MUTANT STRAIN OF THE ALGA NANNOCHLOROPSIS AND METHOD OF PRODUCTION OF THE SAME, ITS USE IN THE PRODUCTION OF ASTAXANTHIN AND OMEGA-3 AND RELATED COMPOSITIONS

Abstract

The mutant strain of the alga Nannochloropsis is provided. A method for producing the mutant strain of the alga Nannochloropsis is also provided. Use of compounds produced from the mutant strain of the alga Nannochloropsis is further provided.

Claims

1. The mutant algal strain ASTAOMEGA (formerly Nannochloropsis gaditana D23) deposited in the Culture Collection of Algae and Protozoa (CCAP) (SAMS Limited Scottish Marine Institute; OBAN, Argyll, PA37 1QA, UK) on 28 Jan. 2016 under the Accession Number given by the International Depositary Authority CCAP 849/16.

2. The mutant algal strain of claim 1, characterized by 504 mutations as illustrated in FIG. 2.

3. A method for obtaining the mutant algal strain ASTAOMEGA (formerly Nannochloropsis gaditana D23) deposited in the Culture Collection of Algae and Protozoa (CCAP) (SAMS Limited Scottish Marine Institute; OBAN, Argyll, PA37 1QA, UK) on 28 Jan. 2016 under the Accession Number given by the International Depositary Authority CCAP 849/16, the method comprising subjecting the alga Nannochloropsis gaditana W.T. to a random chemical mutation phase by exposure to the mutagenic agent ethyl methanesulfonate.

4. The method of claim 3, wherein said exposure to the mutagenic agent ethyl methanesulfonate at a final weight/volume percentage is of about 2%.

5. The method of claim 3, wherein said exposure is protracted for a time of 2 hours.

6. A process for producing astaxanthin and omega-3 fatty acids, particularly eicosapentaenoic acid, the process comprising culturing the mutant algal strain of claim 1.

7. The process of claim 6, wherein said cultivation is conducted in the presence of glucose, glycerol, or ethanol.

8. The process of claim 7, wherein said cultivation is conducted in the presence of glucose in an amount of about 1.5-40 g/L.

9. The process of claim 6, wherein said cultivation is conducted employing a light emitting white light varying from 20 to 1000 mol photons m.sup.2 s.sup.1.

10. The process of claim 6, wherein said cultivation is conducted at a temperature between about 20 C. and 35 C.

11. The process of claim 6, wherein said cultivation is conducted in the presence of up to about 15% CO.sub.2.

12. The process of claim 6, wherein said process further produces an algal biomass.

13. A mixture of astaxanthin and eicosapentaenoic acid obtained according to the process of claim 6.

14. The mixture of claim 13, comprising an eicosapentaenoic acid/astaxanthin ratio of about 4.4-7.9.

15. An algal biomass obtained according to the process of claim 12.

16. A food, pharmaceutical, nutraceutical or cosmetic composition or a zootechnical supplement or feed comprising the mixture of astaxanthin and eicosapentaenoic acid of claim 13.

17. A method for producing astaxanthin and omega-3 eicosapentaenoic acid (EPA), the method comprising using the mutant algal strain of claim 1.

18. The mixture of astaxanthin and eicosapentaenoic acid of claim 13, wherein the mixture is used in food, pharmaceutical, nutraceutical, or cosmetic products.

19. The mixture of astaxanthin and eicosapentaenoic acid of claim 13, wherein the mixture is used in aquaculture.

20. The algal biomass of claim 12, wherein the algal biomass is used as feed.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0070] FIG. 1 shows the copy of the certificate of deposit of the ASTAOMEGA (formerly Nannochloropsis gaditana D23) mutant strain of this invention with the CCAP-SAMS International Depositary Authority.

[0071] FIG. 2 shows the list of identified mutations of the ASTAOMEGA mutant strain of this invention.

SUBJECT MATTER OF THE INVENTION

[0072] In a first subject matter, this invention describes a mutant strain of the seaweed Nannochloropsis.

