A method for constructing a high-fold sweet protein variant engineering algal strain of chlamydomonas reinhardtii and application thereof

Abstract

This invention discloses a method for constructing an engineered algal strain of high-intensity sweet protein variants derived from *Chlamydomonas reinhardtii* and its application. First, the sweeteners Thaumatin and Brazzein are genetically modified. Then, through codon optimization based on *Chlamydomonas reinhardtii* preference, the encoding genes for CrThaumatin and CrBrazzein are synthesized and cloned into the expression vector pGM6. The recombinant expression vector is introduced into wild-type *Chlamydomonas reinhardtii* sp. algal strains. Using the paromomycin resistance marker carried by the expression vector, the engineered algal strains are successfully obtained. The engineered algal strains are fermented to produce dried, stable *Chlamydomonas reinhardtii* powder rich in the target sweet protein. Finally, oral administration tests after protein purification show that its sweetness is 3-5 times that of natural Thaumatin and Brazzein proteins.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a method for constructing an engineered algal strain of high-intensity sweet protein variant derived from Chlamydomonas reinhardtii and its application. Background Technology

[0002] Sweetness, a widely enjoyed taste sensation, traditionally originates primarily from carbohydrates such as sugars. However, long-term excessive intake of these substances is significantly associated with health risks such as high blood sugar, type 2 diabetes, and obesity. With the increasing popularity of health-conscious consumption, the market demand for low-energy, low-sugar foods is growing. Currently, alternatives used in the food industry, such as low-molecular-weight sugars, sugar alcohols, or artificial sweeteners, generally suffer from insufficient sweetness, high calorie content, or potential health risks, failing to fully meet health requirements. Therefore, developing new sugar-free, high-sweetness, safe, and natural sweeteners has become an important technical challenge in food science and the health industry.

[0003] Sweeteners are a class of proteins that impart a sweet taste to food or feed, possessing advantages such as low calories, high sweetness, biosafety, and non-toxicity. Globally, sweeteners are widely used; currently, there are over 20 types commonly used in domestic and international markets. In China, with the booming development of the food industry and increased emphasis on food safety and health, approximately 15 types of sweeteners have been approved for use. These sweeteners have become the world's most consumed type of food additive, primarily used in pastries, beverages, preserved fruits, and other food products. Compared to traditional sugar-based sweeteners, sweeteners, due to their unique low-calorie, high-sweetness, and biosafety properties, have extremely broad application prospects. Especially with the increasing health awareness of people today, they are expected to become a new type of health-oriented sweetener, bringing new changes to the food and feed industries and helping people enjoy delicious food while moving towards a healthier lifestyle.

[0004] Natural plant sweet proteins, as a very special and valuable class of substances, refer to proteins that exist naturally in nature, possess a sweet taste themselves, or can induce sweetness in organisms. Their sources are somewhat limited, mainly originating from the fleshy arils of the berries, fruits, and seeds of specific shrubs in West Africa, Malaysia, and subtropical regions. The unique climatic conditions, soil environments, and ecosystems of these regions have nurtured plants capable of synthesizing these special proteins. Currently, eight plant sweet proteins have been discovered, including Thaumatin, Monellin, Mabinlin, Brazzein, Pentadin, Miraculin, Neoculin, and Curculin. To be precise, only five of them—Thaumatin, Monellin, Mabinlin, Brazzein, and Pentadin—are sweet proteins. Miraculin and Neoculin are sweetness-inducing proteins, which have the function of correcting tastes and can change the perceived sour taste into a sweet taste. Curculin, on the other hand, has the characteristics of the first two types of proteins. Curculin protein is not only a sweet protein, but also has the characteristics of a sweetness-inducing protein.

[0005] In today's society, health issues are receiving increasing attention. In recent years, the incidence of diabetes has shown a year-on-year upward trend, cardiovascular disease has become one of the major killers threatening human life and health, and various chronic diseases are troubling many people. At the same time, the number of obese people is also constantly rising. According to relevant data, the proportion of obese people globally has increased significantly in the past few decades. The emergence of these health problems is largely related to people's dietary habits, with high-sugar diets considered a major contributing factor. Therefore, people's demand for low-calorie, low-energy, and low-sugar foods is becoming increasingly urgent.

[0006] Natural sweeteners, as a superior alternative to sucrose, are gradually gaining popularity. They possess numerous significant advantages, not only exhibiting excellent functional activity but also imparting sweetness to food while enhancing its unique flavor and quality. More importantly, natural sweeteners can be digested and broken down by pepsin into various essential amino acids needed by the human body, supplementing nutrition and satisfying people's sweetness needs while ensuring health. However, currently, the raw materials for producing natural sweet proteins are mostly specific types of shrubs. These plants are extremely demanding in their growing environment, only thriving under specific local climates and soil conditions, making cross-regional cultivation difficult. This strict limitation on growing locations makes the isolation and extraction of natural sweet proteins from plant berries and seeds very challenging, severely restricting the large-scale production and widespread application of natural sweet proteins.

[0007] Against this backdrop, the modification and screening of sweet proteins using genetic engineering technology has become a hot topic in scientific research and industry. Genetic engineering technology holds the promise of overcoming the geographical limitations of plant growth by introducing genes encoding sweet proteins into easily cultured and propagated microorganisms or plant cells, enabling the efficient production of sweet proteins. This could not only meet market demand for low-sugar, healthy sweeteners but also potentially bring new development opportunities to the food and pharmaceutical industries. Based on this, this study aims to provide a method for screening engineered algal strains of high-sweetness sweet proteins using *Chlamydomonas reinhardtii* as a substrate.

[0008] Currently, recombinant protein production mainly relies on substrates such as mammalian cells, insect cells, microorganisms (e.g., E. coli, yeast), and higher plants. However, each of these substrates has its limitations: mammalian and insect cell culture is costly and susceptible to contamination by animal-derived pathogens; prokaryotic systems lack eukaryotic-specific post-translational modification capabilities and often produce endotoxins; yeast's glycosylation pattern differs from that of humans; and higher plant systems suffer from long growth cycles, susceptibility to seasonal influences, and the risk of transgenic spread. Therefore, there is an urgent need to develop a novel production substrate that is cost-effective, highly safe, and possesses comprehensive protein processing capabilities.

[0009] *Chlamydomonas reinhardtii* is a single-celled eukaryotic photosynthetic microorganism widely distributed in various environments, including freshwater, soil, and marine environments. The algal cells are approximately 5-10 micrometers in diameter and possess two flagella of equal length as locomotion organs. Although its structure is relatively simple, it possesses a complete eukaryotic organelle system, including prominent large cup-shaped chloroplasts and typical endoplasmic reticulum and Golgi apparatus, thus enabling it to perform complex post-translational modifications of eukaryotic proteins and achieve efficient expression of exogenous proteins. Furthermore, *Chlamydomonas reinhardtii* has advantages such as low production cost, short growth cycle, and no need to occupy arable land. In summary, *Chlamydomonas reinhardtii* is an ideal platform for recombinant protein expression. Summary of the Invention

[0010] The purpose of this invention is to provide a method for constructing an engineered algal strain of high-intensity sweet protein variant derived from Chlamydomonas reinhardtii and its application, providing a feasible technical path for the green and sustainable production of high-intensity sweeteners.

[0011] This invention first modifies the genes of the sweeteners Thaumatin and Brazzein, and then synthesizes the encoding genes of CrThaumatin and CrBrazzein through codon optimization of Chlamydomonas reinhardtii. These genes are then cloned into the independently modified Chlamydomonas reinhardtii-specific expression vector pGM6. The recombinant expression vector is introduced into wild-type Chlamydomonas sp. algal strains using electroporation transformation. Screening is performed using the paromomycin (Paro) resistance marker carried by the expression vector, and immunoblotting experiments confirm the successful acquisition of engineered algal strains that stably and efficiently express the target sweet proteins. The engineered algal strains with high expression of the sweeteners CrThaumatin and CrBrazzein proteins are then fermented on a large scale and dehydrated using vacuum freeze-drying technology to obtain dry, stable Chlamydomonas reinhardtii algal powder rich in the target sweet proteins CrThaumatin and CrBrazzein. The obtained algal powder, after protein purification and oral administration tests, shows a sweetness 3-5 times that of natural Thaumatin and Brazzein proteins.

