A method for constructing and applying an engineered algal strain of high-concentration sweetness-inducible protein variant derived from Chlamydomonas reinhardtii.

Abstract

This invention discloses a method for constructing an engineered algal strain of high-concentration sweetness-inducing protein variants from *Chlamydomonas reinhardtii* and its applications. First, the protein coding sequences of the sweetness-inducing proteins Miraculin and Neoculin are modified and optimized to obtain optimized CrMiraculin and CrNeoculin gene sequences. These sequences are then cloned into the expression vector pGM6 to construct a recombinant expression vector. This recombinant expression vector is introduced into wild-type *Chlamydomonas reinhardtii* strains, and the transformed algal strains are selected using a paromomycin resistance marker carried by the vector. The obtained engineered algal strains can stably inherit and efficiently express the target sweetness-inducing proteins. The engineered algal strains are then fermented to obtain *Chlamydomonas reinhardtii* algal powder rich in CrMiraculin and CrNeoculin sweetness-inducing proteins. Finally, after protein purification, mixing with acidic substances can induce a sweet taste, thus demonstrating significant application potential in the food, condiment, or health product fields.

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 a high-sweetness-inducible protein variant derived from Chlamydomonas reinhardtii and its application. Background Technology

[0002] Sweetness is a widely enjoyed taste sensation, but the application of traditional carbohydrate sweeteners, represented by sugars, is increasingly limited due to the health risks of high blood sugar, type 2 diabetes, and obesity associated with excessive intake. While the food industry has attempted to use low-molecular-weight sugars, sugar alcohols, or artificial sweeteners as alternatives as consumer preferences shift towards healthier, lower-energy, and lower-sugar options, these traditional substitutes generally have lower sweetness levels, higher calorie values, or potential risks of cardiovascular disease, obesity, diabetes, and tooth decay, failing to fully meet health demands. Therefore, developing new sugar-free, high-sweetness, safe, and natural sweeteners has become a critical issue that urgently needs to be addressed in food science and the health industry.

[0003] Natural sweeteners are a class of protein-based substances that impart a sweet taste to food or feed, possessing characteristics such as low calories, high sweetness, high biocompatibility, and no toxicity. Currently, sweeteners are widely used globally, with over twenty types commonly found in the international market. In China, with the continuous development of the food industry and the increasing demand for food safety and nutritional health, approximately fifteen types of sweeteners have been approved for use, making it one of the world's largest categories of food additives, widely used in pastries, beverages, preserved fruits, and many other foods. Compared to traditional sugar-based sweeteners, these protein-based sweeteners, with their low calories, high sweetness, and good biocompatibility, show broad application prospects. Against the backdrop of increasing health awareness, these sweeteners are expected to develop into novel health-functional sweetener ingredients, providing technological innovation directions for the food and feed industries and helping the public achieve healthy dietary goals while pursuing delicious experiences.

[0004] Natural plant sweet proteins are a class of special proteins derived from specific plants that can impart or trigger the perception of sweetness. These proteins are mainly distributed in specific shrubs in subtropical regions such as West Africa and Malaysia, with their berries, fruits, and arils being the primary sources. Currently, eight main types of plant sweet proteins have been discovered, which can be divided into three categories according to their mechanisms of action: Thaumatin, Monellin, Mabinlin, Brazzein, and Pentadin are sweet proteins; Miraculin and Neoculin are sweetness-inducing proteins, possessing the ability to convert sour tastes into sweet tastes; while Curculin possesses both sweetness-imparting and sweetness-inducing properties.

[0005] Currently, health issues are increasingly attracting global attention. The incidence of diabetes continues to rise, cardiovascular disease has become one of the leading causes of death, and various chronic diseases pose a persistent threat to public health. At the same time, the number of obese people is showing a significant upward trend globally. Numerous studies and statistics indicate that the occurrence of these health problems is closely related to daily dietary structure, with high sugar intake widely considered a key risk factor. Against this backdrop, the market demand for low-calorie, low-sugar healthy foods is becoming increasingly urgent. Natural sweeteners, as ideal substitutes for sucrose, have attracted much attention due to their unique advantages. These substances not only possess good functional activity, effectively imparting sweetness to food and improving its flavor quality, but can also be degraded into various essential amino acids in the body by pepsin, providing nutritional supplementation while satisfying taste enjoyment, combining safety and health value. However, currently known natural sweet proteins mainly come from specific shrub plants, which are generally highly dependent on the climate, soil, and other habitat conditions of their native habitat, making large-scale cultivation in different locations difficult. This geographical limitation severely restricts the direct extraction of sweet proteins from plant fruits and seeds, thus limiting the stable supply and widespread application of natural sweet proteins.

[0006] In the current technological context, the use of genetic engineering to modify and screen sweeteners has become a focal point of research and industry. This technology can effectively overcome the dependence of natural plants on specific growing regions by introducing the target sweetener encoding gene into easily scalable microbial or plant expression systems, achieving efficient and stable production of sweet proteins. This approach not only helps meet the growing market demand for low-sugar, healthy sweeteners but also provides new possibilities for innovative development in the food, pharmaceutical, and other related industries.

[0007] Currently, the production of recombinant proteins mainly relies on the following expression systems: mammalian cells, insect cells, microbial systems (such as Escherichia coli and yeast), and higher plants. However, all of these systems have inherent limitations: mammalian and insect cell systems are not only costly to cultivate, but also carry the risk of contamination by animal-derived pathogens; prokaryotic systems such as E. coli lack eukaryotic-specific protein post-translational modification mechanisms and are prone to producing endotoxins; although yeast is a eukaryotic expression system, its glycosylation pattern differs from that of humans, which may affect the applicability of recombinant proteins; and higher plant systems face challenges such as long growth cycles, susceptibility to environmental and seasonal constraints, and potential ecological safety issues such as the spread of transgenic pollen. Therefore, developing a novel expression platform that combines cost advantages, high biosafety, and eukaryotic protein processing capabilities has become an urgent technical problem to be solved in this field.

[0008] *Chlamydomonas reinhardtii* is a single-celled eukaryotic photosynthetic microorganism widely distributed in various natural environments, including freshwater, soil, and ocean. The algae's cells are approximately 5-10 micrometers in diameter and possess two flagella of equal length. While its cellular structure is relatively simple, it possesses a complete eukaryotic organelle system, including prominent large cup-shaped chloroplasts, a typical endoplasmic reticulum, and a Golgi apparatus. This allows it to perform complex post-translational modifications of eukaryotic proteins and achieve efficient expression of exogenous proteins. Furthermore, *Chlamydomonas reinhardtii* offers advantages such as low cultivation cost, short growth cycle, and no need for arable land, making it an ideal chassis system for recombinant protein expression. Summary of the Invention

[0009] The purpose of this invention is to provide a method for constructing an engineered algal strain of high-sweetness-inducible protein variant derived from Chlamydomonas reinhardtii and its application, which has the advantages of high production efficiency, good safety and low cost.

[0010] To achieve the above objectives, this application adopts the following technical solution:

[0011] In a first aspect, the present invention provides a method for constructing the recombinant plasmid pGM6-CrMiraculin-HA-aph8 / pGM6-CrNeoculin-HA-aph8, comprising the following steps:

[0012] Step 1, construction of the pGM6-H-HA-aph8 plasmid, includes the following steps:

[0013] Step 11: Submit the nucleotide sequence of 3 x HA to a gene synthesis company, synthesize it into the universal vector pUC57, and obtain the pUC57-3 x HA plasmid. Use primers pG-HA-S / AS to amplify the HA fragment by PCR.

[0014] The nucleotide sequence of the HA is shown in SEQ ID NO.1, and the nucleotide sequences of the primer pG-HA-S / AS are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0015] Step 12: Use primers pM-S / AS to perform PCR amplification on the universal vector pMO508 to clone and obtain the target vector fragment.

