METHOD FOR SYNTHESIS OF FATTY ACIDS

Abstract

Disclosed is a method for synthesis of fatty acids by culturing a eukaryotic microorganism from the fungi kingdom, that is naturally oleaginous or rendered oleaginous. The culture is performed in the presence a fatty acid synthase inhibitor in the culture medium.

Claims

1. Method for synthesis of short-chain or medium-chain fatty acids by culturing a eukaryotic microorganism from the kingdom of fungi, that is naturally oleaginous or originates from the yeast strain JMY3501, wherein the culture is performed in the presence of a fatty acid synthase inhibitor in the culture medium.

2. Method for synthesis of short-chain or medium-chain fatty acids by culturing a eukaryotic microorganism from the kingdom of fungi, that is naturally oleaginous, wherein the culture is performed in the presence of a fatty acid synthase inhibitor in the culture medium.

3. Method according to claim 1, wherein the short or medium chain of the fatty acids has between 4 and 15 carbon atoms.

4. Method according to claim 1, wherein the fatty acid synthase inhibitor is selected from cerulenin and analogues thereof, triclosan (5-chloro-2-(2,4-dichlorophenoxy) phenol), TOFA (5-(tetradecyloxy)-2-20 furancarboxylic acid), bischloroanthrabenzoxocinone, thiolactomycin, platensimycin and also the analogues of these molecules selected from C75 (4-methylene-2-octyl-5-oxo-tetrahydrofuran-3-carboxylic acid), C93 (or FAS93), FAS31, orlistat (N-formyl-L-leucine (1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]methyl]dodecyl ester), GSK837149A (dibenzenesulfonamide urea), isoniazid, platencin, pyrazinamide, ethionamide, diazoborine, hexachlorophene, diclofenac, epigallocatechin-3-gallate (EGCG), luteolin, taxifolin, kaempferol, quercetin, apigenin, anthecotulide, anthecularin, 4-hydroxyanthecotulide, 4-acetoxyanthecotulide, and C247.

5. Method according to claim 1, wherein the fatty acid synthase inhibitor is cerulenin.

6. Method according to claim 1, wherein the microorganism is of the Yarrowia, Saccharomyces, Rhodotorula, or Rhodosporidiu genus.

7. Method according to claim 6, wherein the microorganism is the yeast Yarrowia lipolytica or Rhodotorula glutinis.

8. Method according to claim 6, wherein the microorganism is the yeast Yarrowia lipolytica.

9. Method according to claim 4, wherein the cerulenin is introduced into the culture medium either by continuous addition, or by pulsed addition, one-time addition, or multiple and successive additions.

10. Method according to claim 9, wherein the concentration of cerulenin varies from 0.01 to 25 mg/g of dry yeast.

11. Method according to claim 9, wherein the concentration of cerulenin varies from 1 to 25 mg/g of dry yeast.

12. Method according to claim 9, wherein the concentration of cerulenin varies from 0.01 to 1 mg/g of dry yeast.

13. Method according to claim 1, wherein the ratio between the rate of carbon consumption and the rate of nitrogen consumption (rC/rN) has a value between 5 and 100 moles of carbon consumed per mole of nitrogen consumed.

14. Method according to claim 1, wherein the ratio between the rate of carbon consumption and the rate of nitrogen consumption (rC/rN) has a value between 16 and 100 moles of carbon consumed per mole of nitrogen consumed.

15. Method according to claim 1, wherein the ratio between the rate of carbon consumption and the rate of nitrogen consumption (rC/rN) has a value between 12 and 50 moles of carbon consumed per mole of nitrogen consumed.

16. Method according to claim 1, wherein the content of phosphorus in the culture medium could be adjusted so as to keep the level of intracellular phosphorus of the yeast at a value varying from 4 to 27 mg/g of biomass.

17. Method according to claim 1, wherein the short-chain or medium-chain fatty acids are obtained in the form of a mixture of free fatty acids and triglycerides.

