Recombinant algal strain for expressing AGAT, BHAA, BHAD and ISR as well as construction method and application of recombinant algal strain

Abstract

The invention provides a recombinant algal strain for expressing AGAT, BHAA, BHAD and ISR as well as a construction method and application of the recombinant algal strain, and belongs to the technical field of gene engineering. The invention provides a recombinant microalgae strain, which is a microalgae strain for expressing aspartic acid-glyoxylate transaminase, beta-hydroxyaspartic acid aldolase, beta-hydroxyaspartic acid dehydratase and iminosuccinic acid reductase. The invention further provides a preparation method of the recombinant microalgae strain. The recombinant microalgae strain can decompose a light respiration by-product glycollic acid into carbon dioxide, the carbon dioxide concentration of chloroplast is improved, the carbon sequestration efficiency of microalgae is effectively improved, and fixed CO2 can be converted into biomass, pyruvic acid, starch, grease and other metabolites and bioactive substances. The recombinant microalgae strain disclosed by the invention has a wide application prospect in the aspects of biological carbon sequestration, synthesis of microalgae metabolites and preparation of microalgae bioactive substances.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant algal strain expressing AGAT, BHAA, BHAD and ISR, and a construction method and application thereof. Background Art

[0002] With the development of human society, the advancement of industrialization and the increase in population, the emission of CO2 is increasing day by day, and the greenhouse effect is becoming increasingly serious. Therefore, how to efficiently fix carbon is the key to solving global warming. The technology of microalgae for fixing CO2 is one of the effective ways of carbon fixation. Microalgae have a high photosynthesis efficiency. Microalgae efficiently fix carbon dioxide through the carbon dioxide concentrating mechanism (CCM), and the carbon fixation efficiency is 10-50 times that of other terrestrial plants. At the same time, microalgae have a fast growth rate, and microalgae reproduce rapidly (reproducing once every few hours), which is much higher than that of higher plants, and it is an excellent organism for realizing carbon capture, utilization and storage (CCUS).

[0003] In order to further improve the carbon fixation efficiency of microalgae, the existing technology mainly improves the microalgae culture conditions, screens and domesticates excellent algal strains, and modifies microalgae cells at the molecular level by applying genetic engineering means. Among them, constructing engineered microalgae with high-efficiency CO2 fixation by genetic engineering methods is an effective way to further improve the CO2 fixation ability of microalgae strains. However, at present, the strains obtained by genetic engineering have problems of unstable carbon fixation efficiency and low growth rate of microalgae cells. Summary of the Invention

[0004] In view of this, the present invention provides a recombinant microalgae strain expressing aspartate-glyoxylate aminotransferase, β-hydroxyaspartate aldolase, β-hydroxyaspartate dehydratase and iminosuccinic acid reductase. In the recombinant microalgae strain, a new photorespiratory bypass BHAC pathway is introduced, which can achieve efficient carbon fixation and has a high growth rate of microalgae cells.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a recombinant microalgae strain, which is a microalgae strain expressing aspartate-glyoxylate aminotransferase, β-hydroxyaspartate aldolase, β-hydroxyaspartate dehydratase and iminosuccinic acid reductase.

[0007] Preferably, the amino acid sequence of the aspartate-glyoxylate aminotransferase is as shown in SEQ ID NO: 1;

[0008] The amino acid sequence of the β-hydroxyaspartate aldolase is as shown in SEQ ID NO: 2;

[0009] The amino acid sequence of the β-hydroxyaspartate dehydratase is as shown in SEQ ID NO: 3;

[0010] The amino acid sequence of the iminosuccinic acid reductase is as shown in SEQ ID NO: 4.

[0011] Preferably, the microalgae include marine microalgae and / or freshwater microalgae;

[0012] Preferably, the freshwater microalgae include Chlamydomonas reinhardtii.

[0013] The present invention provides a method for constructing the recombinant microalgae strain, comprising the following steps:

[0014] Fusing the aspartate-glyoxylate aminotransferase gene, β-hydroxyaspartate aldolase gene, β-hydroxyaspartate dehydratase gene, and iminosuccinic acid reductase gene separately or simultaneously with the PsaD promoter for expression in microalgae.

[0015] Preferably, one, two, three, or four genes of the aspartate-glyoxylate aminotransferase gene, β-hydroxyaspartate aldolase gene, β-hydroxyaspartate dehydratase gene, and iminosuccinic acid reductase gene are transformed into microalgae in the form of a recombinant vector with the PsaD promoter.

[0016] Preferably, the recombinant vector includes a recombinant vector expressing aspartate-glyoxylate aminotransferase and β-hydroxyaspartate aldolase, and a recombinant vector expressing β-hydroxyaspartate dehydratase and iminosuccinic acid reductase.

[0017] The present invention provides the application of the recombinant microalgae strain or the recombinant microalgae strain constructed by the construction method in CO2 fixation.

[0018] The present invention provides the application of the recombinant microalgae strain or the recombinant microalgae strain constructed by the construction method in the preparation of at least one of the following products: feed, fertilizer, food, cosmetics, biofuel, and bioactive substances.

[0019] The present invention provides a method for improving the carbon fixation efficiency of microalgae, comprising the following steps:

[0020] Expressing aspartate-glyoxylate aminotransferase, β-hydroxyaspartate aldolase, β-hydroxyaspartate dehydratase, and iminosuccinic acid reductase in microalgae.

