture conditions Zinc concentration Defensin AhPDF1.4 Yield (mg/gDW) 2.5 μM No expression 2.5 2.5 μM Expression 1.5 20 mM No expression 1 20 mM Expression 41.5
[0120] In this case, too, the yield of production of ergosterol is considerably increased for the combination “excess of zinc in the culture medium—expression of defensin”.
EXAMPLE 3—DEFENSINS TaDEF and MsDEF1 AND EXCESS OF ZINC
[0121] Strains of Saccharomyces cerevisiae were modified, according to the method described in example 1 above, to express, respectively: [0122] the plant defensin of common wheat Triticum aestivum TaDEF (nucleotide sequence SEQ ID NO:5, GenBank accession number AB089942; amino acid sequence SEQ ID NO:6, GenBank accession number BAC10287, this defensin has a CSαβ type motif and a gamma-core loop, and has 33% amino acid identity with AhPDF1.1b in the mature protein); or [0123] the plant defensin of the Medicago sativa plant MsDEF1 (nucleotide sequence SEQ ID NO:7, GenBank accession number AY681971; amino acid sequence SEQ ID NO:8, GenBank accession number AAV85436; this defensin has a CSαβ type motif and a gamma-core loop, and has 29% amino acid identity with AhPDF1.1b in the mature protein).
[0124] The strains modified in this way were cultured in the presence of an excess of zinc (20 mM), in accordance with the present invention, following the same protocol as that described in example 1 above.
[0125] By way of comparison, a strain of Saccharomyces cerevisiae modified to express the defensin AhPDF1.1b was also cultured in the same culture medium containing an excess of zinc (20 mM).
[0126] For each experiment, when the diameter of the colonies has reached 0.48±0.02 mm on average, which corresponds to an exponential growth phase, the yeasts were harvested, rinsed twice in pure water and then lyophilized.
[0127] The lyophilized samples were subjected to a step of saponification before extraction and analysis. For this purpose, a few tens of mg of sample had added to them 400 μl of a 10% (w/v) methanolic KOH solution, 0.2 ml of microbeads 0.1 mm in diameter and 6 mg of 7-dihydrocholesterol (7-DHC) used as internal standard. The mixture was then incubated at 85° C. for 2 hours with vigorous stirring. After cooling to room temperature, 300 μl of water were added. The samples were extracted three times with 700 μl of hexane. The hexane phases containing the unsaponifiable lipids were brought back together and dried, and then taken up in 200 μl of chloroform.
[0128] A Shimatzu GC 2010 Plus gas chromatograph, fitted with an automatic injector, a ZEBRON® ZB-5HT INFERNO® capillary column and a flame ionization detector, was used to assay the ergosterol, with reference to a standard range of commercial ergosterol (Sigma, purity ≧95%) and to an internal standard (7-DHC). The volume injected was 0.2 μl with a ratio of division of 1/25. The temperature of the injector and the detector was 310° C. That of the oven was made to vary from 270° C. to 310° C. in 10 min, then maintained at 310° C. for 10 min.
[0129] The yields of production of ergosterol by yeasts, expressed in μg per g of dry weight of yeasts (mg/gDW) were determined for each of the culture conditions. The results are indicated in table 4 below.
TABLE-US-00004 TABLE 4 Yields of production of ergosterol by yeasts as a function of the defensin employed (in the presence of 20 mM of zinc) Defensin Yield (mg/gDW) Control without defensin 2.0 AhPDF1.1b 13.7 TaDEF 6.5 MsDEF 10.5
[0130] As can be observed, in the presence of an excess of zinc (20 mM), the yeasts which express the defensins TaDEF and MsDEF have an increase in the ergosterol content by a factor of greater than 3 and greater than 5, respectively, compared to the yeasts which do not express defensin.
[0131] These defensins TaDEF and MsDEF also enable, as does AhPDF1.1b, the overproduction of ergosterol by yeasts.
EXAMPLE 4—DEFENSIN AhPDF1.1b AND EXCESS OF CADMIUM
[0132] A strain of Saccharomyces cerevisiae was modified to express the plant defensin of Arabidopsis thaliana AhPDF1.1b then cultured in the presence of an excess of cadmium (10 or 20 μM) following the same protocol as that described in example 1 above.
[0133] The protocol for saponification, for extraction of the unsaponifiables and the GC FID analysis is the same as for example 3 presented above.
[0134] The yields of production of ergosterol by yeasts, expressed in μg per g of dry weight of yeasts (μg/gDW) were determined for each of the culture conditions. The results are indicated in table 5 below.
TABLE-US-00005 TABLE 5 Yields of production of ergosterol by yeasts Cadmium concentration Expression of (μM) AhPDF1.1b Yield (μg/gDW) 10 No expression 21.3 10 Expression 43.7 20 No expression 35.6 20 Expression 91.5
[0135] It is observed that under the conditions of expression of defensin and in the presence of cadmium, the production of ergosterol is significantly increased compared to the other conditions, this effect being more pronounced at a concentration of cadmium in the culture medium of 20 μM.
[0136] The defensin AhPDF1.1b is thus capable of inducing an increase in the production of ergosterol when an excess of cadmium is provided in the medium.