[0012] Compared with traditional production methods, the advantages of this invention are as follows: Utilizing *Chlamydomonas reinhardtii* as a recombinant protein expression host effectively solves the problems of high cost and low production capacity caused by the limited availability of plant raw materials and complex extraction processes for natural Thaumatin and Brazzein; Simultaneously, compared with eukaryotic expression systems such as animal cells and insect cells, the cultivation cost of *Chlamydomonas reinhardtii* is significantly reduced, and the potential risk of pathogen contamination from animal-derived hosts is completely avoided; compared with microbial systems such as bacteria and yeast, *Chlamydomonas reinhardtii* possesses the post-translational modification capabilities unique to eukaryotes, enabling it to correctly complete the folding and post-translational processing of complex proteins; compared with higher plant cell systems, *Chlamydomonas reinhardtii* has advantages such as a short cultivation cycle, high genetic stability, and good batch-to-batch consistency, and its cultivation process does not rely on arable land resources, enabling uninterrupted and controllable production throughout the year, effectively avoiding the seasonal and regional limitations of agricultural production.

[0013] To achieve the above objectives, this application adopts the following technical solution:

[0014] In a first aspect, the present invention provides a method for constructing the recombinant plasmid pGM6-CrThaumatin-HA-aph8 / pGM6-CrBrazzein-HA-aph8, comprising the following steps:

[0015] Step 1, construction of the pGM6-H-HA-aph8 plasmid, includes the following steps:

[0016] Step 1A: Using the universal vector pMO508 as a template, clone the vector fragment using primers pM-S / AS. The nucleotide sequences of the primers pM-S / AS are shown in SEQ ID NO.6 and SEQ ID NO.7.

[0017] Step 1B: The nucleotide sequence of 3 x HA is synthesized into the universal vector pUC57 to obtain the pUC57-3 x HA plasmid. Using this plasmid as a template, the target fragment is cloned using primers pG-HA-S / AS. The nucleotide sequence of HA is as shown in SEQ ID NO.5, and the nucleotide sequence of primers pG-HA-S / AS is as shown in SEQ ID NO.8 and SEQ ID NO.9.

[0018] Step 1C: The target fragment obtained in Step 1B and the vector fragment obtained in Step 1A are analyzed by agarose gel electrophoresis. Then, the target bands are cut under UV light and the DNA is purified and recovered using a gel extraction kit.

[0019] Step 1D: The target fragment purified in Step 1C is mixed with the vector fragment at the optimal molar ratio, and then recombinase is added to carry out homologous recombination reaction to obtain recombinant plasmid;

[0020] Step 1E: The recombinant plasmid obtained in Step 1D is transformed into DH5α Escherichia coli competent cells, placed on ice, and then subjected to heat shock and recovery on ice.

[0021] Step 1F: Add antibiotic-free LB liquid medium to each tube and incubate. Then, take an appropriate amount of bacterial culture and spread it on an LB agar plate containing Amp, and incubate it upside down overnight.

[0022] Step 1G: Randomly select multiple regular single colonies from the plate and inoculate them into LB liquid medium containing Amp, and culture them until the late logarithmic growth stage.

[0023] Step 1H: Take an appropriate amount of bacterial culture, extract the recombinant plasmid using a plasmid miniprep kit, and send the plasmid for sequencing verification to obtain the pGM6-H-HA-aph8 plasmid.

[0024] Step 2, the construction of recombinant plasmids pGM6-CrThaumatin-HA-aph8 and pGM6-CrBrazzein-HA-aph8, includes the following steps:

[0025] Step 2A: First, site-directed mutagenesis was performed on the Thaumatin protein sequence to obtain a Thaumatin-encoding protein sequence with arginine at position 30 mutated to lysine (R30K), aspartic acid at position 43 mutated to asparagine (D43N), aspartic acid at position 47 mutated to asparagine (D47N), and arginine at position 51 mutated to lysine (R51K). Simultaneously, the Brazzein protein-encoding sequence was modified to obtain a Brazzein-encoding protein sequence with the first glutamine (ΔQ1) deleted at the N-terminus, arginine at position 33 mutated to lysine (R33K), arginine at position 43 mutated to lysine (R43K), and isoleucine at position 48 mutated to leucine (I48L). Based on the codon bias of Chlamydomonas reinhardtii, the codons of the two mutant protein sequences were optimized to obtain the optimized CrThaumatin and CrBrazzein nucleotide sequences. Subsequently, the optimized nucleotide sequences were cloned into the universal vector pUC57 to construct the recombinant plasmid pUC57-CrThaumatin / pUC57-CrBrazzein. Using the constructed recombinant plasmid as a template, the CrThaumatin / CrBrazzein target gene fragments were cloned using primers pGM6-CrThaumatin-S / AS and pGM6-CrBrazzein-S / AS, respectively.

[0026] The nucleotide sequence of CrThaumatin is shown in SEQ ID NO.3, the nucleotide sequence of CrBrazzein is shown in SEQ ID NO.4, the nucleotide sequence of the pGM6-CrThaumatin-S primer is shown in SEQ ID NO.10, the nucleotide sequence of the pGM6-CrThaumatin-AS primer is shown in SEQ ID NO.11, the nucleotide sequence of the pGM6-CrBrazzein-S primer is shown in SEQ ID NO.12, and the nucleotide sequence of the pGM6-CrBrazzein-AS primer is shown in SEQ ID NO.13.

[0027] Step 2B: Using the pGM6-H-HA-aph8 plasmid prepared in Step 1 as a template, clone the expression vector fragment pGM6 using primers pGM6-HA-S / AS.

[0028] The nucleotide sequence of the primer pGM6-HA-S is shown in SEQ ID NO.14, and the nucleotide sequence of the primer pGM6-HA-AS is shown in SEQ ID NO.15.

[0029] Step 2C: The CrThaumatin / CrBrazzein target gene fragment obtained in Step 2A and the expression vector fragment pGM6 obtained in Step 2B are analyzed by agarose gel electrophoresis. Under ultraviolet light, the gel fragment containing the target band is accurately excised. Then, the DNA fragment is purified and recovered using a gel recovery kit.

[0030] Step 2D: The CrThaumatin / CrBrazzein target gene fragment purified in Step 2C is mixed with the expression vector fragment pGM6 at the optimal molar ratio, and then recombinase is added for homologous recombination to obtain recombinant plasmids;

[0031] Step 2E: The recombinant plasmids obtained in Step 2D are introduced into DH5⍺ Escherichia coli competent cells, and the mixture is placed on ice to allow the DNA to fully contact the cells, followed by heat shock and ice recovery treatment.

[0032] Step 2F: Add antibiotic-free LB liquid medium to each tube, shake and incubate to allow the bacteria to recover and express the resistance gene encoded by the plasmid. Take an appropriate amount of the recovered bacterial culture and spread it evenly on an LB agar plate containing Amp resistance, and incubate at 37°C overnight.

[0033] Step 2G: Randomly select multiple single colonies that are morphologically regular and well separated and grow on selective plates, and inoculate them into LB liquid medium containing the corresponding Amp resistance, and culture with shaking until the stationary phase;

[0034] Step 2H: Extract recombinant plasmids from the amplified bacterial culture using a plasmid miniprep kit;

[0035] Step 2I: Sequencing was performed on the recombinant plasmid to confirm that the recombinant plasmid pGM6-CrThaumatin-HA-aph8 / pGM6-CrBrazzein-HA-aph8 was successfully constructed.

[0036] Secondly, the present invention provides recombinant plasmids pGM6-CrThaumatin-HA-aph8 / pGM6-CrBrazzein-HA-aph8, which are constructed using the above-described construction method.

[0037] Thirdly, this invention provides a method for constructing an engineered Chlamydomonas strain with high expression of CrThaumatin / CrBrazzein protein. The recombinant plasmid pGM6-CrThaumatin-HA-aph8 / pGM6-CrBrazzein-HA-aph8 is introduced into wild-type Chlamydomonas sp. cells via electroporation. Algal strains with high expression of CrThaumatin / CrBrazzein protein are screened by Western blotting, thus obtaining the engineered Chlamydomonas strain with high expression of CrThaumatin / CrBrazzein protein.

