New strain of the microalga chlorella vulgaris as a sustainable source for producing zeaxanthin and lutein

Abstract

The present invention relates to the use of a new strain of the microalgal species Chlorella vulgaris for producing lutein and zeaxanthin, as well as to the method for obtaining it.

Description

[0001] "New strain of the microalga Chlorella vulgaris as a sustainable source for producing zeaxanthin and lutein" DESCRIPTION

[0002] Chlorella vulgaris strains are already commercially available and are capable of accumulating very low amounts of zeaxanthin, unless strongly stressed in intense light.

[0003] Chlorella vulgaris is already consumed as a food in the EU and many other countries as it is rich in proteins, vitamins and lutein (an important pigment for eye health).

[0004] Carotenoids are C40 compounds with conjugated double bonds which give rise to high light absorption and are characterized by strong antioxidant properties. Five main carotenoids accumulate in terrestrial plants and green microalgae: p-carotene and xanthophylls (of which lutein is the most abundant), zeaxanthin, violaxanthin and neoxanthin. Most of these carotenoids are bound to proteins, which form protein complexes called Photosystems, which also bind the chlorophylls responsible for light absorption. The ratio between the different carotenoids bound by photosynthetic complexes is generally maintained in many different species, with the exception of violaxanthin and zeaxanthin, which undergo what is called the xanthophyll cycle depending on the light conditions. The zeaxanthin produced by beta-carotene does not accumulate and is rapidly converted to violaxanthin by the enzyme zeaxanthin epoxidase (ZEP). Under high light conditions, the enzyme violaxanthin deepoxidase (VDE) is activated and converts violaxanthin to zeaxanthin for photoprotection: in fact, the accumulation of zeaxanthin allows different photosynthetic organisms such as plants and microalgae to be more resistant to exposure to light stresses. The zeaxanthin produced is then reconverted to violaxanthin by the enzyme ZEP when the high light stress ends. Carotenoids play a fundamental role in both light absorption and photoprotection. They broaden the range of visible light directed towards chlorophyll a, by virtue of the absorption thereof in the range 480-550 run, where chlorophyll absorption is weak. Furthermore, they are structural components of the two photosystems, fundamental for the correct assembly thereof and they protect photosynthetic membranes from oxidative photodamage .

[0005] Lutein and zeaxanthin are two carotenoids characterized by strong antioxidant properties. They are used in a wide range of applications, including cosmetics, for their ability to improve skin tone, and animal feed (as an additive in the poultry industry, to improve the yellow color of egg yolks). However, their main use is in the nutraceutical and pharmaceutical sectors; in fact, lutein and zeaxanthin play an important role in eye health, reducing the incidence of diseases such as age-related macular degeneration (AMD) and cataract. Furthermore, numerous research studies have demonstrated a peculiar role of lutein and zeaxanthin in the development and protection of the eye in the neonatal period. Although both lutein and zeaxanthin are present in the human diet, and especially in fruits and vegetables, it has been estimated that EU citizens consume on average 1-2 mg / day of lutein and 0.2-0.9 mg / day of zeaxanthin from the diet, against a recommended dose of at least 10 mg / day of lutein and 2 mg / day of zeaxanthin, thus highlighting the need to increase the daily intake thereof by means of dietary supplements. Nowadays, lutein and zeaxanthin are extracted from the petals of calendula flowers (Tagetes erecta).

[0006] However, their production has some drawbacks: it is impossible to provide a stable supply of these carotenoids, because the yield is seasonal and highly dependent on weather conditions. Furthermore, the cultivation of calendula is expensive, requires a lot of labor and large agricultural areas. Despite this, extraction from calendula flowers is still considered the gold standard for lutein and zeaxanthin production, due to a lack of alternatives. The chemical synthesis of zeaxanthin leads to a product that actually shows little biological activity, limiting applications mainly to the animal feed segment, where it is used as a pigment.