[0021] Preferably, the amino acid sequence of the aspartate-glyoxylate aminotransferase is as shown in SEQ ID NO: 1;

[0022] The amino acid sequence of the β-hydroxyaspartate aldolase is as shown in SEQ ID NO: 2;

[0023] The amino acid sequence of the β-hydroxyaspartate dehydratase is as shown in SEQ ID NO: 3;

[0024] The amino acid sequence of the iminosuccinate reductase is as shown in SEQ ID NO: 4.

[0025] The present invention has the following advantages compared with the prior art:

[0026] The present invention provides a recombinant microalgae strain, which is a microalgae strain expressing aspartate-glyoxylate aminotransferase, β-hydroxyaspartate aldolase, β-hydroxyaspartate dehydratase and iminosuccinate reductase. In the recombinant microalgae strain of the present invention, aspartate-glyoxylate aminotransferase catalyzes the oxidative decomposition of glycolate to form glyoxylate and oxalic acid, and the efficient β-hydroxyaspartate dehydratase and iminosuccinate reductase catalyze the formation of the C4 compound oxaloacetate, increasing the CO2 concentration in the chloroplast while reducing carbon loss. The BHAC pathway constructed by the four enzymes significantly improves the carbon fixation efficiency of the recombinant microalgae strain of the present invention compared with the wild-type strain. The recombinant microalgae strain can decompose the photorespiratory by-product glycolate into carbon dioxide, increase the carbon dioxide concentration in the chloroplast, effectively improve the carbon fixation efficiency of microalgae, and contribute to the fixation of CO2 into metabolites and bioactive substances such as biomass, pyruvate, starch and oil. It has broad application prospects in biological carbon fixation, synthesis of microalgae metabolites and preparation of microalgae bioactive substances.

[0027] The present invention provides a method for constructing the recombinant microalgae strain, comprising the following steps: separately or simultaneously fusing the aspartate-glyoxylate aminotransferase gene, β-hydroxyaspartate aldolase gene, β-hydroxyaspartate dehydratase gene and iminosuccinate reductase gene with the PsaD promoter for expression in microalgae. The method of the present invention can optimize the photorespiratory bypass of microalgae, establish the BHAC pathway of the C4 cycle in microalgae, increase the carbon dioxide concentration in the chloroplast, and promote the growth and photosynthetic carbon fixation efficiency of microalgae. In an embodiment of the present invention, it is shown that compared with the Chlamydomonas reinhardtii strain CC-849, the growth rate, biomass, and contents of organic acids and alcohols of the recombinant microalgae strain are significantly increased; after detecting the dry weight, when cultured only with acetate as the carbon source, the recombinant Chlamydomonas reinhardtii strain is increased by 32%; when cultured only with carbon dioxide as the carbon source, the recombinant Chlamydomonas reinhardtii strain is increased by 9%; when using acetate and carbon dioxide simultaneously, the recombinant Chlamydomonas reinhardtii strain is increased by 39%. Description of the Drawings

[0028] Figure 1 It is the plasmid map of the pDb124-cre-AGAT-2A-cre-BHAA recombinant vector;

[0029] Figure 2It is the plasmid profile diagram of the pDh124-cre-BHAD-2A-cre-ISR recombinant vector;

[0030] Figure 3 It is the detection result diagram of the transcriptional level of the recombinant Chlamydomonas reinhardtii strain; A is the detection result of the transcriptional level of the Chlamydomonas reinhardtii strain CC-849: the M lane is the RNA Marker; the 1-5 lanes are the PCR products of the internal reference actin, AGAT PCR product, BHAAPCR product, BHAD PCR product, and ISRPCR product respectively; B is the detection result of the transcriptional level of the recombinant Chlamydomonas reinhardtii strain: the M lane is the RNA Marker; the 1-5 lanes are the PCR products of the internal reference actin, AGAT PCR product, BHAAPCR product, BHAD PCR product, and ISRPCR product respectively;

[0031] Figure 4 It is the growth curve diagram of the recombinant Chlamydomonas reinhardtii strain under different culture conditions;

[0032] Figure 5 It is the detection result diagram of the phenotypic characteristics of the recombinant Chlamydomonas reinhardtii strain under different culture conditions. Detailed implementation mode

[0033] The present invention provides a recombinant microalgae strain, which is a microalgae strain expressing aspartate-glyoxylate aminotransferase, β-hydroxyaspartate aldolase, β-hydroxyaspartate dehydratase, and iminosuccinate reductase.

[0034] In the present invention, the microalgae include marine microalgae and / or freshwater microalgae. The freshwater microalgae preferably include Chlamydomonas reinhardtii. In the embodiment of the present invention, the Chlamydomonas reinhardtii strain CC-849 is used as the host microalgae strain to illustrate the carbon fixation efficiency advantage of the recombinant microalgae strain.

[0035] In the present invention, the aspartate-glyoxylate aminotransferase is derived from rice, GeneBank accession number: WP_011241925.1, and the amino acid sequence is preferably as shown in SEQ ID NO: 1; the β-hydroxyaspartate aldolase is derived from Escherichia coli, GeneBank accession number: WP_011241927.1, and the amino acid sequence is preferably as shown in SEQ ID NO: 2; the β-hydroxyaspartate dehydratase is derived from Escherichia coli, GeneBank accession number: WP_011241926.1, and the amino acid sequence is as shown in SEQID NO: 3; the iminosuccinate reductase is derived from Escherichia coli, GeneBank accession number: AAV97283.1, and the amino acid sequence is preferably as shown in SEQ ID NO: 4.