18. Method according to claim 9, wherein the concentration of cerulenin varies from 0.01 to 14 mg/g of dry yeast.

19. Method according to claim 9, wherein the concentration of cerulenin varies from 0.05 to 14 mg/g of dry yeast.

20. Method according to claim 9, wherein the concentration of cerulenin varies from 1 to 14 mg/g of dry yeast.

Description

[0060] The drawings and the following examples aim to further illustrate the present invention, without limiting the scope of the invention in any way.

[0061] FIG. 1: Detail of the central anabolism of the fatty acids in the case of Yarrowia lipolytica.

[0062] FIG. 2: Development of the concentration of biomass (g.sub.x.Math.l.sup.−1) over time (hours, h) during the culture in fed-batch mode of Y. lipolytica with introduction of a nitrogen limitation (symbol +) and injection of a pulse of DMSO (10 mL) (symbol X) (Culture A).

[0063] FIG. 3: Development of the fatty acids profile during the phase of lipid accumulation before (−2 h), during (0 h) and after (15 mn, 1 h, 3 h) a pulse of DMSO (10 mL) during a fed-batch culture of Y. lipolytica (Culture A).

[0064] FIG. 4: Development of the concentration of biomass (g.sub.x.Math.l.sup.−1) over time (h) during the culture in fed-batch mode of Y. lipolytica with the introduction of a nitrogen limitation (symbol +) and the injection of pulses of cerulenin of 7 mg.sub.cerulenin.Math.g.sub.x.sup.−1 (symbol X) (Culture B).

[0065] FIG. 5: Development of the rate of growth calculated on the basis of data of the capacitance probe [h−1] over time [h] during the culture in fed-batch mode of Y. lipolytica with the introduction of a nitrogen limitation (symbol +) and the injection of pulses of cerulenin of 7 mg.sub.cerulenin.Math.g.sub.x.sup.−1 (symbol X). (Culture B)

[0066] FIG. 6: Development of the fatty acids profile during the phase of lipid accumulation before (−2 h), during (0 h) and after (15 mn, 1 h, 3 h) a pulse of 7 mg.sub.cerulenin.Math.g.sub.x.sup.−1 during a fed-batch culture of Y. lipolytica. (Culture B)

[0067] FIG. 7: Development of the mass content [g.sub.AGi.Math.g.sub.x.sup.−1] of the different fatty acids predominantly present in Y. lipolytica during the phase of lipid accumulation before (−2 h) and after (3 h) a pulse of 7 mg.sub.cerulenin.Math.g.sub.x.sup.−1 during a fed-batch culture. (Culture B)

[0068] FIG. 8: Profile of the fatty acids accumulated by Y. Lipolytica 2 h after a pulse of cerulenin of 7 mg.sub.cerulenin.Math.g.sub.x.sup.−1 during a fed-batch culture with nitrogen limitation (Culture B).

[0069] FIG. 9: Comparison of the fatty acid profiles during the phase of lipid accumulation 3 h after pulses 1 and 2 during a fed-batch culture of Y. lipolytica. (Culture B).

[0070] FIG. 10: Development of the fatty acid profile before and after a pulse of ethanol during a fed-batch culture of Y. lipolytica JMY3501 (Culture C). The arrow indicates the point in time at which the pulse of ethanol was provided.

[0071] FIG. 11: Development of the fatty acid profile before and after a pulse of 0.25 mg.sub.cerulenin.Math.g.sub.x.sup.−1 during a fed-batch culture of Y. lipolytica JMY3501 (Culture D). The arrow indicates the point in time at which the pulse of cerulenin was provided.