[0007] The metabolic engineering of microorganisms such as bacteria and yeasts to produce lutein and zeaxanthin has been successfully reported, but the GMO origin of the carotenoids produced hinders the application thereof in the food and nutraceutical sectors. Photosynthetic microorganisms, such as green microalgae, naturally produce and accumulate lutein and zeaxanthin: several attempts have been made to extract lutein and zeaxanthin from microalgae in recent years. Against calendula flowers, microalgae (such as Chlorella, Monoraphidium or Scenedesmus species) can be grown in controlled environments (typically a bioreactor), eliminating problems related to seasonality, dependence on weather conditions and possible contamination, ensuring stable and scalable production.

[0008] For these reasons, microalgae are considered the most valid alternative to the production of calendula flowers. However, the adoption of this solution is hampered by the very low content of lutein and zeaxanthin and the extremely high extraction costs.

[0009] To date, however, there are no products on the market based on lutein and zeaxanthin derived from microalgae.

[0010] International patent application WO 2022 / 158814 describes obtaining a strain of Chlorella by employing EMS as a mutagenic agent and selection based on the color of the plated colonies.

[0011] European patent application EP 2,157,167 describes a process for obtaining lutein from particular strains of Chlorella sorokiniana .

[0012] International patent application WO 2020 / 105001 describes a method for obtaining Chlorella vulgaris strains which produce low amounts of chlorophyll.

[0013] Summary of the invention

[0014] The inventors of the present patent application have developed a new method based on microalgae, which allows the production of lutein and zeaxanthin in a scalable, sustainable and economically advantageous manner.

[0015] Brief description of the drawings

[0016] Figure 1 shows the phylogenetic tree generated from the 18S sequence of different chlorella species. The mutant fm53 was generated using Chlorella vulgaris SAG 211 / 11P as a comparison.

[0017] Figure 2 shows the screening of C. vulgaris NPQ mutants. (A) Example of agar plate screening after UV mutagenesis. (B) NPQ (non- photochemical quenching) achieved after ten minutes of lighting; the average of the Wild type (Wt) inserted as a control in each plate is shown. (C) Chlorophyll fluorescence image after ten minutes of lighting. (D) NPQ traces of the Wt and three selected lines. The values refer to means ± SD (n=3).

[0018] Figure 3 shows the result of HPLC analysis of pigments extracted from wild-type (A) and fm (B-D) mutants in LL-adapted cells. The numbers correspond to 1-neoxanthin, 2-violaxanthin; 3-lutein; 4- zeaxanthin, 5-chlorophyll b; 6-chlorophyll a; 7-p carotene.

[0019] Figure 4 shows: (A) Diagrammatic depiction of a single point mutation in the 5'-UTR region of ZEP (zeaxanthin epoxidase) gene g9297. (B) Analysis of the relative amount of ZEP and VDE transcription with respect to wild type. The transcription levels were calculated using the 2-AACT method, related to the wild-type expression level set to 1. (C) Immunodetection of ZEP; from left to right the lanes are loaded with molecular weight (MW), total protein extract of wild-type (Wt) and fm53 and recombinant Chlorella vulgaris ZEP. Ponceau Red staining of the filter confirms the same quantity of proteins loaded for the two genotypes (left). Western blot with an a-ZEP antibody (right). Blue arrow indicates the missing ZEP band in the mutant.

[0020] Figure 5 shows the type of mutations found in the mutant of the invention.

[0021] Figure 6 shows the specific mutations of the mutant of the invention.

[0022] Figure 7 shows in detail the single nucleotide variants (SNV) and insertion deletion mutations (INDELS) analyzed by whole genome sequencing on fm53 with respect to the wild-type genome.

[0023] Figure 8 shows: (A) Volumetric (left) and daily volumetric (right) productivity of the biomass (A,B), lutein (C,D) and zeaxanthin (E,F) for wild-type (Wt black) and fm53 (grey) at the end of growth shown in Figure 10. Values refer to means ± SD (n=3). * indicates significantly different values (P < 0.05) between wild- type and fm53.

[0024] Figure 9 shows the carotenoid content at the end of growth at the different light intensities shown in Figure 8 for wild-type (Wt) and fm53 blown with 0.04% CO2 (air) or 3% CO2 (CO2). The values are shown as mg of carotenoids per g of dry biomass. The data are expressed as mean ± SD (n = 3).