[0036] In the present invention, the nucleotide sequence of the aspartate-glyoxylate aminotransferase gene is 1,188 bases in length, encoding 396 amino acids with a molecular weight of 42.1 kDa; the nucleotide sequence of the β-hydroxyaspartate aldolase gene is 1,161 bases in length, encoding 387 amino acids with a molecular weight of 41.8 kDa; the nucleotide sequence of the β-hydroxyaspartate dehydratase gene is 951 bases in length, encoding 317 amino acids with a molecular weight of 34.2 kDa. The nucleotide sequence of the iminosuccinic acid reductase gene is 876 bases in length, encoding 292 amino acids with a molecular weight of 30.6 kDa.

[0037] Green plant cells rely on ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco) for CO2 fixation, which produces toxic oxidation product glycolate, reducing photosynthetic efficiency. As a byproduct of photorespiration, glycolate releases CO2 during decomposition but does not generate energy ATP, accounting for nearly 20% of the energy produced by photosynthesis being wasted. In the recombinant microalgae strain of the present invention, aspartate-glyoxylate aminotransferase, β-hydroxyaspartate aldolase, β-hydroxyaspartate dehydratase, and iminosuccinic acid reductase are four key enzymes in the β-hydroxyaspartate cycle (BHAC pathway). Aspartate-glyoxylate aminotransferase catalyzes the oxidative decomposition of glycolate to form glyoxylate and oxalic acid, and efficient β-hydroxyaspartate dehydratase and iminosuccinic acid reductase catalyze the formation of the C4 compound oxaloacetate, increasing the CO2 concentration in the chloroplast while reducing carbon loss. The BHAC pathway constructed by the four enzymes significantly improves the carbon fixation efficiency of the recombinant microalgae strain of the present invention compared to the wild-type strain. The recombinant microalgae strain of the present invention expresses the aspartate-glyoxylate aminotransferase gene (AGAT), β-hydroxyaspartate aldolase gene (BHAA), β-hydroxyaspartate dehydratase (BHAD), and iminosuccinic acid reductase (ISR) in the chloroplast, establishing a new photorespiratory bypass BHAC pathway in the chloroplast, which can convert the photorespiratory byproduct glycolate into oxaloacetate, and then decompose it into carbon dioxide, reducing carbon and nitrogen losses, increasing the carbon dioxide concentration in the chloroplast, effectively improving the carbon fixation efficiency of microalgae, and contributing to the fixation of CO2 into metabolites and bioactive substances such as biomass, pyruvate, starch, and oil. It has broad application prospects in biological carbon fixation, synthesis of microalgae metabolites, and preparation of microalgae bioactive substances.

[0038] The present invention provides a method for constructing the recombinant microalgae strain, comprising the following steps:

[0039] Fusing and expressing the aspartate-glyoxylate aminotransferase gene, β-hydroxyaspartate aldolase gene, β-hydroxyaspartate dehydratase gene, and iminosuccinic acid reductase gene separately or simultaneously with the PsaD promoter in microalgae.

[0040] In the present invention, one, two, three or four genes of the aspartate-glyoxylate aminotransferase gene, β-hydroxyaspartate aldolase gene, β-hydroxyaspartate dehydratase gene, and iminosuccinic acid reductase gene are transformed into microalgae in the form of a recombinant vector with the PsaD promoter.

[0041] In the present invention, the aspartate-glyoxylate aminotransferase gene (cre-AGAT), β-hydroxyaspartate aldolase gene (cre-BHAA), β-hydroxyaspartate dehydratase gene (cre-BHAD), and iminosuccinate reductase gene (cre-ISR) are preferably obtained by optimizing sequences according to the codon preference of Chlamydomonas reinhardtii. The nucleotide sequence of the aspartate-glyoxylate aminotransferase gene is preferably as shown in SEQ ID NO: 5; the nucleotide sequence of the β-hydroxyaspartate aldolase gene is preferably as shown in SEQ ID NO: 6; the nucleotide sequence of the β-hydroxyaspartate dehydratase gene is preferably as shown in SEQ ID NO: 7; the nucleotide sequence of the iminosuccinate reductase gene is preferably as shown in SEQ ID NO: 8.The nucleotide sequence of the PsaD promoter gene is preferably as shown in SEQ ID NO: 9 (GATCCCACACACCTGCCCGTCTGCCTGACAGGAAGTGAACGCATGTCGAGGGAGGCC TCACCAATCGTCACACGAGCCCTCGTCAGAAACACGTCTCCGCCACGCTCTCCCTCTCACGGCCGACCCCGCAGCCCTTTTGCCCTTTCCTAGGCCACCGACAGGACCCAGGCGCTCTCAGCATGCCTCAACAACCCGTACTCGTGCCAGCGGTGCCCTTGTGCTGGTGATCGCTTGGAAGCGCATGCGAAGACGAAGGGGCGGAGCAGGCGGCCTGGCTGTTCGAAGGGCTCGCCGCCAGTTCGGGTGCCTTTCTCCACGCGCGCCTCCACACCTACCGATGCGTGAAGGCAGGCAAATGCTCATGTTTGCCCGAACTCGGAGTCCTTAAAAAGCCGCTTCTTGTCGTCGTTCCGAGACATGTTAGCAGATCGCAGTGCCACCTTTCCTGACGCGCTCGGCCCCATATTCGGACGCAATTGTCATTTGTAGCACAATTGGAGCAAATCTGGCGAGGCAGTAGGCTTTTAAGTTGCAAGGCGAGAGAGCAAAGTGGGACGCGGCGTGATTATTGGTATTTACGCGACGGCCCGGCGCGTTAGCGGCCCTTCCCCCAGGCCAGGGACGATTATGTATCAATATTGTTGCGTTCGGGCACTCGTGCGAGGGCTCCTGCGGGCTGGGGAGGGGGATCTGGGAATTGGAGGTACGACCGAGATGGCTTGCTCGGGGGGAGGTTTCCTCGCCGAGCAAGCCAGGGTTAGGTGTTGCGCTCTTGACTCGTTGTGCATTCTAGGACCCCACTGCTACTCACAACAAGCCAAA). The two adjacent genes are linked by a 2A peptide gene, and the nucleotide sequence of the 2A peptide gene is preferably as shown in SEQ ID NO: 11.