A—PRACTICAL EXAMPLES OF THE INVENTION WITH THE STRAIN OF YARROWIA LIPOLYTICA W29

1—Materials and Methods

[0072] 1.1—Strain Yarrowia lipolytica W29 and Culture Media

[0073] The strain Yarrowia lipolytica W29 is a wild-type strain. Stocks of the strain were produced from axenic pre-cultures produced in baffled Erlenmeyer flasks placed on a rotary stirring table, with a rich medium having an initial concentration of glucose of 10 g/L. In the middle of the exponential phase, samples of 1 mL were taken and mixed with sterile glycerol (30% volume/volume). These stocks were then stored in sterile vials at −80° C. These frozen concentrated cultures were used to seed the various pre-cultures for the purposes of fed-batch culture. The pre-cultures of yeast were performed in two 100 mL Erlenmeyer flasks containing 8 mL of rich medium LB at 30° C. for 16 h on a rotary stirring table (100 rpm). The cultures were transferred to two 250 mL Erlenmeyer flasks containing 72 mL of mineral medium (pH 5.6) having an initial concentration of glucose of 10 g/L. After 12 h at 30° C., the cultures of 80 mL volume were used to seed two 5 L Erlenmeyer flasks containing 710 mL of mineral medium with vitamins. These flasks were incubated at 30° C. for 12 h, with an initial concentration of glucose of 10 g/L. The content of one of the flasks from the last culture was used to seed 8 L of mineral medium in a 20 L bioreactor. The series of pre-cultures performed in parallel was used to check the reproducibility of the pre-cultures.

[0074] The composition of the medium of type LB was as follows: casein peptone 10 g/L; NaCl 9 g/L; autolysed yeast extract 5 g/L with glucose at a concentration of 10 g/L.

[0075] The composition of the mineral medium was as follows: K.sub.2HPO.sub.4: 3 g/L; (NH.sub.4).sub.2SO.sub.4: 3 g/L; NaH.sub.2PO.sub.4,H.sub.2O: 3 g/L; MgSO.sub.4,7H.sub.2O: 1 g/L; ZnSO.sub.4,7H.sub.2O: 0.04 g/L; FeSO.sub.4,7H.sub.2O: 0.0163 g/L; MnSO4,H.sub.2O: 0.0038 g/L; CoCl.sub.2,6H.sub.2O: 0.0005 g/L; CuSO.sub.4,5H.sub.2O: 0.0009 g/L; Na.sub.2MoSO.sub.4,2H.sub.2O: 0.00006 g/L; CaCl.sub.2,2H.sub.2O: 0.23 g/L; H.sub.3BO.sub.3: 0.03 g/L; and 10 mL of a solution of vitamins. The solution of vitamins had been prepared at the following concentration by a factor of 1000: d-biotin: 0.05 g/L, thiamine chlorohydrate: 1 g/L, panthotenic acid: 1 g/L, pyridoxol chlorohydrate: 1 g/L; nicotinic acid: 1 g/L, p-aminobenzoic acid: 0.2 g/L, myo-inositol: 25 g/L. Before sterilisation, the pH of this medium was adjusted to 4.5 with a solution of H.sub.3PO.sub.4 and to a working pH (5.5) with an ammonia solution.

[0076] 1.2—Cultures

[0077] Fed-batch cultures (8 L) were produced in a 20 L bioreactor (total volume) using the Braun Biostat E culture system (Braun, Melsungen, Germany) without oxygen limitation.

[0078] The temperature was controlled to 28° C. and the pH to 5.5 by addition of a 10 mol/L solution of NH.sub.3 (growth phase) or of a solution of KOH (lipid accumulation phase). A software developed in the laboratory of the inventors made it possible to acquire and control the operating parameter values, such as the stirring speed, pH, temperature, partial pressure of dissolved oxygen (DO), and volumes and flow rates of the feed of the bases and of the anti-foaming agent.

[0079] The pressure in the bioreactor was regulated to 0.3 bar (relative pressure).

[0080] The maximum amount of anti-foaming agent (Struktol) added was equal to 0.5 mL per culture.