[0025] Figure 10 shows data related to the lutein / zeaxanthin molar ratio in the mutant fm53 pigment extract from cells grown at different light intensities (100 to 2000 μmol-2s-1) at atmospheric CO2 concentration (0.04%, AIR) or at 3% CO2.

[0026] Object of the invention

[0027] In a first object, the present invention describes a mutant strain of the microalga Chlorella vulgaris.

[0028] In a second object, the present invention describes a method for obtaining the mutant strain of the microalga Chlorella vulgaris.

[0029] In a third object, the present invention describes a method for producing zeaxanthin and lutein by culturing the mutant strain of the microalga Chlorella vulgaris of the invention.

[0030] The zeaxanthin and lutein pigments obtained by culturing the mutant strain of the microalga Chlorella vulgaris represent further objects of the present invention.

[0031] The biomass obtained according to the described method and comprising the compounds zeaxanthin and lutein represents a further object of the invention. In a fourth object, the present invention describes a food, pharmaceutical, nutraceutical, zoo-technical or cosmetic product comprising the biomass obtained by the method of the invention. Detailed description of the invention In a first object, the present invention describes a mutant strain of the microalga Chlorella vulgaris.

[0032] In particular, such a strain is characterized by comprising, with respect to the wild-type strain, the mutation on the gene Gene_ID g9297 c.-lllO T; such a mutation silences the expression of the gene encoding the enzyme zeaxanthin epoxidase.

[0033] According to a particular aspect of the invention, the mutant strain of the microalga Chlorella vulgaris of the invention is further characterized by one or more of the further mutations:

[0034] In particular, these are all the mutations indicated in the table above.

[0035] According to an even more particular aspect of the invention, the mutant strain of the microalga Chlorella vulgaris of the invention is further characterized by one or more of the mutations indicated in Figure 5.

[0036] In particular, these are all the mutations indicated in Figure 5.

[0037] In a second object, the present invention describes a method for obtaining the mutant strain of the microalga Chlorella vulgaris.

[0038] In particular, such a method includes a mutation step.

[0039] Prior to the mutation step, a culturing step can be carried out.

[0040] In particular, the strain is cultured in a suitable medium.

[0041] To this end, TAP (Tris Acetate Phosphate) or BG-11 medium or another suitable medium can be used.

[0042] In particular, the culturing conditions include an alternating "light / dark" regime of: 16 hours light / 8 hours dark.

[0043] In particular, the culturing conditions include a temperature of:

[0044] -20°C to 25°C and preferably 22°C in the light, and

[0045] -16°C to 20°C and preferably 18°C in the dark.

[0046] In particular, the culturing conditions in this first step include a light exposure of 70 pmol-2s-1.

[0047] As instead regards the mutation step, this comprises the exposure of a microalgal strain of Chlorella vulgaris to mutagenic conditions.

[0048] For the purposes of the present invention, the mutagenic conditions are represented by exposure to UV rays.

[0049] In particular, the mutation step comprises exposing a microalgal strain of Chlorella vulgaris to UV at the wavelength of 254 nm. In particular, the exposure time is 2-5 minutes, determined based on the ability of the treatment to induce mortality for 90% of the treated cells.

[0050] For the purposes of the present invention, the strain of microalga Chlorella vulgaris subjected to mutation is represented by the strain Chlorella vulgaris 211 / 11P obtained from the Culture Collection of Algae of the University of Gottingen (Germany, https: / / sagdb .uni-goettingen.de / ).

[0051] In particular, a quantity of cells of about 108is subjected to mutation.

[0052] The mutation step results in the death of about 90% of the cells.

[0053] After the mutation induction step, the surviving cells are subjected to a stabilization step.

[0054] For the purposes of the present invention, said step comprises leaving the cells in the dark for a certain time period so as to prevent their reactivation; for example, the cells can be stabilized for a time period of about 1-10 hours and preferably for about 2 hours.

[0055] After the mutation and stabilization step, the cells are subjected to a selection step.

[0056] For the selection step, the stabilized cells are plated with an appropriate dilution and on an appropriate medium.

[0057] To this end, the cells can be, for example, diluted 1:100.

[0058] To this end, TAP (Tris Acetate Phosphate) liquid medium or BG- 11 liquid medium or another suitable medium may be used. With regard to the selection step, the colonies are selected by measuring photosynthetic parameters by acquiring the fluorescence emitted by the chlorophylls in vivo following exposure to different lights.