[0042] In the present invention, when constructing a recombinant vector by ligating two of the aspartate-glyoxylate aminotransferase gene, β-hydroxyaspartate aldolase gene, β-hydroxyaspartate dehydratase gene, and iminosuccinic acid reductase gene with the PsaD promoter, it is preferably to ligate the aspartate-glyoxylate aminotransferase gene and the β-hydroxyaspartate aldolase gene to construct a recombinant vector expressing aspartate-glyoxylate aminotransferase and β-hydroxyaspartate aldolase, and ligate the β-hydroxyaspartate dehydratase gene and the iminosuccinic acid reductase gene to construct a recombinant vector expressing β-hydroxyaspartate dehydratase and iminosuccinic acid reductase. The backbone vector of the recombinant vector preferably includes the pDb124 vector and the pDh124 vector. The pDb124 vector contains the PsaD promoter and the bleomycin resistance gene, and the pDh124 vector contains the PsaD promoter and the hygromycin resistance gene. The construction of the pDb124 vector and the pDh124 vector refers to the prior art Wang Y, Jiang X, Hu C, et al. Optogenetic regulation of artificial microRNA improves H2 production in green alga Chlamydomonas reinhardtii[J]. Biotechnology for biofuels, 2017, 10: 1-9.. In the examples of the present invention, the chloroplast targeting peptide gene, aspartate-glyoxylate aminotransferase gene, 2A peptide gene, and β-hydroxyaspartate aldolase gene are ligated in sequence to obtain the fusion gene cre-AGAT-2A-cre-BHAA, and the fusion gene cre-AGAT-2A-cre-BHAA is cloned into the Pml I and Nhe I multiple cloning sites of the backbone vector pDb124 to obtain a recombinant vector (pDb124-cre-AGAT-2A-cre-BHAA) expressing aspartate-glyoxylate aminotransferase and β-hydroxyaspartate aldolase; the chloroplast targeting peptide gene, β-hydroxyaspartate dehydratase gene, 2A peptide gene, and iminosuccinic acid reductase gene are ligated in sequence to obtain the fusion gene cre-BHAD-2A-cre-ISR, and the fusion gene cre-BHAD-2A-cre-ISR is cloned into the Pml I and Nhe I multiple cloning sites of the backbone vector pDh124 to obtain a recombinant vector (pDh124-cre-BHAD-2A-cre-ISR) expressing β-hydroxyaspartate dehydratase and iminosuccinic acid reductase. The present invention does not specifically limit the construction method of the fusion gene, and the conventional fusion gene construction methods in the art can be used.In the embodiments of the present invention, Nanjing Genscript Biotech Co., Ltd. was commissioned to synthesize the fusion gene cre-AGAT-2A-cre-BHAA and the fusion gene cre-BHAD-2A-cre-ISR, and restriction enzyme digestion sites of PmlI and Nhe I were added during the synthesis.

[0043] In the present invention, the fusion expression in microalgae preferably includes microalgae transformation and microalgae screening. The microalgae transformation preferably involves transforming the constructed recombinant vectors pDb124-cre-AGAT-2A-cre-BHAA and pDh124-cre-BHAD-2A-cre-ISR into microalgae. The present invention does not specifically limit the transformation method, and conventional microalgae transformation methods in the art can be used, such as bead milling or electroporation. The microalgae are preferably marine microalgae and / or freshwater microalgae; the freshwater microalgae preferably include Chlamydomonas reinhardtii, and more preferably include the Chlamydomonas reinhardtii strain CC-849. Microalgae screening is performed after the transformation. The microalgae screening is preferably through plate screening, and the culture medium used for the plate screening is preferably a TAP solid medium containing antibiotics. The antibiotics preferably include bleomycin 10 mg / L, hygromycin 10 mg / L, and ampicillin 100 mg / L. After the plate screening, the aspartate-glyoxylate aminotransferase gene, β-hydroxyaspartate aldolase gene, β-hydroxyaspartate dehydratase gene, and iminosuccinate reductase gene in the recombinant microalgae strains are preferably identified by molecular methods. The primers for identifying the aspartate-glyoxylate aminotransferase gene and β-hydroxyaspartate aldolase gene include a forward primer with a nucleotide sequence as shown in SEQ ID NO: 12 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 13; the primers for identifying the β-hydroxyaspartate dehydratase gene and iminosuccinate reductase gene include a forward primer with a nucleotide sequence as shown in SEQ ID NO: 14 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 15. After molecular identification, recombinant microalgae strains with successful transformation are obtained.