[0038] Fourthly, this invention provides an engineered Chlamydomonas reinhardtii strain that highly expresses CrThaumatin / CrBrazzein protein, which is constructed using the above-described method.

[0039] Fifthly, the present invention provides Chlamydomonas reinhardtii algal powder with high expression of CrThaumatin / CrBrazzein protein, characterized in that it is produced by large-scale fermentation of the above-mentioned Chlamydomonas reinhardtii engineered algal strain with high expression of CrThaumatin / CrBrazzein protein.

[0040] Sixthly, the present invention provides the application of the above-mentioned Chlamydomonas reinhardtii algal powder with high expression of CrThaumatin / CrBrazzein protein as a food sweetener.

[0041] In a seventh aspect, the present invention provides the application of the above-mentioned Chlamydomonas reinhardtii algal powder with high expression of CrThaumatin / CrBrazzein protein as a flavor modifier.

[0042] Eighthly, the present invention provides the application of the above-mentioned Chlamydomonas reinhardtii algal powder with high expression of CrThaumatin / CrBrazzein protein as a fermentation nutrient aid.

[0043] Algae powder can be added to food, beverages, pharmaceuticals, health products, or animal feed to increase sweetness, mask or neutralize unpleasant flavors, and achieve nutritional fortification. Additionally, algae powder can be added to baking ingredients for co-fermentation, which can improve dough fermentation characteristics and product texture while reducing sugar content, thereby producing low-sugar, healthy baked goods with excellent taste.

[0044] The beneficial effects of this invention are as follows:

[0045] 1) The screening system developed in this invention, which uses Chlamydomonas reinhardtii as a chassis cell, enriches and changes the existing production methods of recombinant protein drugs. Compared with other synthetic biology chassis systems, Chlamydomonas reinhardtii as a chassis cell has advantages such as biosafety, low production cost, short production cycle, stable production batches, no occupation of arable land, post-translational modification, and clear genetic background.

[0046] 2) The Chlamydomonas reinhardtii engineered algal strain screening system provided by this invention has unique advantages in screening Chlamydomonas reinhardtii engineered algal strains with high expression of recombinant proteins, due to its simple and fast operation, wide applicability and high efficiency.

[0047] 3) The screening system using *Chlamydomonas reinhardtii* as the substrate cell developed in this invention exhibits significant advantages in acceptability compared to screening systems using bacteria, yeast, or other microorganisms as substrate cells. While bacterial substrates possess characteristics such as rapid growth and relatively simple genetic manipulation, their fermentation process is susceptible to phage contamination, and some bacterial proteins may contain endotoxin residues. Yeast substrate production may generate metabolic byproducts such as ethanol, affecting protein expression and activity. Therefore, the screening system using *Chlamydomonas reinhardtii* as the substrate cell better meets the public's demand for safe, efficient, and green screening in industrial production, biomedicine, and food fields, and is thus more easily accepted by the public. Attached Figure Description

[0048] The embodiments of the present invention will now be described with reference to the accompanying drawings. These drawings are for illustrative purposes only and illustrate some embodiments of the present invention, but should not be construed as limiting the scope of protection of the present invention.

[0049] Figure 1 The image shows a comparison between the sequences obtained by mutating the coding sequences of the natural sweet proteins Thaumatin and Brazzein according to the present invention and optimizing them according to the codon bias of Chlamydomonas reinhardtii, and the natural sequences.

[0050] Figure 2 The figure shown is a result of the PCR amplification of the target gene coding sequences of the sweet proteins CrThaumatin and CrBrazzein described in this invention and their corresponding expression vectors, verified by agarose gel electrophoresis.

[0051] Figure 3 The image shows the results of agarose gel electrophoresis after linearization of the recombinant plasmids pGM6-CrThaumatin-HA-aph8 and pGM6-CrBrazzein-HA-aph8 constructed in this invention by single-enzyme digestion with restriction endonuclease KpnI.

[0052] Figure 4The image shows the Western Blot results of protein-level screening of the transformants obtained after successfully introducing the recombinant expression plasmids pGM6-CrThaumatin-HA-aph8 and pGM6-CrBrazzein-HA-aph8 into wild-type Chlamydomonas sp. via electroporation.

[0053] Figure 5 The image shows a comparison of the sweetness of the sweet proteins CrThaumatin and CrBrazzein of this invention with that of natural sweet proteins Thaumatin and Brazzein.

[0054] Figure 6 The image shown is a photograph of Chlamydomonas reinhardtii algal powder, which is rich in sweet protein, obtained by large-scale cultivation, collection, and vacuum freeze-drying of the CrThaumatin and CrBrazzein-expressing engineered algal strains of the present invention. Detailed Implementation

[0055] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below in conjunction with specific embodiments. This invention can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art. This invention will be defined only by the claims.

[0056] Unless otherwise specified, the test methods or experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are obtained from conventional commercial sources or prepared by conventional methods.

[0057] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0058] This invention provides a method for constructing an engineered algal strain of a high-sweetness protein variant derived from *Chlamydomonas reinhardtii* and its application. The protein engineering modification involves site-directed modification of two high-sweetness proteins. For Thaumatin, the modification involves mutating arginine at position 30 to lysine (R30K), aspartic acid at position 43 to asparagine (D43N), aspartic acid at position 47 to asparagine (D47N), and arginine at position 51 to lysine (R51K). For Brazzein, the first glutamine molecule is deleted from the N-terminus. (ΔQ1) Arginine at position 33 was mutated to lysine (R33K), arginine at position 43 was mutated to lysine (R43K), and isoleucine at position 48 was mutated to leucine (I48L). Then, through Chlamydomonas reinhardtii codon optimization, the optimized CrThaumatin / CrBrazzein nucleotide sequences were submitted to a gene synthesis company and synthesized into the universal vector pUC57, resulting in pUC57-CrThaumatin and pUC57-CrBrazzein plasmids. Subsequently, using pUC57-CrThaumatin and pUC57-CrBrazzein plasmids as templates, they were cloned into the self-modified Chlamydomonas reinhardtii-specific expression vector pGM6-H-HA-aph8 via polymerase chain reaction (PCR), resulting in pGM6-CrThaumatin-HA-aph8 and pGM6-CrBrazzein-HA-aph8 plasmids. The recombinant expression plasmids pGM6-CrThaumatin-HA-aph8 and pGM6-CrBrazzein-HA-aph8 were introduced into wild-type *Chlamydomonas* sp. using electroporation. The plasmids were screened using a paromomycin (Paro) resistance marker, and the results were verified by Western blotting. Successful engineered algal strains capable of stably and efficiently expressing the target sweet proteins CrThaumatin and CrBrazzein were obtained. Subsequently, algal powder with high CrThaumatin and CrBrazzein protein expression was obtained through large-scale fermentation and freeze-drying. Oral administration of purified proteins confirmed that the proteins from the *Chlamydomonas* strain with high CrThaumatin and CrBrazzein protein expression were 3-5 times sweeter than the natural sweeteners CrThaumatin and CrBrazzein. This invention utilizes Chlamydomonas reinhardtii as a host cell to express recombinant proteins, effectively solving the problems of high cost and low production capacity of natural Thaumatin and Brazzein due to limited plant raw material sources and complex extraction processes.

[0059] In this invention, the sweet proteins CrThaumatin and CrBrazzein possess characteristics such as low calories and high sweetness. Chlamydomonas reinhardtii, as a recognized safe photosynthetic microorganism, produces recombinant high-sweetness proteins that have significant advantages in terms of biosafety and consumer acceptance, providing a feasible technical path for the green and sustainable production of high-sweetness sweeteners.

