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

For commercial production of carotenoids, such as e.g. lycopene or betacarotene, both chemical and biotechnological synthesis is competing. In nature, carotenoids are synthesized by certain bacteria, fungi and photosynthetic organisms. Whereas chemically produced beta-carotene is only available in all- trans configuration, naturally produced beta-carotene is available in various configurations, such as all-trans, mono-cis, di-cis and poly-cis. The most commonly used host systems in biotechnological production of beta-carotene with commercial relevance are algae, such as e.g. Dunaliella salina, or Haematococcus pluvialis, and fungi, such as e.g. Blakeslea trispora (for an overview, see e.g. Mussagy et al., Appl Microbiol Biotechnol 103:1095-1114 (2019).

Besides production of beta-carotene, further pathway intermediates or sideproducts are accumulating during beta-carotene fermentation, such as e.g. lycopene, gamma-carotene or 7,8 dihydro beta-carotene, which have a negative impact on the production process in many ways (yield, down-stream processing, etc.).

Thus, it is an ongoing task to look for fermentation methods and strains suitable for beta-carotene production, wherein the formation of undesired side-products is reduced.

Surprisingly, we now found that the purity of a carotenoid-mix obtained by fermentation, particularly using B. trispora as host cell, can be increased in the presence of low concentrations of lycopene cyclase inhibitors. Particularly, the present invention is directed to production of carotenoids in a carotenoid-producing host cell as defined herein, such as e.g. in B. trispora as host system, wherein the fermentation is performed in the presence of low concentrations of a lycopene cyclase inhibitor, with formation of undesired sideproducts, particularly 7,8 dihydro beta-carotene, reduced to about 5% or less based on total carotenoids.

Suitable lycopene cyclase inhibitors might be selected from nitrogen containing compounds, either aliphatic or heterocyclic compounds, including but not limited to pyridine, triethylamine, tripropylamine, imidazole and derivatives thereof, such as e.g. 2-methylpyridine, 2-acetylpyridine, 2-aminopyridine, 2- hydroxypyridine, 2,4-dimethylpyridine, 2,6-dimethylpyridine, 2,4,6-trimethyl pyridine, 1-methylimidazole, 2-methylimidazole, 2-phenylimidazole, 1 -ethyl-2- methylimidazole, 2-isopropylimidazole, creatinine, piperidine, nicotine, quinoline, 2-(4-chlorophenylthio)triethylamine hydrochloride, tripropylamine hydrochloride, diphenylamine, trimethylamine, isopropylamine, or diisopropylamine. Particularly useful for the purpose of the present invention are compounds selected from pyridine, triethylamine, tripropylamine, imidazole or derivatives thereof.

As used herein, the term "low concentration" in connection with lycopene cyclase inhibitors refers to an amount of up to about 1000 mg/l, such as e.g. about 500, 300, 200, 100, 50, 30, 20, 10, 8, 7, 5, 2, 1 mg/L, of the respective compounds, such as in particular e.g. pyridine, triethylamine, tripropylamine, imidazole, and/or derivatives thereof present in the fermentation broth /cultivation medium, wherein typically about 1 mg/L to about 100 mg/L - depending on the compound - are sufficient. Particularly, said compounds are added at the start of the fermentation but might also be added at a later stage, i.e. before carotenoid production is starting. Optionally, the lycopene cyclase inhibitors as defined herein are added at any timepoint from 0 to 24h after the inoculation of the production fermenters, such as e.g. after about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22 or 24h after inoculation. Typically, the fermentations run for 100 to 120 h, with production of at least about 2 to 4 g/l of betacarotene.

In one aspect, the present invention is directed to a process for reducing the formation of undesired side-products, particularly wherein the production of 7,8-dihydro-beta-carotene is reduced, said process comprising production of a carotenoid-mix in a carotenoid-producing host cell, such as e.g. B. trispora, in the presence of low concentrations of a lycopene cyclase inhibitor as defined herein, particularly selected from pyridine, triethylamine, tripropylamine, imidazole, and/or derivatives thereof, and wherein the percentage of 7,8- dihydro-beta-carotene is reduced to about 5% or less based on total carotenoids, preferably reduced to about 2.5% or less, such as e.g. about 4.5, 4,

3.5, 3, 2.5, 2, 1.5, 1, 0.5 or up to about 0% based on total carotenoids.

