METHODS FOR IMPROVING TITER AND PURITY OF BETA CAROTENE FERMENTATION IN BLAKESLEA TRISPORA (BETA-CAROTENE FERMENTATION METHOD)

Abstract

The present invention is related to the production of bio-based carotenoids, particularly to methods for enhancing the titer and purity of beta-carotene fermentation in a suitable host cell.

Claims

1. A process for fermentative production of carotenoids in a carotenoid-producing host cell, preferably a native carotenoid producer, more preferably Blakeslea trispora, wherein the fermentation is performed in the presence of an effective amount of curcumin being added during the fermentation, preferably curcumin concentrations below about 1.0% (w/v) is added.

2. A process according to claim 1, wherein the curcumin concentration added during fermentation is in the range of about 0.01 to 1.0% (w/v), preferably in the range of about 0.3 to 1.0% (w/v).

3. A process according to claim 1, wherein the production of beta-carotene is increased by at least about 12% compared to fermentation without addition of curcumin.

4. A process according to claim 1, wherein the formation of 7,8-dihydro-beta-carotene during the fermentation is reduced.

5. The process according to claim 4, wherein the percentage of 7,8-dihydro-beta-carotene based on total carotenoids formed during the fermentation is reduced to about 7% or less, preferably to about 3% based on total carotenoids.

6. The process according to claim 4, wherein the percentage of 7,8-dihydro-beta-carotene formed during the fermentation is reduced by about 70% compared to fermentation without addition of curcumin.

7. A process according to claim 1, wherein the percentage of beta-carotene based on total carotenoids formed during the fermentation is increased, preferably increased by about 3 to 11%.

8. The process according to claim 7, wherein the percentage of beta-carotene based on total carotenoids formed during fermentation is in the range of about 90% or more compared to fermentation without addition of curcumin.

9. The process according to claim 1, wherein the fermentation is performed without addition of isoniazid during the fermentation.

10. Use of curcumin added in an effective amount during fermentation of a carotenoid-producing host cell, preferably a native carotenoid producer, more preferably Blakeslea trispora, for increasing the percentage of beta-carotene based on total carotenoids, preferably increasing the percentage by at least about 3%.

11. Use of curcumin added in an effective amount during fermentation of a carotenoid-producing host cell, preferably a native carotenoid producer, more preferably Blakeslea trispora, preferably in concentrations of 1.0% of less (w/v) added during the fermentation, for reducing the percentage of 7,8-dihydro-beta-carotene based on total carotenoids, preferably reducing the percentage by about 70%.

12. Use of curcumin according to claim 10, wherein concentrations of about 0.1 to 1.0% (w/v) curcumin is added during fermentation.

Description

[0026] Particularly, the present invention features the following embodiments:

[0027] (1) A method for enhancing the titer of beta-carotene production in a fermentation process of producing beta-carotene in Blaskeslea trispora, wherein the method comprises adding an effective amount of curcumin during the fermentation.

[0028] (2) The method as of embodiment (1), wherein said curcumin is added in the amount of 1.0% or less by weight of the fermentation medium.

[0029] (3) The method as of embodiments (1) or (2), wherein said curcumin is added in the amount of 0.3% or less by weight of the fermentation medium.

[0030] (4) The method as of embodiments (1), (2), or (3), wherein said curcumin is added in the amount of 0.1% or less by weight of the fermentation medium.

[0031] (5) The method as of embodiments (1), (2), (3), or (4), wherein said curcumin is added in the amount of 0.03% or less by weight of the fermentation medium.

[0032] (6) The method of as of embodiments (1), (2), (3), (4), or (5), wherein said method further reduces the amount of 7,8-dihydro-beta-carotene produced during the fermentation.

[0033] (7) The method as of embodiments (1), (2), (3), (4), (5) or (6), wherein the amount of 7,8-dihydro-beta-carotene generated during the fermentation is less than 7% by weight of the fermentation medium.

[0034] (8) The method as of embodiments (1), (2), (3), (4), (5), (6) or (7), wherein isoniazid is not added during the fermentation.

[0035] (9) A method for reducing the amount of 7,8-dihydro-beta-carotene generated in a fermentation process of producing beta-carotene in Blaskeslea trispora, wherein the method comprises adding an effective amount of curcumin during the fermentation.

[0036] (10) The method as of embodiment (9), wherein said curcumin is added in the amount of 1.0% or less by weight of the fermentation medium.

[0037] (11) The method as of embodiments (9) or (10), wherein said curcumin is added in the amount of 0.3% or less by weight of the fermentation medium.

[0038] The following examples are illustrative only and are not intended to limit the scope of the invention in any way. The contents of all references, patent applications, patents and published patent applications, cited throughout this application are hereby incorporated by reference, in particular WO2008042338, WO2014096992, WO2006102342, US20070166782, EP1367131, WO2002010429.

EXAMPLES

Example 1: General Methods, Strains and Plasmids

[0039] Shake flask seed medium MIBLT-1. 23.5 g/l corn meal, 23 g/l soybean meal, 10 g/l glucose, 0.5 g/l KH.sub.2PO.sub.4 are mixed, the medium pH is adjusted to 6.3, homogenized with an Ultra-Turrax® blender (Ika) for 8 minutes at 13500 rpm, and heated at 70° C. for 45 minutes prior to autoclaving.