[0060] The sweetener encoding the protein sequence of this invention is as follows:

[0061] CrThaumatin encoding protein sequence: (SEQ ID NO.1)

[0062] MAATTCFFFLFPFLLLLTLSRAATFEIVNKCSYTVWAAASKGNAALNAGGKQLNSGESWTINVEPGTNGGKIWARTDCYFDDSGSGICKTGDCGGLLRCKRFGRPPTTLAEFSLNQY GKDYIDISNIKGFNVPMDFSPTTRGCRGVRCAADIVGQCPAKLKAPGGGCNDACTVFQTSEYCCTTGKCGPTEYSRFFKRLCPDAFSYVLDKPTTVTCPGSSNYRVTFCPTALELEDE

[0063] CrBrazzein encoded protein sequence: (SEQ ID NO.2)

[0064] MDKCKKVYENYPVSKCQLANQCNYDCKLDKHAKSGECFYDEKKNLQCLCDYCEY

[0065] Codon-optimized sweetener nucleotide sequence:

[0066] CrThaumatin nucleotide sequence (SEQ ID NO.3)

[0067] ATGGCCGCCACCACCTGCTTCTTCTTCCTCTTCCCCTTTCTGCTGCTGCTGACCCTGAGCCGCGCCGCCACCTTCGAGATCGTCAACAAGTGCAGCTATACCGTGTGGGCTGCTGCCAGCAAGGGCAACGCTGCGCTGAACGCTGGCGGCAAGCAGCTGAACTCGGGCGAGTCCTGGACCATCAACGTGGAGCCGGGCACCAACGGGGGCAAGATCTGGGCTCGGACCGACTGCTACTTCGACGACAGCGGGTCCGGTATCTGCAAGACCGGCGACTGCGGCGGCCTGCTGCGCTGCAAGCGCTTCGGGCGCCCGCCCACCACCCTGGCCGAGTTTTCCCTCAACCAGTACGGCAAGGACTACATCGACATCAGCAACATCAAGGGCTTCAACGTCCCGATGGACTTCTCCCCCACCACCCGCGGTTGCCGCGGCGTGCGCTGCGCTGCGGACATCGTGGGGCAGTGCCCTGCCAAGCTGAAGGCCCCCGGTGGCGGCTGCAACGATGCGTGCACCGTCTTCCAGACCTCCGAGTATTGCTGCACCACCGGCAAGTGCGGCCCCACCGAGTATTCGCGCTTCTTCAAGCGCCTGTGCCCGGACGCGTTCTCGTACGTGCTGGACAAGCCCACCACCGTGACCTGCCCCGGTAGCTCCAACTACCGCGTGACCTTCTGCCCCACCGCCCTGGAGCTGGAGGACGAG

[0068] Nucleotide sequence of CrBrazzein: (SEQ ID NO.4)

[0069] ATGGATAAGTGCAAGAAGGTGTACGAGAACTACCCCGTGAGCAAGTGCCAGCTGGCCAACCAGTGCAACTACGACTGCAAGCTGGACAAGCATGCTAAGTCGGGGGAGTGCTTCTACGACGAGAAGAAGAACCTGCAGTGCCTGTGCGACTACTGCGAGTAC

[0070] The method for constructing recombinant plasmids containing the genes for the sweeteners CrThaumatin and CrBrazzein, as described in this invention, specifically comprises two steps:

[0071] The first step is the construction of the pGM6-H-HA-aph8 plasmid.

[0072] The expression vector was constructed by replacing the 3 x FLAG tag protein in the universal vector pMO508 with 3 x HA, resulting in the self-modified plasmid pGM6-H-HA-aph8.

[0073] 1. Using the universal vector pMO508 as a template, clone the vector fragment pMO508 with primers pM-S / AS.

[0074] The nucleotide sequence of primer pM-S is shown in SEQ ID NO.6; the nucleotide sequence of primer pM-AS is shown in SEQ ID NO.7.

[0075] 2. Submit the nucleotide sequence of 3 x HA to a gene synthesis company, synthesize 3 x HA into the universal vector pUC57 to obtain the plasmid pUC57-3 x HA. Use this plasmid as a template to clone the target fragment HA using primers pG-HA-S / AS.

[0076] HA sequence: (SEQ ID NO.5)

[0077] tcgcgatacccctacgacgtgcccgactacgcctacccctacgacgtgcccgactacgccgatcgatccggaccgtacccctacgacgtgcccgactacgcccgctccgtgtga

[0078] The nucleotide sequence of primer pG-HA-S is shown in SEQ ID NO.8, and the nucleotide sequence of primer pG-HA-AS is shown in SEQ ID NO.9.

[0079] 3. The target fragment and the vector fragment were analyzed by agarose gel electrophoresis. The target band was then cut under UV light and the DNA was purified and recovered using a gel extraction kit.

[0080] 4. Mix the target fragment HA with the vector at the optimal molar ratio, and then add recombinase to carry out homologous recombination reaction.

[0081] 5. Transform the recombinant plasmid into DH5α Escherichia coli competent cells and incubate on ice for 30 minutes; then subject to heat shock at 42°C and recovery on ice.

[0082] 6. Add 500 µL of antibiotic-free LB liquid medium to each tube and incubate at 37°C and 150 rpm for 45 min. Then, take an appropriate amount of bacterial culture and spread it on LB agar plates containing Amp, and incubate upside down at 37°C overnight.

[0083] 7. Randomly select 2-3 regular single colonies from the plate and inoculate them into 3-5 mL of LB liquid medium containing Amp. Incubate at 37℃ and 200 rpm for 12-16 hours until the late logarithmic growth stage.

[0084] 8. Take an appropriate amount of bacterial culture, extract the recombinant plasmid using a plasmid mini-prep kit, and send 300 ng of the plasmid for sequencing verification. The plasmid pGM6-H-HA-aph8 has been successfully constructed.

[0085] The second step involves the construction of the final plasmids pGM6-CrThaumatin-HA-aph8 and pGM6-CrBrazzein-HA-aph8.

[0086] 1) First, site-directed mutagenesis was performed on the Thaumatin protein sequence to obtain a Thaumatin-encoded protein sequence with arginine at position 30 mutated to lysine (R30K), aspartic acid at position 43 mutated to asparagine (D43N), aspartic acid at position 47 mutated to asparagine (D47N), and arginine at position 51 mutated to lysine (R51K). Simultaneously, the Brazzein protein-encoded sequence was modified to obtain a Brazzein-encoded protein sequence with the first glutamine (ΔQ1) deleted at the N-terminus, arginine at position 33 mutated to lysine (R33K), arginine at position 43 mutated to lysine (R43K), and isoleucine at position 48 mutated to leucine (I48L). Based on the codon bias of Chlamydomonas reinhardtii, the codons of the two mutant protein sequences were optimized to obtain the optimized CrThaumatin and CrBrazzein nucleotide sequences. Subsequently, the optimized nucleotide sequences were submitted to a gene synthesis company and cloned into the universal vector pUC57 to construct the recombinant plasmids pUC57-CrThaumatin and pUC57-CrBrazzein. Using these plasmids as templates, the CrThaumatin / CrBrazzein target gene fragments were cloned using primers pGM6-CrThaumatin-S / AS and pGM6-CrBrazzein-S / AS, respectively.

[0087] The nucleotide sequence of the pGM6-CrThaumatin-S primer is shown in SEQ ID NO.10, the nucleotide sequence of the pGM6-CrThaumatin-AS primer is shown in SEQ ID NO.11, the nucleotide sequence of the pGM6-CrBrazzein-S primer is shown in SEQ ID NO.12, and the nucleotide sequence of the pGM6-CrBrazzein-AS primer is shown in SEQ ID NO.13.

[0088] 2) Using the pGM6-H-HA-aph8 plasmid constructed in step 1 as a template, primers pGM6-HA-S / AS were used to clone the expression vector fragment pGM6.

[0089] 3) The target gene fragment and expression vector fragment were analyzed separately by agarose gel electrophoresis. Under UV light, the gel fragment containing the target band was precisely excised. Subsequently, the DNA fragment was purified and recovered using a gel recovery kit, strictly following the manufacturer's operating procedures.

[0090] 4) Mix the CrThaumatin / CrBrazzein target gene fragments with the expression vector fragment pGM6 at the optimal molar ratio, and then add recombinase for homologous recombination.

[0091] 5) The recombinant plasmid was introduced into DH5⍺ Escherichia coli competent cells. The mixture was placed on ice for 30 minutes to allow the DNA to fully contact the cells. The cells were then subjected to heat shock at 42°C and recovery on ice.

