novel strains of the microalga chlorella vulgaris as a sustainable source of zeaxanthin and lutein

Abstract

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

Description

Background Technology

[0001] Common Chlorella ( Chlorella vulgaris The plants are already commercially available and can accumulate very small amounts of zeaxanthin unless subjected to severe stress under strong light.

[0002] Common Chlorella ( Chlorella vulgaris It is rich in protein, vitamins, and lutein (a pigment that is important for eye health), and is therefore consumed as food in the EU and many other countries.

[0003] Carotenoids are C40 compounds with conjugated double bonds, characterized by high light absorption and strong antioxidant properties. Five major carotenoids accumulate in terrestrial plants and green algae: β-carotene and xanthophylls (with xanthophylls being the most abundant), zeaxanthin, azadirachtin, and neoxanthin. Most of these carotenoids bind to proteins, forming a protein complex called the photosystem, which also binds to chlorophyll, responsible for light absorption. Except for azadirachtin and zeaxanthin, the ratios of different carotenoids bound by the photosynthetic complex generally remain constant across many different species. Azadirachtin and zeaxanthin undergo what is known as the xanthophylline cycle depending on light conditions. Zeaxanthin, produced from β-carotene, does not accumulate but is rapidly converted to azadirachtin by zeaxanthin cyclooxygenase (ZEP). Under high light conditions, zeaxanthin decyclooxygenase (VDE) is activated, converting zeaxanthin into zeaxanthin for photoprotection: in fact, the accumulation of zeaxanthin makes various photosynthetic organisms, such as plants and microalgae, more tolerant of light stress exposure. When high light stress ends, the produced zeaxanthin is reconverted into zeaxanthin by the enzyme ZEP. Carotenoids play a fundamental role in both light absorption and photoprotection. Due to their absorption in the 480-550 nm range, they broaden the visible light range for chlorophyll a, in which chlorophyll absorption is weaker. Furthermore, they are structural components of both photosystems, fundamental to their proper assembly, and they protect the photosynthetic membrane from oxidative photodamage.

[0004] Lutein and zeaxanthin are two carotenoids with strong antioxidant properties. They have wide applications, including in cosmetics (as an additive in poultry to improve the yellow color of egg yolks) and animal feed (as an additive in poultry farming). However, their primary uses are in the nutritional and pharmaceutical fields; indeed, lutein and zeaxanthin play a significant role in eye health, thereby reducing the incidence of diseases such as age-related macular degeneration (AMD) and cataracts. Furthermore, numerous studies have demonstrated the specific role of lutein and zeaxanthin in the development and protection of the eyes in newborns. Although both lutein and zeaxanthin are present in the human diet, particularly in fruits and vegetables, it is estimated that EU citizens ingest an average of 1–2 mg / day of lutein and 0.2–0.9 mg / day of zeaxanthin from their diet, while the recommended intake is at least 10 mg / day of lutein and 2 mg / day of zeaxanthin, thus highlighting the need to increase their daily intake through dietary supplements. Currently, lutein and zeaxanthin are derived from marigolds (also known as calendula or calendula flower). Tagetes erecta Extracted from the petals of ))

[0005] However, their production has some drawbacks: a stable supply of these carotenoids is impossible because yields are seasonal and highly dependent on weather conditions. Furthermore, marigold cultivation is expensive, requiring a large labor force and extensive agricultural land. Nevertheless, due to the lack of alternatives, extraction from marigolds is still considered the gold standard for lutein and zeaxanthin production. The chemical synthesis of zeaxanthin has resulted in a product with virtually no biological activity, thus limiting its application primarily to animal feed, where it is used as a pigment.

[0006] Metabolic engineering of lutein and zeaxanthin by microorganisms (such as bacteria and yeast) has been successfully reported, but the GMO source of the resulting carotenoids hinders their application in the food and nutrition fields. Photosynthetic microorganisms, such as green algae, naturally produce and accumulate lutein and zeaxanthin: In recent years, some attempts have been made to extract lutein and zeaxanthin from microalgae. Compared to calendula, microalgae (such as Chlorella vulgaris)... Chlorella ), Single-disc algae ( Monoraphidium ) or Scenedesmus ( Scenedesmus These species can be grown in controlled environments (typically bioreactors), eliminating problems associated with seasonality, dependence on weather conditions, and potential pollution, which ensures stable and scalable production.

