A single-cell green alga chlorophyll synthesis-deficient mutant strain and application thereof

Abstract

The application discloses a single-cell green alga chlorophyll synthesis-defective mutant strain and application thereof. The mutant strain includes Parachlorella kessleri SCUT-ACB 4 and SCUT-ACB 4-65, and the preservation numbers are CCTCC NO: M 2022518 and CCTCC NO: M 2022519, which are preserved in the China Center for Type Culture Collection of Wuhan University in Wuhan, China on April 28, 2022. Compared with wild-type algae strains, the mutant strain in the application fundamentally cuts off non-light-dependent chlorophyll synthesis, and the content of chlorophyll a is reduced by more than 98%, and the content of chlorophyll b is reduced by 100%, and the algal body is golden yellow, and the mutant strain can be used for producing protein, and provides high-yield and high-quality fermentation algae for replacing protein in precise fermentation production.

Description

Technical Field

[0001] This invention belongs to the field of microbial engineering, and specifically relates to a chlorophyll synthesis defective mutant strain of single-celled green algae and its application. Background Technology

[0002] With social development and rising living standards, the demand for animal protein is gradually increasing. Traditionally, animal protein comes from livestock farming; however, the development of livestock farming leads to shortages of water, soil, and food resources, as well as increasing environmental degradation. In particular, methane and CO2 emissions account for 26% of greenhouse gas emissions from food production systems, exacerbating the greenhouse effect. Excessive consumption of animal meat can easily lead to obesity, cardiovascular disease, and other health risks. Therefore, the market for non-animal-derived alternative proteins has experienced explosive growth in the past two years. Developing new categories of nutritionally superior, cost-effective, and sustainably produced alternative proteins has become a focus of attention for people concerned about food security, nutritional health, and environmental friendliness.

[0003] Single-celled green algae (such as Chlorella, Scenedesmus, and Chlorella) have extremely high nutritional value, with a protein content as high as 50% to 65%. They are rich in 18 kinds of amino acids, polyunsaturated fatty acids, polysaccharides, vitamins, minerals and other complex nutrients. In particular, they can be easily fermented at high density in industrial fermentation systems, with short production cycles and high production efficiency. Therefore, they are regarded as high-quality microbial germplasm to replace protein precision fermentation production.

[0004] However, the practical application of wild-type single-celled green algae is limited by factors such as high chlorophyll content and a strong algal odor, which seriously affects its sensory appeal in food, suppresses market demand, and greatly restricts its widespread use in the food industry. Therefore, the chlorophyll removal process for food-grade green algae powder is essential. Traditional chlorophyll removal methods typically employ solvent extraction, which is not only complex and costly but also causes environmental pollution and the loss of other nutrients. Therefore, the presence of chlorophyll and the odor in single-celled green algae is the biggest limitation to its market as a protein substitute and food ingredient; currently, a highly efficient, low-energy-consumption, and environmentally friendly solution for decolorization and deodorization has not yet been found.

[0005] Research on chlorophyll removal from single-celled green algae mainly focuses on two directions. The first is optimizing and exploring solvent extraction methods for chlorophyll, such as supercritical CO2 extraction, adsorption resin extraction, and organic solvent extraction. Currently, various parameters have been gradually optimized, but 100% removal is still difficult to achieve and does not meet the requirements for product application. The second direction involves using gene manipulation and mutagenesis to genetically modify single-celled green algae, inhibiting or blocking the intracellular chlorophyll biosynthesis pathway. This breaks through the bottleneck in algal strain phenotypic expression at the genetic level, fundamentally solving the problems of chlorophyll synthesis and the production of odorous substances. This results in fermented green algae powder that is no longer green and has a significantly reduced odor, making it more suitable for widespread application in the food industry.

[0006] A novel ambient-pressure room-temperature plasma (ARTP) biomutation breeding technology, jointly developed by Tsinghua University and Beijing Siqingyuan Biotechnology Co., Ltd., offers advantages such as low jet temperature, uniform distribution of active particles, ease of operation, and no harm to humans or the environment. It provides an innovative platform for constructing target strains using systems biology and synthetic biology techniques, and represents a new approach to reduce or eliminate chlorophyll synthesis and odor formation in single-celled green algae. However, there are currently no reports on using ARTP mutagenesis to screen for chlorophyll synthesis-deficient microalgal mutants. Summary of the Invention

[0007] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a mutant strain of single-celled green algae with chlorophyll synthesis defects.

[0008] Another object of the present invention is to provide the application of the single-celled green algae chlorophyll synthesis defective mutant strain.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] A mutant strain of chlorophyll synthesis defect in a single-celled green algae, comprising at least one of the mutant strains P. ks SCUT-ACB 4 (hereinafter referred to as P. ks 4) and P. ks SCUT-ACB 4-65 (hereinafter referred to as P. ks 4-65); wherein,

[0011] The mutant strain P.ks SCUT-ACB 4, named Parachlorella kessleri SCUT-ACB 4, with accession number CCTCC NO: M 2022518, was deposited on April 28, 2022, at the China Center for Type Culture Collection (CCTCC) of Wuhan University, Wuhan, China.

[0012] The mutant strain P.ks SCUT-ACB 4-65, named Parachlorella kessleri SCUT-ACB 4-65, with accession number CCTCC NO: M 2022519, was deposited on April 28, 2022, at the China Center for Type Culture Collection (CCTCC) of Wuhan University, Wuhan, China.

[0013] The *Parachlorella kessleri*, formerly known as *Chlorella kessleri*, provided by this invention, possesses the characteristic structures of a typical phylum (Chlorophyta), class (Chlorophyceae), order (Chlorococcalales), family (Chlorellaceae), and genus (Chlorella). When cultured under suitable conditions in sterile media such as Basal, BG-11, and BBM, its vegetative cells are approximately 5–10 μm in size, spherical, and green. It reproduces asexually via spores, with each cell producing 2, 4, 8, or 16 spore-like cells. It can grow autotrophically, heterotrophically, and multitrophically, with autotrophic growth being the slowest, followed by heterotrophic growth, and multitrophic growth being the fastest.

[0014] Among the chlorophyll synthesis defective mutant strains of the single-celled green algae described above, the 18S rRNA gene of mutant strain P.ks 4 is shown in SEQ ID NO.1, and the 18S rRNA gene of mutant strain P.ks 4-65 is shown in SEQ ID NO.2.

[0015] A method for culturing the chlorophyll synthesis defective mutant strain of the single-celled green algae, the specific steps of which are: inoculating the chlorophyll synthesis defective mutant strain of the single-celled green algae into a culture medium and culturing it at 28-30℃.

[0016] The culture medium is at least one of Basal medium, BG-11 medium and BBM medium; preferably Basal medium containing carbon and nitrogen sources; more preferably Basal medium containing 2.5-5 g / L sodium nitrate and 10-30 g / L glucose; and even more preferably Basal medium containing 5 g / L sodium nitrate and 30 g / L glucose.

[0017] The culture medium has the following formula: NaNO3 2.5-5 g / L, glucose 10-30 g / L, H3BO3 114.2 mg / L, MoO3 7.1 mg / L, KH2PO4 1250 mg / L, CaCl2·2H2O 111 mg / L, MnCl2·4H2O 14.2 mg / L, MgSO4·7H2O 1000 mg / L, FeSO4·7H2O 49.8 mg / L, CuSO4·5H2O 15.7 mg / L, EDTA 500 mg / L, ZnSO4·7H2O 88.2 mg / L, CoNO3·6H2O 6.1 mg / L.

[0018] The culture is preferably carried out in a shaker at a speed of 125-150 rpm.

[0019] The cultivation is carried out under light (co-trophic) or darkness (heterotrophic) conditions; preferably, it is carried out under darkness conditions.

[0020] The preferred light intensity is 10 μmol / m². 2 / s.