[0059] For the purposes of the present invention, the colonies were first adapted to the dark for at least 20 minutes and then lit with a low intensity measuring light (preferably less than 7 pmol photons m-2s-1) and an actinic light of 1200 pmol photons m-2s-1for an appropriate time; for example, the exposure can be continued for 10 minutes.

[0060] At regular intervals, such as every minute, the cells were exposed to saturating light of intensity preferably greater than 5000 pmol photons m-2s-1.

[0061] Exposure to such a saturating light in cells lit with the measuring light allows the parameter Fm (maximum fluorescence emitted by chlorophylls in cells adapted to the dark) to be acquired, while in cells lit with actinic light it allows the parameter Fm' (maximum fluorescence emitted by chlorophylls in cells exposed to actinic light) to be measured.

[0062] By means of the values of Fm and Fm', it is possible to calculate the NPQ parameter as (Fm-Fm') / Fm', indicating the induction of a light-dependent photoprotection mechanism by thermal dissipation of a part of the absorbed light energy.

[0063] The Non-Photochemical Quenching ("NPQ) results were evaluated so as to select mutant lines with reduced NPQ activation. The selected lines were then possibly confirmed by measuring the NPQ parameter at different actinic lights (from 250 to 5000 pmol photons m-2s-1).

[0064] In a third object, a method is described for producing zeaxanthin and lutein by culturing the mutant strain of the microalga Chlorella vulgaris selected in accordance with the present invention.

[0065] In particular, the method of the present invention comprises the step of culturing the mutant strain of the microalga Chlorella vulgaris under suitable growth conditions.

[0066] In a preferred aspect, the microalga of the invention is cultured in a culture medium.

[0067] For example, BG-11 or TAP (Tris Acetate Phosphate) culture medium can be used.

[0068] For the purposes of the present invention, the microalga of the invention is cultured under light intensity conditions between 50 and 2000 pmol photons m-2s-1, preferably from 50-100 pmol m-2s-1to 800 pmol m-2s-1and even more preferably from 500 pmol m-2s-1to 800 pmol m-2s-1.

[0069] In a preferred aspect, the microalga of the invention is grown under light intensity conditions of 800 pmol m-2s-1.

[0070] In a preferred aspect of the invention, the culturing is carried out in the presence of a concentration of carbon dioxide between 0.04 (air) and 3% CO2 (v / v).

[0071] In a preferred aspect, the culturing is carried out in the presence of a carbon dioxide concentration of 3% (v / v). For the purposes of the present invention, the culturing is carried out at a biomass concentration initially of about 0.05 g / L, which can rise to a biomass concentration of about 0.4-3 g / L.

[0072] By appropriately modulating the process conditions, for example by modifying the growth irradiation and the availability of CO2, it is possible to push the production more towards obtaining zeaxanthin or zeaxanthin and lutein (improving the ratio between zeaxanthin and lutein, in favor of the former).

[0073] For example, a relative accumulation of zeaxanthin and lutein can be obtained with a zeaxanthin:lutein ratio from 0.5 to 1.4.

[0074] In particular, the production is pushed towards obtaining lutein (with respect to zeaxanthin) using a relatively high light intensity (500-2000 pmol photons m-2s-1) in aerated photobioreactors without enrichment of CO2, i.e., with air ventilation.

[0075] The production of zeaxanthin (with respect to lutein) is favored under enriched CO2 conditions and under low light intensity conditions (50-100 pmol photons m-2s-1) or, always under enriched CO2 conditions, and at the usable light intensity (2000 pmol photons m- 2s-1).

[0076] Therefore, in a first aspect, the method of the invention allows obtaining a greater daily production of zeaxanthin and lutein, by culturing the fm53 strain under conditions of 500 pmol photons m-2s_1and in the presence of a CO2 concentration greater than 1% and preferably between 3 and 5% (v / v).