[0073] In a second subject matter, this invention describes a method for obtaining it.

[0074] In a third subject matter, this invention describes a process for the production of Astaxanthin, ketocarotenoids, and Omega-3 (EPA), comprising the use of said mutated strain.

[0075] In a fourth subject matter, this invention describes food and nutraceutical compositions comprising compounds produced by the mutated strain.

[0076] In other subjects, this invention describes the use of compounds produced from the mutated strain for use in the food supplement and nutraceutical industry, the pharmaceutical and/or cosmetic industry, and the aquaculture industry.

DETAILED DESCRIPTION OF THE INVENTION

[0077] According to a first subject matter, this invention describes a mutant strain of the seaweed Nannochloropsis.

[0078] This ASTAOMEGA mutant strain has been created and selected at the Department of Biotechnology of the University of Verona, by the group directed by Prof. Matteo Ballottari.

[0079] Said strain has been deposited with the CCAP-SAMS International Depositary Authority (CULTURE COLLECTION OF ALGAE AND PROTOZOA (CCAP)SAMS Limited Scottish Marine 18 Institute, OBAN, Argyll, PA37 1QA, UK) on Jan. 28, 2016, and registered under CCAP Access Number 849/16 (the name indicated Nannochloropsis gaditana D23 was the identifying abbreviation initially assigned by the authors to the strain, later changed by said authors to ASTAOMEGA, as used for convenience in this description).

[0080] According to a second subject matter, this invention describes a method for obtaining the aforementioned mutated strain.

[0081] In particular, this method comprises the step of random chemical mutagenesis carried out by exposing N. gaditana W.T. (strain obtained from the CCAP-SANS Institute, Access Number CCAP849/5) to a mutagenic agent represented by EMS (ethyl methanesulfonate or ethyl mesylate; Merck Index, 11th Ed, 3782) following the procedure described in Cecchin et al 2020 (Improved lipid productivity in Nannochloropsis gaditana in nitrogen-replete conditions by selection of pale green mutants, Cecchin M, Berteotti S, Paltrinieri S, Vigilante I, Iadarola B, Giovannone B, Maffei M E, Delledonne M, Ballottari M. Biotechnol Biofuels. 2020 Apr. 21; 13:78. doi: 10.1186/s13068-020-01718-8. eCollection 2020, which is incorporated herein in its entirety as reference).

[0082] Specifically, the EMS compound was added to 10.sup.8 cells/mL at final weight/volume percentages of 0.75%, 1.5%, 2%, and 2.5%.

[0083] Samples were incubated for 2 hours in the dark and then diluted in 10% sodium thiosulfate solution to inactivate the mutagen activity.

[0084] The cells were then centrifuged at 6000 g, washed twice with 1 M NaCl, dissolved in 500 l of f/2 growth medium (commercially available), and maintained overnight under low light conditions.

[0085] The cells were then plated on solid f/2 medium and kept under low-light conditions (50 mol m.sup.2 s.sup.1) for at least 2 weeks.

[0086] The cells treated with EMS concentrations that induce 95% mortality (determined as the number of colonies on plate in the EMS-treated cells compared with the number of colonies on plate of the sample not exposed to the mutagen) were used for the subsequent screening procedure.

[0087] This concentration was found to be 2% EMS.

[0088] The EMS treatment generated variants in the genome of early N. gaditana W.T. creating a library of mutants.

[0089] The different strains obtained from single colony on plate were classified and selected according to the different pigment composition.

[0090] Specifically, strains with different carotenoid/chlorophyll ratios were selected and further characterized based on the 500/680 nm absorption ratio of the total pigments extracted.

[0091] The ASTAOMEGA CCAP 849/16 mutant (formerly Nannochloropsis gaditana D23) was particularly notable for having a high 500/680 nm ratio, due to a high carotenoid/chlorophyll ratio, with an accumulation of Astaxanthin up to 1% of its dry weight, as subsequently verified by HPLC.

[0092] According to a particular aspect of this invention, the carotenoid/chlorophyll ratio is increased up to 150% with respect to the wild-type strain.