[0044] In the present invention, the genes of aspartate-glyoxylate aminotransferase, β-hydroxyaspartate aldolase, β-hydroxyaspartate dehydratase and iminosuccinic acid reductase are respectively or simultaneously fused and expressed with the PsaD promoter in microalgae, which can optimize the photorespiratory bypass, establish the BHAC pathway in microalgae, increase the carbon dioxide concentration, promote the growth and development of microalgae and the photosynthetic carbon fixation efficiency, and promote the synthesis of starch and lipids. The BHAC pathway provided by the present invention has the characteristics of high catalytic efficiency and participation in carbon dioxide formation, and can directly decompose the by-product glycolic acid generated by photorespiration completely into CO2 in the chloroplast, directly increasing the CO2 concentration in the chloroplast, thereby forming a photosynthetic CO2 concentration mechanism similar to the C4 cycle, improving the carbon fixation efficiency, and contributing to the fixation of CO2 into metabolites and biomass such as glucose, sucrose, starch and oil. An embodiment of the present invention shows that compared with the Chlamydomonas reinhardtii strain CC-849, the growth rate, biomass and organic acid content of the recombinant microalgae strain are significantly increased, the sugar content is significantly decreased, and the glycerol content of the recombinant microalgae strain is significantly increased under the culture condition of using 5% CO2. The method of the present invention has broad application prospects in improving the biomass of Chlamydomonas and the synthesis of its metabolites and in increasing the carbon fixation efficiency of microalgae.

[0045] The present invention provides an application of the recombinant microalgae strain or the recombinant microalgae strain constructed by the construction method in CO2 fixation.

[0046] In the present invention, the method for CO2 fixation is to culture the recombinant microalgae strain in an environment containing a carbon source. The carbon source preferably includes organic carbon and / or inorganic carbon for culturing the recombinant microalgae strain. The organic carbon preferably includes acetate. The inorganic carbon preferably includes 5% CO2. The culture medium for culturing the recombinant microalgae strain preferably includes TAP medium or TP medium. During the culture period, 5% CO2 is preferably introduced into the culture medium. The culture medium is beneficial to improving the CO2 fixation performance of the recombinant microalgae strain and promoting the conversion of CO2 by the recombinant microalgae strain into different metabolites. The CO2 fixation preferably includes converting carbon into at least one of the following: biomass, sugars, organic acids and alcohols.

[0047] The recombinant microalgae strain of the present invention can more efficiently increase the carbon dioxide concentration in the chloroplast, improve the photosynthetic efficiency, promote the conversion of carbon dioxide into more biomass, and increase the growth rate, pyruvate content, starch content and glycerol content of Chlamydomonas. Through measurement, the biomass of the recombinant microalgae strain of the present invention is significantly increased, and the pyruvate content, starch content and the number of starch grains are significantly increased. Therefore, the recombinant microalgae strain of the present invention has broad application prospects in the culture of CO2 fixation, improving the biomass of Chlamydomonas, pyruvate content and starch content.

[0048] Based on the fact that the recombinant microalgae strain contains various biomasses, the present invention provides an application of the recombinant microalgae strain obtained by the construction method in the preparation of at least one of the following products: feed, fertilizer, food, cosmetics, biofuel, and bioactive substances.

[0049] The present invention provides a method for improving the carbon fixation efficiency of microalgae, comprising the following steps:

[0050] Express aspartate-glyoxylate aminotransferase, β-hydroxyaspartate aldolase, β-hydroxyaspartate dehydratase, and iminosuccinate reductase in microalgae.

[0051] In the present invention, the amino acid sequence of the aspartate-glyoxylate aminotransferase is preferably as shown in SEQ ID NO: 1; the amino acid sequence of the β-hydroxyaspartate aldolase is preferably as shown in SEQ ID NO: 2; the amino acid sequence of the β-hydroxyaspartate dehydratase is as shown in SEQ ID NO: 3; the amino acid sequence of the iminosuccinate reductase is preferably as shown in SEQ ID NO: 4. The method for expressing the aspartate-glyoxylate aminotransferase, β-hydroxyaspartate aldolase, β-hydroxyaspartate dehydratase, and iminosuccinate reductase in microalgae in the present invention is the same as the construction method of the recombinant microalgae strain in the above technical solution, and will not be elaborated here.

[0052] The present invention can more efficiently increase the carbon dioxide concentration in the chloroplast of microalgae, improve the photosynthetic efficiency, and promote the conversion of carbon dioxide into more biomasses by expressing aspartate-glyoxylate aminotransferase, β-hydroxyaspartate aldolase, β-hydroxyaspartate dehydratase, and iminosuccinate reductase in microalgae. In an embodiment of the present invention, the growth indexes and the contents of sugars, organic acids, and alcohols of the recombinant Chlamydomonas reinhardtii strain were measured. The results showed that compared with the Chlamydomonas reinhardtii strain CC-849 (wild type), the growth rate and biomass of the recombinant microalgae strain were significantly improved. After detecting the dry weight, compared with the wild type strain, when cultured with acetate as the carbon source only (cultured in TAP medium), the recombinant microalgae strain increased by 32%; when cultured with carbon dioxide as the carbon source only (cultured in TP medium + 5% CO2), the recombinant microalgae strain increased by 9%; when using acetate and carbon dioxide simultaneously (cultured in TAP medium + 5% CO2), the recombinant microalgae strain increased by 39%. It shows that expressing aspartate-glyoxylate aminotransferase, β-hydroxyaspartate aldolase, β-hydroxyaspartate dehydratase, and iminosuccinate reductase in microalgae can effectively improve the carbon fixation efficiency of microalgae.