[0021] The single-celled green algae chlorophyll synthesis defect mutants P.ks 4 and P.ks 4-65 provided by this invention exhibit a golden-yellow thallus under heterotrophic conditions, lacking a green phenotype. They contain only one carotenoid—lutein, with chlorophyll a decreasing by 99.54% and 98.16% respectively compared to the wild type, and neither contains chlorophyll b. The protein content in the heterotrophic fermented algal powder reaches 39.25% and 46.62% respectively, with P.ks 4-65 showing a 25.05% increase in protein yield compared to the wild type, significantly improving the production efficiency of the algae.

[0022] The application of the single-celled green algae chlorophyll synthesis defective mutant strain in protein production.

[0023] The application of the single-celled green algae chlorophyll synthesis defective mutant strain in protein production involves inoculating the activated single-celled green algae chlorophyll synthesis defective mutant strain into a culture medium containing carbon and nitrogen sources, culturing it at 28-30°C in the dark, collecting the algal liquid by centrifugation, washing, and freeze-drying to obtain protein-rich algal powder.

[0024] The carbon and nitrogen source-containing culture medium is at least one of Basal medium, BG-11 medium, and BBM medium; preferably Basal medium containing carbon and nitrogen source; more preferably Basal medium containing 2.5-5 g / L sodium nitrate and 10-30 g / L glucose; and even more preferably Basal medium containing 5 g / L sodium nitrate and 30 g / L glucose.

[0025] The formulation of the culture medium containing carbon and nitrogen sources is as follows: NaNO3 2.5-5 g / L, glucose 10-30 g / L, H3BO3 114.2 mg / L, MoO3 7.1 mg / L, KH2PO4 1250 mg / L, CaCl2·2H2O 111 mg / L, MnCl2·4H2O 14.2 mg / L, MgSO4·7H2O 1000 mg / L, FeSO4·7H2O 49.8 mg / L, CuSO4·5H2O 15.7 mg / L, EDTA 500 mg / L, ZnSO4·7H2O 88.2 mg / L, CoNO3·6H2O 6.1 mg / L.

[0026] The culture is preferably carried out in an Erlenmeyer flask with a shaking speed of 125-150 rpm.

[0027] The inoculation density is 1×10 6 ~1×10 7 cells / mL; preferably 2×10 6 ~1×10 7 cells / mL.

[0028] The protein described is a substitute protein. When the mutant strains P.ks 4 and P.ks 4-65 are heterotrophically cultured under dark conditions, the algae are golden yellow, and the chlorophyll a content is reduced by 99.54% and 98.16% respectively compared to the wild type, with no chlorophyll b. In practical applications, traditional decolorization processes can be eliminated. Furthermore, the protein content in the heterotrophically fermented algal powder from mutant strains P.ks 4 and P.ks 4-65 reaches 39.25% and 46.62% respectively, containing all eight essential amino acids with amino acid scores significantly higher than the wild type. The protein yield of mutant strain P.ks 4-65 is 25.05% higher than the wild type, significantly improving the production efficiency of the algal strain. Therefore, using the mutant strains P.ks 4 or P.ks 4-65 of this invention as fermentation algae strains eliminates the need for decolorization and deodorization treatments, offering significant quality and cost advantages for the precise fermentation production of substitute proteins.

[0029] The present invention has the following advantages and effects compared with the prior art:

[0030] (1) This invention uses ARTP mutagenesis to select mutant strains of *Chlorella keckii*. First, the optimal mutagenesis conditions for atmospheric pressure and room temperature plasma were determined using lethality curves. Based on this, multiple rounds of mutagenesis were conducted to obtain chlorophyll synthesis-deficient mutant strains, which were then systematically evaluated. This evaluation comprehensively assessed fermentation performance and protein and carotenoid quality characteristics, ultimately yielding high-quality yellow mutant strains, namely the chlorophyll synthesis-deficient *Chlorella keckii* mutant strains P.ks4 and P.ks4-65. Compared to wild-type strains, this invention fundamentally interrupts non-light-dependent chlorophyll synthesis, reducing chlorophyll a content by over 98% and chlorophyll b content by 100%, resulting in a golden-yellow algae. This breakthrough overcomes the technical bottleneck at its source, reducing the inefficient, high-cost, and highly polluting decolorization and deodorization steps in industrial production, greatly simplifying the process and reducing production costs.

[0031] (2) This invention reveals the effects of different nutrient modes and carbon and nitrogen source concentrations on the growth and protein accumulation of wild-type and mutant strains, and determines the nutrient mode in which the heterotrophic mutant strain P.ks 4 can achieve a better pigment composition. The optimal carbon and nitrogen source concentrations for efficient protein accumulation in mutant strain P.ks 4 are 5 g / L initial sodium nitrate concentration and 30 g / L initial glucose concentration, which significantly improves protein content and yield. Compared with the initial sodium nitrate concentration of 2.5 g / L and the initial glucose concentration of 10 g / L, the protein content and yield of P.ks 4 increased by 96.34% and 99.56% respectively, achieving high-yield protein fermentation regulation, which can be applied to the field of large-scale precision fermentation production of alternative proteins.

[0032] (3) This invention uses the mutant strain P.ks 4 as the starting algal strain and performs secondary induction to obtain a chlorophyll synthesis defective mutant strain P.ks 4-65 of single-celled green algae. Under the above-mentioned optimal culture conditions, the net biomass yield of P.ks 4-65 is 3.63% and 4.18% higher than that of the wild type and the mutant strain P.ks 4, respectively. The protein content and protein yield are 20.46% and 25.05% higher than those of the wild type, respectively, and 18.77% and 24.10% higher than those of the mutant strain P.ks 4, respectively, which greatly improves the production efficiency. Compared to the wild type, the mutant strain P.ks 4-65 has a better amino acid composition and richer content, fundamentally solving the application bottlenecks of wild-type Chlorella vulgaris, which is affected by the synthesis of large amounts of chlorophyll, thus affecting its color and odor sensory characteristics, and by the presence of limiting amino acids, thus affecting its nutritional value. It provides a high-yield, high-quality fermentation algae strain for the precise fermentation production of alternative proteins, reducing the decolorization and deodorization steps in actual production, and laying the foundation for promoting the application of alternative proteins in the food industry and the development of the market. Attached Figure Description

[0033] Figure 1The graphs show the ARTP mutagenesis mortality rates of wild-type P. ks of Chlorella vulgaris under different trophic conditions in Example 1; where (a) represents multitrophic and (b) represents heterotrophic.

[0034] Figure 2 These are photographs of algal colonies and algal blooms of the chlorophyll synthesis-deficient mutant strains (P.ks 1-4) in Example 1; where a-d represent the mutant strains on the dioecious screening plate; e-h represent the mutant strains on the dioecious purification plate; and i-l represent the mutant strains on the heterotrophic purification plate.

[0035] Figure 3 These are photographs of algal solutions from the wild-type and mutant strains in Example 2; where a to e represent heterotrophic algal solutions and f to j represent fasciotrophic algal solutions.

[0036] Figure 4 This is a graph showing the biomass concentration of the wild-type and mutant strains in the diversification culture of Example 2.

[0037] Figure 5 This is a graph showing the biomass concentration of the wild-type and mutant strains in heterotrophic culture in Example 2.

[0038] Figure 6 This is a statistical chart showing the protein content, yield, and productivity of wild-type and mutant strains under heterotrophic and multitrophic conditions in Example 2.

[0039] Figure 7 This is a graph showing the xanthophyll content and the relative contents of chlorophyll a and b in the wild-type and mutant strains under heterotrophic and ditrophic conditions in Example 2.

[0040] Figure 8 This is a statistical chart of the net biomass yield of wild-type and mutant strains under different sugar supplementation modes and different nitrogen source concentrations in Example 3.

[0041] Figure 9 This is a graph showing the xanthophyll content and relative chlorophyll a and b content of wild-type and mutant strains under different sugar supplementation modes in Example 3 (in the graph, the initial nitrogen source concentration is 2.5 g / L NaNO3).