[0016] The nucleotide sequences of the primer pM-S / AS are shown in SEQ ID NO.4 and SEQ ID NO.5;

[0017] Step 13: The HA fragment and the target vector fragment are analyzed by agarose gel electrophoresis, and purified and recovered using a gel recovery kit;

[0018] Step 14: Mix the purified HA fragment with the target vector fragment at the optimal molar ratio, and perform homologous recombination ligation in the presence of recombinase to obtain a recombinant plasmid.

[0019] Step 15: The recombinant plasmid was introduced into Trans5 α Escherichia coli competent cells, placed on ice, then subjected to heat shock treatment and transferred to ice for static incubation; then antibiotic-free LB liquid medium was added and cultured with shaking; the revived bacterial culture was spread on LB agar plates containing Amp resistance and incubated at constant temperature.

[0020] Step 16: Randomly select several single colonies with regular morphology and good separation on the plate, and inoculate them into LB liquid medium containing Amp. Shake and culture until the bacterial culture reaches the stationary phase.

[0021] Step 17: Use a plasmid micro-extraction kit to extract the recombinant plasmid from the amplified bacterial culture, and verify it by sequencing to obtain the pGM6-H-HA-aph8 plasmid.

[0022] Step 2, the construction of recombinant plasmids pGM6-CrMiraculin-HA-aph8 / pGM6-CrNeoculin-HA-aph8, includes the following steps:

[0023] Step 21: First, site-directed mutagenesis was performed on the Miraculin protein sequence to obtain the Thaumatin-encoding protein sequence with isoleucine at position 39 mutated to leucine, isoleucine at position 52 mutated to leucine, histidine at position 59 mutated to lysine, and glutamine at position 82 mutated to leucine. Simultaneously, the Neoculin protein-encoding sequence was modified. Neoculin is a dimer composed of A and B chains. First, tyrosine at position 9 of chain A was mutated to lysine, and threonine at position 43 was mutated to aspartic acid to obtain the mutated A chain. Then, isoleucine at position 25 of chain B was mutated to leucine, tryptophan at position 41 was mutated to serine, and glycine at position 60 was mutated to asparagine to obtain the mutated B chain. The A and B chains were then linked together to obtain the mutated chain. Based on the codon bias of Chlamydomonas reinhardtii, the two mutant protein sequences encoded by Neoculin were codon optimized to obtain optimized CrMiraculin and CrNeoculin nucleotide sequences. Subsequently, the optimized nucleotide sequences were cloned into the universal vector pUC57 to construct the recombinant plasmid pUC57-CrMiraculin / pUC57-CrNeoculin. Using the constructed recombinant plasmid as a template, the target gene fragments of CrMiraculin and CrNeoculin were obtained by PCR amplification using specific primers pGM6-CrMiraculin-S / AS or pGM6-CrNeoculin-S / AS.

[0024] The nucleotide sequences of the pGM6-CrMiraculin-S / AS are shown in SEQ ID NO.6 and SEQ ID NO.7, and the nucleotide sequences of the pGM6-CrNeoculin-S / AS are shown in SEQ ID NO.8 and SEQ ID NO.9.

[0025] Step 22: Using the pGM6-H-HA-aph8 plasmid prepared in step 1 as a template, the expression vector fragment pGM6 is obtained by PCR amplification using primers pGM6-HA-S / AS.

[0026] The nucleotide sequences of the pGM6-HA-S / AS are shown in SEQ ID NO.10 and SEQ ID NO.11;

[0027] Step 23: Separate the CrMiraculin / CrNeoculin target gene fragment and the expression vector fragment pGM6 by agarose gel electrophoresis, and cut the gel region containing the target band under ultraviolet light to purify and recover the corresponding DNA fragment;

[0028] Step 24: The purified CrMiraculin / CrNeoculin target gene fragment is mixed with the purified expression vector fragment pGM6 at the optimized molar ratio, and recombinase is added for homologous recombination ligation to obtain recombinant plasmids;

[0029] Step 25: The recombinant plasmid is introduced into Trans5⍺ Escherichia coli competent cells, placed on ice to allow the DNA to fully contact the cells, and then subjected to heat shock followed by ice recovery treatment.

[0030] Step 26: Add LB medium without resistance, shake and incubate, take an appropriate amount of the recovered bacterial culture and spread it on LB agar plates containing Amp resistance, and incubate overnight;

[0031] Step 27: Randomly select several single colonies that grow well and have regular morphology on the plate, and inoculate them into LB liquid medium containing Amp. Shake and culture until the bacterial culture reaches the stationary phase.

[0032] Step 28: Use a plasmid micro-extraction kit to extract recombinant plasmids from the propagation bacterial culture, and sequence them for verification. The recombinant plasmids pGM6-CrMiraculin-HA-aph8 / pGM6-CrNeoculin-HA-aph8 were successfully constructed.

[0033] Secondly, the present invention provides recombinant plasmids pGM6-CrMiraculin-HA-aph8 / pGM6-CrNeoculin-HA-aph8, which are constructed using the above-described construction method.

[0034] Thirdly, this invention provides a method for constructing an engineered Chlamydomonas strain with high expression of CrMiraculin / CrNeoculin sweet-inducible protein. The recombinant plasmid pGM6-CrMiraculin-HA-aph8 / pGM6-CrNeoculin-HA-aph8 is linearized by single-enzyme digestion with the restriction endonuclease ScaI, and the linearized plasmid is obtained after purification using a purification kit. The linearized plasmid is introduced into wild-type Chlamydomonas sp. cells via electroporation. After transformation, the algae are transferred to TAP medium containing sorbitol and cultured overnight under low light conditions to complete cell repair. The repaired algal solution is spread on Paro-containing solid selective medium for preliminary resistance screening. After culture, multiple single-clone algal strains are randomly selected and inoculated into well plates, and cultured to the logarithmic growth phase. The protein expression level of the obtained algal strains is detected by Western blotting, and finally, the engineered Chlamydomonas strain with high expression of CrMiraculin / CrNeoculin sweet-inducible protein is obtained.

[0035] Fourthly, this invention provides an engineered Chlamydomonas reinhardtii strain that highly expresses CrMiraculin / CrNeoculin sweetness-inducing proteins, which is constructed using the above-described method.

[0036] Fifthly, the present invention provides Chlamydomonas reinhardtii algal powder with high expression of CrMiraculin / CrNeoculin sweetness-inducing protein, which is obtained by large-scale fermentation production of the above-mentioned Chlamydomonas reinhardtii engineered algal strain with high expression of CrMiraculin / CrNeoculin protein.

[0037] Sixthly, the present invention provides the application of Chlamydomonas reinhardtii algal powder with high expression of the above-mentioned CrMiraculin / CrNeoculin sweetness-inducing protein as a natural raw material with flavor-modifying function.

[0038] In a seventh aspect, the present invention provides the application of Chlamydomonas reinhardtii algal powder with high expression of the above-mentioned CrMiraculin / CrNeoculin sweetness-inducing protein as a sweetness inducer.

[0039] Eighthly, this invention provides the application of Chlamydomonas reinhardtii algal powder with high expression of the above-mentioned CrMiraculin / CrNeoculin sweetness-inducing protein in food processing, beverage manufacturing, pharmaceutical preparations and cosmetic development.

[0040] The beneficial effects of this invention are as follows:

[0041] 1) The Chlamydomonas reinhardtii chassis cell expression system developed in this invention provides an innovative technical pathway for the production of recombinant protein drugs. Compared with other existing synthetic biology chassis systems, the Chlamydomonas reinhardtii system exhibits several comprehensive advantages: it possesses complete post-translational modification capabilities for eukaryotic proteins, ensuring the bioactivity of the protein products; its photosynthetic autotrophic characteristics significantly reduce cultivation costs and it does not rely on arable land resources, making it easy to achieve large-scale and controllable production; as a recognized safe microorganism, it has no risk of endotoxin or animal-derived pathogen contamination, resulting in high biosafety; and it also features a clear genetic background, short growth cycle, and good batch-to-batch stability, providing an ideal technical platform for the efficient, safe, and economical preparation of recombinant protein drugs.