[0093] The characterization of the ASTAOMEGA genotype by whole-genome sequencing revealed the presence of 504 mutations.

[0094] The list of mutations identified is shown in FIG. 2.

[0095] Among the 504 variants identified, a missense mutation (Naga_100050g23) on the carotenoid oxygenase enzyme could be responsible for an altered carotenoid biosynthetic pathway, thus leading to the increased production of Astaxanthin and Cantaxanthin as observed in this mutant with respect to the wild-type form.

[0096] Moreover, another missense mutation on the enzyme glutamate synthase (Naga_100005g23) suggests possible reduced activity for this key enzyme for nitrogen assimilation and chlorophyll biosynthesis (Gomez-Silva et al., Planta 1985), thus making this mutation likely responsible for the reduced chlorophyll content and increased lipid accumulation phenotype observed in the ASTAOMEGA mutant.

[0097] A mutation on the chloroplast RNA polymerase subunit (Naga_1Chloroplast7) was also identified; this mutation generated reduced chloroplast transcription, resulting in reduced accumulation of chlorophyll-binding subunits.

[0098] According to a third subject matter, this invention describes a process for the production of Astaxanthin, ketocarotenoids, and Omega-3 (EPA) using the mutated strain.

[0099] In particular, the ASIAOMEGA mutant strain may be grown in growth media suitable for the cultivation of marine algae, such as, for example, f/2 medium (Guillard, R. R. L. & Ryther, J. H. Studies of marine planktonic diatoms, I, Cyclotella nanna (Hustedt) and Detonula convervacea (Cleve). Can. J. Microbiol. (1962)) in closed (e.g., photobioreactors) or open (commonly referred to as open ponds or raceway ponds) culture systems.

[0100] Cultivation may also be conducted in saline waters.

[0101] Production may occur under different light conditions, at different temperatures, and at different CO.sub.2 concentrations.

[0102] Said cultivation may then be conducted under one or more of the following conditions: [0103] light conditions between 20-1000 mol photons m.sup.2 s.sup.1, [0104] temperature between about 20-35 C. and preferably 20-25 C., [0105] CO.sub.2 concentrations up to 15% and preferably between 0.03%-3% (v/v).

[0106] CO.sub.2 could also be made available directly in the growth medium, for example, in the form of carbonate.

[0107] Glucose, or another reduced carbon source such as glycerol or ethanol, may be added to the growth medium to improve productivity.

[0108] Cultivation may be conducted until the saturation phase is reached, such as in 4-8 days.

[0109] Longer cultivation in the saturation phase results in increased ketocarotenoid content.

[0110] Pigment analysis demonstrates the production of Astaxanthin, Cantaxanthin, and other trace ketocarotenoids.

[0111] The productivity values obtained averaged out to a productivity of:

TABLE-US-00001 0.14-0.17 g/L/day biomass 0.5-0.7 mg/L/day ASX (and ketocarotenoids) 2.97-4.54 mg/L/day EPA

[0112] Glucose may increase biomass productivity by up to 0.22 g/L/day, but may reduce the percentage of ketocarotenoids, resulting in a ketocarotenoid productivity of 0.75 mg/L/day.

[0113] Astaxanthin and lipids may be extracted from the cells according to the same methodologies used to extract Astaxanthin produced by Hematococcus pluvialis.

[0114] According to a preferred aspect of this invention, the ASTAOMEGA strain may be grown under one or more of the following conditions: [0115] in commercially available f/2 culture medium and preferably in photobioreactors with volumes ranging from 80 mL to 20 L; [0116] by air insufflated from the bottom of the photobioreactor enriched with varying concentrations of CO.sub.2, preferably between 300 ppm and 30,000 ppm. In any case, the enrichment of the insufflated air with CO.sub.2 may be modulated based on the pH of the growth medium as an index of CO.sub.2 consumption by the cultured micro algae.