[0042] Figure 10 This is a graph showing the xanthophyll content and relative chlorophyll a and b content of wild-type and mutant strains under different sugar supplementation modes in Example 3 (in the graph, the initial nitrogen source concentration is 3.75 g / L NaNO3).

[0043] Figure 11 This is a graph showing the xanthophyll content, chlorophyll a and b relative contents of wild-type and mutant strains under different sugar supplementation modes in Example 3 (in the graph, the initial nitrogen source concentration is 5 g / L NaNO3).

[0044] Figure 12This is a statistical graph showing the protein yield of wild-type and mutant strains under different sugar supplementation modes and nitrogen source concentrations in Example 3.

[0045] Figure 13 This is a lethality curve of the P.ks4 mutant strain under ARTP secondary mutagenesis at different heterotrophic cell stages in Example 4.

[0046] Figure 14 This is a photograph of the algal colony and algal bloom of the P.ks 4-65 mutant strain obtained through secondary mutagenesis on a heterotrophic purification plate.

[0047] Figure 15 This is a statistical chart showing the specific growth rate and net biomass yield of wild-type P.ks, mutant P.ks 4, and P.ks 4-65 in Example 4.

[0048] Figure 16 This is a statistical chart showing the lutein content, relative chlorophyll a and b content, protein content, yield, and productivity of wild-type P.ks, mutant P.ks 4, and P.ks 4-65 in Example 4; where (A) represents the lutein content and the relative chlorophyll a and b content; and (B) represents the protein content, yield, and productivity. Detailed Implementation

[0049] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to the prior art. Reagents, instruments and equipment used without specifying the manufacturer are considered to be conventional products that can be purchased commercially.

[0050] The detection method adopted in this embodiment of the invention can be described as follows:

[0051] (I) Cell density measurement

[0052] Cell density was determined using flow cytometry. 2 mL of cell culture medium was aspirated, centrifuged to remove the supernatant, washed with ultrapure water, and resuspended to the flow cytometry concentration (approximately 1–5 × 10⁻⁶). 6 Cells / mL were filtered through a 300-mesh nylon sieve and then subjected to absolute cell density counting (3 replicates).

[0053] (II) Biomass Concentration Determination

[0054] Biomass concentration was determined using the drying differential weight method. The weight of a centrifuge tube dried to constant weight was recorded as the empty tube weight W1. 2 mL of algal solution was added to this centrifuge tube, centrifuged at 8000 rpm for 3 min, the supernatant was discarded, and the tube was washed with distilled water and centrifuged twice. The algal sludge was then dried in an oven at 80℃ to constant weight, and the total weight W2 was measured and recorded. Three replicates were set up for each sample. The average value and standard deviation were calculated, and the biomass concentration and net biomass yield were calculated using the following formula:

[0055]

[0056]

[0057] In the formula: W1 is the weight of the empty tube, in grams;

[0058] W2 is the total weight of the dried centrifuge tubes and microalgae biomass, in grams;

[0059] V is the sample volume, in liters (L).

[0060] t represents the incubation time, expressed in days (d).

[0061] Net biomass yield is the biomass concentration on day t after the end of cultivation minus the biomass concentration on day 0.

[0062] (III) Protein content determination – Kjeldahl method

[0063] (1) Collect algal cells by centrifuging fresh algal solution at 4℃ and 8000r / min for 5min. Wash with distilled water, centrifuge twice, and freeze-dry to obtain lyophilized algal powder. Weigh 100mg of lyophilized algal powder into a Kjeldahl flask, add 6.4g of mixed catalyst (0.4g of anhydrous copper sulfate and 6g of sodium sulfate, ground and mixed well) and 12mL of 18.4mol / L concentrated sulfuric acid, and place the sample in a two-stage heating digestion process on an electric furnace: 150℃ for 40min and 420℃ for 150min; until the sample turns transparent blue-green, cool to room temperature, and prepare a blank sample at the same time.

[0064] (2) Place the above sample in a Kjeldahl nitrogen analyzer, set the alkali addition to 75 mL (40% (w / v) NaOH solution), the boric acid absorption solution to 20 mL (10% (w / v) boric acid solution), the dilution water to 10 mL, and the distillation time to 5 min.

[0065] (3) After the reaction is complete, add three drops of mixed indicator (equal volumes of methyl red ethanol solution and bromocresol green ethanol solution) to the receiving flask. The methyl red ethanol solution is prepared by weighing 0.1 g of methyl red, dissolving it in ethanol and diluting it to 100 mL; the bromocresol green ethanol solution is prepared by weighing 0.5 g of bromocresol green, dissolving it in ethanol and diluting it to 100 mL) and titrate with 0.1 mol / L hydrochloric acid standard solution. When the solution changes from blue-green to gray-red, the titration endpoint is reached, and the titration volume is recorded.

[0066] The crude protein content in the sample is expressed as a mass fraction (%), calculated using the following formula:

[0067]

[0068] In the formula:

[0069] V2—The volume of hydrochloric acid standard titration solution consumed in titrating the sample, in milliliters (mL);

[0070] V1—The volume of standard hydrochloric acid solution consumed in the titration of the blank, in milliliters (mL);

[0071] c — Concentration of the hydrochloric acid standard titration solution, in moles per liter (mol / L);

[0072] m — Sample mass, in grams (g);

[0073] 14 — Molar mass of nitrogen, in grams per mole (g / mol);

[0074] 6.25 — Average coefficient for converting nitrogen to crude protein.

[0075] Two parallel samples were taken for each test, and the arithmetic mean of the two samples was used as the test result.

[0076] (IV) Amino Acid Analysis – Automated Amino Acid Analyzer Method

[0077] Amino acid analysis was performed using an amino acid analyzer. The specific steps are as follows:

[0078] (1) 17 amino acids (Asp, Thr, Ser, Glu, Gly, Ala, Cys, Val, Met, Ile, Leu, Tyr, Phe, Lys, His, Arg, Pro) were extracted by conventional acid hydrolysis.

[0079] Accurately weigh 50–100 mg of lyophilized algae powder into a hydrolysis tube, prepare three parallel sets, accurately add 10 mL of 6 mol / L hydrochloric acid hydrolysis solution, and tighten the cap. Place the hydrolysis tube in a constant temperature drying oven at 110℃±2℃ for 22–24 h. Cool, mix well, open the tube, and filter. Accurately pipette an appropriate amount of filtrate and dry it in a petri dish. Add 3–5 mL of sodium citrate buffer solution (pH=2.2) to reconstitute the sample, so that the amino acid concentration in the sample reaches 50–250 nmol / mL. Shake well, filter or centrifuge, and analyze the supernatant using an L-8900 high-speed amino acid analyzer. The injection volume is 20 μL, and the absorbance is measured and integrated simultaneously at dual wavelengths of 570 nm and 440 nm.

[0080] (2) Alkaline hydrolysis method for extracting tryptophan (Trp)

[0081] Accurately weigh 50–100 mg of lyophilized algae powder into a polytetrafluoroethylene (PTFE) hydrolysis tube, prepare three parallel sets, add 1.5 mL of 4 mol / L lithium hydroxide solution, purge with nitrogen for 1 min, tighten the seal cap, and place in a constant temperature drying oven at 110℃±2℃ for 20 h of hydrolysis. Remove the hydrolysis tube and cool to room temperature. Quantitatively transfer the hydrolysate to a 25 mL volumetric flask using sodium acetate buffer (pH = 4.5), and dilute to volume with the aforementioned buffer. After centrifugation or filtration, analyze the supernatant using an L-8900 high-speed amino acid analyzer, simultaneously measuring and integrating the absorbance at both 570 nm and 440 nm wavelengths.