[0042] 2) The Chlamydomonas reinhardtii engineered algal strain screening system provided by this invention has outstanding advantages such as simple operation, high efficiency and wide applicability. It can quickly and accurately screen and obtain engineered algal strains that highly express the target recombinant protein, showing significant technical advantages and application potential in the large-scale screening and preparation of highly efficient algal strains.

[0043] 3) The Chlamydomonas reinhardtii chassis cell screening system developed in this invention exhibits significant advantages over traditional microbial chassis such as bacteria and yeast in terms of biosafety and public acceptance. While bacterial systems are characterized by rapid growth and ease of operation, their fermentation process carries the risk of phage contamination, and the expressed proteins may carry endotoxins. Yeast systems, on the other hand, may suffer from the accumulation of metabolic byproducts such as ethanol, which can affect the activity and yield of the target protein. The Chlamydomonas reinhardtii system not only effectively avoids these risks but also, due to its photosynthetic autotrophy, absence of animal-derived pathogens, and lack of endotoxin production, aligns better with green and safe production principles in fields with stringent safety requirements, such as industrial production, biomedicine, and food. Therefore, it possesses greater market acceptance potential and application promotion value. Attached Figure Description

[0044] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be noted that the accompanying drawings are merely schematic illustrations, used to help illustrate the technical solutions and preferred embodiments of the present invention, and do not constitute a limitation on the scope of protection of the technical solutions of the present invention. Within the scope defined by the claims of the present invention, any equivalent transformations or modifications based on the principles of the present invention should be considered to fall within the protection scope of the present invention.

[0045] Figure 1 The diagram shows a comparison between the coding sequences of CrMiraculin and CrNeoculin sweet-tasting proteins after mutation and codon optimization by Chlamydomonas reinhardtii in this invention and their corresponding natural sequences.

[0046] Figure 2The image shown is an identification map of the PCR amplification products of the gene fragments encoding the sweetness-inducing proteins CrMiraculin and CrNeoculin and their corresponding expression vectors, verified by agarose gel electrophoresis.

[0047] Figure 3 The image shown is an agarose gel electrophoresis pattern of the recombinant plasmids pGM6-CrMiraculin-HA-aph8 and pGM6-CrNeoculin-HA-aph8 constructed in this invention after linearization with ScaI restriction endonuclease.

[0048] Figure 4 The figure shown is a detection diagram of the expression of sweetness-inducible protein in Chlamydomonas reinhardtii in an embodiment of the present invention, which is verified by Western blotting technology. It shows the expression characteristics of the target protein in the transformed algal strain after the recombinant expression plasmids pGM6-CrMiraculin-HA-aph8 (4A) and pGM6-CrNeoculin-HA-aph8 (4B) were introduced into wild-type Chlamydomonas reinhardtii sp. by electroporation.

[0049] Figure 5 The diagram shows the induction of sweetness by the sweetness-inducing proteins CrMiraculin and CrNeoculin of the present invention under acidic conditions in the presence of vinegar (5A) and lemon juice (5B).

[0050] Figure 6 The image shown is a physical representation of the Chlamydomonas reinhardtii algal powder rich in sweetness-inducing proteins CrMiraculin (6A) and CrNeoculin (6B) prepared according to the present invention. Detailed Implementation

[0051] 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.

[0052] 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.

[0053] 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.

[0054] This invention discloses a gene synthesis method, recombinant protein expression system, and applications of the sweetness-inducing proteins CrMiraculin and CrNeoculin derived from *Chlamydomonas reinhardtii*. CrMiraculin and CrNeoculin proteins possess the function of regulating taste perception in acidic environments, effectively reducing sour taste perception and converting sour tastes into sweet tastes. *Chlamydomonas reinhardtii* is a recognized safe model photosynthetic microorganism, and the recombinant proteins expressed by it have significant advantages in terms of biosafety and public acceptance, providing an environmentally friendly and sustainable biomanufacturing pathway for sweetness inducers.

[0055] This invention mutates the coding sequences of the sweetness-inducing proteins Miraculin and Neoculin. For the Miraculin protein sequence, isoleucine at position 39 is mutated to leucine, isoleucine at position 52 is mutated to leucine, histidine at position 59 is mutated to lysine, and glutamine at position 82 is mutated to leucine. Simultaneously, for the Neoculin protein coding sequence, Neoculin is a dimer composed of A and B chains. The mutated A chain is obtained by mutating tyrosine at position 9 of the A chain to lysine and threonine at position 43 to aspartic acid. Then, isoleucine at position 25 of the B chain is mutated to leucine, and histidine at position 82 is mutated to leucine. The mutated B chain was obtained by mutating tryptophan at position 41 to serine and glycine at position 60 to asparagine. The A and B chains were then linked together to obtain the mutated Neoculin-encoded protein sequence. Based on the codon bias of *Chlamydomonas reinhardtii*, the codons of the two mutated protein sequences were optimized to obtain the optimized CrMiraculin and CrNeoculin nucleotide sequences. Subsequently, the optimized nucleotide sequences were cloned into the universal vector pUC57 to construct the recombinant plasmids pUC57-CrMiraculin / pUC57-CrNeoculin. Using the constructed recombinant plasmids pUC57-CrMiraculin / pUC57-CrNeoculin as templates, they were cloned into the self-modified *Chlamydomonas reinhardtii*-specific expression vector pGM6-H-HA-aph8 to obtain the plasmids pGM6-CrMiraculin-HA-aph8 and pGM6-CrNeoculin-HA-aph8. Recombinant plasmids pGM6-CrMiraculin-HA-aph8 and pGM6-CrNeoculin-HA-aph8 were introduced into wild-type *Chlamydomonas* sp. using electroporation. Transformed algal strains were screened using the paromomycin (Paro) resistance marker carried by the vector, and the expression of the target protein was verified by Western blotting analysis. Finally, engineered algal strains capable of stably and efficiently expressing the sweet-tasting inducible protein were obtained. Furthermore, the engineered algal strains highly expressing CrMiraculin and CrNeoculin were subjected to large-scale fermentation culture. After collecting the algal cells, they were freeze-dried to obtain dehydrated, stable *Chlamydomonas* powder rich in the target sweet-tasting inducible protein. Finally, after protein purification, the powder was mixed with acidic substances such as lemon juice and vinegar, and oral administration tests confirmed that it could induce a sweet taste from the sour taste.

[0056] The technical solution provided by this invention has the following significant advantages over traditional production methods: By using *Chlamydomonas reinhardtii* as the recombinant protein expression platform, it not only effectively overcomes the bottleneck of production capacity and high cost caused by the limited plant raw materials and complex extraction process in natural extraction routes, but also completely eliminates the risk of pathogen contamination that may exist in eukaryotic expression systems such as animal cells and insect cells, while significantly reducing culture costs; in terms of protein expression performance, compared with microbial systems such as bacteria and yeast, *Chlamydomonas reinhardtii* has complete eukaryotic post-translational modification capabilities, which can ensure the correct folding and functional maturation of the target protein; in addition, compared with higher plant cell systems, this system has the advantages of short culture cycle, high genetic stability, and good batch consistency, and does not occupy arable land resources, enabling continuous and controllable production throughout the year, fundamentally breaking through the seasonal and regional limitations of traditional agricultural production.

[0057] The protein sequences encoded by the sweetness inducers CrMiraculin / CrNeoculin are as follows:

[0058] The protein sequence encoded by CrMiraculin is shown in SEQ ID NO.12, and the protein sequence encoded by CrNeoculin is shown in SEQ ID NO.13.