In one aspect, the present invention is directed to a process for reducing the formation of undesired side-products, particularly wherein the production of 7,8-dihydro-beta-carotene is reduced and/or lycopene is reduced, said process comprising production of a carotenoid-mix in a carotenoid-producing host cell, such as e.g. B. trispora, in the presence of low concentrations of a lycopene cyclase inhibitor as defined herein, particularly selected from pyridine, triethylamine, tripropylamine, imidazole, and/or derivatives thereof, and wherein the percentage of lycopene is reduced to about 5% or less based on total carotenoids, preferably reduced to about 2.5% or less, such as e.g. about

4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5 or up to about 0% based on total carotenoids.

In another aspect, the present invention is directed to a process for reducing the formation of undesired side-products, particularly wherein the production of 7,8-dihydro-beta-carotene is reduced and/or lycopene is reduced and/or gamma-carotene is reduced, said process comprising production of a carotenoid-mix in a carotenoid-producing host cell, such as e.g. B. trispora, in the presence of low concentrations of a lycopene cyclase inhibitor as defined herein, particularly selected from pyridine, triethylamine, tripropylamine, imidazole, and/or derivatives thereof, and wherein the percentage of lycopene is reduced to about 5% or less based on total carotenoids, preferably reduced to about 2.5% or less, such as e.g. about 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5 or up to about 0% based on total carotenoids.

Particularly, the process according to the present invention is related to betacarotene fermentation in a suitable host cell, particularly B. trispora, wherein the formation of 7,8-dihydro-beta-carotene and lycopene and gamma-carotene is reduced, such as e.g. to about 5% or less, such as e.g. about 2.5% or less, for each of the side-products based on total carotenoids and wherein the fermentation is performed in the presence of low concentration of lycopene cyclase inhibitors as defined herein, i.e. with up to 1000 mg/l, particularly a range of about 1 to 100 mg/L, preferably about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mg/L, of said lycopene cyclase inhibitors added during the fermentation.

In one particular embodiment, said process for reduction of 7,8-dihydo-beta- carotene and/or lycopene and/or gamma-carotene as described herein comprises addition of about 1 to 100 mg/L of a lycopene cyclase inhibitor as defined herein during the fermentation, such as e.g. about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mg/L, wherein the percentage of beta-carotene based on total carotenoids is maintained at the same level as without the addition of the lycopene cyclase inhibitors or is increased by at least about 1%, such as e.g. at least about 5, 10, 12, 13, 14, 15, 16, 17, 20, 25, 30% compared to a process without the addition of said lycopene cyclase inhibitors.

With regards to pyridine, a preferred range is about up to 100 mg/L present during the fermentation. With regards to triethylamine, a preferred range is about up to 30 mg/L present during the fermentation. With regards to tripropylamine, a preferred range is about up to 30 to 100 mg/L present during the fermentation. With regards to imidazole, the preferred range is about 1 mg/L present during the fermentation.

In one aspect, the present invention is directed to a process for production of a carotenoid-mix in a carotenoid-producing host cell, such as e.g. B. trispora, in the presence of low concentrations of a lycopene cyclase inhibitor as defined herein, particularly selected from pyridine, triethylamine, tripropylamine, imidazole, and/or derivatives thereof, wherein the percentage of beta-carotene based on total carotenoids is in the range of at least about 90%, such as e.g. about 92, 94, 95, 98, 99 or 100% based on total carotenoids, preferably in the form of trans beta-carotene.