[0082] The amino acid content in the sample, expressed as a mass fraction ω (%), is calculated using the following formula:

[0083]

[0084] In the formula:

[0085] n—the dilution factor of the sample hydrolysis solution;

[0086] A i —The peak area of ​​the corresponding amino acid in the sample;

[0087] V—Volume of hydrolysis solution in the sample, in mL;

[0088] c — the concentration of the corresponding amino acid in the standard working solution, in nmol / mL;

[0089] M – Molar mass of amino acid, in g / mol;

[0090] V i —Sample injection volume, in μL;

[0091] A st —Peak area of ​​amino acid standard solution;

[0092] m—Sample mass, in mg;

[0093] V st —Injection volume of amino acid standard solution, in μL.

[0094] (V) Natural Pigments – High Performance Liquid Chromatography

[0095] The determination of natural pigments was performed using high-performance liquid chromatography (HPLC). The specific steps are as follows:

[0096] (1) Preparation of the standard curve: Accurately weigh 10 mg of lutein standard and dissolve it in 10 mL of volumetric precipitate (methanol: tert-butyl methyl ether (MTBE) = 1:1, v / v; containing 0.1% (w / v) 2,6-di-tert-butyl-p-cresol (BHT)) to prepare a standard solution. Then dilute it to different concentration gradients with volumetric precipitate and filter it through a 0.22 μm organic filter membrane into a liquid chromatography vial for analysis. The HPLC system used a Waters dual 1525 pump, a Waters 2996 diode array (PDA) detector, and a YMC carotenoid column C30 (4.6 × 150 mm, 3 μm). The mobile phase consisted of methanol A and tert-butyl methyl ether B. Elution conditions were as follows: 0–2 min, 90%→80%A, 10%→20%B; 2–6 min, 80%→60%A, 20%→40%B; 6–15 min, 60%→50%A, 40%→50%B; 15–16 min, 50%→90%A, 50%→10%B. The flow rate was 0.8 mL / min, the injection volume was 20 μL, the detection wavelength was 445 nm, and the column temperature was 25 °C. A standard curve of lutein concentration (mg / L, y) versus peak area (x) was plotted.

[0097] (2) Determination of lutein content

[0098] Accurately weigh 10 mg of lyophilized algal powder sample and place it in a cryovial. Add an appropriate amount of grinding beads and 1 mL of extraction reagent (methanol:acetone = 1:1, v / v; containing 0.1% (w / v) BHT). Quickly freeze in liquid nitrogen, then rapidly shake in a grinder (70 kHz, 60 s), repeating four times. Centrifuge and collect the supernatant into a 15 mL centrifuge tube, repeating several times until the algal powder turns completely white. Dry the organic solvent in the 15 mL centrifuge tube with nitrogen, add a volumetric precipitant (methanol: tert-butyl methyl ether MTBE = 1:1, v / v; containing 0.1% (w / v) BHT), accurately dilute to 1 mL, filter through a 0.22 μm organic filter membrane into a liquid chromatography vial for testing (method as in step (1)). Calculate the lutein concentration in the liquid using a standard curve. Then convert it to the lutein content in the algal powder.

[0099] (3) Calculate the relative contents of chlorophyll a and chlorophyll b in the mutant strain.

[0100] The extraction and elution gradients for chlorophyll a and chlorophyll b were the same as for xanthophyll. The PDA detection wavelengths on the HPLC were set to 663 nm and 645 nm. The ratio of the peak area corresponding to chlorophyll a in the mutant algal powder at 663 nm to that in the wild-type algal powder was calculated, and the peak area corresponding to chlorophyll a in the wild-type algal powder was set as 100% to obtain the relative content of chlorophyll a in the mutant. Similarly, the ratio of the peak area corresponding to chlorophyll b in the mutant algal powder at 645 nm to that in the wild-type algal powder was calculated, and the peak area corresponding to chlorophyll b in the wild-type algal powder was set as 100% to obtain the relative content of chlorophyll b in the mutant.

[0101] Example 1: ARTP mutagenesis to breed chlorophyll synthesis defective mutants

[0102] This embodiment uses *Parachlorella kessleri* as an example to illustrate this method in detail. *Parachlorella kessleri* was purchased from Carolina Biological Supply Company (USA) and preserved by slant transfer on Basal medium at a temperature of 4°C. The Basal medium formulation is shown in Table 1. The ARTP (Ambient Temperature Plasma) mutagenesis breeding instrument used was from Wuxi Yuanqing Tianmu Biotechnology Co., Ltd.

[0103] Table 1. Basal medium formulation

[0104] Components Content (mg / L) Components Content (mg / L) Components Content (mg / L) NaNO3 1250 H3BO3 114.2 MoO3 7.1 KH2PO4 1250 CaCl2·2H2O 111 MnCl2.4H2O 14.2 MgSO4.7H2O 1000 FeSO4.7H2O 49.8 <![CDATA[CuSO4·5H2O]]> 15.7 EDTA 500 <![CDATA[ZnSO4·7H2O]]> 88.2 <![CDATA[CoNO3·6H2O]]> 6.1

[0105] Note: If preparing solid Basal medium, add 2% (w / v) agar; EDTA is ethylenediaminetetraacetic acid.

[0106] 1.1 Chlorella culture

[0107] 10 mL of Basal medium (containing 10 g / L glucose and 1.25 g / L sodium nitrate, pH adjusted to 6.14 ± 0.05) was placed in a 50 mL Erlenmeyer flask. After autoclaving at 121 °C for 15 min, the medium was cooled to room temperature. Single colonies of *Chlorella vulgaris* were picked from solid slant or plate culture medium using an inoculation loop and inoculated. The flasks were then placed in a constant-temperature shaking incubator at 30 °C and a rotation speed of 150 rpm. Two groups were set up: a co-culture group (placed under 10 μmol / L light) and a ditrophic group. 2 / s) and heterotrophic (dark) group cultivation.

[0108] 1.2 Pretreatment before mutagenesis

[0109] Take the early, middle, and late logarithmic stages of *Chlorella vulgaris* seed culture in both multitrophic and heterotrophic forms, and dilute with sterile water to a concentration of 10:1. 6~ 10 7 Cells / mL. On a clean bench, mix 500 μL of the diluent with 500 μL of 10% (v / v) glycerol solution to achieve a final glycerol concentration of 5% (v / v), which will serve as a mutagenic protectant. Spread 10 μL of the above mixed algal solution evenly onto a sterilized metal slide.

[0110] 1.3 Specific steps of the mutagenesis operation:

[0111] The operating conditions of the ARTP mutagenesis breeding instrument were set as follows: RF power 120W, helium gas flow rate of 10 L / min (99.99% purity), treatment distance (distance between the plasma emission source and the metal slide) 2 mm, and operating temperature room temperature. Treatment times were set to 0, 15, 20, 25, 30, 40, and 50 s. After treatment, the metal slide was immediately placed in a centrifuge tube containing 1 mL of Basal medium and eluted for 1 min. The tube was then incubated in the dark for 3 h to prevent photorecovery. The eluent was serially diluted to appropriate concentrations, and 100 μL of each eluent was spread onto Basal medium plates and incubated at 30℃ under light for 10 days (other incubation conditions were the same as in 1.1). Three replicates were prepared for each group. Unmutated algal solution was used as a blank control. The colony counts in each group were calculated and compared with the control group. The lethality rate was calculated (lethality rate (%) = (1 - number of colonies in the treatment group / number of colonies in the control group) × 100%). The optimal mutagenesis time was selected when the lethality rate was above 95%. Based on the results, the processing time was further refined to 0, 15, 17, 19, 21, and 23 seconds, and the above steps were repeated.

[0112] The mortality curves of ARTP against concomitant and heterotrophic *Chlorella vulgaris* are as follows: Figure 1 As shown, heterotrophic *C. kappa* exhibits higher sensitivity to ARTP, with higher sensitivity in the early and late logarithmic phases than in the mid-logarithmic phase. Ultimately, the optimal mutagenesis times for the disotrophic *C. kappa* were determined to be: early logarithmic phase: 15 s; mid-logarithmic phase: 19 s; late logarithmic phase: 15 s, with lethalities of 98.91%, 97.17%, and 96.36%, respectively. For the heterotrophic *C. kappa*, the optimal mutagenesis times were: early logarithmic phase: 15 s; mid-logarithmic phase: 19 s; late logarithmic phase: 17 s, with lethalities of 99.57%, 98.73%, and 99.77%, respectively. Mutagenesis of the corresponding algal solutions under these determined optimal mutagenesis times is beneficial for increasing the positive mutation rate.