[0081] The bioreactor was equipped with three sterile feed systems (carbon source advantageously from glucose alone, salt, ammonia or potassium hydroxide) using peristaltic pumps (Masterflex and Gilson). The concentration of the feed of carbon source, advantageously from glucose alone, was equal to 730 g/L. The masses of the carbon source solution and of the ammonia (or potassium hydroxide) solution introduced into the bioreactor were measured continuously by monitoring the masses of the flasks containing the solution stocks (Sartorius scales). The concentrations of carbon source and of nitrogen in the fermenter were estimated according to the carbon balance and redox balance equations. The rate of evaporation was estimated on the basis of the culture temperature, the efficacy of the condenser of the fermenter, and the aeration flow rate. The culture volume was calculated according to a material balance realised on the basis of the inputs of substrate, salt, ammonia, base, vitamins and anti-foaming agent and the outputs by evaporation and sampling with and without biomass.

[0082] 1.3—Chemical Agents

[0083] The chemical products (glycerol, salts, oligoelements, orthophosphoric acid and NH.sub.3) were provided by Prolabo (France), and the vitamins were provided by Sigma (E.U.A.). All of these products were of the highest analytical quality available. The cerelose for the fed-batch cultures was provided by Roquette (France).

[0084] 1.4—Strategy for Feeding Glucose

[0085] During the growth phase, an exponential profile of the flow rate of the pump feeding the carbon source made it possible to maintain a constant growth rate.

[0086] During the phase of accumulation, a constant growth rate was maintained in the most stable manner possible by an exponential flow rate of the carbon source.

[0087] 1.5—Strategy for Feeding Concentrated Salts

[0088] The bioreactor was fed by a flow rate of a solution of concentrated salts corresponding to 1/10 of the flow rate feeding the substrate. The composition of the solution of concentrated salts was as follows: KCl: 20 g/L, CuSO.sub.4,5H.sub.2O: 0.6 g/L, NaCl: 20 g/L, Na.sub.2MoO.sub.4,2H.sub.2O: 0.094 g/L, MgSO.sub.4,7H.sub.2O: 27 g/L, CaCl.sub.2,2H.sub.2O: 6.4 g/L, ZnSO.sub.4,7H.sub.2O: 7.7 g/L, FeSO.sub.4,7H.sub.2O: 3.97 g/L, MnSO.sub.4,H.sub.2O: 0.47 g/L, H.sub.3BO.sub.3: 0.3 g/L, CoCl.sub.2,6H.sub.2O: 0.3 g/L, H.sub.3PO.sub.4: 46.7 g/L.

[0089] 1.6—Strategy for Feeding Vitamins

[0090] All of the cultures were performed with a sequenced feed of vitamins as a function of the growth rate: quantities of 0.1% (vol/vol) of solution of vitamins were added during production of 10 g/L of biomass.

[0091] 1.7—Strategy for Feeding Ammonium

[0092] During the growth phase, the nitrogen was added with the aid of the base pump in order to regulate the pH to a constant value equal to 5.5. During the lipid production phase, the addition of nitrogen was controlled by a peristaltic pump with an exponential flow rate, varying from 0.00014 L.Math.h.sup.−1 to 0.004 L.Math.h.sup.−1, of solution of NH.sub.3 (5 mol/L) in order to maintain a constant specific growth rate; the pH was regulated by addition of a solution of KOH (10 mol/L).

2. Analytical Methods

[0093] 2.1—Quantification and Qualification of the Biomass

[0094] The concentration of yeast was determined by spectrophotometric measurements at 600 nm in a HITACHI U-1100 spectrophotometer in a quartz cell having an optical path of 0.2 cm. Dilutions of the sample were performed such that the optical density was within the range of 0.1 to 0.6 AU. For each sample, the average of three measurements was calculated. In order to determine the dry mass of the cells, culture samples (5 to 10 ml) were collected by filtration over a 0.45 mm membrane (Sartorius) and were dried at 200 mm Hg and 60° C. for 48 h until a constant mass was obtained.