[0059] The nucleotide sequence of the codon-optimized sweetness inducer CrMiraculin is shown in SEQ ID NO.14, and the nucleotide sequence of CrNeoculin is shown in SEQ ID NO.15.

[0060] This invention provides a method for constructing the recombinant plasmid pGM6-CrMiraculin-HA-aph8 / pGM6-CrNeoculin-HA-aph8, comprising the following steps:

[0061] Step 1, construction of the pGM6-H-HA-aph8 plasmid, includes the following steps:

[0062] Step 11: Using the 3×HA sequence plasmid as a template, the HA fragment was obtained by PCR amplification using primers pG-HA-S / AS;

[0063] The nucleotide sequence of HA is shown in SEQ ID NO.1, and the nucleotide sequences of primer pG-HA-S / AS are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0064] Step 12: Use primers pM-S / AS to perform PCR amplification on the universal vector pMO508 to clone and obtain the target vector fragment.

[0065] The nucleotide sequences of primers pM-S / AS are shown in SEQ ID NO.4 and SEQ ID NO.5;

[0066] Step 13: Perform agarose gel electrophoresis analysis on the HA fragment and the target vector fragment, and purify and recover them using a gel recovery kit;

[0067] Step 14: Mix the purified HA fragment with the target vector fragment at the optimal molar ratio, and perform homologous recombination ligation in the presence of recombinase to obtain the recombinant plasmid.

[0068] Step 15: The recombinant plasmid was introduced into Trans5 α Escherichia coli competent cells, placed on ice, and then subjected to heat shock before being transferred to ice and placed statically. Then, antibiotic-free LB liquid medium was added and cultured with shaking. The revived bacterial culture was spread onto LB agar plates containing Amp resistance and incubated at constant temperature.

[0069] Step 16: Randomly select several single colonies with regular morphology and good separation on the plate, and inoculate them into LB liquid medium containing Amp. Shake and culture until the bacterial culture reaches the stationary phase.

[0070] Step 17: Use a plasmid micro-extraction kit to extract recombinant plasmids from the amplified bacterial culture, and verify by sequencing to obtain pGM6-H-HA-aph8 plasmid.

[0071] Step 2, the construction of recombinant plasmids pGM6-CrMiraculin-HA-aph8 / pGM6-CrNeoculin-HA-aph8, includes the following steps:

[0072] Step 21: First, site-directed mutagenesis was performed on the Miraculin protein sequence to obtain the Thaumatin-encoding protein sequence with isoleucine at position 39 mutated to leucine, isoleucine at position 52 mutated to leucine, histidine at position 59 mutated to lysine, and glutamine at position 82 mutated to leucine. Simultaneously, the Neoculin protein-encoding sequence was modified. Neoculin is a dimer composed of A and B chains. First, tyrosine at position 9 of chain A was mutated to lysine, and threonine at position 43 was mutated to aspartic acid to obtain the mutated A chain. Then, isoleucine at position 25 of chain B was mutated to leucine, tryptophan at position 41 was mutated to serine, and glycine at position 60 was mutated to asparagine to obtain the mutated B chain. The A and B chains were then linked together to obtain the mutated chain. Based on the codon bias of Chlamydomonas reinhardtii, the two mutant protein sequences encoded by Neoculin were codon optimized to obtain optimized CrMiraculin and CrNeoculin nucleotide sequences. Subsequently, the optimized nucleotide sequences were cloned into the universal vector pUC57 to construct the recombinant plasmid pUC57-CrMiraculin / pUC57-CrNeoculin. Using the constructed recombinant plasmid as a template, the target gene fragments of CrMiraculin and CrNeoculin were obtained by PCR amplification using specific primers pGM6-CrMiraculin-S / AS or pGM6-CrNeoculin-S / AS.

[0073] The nucleotide sequences of pGM6-CrMiraculin-S / AS are shown in SEQ ID NO.6 and SEQ ID NO.7, and the nucleotide sequences of pGM6-CrNeoculin-S / AS are shown in SEQ ID NO.8 and SEQ ID NO.9.

[0074] Step 22: Using the pGM6-H-HA-aph8 plasmid prepared in step 1 as a template, the expression vector fragment pGM6 is obtained by PCR amplification using primers pGM6-HA-S / AS.

[0075] The nucleotide sequences of pGM6-HA-S / AS are shown in SEQ ID NO.10 and SEQ ID NO.11.

[0076] Step 23: Separate the CrMiraculin / CrNeoculin target gene fragment and the expression vector fragment pGM6 by agarose gel electrophoresis. Under ultraviolet light, cut the gel region containing the target band and purify and recover the corresponding DNA fragment.

[0077] Step 24: The purified CrMiraculin / CrNeoculin target gene fragments are mixed with the purified expression vector fragment pGM6 at the optimized molar ratio, and recombinase is added for homologous recombination ligation to obtain recombinant plasmids.

[0078] Step 25: The recombinant plasmid is introduced into Trans5⍺ E. coli competent cells, placed on ice to allow the DNA to fully contact the cells, and then subjected to heat shock followed by ice recovery treatment.

[0079] Step 26: Add LB medium without resistance, shake and incubate, take an appropriate amount of the recovered bacterial culture and spread it on LB agar plates containing Amp resistance, and incubate overnight.

[0080] Step 27: Randomly select several single colonies that grow well and have regular morphology on the plate, and inoculate them into LB liquid medium containing Amp. Shake and culture until the bacterial culture reaches the stationary phase.

[0081] Step 28: Use a plasmid micro-extraction kit to extract recombinant plasmids from the propagation bacterial culture, and sequence them for verification. The recombinant plasmids pGM6-CrMiraculin-HA-aph8 / pGM6-CrNeoculin-HA-aph8 were successfully constructed.

[0082] This invention provides a method for constructing an engineered Chlamydomonas reinhardtii algal strain that highly expresses CrMiraculin / CrNeoculin sweet-inducible proteins. The recombinant plasmid pGM6-CrMiraculin-HA-aph8 / pGM6-CrNeoculin-HA-aph8 was linearized by single-enzyme digestion with the restriction endonuclease ScaI, and the linearized plasmid was obtained after purification using a purification kit. The linearized plasmid was introduced into wild-type Chlamydomonas sp. cells via electroporation. After transformation, the algal cells were transferred to TAP medium containing sorbitol and cultured overnight under low light conditions to complete cell repair. The repaired algal solution was spread on Paro-containing solid selective medium for preliminary resistance screening. After culture, multiple single-clone algal strains were randomly selected and inoculated into well plates, and cultured to the logarithmic growth phase. The protein expression level of the obtained algal strains was detected by Western blotting, and finally, an engineered Chlamydomonas reinhardtii algal strain with high expression of CrMiraculin / CrNeoculin sweet-inducible proteins was obtained.

[0083] This invention also provides Chlamydomonas reinhardtii algal powder with high expression of CrMiraculin / CrNeoculin sweet-inducing protein, which is produced by large-scale fermentation of an engineered Chlamydomonas reinhardtii algal strain with high expression of CrMiraculin / CrNeoculin sweet-inducing protein.

[0084] First, the algal 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 scale-up fermentation, which lasted approximately 7 days. After fermentation, the algal cells were collected and freeze-dried to obtain dried *Chlamydomonas reinhardtii* powder rich in CrMiraculin and CrNeoculin. Finally, after protein purification, the powder was mixed with acidic substances such as lemon juice and vinegar. Oral testing confirmed that it could induce a sweet taste by converting sourness into sweetness. These results demonstrate that the engineered algal strain constructed based on *Chlamydomonas reinhardtii* basal cells can effectively express and accumulate sweet-tasting proteins with complete biological activity, providing a feasible technical pathway and material basis for developing novel flavor-modifying products.