In one particular aspect, the present invention is directed to a process for production of a carotenoid-mix in a carotenoid-producing host cell, such as e.g. B. trispora, in the presence of low concentrations of a lycopene cyclase inhibitor as defined herein, particularly selected from pyridine, triethylamine, tripropylamine, imidazole, and/or derivatives thereof, wherein the carotenoidmix produced via such process comprises beta-carotene with a percentage in the range of at least about 90%, such as e.g. about 92, 94, 95, 98, 99 or 100%, preferably trans beta-carotene, and wherein furthermore the carotenoid-mix comprises lycopene and/or gamma-carotene with a percentage for each sideproduct of about 5% or less, preferably about 2.5% or less, such as e.g. about 5, 4, 3, 2.5, 2, 1, 1.5 or 0%, preferably about 2.5% or less based on total carotenoids, and wherein furthermore the carotenoid-mix comprises 7,8-dihydro-beta- carotene with a percentage of about 5% or less, preferably about 2.5% or less, such as e.g. about 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1 or even 0%, all based on total carotenoids. Such process leads to an increase in beta-carotene production in the range of at least about 1 to 20% compared to beta-carotene production in the absence of lycopene cyclase inhibitor as defined herein.

As used herein, a "carotenoid-mix" comprises carotenoids selected from betacarotene, lycopene, neurosporene (7,8-dihydro-lycopene), gamma-carotene and/or 7, 8-dihydro-beta-carotene, particularly trans beta-carotene, 13-cis beta carotene, 15-cis beta carotene, neurosporene, lycopene, gamma-carotene, 7,8- dihydro-beta-carotene and/or mixtures thereof. The skilled person knows how to measure formation of carotenoids as described herein, including measurement via HPLC and the like. The terms "carotenoid-mix" or "total carotenoids" is used interchangeably herein.

The term "carotenoids" as used herein is well known in the art. It includes long, 40 carbon conjugated isoprenoid polyenes that are formed in nature by the ligation of two 20 carbon geranylgeranyl pyrophosphate molecules. These include but are not limited to phytoene, lycopene, and carotene, such as e.g. beta-carotene, which can be oxidized on the 4-keto position or 3-hydroxy position to yield canthaxanthin, zeaxanthin, or astaxanthin. Cultivation and isolation of beta-carotene-producing host cells selected from Yarrowia and Saccharomyces is described in e.g. W02008042338 or WO2014096992.

Biosynthesis of carotenoids using fungal host is described in e.g. W02006102342. With regards to production of beta-carotene in E. coli as host cell, methods are described in e.g. US20070166782. Carotenoid production in Blakeslea trispora is described in e.g. EP1367131 or W02002010429.

The bio-based carotenoids, particularly beta-carotene, as described herein can be used as ingredients, such as e.g. in the form of crystals, in the food, feed, cosmetic or pharma industry known to the skilled person.

In one embodiment, the process according to the present invention for production of bio-based beta-carotene in a carotenoid-producing host cell, such as e.g. B. trispora, comprises addition of low concentration of a lycopene cyclase inhibitor as specified herein, particularly 1 to 1000 mg/l, such as e.g. 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900 mg/L of the defined lycopene cyclase inhibitor, particularly to be added at the start of the fermentation or within 24h as defined herein, resulting in a carotenoid-mix comprising undesired side-products including but not limited to 7,8-dihydro- beta-carotene, lycopene and/or gamma-carotene with a percentage of about 5% or less, preferably about 2.5% or less, such as e.g. about 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1 or even 0%, based on total carotenoids.

Suitable host cells to be used for a process according to the present invention include microorganisms capable of carotenoid biosynthesis selected from bacteria, algae or fungi, particularly yeast, such as e.g. selected from Escherichia, Streptomyces, Pantoea (Erwinia), Bacillus, Flavobacterium, Synechococcus, Lactobacillus, Corynebacterium, Micrococcus, Mixococcus, Brevibacterium, Bradyrhizobium, Gordonia, Dietzia, Muricauda, Sphingomonas, Synochocystis, Paracoccus, Saccharomyces, Aspergillus, Pichia, Hansenula, Yarrowia, Phycomyces, Mucor, Rhodotorula, Sporobolomyces, Xanthophyllomyces, Phaffia, Blakeslea, Haematococcus, Chlorella, Du naliella, Neospongicoccum, Chlaydomonas, Murielopsus, or Scenedesmus, e.g. selected from Escherichia coli, Paracoccus xeaxanthinifaciens, Saccharomyces cerevisiae, Aspergillus niger, Pichia pastoris, Hansenula polymorpha, Phycomyces blakesleanus, Blakeslea trispora, Yarrowia lipolytica or Dunaliella salina. Preferably, the host cell is selected from Blakeslea, more preferably from B. trispora.