[0095] An on-line estimation of the concentration of active cells was performed using a capacitance probe (Fogale). This technology is based on the correlation between the volume of the viable catalytic biomass and the variation of the dielectric permittivity of the medium in which the cells are dispersed.

[0096] All the cell concentrations were expressed in g.sub.ms/L, that is to say the dry mass of yeast per unit of volume of culture. The amount of ash was determined after two processes of total combustion of the dry mass filters with biomass in the presence of 200 mL of 20 g/L solution of NH.sub.4NO.sub.3 in a muffle furnace at 550° C. for 12 h each time. The formula of the biomass was determined at ENSIACET (Toulouse, France) by elementary analysis of C, H, O and N and the ashes. Due to a significant accumulation of lipids, the formulas of the biomass varied during the course of the culture from CH.sub.1.86O.sub.0.52N.sub.0.13 (growth phase) to CH.sub.2.00O.sub.0.59N.sub.0.07 (accumulation phase).

[0097] 2.2—Sampling

[0098] Every 20 minutes, a sample of supernatant was collected by a tangential filtration system connected to an automated fraction collector. A sample of culture medium was collected every hour directly by means of a septum. All of the samples were stored at −20° C.

[0099] 2.3—Analysis of the Outlet Gases of the Reactor

[0100] The outlet gases of the fermenter were analysed every 20 seconds by mass spectroscopy at the outlet of the gas condenser of the fermenter. The mass spectrometer (PRIMA 600s; VG Gas, Manchester, United Kingdom) was used due to its accuracy in measuring the compositions of CO.sub.2, O.sub.2, N.sub.2 and Ar.

[0101] The rate of O.sub.2 consumption and the rate of CO.sub.2 production were calculated according to the material balances, combining the volume of the gases in the reactor, the flow rate of inflowing air (measured by a mass flowmeter), the temperature, humidity, and the pressure and composition of the inlet and outlet gases.

[0102] 2.4—Extraction and Quantification of the Lipids

[0103] The total cellular lipids were extracted in accordance with the technique of Cescut J. et al. (PloS one; 6 (11): e27966, 2011), which is an automisation of the working method of Bligh and Dyer, as follows: the gradient extraction of solvent was performed in a pressurised liquid extractor (SPE). 500 mg of lyophilisates were placed in the extraction cells. Three different solvent mixtures were injected under pressure and heat into the cell (100° C., 100 bars). The successive solvent mixtures were: methanol/chloroform (2:1, vol/vol), (1:1, vol/vol) and lastly (1:2, vol/vol).

[0104] The three organic phases were mixed and washed twice with a 25% (vol/vol) solution of a 0.88% solution of KCl (mass/volume) for 15 minutes under gentle stirring. The organic phase was recovered by liquid/liquid separation after centrifugation (5000×g, 10 min).

[0105] Lastly, the lipids were collected after the evaporation of the solvents in a centrifugal evaporator (45° C.; 500 g) from the Genevac brand. The total content of lipids was quantified by a gravimetric method. The extract of lipids was held in a chloroform/methanol mixture at −20° C.

[0106] 2.5—Evaluation of the Fatty Acid Profiles

[0107] The free or bonded fatty acids were methylated in fatty acid methyl ester (FAME) using trimethylsufonium hydroxide (TMSH, 0.2 M in methanol, Macherey-Nagel, Germany). The analysis was performed using a Hewlett-Packard 5890 gas phase chromatography apparatus equipped with a WCOT fused silica column measuring 50 m×250 mm×25 mm in size (VARIAN, E.U.A.) and equipped with an FID, under the following conditions: mobile phase: N.sub.2, flow rate 50 mL.Math.min.sup.−1, temperature of the furnace: 50-75° C. at 9° C..Math.min.sup.−1, then 75-140° C. at 13° C..Math.min.sup.−1, then 140-180° C..Math.min.sup.−1 at 1.5° C..Math.min.sup.−1, then 180-240° C. at 4.5° C..Math.min.sup.−1, injector temperature 140° C., detector temperature 250° C.