[0085] The Chlamydomonas reinhardtii engineered algal strain and powder of Chlamydomonas reinhardtii with high expression of CrMiraculin / CrNeoculin sweetness-inducing protein can be applied in the fields of food, condiments or health products. They can effectively improve the unpleasant flavor of food, give it a sweet taste, and help reduce the health risks caused by sugar intake while reducing the amount of traditional added sugar.

[0086] This invention constructs a complete production technology system using *Chlamydomonas reinhardtii* as a bioreactor, encompassing the entire process from gene construction, engineered algal strain screening, large-scale fermentation to end-product preparation. This system aims to achieve efficient expression and stable accumulation of the sweetness-inducing proteins CrMiraculin and CrNeoculin in *Chlamydomonas reinhardtii*, and based on this, prepare *Chlamydomonas reinhardtii* algal powder products rich in active sweetness-inducing proteins. The algal powder can serve as a natural raw material with flavor-modifying functions, directly applicable to multiple industrial fields such as food processing, beverage manufacturing, pharmaceutical preparations, and cosmetic development. It provides a comprehensive solution from technology to materials for developing safe and healthy novel sweetness inducers, thereby promoting systematic progress in product innovation, quality improvement, and green sustainable development in related industries.

[0087] Example 1: Construction of recombinant plasmids pGM6-CrMiraculin-HA-aph8 / pGM6-CrNeoculin-HA-aph8

[0088] The construction method includes the following specific steps:

[0089] 1. Construction of pGM6-H-HA-aph8 plasmid

[0090] (1) The nucleotide sequence of 3 x HA was submitted to a gene synthesis company, and the 3 x HA was synthesized into the universal vector pUC57 to obtain the pUC57-3 x HA plasmid. Using this plasmid as a template, the desired HA fragment was amplified using the specific primers pG-HA-S / AS. The specific primer information is as follows:

[0091] HA sequence (SEQ ID NO. 1):

[0092] Tcgcgatacccctacgacgtgcccgactacgcctacccctacgacgtgcccgactacgccgatcgatccggaccgtacccctacgacgtgcccgactacgcccgctccgtgtga

[0093]

[0094] The synthesized HA plasmid DNA was diluted to 10 ng / µl with ddH2O and used as a template. PCR amplification was performed using primers pG-HA-S / AS to obtain the target gene fragment HA.

[0095] The PCR system is as follows:

[0096]

[0097] The PCR procedure is as follows:

[0098]

[0099] (2) Using the universal vector pMO508 as a template, the vector fragment was cloned using pM-S / AS primers. Specific primer information is as follows:

[0100]

[0101] The universal vector pMO508 was diluted to a final concentration of 10 ng / μL with ddH2O and used as a template. The vector fragment was obtained by PCR amplification using forward primer pM-S and reverse primer pM-AS.

[0102] The PCR system is as follows:

[0103]

[0104] The PCR procedure is as follows:

[0105]

[0106] (3) The HA fragment and the vector fragment were separated by agarose gel electrophoresis. Then, the gel region containing the target band was cut out and the corresponding DNA fragment was purified and recovered using a gel recovery kit.

[0107] (4) The purified HA target fragment and the pMO508 universal vector were mixed in the optimized molar ratio, and homologous recombinase was added to carry out the ligation reaction at a suitable temperature to construct the recombinant plasmid.

[0108] (5) The recombinant plasmid was introduced into Trans5α Escherichia coli competent cells, and after being incubated on ice for 30 minutes, it was heat-shocked in a water bath at 42°C for 30 seconds, and then immediately transferred to ice and placed on ice for 2 minutes. Then, 500 μL of antibiotic-free LB liquid medium was added, and the cells were incubated at 37°C with shaking at 150 rpm for 60 minutes. An appropriate amount of bacterial culture was spread on LB agar plates containing Amp and incubated at 37°C for 12-16 hours.

[0109] (6) Using a sterile toothpick, randomly select 3 single colonies with regular morphology and good isolation, and inoculate them into 5 mL of LB liquid medium containing Amp. Incubate at 37℃ and 200 rpm for 16 hours until the late logarithmic growth phase is reached.

[0110] (7) The recombinant plasmid was extracted from the propagated bacterial culture using a plasmid micro-extraction kit, and 300 ng of the extraction product was sequenced for verification to obtain the pGM6-H-HA-aph8 plasmid.

[0111] 2. Construction of recombinant plasmid pGM6-CrMiraculin / CrNeoculin-HA-aph8

[0112] (1) Obtaining the target gene fragment of CrMiraculin / CrNeoculin

[0113] Site-directed mutagenesis was performed on the Miraculin protein sequence to obtain Thaumatin-encoding sequences with isoleucine at position 39 replaced by leucine, isoleucine at position 52 replaced by leucine, histidine at position 59 replaced by lysine, and glutamine at position 82 replaced by leucine. Simultaneously, the Neoculin protein-encoding sequence was modified. Neoculin is a dimer composed of A and B chains. First, tyrosine at position 9 of chain A was mutated to lysine, and threonine at position 43 was mutated to aspartic acid to obtain the mutated A chain. Then, the mutated B chain was modified... The mutated B chain was obtained by mutating isoleucine at position 25 to leucine, tryptophan at position 41 to serine, and glycine at position 60 to asparagine. The A and B chains were then linked together to obtain the mutated Neoculin-encoded protein sequence. Based on the codon bias of *Chlamydomonas reinhardtii*, codon optimization was performed on both mutated protein sequences to obtain the optimized CrMiraculin and CrNeoculin nucleotide sequences. The results of comparing the codon-optimized sequences with the protein-encoded sequences of natural sweetness inducers are shown below. Figure 1 Subsequently, the optimized nucleotide sequences were cloned into the universal vector pUC57 to construct the recombinant plasmids pUC57-CrMiraculin / pUC57-CrNeoculin.

[0114] Primers pGM6-CrMiraculin-S / AS and pGM6-CrNeoculin-S / AS were designed based on the target gene. Specific primer information is as follows:

[0115]

[0116] The synthesized CrMiraculin and CrNeoculin plasmid DNAs were diluted to 10 ng / µl with ddH2O and used as templates. PCR amplification was performed using primers pGM6-CrMiraculin-S / AS and pGM6-CrNeoculin-S / AS to obtain the target gene fragments.

[0117] The PCR system is as follows:

[0118]

[0119] The PCR procedure is as follows:

[0120]

[0121] (2) Obtaining the expression vector fragment pGM6

[0122] Amplification primers pGM6-HA-S and pGM6-HA-AS were designed based on the expression vector. Specific primer information is as follows:

[0123]

[0124] The pGM6-H-HA-aph8 plasmid was diluted to a final concentration of 10 ng / μL with ddH2O and used as a PCR template. The forward primer pGM6-HA-S and the reverse primer pGM6-HA-AS were selected, and the expression vector fragment pGM6 was obtained by PCR amplification.

[0125] The PCR system is as follows:

[0126]

[0127] The PCR procedure is as follows:

[0128]

[0129] (3) Gel recovery and purification of target gene fragment and expression vector fragment

[0130] The PCR amplification products of the target gene fragment and the expression vector fragment pGM6 were separated by 1% agarose gel electrophoresis, and the results were recorded using a Tanon 1600 series gel imaging system. Figure 2As shown. After confirming that the target band size met expectations by comparing it with the DNA molecular weight standard, the corresponding gel region was excised, and the DNA fragment was recovered and purified using the Sangon Biotech SanPrep Column DNAGel Extraction Kit (catalog number: J92KA0972). The specific operation was performed according to the kit instructions.

[0131] (4) The target gene fragment is ligated to the expression vector fragment to obtain recombinant plasmid DNA.