[0092] 6) Add 500 µL of antibiotic-free LB liquid medium to each tube. Place the centrifuge tubes in a shaker at 37°C and shake at 150-200 rpm for 45-60 minutes to allow the bacteria to recover and express the plasmid-encoded resistance gene. Spread an appropriate amount of the recovered bacterial culture evenly onto LB agar plates containing Amp resistance and incubate overnight at 37°C.

[0093] 7) Using a sterile toothpick or pipette tip, randomly select 2 to 3 single colonies that are growing on selective plates, have regular morphology and are well separated, and inoculate them into 3-5 mL of LB liquid medium containing the corresponding Amp resistance. Incubate at 37°C and 220 rpm for 12-16 hours with shaking until the stationary phase.

[0094] 8) Extract recombinant plasmids from the amplified bacterial culture using a plasmid miniprep kit.

[0095] 9) Take 300 ng of recombinant plasmid for sequencing confirmation, that is, the pGM6-CrThaumatin-HA-aph8 and pGM6-CrBrazzein-HA-aph8 plasmids were successfully constructed.

[0096] Furthermore, the method for constructing the Chlamydomonas reinhardtii engineered algal strain with high expression of CrThaumatin / CrBrazzein protein in this invention is as follows:

[0097] The recombinant plasmids containing the sweeteners CrThaumatin and CrBrazzein genes were digested with the restriction endonuclease KpnI and then purified using a purification kit to obtain linearized expression plasmids. Subsequently, the highly efficient gene transfer technique of electroporation was used to introduce the processed expression plasmids into wild-type *Chlamydomonas* sp. cells. After electroporation, the cells were transferred to TAP + 60 mM sorbitol medium for overnight remediation. The remediated algae were then plated on Paro-resistant solid culture plates for initial screening. After a period of culture, 100 single-clone algal strains were randomly selected and cultured in 24-well plates to the logarithmic growth phase. Western blotting was then used for further screening to accurately identify engineered *Chlamydomonas* strains with high expression of CrThaumatin and CrBrazzein proteins.

[0098] The synthesized Chlamydomonas reinhardtii strain expressing CrThaumatin / CrBrazzein proteins was subjected to large-scale fermentation to prepare Chlamydomonas reinhardtii algal powder expressing CrThaumatin / CrBrazzein proteins. Specifically, the strain was expanded on a large scale. The initial strain was inoculated into 1 L Erlenmeyer flasks for pre-culture. After reaching the logarithmic growth phase, it was transferred to a fermentation tank system for approximately 7 days of scale-up fermentation. After fermentation, the algal broth rich in the target sweet proteins was collected and dehydrated using vacuum freeze-drying technology to obtain dry, stable Chlamydomonas reinhardtii algal powder rich in the target sweet proteins CrThaumatin and CrBrazzein. Sensory evaluation experiments were conducted through oral protein purification tests. The results showed that the Chlamydomonas reinhardtii algal powder expressing CrThaumatin and CrBrazzein could elicit a very strong sweet taste sensation, indicating that the expressed sweet proteins not only accumulated in large quantities but also maintained their native conformation and biological activity. This result fully demonstrates that the engineered algal strain constructed using Chlamydomonas reinhardtii as the chassis cell can efficiently express and accumulate functionally active sweet proteins CrThaumatin and CrBrazzein, providing a reliable technical path and material basis for the development of novel sweeteners.

[0099] This invention constructs a complete technical system from gene to product, using Chlamydomonas reinhardtii as a bioreactor to obtain engineered algal strains that can efficiently express the high-sweetness proteins CrThaumatin and CrBrazzein.

[0100] Example 1: Construction of expression plasmids pGM6-CrThaumatin-HA-aph8 and pGM6-CrBrazzein-HA-aph8 for the sweeteners CrThaumatin and CrBrazzein genes

[0101] The construction of the final expression plasmids involves two steps. The first step yields the intermediate plasmid pGM6-H-HA-aph8, and the second step yields the final plasmids pGM6-CrThaumatin-HA-aph8 and pGM6-CrBrazzein-HA-aph8. The specific steps are as follows:

[0102] Step 1: Construction of pGM6-H-HA-aph8 plasmid

[0103] Step 1A: Obtaining the pMO508 vector fragment

[0104] Amplification primers pM-S and pG-AS were designed based on the expression vector. Specific primer information is as follows:

[0105]

[0106] The universal plasmid pMO508 was diluted to 10 ng / µl with ddH2O and used as a template. PCR amplification was performed using the forward primer pM-S and the reverse primer pM-AS to obtain the vector fragment pMO508.

[0107] The PCR system is as follows:

[0108]

[0109] The PCR procedure is as follows:

[0110]

[0111] Step 1B: Obtaining the HA target fragment

[0112] The nucleotide sequence of 3 x HA was submitted to a gene synthesis company, and 3 x HA was synthesized into the universal vector pUC57 to obtain the pUC57-3 x HA plasmid.

[0113] Primers pG-HA-S / AS were designed based on the target gene. Specific primer information is as follows:

[0114]

[0115] The synthesized pUC57-3 x HA plasmid DNA was diluted to 10 ng / µl with ddH2O and used as a template. PCR amplification was performed using the forward primer pG-HA-S and the reverse primer pG-HA-AS to obtain the target gene fragment HA.

[0116] The PCR system is as follows:

[0117]

[0118] The PCR procedure is as follows:

[0119]

[0120] Step 1C: Purification of the target gene fragment HA and the vector using pMO508 gel extraction.

[0121] PCR products containing the target gene and expression vector were separated by 1% agarose gel electrophoresis and analyzed using a Tanon 1600 series gel imaging system. The target band size was found to be within expectations. The target band was then excised from the corresponding location, and DNA recovery and purification were performed using the SanPrep Column DNA Gel Extraction Kit (batch number: J92KA0972), following the kit's instruction manual.

[0122] Step 1D: The target gene fragment HA is reacted with the vector pMO508 to obtain the recombinant plasmid pGM6-H-HA-aph8.

[0123] The target gene fragment HA obtained from gel recovery and the vector fragment pMO508 were reacted with the Novavit Transcript Recombinant Kit ClonExpress II One Step Cloning Kit (lot# C12-01) at 37°C for 30 min. The ligation system is as follows:

[0124]

[0125] Step 1E: Recombinant plasmid pGM6-H-HA-aph8 is transduced into competent E. coli cells.

[0126] Take 50 µl of DH5α E. coli competent cells and thaw them on ice. Add the ligation product DNA obtained in step 1D to the competent cells and incubate on ice for 30 minutes. Then, perform heat shock treatment in a 42°C water bath for 30 seconds, and immediately return to ice and let stand for 2 minutes. Add 500 µl of antibiotic-free LB liquid medium and incubate at 37°C with shaking for 1 hour to achieve cell recovery. Finally, spread the bacterial culture on solid agar plates containing ampicillin (Amp) resistance and incubate upside down at 37°C overnight.

[0127] Step 1F: Screening for positive recombinant colonies

[0128] Remove the plates from the 37°C incubator after the above culture is complete. Using a sterile inoculation needle, select 2 to 3 morphologically distinct single colonies and inoculate them into 5 ml of LB liquid medium containing Amp resistance. Incubate at 37°C with shaking for 12 to 16 hours.

[0129] Step 1G: Extraction of recombinant plasmid pGM6-H-HA-aph8

[0130] The bacterial culture that had been cultured overnight was removed from 37°C and the recombinant plasmids were isolated and purified using the SanPrep Column Plasmid Mini-Preps Kit (batch number: IB23KA7480). The specific operating steps were performed according to the instructions of the kit.

[0131] Step 1H: Sequencing verification of recombinant plasmid pGM6-H-HA-aph8

[0132] The plasmid sample extracted in step 1G was sent to Sangon Biotech for DNA sequencing analysis. The sequencing primers used were P1: gaagcgcgaccacatggtgc. The obtained sequencing results were compared with the expected recombinant plasmid sequence, confirming the successful construction of the intermediate plasmid pGM6-H-HA-aph8.