[0007] For these reasons, microalgae are considered the most effective alternative to calendula production. However, the adoption of this solution is hampered by the extremely low content of lutein and zeaxanthin and the extremely high extraction costs.

[0008] However, to date, there are no products on the market based on lutein and zeaxanthin derived from microalgae.

[0009] International patent application WO 2022 / 158814 describes obtaining Chlorella by using EMS as a mutagen and selecting based on the color of the plated colony. Chlorella ) strain.

[0010] European patent application EP 2,157,167 describes the process of obtaining Chlorella sorokinense ( Chlorella sorokiniana Methods for obtaining lutein from specific plants.

[0011] International patent application WO 2020 / 105001 describes obtaining Chlorella that produces low amounts of chlorophyll ( Chlorella vulgaris (The method of) plant. Summary of the Invention

[0012] The inventors of this patent application have developed a novel microalgae-based method that allows for the scalable, sustainable, and economically viable production of lutein and zeaxanthin. Attached Figure Description

[0013] Figure 1 A phylogenetic tree is shown, generated from the 18S sequences of different Chlorella species. Using common Chlorella (… Chlorella vulgaris As a comparison, SAG 211 / 11P produced the mutant fm53.

[0014] Figure 2 It shows common Chlorella ( C. vulgaris Screening of NPQ mutants. (A) Example of screening on agar plates after UV mutagenesis. (B) NPQ (non-photochemical quenching) achieved after 10 minutes of illumination; showing the mean of wild-type (Wt) inserted as a control in each plate. (C) Chlorophyll fluorescence image after 10 minutes of illumination. (D) NPQ traces of Wt and three selected lines. Numerical values ​​represent mean ± SD (n=3).

[0015] Figure 3 HPLC analysis results of pigments extracted from wild-type (A) and fm (BD) mutants in LL-adapted cells are shown. The numbers correspond to 1-neoxanthin, 2-purpurichin; 3-lutein; 4-zeaxanthin, 5-chlorophyll b; 6-chlorophyll a; and 7-β-carotene.

[0016] Figure 4Display: (A) Illustration of a single-point mutation in the 5'-UTR region of the ZEP (zeaxanthin cyclooxygenase) gene g9297. (B) Analysis of the relative amounts of ZEP and VDE transcription relative to wild type. Transcriptional levels were calculated using the 2-ΔΔCT method and correlated with wild-type expression levels set to 1. (C) Immunoassay of ZEP; lanes from left to right loaded with total protein extracts of molecular weight (MW), wild type (Wt), and fm53, as well as recombinant Chlorella vulgaris (…). Chlorella vulgaris ZEP. Ponceau S staining of the filter confirmed that the amount of protein loaded in both genotypes was the same (left panel). Western blotting with α-ZEP antibody (right panel). Blue arrows indicate the missing ZEP bands in the mutants.

[0017] Figure 5 The mutation types present in the mutants of this invention are shown.

[0018] Figure 6 Specific mutations of the mutants of the present invention are shown.

[0019] Figure 7 The study details the single nucleotide variants (SNVs) and insertion / deletion mutations (INDELs) of fm53 as a whole genome relative to the wild-type genome.

[0020] Figure 8 Display: (A) as shown Figure 10 As shown, at the end of growth, the volume (left panel) and daily volume (right panel) production capacity of biomass (A, B), lutein (C, D), and zeaxanthin (E, F) for wild type (Wt black) and fm53 (grey) are presented. Numerical values ​​represent mean ± SD (n=3). This indicates a significant difference between wild-type and fm53 (P<0.05).

[0021] Figure 9 Shown in Figure 8 Carotenoid content of wild-type (Wt) and fm53 at the end of growth under different light intensities, after aeration with 0.04% CO2 (air) or 3% CO2 (CO2). Values ​​are shown in milligrams of carotenoid per gram of dry biomass. Data are expressed as mean ± standard deviation (n=3).

[0022] Figure 10 The results show the effects of different light intensities (100 to 2000 μmol m) at atmospheric CO2 concentrations (0.04%, air) or 3% CO2. -2 s -1 Data related to the lutein / zeaxanthin molar ratio in the pigment extract of mutant fm53 cells grown under )

[0023] Invention Objective

[0024] In the first objective, the present invention describes the microalgae Chlorella vulgaris ( Chlorella vulgaris () mutant strain.