[0053] To further illustrate the present invention, a recombinant algal strain expressing AGAT, BHAA, BHAD, and ISR provided by the present invention, its construction method and application will be described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0054] In this embodiment, the methods used are all operated according to conventional methods unless otherwise specified, and the reagents used are all conventional reagents or reagents prepared according to conventional methods unless otherwise specified.

[0055] Example 1

[0056] The cre-AGAT gene, cre-BHAA gene, cre-BHAD gene, and cre-ISR gene were obtained by screening through database comparison

[0057]

[0058] The reported protein sequence GeneBank accession number of marine Proteus β-hydroxyaspartate aldolase: WP_011241927.1, and the amino acid sequence is as shown in SEQ ID NO: 2 (MKDMTNLDNFEVGFDIPAKPGMDAAEIQTPCLVLDLDALERNVKKMGDYAKAHGMRH RVHGKMHKSVDVAKLQERLGGAVGVCCQKVSEAEVFVRGGIKDVLVSNQVRDPLKIDRLAQLPKLGSRIIVCVDDLANVADLSAAALRHGTELEVLIEIDCGAGRCGVGTTQDVVAIARAVDAAENLKFTGIQAYQGAMQHLDKYEDRKAKLDIAIEMVESAVQGLKAVGLDPELVSGGGTGSYYFEANSGVYNELQCGSYAFMDADYGRILDQNGQRIDKGEWENAMFILTQVMSHAKADKAIVDAGLKAQSVDSGLPFVHGRDDVEYIKCSDEHGVVADPNGVLKVGDKLRLVPGHCDPTANVHDWYVGVRGGKVECVWPVSARGKAY). The coding region length of the β-hydroxyaspartate aldolase gene is 1161bp. Using the online analysis of conserved domains Conserved domains (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi?INPUT_TYPE=live&SEQUENCE=WP_011241927.1) to analyze the protein domain of the deduced amino acid sequence of the cre-BHAA gene, it was found that the protein contains a type III pyridoxal 5-phosphate (PLP)-dependent enzyme low-specificity D-threonine aldolase domain located at 25-384, which is consistent with the characteristics of the β-hydroxyaspartate aldolase gene. The ORF region sequence of the β-hydroxyaspartate aldolase gene was optimized according to the codon preference of Chlamydomonas reinhardtii to obtain the optimized BHAA gene (cre-BHAA gene) nucleotide sequence of the β-hydroxyaspartate aldolase gene, as shown in SEQ ID NO: 6 (ATGAAGGACATGACCAACCTGGACAACTTCGAGGTGGGCTTCGACATCCCGGCCAAGCCCGGCATGGACGCCGCAGAGATTCAAACCCCGTGCCTCGTGCTGGACCTGGATGCACTGGAGCGGAATGTCAAGAAGATGGGCGACTATGCCAAGGCGCATGGCATGCGCCACCGCGTGCACGGCAAGATGCACAAGTCGGTGGACGTGGCTAAGCTACAGGAGCGCCTCGGTGGGGCAGTAGGTGTGTGTTGCCAGAAGGTGTCAGAAGCGGAGGTGTTTGTTAGAGGCGGCATCAAGGACGTTCTAGTGTCCAACCAGGTGCGTGACCCTCTCAAGATCGATCGGCTGGCGCAACTGCCCAAGCTGGGCAGTAGGATCATCGTATGTGTGGACGATTTGGCGAATGTGGCTGACCTGTCGGCGGCGGCGCTGCGCCATGGCACGGAGTTGGAAGTGCTGATCGAGATTGACTGCGGAGCCGGCCGCTGCGGCGTTGGCACCACCCAGGACGTGGTGGCGATTGCGCGCGCCGTGGATGCGGCAGAGAACCTCAAGTTCACGGGAATACAGGCCTACCAGGGGGCGATGCAGCACCTGGACAAGTACGAGGACCGCAAGGCGAAGCTGGACATCGCTATTGAGATGGTGGAGAGCGCGGTGCAGGGCCTTAAGGCCGTCGGTCTTGACCCGGAGCTGGTCTCTGGCGGTGGCACTGGCAGCTACTACTTTGAGGCCAACAGCGGCGTGTACAACGAGCTGCAGTGCGGCTCGTACGCCTTCATGGACGCGGACTACGGCAGGATCCTGGACCAGAACGGCCAGCGGATCGACAAGGGCGAGTGGGAGAACGCCATGTTCATCCTGACGCAGGTGATGAGCCACGCCAAGGCCGATAAGGCTATCGTGGACGCAGGGCTGAAAGCTCAGAGCGTGGACAGCGGGCTGCCGTTCGTGCACGGTCGCGACGACGTGGAGTACATCAAATGCTCCGACGAGCACGGGGTGGTCGCCGACCCCAACGGCGTCCTGAAGGTGGGCGATAAACTGCGGCTGGTGCCGGGCCACTGCGACCCAACAGCGAACGTCCACGACTGGTACGTGGGCGTTCGCGGTGGGAAGGTCGAGTGCGTGTGGCCCGTCTCCGCTCGGGGCAAGGCGTAC) as shown.