[0117] The function of CO.sub.2-enriched air insufflation is both to promote gas exchange in the culture medium, by supplying CO.sub.2 and reducing the O.sub.2 concentration so as to promote photosynthetic activity of the cells, and to prevent, or reduce, cell sedimentation.

[0118] Cultivation in photobioreactors is conducted for a variable cultivation time, preferably until the saturation phase is reached (3 to 10 days, preferably 4 to 8 days), achieving biomass, ketocarotenoid, Astaxanthin, and EPA production yields in line with the above.

[0119] According to a particular aspect of the invention, the culture of the ASTAOMEGA strain may also be carried out in the presence of an appropriate amount of a carbon source, such as, for example, glucose, preferably in an amount of about 0.5-40 g/L, or a similar amount of a reduced carbon source such as glycerol or ethanol, in order to improve productivity.

[0120] A number of illustrative variants briefly set forth below may be applied to the process conditions according to this invention.

[0121] The production may be done by considering one or more of the following variants: [0122] in closed photobioreactors or in open systems (open ponds or raceway ponds), as well as in other devices developed for microalgae cultivation such as hybrid systems or biofilm cultivation systems; [0123] with discontinuous (batch), semi-discontinuous (semi-batch), continuous or semi-continuous cultivation methods; [0124] indoors or outdoors; [0125] by means of LED lighting.

[0126] In addition, the mutated strain preparation of this invention may also be achieved by genome-specific editing of the N. gaditana genome by reproducing all or parts of the introduced mutations.

[0127] Thus, according to another subject matter of this invention, the same mutations of the ASTAOMEGA strain of this patent application are described to induce other microalgae species (marine and/or non-marine species) to produce Astaxanthin.

[0128] The ASTAOMEGA technology of this invention may be extended to all the different applications in which Astaxanthin is required or involved, including those in which Astaxanthin is a metabolic intermediate or by-product.

[0129] An automatic algae harvesting phase may also be integrated into the process to further reduce and optimize production costs.

[0130] Thus, the process of this invention enables the production of a mixture of astaxanthin and eicosapentaenoic acid.

[0131] More specifically, this mixture has an eicosapentaenoic acid/astaxanthin ratio by weight in the range of 4.4 to 7.9.

[0132] According to one particular aspect, the described process also allows an algal biomass to be obtained, which is rich in astaxanthin and eicosapentaenoic acid.

[0133] More specifically, this biomass has an eicosapentaenoic acid/astaxanthin weight ratio in the range of 4.4 to 7.9.

[0134] In a fourth subject matter, this invention describes food, pharmaceutical, nutraceutical, or cosmetic compositions comprising the mixture of compounds produced by the mutated strain.

[0135] According to other subject matters of this invention, the use of the compounds produced by the mutated strain in the food, pharmaceutical, nutraceutical, and cosmetic industries is described.

[0136] The compositions or formulations are achievable by the person skilled in the art by using the common technologies of pharmaceutical preparative technique known in the art, with the addition or not of the appropriate additives, carriers, excipients, and/or active ingredients, depending on the type of product and/or form of administration desired.

[0137] According to a particular aspect of this invention, the produced compounds find application in the aquaculture industry.

[0138] Said compounds are, in fact, responsible for fish pigmentation, which is recognized as a difficult quality trait to achieve in farm-raised fish.

[0139] The biomass obtained from the culture process may also be used in aquaculture and, in particular, as fish feed.

[0140] For this object, an automatic algae harvesting phase may be integrated into the process to further reduce and optimize production costs.

[0141] In this way, the oil enriched in Astaxanthin and EPA may be put to the most valuable uses, while the biomass remaining after oil extraction, which in each case is enriched in Astaxanthin and EPA, may be used as fish feed.