[0077] The biomass must be collected upon reaching the growth phase defined as "stationary" in which cell density reaches saturation; this phase is normally obtained after about 4 days of culturing. In accordance with a second aspect, the method of the invention allows maximizing the zeaxanthin :lutein ratio in the biomass produced, by culturing strain fm53 with a CO2 concentration condition greater than 1% and preferably between 3-5% (v / v) and with exposure conditions between 50 and 100 pmol photons m-2s-1or 2000 pmol photons m-2s-1.

[0078] A biomass comprising the compounds zeaxanthin and lutein or a mixture thereof in different proportions obtained by culturing the mutant strain of the microalga Chlorella vulgaris according to the described method represent further objects of the present invention.

[0079] The compounds zeaxanthin and lutein or a mixture thereof in different proportions obtained by culturing the mutant strain of the microalga Chlorella vulgaris according to the described method represent further objects of the present invention.

[0080] The biomass obtained according to the described method and comprising the compounds zeaxanthin and lutein represents a further object of the invention.

[0081] In a fourth object, the present invention describes the use of the pigments obtained in the food, pharmaceutical, nutraceutical, zoo-technical and cosmetic sectors.

[0082] To this end, the present invention describes a food, pharmaceutical, nutraceutical, zoo-technical or cosmetic product comprising the biomass obtained by the method of the invention.

[0083] For the purposes of the present invention, possible applications in the food sector include the use as a dye by virtue of the presence of the pigments lutein and zeaxanthin, and the use as a protein source (about 50-60% of the biomass produced), and unsaturated fatty acids of plant origin (such as hexadecadienoic acid (16:2), hexadecatrienoic acid (16:3), linoleic acid (18:2) and a-linolenic acid (18:3, oleic acid (18:1)), enriched in antioxidant pigments such as lutein and zeaxanthin.

[0084] For the purposes of the present invention, possible applications in the pharmaceutical field include use as an antioxidant, dye and as a source of lutein and zeaxanthin, pigments necessary for eye health.

[0085] According to a preferred aspect, lutein and zeaxanthin, as well as a mixture of the two pigments, obtained according to the method of the present invention can have a medical use.

[0086] For example, lutein and zeaxanthin or a mixture of the two obtained according to the method of the present invention can be used for the prevention and / or treatment of ophthalmic diseases.

[0087] In particular, such diseases comprise: age-related macular degeneration, cataract or for correct neonatal ocular development.

[0088] For the purposes of the present invention, possible applications in the nutraceutical field include use as an antioxidant, protein supplement, unsaturated fatty acid supplement with lutein and zeaxanthin added.

[0089] For the purposes of the present invention, possible applications in the zoo-technical field include use as feed (also with the aim of improving the coloring of the egg yolk),

[0090] For the purposes of the present invention, possible applications in the cosmetic field include use as antioxidant, dye.

[0091] Experimental section The present invention is based on a new non-GMO strain of the green microalga Chlorella vulgaris called mutant fm53 obtained at the SOLE- LAB of Prof. Matteo Ballottari, as part of the project ERC-StG-2015 SOLENALGAE .

[0092] During the SOLENALGAE project, different species of microalgae were studied in terms of photoprotective properties and libraries of mutant strains were generated by random insertional or physical / chemical mutagenesis.

[0093] One of the species investigated was Chlorella vulgaris. In this species, mutant strains were generated by exposure to UV rays inducing random mutations at the DNA level and among the mutant strains selected to have a reduced photosensitivity with respect to the WT strain, the mutant strain called fm53, showed a high constitutive accumulation of zeaxanthin, while in the case of WT this carotenoid is accumulated only transiently after exposure to strong light (see Figure 2).

[0094] The mutant fm53 was therefore capable of accumulating high amounts of both lutein and zeaxanthin.

[0095] The genotype underlying the phenotype of the mutant fm53 was studied by genome sequencing, retrieving a specific mutation which causes an altered translation of a zeaxanthin epoxidase gene, encoding the enzyme which uses zeaxanthin as a substrate.