[0113] 1.4 Screening of mutant strains

[0114] Single algal colonies that grew after mutagenesis were initially screened using visual colony color as an indicator. Compared with wild-type colonies, colonies with a visible lightening of the green color (e.g., light green, pale green, or yellow) were selected. Their growth rate, protein content, and biomass yield were systematically evaluated to identify mutant strains with significant growth advantages and high protein content, ultimately constructing a chlorophyll synthesis-deficient *Chlorella keckii* mutant library. After initial screening, using the dark green wild-type *Chlorella keckii* P.ks as the starting strain, four color mutant strains, P.ks 1–4, were obtained under optimal conditions. Under multitrophic conditions, all showed light green colonies, while under heterotrophic conditions, P.ks 1 was light green, and P.ks 2–4 were yellow. Photos of colonies and algal blooms on screening plates, multitrophic purification plates, and heterotrophic purification plates are shown below. Figure 2 .

[0115] 1.5 Passage Culture

[0116] The mutant strains P.ks 1–4 were cultured eight times using streak plating to verify their genetic stability by visualizing the color of the algal colonies. The results showed that these mutant strains have good genetic stability.

[0117] Example 2 Evaluation of the production performance and quality characteristics of chlorophyll synthesis-deficient mutants under different nutritional conditions

[0118] 2.1 Activation of algal strains

[0119] Single colonies of wild-type *P. ks* and mutant strains P. ks 1–4 were picked from solid culture medium and inoculated into Basal medium (pH 6.14 ± 0.05) containing 10 g / L glucose, sterilized at 121 °C for 15 min. Heterotrophic (dark) and cotrophic (light intensity 10 μmol / m²) colonies were then subjected to inoculation. 2 Seed culture was carried out in a constant temperature shaker at 30°C and 150 rpm until the logarithmic growth phase.

[0120] 2.2 Cultivation under different nutritional modes

[0121] Prepare Basal medium with an initial sodium nitrate concentration of 3.75 g / L and an initial glucose concentration of 30 g / L, at pH 6.14, and sterilize at 121°C for 15 min. Then, take a certain volume of the logarithmic growth phase seed culture obtained in step 2.1 above into a sterile centrifuge tube, centrifuge at 4000 rpm for 3 min, discard the supernatant, resuspend the cell pellet in sterile Basal medium, and inoculate it into a newly prepared medium at an inoculation density of 2 × 10⁶ cells / mL. 6 Cells / mL were used for heterotrophic and cotrophic culture, with a culture period of 4 days until the carbon or nitrogen source was exhausted.

[0122] 2.3 Analysis and Testing

[0123] Every 24 hours, 2 mL of sample was taken to measure cell density and biomass concentration. After the culture was completed, the algal solution was collected, centrifuged, washed, and freeze-dried to obtain algal powder, which was then stored at -20°C for analysis of pigment composition and protein content, and calculation of protein yield and productivity.

[0124] Data were processed using Origin 9.0 and SPSS software. Statistical analysis was performed using one-way ANOVA and paired data t-tests for significance testing. Different letters indicate significant differences between groups (P<0.05).

[0125] 2.4 Results of Production Performance and Quality Characteristics Analysis

[0126] Using the same carbon and nitrogen source concentrations, the biomass concentrations of wild-type and mutant strains under heterotrophic and ditrophic conditions are shown in [reference needed]. Figure 4 and Figure 5 As shown in the figure, under mixed-trophic conditions, the specific growth rates of the wild-type and mutant strains were similar and showed no significant difference, but both were significantly higher than the specific growth rates under heterotrophic conditions (P<0.05). Among them, the mutant strain P.ks 3 had the highest biomass concentration and net yield under mixed-trophic conditions, at 17.0 g / L and 3.75 g / L / d, respectively, which were 13.33% and 16.80% higher than those of the wild-type. Under heterotrophic conditions, the mutant strain P.ks 3 achieved the highest biomass concentration and net yield, at 14.42 g / L and 3.24 g / L / d, respectively, which were 41.03% and 41.36% higher than those of the wild-type.

[0127] Using the same carbon and nitrogen source concentration, the protein content, yield, and productivity of wild-type and mutant strains under heterotrophic and ditrophic conditions are shown in [reference needed]. Figure 6 As shown in the figure, the protein content of all algal strains under heterotrophic conditions was slightly higher than that under multitrophic conditions. However, due to the higher net biomass yield in multitrophic conditions, the protein yield and efficiency of all algal strains except P. ks 3 were higher than those under heterotrophic conditions. Among them, P. ks 1 had the highest protein yield and efficiency, reaching 4.26 g / L and 1.07 g / L / d, respectively, which was 8.85% higher than that of the wild type.

[0128] Using the same carbon and nitrogen source concentration, the xanthophyll content and relative chlorophyll a and b contents of wild-type and mutant strains under heterotrophic and multitrophic conditions are shown in the figure. Figure 7As shown in the figure, light has a significant impact on the synthesis of xanthophyll and chlorophyll. The xanthophyll content is significantly higher under ditrophic conditions than under heterotrophic conditions. The relative contents of chlorophyll a and b are even more pronounced. Under heterotrophic conditions, chlorophyll synthesis in mutants is largely inhibited, with mutant P.ks 4 exhibiting the lowest chlorophyll content, with chlorophyll a only 0.46% of the wild type and no chlorophyll b. Under ditrophic conditions, chlorophyll synthesis in mutants is similar to that of the wild type, with mutants P.ks 2–3 showing higher chlorophyll a content, at 118.9% and 103.6% of the wild type, respectively. Therefore, under heterotrophic conditions, the algal solution of mutant P.ks 1 appears light green, while that of mutants P.ks 2–4 appears yellow; however, under ditrophic conditions, both exhibit a deeper dark green color. Figure 3 As shown. Therefore, although cell growth is faster and protein yield is higher under cotrophic conditions, the pigment composition does not meet the breeding objectives, so heterotrophic mode is selected for subsequent culture.

[0129] Example 3: Evaluation of the production performance and quality characteristics of chlorophyll synthesis-deficient mutants under different carbon and nitrogen source concentrations.

[0130] 3.1 Activation of algal strains

[0131] Single colonies of wild-type and mutant strains P.ks 1–4 of Chlorella vulgaris were picked from solid culture medium and inoculated into Basal medium (pH 6.14±0.05) containing 10 g / L glucose, sterilized at 121 °C for 15 min. They were then heterotrophically cultured in the dark at 30 °C and 150 rpm until the logarithmic growth phase.

[0132] 3.2 Heterotrophic culture under different carbon and nitrogen source concentrations

[0133] Basal medium was prepared with initial sodium nitrate concentrations of 2.5, 3.75, and 5 g / L, and initial glucose concentrations of 30 g / L (added all at once) and 10 g / L (added in batches: glucose concentration was monitored daily, and when depleted, glucose was added back to the medium to 10 g / L, for a total of two additions). The pH was 6.14, and the medium was sterilized at 121°C for 15 min. A certain volume of the logarithmic growth phase seed culture obtained in step 3.1 was taken into a sterile centrifuge tube, centrifuged at 4000 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in sterile Basal medium with different carbon and nitrogen source concentrations and inoculated into the prepared medium at an inoculation density of 2 × 10⁶ cells / mL. 6cells / mL. Wild-type and mutant strains P. ks 1–4 were cultured with initial sodium nitrate concentrations of 2.5, 3.75, and 5 g / L, and initial glucose concentrations of 30 g / L and 10 g / L, respectively. Six experimental groups were set up for each strain (30 groups in total), with two biological replicates per group. Heterotrophic culture was conducted in a constant-temperature shaker at 30℃ and 150 rpm for 4 days until the carbon or nitrogen source was depleted.