3. Results

[0108] According to preliminary studies, in which the mass of cerulenin (antibiotic) per mass unit of biomass (x) varied between 1 mg.sub.cerulenin.Math.g.sub.x.sup.−1 and 25 mg.sub.cerulenin.Math.g.sub.x.sup.−1, it was found that a dose of 15 mg.sub.cerulenin.Math.g.sub.x.sup.−1 completely inhibits growth. A dose of 7 μg.sub.cerulenin.Math.mg.sub.x.sup.−1 was thus retained for partial inhibition of the growth and the quantification of the modulation of the rate of elongation of the fatty acids of Y. lipolytica. Two cultures were performed.

[0109] Culture A, referred to as the control culture, made it possible to identify the influence of the DMSO, solvent of the antibiotic, on the physiology of the yeast. DMSO is a solvent which is indispensable for dissolving the antibiotic that was added during a pulse in the culture B.

[0110] All the operating conditions were identical during these two cultures.

Culture A

[0111] The results of culture A are shown by FIG. 2 and FIG. 3; they show the development of the growth and of the fatty acid profiles as a function of the culture time. It would appear that the growth dynamic is not influenced by the injection of DMSO. The stability of the fatty acid profile during the period between +15 min and +3 h relative to the DMSO pulse completes the analysis, revealing that DMSO does not affect the metabolism of lipid accumulation.

Conclusion: In control culture A, the DMSO pulse influences neither the rate of growth of the yeast nor the fatty acid profile.

Culture B

[0112] A pulse of cerulenin was introduced into the culture B at 28.2 h (FIG. 4), i.e. 11 h after the start of the nitrogen limitation phase, this pulse triggering the induction of lipid biosynthesis with a growth rate maintained at 0.045 h.sup.−1 (maximum variation 5%), when a cell concentration of 6.9 g.sub.x.Math.L.sup.−1 was reached.

[0113] With regard to the development of the cell concentration over time, the growth dynamic was not influenced by the cerulenin pulse during the 10 h of culture following the injection. As shown in FIG. 5, the variation of the growth rate during the 10 h following the first injection of cerulenin was less than 5%. It is shown that the supply of a cerulenin dose of 7 μg.sub.cerulenin.Math.mg.sub.x.sup.−1 during a culture of Y. lipolytica under nitrogen limitation conditions had no effect on the growth dynamic of the yeast.

[0114] Throughout the nitrogen limitation phase, an accumulation of total fatty acids was quantified on the basis of the imposed flow rate of the substrate in accordance with previous works (Cescut et al., PloS one; 6 (11): e27966, 2011) with an absence of citric acid secretion: 20% total fatty acids were accumulated during the entire nitrogen limitation phase (50 h), 3% of which were accumulated during the 10 h following the cerulenin pulse. With regard to the kinematic behaviour, a reduction of the specific speed of production of fatty acid from 0.004 g.sub.AG.Math.g.sub.x.Math.h.sup.−1 to 0.0017 g.sub.AG.Math.g.sub.x.Math.h.sup.−1 was observed following the addition of cerulenin.

[0115] With regard to the lipid profile, significant developments of the fatty acid composition of the lipids accumulated before and after the cerulenin pulse were observed. Looking at 0 h, the time of injection (culture time 28.2 h), it would appear that the fatty acid profile before the addition of cerulenin is composed primarily of C16:1 (17%), C18:2 (31%) and C16:0 (29%) with short-chain or medium-chain fatty acid levels (less than 15 carbon atoms) being less than 1%. The degree of unsaturation, defined by the ratio between the number of moles of unsaturation and the number of fatty acid moles, is 0.95.sup.+/.sub.−2% and the average length of the carbon chain, defined by the average carbon number of all the fatty acids, is 16.98.sup.+/.sub.−2%.