[0025] In the second objective, the present invention describes obtaining the microalgae Chlorella vulgaris ( Chlorella vulgaris Methods for mutant strains.

[0026] In the third objective, the present invention describes the cultivation of the microalgae Chlorella vulgaris of the present invention (Chronicella spp.). Chlorella vulgaris A method to produce zeaxanthin and lutein by using mutant strains of zeaxanthin.

[0027] By cultivating microalgae, common Chlorella ( Chlorella vulgaris The zeaxanthin and lutein pigments obtained from mutant strains of ) represent other objectives of this invention.

[0028] The biomass obtained according to the method and containing the compounds zeaxanthin and lutein represents other objectives of the present invention.

[0029] In the fourth objective, the present invention describes food, pharmaceutical, nutritional, animal technology, or cosmetic products comprising biomass obtained by the method described herein. Detailed Implementation

[0030] In the first objective, the present invention describes the microalgae Chlorella vulgaris ( Chlorella vulgaris () mutant strain.

[0031] Specifically, this strain is characterized by a mutation in the gene Gene_ID g9297 c.-111C >T, relative to the wild-type strain; this mutation silences the expression of the gene encoding maize xanthine cyclooxygenase.

[0032] According to one specific aspect of the present invention, the microalgae Chlorella vulgaris (commonly known as Chlorella vulgaris) Chlorella vulgaris The mutant strain is also characterized by one or more other mutations:

[0033] Specifically, these are all the mutations specified in the table above.

[0034] According to even more specific aspects of the present invention, the microalgae Chlorella vulgaris (commonly known as Chlorella vulgaris) Chlorella vulgaris The mutant strain is also characterized by Figure 5 One or more mutations specified in the document: Specifically, these are Figure 5 All mutations specified in the text.

[0035] In the second objective, the present invention describes obtaining the microalgae Chlorella vulgaris ( Chlorella vulgaris Methods for mutant strains.

[0036] Specifically, this method includes a mutation step.

[0037] A culture step can be performed before the mutation step.

[0038] Specifically, the strain is cultured in a suitable culture medium.

[0039] Therefore, TAP ( Tris-acetate-phosphate (or BG-11 medium or another suitable medium.)

[0040] Specifically, the culture conditions include an alternating light / dark schedule: 16 hours of light / 8 hours of darkness.

[0041] Specifically, the culture conditions include the following temperatures: Under illumination, 20°C to 25°C, and preferably 22°C, and In the dark, 16°C to 20°C, and preferably 18°C.

[0042] Specifically, the culture conditions in this first step include 70 μmol m -2 s -1 Light exposure.

[0043] Conversely, regarding the mutation steps, this includes converting common Chlorella ( Chlorella vulgaris Microalgae strains were exposed to mutagenic conditions.

[0044] For the purposes of this invention, the mutagenic conditions are represented by exposure to UV rays.

[0045] Specifically, the mutation steps include converting common Chlorella ( Chlorella vulgaris The microalgae strain was exposed to UV light at a wavelength of 254 nm.

[0046] Specifically, the exposure time was determined to be 2-5 minutes based on the treatment's ability to induce 90% cell death.

[0047] For the purposes of this invention, a mutated microalgae, *Chlorella vulgaris* (also known as common chlorella), was developed. Chlorella vulgaris The strain was obtained from the University of Göttingen (Germany, https: / / sagdb.uni-goettingen.de / ). Culture Center Collection of Algae Common Chlorella obtained Chlorella vulgaris ) strain 211 / 11P express.

[0048] Specifically, for about 10 8 The cell count was mutated.

[0049] The mutation process leads to approximately 90% cell death.

[0050] Following the mutation induction step, the surviving cells undergo a stabilization step.

[0051] For the purposes of this invention, the steps include placing the cells in darkness for a specific period of time to prevent their reactivation; for example, the cells may be stable for about 1-10 hours, and preferably for about 2 hours.

[0052] Following the mutation and stabilization steps, a cell selection step is performed.

[0053] For the selection step, stable cells are plated with an appropriate dilution onto a suitable culture medium.

[0054] For this purpose, cells can be diluted (for example) at a ratio of 1:100.

[0055] For this purpose, TAP (Tris-acetate-phosphate) liquid medium, BG-11 liquid medium, or another suitable medium can be used.