EXAMPLE 5—OVERPRODUCTION OF INTERMEDIATE COMPOUNDS OF THE STEROL BIOSYNTHESIS PATHWAY IN YEAST
[0137] The following strains of Saccharomyces cerevisiae were used: [0138] mutant ERG2 (genotype BY4741; Mat a; his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, YMR202w::kanMX4), available from the EUROSCARF collection center (http://web.uni-frankfurt.de/fb15/mikro/euroscarf/about.html) under the accession number Y00788. In this mutant the sterol biosynthesis pathway is blocked to enable the accumulation of fecosterol, by inhibition of the enzyme activity associated with the gene ERG2; [0139] mutant ERG3 (genotype BY4741; Mat a; his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, YLR056w::kanMX4), available from the EUROSCARF collection center (http://web.uni-frankfurt.de/fb15/mikro/euroscarf/about.html) under the accession number Y02667. In this mutant the sterol biosynthesis pathway is blocked to enable the accumulation of episterol, by inhibition of the enzyme activity associated with the gene ERG3; [0140] mutant NCP1 (genotype W303-1B, MATalpha, leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 YHR042w::TRP1). In this mutant the sterol biosynthesis pathway is blocked to enable the accumulation of squalene, by inhibition of the enzyme activity associated with the gene NCP1.
[0141] Each of these mutants was modified to express the plant defensin of Arabidopsis halleri AhPDF1.1b, then cultured in the presence of an excess of zinc in accordance with the present invention, according to the protocol indicated in example 1 above, to overproduce the associated intermediate compound of interest of the sterol biosynthesis pathway, that is to say fecosterol, episterol and squalene, respectively.
EXAMPLE 6—OVERPRODUCTION OF COMPOUNDS IN THE MOSS PHYSCOMITRELLA PATENS
[0142] The coding sequence of the defensin AhPDF1.1b was introduced into either one of the plasmids proAct1in108Kan and BNRr-108-3′-5′-HSP-CaMV, under the control of the rice constitutive actin promoter and the soy inducible heat shock protein (HSP) promoter, respectively.
[0143] The plasmids were integrated at the locus 108 of the genome of the moss Physcomitrella patens by genetic transformation, as described in the publication by Schaefer and Zryd, 1997.
[0144] The clones were selected in the presence of kanamycin in the medium (selection marker).
[0145] For the overproduction of compounds of the sterol biosynthesis pathway, the clones are cultured in the following culture medium referred to as PPNH.sub.4, enriched with 4 mM of zinc: 3.3 mM Ca(NO.sub.3).sub.2, 1 mM MgSO.sub.4, 0.5 mM FeSO.sub.4, 0.2 mM KPO.sub.4 pH 7, 0.3 mM ammonium tartrate, 10 μM H.sub.3BO.sub.3, 15 μM MnCl.sub.2, 0.2 μM CuSO.sub.4, 2 μM ZnSO.sub.4, 0.2 μM KI, 0.2 μM CoCl.sub.2, 0.1 μM Na.sub.2MoO.sub.4, 7 g/l agar.
BIBLIOGRAPHIC REFERENCES
[0146] Boman, 1998, Scandinavian Journal of Immunology 48: 15-25 [0147] Burden et al., 1989, Phytochemistry, 28: 1791-1804 [0148] Burg et al., 2011, Progress in Lipid Research 50: 403-410 [0149] Chomczynski et Sacchi, 1987, Anal Biochem. 162:156-159 [0150] Cornet et al., 1995, Structure 3: 435-48 [0151] Duport et al., 1998, Nature Biotechnology 16: 186-189 [0152] Ganz et al., 1995, Pharmacology & Therapeutics 66: 191-205 [0153] Gietz & Woods, 2002, Methods in Enzymology 350: 87-96 [0154] Husselstein et al., 1996, FEBS Letters 381: 87-92 [0155] Kuhn et al., 1979, Eur. J. Appl. Microbiol. Biotechnol. 6: 341-349 [0156] Lay et al., 2005, Curr Protein Pept Sci 6: 85-101 [0157] Lomascolo et al., 1994, Revue Canadienne de Microbiologie 40: 724-729 [0158] Medaglia et Panke, 2010, Appl. Microbiol. Biotechnol. 87: 145-157 [0159] Mirouze et al., 2006, The Plant Journal 47: 329-342 [0160] Munoz et al., 2014, Mol Microbiol 92: 1357 [0161] Nowosielski et al., 2011, Journal of Chemical Information and Modeling 51: 455-462 [0162] Sagaram et al., 2011, PLoS One 6(4): e18550 [0163] Schaefer et Zryd, 1997, Plant Journal 11: 1195-1206 [0164] Shahzad et al., 2013, New Phytologist doi: 10.1111/nph.12396 [0165] Shobayashi et al., 2005, Biosci Biotechnol Biochem 69: 2381-2388 [0166] Talke et al., 2006, Plant Physiology, 142: 148-167 [0167] Terras et al., 1995, Plant Cel. 7(5): 573-588 [0168] Van der Weerden et al., 2013a, Fungal Biology Reviews 26: 121-131 [0169] Van der Weerden et al., 2013b, Cel. Mol. Life Sci. 70: 3545-3570 [0170] Wriessnegger et al., 2013, Progress in Lipid Research 52: 277-293