[0096] Obtaining the mutant Chlorella vulgaris

[0097] The strain Chlorella vulgaris (CV) 211 / 11P was obtained from the Culture Collection of Algae of the University of Gottingen (Germany, http: / / sagdb.uni-goettingen.de / ) and used for generating mutants by exposure to UV light. The WT and mutant strains were cultured for physiological characterization using TAP (Tris Acetate Phosphate) culture medium (Gorman & Levine, 1965) or BG-11 minimal medium (Allen, M.M., Stanier, 1968) in a 16 h light / 8 h dark climate chamber at 22°C / 18°C and light intensity (70 gmol m-2s-1PPFD). The UV mutagenesis was carried out as described below: 10 mL of a mid-log culture (about 108cells) were subjected to UV irradiation using a 6 W UV lamp (254 nm), so as to induce 90% cell mortality. The surviving cells were allowed to stand in the dark for 2 hours to prevent photoreactivation, followed by plating at a 100-fold dilution on acetate-containing medium (TAP). TAP agar plates were screened by in vivo chlorophyll-emitted fluorescence measurements to select mutants with altered photosynthetic properties. The colonies were lit with a light of 1200 gmol photons m-2s-1for ten minutes and traces of NPQ were recorded after exposure to a supersaturated light of 10000 gmol photons m-2s-1. Approximately 2500 colonies were selected and compared with wild type distributed on the plates. Although many colonies fell within the control mean, some showed significantly different NPQ values (Figure 2). These lines were transferred to new plates to repeat analysis, confirm fluorescence traces and avoid false positives. After these screening cycles, three lines were identified, called fm (Fluorescent Mutants), which showed significant differences in NPQ traces with respect to wild type (Figure 2). Two lines, fm53 and fm275, showed a lower NPQ level, while fml79 showed a 2-fold increase in NPQ with respect to the wild- type.

[0098] Since the distribution of pigments is closely related to NPQ, the content of chlorophylls and carotenoids was analyzed in these three mutants by HPLC. The line fm53 showed a strongly altered carotenoid pattern: neoxanthin and violaxanthin were completely absent and zeaxanthin was constitutively accumulated in low-light conditions when the pigment was not present in the wild-type. The other two mutants showed a distribution of pigments similar to the wild type

[0099] (Figure 3).

[0100] In order to identify the mutations underlying the new phenotype observed, the genome fm53 was sequenced by Illumina next generation sequencing (NGS) to identify all the mutations present. The wild- type and fm53 strains were compared to the reference genome (Cecchin, et al. 2019), obtaining a mapped coverage of 62X and 50X, respectively (Table in Figure 5). The total number of single nucleotide variants (SNV) in the fm53 and insertion deletion mutations (INDELS) was 36 and 4, respectively. Among the 36 SNPs observed in the genome fm53, 18 were predicted as missense variant, 1 as missense variant close to a splicing site, 1 as stop, and 16 as synonymous effects. This resulted in 18 moderate- and high-impact variants affecting 16 genes (table in Figure 6). Among these genes, a point mutation was observed upstream of the initial codon of the g9297 gene encoding ZEP, validating the HPLC analysis. This substitution mutation C>T is located 111 bp upstream of the initial codon (see Figure 6) and in the 177 bp of the transcript considered 5'UTR (Cecchin eta 1., 2019). qRT-PCR showed no differences at the ZEP transcription level (see Figure 2C). The transcription of the VDE gene was also monitored without showing any difference. These results indicate that the C117T substitution did not affect mRNA transcription. The effect of the mutation was instead observed at the protein level, determined by Western blot (see Figure 4). The protein was detected using the a-ZEP antibody generated using two synthetic peptides from Dunaliella tertiolecta (Kim, et al. 2018); one of the two peptides had 100% homology with the C. vulgaris sequence. The ZEP of C. vulgaris was cloned, expressed in Escherichia coll and purified to test it with the antibody and confirm cross- reactivity. The antibody reacted correctly with the recombinant protein; at the same height a band was present in the wild type but it was completely absent in the mutant.

[0101] The effect of a single substitution in the 5'UTR obtaining a null protein translation could be due to an altered formation of the secondary structure leading to incorrect ribosomal binding and expression. To better understand whether the point mutation in the 5'UTR induced a different conformation of the RNA secondary structure, the minimum free energy of 5'UTR of wild-type and fm53 strains was analyzed obtaining the same value of -82.40 kcal / mol for both sequences, although a secondary structure with lower center of gravity for the wild-type, -53.40 with respect to -26.60 kcal / mol of fm53, and a lower entropy could suggest a possible perturbation of the structure (Lorentz 1976; Mathews, et al 2004).