[0056] Regarding the selection process, colonies are selected by measuring photosynthetic parameters, which are measured by collecting fluorescence emitted by chlorophyll in the cells after exposure to different light.

[0057] For the purposes of this invention, the colony is first acclimatized to darkness for at least 20 minutes, and then measured with low-intensity light (preferably less than 7 μmol photons per minute). -2 s -1 ) and 1200 μmol photons m -2 s -1 Expose the light to photochemical light for an appropriate period of time; for example, you can continue exposure for 10 minutes.

[0058] Cells are exposed to photons with an intensity preferably greater than 5000 μmol m at regular intervals, such as every minute. -2 s -1 saturated light.

[0059] Exposing cells to this saturated light in cells irradiated with measurement light allows the acquisition of parameter Fm (the maximum fluorescence emitted by chlorophyll in cells adapted to darkness), while in cells irradiated with photochemical light, it allows the measurement of parameter Fm' (the maximum fluorescence emitted by chlorophyll in cells exposed to photochemical light).

[0060] By using the values ​​of Fm and Fm', it is possible to calculate the NPQ parameter as (Fm-Fm') / Fm', which indicates that a light-dependent photoprotection mechanism is induced by the dissipation of a portion of the absorbed light energy in the form of heat.

[0061] Evaluation of non-photochemical quenching (NPQ) results was conducted to select mutant lines with reduced NPQ activation.

[0062] Then, it is possible to use different photochemical light (from 250 to 5000 μmol photons m -2 s -1 The selected system can be confirmed by measuring the NPQ parameters.

[0063] In the third objective, it is described how to cultivate the microalga *Chlorella vulgaris* selected according to the present invention (…). Chlorella vulgaris A method to produce zeaxanthin and lutein by using mutant strains of zeaxanthin.

[0064] Specifically, the method of the present invention includes culturing the microalgae Chlorella vulgaris (commonly known as Chlorella) under appropriate growth conditions. Chlorella vulgaris The steps for mutant strains.

[0065] In a preferred embodiment, the microalgae of the present invention are cultured in a culture medium.

[0066] For example, BG-11 or TAP (Tris-acetate-phosphate) medium can be used.

[0067] For the purposes of this invention, the microalgae of this invention are used in applications with 50 to 2000 μmol photons per minute. -2 s -1 Preferably 50-100 μmol m -2 s -1 Up to 800 μmol m -2 s -1 And even more preferably 500 μmol m -2 s -1 Up to 800 μmol m -2 s -1 Cultivate under light intensity conditions.

[0068] In a preferred embodiment, the microalgae of the present invention are at 800 μmol m -2 s -1 It grows under light intensity conditions.

[0069] In a preferred aspect of the invention, cultivation is carried out in the presence of a carbon dioxide concentration between 0.04% (air) and 3% CO2 (v / v).

[0070] In a preferred embodiment, cultivation is carried out in the presence of a carbon dioxide concentration of 3% (v / v).

[0071] For the purposes of this invention, cultivation is carried out at an initial biomass concentration of about 0.05 g / L, which may be increased to about 0.4-3 g / L.

[0072] By appropriately adjusting process conditions, such as by changing growth irradiation and CO2 availability, it is possible to push production more towards obtaining zeaxanthin or zeaxanthin and lutein (improving the ratio of zeaxanthin to lutein to favor the former).

[0073] For example, when the ratio of zeaxanthin to lutein is 0.5 to 1.4, a relative accumulation of zeaxanthin and lutein can be achieved.

[0074] Specifically, using relatively high light intensity (500-2000 μmol photons / m²) -2 s -1 In a photobioreactor without CO2 enrichment (i.e., with air ventilation), lutein (as opposed to zeaxanthin) will be produced.

[0075] Zeaxanthin (as opposed to lutein) is produced under conditions of CO2 enrichment and low light intensity (50-100 μmol photons / m²). -2 s -1 Or, always under CO2 enrichment conditions and at available light intensity (2000 μmol photons / m²), -2 s -1 This is advantageous.

[0076] Therefore, in a first aspect, the method of the present invention allows for the use of 500 μmol photons m -2 s -1 Under certain conditions and in the presence of CO2 concentrations greater than 1%, and preferably between 3% and 5% (v / v), the fm53 strain was cultured to obtain greater daily yields of zeaxanthin and lutein.