[0132] The target gene fragment obtained from gel extraction and the expression vector fragment pGM6 were ligated using the Novavit plasmid DNA ligation kit (ClonExpress II One Step Cloning Kit, lot # C12-01) at 37˚C for 30 min to obtain recombinant plasmid DNA. The ligation system is as follows:

[0133]

[0134] (5) Introduction of recombinant plasmid DNA into competent E. coli cells

[0135] Thaw 50 μL of Trans5 α Escherichia coli competent cells on ice, add the ligation product from (4), and incubate on ice for 30 minutes; after heat shock in a 42℃ water bath for 30 seconds, immediately incubate on ice for 2 minutes; then add 500 μL of antibiotic-free LB liquid medium, and revive at 37℃ and 150 rpm for 1 hour; finally, spread an appropriate amount of bacterial solution on LB agar plates containing Amp, and incubate at 37℃ for 12-16 hours.

[0136] (6) Screening of plaques with recombinant plasmid DNA

[0137] Remove the overnight culture plate from 37˚C, then pick 3 morphologically healthy monoclonal clones and place them in 5 ml of LB containing Amp resistance, and incubate overnight at 37˚C.

[0138] (7) Extraction of recombinant plasmids

[0139] Collect the bacterial culture that has been incubated overnight at 37°C, and extract plasmids using the Sangon Biotech SanPrep Column Plasmid Mini-Preps Kit (catalog number: IB23KA7480). Follow the kit instructions for specific procedures.

[0140] (8) Confirmation by recombinant plasmid sequencing

[0141] The plasmid sample extracted in step (7) was sent to Sangon Biotech for DNA sequencing. The sequence obtained was confirmed by sequence alignment analysis to be completely consistent with the theoretically designed recombinant plasmid sequence, thus verifying that the recombinant plasmids pGM6-CrMiraculin-HA-aph8 and pGM6-CrNeoculin-HA-aph8 were successfully constructed.

[0142] Example 2: Construction of engineered algal strains with high expression of sweetness inducers CrMiraculin and CrNeoculin in Chlamydomonas reinhardtii

[0143] The construction method includes the following specific steps:

[0144] 1. Preparing to insert a segment

[0145] The recombinant plasmids pGM6-CrMiraculin-HA-aph8 and pGM6-CrNeoculin-HA-aph8 successfully constructed in Example 1 were digested with ScaI restriction endonuclease at 37°C for 2 hours. The digestion products were separated by 1% agarose gel electrophoresis and analyzed using a Tanon 1600 series gel imaging system. The results... Figure 3 As shown. After confirming that the linearized band size met expectations by comparing it with the DNA molecular weight standard, the gel region corresponding to the target band was excised, and the DNA fragment was recovered and purified using the Sangon Biotech SanPrepColumn DNA Gel Extraction Kit (catalog number: J92KA0972). The specific operation was performed according to the kit instructions.

[0146] The enzyme digestion system is as follows:

[0147]

[0148] 2. Chlamydomonas competent cell culture

[0149] Wild-type Chlamydomonas reinhardtii was transferred from TAP solid plates to TAP liquid medium and cultured for 3-4 days under continuous light and aeration until the cell density reached 1×10⁻⁶. 7 cells / mL. Take 10 mL of this algal culture and inoculate it into an Erlenmeyer flask containing 100 mL of TAP liquid medium, adjusting the initial cell density to 1 × 10⁻⁶ cells / mL. 6 The cells were then placed in a shaker at 200 rpm under continuous light for approximately 20 hours until the cell density reached 5 × 10⁶ cells / mL. 6 Electroporation was performed immediately when the concentration of cells / mL was reached.

[0150] 3. Obtaining transformants of Chlamydomonas rhinelandii engineered algal strains

[0151] (1) Resuspension and preparation of wild-type Chlamydomonas cells:

[0152] In the clean bench, the density reaches 5×10 6 Chlamydomonas cells / mL were collected in 50 mL sterile centrifuge tubes, and 30 mL of cells were collected. The tubes were centrifuged at 2500 rpm for 3 min at room temperature. The cell pellet was resuspended in 1 mL of pre-chilled TAP + 60 mM sorbitol solution. The volume was then adjusted to 20 mL with pre-chilled TAP + 60 mM sorbitol solution, and centrifuged at 2500 rpm for 3 min at room temperature. The supernatant was carefully discarded, and the cells were resuspended in 1 mL of pre-chilled TAP + 60 mM sorbitol solution. The final concentration of Chlamydomonas cells was then adjusted to approximately 1-2 × 10⁻⁶ cells / mL with TAP + 60 mM sorbitol. 8 Take 10 cells and incubate the prepared cell suspension on ice for 10 min.

[0153] (2) Cleaning and pre-cooling of the electric shock cup:

[0154] To ensure the high efficiency of the electroporation process and the sterility of the operating system, all electroporation cups were cleaned in a laminar flow hood. The specific steps were as follows: First, the cups were rinsed once with anhydrous ethanol to remove organic residues and to achieve rapid drying due to its volatility; then, they were rinsed three times consecutively with TAP + 60 mM sorbitol solution, inverting the cups five times each time to match the internal environment of the electroporation cups with the osmotic pressure conditions for subsequent electroporation; finally, the treated electroporation cups were pre-cooled at -20°C for later use.

[0155] (3) Preparation of electroporation conversion mixture:

[0156] Remove the electroporation cuvettes from -20℃ and open them in a clean bench. Add 250 μl of prepared Chlamydomonas cells to each pre-cooled electroporation cuvette, followed by 100-150 ng of pGM6-CrMiraculin-HA-aph8 or pGM6-CrNeoculin-HA-aph8 expression plasmid fragments linearized with ScaI restriction enzyme into the electroporation cuvettes. Gently pipette and mix to ensure thorough mixing of the cells and expression vector DNA fragments. Place the mixture on ice for 10 min.

[0157] (4) Electric shock operation:

[0158] Adjust the parameters of the stun gun (model BTX ECM630): set the voltage to 800 V, the resistance to 1575 Ω, and the capacitance to 50 μF. First, test all parameters of the stun gun with an empty stun cup. Once these parameters are stable, gently wipe the stun cup's outer shell dry with absorbent paper. Secure the stun cup by tightly contacting the two sides with the metal strip with the stun gun's clamps. Then, lower the stun gun's safety cover and start the stun program. To ensure stun efficiency, the stun time must be strictly controlled between 10 and 14 ms. After the stun is complete, immediately place the stun cup back on the ice and leave it for 10 minutes.

[0159] (5) Obtaining the electroconverter

[0160] Inside a clean bench, Chlamydomonas cells that had undergone electroporation were transferred to 50 mL sterile centrifuge tubes containing 10 mL TAP + 60 mM sorbitol solution. The centrifuge tubes were wrapped with aluminum foil and placed on a shaker at 100 rpm under low light conditions for 12-16 hours for recovery culture. The next day, the Chlamydomonas cells were collected by centrifugation at 2500 rpm for 3 minutes at room temperature.

[0161] Simultaneous preparation of corn starch suspension: Take an appropriate amount of corn starch and wash it once with anhydrous ethanol, three times with sterile ultrapure water (ddH2O), and four times with TAP + 60 mM sorbitol solution; finally, resuspend the treated starch with TAP + 60 mM sorbitol solution to prepare a 20% (w / v) suspension.

[0162] Add 1 mL of the above 20% corn starch solution to the collected Chlamydomonas cells, gently mix by pipetting, and then spread evenly on a TAP solid screening plate containing Paro. Let it stand in a clean bench until the liquid surface dries naturally, seal with sealing film, invert the plate, and incubate on a photoperiodic culture rack for 4-5 days until the transformed algal colonies are clearly visible.