[0059] The protein sequence GeneBank accession number of the reported marine Proteus β-hydroxyaspartate dehydratase: WP_011241926.1, and the amino acid sequence is as shown in SEQ ID NO: 3 (MYIPTFEDMLAAHARIEPHIRRTPVRVSDYLNELTGAQLFFKCENFQEPGAFKIRGATNAVFGLDDAQAAKGVATHSSGNHASCLSYAAMLRGIPCNVVMPSTAPQAKKDTVRRYGGVITECAPSTSAREETFARVQAQTGGDFVHPYNDPRVIAGQGTCARELMEQTDGLDIVVAPIGGGGMISGTCLTLSTLAPETRVIAAEPEQADDAYRSFKAGHIIADDAPKTIADGLLVPLKDLTWHFVSNHVAEIYTASEQEIIEAMKLTWKHLRVVMEPSSAVPLATILKNRKAFAGKRVGVIITGGNVDLDKLPWMNG). The coding region length of the β-hydroxyaspartate dehydratase gene is 951 bp. The protein domain analysis of the deduced amino acid sequence of the β-hydroxyaspartate dehydratase gene was performed using the online analysis of conserved domains Conserved domains (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi?INPUT_TYPE=live&SEQUENCE=WP_011241926.1). It was found that the protein contains a PALP-containing domain of the tryptophan synthase β superfamily (fold type ii) located at 2-312, which is consistent with the characteristics of the β-hydroxyaspartate dehydratase gene. The ORF region sequence of the β-hydroxyaspartate dehydratase gene was optimized according to the codon preference of Chlamydomonas reinhardtii to obtain the optimized BHAD gene (cre-BHAD gene).The nucleotide sequence is as shown in SEQ ID NO: 7 (ATGTACATCCCGACCTTCGAGGACATGCTGGCGGCGCACGCGCGCATCGAGCCGCAC ATTCGCCGCACACCAGTTCGTGTGAGCGACTACCTGAACGAGCTCACTGGTGCGCAGCTGTTCTTCAAGTGCGAGAACTTCCAGGAGCCGGGTGCATTCAAGATCCGCGGCGCGACCAACGCAGTGTTCGGACTCGATGACGCCCAGGCGGCCAAGGGGGTGGCTACGCATAGCTCCGGCAACCACGCCAGTTGTCTGAGCTACGCCGCCATGCTTCGGGGCATCCCCTGCAACGTGGTCATGCCGTCGACCGCGCCGCAGGCGAAGAAGGACACGGTGCGGCGCTATGGCGGAGTCATCACGGAGTGCGCGCCAAGCACATCGGCGCGGGAGGAGACGTTTGCGCGTGTGCAGGCCCAGACTGGCGGCGACTTCGTGCACCCCTACAACGACCCGCGGGTAATCGCGGGCCAGGGGACCTGCGCCCGAGAACTGATGGAGCAGACGGACGGGCTGGACATCGTGGTGGCACCTATTGGCGGCGGCGGCATGATCAGCGGCACCTGCCTGACGCTGTCAACACTGGCTCCCGAGACCAGGGTGATTGCAGCGGAACCTGAGCAGGCTGACGACGCCTACCGCAGCTTTAAAGCTGGCCACATCATAGCTGACGATGCGCCCAAGACCATAGCGGACGGCCTGCTGGTGCCCCTAAAGGATTTGACCTGGCACTTCGTCTCCAACCACGTCGCGGAGATTTACACCGCCTCTGAGCAAGAGATTATCGAGGCGATGAAGCTGACCTGGAAGCACCTGCGGGTGGTGATGGAGCCCTCCTCGGCCGTGCCGCTGGCCACCATCCTCAAGAACCGCAAGGCCTTCGCCGGCAAGCGCGTCGGGGTCATCATCACCGGCGGTAATGTGGACCTGGACAAGCTGCCCTGGATGAACGGC).

[0060] The protein sequence GeneBank accession number of the reported marine Proteus iminosuccinate reductase is: AAV97283.1, and the amino acid sequence is as shown in SEQ ID NO: 4 (MAAGTARNFPVIREAIGHADALYGFKSGFDRQALNLGLKSGGYWPGNADKGLTNHQSTVFLFDADTGRCQAVVGGNLLTALRTAAASSVSIKHLARQDAKVIGMIGAGHQAKFQLRAALEQRNFEKVIGWNLHPEMLVNLQEVADEAGLPFEAVELDGMREADVIITITSSFDAILKADQVSPGTHIACMGTDTKGKQEVDPQLLVMADVFTDEVAQSISIGEAQHAVAHGLIQAADVAQIGAVINGTNPGRTSDAQITLFDGTGVGLQDLAVAATVVERARAAGAGTEIAF). The coding region length of the iminosuccinate reductase gene is 876 bp. Using the online analysis of conserved domains Conserved domains (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi?INPUT_TYPE=live&SEQUENCE=AAV97283.1) to analyze the protein domain of the deduced amino acid sequence of the iminosuccinate reductase gene, it was found that this protein contains an iminosuccinate reductase domain located at 1-290, which is consistent with the characteristics of the iminosuccinate reductase gene.The ORF region sequence of the iminosuccinic acid reductase gene was optimized according to the codon preference of Chlamydomonas reinhardtii to obtain the optimized ISR gene (cre-ISR gene), and the nucleotide sequence is as shown in SEQ ID NO: 8 (ATGGCGGCCGGCACCGCCCGCAACTTCCCGGTGATCCGGGAAGCGATCGGACACGCGGACGCACTGTACGGCTTTAAGAGCGGCTTCGACCGCCAGGCTCTCAACCTCGGCTTGAAATCCGGGGGCTACTGGCCCGGCAACGCCGACAAGGGGCTTACAAACCACCAATCAACGGTGTTCCTGTTCGATGCGGACACGGGCCGCTGCCAGGCGGTGGTGGGTGGCAACCTCCTAACGGCGCTGCGCACCGCTGCGGCGAGTTCTGTGTCCATCAAGCACCTGGCCAGGCAGGACGCGAAGGTCATTGGCATGATCGGCGCGGGGCACCAGGCCAAGTTCCAGCTGCGGGCGGCCCTGGAGCAGCGCAATTTCGAGAAGGTCATTGGCTGGAACCTGCATCCGGAGATGCTGGTCAACCTGCAGGAGGTGGCCGACGAGGCAGGTCTGCCCTTTGAGGCTGTGGAGCTGGACGGCATGCGCGAGGCAGACGTAATAATCACCATCACTTCGTCGTTTGACGCCATCCTGAAGGCGGACCAGGTGTCGCCTGGGACGCACATCGCCTGCATGGGCACCGACACCAAGGGCAAGCAGGAGGTCGATCCGCAGCTGCTGGTGATGGCCGACGTGTTCACGGACGAGGTTGCTCAGAGCATCAGCATCGGCGAGGCGCAGCACGCCGTGGCGCACGGCCTGATCCAGGCAGCCGACGTGGCGCAGATCGGTGCCGTCATCAACGGGACCAACCCAGGCCGTACCAGCGATGCGCAGATTACACTTTTCGACGGCACTGGCGTGGGATTGCAAGACCTGGCGGTGGCCGCCACCGTGGTGGAGCGGGCGCGGGCGGCTGGTGCAGGCACGGAGATTGCGTTC).