[0077] Biomass must be collected when the cell density reaches a “quiescent” growth phase, which is defined as the period in which the cell density reaches saturation; this growth phase is typically obtained after about 4 days of culture.

[0078] According to the second aspect, the method of the present invention allows for the use of CO2 concentration conditions greater than 1%, and preferably between 3-5% (v / v), and 50 to 100 μmol photons. -2 s -1 Between or 2000 μmol photons m -2 s -1 The exposure conditions of strain FM53 were used to maximize the ratio of zeaxanthin to lutein in the biomass produced.

[0079] According to the method described, microalgae such as Chlorella vulgaris (commonly known as common Chlorella) are cultured. Chlorella vulgaris Biomass containing the compounds zeaxanthin and lutein or mixtures thereof in different proportions obtained from mutant strains of zeaxanthin represents other objectives of the present invention.

[0080] According to the method described, microalgae such as Chlorella vulgaris (commonly known as common Chlorella) are cultured. Chlorella vulgaris The compounds zeaxanthin and lutein, or mixtures thereof in different proportions, obtained from mutant strains of zeaxanthin represent other objectives of this invention.

[0081] The biomass obtained according to the method and containing the compounds zeaxanthin and lutein represents other objectives of the present invention.

[0082] In the fourth objective, the present invention describes the use of the obtained pigments in the fields of food, pharmaceuticals, nutritional products, animal technology products, and cosmetics.

[0083] Therefore, the present invention describes food, pharmaceutical, nutritional, animal technology, or cosmetic products comprising biomass obtained by the method described herein.

[0084] For the purposes of this invention, possible applications in the food field include use as a coloring agent due to the presence of pigments lutein and zeaxanthin, and as a protein source (approximately 50-60% of the biomass produced), and plant-derived unsaturated fatty acids (such as hexadecadienoic acid (16:2), hexadecadienoic acid (16:3), linoleic acid (18:2), α-linolenic acid (18:3), oleic acid (18:1)) rich in antioxidant pigments such as lutein and zeaxanthin.

[0085] For the purposes of this invention, possible applications in the pharmaceutical field include use as an antioxidant, a colorant, and a source of lutein and zeaxanthin (pigments essential for eye health).

[0086] According to a preferred aspect, the lutein and zeaxanthin obtained by the method according to the invention, as well as mixtures of the two pigments, can have medical uses.

[0087] For example, lutein and zeaxanthin, or a mixture of both, obtained according to the method of the present invention can be used for the prevention and / or treatment of ophthalmic diseases.

[0088] Specifically, these conditions include age-related macular degeneration, cataracts, or conditions that affect the normal development of the newborn's eye.

[0089] For the purposes of this invention, possible applications in the field of nutrition include use as an antioxidant, a protein supplement, and an unsaturated fatty acid supplement fortified with lutein and zeaxanthin.

[0090] For the purposes of this invention, possible applications in the field of animal technology include use as feed (and also for improving the coloring of egg yolks).

[0091] For the purposes of this invention, possible applications in the cosmetics field include use as an antioxidant and a colorant.

[0092] Experimental Section

[0093] As part of project ERC-StG-2015 SOLENALGAE, this invention is based on the green algae Chlorella vulgaris, known as mutant fm53, obtained at Professor Matteo Ballottari's SOLE-LAB. Chlorella vulgaris A novel non-GMO strain.

[0094] During the SOLENALGAE project, the photoprotective properties of different microalgae species were studied, and mutant libraries were generated through random insertion or physical / chemical mutagenesis.

[0095] One of the species studied is Chlorella vulgaris ( Chlorella vulgaris In this species, mutants were generated by exposure to UV light that induced random mutations in DNA levels, and among the selected mutants exhibiting reduced photosensitivity relative to the WT strain, a mutant known as fm53 showed high constitutive accumulation of zeaxanthin, whereas in the WT case, this carotenoid only accumulated transiently after exposure to strong light (see [link to relevant documentation]). Figure 2 ).

[0096] Therefore, the mutant fm53 is able to accumulate large amounts of lutein and zeaxanthin.

[0097] Genome sequencing was used to investigate the genotype behind the mutant fm53 phenotype and to identify a specific mutation that leads to altered translation of the zeaxanthin cyclooxygenase gene, which encodes an enzyme that uses zeaxanthin as a substrate.