[0163] 4. Screening of engineered Chlamydomonas reinhardtii strains with high expression of CrMiraculin / CrNeoculin proteins

[0164] (1) Sample collection

[0165] After the transformed algal colonies reached a stable growth state through 4-5 days of selective culture, aseptic procedures were performed in a clean bench: Regularly shaped, clearly edged monoclonal transformants were precisely separated using sterile toothpicks or inoculation loops and transferred to pre-prepared antibiotic-free TAP solid medium plates, which were then permanently numbered. The inoculated plates were placed on a photoperiodic culture rack and cultured under constant temperature and moderate light conditions until the algal colony diameter expanded to 1-2 mm and its morphological characteristics stabilized.

[0166] The subsequent liquid amplification stage involves using sterile toothpicks to inoculate the target algal colonies from the solid agar plates into individual wells of a 24-well plate containing 1.5 mL of TAP liquid medium, ensuring strict correspondence between each well and the plate label number. The 24-well plates are then placed in a temperature-controlled shaker culture system at 200 rpm and cultured continuously for 2-3 days under a photoperiod of 14:10 (light:dark) until the culture medium turns a deep green and the cell density reaches 5-10 × 10⁶ cells / well. 6 The cells / mL method enables the exponential proliferation of engineered algal strains in liquid systems.

[0167] After the predetermined culture cycle was completed in the 24-well plates, the engineered Chlamydomonas reinhardtii algal strains were systematically collected from the wells. Using a precision pipette, the cell suspension from each well was quantitatively aspirated and dispensed into 1.5 mL centrifuge tubes, ensuring that the total cell count in each tube reached 1 × 10⁻⁶ cells. 7 Each sample tube was placed in a high-speed centrifuge and centrifuged at 14,000 rpm for 1 minute at room temperature to achieve efficient separation of cells from the culture medium. After centrifugation, the supernatant was carefully removed, keeping the pipette tip away from the cell pellet at the bottom of the tube to ensure the integrity of the cell clumps. The centrifuge tubes containing the cell pellet were then immediately flash-frozen in liquid nitrogen and finally transferred to an ultra-low temperature freezer at -80°C for long-term storage.

[0168] (2) Sample preparation

[0169] Pre-prepared Chlamydomonas reinhardtii cell samples were removed from an ultra-low temperature freezer at -80°C, and the entire process was performed on ice to maintain the low-temperature environment. 100 μL of pre-chilled cell lysis buffer A (containing protease inhibitors) was precisely added to each sample, and the mixture was gently pipetted to ensure complete cell lysis while avoiding vigorous shaking that could generate air bubbles and prevent protein denaturation or degradation.

[0170] After lysis, protein denaturation was performed: 100 μL of 2×SDS protein loading buffer was added to each lysed sample, and the mixture was thoroughly vortexed to ensure complete binding of the protein to SDS. The mixed sample was then incubated precisely in a 100°C metal bath for 10 minutes to allow complete protein denaturation. Immediately after heat denaturation, the sample was transferred to ice and rapidly cooled for 2 minutes to terminate the reaction.

[0171] Finally, the sample is briefly centrifuged (10,000 × g, 30 seconds), the condensate on the tube wall is collected and a small amount of insoluble matter settles. The resulting supernatant is the total protein sample, which can be stored at -20°C for subsequent protein analysis experiments such as Western blotting.

[0172] (3) Western Blot assay

[0173] 1) Gel preparation: Based on the molecular weight characteristics of the target proteins CrMiraculin-HA (approximately 28 kDa) and CrNeoculin-HA (approximately 24 kDa), a 15% separating gel was selected for SDS-PAGE analysis. First, appropriate volumes of 30% acrylamide mixture, 1.5 M Tris-HCl (pH 8.8), 10% SDS, 10% ammonium persulfate, and TEMED were added sequentially to 50 mL centrifuge tubes. The tubes were gently shaken 8-10 times to homogenize the solution. The separating gel solution was then slowly poured along the inner wall of a 1.5 mm thick glass plate. Ultrapure water was then slowly added along the wall using a micropipette for liquid sealing. The mixture was allowed to stand at room temperature for 25 minutes until the oil immersion lens interface became clearly visible, indicating complete gel polymerization.

[0174] After the separating gel has cured, prepare a 5% stacking gel according to the formula: mix the corresponding volume of 30% acrylamide mixture, 0.5 M Tris-HCl (pH 6.8), 10% SDS, 10% ammonium persulfate, and TEMED in a 15 mL centrifuge tube. Discard the upper aqueous phase of the separating gel and blot away any remaining droplets with filter paper. Pour the stacking gel solution into the mold at a constant flow rate, and immediately insert a 15-well gel casting comb at a 45° angle. Allow it to stand at room temperature for 20 minutes to ensure complete cross-linking of the stacking gel, thus obtaining a complete SDS-PAGE gel. This gel can be used immediately or sealed and stored in a humid environment at 4°C.

[0175] 2) Sample Loading: After the stacking gel has fully polymerized, carefully remove the gel comb vertically upwards and mount the gel and glass plate together in the electrophoresis tank holder. Pour in 1× electrophoresis buffer until the liquid level completely covers the sample wells. Using a micropipette, add the pre-stained protein molecular weight standards and the denatured total protein sample sequentially to the sample wells. To maintain electric field uniformity, add an equal volume of 1×SDS loading buffer to the blank sample wells. All sample loading operations should be completed within 10 minutes to minimize sample diffusion and edge effects.

[0176] 3) Electrophoresis: SDS-PAGE electrophoresis is performed in constant voltage mode. Initially, the voltage is set to 80 V to allow the sample to aggregate in the stacking gel. When the bromophenol blue indicator migrates to the separating gel interface, the voltage is increased to 120 V to achieve precise separation of the target protein according to its molecular weight. Electrophoresis is continued until the bromophenol blue front reaches the bottom of the gel, at which point the process is terminated.

[0177] 4) After electrophoresis, precisely cut the gel region containing the target protein molecular weight range (20-35 kDa) according to the pre-stained protein markers. In pre-cooled transfer buffer, assemble the transfer membrane structure in the following order: cathode plate - sponge - three layers of filter paper - separating gel - PVDF membrane - three layers of filter paper - sponge - anode plate. During assembly, remove air bubbles layer by layer to ensure tight adhesion and alignment between the PVDF membrane and the gel interface.

[0178] Insert the complete transfer jacket vertically into the transfer tank and inject pre-cooled transfer buffer until the device is completely submerged. Perform transfer at a constant current of 350 mA for 60 minutes. These transfer conditions ensure efficient transfer of target proteins with molecular weights in the range of 20-35 kDa to the PVDF membrane.

[0179] 5) After the transfer is complete, remove the PVDF membrane and accurately label the region where the target protein is located according to the pre-stained protein molecular weight standard. Immerse the membrane completely in TBST blocking solution containing 10 mL of 5% skim milk, place it on a shaker at 100 rpm, and block it at room temperature for 1 hour to block non-specific binding sites on the membrane.

[0180] 6) Primary antibody hybridization: The mouse anti-HA tag monoclonal antibody was diluted 1:2000 using TBST solution containing 3% skim milk. The blocked PVDF membrane was transferred to the primary antibody, ensuring complete coverage. The membrane was incubated on a shaker at room temperature for 1 hour to allow the antibody to specifically bind to the target proteins CrMiraculin-HA / CrNeoculin-HA. After incubation, the PVDF membrane was washed three times with TBST buffer on a shaker for 5 minutes each time to thoroughly remove any unbound primary antibody.

[0181] 7) Secondary antibody hybridization: Dilute the secondary antibody 1:5000 using TBST solution containing 3% skim milk. Transfer the PVDF membrane, after primary antibody hybridization and washing, to the secondary antibody and incubate at 100 rpm for 1 hour at room temperature. After incubation, wash the PVDF membrane three times with TBST buffer on a shaker for 5 minutes each time to completely remove unbound secondary antibody and prepare for subsequent chemiluminescence detection.