With regards to the present invention, it is understood that organisms, such as e.g. microorganisms, fungi, algae or plants also include synonyms or basonyms of such species having the same physiological properties, as defined by the International Code of Nomenclature of Prokaryotes or the International Code of Nomenclature for algae, fungi, and plants (Melbourne Code).

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 W02008042338, WO2014096992, W02006102342, US20070166782, EP1367131, and W02002010429. Examples

Example 1: General Methods, strains and plasmids

All basic molecular biology and DNA manipulation procedures described herein are generally performed according to Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: New York (1989) or Ausubel et al. (eds). Current Protocols in Molecular Biology. Wiley: New York (1998).

Shake flask seed medium MIBLT-1. 23.5 g/l corn meal, 23 g/l soya flour, 10 g/ 1 glucose, 0.5 g/l KH2PO4 are mixed, the medium pH is adjusted to 6.3 and homogenization is performed with an Ultra-Turrax® blender (Ika) for 8 min at 13500 rpm. After heating at 70°C for 45 min, the medium is autoclaved.

Shake flask seed medium MIBLT-2. 17.5 g/l corn meal, 40 g/l soya flour, 10 g/l soy lecithin, 0.5 g/l KH2PO4, 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 and homogenization is performed with an Ultra-Turrax® blender (Ika) for 8 min at 13500 rpm. After heating at 70°C for 45 min, the medium is autoclaved.

Shake Flask Process. An Erlenmeyer flask (250 or 300mL volume) containing 30 ml of seed medium is inoculated with spores of B. trispora (-) strain to a concentration of 4.5 x 10 ³ spores/ml. A second Erlenmeyer flask containing 30 ml of seed medium is inoculated with spores of B. trispora (+) strain to a concentration of 1.5 x 10 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 300mL volume) containing 20 ml of production medium 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 20mL production cultures, 40pL 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.

Sample preparation for HPLC-analvsis. 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 (dichloromethane / methanol, 1:1) 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.

HPLC-analvsis. Samples from the above extract (10 pl) 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, 25ml 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: Carotenoid-fermentation with B. trispora in the presence of low pyridine concentration

Fermentations were run with different concentrations of pyridine as indicated in Table 1. Percentage of beta-carotene in the presence of no pyridine (control) was set with 100% and the change in percentage measured and indicated as BC (% control).

Table 1: Effect of pyridine (P) on beta-carotene titer (BC), 7,8-dihydro-beta- carotene (7,8-DH), lycopene (Lyc), or gamma-carotene (GC). For more details, see text. Example 3: Carotenoid-fermentation with B. trispora in the presence of low triethylamine concentration

Fermentations were run with different concentrations of triethylamine as indicated in Table 2. Percentage of beta-carotene in the presence of no triethylamine (control) was set with 100% and the change in percentage measured and indicated as BC (% control).

Table 2: Effect of triethylamine (TEA) on beta-carotene titer (BC), 7,8-dihydro- beta-carotene (7,8-DH), lycopene (Lyc), or gamma-carotene (GC). For more details, see text.

Example 4: Carotenoid-fermentation with B. trispora in the presence of low tripropylamine concentration

Fermentations were run with different concentrations of tripropylamine as indicated in Table 3. Percentage of beta-carotene in the presence of no tripropylamine (control) was set with 100% and the change in percentage measured -and indicated as BC (% control).

Table 3: Effect of triproylamine (TPA) on beta-carotene titer (BC), 7,8-dihydro- beta-carotene (7,8-DH), lycopene (Lyc), or gamma-carotene (GC). For more details, see text.

Example 5: Carotenoid-fermentation with B. trispora in the presence of low imidazole concentration

Imidazole was added 24h after the fermentation start at different concentrations as indicated in Table 4.

Table 4: Effect of imidazole (l) on percent beta-carotene (% BC), 7,8-dihydro- beta-carotene (7,8-DHBC), lycopene (Lyc), or gamma-carotene (GC). For more details, see text.