[0098] Obtain mutant Chlorella vulgaris ( Chlorella vulgaris )

[0099] Common Chlorella ( Chlorella vulgaris (CV) 211 / 11P strain was obtained from the University of Göttingen (Germany, http: / / sagdb.uni-goettingen.de / ). Algal Culture Preservation Center (Culture Collection of Algae) This was used to generate mutants by exposure to UV light. TAP (Tris-acetate-phosphate) medium (Gorman & Levine, 1965) or BG-11 minimal medium (Allen, MM, Stanier, 1968) were used in a 16h light / 8h dark climate chamber at 22°C / 18°C and a light intensity of 70 μmol / m². -2 s -1WT and mutant strains were cultured under PPFD for physiological identification. UV mutagenesis was performed as follows: 10 mL of logarithmic mid-phase culture (approximately 10 μL) was mutagenized using a 6W UV lamp (254 nm). 8 (Number of cells) were subjected to UV irradiation to induce 90% cell death. Surviving cells were placed in the dark for 2 hours to prevent photoreactivation, and then plated 100-fold diluted TAP agar plates. TAP agar plates were screened by fluorescence measurement of in vivo chlorophyll emission to select mutants with altered photosynthetic characteristics. 1200 μmol photons were used... -2 s -1 The colony was irradiated with light for 10 minutes and exposed to 10,000 μmol of photons. -2 s -1 NPQ traces were recorded after supersaturated light. Approximately 2500 colonies were selected and compared with wild-type colonies distributed on the plate. Although many colonies were within the control mean, some showed significantly different NPQ values ​​( Figure 2 These lines were transferred to new plates for repeated analysis to confirm the fluorescence traces and avoid false positives. After these screening cycles, three lines, termed fm (fluorescent mutants), were identified that showed significant differences in the NPQ traces relative to the wild type. Figure 2 The two lines, fm53 and fm275, showed lower NPQ levels, while fm179 showed a 2-fold increase in NPQ relative to the wild type.

[0100] Since pigment distribution is closely related to NPQ, the chlorophyll and carotenoid contents of these three mutants were analyzed by HPLC. The fm53 line showed a significantly altered carotenoid pattern: neoxanthin and zeaxanthin were completely absent, and constitutively accumulated zeaxanthin under low light conditions, under which this pigment was absent in the wild type. The other two mutants showed pigment distributions similar to the wild type. Figure 3 ).

[0101] To identify potential mutations in the observed novel phenotype, the fm53 genome was sequenced using Illumina next-generation sequencing (NGS) to identify all present mutations. Wild-type and fm53 strains were compared with a reference genome (Cecchin et al., 2019) to obtain contrast coverage of 62x and 50x, respectively. Figure 5The total number of single nucleotide variants (SNVs) and insertion / deletion mutations (INDELs) in fm53 are 36 and 4, respectively. Of the 36 SNPs observed in the fm53 genome, 18 were predicted as missense variants, 1 as a missense variant near the splice site, 1 as a termination variant, and 16 as synonymous effects. This resulted in 18 intermediate and high-impact variants affecting 16 genes. Figure 6 (See table in [reference]). Among these genes, a point mutation was observed upstream of the start codon in the g9297 gene encoding ZEP, confirming the HPLC analysis. This substitution mutation, C>T, is located 111 bp upstream of the start codon (see [reference]). Figure 6 ), and located at 177 bp in what is believed to be a 5' UTR transcript (Cecchin et al., 2019). qRT-PCR showed no difference at the ZEP transcriptional level (see [link to qRT-PCR]). Figure 2 C). Transcription of the VDE gene was also monitored, and no differences were found. These results indicate that the C117T substitution does not affect mRNA transcription. Instead, the effect of the mutation was observed at the protein level, as determined by Western blotting (see [link to article]). Figure 4 Using algae from Dunaliella salina ( Dunaliella tertiolecta (Kim et al., 2018) α-ZEP antibody generated from two synthetic peptides was used to detect this protein; one of the two peptides was similar to that of common Chlorella ( C. vulgaris The sequence has 100% homology. The common Chlorella ( ) was cloned. C. vulgaris ZEP in Escherichia coli ( Escherichia coli The protein was expressed and purified in [a specific culture medium] to test and confirm its cross-reactivity with the antibody. The antibody reacted correctly with the recombinant protein; at the same altitude, a band was present in the wild type but not at all in the mutant.