[0102] Example

[0103] The mutant fm53 and the wild-type strain were cultured in BG-11 medium in a wide range of light intensities (50 to 2000 gmol photons m-2s-1) inside aerial transport photobioreactors bubbling with air (0.04% CO2) or 3% CO2. Growth was monitored by recording the OD at 720 nm. In the test tubes with air, the samples grew more slowly and reached a lower OD (always less than 0.5) while in the samples with 3% C02the strains grew with faster kinetics and reached much higher cell concentrations. In the air samples (i.e., characterized by an atmospheric C02concentration of about 0.04%) no significant effect of light intensity occurred, since the curve was similar for all the different light regimes used. In contrast, in the sample with added CO2, the growth kinetics and density achieved also depended on light intensities and increased with increasing light; the tubes grown with a light greater than 100 gmol photons m-2s-1reached a strongly higher concentration with respect to the respective tubes bubbling with air. The biomass and carotenoid content was quantified at the end of growth (see Figure 8). In the tubes aerated using compressed air, the final biomass reached about 0.5 g / L with 100 gmol photons m-2s-1and did not increase further even with a higher light intensity, actually decreasing to about 0.3 g / L at light intensity equal to 800 or 2000 gmol photons m-2s-1. In the photobioreactors fed with 3% CO2, the biomass concentration was higher in all light conditions, reaching the concentration of 3 g / L at the highest lights tested. For the different growth experiments, the biomass yield calculated in grams per liter per day was also monitored (see Figure 8). The cell cultures grown at atmospheric CO2 concentration reached a productivity of about 0.25 g / L / day at 500 gmol photons m-2s-1and then the value started to decrease. The cell cultures grown by providing 3% CO2, reached a value around 0.8-1 g / L / day, which did not decrease more than 500 gmol m-2s-1photons, but instead continued to increase although not significantly. Under all these conditions, the strain fm53 showed similar productivity to the wild-type strain. The carotenoids were also quantified, as measured by acetone spectra and HPLC analysis (Figure 8 C,D) (Cazzaniga, et al. 2020). The total carotenoids (mg pigments per dry weight of cells) were similar between the two genotypes for all the conditions tested. This value was higher with lower light intensities (50-100 gmol photons m-2s-1) and decreased with higher light without significant differences between air-supplied sample or enriched CO2 concentration. The yield in carotenoids (mg / l / day) was instead higher in the samples supplied with 3% CO2, with the maximum productivity obtained at 500 gmol photons m-2s-1, but still without difference between the two genotypes. While the total carotenoid values were similar, the zeaxanthin content and yield were higher in the mutant. At 50 and 100 gmol m-2s-1photons, the strain fm53 accumulated zeaxanthin, while it was completely absent in the wild type strain. With higher light intensities, the wild type strain also began to accumulate zeaxanthin, but the productivity was even higher in the mutant, reaching a maximum value of 1.8 mg / L / day at 800 gmol photons m-2s-1. The analysis of carotenoid content was performed by HPLC: the pigments extracted from intact cells in organic solvent were loaded onto reverse-phase HPLC equipped with C18 column, using a 15-minute gradient of ethyl acetate (0 to 100%) in acetonitrile-water- triethylamine (9:1:0.01, vol / vol / vol) at a flow rate of 1.5 mL / min. Pigment detection was conducted with a 350-750 nm Thermo-Fisher diode array detector. The distribution as mg of pigment per g of dry biomass for all carotenoids is shown in Figure 9, where "neo" is neoxanthin, "viola" is violaxanthin, "antera" is anteraxanthin, "lute" is lutein, "zea" is zeaxanthin, and "b car" is beta-carotene. Based on the lutein and zeaxanthin content, the zeaxanthin:lutein ratios reported in Figure 10 were then determined.

[0104] The culturing of the mutant fm53 allows producing a biomass of Chlorella vulgaris constitutively enriched in zeaxanthin, while to obtain the same result using the wild-type strain, exposure to a light source must activate the photoprotective mechanism known as the "xanthophyll cycle", by means of which violaxanthin is converted into zeaxanthin by the enzyme VDE.