[0134] 3.3 Analysis and Testing

[0135] Cell density was measured by taking 2 mL samples every 24 hours. After centrifugation and washing, biomass concentration was measured. After cultivation, the algal solution was centrifuged, washed, and freeze-dried to obtain algal powder, which was stored at -20°C for analysis of pigment composition, protein content, and amino acid composition. Protein yield and productivity were calculated, along with the amino acid score (AAS = (nitrogen or amino acid content per gram of the tested food protein (mg) / nitrogen or amino acid content per gram of the reference protein (mg)) × 100. The reference protein could be the WHO essential amino acid model for humans.

[0136] 3.2.3 Results of Production Performance and Quality Characteristics Analysis

[0137] The net biomass yields of wild-type P. ks and mutant strains P. ks 1–4 under different initial carbon and nitrogen source concentrations in different culture media are shown in the figure. Figure 8 As shown in the figure, when the initial nitrogen source concentration remained consistent, the net biomass yield of both wild-type and mutant strains was significantly higher when glucose was added in a single batch than when glucose was added in batches (P<0.05). When glucose was added, the overall trend of net biomass yield was a decrease with increasing initial nitrogen source concentration, indicating that higher nitrogen source concentrations inhibit cell growth.

[0138] The content of xanthophyll and the relative contents of chlorophyll a and b under different initial nitrogen source concentrations are shown in the figure. Figures 9-11 As shown in the figure, under the same initial sodium nitrate concentration, adding glucose all at once is more beneficial to the synthesis of xanthophyll, chlorophyll a, and chlorophyll b than adding glucose in batches. Under the same initial glucose concentration, a higher initial nitrogen source concentration promotes chlorophyll synthesis, while the effect on xanthophyll shows a trend of first increasing and then decreasing. Under different carbon and nitrogen source concentrations, the mutant strain P.ks 4 had chlorophyll a and chlorophyll b reduced by 98.78%–99.54% and 100% respectively compared to the wild type, making it the mutant with the lowest chlorophyll content among P.ks 1–4. Meanwhile, its xanthophyll content reached 0.62 mg / g at an initial glucose concentration of 30 g / L and an initial sodium nitrate concentration of 5 g / L, which is 115.9% higher than the culture condition of 2.5 g / L sodium nitrate, and superior to other mutant strains. Therefore, the pigment composition of the mutant strain P.ks 4 is most consistent with expectations.

[0139] Protein content, yield, and productivity at different initial carbon and nitrogen source concentrations in different culture media are shown in the figure. Figure 12 As shown in the figure, when the initial nitrogen source concentration was consistent, the protein yield of wild-type and mutant strains was significantly higher when glucose was added all at once than when glucose was added in batches (p<0.05). With increasing nitrogen source concentration, protein content and yield increased significantly. The mutant strain P.ks 4 performed best, achieving a protein content of 39.25% at an initial sodium nitrate concentration of 5 g / L, a 96.34% increase compared to an initial sodium nitrate concentration of 2.5 g / L; and a protein yield of 1.15 g / L / d, an 86.75% increase compared to sodium nitrate at 2.5 g / L, representing the highest level among all algal strains under all culture conditions.

[0140] Amino acid composition analysis was performed on algal powder cultured at an initial glucose concentration of 30 g / L and an initial sodium nitrate concentration of 5 g / L. The results are shown in Table 2. As shown in Table 2, all algal strains contained eighteen amino acids, including the eight essential amino acids for humans. Glutamic acid, leucine, alanine, aspartic acid, and proline were present in higher amounts, while histidine, methionine, and cysteine ​​were present in lower amounts. P. ks 4 had the highest total amino acid content, reaching 340.55 mg / g, an increase of 6.33% compared to the wild type. The content of the eight essential amino acids (Lys, His, Trp, Phe, Met, Ile, Leu, Val, Thr) was 135.69 mg / g, an increase of 14.86% compared to the wild type. The total content of branched-chain amino acids (Val, Ile, Leu) was 63.96 mg / g, an increase of 17.76% compared to the wild type.

[0141] Table 2. Amino acid composition of algae powder added with 5 g / L NaNO3 and glucose in a single addition.

[0142]

[0143]

[0144] A comparison of the essential amino acids contained in the above-mentioned strains with the FAO / WHO recommended model yielded the amino acid score table shown in Table 3. The amino acid composition of P.ks and mutant strain P.ks 1 is relatively unbalanced, containing two limiting amino acids: methionine + cysteine ​​and isoleucine, with amino acid scores of only 49.05 and 51.94 respectively, which is not conducive to human digestion and absorption. The amino acid compositions of mutant strains P.ks2 and P.ks3 are similar to the FAO / WHO recommended model, with sulfur-containing amino acids as the first limiting amino acid, achieving scores of 94.51 and 78.74 respectively. Mutant strain P.ks 4 has the best amino acid composition, with all components superior to the FAO / WHO recommended model. It contains no limiting amino acids and has a score as high as 101.34, representing a significant improvement over the wild-type strain and meeting the standards for high-quality protein.

[0145] Table 3. Amino acid score of algae powder with 5 g / L NaNO3 and after single addition of glucose.

[0146]

[0147] In summary, the mutant strain P.ks 4 exhibits the best pigment composition, protein yield, and amino acid composition, thus best representing the expected chlorophyll synthesis-deficient mutant algae strain. Furthermore, initial glucose concentrations of 30 g / L and initial sodium nitrate concentrations of 5 g / L are the optimal conditions for achieving efficient protein accumulation.

[0148] The 18S rRNA gene of the mutant strain P.ks 4 was sequenced and analyzed (Guangdong Provincial Center for Microbiology Analysis and Testing), and the sequence is shown in SEQ ID NO.1. According to sequence alignment, the 18S rRNA of the mutant strain P.ks 4 has 99.88% homology with the published Chlorella strain.

[0149] Based on the colony morphology and molecular identification results of the mutant strain P.ks 4, the mutant strain P.ks 4 was named Parachlorella kessleri SCUT-ACB 4, and was deposited on April 28, 2022, at the China Center for Type Culture Collection (CCTCC) of Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2022518.

[0150] Example 4: Systematic evaluation of secondary mutagenesis and various properties of mutant strain P.ks4

[0151] In this embodiment, the optimal mutant strain P.ks 4 was used as the starting strain for secondary mutagenesis.

[0152] 4.1 Seed culture

[0153] The method is the same as in 1.1.

[0154] 4.2 Pretreatment before mutagenesis

[0155] The method is the same as 1.2.

[0156] 4.3 Specific steps of the mutagenesis operation:

[0157] The method is the same as in 1.3.

[0158] The mortality rate curve is shown below. Figure 13 As shown in the figure, *P. ks* 4 showed higher sensitivity to ARTP during the early and late logarithmic phases than during the mid-logarithmic phase. The optimal mutagenesis times for *P. ks* 4 were determined to be: early logarithmic phase: 15 s; mid-logarithmic phase: 17 s; late logarithmic phase: 15 s, with lethality rates of 98.01%, 99.84%, and 97.96%, respectively. Mutagenesis was then induced in the corresponding algal solutions at these optimal mutagenesis times.

[0159] 4.4 Screening of mutant strains

[0160] After mutagenesis, the initial screening and ranking were performed using the time of emergence of single algal colonies on solid screening plates as the growth rate indicator. The earliest single algal colonies to emerge on days 6-7 after mutagenesis were selected as target strains, which were then transferred to purification plates. Finally, the 22 resulting mutant strains were placed in 48-well plates for high-throughput heterotrophic culture (in darkness). A second screening was performed using biomass yield as the indicator, yielding the fastest-growing mutant strain, P.ks 4-65. This mutant was streaked heterotrophically onto solid plates, and the color characteristics of its algal colonies and algal blooms are shown in [the figure]. Figure 14 .