[0133] Step 2: Construction of the final expression plasmids pGM6-CrThaumatin-HA-aph8 and pGM6-CrBrazzein-HA-aph8.

[0134] Step 2A: Obtaining the CrThaumatin / CrBrazzein target gene fragment

[0135] First, site-directed mutagenesis was performed on the Thaumatin protein sequence to obtain a Thaumatin-encoded protein sequence with arginine at position 30 mutated to lysine (R30K), aspartic acid at position 43 mutated to asparagine (D43N), aspartic acid at position 47 mutated to asparagine (D47N), and arginine at position 51 mutated to lysine (R51K). Simultaneously, the Brazzein protein-encoded sequence was modified to obtain a Brazzein-encoded protein sequence with the first glutamine (ΔQ1) deleted at the N-terminus, arginine at position 33 mutated to lysine (R33K), arginine at position 43 mutated to lysine (R43K), and isoleucine at position 48 mutated to leucine (I48L). Based on the codon bias of *Chlamydomonas reinhardtii*, codon optimization was performed on the two mutated protein sequences to obtain the optimized CrThaumatin and CrBrazzein nucleotide sequences. The comparison results between the codon-optimized sequences and the protein-encoded sequences of natural sweeteners are shown below. Figure 1 Subsequently, the optimized nucleotide sequences were submitted to a gene synthesis company and cloned into the universal vector pUC57, thereby constructing the recombinant plasmids pUC57-CrThaumatin and pUC57-CrBrazzein.

[0136] Primers pGM6-CrThaumatin-S / AS and pGM6-CrBrazzein-S / AS were designed based on the target gene. Specific primer information is as follows:

[0137]

[0138] The synthesized pUC57-CrThaumatin and pUC57-CrBrazzein plasmids were diluted to 10 ng / µl with ddH2O and used as templates. PCR amplification was performed using the forward primer pGM6-CrThaumatin / CrBrazzein-S and the reverse primer pGM6-CrThaumatin / CrBrazzein-AS to obtain the target gene fragments.

[0139] The PCR system is as follows:

[0140]

[0141] The PCR procedure is as follows:

[0142]

[0143] Step 2B: Obtaining the expression vector fragment pGM6

[0144] Amplification primers pGM6-HA-S and pGM6-HA-AS were designed based on the expression vector. Specific primer information is as follows:

[0145]

[0146] The pGM6-H-HA-aph8 plasmid was diluted to 10 ng / µl with ddH2O and used as a template. PCR amplification was performed using the forward primer pGM6-HA-S and the reverse primer pGM6-HA-AS to obtain the expression vector fragment pGM6.

[0147] The PCR system is as follows:

[0148]

[0149] The PCR procedure is as follows:

[0150]

[0151] Step 2C: Gel extraction and purification of the target gene fragment CrThaumatin / CrBrazzein with the expression vector pGM6.

[0152] The PCR products of the target gene and expression vector were electrophoresed on a 1% agarose gel, and then imaged using a Tanon 1600 series multi-functional gel imaging system. The results are as follows: Figure 2 As shown. Observe whether the band size matches the target size, cut out the target band of the corresponding size, and use SanPrep Column DNA Gel Extraction Kit (LOT# J92KA0972) from Sangon Biotech for DNA recovery and purification. Refer to the kit instructions for specific steps.

[0153] Step 2D: The target gene fragment CrThaumatin / CrBrazzein is expressed with the expression vector pGM6 to obtain recombinant plasmids pGM6-CrThaumatin-HA-aph8 and pGM6-CrBrazzein-HA-aph8.

[0154] The target gene fragment obtained from gel extraction and the expression vector fragment were ligated using the Novavit ClonExpress II One Step Cloning Kit (lot# C12-01) at 37°C for 30 min. The ligation system is as follows:

[0155]

[0156] Step 2E: Introduction of recombinant plasmid DNA into E. coli competent cells

[0157] Take 50 µl of DH5⍺ Escherichia coli competent cells and thaw them on ice. Add the ligation product of the target gene fragment and expression vector from step 2D to the competent cells, incubate on ice for 30 min, heat shock at 42℃ for 30 s, place on ice for 2 min, add 500 µl of antibiotic-free LB liquid, recover at 37℃ for 1 h, and then plate on culture plates with Amp resistance and incubate overnight (about 12-16 h).

[0158] Step 2F: Screening for positive plaques

[0159] Remove the culture plate that has been cultured overnight from 37°C, and then pick 2-3 single clones into 5 ml of LB containing Amp resistance, and culture at 37°C overnight (about 12-16 h).

[0160] Step 2G: Extraction of recombinant plasmids pGM6-CrThaumatin-HA-aph8 and pGM6-CrBrazzein-HA-aph8

[0161] Remove the bacterial culture that has been cultured overnight at 37°C and extract plasmids using the SanPrep Column Plasmid Mini-Preps Kit (LOT# IB23KA7480) from Sangon Biotech. For detailed instructions, please refer to the kit manual.

[0162] Step 2H: Sequencing confirmed the recombinant plasmids pGM6-CrThaumatin-HA-aph8 and pGM6-CrBrazzein-HA-aph8.

[0163] The plasmids extracted in step 2G were sent to Sangon Biotech for sequencing. The sequencing primers were p2:ggctcctctgtcgctgtctc. The sequencing results were then compared with the recombinant plasmid sequences to confirm that the pGM6-CrThaumatin-HA-aph8 and pGM6-CrBrazzein-HA-aph8 plasmids were successfully constructed.

[0164] Example 2: Obtaining an engineered algal strain with high expression of the sweeteners CrThaumatin and CrBrazzein proteins from Chlamydomonas reinhardtii

[0165] 1. Preparing to insert a segment

[0166] The pGM6-CrThaumatin-HA-aph8 and pGM6-CrBrazzein-HA-aph8 plasmids successfully constructed in Example 1 were digested with the restriction endonuclease KpnI at 37℃ for 2 h. The digestion products were then subjected to DNA electrophoresis on a 1% agarose gel and imaged using a Tanon 1600 series multi-functional gel imaging analyzer. The results are shown below. Figure 3 As shown. Observe whether the size of the enzyme digested band matches the target size. Cut the target band of the corresponding size and use the SanPrep Column DNA Gel Extraction Kit (LOT# J92KA0972) to recover and purify the DNA. Refer to the kit instructions for specific steps.

[0167] The enzyme digestion system is as follows:

[0168]

[0169] 2. Chlamydomonas competent cell culture

[0170] Wild-type algal strain (Chlamydomonas sp.) was inoculated from TAP solid plates into TAP liquid medium and cultured under continuous light with aeration for 3-4 days until the cell concentration reached 1×10⁻⁶. 7 Cells / ml. Take 10 ml of the Chlamydomonas cell suspension and transfer it to an Erlenmeyer flask containing 100 ml of TAP liquid medium to adjust the initial cell concentration to 1×10⁻⁶ cells / ml. 6 Cells / ml. The Erlenmeyer flask was then placed on a shaker (200 rpm) and cultured under continuous light for approximately 20 h until the Chlamydomonas cell concentration reached 5 × 10⁻⁶ cells / ml. 6 When the cell / ml ratio is reached, electroporation transformation experiments should be carried out immediately.

[0171] 3. Electroporation transformation of Chlamydomonas reinhardtii cells to obtain transformants

[0172] (1) Chlamydomonas cell resuspension and preparation:

[0173] Collect *Chlamydomonas reinhardtii* cells using 50 ml sterile centrifuge tubes. Centrifuge at 2500 rpm for 3 min at room temperature. Resuspend the precipitate in 1 ml of pre-chilled TAP + 60 mM sorbitol solution. Then, bring the volume to 15 ml with pre-chilled TAP + 60 mM sorbitol solution. Centrifuge at 2500 rpm for 3 min at room temperature. Discard the supernatant. Resuspend the cells again with an appropriate amount of pre-chilled TAP + 60 mM sorbitol solution, adjusting the final concentration of *Chlamydomonas reinhardtii* cells to approximately 1 - 2 × 10⁻⁶ cells / ml. 8 Take 10 cells and incubate the cell suspension on ice for 10 minutes.