[0040] Shake flask production medium MF-BLT-22. 17.5 g/l corn meal, 40 g/l soya flour, 10 g/l soy lecithin, 0.5 g/l KH.sub.2PO.sub.4, 109 g/l soy oil, optionally 0.28 g/l isoniazid, optionally curcumin (1.0, 0.3, 0.1, 0.03 or 0.01% w/v), are mixed, the medium pH is adjusted to 6.3, is homogenized with an Ultra-Turrax® blender (Ika) for 8 minutes at 13500 rpm, and heated at 70° C. for 45 minutes prior to autoclaving.

[0041] Solution A. Dichloromethane and methanol are mixed in 1:1 ratio.

[0042] Shake Flask Process. An Erlenmeyer flask (250 or 300 mL volume) containing 30 ml of seed medium (MIBLT-1) is inoculated with spores of B. trispora (−) strain to a concentration of 4.5×10.sup.3 spores/ml. A second Erlenmeyer flask containing 30 ml of MIBLT-1 is inoculated with spores of B. trispora (+) strain to a concentration of 1.5×10.sup.3 spores/ml. The flasks are incubated (220 rpm, 5 cm displacement, 27° C., protected from light) for 48 hr. Three ml of (+) strain culture are added to 30 ml of (−) strain culture, and the mated strains mixture is incubated (220 rpm, 5 cm throw, 27° C., protected from light) for 45 minutes. Erlenmeyer flasks (250 or 300 mL volume) containing 20 ml of production medium (MF-BLT-22) are inoculated with 2 ml of the mated strains mixture. Flasks are incubated (250 rpm, 5 cm displacement, 25° C., protected from light). At 48 hours after inoculation of the 20 mL production cultures, 40 μL of a 50% v/v sterile beta-ionone solution in absolute ethanol is added to each flask. Cultures are returned to the incubator for a total of 7 days post inoculation.

[0043] Sample preparation for HPLC-analysis. Each flask culture is transferred to a 50 mL conical tube and homogenized with an Ultra-Turrax® blender (Ika) for 2 minutes at 24000 rpm. Weigh out one gram of sample into a 100 ml volumetric flask. Eighty milliliters of Solution A are added to the flask and the flask is sonicated for 5 min. The volume is brought to 100 ml with solution A, and the solution is stirred for 60 minutes and allowed to settle for another hour.

[0044] HPLC-analysis. Samples from the above extract (10 μl) are injected as is on a calibrated HPLC using a Suplex-PKB-100 column (Supelco), with a flow rate of 0.6 ml/min, and column temperature of 30° C. The mobile phase contains (per L) 455 ml acetonitrile, 500 ml methanol, 0.2 ml N-ethyldiisopropylamine, 25 ml aqueous solution of 0.2% ammonium acetate, and 50 mg BHT (dissolved in 20 ml of 2-propanol). Compounds are identified and quantified by UV at 448 nm.

Example 2: Effect on Beta-Carotene Production with B. trispora in the Presence of Curcumin

[0045] Concentration of beta-carotene was measured as described in Example 1, with comparison of different concentrations of curcumin. As shown in Table 1, the highest increase in beta-carotene was observed with 0.3% (w/v) curcumin added to the production medium. Addition of 0.28 g/l isoniazid resulted in 4.17 g beta-carotene per l, thus less than with the addition of 0.3% or 1.0% curcumin.

TABLE-US-00001 TABLE 1 Effect of curcumin on formation of beta-carotene (“BC”). “Increase BC” means the increased percentage in the presence of curcumin compared to beta-carotene production in the control (“None”) with no curcumin/isoniazid present in the medium (average of two measurements). For more details, see text. Curcumin [w/v] Isoniazid [g/l] BC [g/L] Increase BC None None 1.55 n.a. None 0.28 4.17 n.a. 0.01% None 1.74  12% 0.03% None 2.45  58% 0.1% None 3.48 125% 0.3% None 4.99 222% 1.0% None 4.55 194%

Example 3: Effect on Beta-Carotene Purity Profile with B. trispora in the Presence of Curcumin

[0046] Fermentations and analysis were performed as described in Ex. 2, including addition of various concentrations of curcumin. The formation of beta-carotene and 7,8-dihydro-beta-carotene was analyzed by HPLC (see Ex. 1) and as shown in Table 2. Best results were obtained in the presence of 0.3 to 1.0% (w/v) curcumin, with an increase in the percentage of beta-carotene based on total carotenoids of 10.3 to 11.2% and a reduction in the percentage of 7,8-dihydro-beta-carotene based on total carotenoids of 65.4 to 67.7%. Addition of isoniazid resulted in a carotenoid-mix with a percentage of 28.1% 7,8-dihydro-beta-carotene and an increase of less than 10% in the percentage of beta-carotene (both based on total carotenoids).

TABLE-US-00002 TABLE 2 Effect of curcumin on formation of beta-carotene (“BC”), 7,8-dihydro-beta-carotene (“7,8-DH”) shown as percentage of total carotenoids. “None” means no curcumin was present in the medium (average of two measurements). For more details, see text. Curcumin [w/v] BC [%] 7,8-DH [%] None 82.87 9.67 0.01% 81.19 9.98 0.03% 85.59 6.99 0.1% 86.75 5.79 0.3% 91.44 3.35 1.0% 92.17 3.12