[0161] 4.5 Passage Culture

[0162] The mutant strain P.ks 4-65 was cultured eight times using the streak plate method, and its genetic stability was verified by visualizing the color of the algal colonies. The results showed that the mutant strain has good genetic stability.

[0163] 4.6 Evaluation of the production performance and quality characteristics of mutant strain P. ks 4-65

[0164] 4.6.1 Heterotrophic Culture

[0165] P.ks 4-65 was inoculated into Basal medium sterilized at 121℃ for 15 min, with an initial sodium nitrate concentration of 5 g / L, an initial glucose concentration of 30 g / L, and a pH of 6.14. The mixture was then heterotrophically cultured (in the dark) for 4 days in a constant temperature shaker at 30℃ and 150 rpm until the carbon or nitrogen source was depleted.

[0166] 4.6.2 Analysis and Testing

[0167] Every 24 hours, 2 mL of sample was taken to measure cell density and biomass concentration. After cultivation, lyophilized algal powder was prepared and stored at -20℃ for analysis of pigment composition, protein content, and amino acid composition, and protein yield and productivity were calculated, along with amino acid score.

[0168] 4.6.3P.ks 4-65 Production Performance and Quality Characteristics Analysis Results

[0169] Under the same conditions, the specific growth rate and net biomass yield of the mutant strain P.ks 4-65 are shown in the figure. Figure 15 As shown in the figure, under the conditions of an initial glucose concentration of 30 g / L and an initial sodium nitrate concentration of 5 g / L, the specific growth rate and net biomass yield of the mutant strain P.ks 4-65 reached 0.97 and 2.92 g / L / d, respectively, both significantly higher than those of the wild-type P.ks and the starting mutant strain P.ks 4. This indicates that its growth performance was significantly improved after the second round of mutagenesis.

[0170] Figure 16(A) shows the xanthophyll content and relative chlorophyll a and b contents of *P. ks*, mutant strains *P. ks 4*, and *P. ks 4-65* under the same conditions. As shown, under initial glucose concentrations of 30 g / L and initial sodium nitrate concentrations of 5 g / L, the chlorophyll a content of mutant strain *P. ks 4-65* was 98.16% lower than that of the wild type, and chlorophyll b was absent. Meanwhile, the xanthophyll content of mutant strain *P. ks 4-65* reached 0.83 mg / g, which was 27.01% and 33.85% higher than that of *P. ks* and mutant strain *P. ks 4*, respectively.

[0171] Protein content, yield, and fraction of P. ks, mutant P. ks 4, and P. ks 4-65 under the same conditions are shown in the figure. Figure 16 (B). As shown in the figure, under the conditions of an initial glucose concentration of 30 g / L and an initial sodium nitrate concentration of 5 g / L, the protein content, yield, and productivity of the mutant strain P.ks 4-65 were significantly higher than those of P.ks and the mutant strain P.ks 4. Specifically, the protein content of the mutant strain P.ks 4-65 reached 46.62%, which was 20.46% and 18.77% higher than that of P.ks and the mutant strain P.ks 4, respectively; the protein yield and productivity reached 5.44 g / L and 1.36 g / L / d, respectively, which were 25.05% and 18.77% higher than those of P.ks and the mutant strain P.ks 4, respectively. This indicates that protein accumulation in the mutant strain P.ks 4-65 was enhanced after the second round of mutagenesis.

[0172] Amino acid composition analysis was performed on wild-type P. ks, mutant P. ks 4, and P. ks 4-65 under the same conditions, and the results are shown in Table 4. As shown in Table 4, the total amino acid content of P. ks 4-65 reached 414.16 mg / g, which was 29.33% and 21.65% higher than that of the wild-type and P. ks 4, respectively, indicating a richer amino acid content. The content of the eight essential amino acids (Lys, His, Trp, Phe, Met, Ile, Leu, Val, Thr) was 162.24 mg / g, which was 37.34% and 19.56% higher than that of the wild-type and P. ks 4, respectively. The total content of branched-chain amino acids (Val, Ile, Leu) was 80.1 mg / g, which was 47.48% and 25.23% higher than that of the wild-type and P. ks 4, respectively.

[0173] Table 4. Amino acid composition of algae powder added with 5 g / L NaNO3 and glucose in a single addition.

[0174]

[0175]

[0176] The amino acid profiles of the mutant strain P.ks 4-65 were compared with the FAO / WHO recommended model, resulting in the amino acid score table shown in Table 5. The amino acid score of the mutant strain P.ks 4-65 was 70.03, with methionine and cysteine ​​as the first limiting amino acids, and the contents of the remaining amino acids were all better than the FAO / WHO recommended model. As shown in Table 5, the absolute content of cysteine ​​in P.ks 4-65 was significantly lower than that in P.ks 4, hence its amino acid score was lower than that of P.ks 4, but still significantly higher than that of wild-type P.ks. Compared with the wild-type, the amino acid composition of P.ks 4-65 showed a certain degree of improvement.

[0177] Table 5. Amino acid score of algae powder with 5 g / L NaNO3 and after a single addition of glucose.

[0178]

[0179] This embodiment uses the chlorophyll synthesis defective mutant strain P.ks4 as the starting algal strain. After a second round of mutagenesis, the mutant strain P.ks4-65 was obtained, which further improved the growth performance and quality characteristics, providing an invaluable and excellent fermentation algal strain for the field of alternative proteins.

[0180] The 18S rRNA gene of the mutant strain P.ks 4-65 was sequenced and analyzed (Guangdong Provincial Center for Microbiology Analysis and Testing). The gene sequence is shown in SEQ ID NO.2. The 18S rRNA of the mutant strain P.ks 4-65 has 99.94% homology with the published strain of Chlorella kwangsiensis.

[0181] Based on the colony morphology and molecular identification results of the mutant strain P.ks 4-65, the mutant strain P.ks 4-65 was named Parachlorella kessleri SCUT-ACB 4-65, and was deposited on April 28, 2022, at the China Center for Type Culture Collection (CCTCC) of Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2022519.