[0116] After the cerulenin pulse, from 15 min, the appearance of short-chain fatty acids was observed. This accumulation of fatty acid reached 24.5% after 3 h of culture. This was an unexpected result.

[0117] Between the injection and 3 h after the injection of cerulenin, Y. lipolytica synthesised and accumulated neo-synthesised fatty acids with an average degree of unsaturation of 0.6 and an average number of carbon atoms of 12.74 carbon atoms (Table 1).

[0118] The mass contents of fatty acids with a carbon chain length of C4:0-C8:0, C9:0-C12:0 and C13:0-C15:0 increased on the basis of the cerulenin pulse: the variation of mass in relation to the lipid composition prior to the pulse reached, respectively, 0.05 g.sub.AG.Math.g.sub.x.sup.−1, 0.07 g.sub.AG.Math.g.sub.x.sup.−1 and 0.07 g.sub.AG.Math.g.sub.x.sup.−1 in 3 h for the three aforementioned groups (FIG. 6). By contrast, the mass content of palmitic acid (C16:0) increased from 0.014 g.sub.AG.Math.g.sub.x.sup.−1 and that of palmitoleic acid (C16:1) from 0.023 g.sub.AG.Math.g.sub.x.sup.−1 in relation to the lipid composition before the pulse (FIG. 7). The specific rate of synthesis of the short-chain or medium-chain fatty acid is multiplied by a factor of 14 when the dynamics before and 3 h after the pulse are compared.

[0119] By defining F.sub.n,p as the mass fraction of a group of fatty acids of carbon chain length C.sub.n to C.sub.p relative to the total mass of accumulated fatty acid, it would appear that F.sub.4,8 is multiplied by 70 at 3 h, F.sub.9,12 by 28 and F.sub.13,15 by 15. For the fatty acids with a chain length greater than 15, a significant reduction of the mass fraction of fatty acids C16:0 and C18:2 was observed, whereas the mass fraction of the fatty acid C18:3 rose to 6% (FIG. 8). This is translated with regard to the degree of unsaturation into a reduction from 0.95 to 0.75 in 3 h and a reduction of the length of the carbon chain from 16.98 to 15.3.

TABLE-US-00001 TABLE 1 Degree of unsaturation and length of the carbon chains of free or esterified fatty acids present in Y. lipolytica during the phase of lipid accumulation before, during, and after a pulse of 7 mg.sub.cerulenin .Math. g.sub.x.sup.−1 during the course of a fed-batch culture of Y. lipolytica. (Culture B) −2 h 0 h +15 min. +1 h +3 h Degree of unsaturation 0.97 0.95 0.92 0.73 0.75 Length of the carbon chain 16.97 16.98 16.83 16.3 15.3

[0120] The effect of the partial inhibition of the elongation kinetics of the fatty acids by the cerulenin pulse disappeared after 9 h of culture: the fatty acid profile became identical to the profile of accumulated fatty acids before the pulse.

[0121] A complementary experiment in which a second pulse was introduced made it possible to reproduce the same biological phenomena as after the first pulse. The fatty acid profiles 3 h after pulses 1 and 2 are illustrated in FIG. 9. They are both similar for all fatty acids.

Conclusions

[0122] A dose of cerulenin of 7 mg.sub.cerulenin.Math.g.sub.x.sup.−1 makes it possible, during the lipid synthesis phase in Y. lipolytica on glucose in fed-batch mode: [0123] □ to maintain the growth dynamic and the synthesis of lipids, [0124] □ to produce an accumulation of short-chain fatty acids (C4-C15) by partial inhibition of the elongation kinetics of the fatty acids.

[0125] A strategy of sequenced additions of cerulenin doses has proven to be indispensable for maintaining the modulation of the profile of fatty acids synthesised by Y. lipolytica by encouraging the accumulation of short-chain or medium-chain fatty acids.