[0174] (2) Cleaning and pre-cooling of the electric shock cup:

[0175] To ensure the efficiency of electroporation and maintain a sterile environment, all electroporation cups were cleaned in a laminar flow hood. The specific steps were as follows: First, the electroporation cups were rinsed once with anhydrous ethanol to remove trace organic matter and quickly evaporate and dry them; then, they were rinsed three times with a TAP solution containing 60 mM sorbitol to ensure that the cup wall environment was consistent with the osmotic pressure conditions of the subsequent conversion experiments. The treated electroporation cups were then placed at -20°C for pre-cooling.

[0176] (3) Preparation of the electroshock mixture:

[0177] Take 250 μl of a concentration of 1 - 2 × 10⁻⁶. 8 Add 100-150 ng of linearized pGM6-CrThaumatin-HA-aph8 or pGM6-CrBrazzein-HA-aph8 expression plasmid fragments to a pre-chilled electroporation cuvette, and then gently pipette to mix the cells with the expression vector fragment DNA. Place the mixture on ice for 10 min.

[0178] (4) Electric shock operation:

[0179] Adjust the parameters of the stun gun (model BTX ECM630): set the voltage to 800 V, resistance to 1575 Ω, and capacitance to 50 μF. Dry the stun gun cup with absorbent paper, ensuring the sides with the metal strip are firmly in contact with the stun gun's clamps. Lower the safety cover of the stun gun and start the stun program. The stun time must be strictly controlled between 10-14 ms. After the stun, immediately return the stun gun cup to the ice and leave it for 10 minutes.

[0180] (5) Obtaining the transformant

[0181] The Chlamydomonas cells from the electroporation cuvette were transferred to a 50 ml sterile centrifuge tube containing 10 ml of TAP + 60 mM sorbitol solution. The centrifuge tube was then wrapped in aluminum foil and placed on a shaker at 100 rpm under low light conditions for overnight repair culture. The next day, the cells were centrifuged at 2500 rpm for 3 min at room temperature to collect the overnight repaired Chlamydomonas cells. While collecting the cells, a cornstarch suspension was prepared in advance: an appropriate amount of cornstarch was washed once with anhydrous ethanol, then three times with sterile ddH2O, followed by four times with TAP + 60 mM sorbitol solution. Finally, the washed cornstarch was resuspended in TAP + 60 mM sorbitol solution to prepare a 20% cornstarch solution. After cell collection, 1 ml of the prepared 20% cornstarch solution was added, and the cells were gently aspirated with a pipette to thoroughly mix with the starch solution. The mixed cell suspension was evenly spread on a paromomycin (Paro) resistant TAP solid plate and placed in a clean bench. After the surface liquid dried naturally, the plate was sealed with sealing film. The plate was then inverted and incubated under photoperiod conditions. Transformants will grow on the plate after approximately 4-5 days of incubation.

[0182] 4. Screening of engineered algal strains with high expression of sweeteners CrThaumatin and CrBrazzein proteins

[0183] (1) Sample collection

[0184] After electroporation and subsequent culture, the transformants on the plates gradually grow to a healthy state. At this point, using aseptic techniques, a sterile toothpick or inoculation loop is used to precisely pick out a single, clearly defined transformant clone and transfer it to the surface of a freshly prepared antibiotic-free TAP solid medium plate. During this process, clear and accurate numbering should be made on the edge of the plate or the corresponding record sheet for later traceability and experimental procedures. Place the plate with the inoculated clone in a suitable culture environment. Once the newly inoculated transformants have grown sufficiently and stabilized on the antibiotic-free TAP solid medium, the transfer operation is performed again. Use a sterile toothpick to pick the transformants into 24-well plates containing 1.5 ml of TAP medium, with one transformant number per well. Place the 24-well plate in a temperature-controlled shaker at a suitable rotation speed (e.g., 200 rpm) and culture under photoperiodic conditions for 2-3 days to allow the Chlamydomonas reinhardtii cells to proliferate sufficiently in the liquid medium.

[0185] After the culture cycle was completed, the Chlamydomonas cells in the 24-well plates were collected. The cell suspension was carefully aspirated using a pipette, ensuring that each 1.5 ml EP tube contained 1 × 10⁶ cells. 7Place each EP tube in a centrifuge and centrifuge at 10,000 rpm for 1 min at room temperature. After centrifugation, carefully remove the EP tube and aspirate the supernatant using a pipette, avoiding contact with any cells that have settled at the bottom of the tube. Then, quickly place the EP tube in liquid nitrogen and freeze at -80°C to maintain the biological characteristics and protein expression state of the cells.

[0186] (2) Sample preparation

[0187] The pre-collected *Chlamydomonas reinhardtii* samples were retrieved using liquid nitrogen, and each sample was placed on ice to maintain a consistently low-temperature environment. Then, 100 μl of cell lysis buffer A (containing protease inhibitors) was added to each sample, and the mixture was gently pipetted to ensure complete cell lysis while strictly avoiding vigorous shaking to prevent non-specific protein denaturation. After all samples had undergone the above lysis steps, protein denaturation was performed. 50 μl of 2×SDS protein loading buffer was added to each sample, and the mixture was then thoroughly vortexed to ensure complete protein binding to SDS. The samples were then placed in a 100°C metal bath or water bath and incubated for 10 minutes to ensure complete protein denaturation. After denaturation, the samples were immediately transferred to ice and rapidly cooled for 2 minutes. Finally, the sample was briefly centrifuged (approximately 10,000 × g, 30 seconds) to collect the condensate from the tube wall and settle any trace amounts of insoluble matter. The resulting supernatant was the total protein sample, which was stored at -20°C for long-term use in subsequent Western blotting and other analyses.

[0188] (3) Western Blot assay

[0189] 1) Gel Preparation: Prepare a 15% separating gel based on the molecular weight of the target protein. Add the various gel-preparing reagents to a centrifuge tube according to the specified ratio, and gently shake the solution 8 to 10 times to ensure thorough mixing. Then, use a pipette to smoothly pour the separating gel solution between two glass plates along one side, avoiding the formation of air bubbles. Next, slowly add a layer of distilled water on top of the liquid surface to create a liquid seal, expel air, and flatten the gel surface. Let it stand at room temperature for 20 to 30 minutes to allow for complete polymerization. After the separating gel has solidified, prepare the stacking gel using the same method. Discard the distilled water layer on top of the separating gel, and pour the stacking gel solution into the glass plate interlayer using a continuous and steady flow. Then, slowly insert a comb into the gel solution at a certain angle, carefully adjusting the position of the comb teeth to ensure that no air bubbles remain at the tips. Finally, let it stand for at least 30 minutes until the stacking gel has completely polymerized before use.

[0190] 2) Sample Loading: After carefully removing the comb, mount the gel in the electrophoresis tank and inject sufficient electrophoresis buffer. Then, quickly add the protein marker and the sample to be analyzed to the sample wells in sequence. To reduce sample diffusion and edge effects, the sample loading operation should be completed quickly, and the same amount of sample buffer should be added to the blank sample wells.

[0191] 3) Electrophoresis: SDS-PAGE electrophoresis is performed under constant voltage conditions. The stacking gel voltage is typically set to 80 V, and then adjusted to 120 V after the sample enters the separating gel. Electrophoresis continues until the bromophenol blue indicator front reaches the bottom edge of the gel, at which point electrophoresis is stopped.

[0192] 4) After electrophoresis, cut a gel within the desired molecular weight range. Assemble the transfer jacket in transfer buffer in the following order: cathode plate - sponge - filter paper - gel - PVDF membrane - filter paper - sponge - anode plate. Ensure there are no air bubbles between layers and that the membrane and gel are correctly positioned. Place the transfer jacket into the transfer tank and add pre-cooled transfer buffer. Transfer at a constant current of 350 mA for 60 min under ice bath conditions.

[0193] 5) After the transfer is complete, remove the PVDF membrane and mark the band positions according to the pre-stained marker. Then immerse the membrane in 10 mL of 5% skim milk blocking solution and block it on a shaker at room temperature for 1 hour.

[0194] 6) Primary antibody hybridization: Dilute the primary antibody HA 1:2000 with 3% skim milk-TBST solution, place the membrane in the solution, and incubate on a shaker at room temperature for 1 hour. Then wash the membrane 3 times with TBST buffer on a shaker for 5 minutes each time.