[0102] The effect of a single substitution in the 5' UTR on ineffective protein translation may be due to alterations in secondary structure formation, leading to incorrect ribosome binding and expression. To better understand whether point mutations in the 5' UTR induce different conformations of RNA secondary structure, the minimum free energy of the 5' UTR in wild-type and fm53 strains was analyzed, yielding the same value of -82.40 kcal / mol for both sequences. Although the wild-type secondary structure has a lower centroid (-53.40 kcal / mol) compared to fm53 (-26.60 kcal / mol), and the lower entropy may indicate a possible perturbation of the structure (Lorentz 1976; Mathews et al., 2004).

[0103] Example

[0104] In an airlift photobioreactor bubbled with air (0.04% CO2) or 3% CO2, light intensities ranging from 50 to 2000 μmol photons were observed. -2 s -1 Mutant fm53 and wild-type strains were cultured in BG-11 medium. Growth was monitored by recording the OD at 720 nm. In tubes with air, samples grew more slowly and reached lower ODs (consistently less than 0.5), while in samples with 3% CO2, the strains grew with faster kinetics and reached much higher cell concentrations. In air samples (i.e., characterized by an atmospheric CO2 concentration of approximately 0.04%), no significant effect of light intensity was observed, as the curves were similar for all the different light methods used. Conversely, in samples with added CO2, the achieved growth kinetics and density also depended on light intensity and increased with increasing light intensity; with greater than 100 μmol photons m - 2 s -1 The light-grown tubes achieved significantly higher concentrations compared to individual tubes bubbled with air. At the end of growth, the biomass and carotenoid content were quantified (see [link to documentation]). Figure 8 In a tube filled with compressed air, the final biomass was at 100 μmol photons / m. -2 s -1 It reached approximately 0.5 g / L, and did not increase further even at higher light intensities, actually at a concentration equivalent to 800 or 2000 μmol photons / m. -2 s -1 The biomass concentration decreased to approximately 0.3 g / L under varying light intensities. In a photobioreactor fed with 3% CO2, higher biomass concentrations were observed under all light conditions, reaching 3 g / L at the highest light intensity tested. Biomass yield, measured in grams per liter per day, was also monitored for different growth experiments (see [link to relevant documentation]). Figure 8 Cell cultures grown at atmospheric CO2 concentrations under 500 μmol photons -2 s -1 The production capacity reached approximately 0.25 g / L / day, then began to decline. Cell cultures grown with 3% CO2 achieved a value of approximately 0.8–1 g / L / day, which was achieved at concentrations greater than 500 μmol / m³. -2 s -1 The production capacity did not decrease under photon conditions, but continued to increase, albeit not significantly. Under all these conditions, strain fm53 showed a similar production capacity to the wild-type strain.

[0105] Furthermore, carotenoids were quantified, such as those measured by acetone spectroscopy and HPLC analysis. Figure 8 C Figure 8(D) (Cazzaniga et al., 2020). For all test conditions, total carotenoids (milligrams of pigment per unit cell dry weight) were similar between the two genotypes. This value was similar at lower light intensities (50–100 μmol photons / m²). -2 s -1 The yield of carotenoids was higher at higher light intensities and decreased at higher light intensities, while there was no significant difference between air-supplied samples and CO2-enriched samples. Conversely, the yield of carotenoids (mg / L / day) was higher in samples supplied with 3% CO2 at 500 μmol photons. -2 s -1 The mutant achieved maximum production capacity at the same time, but there was still no difference between the two genotypes. With similar total carotenoid values, the mutant had higher maize xanthine content and yield. At 50 and 100 μmol m -2 s -1 Under photon conditions, FM53 plants accumulate zeaxanthin, which is completely absent in wild-type plants. Under higher light intensities, wild-type plants also begin to accumulate zeaxanthin, but the mutants have a higher production capacity, reaching 800 μmol photons. -2 s -1 The maximum concentration was 1.8 mg / L / day. Carotenoid content was analyzed by HPLC: pigments extracted from intact cells in organic solvents were loaded onto a reversed-phase HPLC system equipped with a C18 column and eluted using a 15-minute ethyl acetate gradient (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 performed using a 350–750 nm Thermo-Fisher diode array detector. Figure 9 The distribution of pigment milligrams per gram of dry biomass for all carotenoids is shown, where "neo" is neoxanthin, "viola" is violaxanthin, "antera" is antheraxanthin, "lute" is lutein, "zea" is zeaxanthin, and "b car" is β-carotene. Based on the lutein and zeaxanthin content, then... Figure 10 The ratio of zeaxanthin to lutein in maize as reported in the Chinese study.