B—PRACTICAL EXAMPLES IN ACCORDANCE WITH THE INVENTION WITH THE STRAIN OF YARROWIA LIPOLYTICA JMY3501

1—Materials and Methods

Strain and Culture

[0126] The strain of Yarrowia lipolytica JMY3501 is a strain genetically modified so as to optimise the accumulation of lipids, the culture conditions and the conditions for obtaining said strain being described in (Lazar Z et al., Metabolic Engineering 26 (2014) 89-99).

[0127] The strain of Yarrowia lipolytica JMY3501 can be prepared for example by deriving the strain JMY1233 (Beopoulos et al., Applied and Environmental Microbiology 74 (2008) 7779-7789) as follows: [0128] i. TGL4 is deactivated by introducing the tgl4::URA3ex disruption cassette from the strain JMP1364 (Dulermo et al., Biochimica et Biophysica Acta 1831 (2013) 1486-1495), which produces the strain JMY2179. [0129] ii. An auxotrophic marker, URA3ex, is then removed from the strain JMY2179 using the strain JMP547 (Fickers et al., Journal of Microbiological Methods 55 (2003) 727-737), which produces the strain JMY3122. [0130] iii. The strain JMY3501 is then obtained by introducing, successively to the strain JMY3122, pTEF-DGA2-LEU2ex from the strain JMP1822, and pTEF-GPD1-URA3ex from the strain JMP1128 (Dulermoz and Nicaud, Metabolic Engineering 13 (2011) 482-491). The strain JMP1822 is obtained by replacing the marker URA3ex of the strain JMP1132 (Beopoulos et al. (Beopoulos et al., Applied and Environmental Microbiology 74 (2008) 7779-7789) with LEU2ex.

Culture C

[0131] Culture C was performed in fed-batch mode with the Yarrowia lipolytica yeast strain JMY3501, in a 3 L bioreactor with a usable volume of 1.5 L using the Biostat B. Braum Biotech International culture system (Sartorius AG, Germany) with the acquisition software MFCS/win 2.0. The temperature was regulated to 28° C. and the pH was regulated by addition of a 2.5 mol/L solution of NH.sub.4OH for the growth phase and by addition of a 2.5 mol/L solution of KOH for the nitrogen limitation phase. With the aim of avoiding an oxygen limitation, the amount of inflowing air and the stirring speed were controlled so as to keep the dissolved oxygen above 20% saturation. The compositions of the inflow and outflow air were analysed with the aid of a mass spectrometer (Amatek Process Instruments).

Culture D

[0132] The objective of culture D was to study the impact of cerulenin pulses, in a ratio less than 1 mg/g of dry mass of biomass, on the metabolism of the Yarrowia lipolytica yeast strain JMY3501 in terms of lipid accumulation, fatty acid composition, and citric acid production.

[0133] Culture D was performed under the same culture conditions as culture C.

[0134] The solution of cerulenin was prepared in ethanol and a pulse of 0.25 mg.sub.cerulenin.Math.g.sub.x.sup.−1 was introduced 6 h after the triggering of the nitrogen limitation phase.

2—Result

Culture C

[0135] The results of culture C are shown in FIG. 10; they show the development of the fatty acid profile as a function of the culture time. It would appear that the fatty acid profile is stable before and after the ethanol pulse, indicating that ethanol does not affect the metabolism of lipids.

Culture D

[0136] A significant development of the fatty acid composition of the lipids accumulated before and after the cerulenin pulse (FIG. 11) can be seen. Following the cerulenin pulse, there appears to be an increase in short-chain fatty acids, primarily of C14 and C12. Before the cerulenin pulse, the C14 content in the fatty acid composition was 7%, and that of C12 was 4%, whereas after the cerulenin pulse the C14 content was 14% and that of C12 was 7%.

[0137] It would appear that a cerulenin dose of 0.25 mg.sub.cerulenin.Math.g.sub.x.sup.−1 makes it possible to increase the accumulation of short-chain fatty acids during the lipid accumulation phase in Yarrowia lipolytica JMY3501.