[0182] 8) Thoroughly mix A and B solutions of the Aibotek ECL ultrasensitive chemiluminescence substrate at a 1:1 volume ratio, uniformly cover the reaction area of ​​the PVDF membrane, and incubate at room temperature for 2 minutes. Then place the membrane in a Tanon chemiluminescence imager, set the gradient exposure time according to the signal intensity, and acquire images with the optimal signal-to-noise ratio.

[0183] Through systematic analysis of Western blotting results, the protein expression characteristics of engineered algal strains with different numbers were compared. For example... Figure 4As shown, clear and specific bands were observed at approximately 28 kDa (CrMiraculin-HA fusion protein) and 24 kDa (CrNeoculin-HA fusion protein), with molecular weights highly consistent with theoretical predictions. Notably, no chromogenic bands were observed in the corresponding regions of the empty vector control sample, confirming the signal specificity. Based on the dual verification of band intensity and molecular weight localization, engineered algal strains expressing high levels of CrMiraculin and CrNeoculin were successfully screened. Their target protein expression levels were significantly higher than other transformants, providing a superior algal resource for subsequent large-scale cultivation and application. The engineered algal strains expressing high levels of CrMiraculin and CrNeoculin sweetness-inducing proteins were purified and mixed with acidic substances such as lemon juice and vinegar. Oral administration tests confirmed that they could induce a sweet taste, thus demonstrating significant application potential in the food, condiment, or health product fields. Comparative results are shown below. Figure 5 As shown.

[0184] Example 3: Large-scale fermentation culture of Chlamydomonas reinhardtii engineered algal strains with high expression of CrMiraculin / CrNeoculin proteins

[0185] The specific steps include the following:

[0186] 1. Engineered algal strains that have been validated by Western blotting to efficiently express CrMiraculin and CrNeoculin were inoculated onto freshly prepared TAP solid agar plates using sterile toothpicks to achieve single-clone isolation and activation amplification. The inoculated plates were placed in a photoperiodic culture environment at a constant temperature of 25°C and a photoperiod of 14 hours light / 10 hours dark for 3-4 days until the algal colony reached a diameter of 1-2 mm and a bright green color, thus obtaining activated algal strains in the logarithmic growth phase.

[0187] 2. Using a sterile toothpick, aseptically transfer a suitable amount of the target engineered algal strain, which is well-activated and in the logarithmic growth phase, from a TAP solid plate to a 1 L Erlenmeyer flask containing 500 mL of sterile TAP liquid medium. Place the flask in a photocyclic shaking incubator and amplify the culture under constant temperature of 25℃, a 14h light / 10h dark cycle, and constant shaking at 200 rpm. Monitor cell density every 24 hours. When the cell density in the algal solution reaches approximately 5 × 10⁻⁶ cells / mL... 6 -1×10 7 When the number of cells / mL is high, it indicates that the seed culture is in the mid-logarithmic growth phase and can be immediately used as an inoculum for subsequent large-scale fermentation in a fermenter.

[0188] 3. Pour 3 L of precisely prepared TAP medium into the main body of the 5 L automated fermenter. Simultaneously, dispense 1 L of 10× concentrated fed-batch medium into a dedicated 1 L blue-capped feeding bottle. Also prepare 100 mL of 10% polyether defoamer suspension. Place the fermenter (including the medium), feeding bottle, and defoamer together in an autoclave and sterilize using saturated steam at 121℃ and 0.12 MPa for 20 minutes.

[0189] After the sterilization process is completed, and the system temperature naturally drops below 80℃ and the pressure returns to zero, aseptic connections are made to the fermenter, feed lines, exhaust filtration system, and defoamer injection port in a clean bench environment. Finally, 500 mL of seed culture is added through the inoculation port, and the temperature, pH, dissolved oxygen, and rotation speed control systems are activated to begin the fermentation process.

[0190] 4. The fermentation process employs a multi-parameter synergistic control strategy: The integrated control system of the fermenter maintains a constant culture temperature of 24±0.5℃, and an automatic acid-base titration module stabilizes the pH of the culture medium within the range of 7.7±0.1. Dissolved oxygen is calibrated to 100% air saturation using a dissolved oxygen electrode calibrated with standard solutions before and after inoculation to establish a dissolved oxygen baseline. Based on this baseline, the stirring speed and aeration rate are automatically adjusted to dynamically maintain the dissolved oxygen concentration within the optimal range. This synergistic control mode significantly increases cell density by maintaining the optimal physiological state of algal cells and provides an ideal metabolic environment for the efficient synthesis of sweetness-inducing proteins CrMiraculin and CrNeoculin.

[0191] 5. On day 7 of fermentation, when online monitoring data showed no significant increase in cell density for 12 consecutive hours, the algae were considered to have entered the stationary phase, and fermentation was terminated. All 3 L of fermentation broth was transferred to a 5 L collection container and centrifuged at 4000 rpm for 5 minutes at room temperature to efficiently collect the wet algal sludge. The algal sludge was evenly spread on a freeze-drying tray, with a layer thickness controlled within 5 mm, and placed in a vacuum freeze dryer for dehydration to obtain loose, porous algal powder with a moisture content of less than 5%. Figure 6 As shown. The final product is vacuum-sealed in an aluminum foil bag and can be stably stored under dry conditions at 4°C, maintaining the activity of the sweet protein.

[0192] Compared with existing technologies, the recombinant protein-engineered algal strain based on Chlamydomonas reinhardtii provided by this invention has many advantages such as high production efficiency, good safety and low cost, specifically reflected in:

[0193] 1. Compared to animal or insect cell systems, the Chlamydomonas reinhardtii system is based on photosynthetic autotrophy, consuming only inorganic nutrients and carbon dioxide, which greatly reduces the cost of culture medium. This characteristic also makes it suitable for various large-scale cultivation modes such as open ponds and closed photobioreactors, demonstrating excellent industrial scalability and production controllability.

[0194] 2. Compared to bacterial and yeast systems, the Chlamydomonas reinhardtii system can not only mediate correct folding and post-translational modifications (such as N-linked glycosylation, O-linked glycosylation, and disulfide bond formation) through its complete eukaryotic protein synthesis pathway, thereby producing recombinant proteins with full biological activity; at the same time, the system does not produce endotoxins, does not carry human pathogens, and has a physical barrier against animal viral infections, providing a highly safe biomanufacturing platform for the pharmaceutical field.

[0195] 3. Compared with plant expression systems, Chlamydomonas reinhardtii system has significant advantages in growth rate, production controllability and biosafety: it has the significant characteristics of short culture cycle and easy to achieve rapid iteration and high-throughput screening; it is cultured in a closed and controlled environment, which ensures the high stability and consistency of production batches, while not occupying arable land and completely avoiding the ecological risks of transgenic pollen spread.

[0196] 4. Unique Advantages as an Oral Delivery System: Another key innovation of this invention lies in utilizing Chlamydomonas reinhardtii cells as a natural delivery carrier for oral drugs. Through the physical barrier effect of its cell wall, intracellular recombinant proteins are protected from the harsh environment of the upper digestive tract, delaying their degradation and thus achieving targeted release of active ingredients at specific sites in the intestine. Combined with the inherent safety and edibility of Chlamydomonas reinhardtii, this constitutes its unique advantage as an oral delivery platform.

[0197] In summary, the Chlamydomonas reinhardtii expression chassis developed in this invention combines the research depth of model organisms with the industrial value of engineering applications. It provides a technical solution with significant competitive advantages for the low-cost, high-safety, and sustainable production and delivery of recombinant proteins, vaccines, and other biopharmaceutical products, and shows broad application potential in the fields of industrial biomanufacturing and preventive medicine.

[0198] 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 CrMiraculin protein, characterized in that: The nucleotide sequence encoding the protein is shown as SEQ ID NO.

14.

2. A CrNeoculin protein, characterized by: The nucleotide sequence encoding the protein is shown as SEQ ID NO. 15.

Images