[0106] Culture of mutant fm53 allows for the production of Chlorella vulgaris with constitutively enriched zeaxanthin. Chlorella vulgaris In order to obtain the same results using wild-type plants, exposure to light sources must activate a photoprotective mechanism called the "lutein cycle," through which xanthophyll is converted into zeaxanthin by the enzyme VDE.

[0107] For example, it has been observed that in wild-type plants, maize xanthine only increases after exposure to at least 500 μmol m-2 s -1 It accumulates only after exposure to light, and the amount of zeaxanthin produced per unit of dry biomass is equal to 0.5-1 mg / g (under conditions of 3% CO2, at 500 μmol / m³). -2 s -1 (The maximum value is equal to 1 mg / g), while in the mutant of this invention, zeaxanthin has passed 50 μmol / g. -2 s -1 Constitutive accumulation of the substance upon exposure occurred at levels ranging from 0.75 to 4 mg / g (under conditions of 3% CO2, at 100 μmol / m³). -2 s -1 The maximum value was 4 mg / g, while at 500 μmol m -2 s -1 Upon exposure, a yield of 2.38 mg / g can be obtained.

[0108] Based on the above description, the advantages provided by the present invention will be clear.

[0109] The common Chlorella described Chlorella vulgaris The strain has high biomass production capacity, excellent tolerance to pollutants and high protein content (>50% of dry weight).

[0110] In addition, it is a new food product approved by EFSA and FDA for human consumption, and is currently available as a vitamin-rich protein source suitable for vegan and vegetarian diets.

[0111] This invention provides a disruptive biotechnology solution that enables the production of protein-rich edible biomass in a microalgae species such as Chlorella vulgaris (Chronicella spp.) within a biological system. Chlorella vulgaris It effectively produces zeaxanthin and lutein.

[0112] Regarding zeaxanthin in plants or green algae (including common Chlorella) Chlorella vulgaris Known techniques for transient accumulation of zeaxanthin (from wild-type (WT) strains) allow for the acquisition of stable amounts of zeaxanthin (common carotenoids) comprising 30-40% of total carotenoids in Chlorella vulgaris. Chlorella vulgaris (More than twice the amount accumulated in WT strains).

[0113] Furthermore, the mutants of this invention have shown to accumulate high lutein values, with total lutein + zeaxanthin accounting for more than 80% of the total carotenoids produced.

[0114] Therefore, the strain of the present invention allows the production of “green” biomass derived from CO2 photosynthetic fixation and strongly rich in protein, lutein and zeaxanthin.

[0115] It should be further emphasized that, as mentioned above, the strain of the present invention is not a GMO strain.

[0116] Those skilled in the art will also understand that the method provided by the present invention allows for the production of zeaxanthin or lutein by appropriately changing growth parameters.

Claims

1. Microalgae, common Chlorella ( Chlorella vulgaris The mutant strain, relative to the wild-type strain, contains a mutation on the gene Gene_ID g9297c.-111C >T, which silences the expression of the gene encoding zeaxanthin cyclooxygenase.

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

3. A method for producing zeaxanthin and lutein, comprising in a suitable culture medium and with 50 to 2000 μmol of photons -2 s -1 Between 50-100 μmol m -2 s -1 Up to 800 μmol m -2 s -1 And even more preferably 500 μmol m -2 s -1 Up to 800 μmol m -2 s -1 The steps for culturing the mutant strain of Chlorella vulgaris according to claim 1 under light intensity conditions.

4. The method for producing zeaxanthin and lutein according to claim 3, wherein the method is carried out at a carbon dioxide concentration of between 0.04% and 3% (v / v) of CO2.

5. The method for producing zeaxanthin and lutein according to claim 3 or 4, using 500 μmol photons... -2 s -1 The method is carried out under conditions of CO2 concentration greater than 1%, and preferably between 3% and 5% (v / v).

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

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

8. Food products, pharmaceutical products, nutritional products, animal technology products, or cosmetic products containing the biomass described in claim 7.

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