Example

[0142] The ASTAOMEGA strain was grown in batch airborne photobioreactors under continuous white light at 500 mol photons m.sup.2 s.sup.1 in F/2 medium. The device used for microalgae cultivation was the MC 1000-OD from PSI (Photon Systems Instruments) spol. s r.o. Drsov 470, 664 24 Drasov, Czech Republic. Air enriched with 3% CO.sub.2 was bubbled from the bottom of the photobioreactors.
The composition of the F/2 soil was as follows: 0.092 g/L Guillard's (F/2), seawater enrichment solution (Merk G0154), 32 g/L sea salt (Merck S9883), TRIS-HCl 4.84 g/L, thiamine 0.1 mg/L, biotin 0.5 g/L, vitamin B2 0.5 g/L.
Growth was conducted for 5 days resulting at the end of growth in a total dry biomass of 0.870.02 g/L with a mean daily biomass productivity of 0.170.01 mg/L/day and a maximum daily biomass productivity of 0.350.01 mg/L/day. In this condition, total lipid productivity was 40.394.43 mg/L/day and EPA productivity was 3.220.31 mg/L/day. The productivity of ketocarotenoids is 0.04 mg/L/day.
Under the same conditions, but with the addition of 10 g/L glucose to the culture medium, a total dry biomass of 1.030.14 g/L with a mean daily biomass productivity of 0.210.01 mg/L/day and a maximum daily biomass productivity of 0.390.06 mg/L/day. In this condition, total lipid productivity was 50.6216.7 mg/L/day and EPA productivity was 3.671.16 mg/L/day. Ketocarotenoid productivity was 0.760.12 mg/L/day.

[0143] From the description provided above, the advantages provided by this invention will be apparent to the person skilled in the art.

[0144] In particular, with regard to the mutated strain, it allows for high amounts of astaxanthin and Omega-3 (in particular, EPA) to be obtained.

[0145] This allows for the preparation of numerous formulations enriched in Omega-3 and/or Astaxanthin.

[0146] Of the already known products, in fact, both Omega-3 and Astaxanthin are present only in krill oil, but Astaxanthin is in a concentration generally lower than 0.05%.

[0147] Given that Nannochloropsis algae has recently been proposed in Europe as a novel food for human consumption and is already approved by the FDA (FDA 2015; US Food and Drug AdministrationNew Dietary Ingredient Notification Report #826. http://www.regulations.gov/#!documentDetail;D=FDA-2014-S-0023-0041), the use of the ASTAOMEGA mutant strain appears to be an innovative solution.

[0148] The mutant strain ASTAOMEGA CCAP 849/16 has been identified as a non-GMO (Non-Genetically Modified Organism) and therefore its cultivation is allowed at industrial level without being subject to the restrictions required for GMOs.

[0149] The ASTAOMEGA mutant strain from N. gaditana W.T. is unexpectedly characterized by some unique features, including: [0150] accumulation of Astaxanthin (up to 1% by weight (w/w) per dry weight of algae biomass) and simultaneously Omega-3 EPA fatty acid; [0151] reduced heat dissipation, which is significant for maintaining efficient photosynthesis; [0152] rapid growth of the species with no reduction in biomass production associated with Astaxanthin production; [0153] reduced chlorophyll content, which allows for better light penetration into the photobioreactor (due to reduced pigmentation).

[0154] Regarding the process for producing astaxanthin, the following advantages may be pointed out: [0155] increased productivity, due to the significant accumulation of Astaxanthin (up to 1% (w/w) of its dry weight) with respect to the starting strain Nannochloropsis gaditana wild-type, in which Astaxanthin is normally produced only in trace amounts; [0156] reduction of production costs, based both on the higher productivity of the ASTAOMEGA strain; [0157] reduction of production costs, based on the elimination of the stress phase (red phase), and the possibility of accumulating Astaxanthin; [0158] increased environmental sustainability, both through the elimination of the stress phase mentioned above (which requires a lot of energy in order to provide intense light and high temperature) increased light intensity and temperature) for Astaxanthin production.

[0159] The resulting preparations based on the compounds of the invention further enable compositions and formulations to be offered for human use in the nutraceutical, pharmaceutical, and cosmetic industries.

[0160] The use of the compounds of the invention in the aquaculture industry, on the other hand, gives fish farmers the opportunity to improve fish quality by increasing both pigmentation and Omega-3 content.