[0195] 7) Secondary antibody hybridization: The HRP-labeled secondary antibody Mouse was diluted 1:5000 with 3% skim milk-TBST and incubated on a shaker at room temperature for 1 h. The membrane was then washed 3 times with TBST for 5 min each time.

[0196] 8) Mix Aibotek ECL hypersensitive luminescent liquid A and B in a 1:1 volume ratio, evenly cover the membrane surface, incubate at room temperature for 2 minutes, and then place the membrane in the Tianneng chemiluminescence imager for exposure and image acquisition.

[0197] Through systematic analysis of the immunoblotting results, we compared the bands of engineered algal strains with different numbering. For example... Figure 4As shown, clear and strong specific bands were observed at 26 kDa and 10 kDa (CrThaumatin approximately 26 kDa, CrBrazzein approximately 10 kDa), and the size of these bands was completely consistent with the theoretical molecular weight of the target sweet proteins. Based on both band intensity and molecular weight position, we successfully screened a Chlamydomonas reinhardtii engineered algal strain capable of high-level and stable expression of the CrThaumatin and CrBrazzein sweet proteins from a large number of transformants. Protein purification of the engineered algal strains expressing high levels of CrThaumatin and CrBrazzein proteins was performed, and oral administration validation showed that their sweetness was 3-5 times that of the natural sweeteners Thaumatin and Brazzein. The comparison results are shown below. Figure 5 As shown.

[0198] Example 3: Large-scale fermentation culture of engineered algal strains with high expression of sweeteners CrThaumatin and CrBrazzein proteins:

[0199] 1. The engineered algal strains that have been validated by Western blotting and can efficiently express CrThaumatin and CrBrazzein were streaked onto brand new TAP solid agar plates using a sterile inoculation loop for activation and expansion. The inoculated plates were placed in a photoperiod and cultured for 3 to 4 days under suitable temperature (e.g., 25°C) and standard photoperiod (16 hours light / 8 hours dark).

[0200] 2. The engineered algal strains that have been activated and grown well on TAP plates were aseptically inoculated into 1 L Erlenmeyer flasks containing 500 mL of sterile TAP liquid medium. The flasks were then placed in a photoperiodic shaker incubator and cultured at 25°C, a photoperiod of 16 h light / 8 h dark, and a constant rotation speed of 200 rpm. By periodically monitoring the algal cell density, a highly active, high-density seed culture was obtained when the algae reached the logarithmic growth phase, which was then used for inoculation in subsequent fermenters.

[0201] 3. Add 3 L of prepared culture medium to the 5 L fermenter body. Simultaneously, dispense 1 L of concentrated feed medium into 1 L blue-capped bottles, and prepare 100 mL of defoaming agent separately. Then, place the fermenter (including the culture medium), feed bottles, and defoaming agent together in a high-temperature autoclave for in-situ sterilization using saturated steam. Set the sterilization conditions to 121°C and maintain for 20 minutes to ensure all components reach a sterile state. After sterilization, allow the temperature and pressure to naturally drop to a safe range before removing the system and proceeding with subsequent connections and fermentation operations.

[0202] 4. After the fermenter, feed solution, and defoamer have been sterilized by high temperature and high pressure and naturally cooled to room temperature, they are transferred to a sterile operating area. Subsequently, in the vicinity of the sterile environment created by an alcohol lamp, the pre-prepared high-density seed solution is transferred to the fermenter at a volume ratio of 10%, ensuring that the entire inoculation process is completed under strict aseptic conditions.

[0203] 5. A precise environmental control strategy is employed during the fermentation process: the culture temperature is maintained at a constant 24℃, and the pH of the culture medium is kept at 7.7. Before and after inoculation, 100% saturated dissolved oxygen is measured using a calibrated dissolved oxygen electrode to obtain accurate baseline values. Based on this, an automatic linkage control system adjusts the stirring speed and aeration rate in real time to ensure that the dissolved oxygen concentration is consistently maintained within the optimized range that meets the needs of high-speed algal cell metabolism and product synthesis. Through the coordinated and precise control of these three key parameters—temperature, pH, and dissolved oxygen—the optimal physiological environment is created for the high-density cultivation of *Chlamydomonas reinhardtii* engineered algal strains and the efficient synthesis of the target sweet protein.

[0204] 6. After approximately 7 days of fermentation, when monitoring indicators show that the algal biomass no longer increases significantly and has entered a stable growth plateau, the fermentation process is terminated. Subsequently, all fermentation broth is transferred out of the tank and immediately centrifuged at 4000 rpm for 5 minutes at room temperature to achieve efficient separation of algal cells (algal sludge) from the fermentation supernatant. After discarding the supernatant, the resulting wet algal sludge is evenly spread in a freeze-drying tray and dehydrated using a vacuum freeze dryer. Figure 6 As shown, this process successfully removed most of the moisture from the algal sludge, ultimately yielding a loosely structured, easily stored dried algal powder. This algal powder product was stored long-term at 4°C for subsequent analysis and application.

[0205] This invention provides an innovative system for screening recombinant proteins based on *Chlamydomonas reinhardtii* as the chassis cell. *Chlamydomonas reinhardtii* is a single-celled eukaryotic photosynthetic microorganism widely distributed in freshwater, soil, and marine environments worldwide. Its cells are approximately 5-10 micrometers in diameter and move via two flagella of equal length. Despite its relatively simple structure, its cells contain a complete set of eukaryotic organelles, including a large chloroplast, a typical endoplasmic reticulum, and a Golgi apparatus, enabling it to perform complex functions and become a highly efficient "cell factory."

[0206] The core advantages of the Chlamydomonas reinhardtii chassis-based recombinant protein engineered algal strain screening provided by this invention are reflected in the following aspects:

[0207] 1. Compared to animal / insect cell systems: Chlamydomonas reinhardtii, as a photosynthetic autotroph, requires only light, water, inorganic salts, and carbon dioxide for basic growth, resulting in extremely low culture medium costs. This system is easily capable of large-scale, high-density cultivation in open ponds or closed photobioreactors, exhibiting excellent industrial scalability and controllability.

[0208] 2. Compared to bacterial and yeast systems, *Chlamydomonas reinhardtii* possesses a complete eukaryotic protein synthesis and processing pathway, capable of mediating the correct folding of recombinant proteins and necessary post-translational modifications (such as N-linked glycosylation, O-linked glycosylation, and disulfide bond formation), thereby producing fully biologically active recombinant proteins. Furthermore, this system does not produce endotoxins, does not carry known human pathogens, and its cell wall effectively blocks infection by animal viruses, providing an inherently safe biomanufacturing platform for the production of pharmaceutical proteins and vaccines.

[0209] 3. Compared to plant expression systems, *Chlamydomonas reinhardtii* has an extremely short culture cycle, supporting rapid iteration and high-throughput screening. Cultured in a controlled environment, it ensures stability and consistency between production batches, and the entire process does not occupy arable land resources, eliminating the environmental risk of genetically modified organisms spreading through pollen.

[0210] 4. Unique Advantages as an Oral Delivery System: Another key innovation of this invention lies in utilizing Chlamydomonas reinhardtii cells themselves as "biocapsules" for oral drugs. Their cell walls provide effective physical protection for intracellularly expressed recombinant proteins, delaying their degradation by gastric acid and intestinal enzymes, thereby achieving targeted release of the recombinant proteins at the intestinal immune effector sites. Chlamydomonas reinhardtii itself is safe and edible, giving this chassis a natural advantage in oral drug delivery.

[0211] In summary, the Chlamydomonas reinhardtii chassis provided by this invention integrates the model value of basic research with the application potential of biotechnology. This system offers a highly competitive and innovative technological path for achieving low-cost, high-safety, and sustainable production and delivery of recombinant proteins, vaccines, and biopharmaceutical products, and has broad application prospects in the fields of industrial biomanufacturing and preventive medicine.

[0212] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A CrThaumatin protein, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

3.

2. A CrBrazzein protein, characterized in that: The nucleotide sequence is shown in SEQ ID NO.4.

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