[0105] For example, it has been observed that in the wild-type strain zeaxanthin accumulates only after exposure to at least 500 pmol m-2s-1of light and that the amount of zeaxanthin produced per dry biomass is equal to 0.5-1 mg / g (maximum value equal to 1 mg / g at 500 pmol m-2s-1under the condition of CO2 at 3%), while in the mutant of the present invention, zeaxanthin accumulates constitutively already with an exposure of 50 pmol m-2s-1and in an amount of 0.75 to 4 mg / g (maximum value of 4 mg / g at 100 pmol m-2s-1under the condition of CO2 at 3%, while with an exposure at 500 pmol m-2s-1a production of 2.38 mg / g can be obtained).

[0106] From the above description the advantages offered by the present invention will be clear.

[0107] Chlorella vulgaris strain described has a high biomass productivity, excellent resistance to contaminants and a high protein content (>50% of dry weight).

[0108] Furthermore, it is a product that is approved by both EFSA and FDA as a new food for human consumption and is currently already on the market, as a protein source rich in vitamins suitable for vegan and vegetarian diets.

[0109] The present invention provides a disruptive biotechnological solution which enables the efficient production of zeaxanthin and lutein in the microalgal species Chlorella vulgaris, in a biological system where it is possible to produce protein-rich edible biomass. With respect to the already known technologies, in which zeaxanthin is transiently accumulated in plants or green algae, including the Chlorella vulgaris wild-type (WT) strain, the described methodology allows obtaining zeaxanthin in a stable amount of 30-40% of the total carotenoids (more than double the accumulation in Chlorella vulgaris WT strain).

[0110] Furthermore, the mutant of the present invention has been shown to accumulate high lutein values, with total lutein + zeaxanthin representing more than 80% of the total carotenoids produced.

[0111] Consequently, the strain of the invention allows producing a "green" biomass deriving from the photosynthetic fixation of CO2 strongly enriched in proteins, lutein and zeaxanthin.

[0112] It is further emphasized that, as noted above, the strain of the invention is not a GMO strain.

[0113] The person skilled in the art can also appreciate that the method provided by the present invention allows orienting production towards zeaxanthin or towards lutein by appropriately modifying the growth parameters. ★ ★ ★

Claims

CLAIMS :

1. A mutant strain of the microalga Chlorella vulgaris comprising, with respect to the wild-type strain, a mutation on the gene Gene_ID g9297c.-lllC >T, which silences the expression of the gene encoding the enzyme zeaxanthin epoxidase.

2. A method for obtaining the mutant strain of the microalga Chlorella vulgaris comprising a mutation step comprising exposing the microalgal strain Chlorella vulgaris 211 / 11P obtained from the Culture Collection of Algae of the University of Gottingen to UV at the wavelength of 254 nm.

3. A method for producing zeaxanthin and lutein comprising the step of culturing the mutant strain of the microalga Chlorella vulgaris according to claim 1 in a suitable culture medium and under light intensity conditions between 50 and 2000 pmol photons m-2s-1, preferably from 50-100 pmol m-2s-1to 800 pmol m-2s-1and even more preferably from 500 pmol m-2s-1to 800 pmol m-2s-1.

4. A method for producing zeaxanthin and lutein according to the preceding claim, which is carried out in the presence of a carbon dioxide concentration comprised between 0.04% and 3% (v / v) of CO2.

5. A method for producing zeaxanthin and lutein according to the preceding claim 3 or 4, which is carried out under conditions of 500 pmol photons m-2s-1and with a CO2 concentration greater than 1% and preferably comprised between 3 and 5% (v / v).

6. A method for producing zeaxanthin and lutein according to any one of claims 3 to 5, which is carried out under exposure conditions comprised between 50 and 100 pmol photons m-2s-1or 2000pmol photons m-2s-1and with a CO2 concentration greater than 1% and preferably comprised between 3-5% (v / v).

7. A biomass obtained according to the method of any one of claims 3 to 6, comprising the compounds zeaxanthin and lutein.

8. A food, pharmaceutical, nutraceutical, zoo-technical or cosmetic product comprising the biomass according to claim 7.

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