[0182] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention. sequence list <110> South China University of Technology Guangzhou Algae Energy Biotechnology Co., Ltd. <120> A chlorophyll synthesis-deficient mutant strain of single-celled green algae and its application <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1615 <212> DNA <213> Artificial Sequence <220> <223> P. sk SCUT-ACB 4 18S rRNA gene sequence <400> 1 cccgggttgc caatccgaac acttcaccag cacacccaat cggtaggagc gacgggcggt 60 gtgtacaaag ggcagggacg taatcaacgc aagctgatga cttgcgctta ctaggcattc 120 ctcgttgaag attaataatt gcaataatct atccccatca cgatgcagtt tcaaagatta 180 cccgggcctc tcggccaagg ctaggctcgt tgattgcatc agtgtagcgc gcgtgcggcc 240 cagaacatct aagggcatca cagacctgtt attgcctcat gcttccattg gctagtcgcc 300 aatagtccct ctaagaagtc tgccggcccc cgaggaggcc gtgactattt agcaggctga 360 ggtctcgttc gttaccggaa tcaacctgac aaggcaaccc accaactaag aacggccatg 420 caccaccacc catagaatca agaaagagct ctcaatctgt caatcctcac tatgtctgga 480 cctggtaagt tttcccgtgt tgagtcaaat taagccgcag gctccacgcc tggtggtgcc 540 cttccgtcaa tttcctttaa gtttcagcct tgcgaccata ctccccccgg aacccaaaaa 600 ctttgatttc tcataaggtg ccggcggagt catcgaagaa acatccgccg atccctagtc 660 ggcatcgttt atggttgaga ctaggacggt atctaatcgt cttcgagccc ccaactttcg 720 ttcttgatta atgaaaacat ccttggcaaa tgctttcgca gtagttcgtc tttcgaaaat 780 ccaagaattt cacctctgac atcgaaatac gaatgccccc gactgtccct cttaatcatt 840 actccggtcc tacagaccaa caggataggc cagagtccta tcgtgttatt ccatgctaat 900 gtattcagag cgtaggcctg ctttgaacac tctaatttac tcaaagtaac agcgccgact 960 ccgagtcccg gacagtgaag cccaggagcc cgtccccggc aacaaggcgg gccctgccag 1020 tgcacaccga aacggcggac cggcaggccc cgcccgaaat ccaactacga gcttttaac 1080 tgcagcaact taaatatacg ctattggagc tggaattacc gcggctgctg gcaccagact 1140 tgccctccaa ttgatcctcg ttaaggggtt tagattgtac tcattccaat taccagacct 1200 gaaaaggccc ggtattgtta tttatgtca ctacctccct gtgtcaggat tgggtaattt 1260 gcgcgcctgc tgccttcctt ggatgtggta gccgtttctc aggctccctc tccggaatcg 1320 aaccctaatc ctccgtcacc cgttaccacc atggtaggcc tctatcctac catcgaaagt 1380 tgatagggca gaaatttgaa tgaaacatcg ccggcgcaag gccatgcgat tcgtgaagtt 1440 atcatgattc accgcgagac cggcagagcc gggtcggcct taaatctaat aaatacgtcc 1500 cttccagaag tcgggattta cgcacgtatt agctctagaa ttactacggt tatccggtag 1560 taaggtacca tcaaataaac tataactgat ttaatgagcc attcgcagtt tcaca 1615 <210> 2 <211> 1615 <212> DNA <213> Artificial Sequence <220> <223> P.sk SCUT-ACB 4-65 18s rRNA gene sequence <400> 2 ggaaaccgcc ccgggttgcc aatccgaaca cttcaccagc acacccaatc ggtaggagcg 60 acgggcggtg tgtacaaagg gcagggacgt aatcaacgca agctgatgac ttgcgcttac 120 taggcattcc tcgttgaaga ttaataattg caataatcta tccccatcac gatgcagttt 180 caaagattac ccgggcctct cggccaaggc taggctcgtt gattgcatca gtgtagcgcg 240 cgtgcggccc agaacatcta agggcatcac agacctgtta ttgcctcatg cttccattgg 300 ctagtcgcca atagtccctc taagaagtct gccggccccc gaggaggccg tgactattta 360 gcaggctgag gtctcgttcg ttaccggaat caacctgaca aggcaaccca ccaactaaga 420 acggccatgc accaccaccc atagaatcaa gaaagagctc tcaatctgtc aatcctcact 480 atgtctggac ctggtaagtt ttcccgtgtt gagtcaaatt aagccgcagg ctccacgcct 540 ggtggtgccc ttccgtcaat tcctttaagt ttcagccttg cgaccatact ccccccggaa 600 cccaaaact ttgatttctc ataaggtgcc ggcggagtca tcgaagaaac atccgccgat 660 ccctagtcgg catcgtttat ggttgagact aggacggtat ctaatcgtct tcgagccccc 720 aactttcgtt cttgattaat gaaaacatcc ttggcaaatg ctttcgcagt agttcgtctt 780 tcgaaaatcc aagaatttca cctctgacat cgaaatacga atgcccccga ctgtccctct 840 taatcattac tccggtccta cagaccaaca ggataggcca gagtcctatc gtgttattcc 900 atgctaatgt attcagagcg taggcctgct ttgaacactc taatttactc aaagtaacag 960 cgccgactcc gagtcccgga cagtgaagcc caggagcccg tccccggcaa caaggcgggc 1020 cctgccagtg cacaccgaaa cggcggaccg gcaggccccg cccgaaatcc aactacgagc 1080 tttttaactg cagcaactta aatatacgct attggagctg gaattaccgc ggctgctggc 1140 accagacttg ccctccaatt gatcctcgtt aaggggttta gattgtactc attccaatta 1200 ccagacctga aaaggcccgg tattgttat tattgtcact acctccctgt gtcaggattg 1260 ggtaatttgc gcgcctgctg ccttccttgg atgtggtagc cgtttctcag gctccctctc 1320 cggaatcgaa ccctaatcct ccgtcacccg ttaccaccat ggtaggcctc tatcctacca 1380 tcgaaagttg atagggcaga aatttgaatg aaacatcgcc ggcgcaaggc catgcgattc 1440 gtgaagttat catgattcac cgcgagaccg gcagagccgg gtcggcctta aatctaataa 1500 atacgtccct tccagaagtc gggatttacg cacgtattag ctctagaatt actacggtta 1560 tccggtagta aggtaccatc aaataaacta taactgattt aatgagccat tcgca 1615

Claims

1. A mutant strain of chlorophyll synthesis defective single-celled green algae, characterized in that: The mutant strain is at least one of the mutant strains P. ks SCUT-ACB 4 and P. ks SCUT-ACB 4-65; wherein, The mutant strain P.ks SCUT-ACB 4, named Parachlorella kessleri SCUT-ACB 4, with accession number CCTCC NO: M 2022518, was deposited on April 28, 2022, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China. The mutant strain P.ks SCUT-ACB 4-65, named Parachlorella kessleri SCUT-ACB 4-65, with accession number CCTCC NO: M 2022519, was deposited on April 28, 2022, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China.

2. A method for culturing a chlorophyll synthesis-deficient mutant strain of single-celled green algae as described in claim 1, characterized in that, The specific steps are as follows: Inoculate the chlorophyll synthesis defective mutant strain of single-celled green algae into the culture medium and culture it at 28-30℃.

3. The method according to claim 2, characterized in that: The culture medium is at least one of Basal medium, BG-11 medium and BBM medium; The culture is carried out under light or dark conditions.

4. The method according to claim 3, characterized in that: The culture medium is a Basal medium containing 2.5–5 g / L sodium nitrate and 10–30 g / L glucose; The culture was carried out in the dark.

5. The method according to claim 4, characterized in that: The culture medium has the following formula: NaNO3 2.5-5 g / L, glucose 10-30 g / L, H3BO3 114.2 mg / L, MoO3 7.1 mg / L, KH2PO4 1250 mg / L, CaCl2·2H2O 111 mg / L, MnCl2·4H2O 14.2 mg / L, MgSO4·7H2O 1000 mg / L, FeSO4·7H2O 49.8 mg / L, CuSO4·5H2O 15.7 mg / L, EDTA 500 mg / L, ZnSO4·7H2O 88.2 mg / L, CoNO3·6H2O 6.1 mg / L.

6. The application of the single-celled green algae chlorophyll synthesis defective mutant strain according to claim 1 in protein production.

7. The application according to claim 6, characterized in that: The activated single-celled green algae chlorophyll synthesis defect mutant strain was inoculated into a culture medium containing carbon and nitrogen sources and cultured at 28–30°C in the dark. The algal solution was collected by centrifugation, washed, and freeze-dried to obtain protein-rich algal powder.

8. The application according to claim 7, characterized in that: The carbon and nitrogen source-containing culture medium is at least one of Basal medium, BG-11 medium and BBM medium containing carbon and nitrogen sources. The inoculation density is 1×10 6 ~1×10 7 cells / mL.

9. The application according to claim 8, characterized in that: The culture medium containing carbon and nitrogen sources is Basal medium containing 2.5–5 g / L sodium nitrate and 10–30 g / L glucose.

10. The application according to claim 9, characterized in that: The formulation of the culture medium containing carbon and nitrogen sources is as follows: NaNO3 2.5-5 g / L, glucose 10-30 g / L, H3BO3 114.2 mg / L, MoO3 7.1 mg / L, KH2PO4 1250 mg / L, CaCl2·2H2O 111 mg / L, MnCl2·4H2O 14.2 mg / L, MgSO4·7H2O 1000 mg / L, FeSO4·7H2O 49.8 mg / L, CuSO4·5H2O 15.7 mg / L, EDTA 500 mg / L, ZnSO4·7H2O 88.2 mg / L, CoNO3·6H2O 6.1 mg / L.

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