[0061] Example 2

[0062] Construction method of recombinant Chlamydomonas reinhardtii strain

[0063] The Chlamydomonas reinhardtii strain used in this example is CC-849, which is provided by the Laboratory of Hydrobiology, College of Life and Ocean Sciences, Shenzhen University. It is the same as the strain reported in the prior art Wang Y, Jiang X, Hu C, et al. Optogenetic regulation of artificial microRNA improves H2 production in green alga Chlamydomonas reinhardtii[J]. Biotechnology for biofuels, 2017, 10: 1-9.; The plasmids pDb124 and pDh124 are provided by the Laboratory of Hydrobiology, College of Life and Ocean Sciences, Shenzhen University; The construction of plasmids pDb124 and pDh124 refers to the prior art Wang Y, Jiang X, Hu C, et al. Optogenetic regulation of artificial microRNA improves H2 production in green alga Chlamydomonas reinhardtii[J]. Biotechnology for biofuels, 2017, 10: 1-9..

[0064] The nucleotide sequence of the 2A peptide gene is shown in SEQ ID NO: 11 (gtgaagcagaccctgaacttcgacctgctgaagctggcgggcgacgtggagagcaaccccggcccc); The amino acid sequence of the 2A peptide is shown in SEQ ID NO: 10 (VKQTLNFDLLKLAGDVESNPGP).

[0065] 1) Construction of pDb124-cre-AGAT-2A-cre-BHAA recombinant vector

[0066] Entrust GenScript Biotech Corporation in Nanjing to synthesize the fusion gene cre-AGAT-2A-cre-BHAA of aspartate-glyoxylate aminotransferase gene, 2A peptide gene and β-hydroxyaspartate aldolase gene with Pml I and Nhe I restriction enzyme sites, and respectively ligate them into the NdeI, SpeI sites, KspAI and PmeI sites as shown in Figure 1 pDb124

[0067] The fusion gene cre-AGAT-2A-cre-BHAA and the plasmid pDb124 were digested with Pml I and Nhe I and then ligated to obtain the recombinant vector pDb124-cre-AGAT-2A-cre-BHAA.

[0068] 2) Construction of the recombinant vector pDh124-cre-BHAD-2A-cre-ISR

[0069] The fusion gene cre-BHAD-2A-cre-ISR containing the β-hydroxyaspartate dehydratase gene, 2A peptide gene, and iminosuccinate reductase gene with Pml I and Nhe I restriction sites was synthesized by Nanjing Genscript Biotech Co., Ltd. and ligated into pDh124 at the NdeI, SpeI sites, KspAI, and PmeI sites as shown. Figure 1 as shown.

[0070] The fusion gene cre-BHAD-2A-cre-ISR and the plasmid pDh124 were digested with Pml I and Nhe I and then ligated to obtain the recombinant vector pDh124-cre-BHAD-2A-cre-ISR.

[0071] 3) Construction of recombinant Chlamydomonas reinhardtii strains

[0072] Aspartate-glyoxylate aminotransferase, β-hydroxyaspartate aldolase, β-hydroxyaspartate dehydratase, and iminosuccinate reductase are four key enzymes in the BHAC pathway. The recombinant vectors pDb124-cre-AGAT-2A-cre-BHAA and pDh124-cre-BHAD-2A-cre-ISR contain four key essential genes of the BHAC pathway.

[0073] The constructed recombinant vector pDb124-cre-AGAT-2A-cre-BHAA and the recombinant vector pDh124-cre-BHAD-2A-cre-ISR were transformed into Chlamydomonas reinhardtii. Since the pDb-124 plasmid and the pDh-124 plasmid contain the bleomycin resistance gene and the hygromycin resistance gene respectively, the pDb-124 plasmid has bleomycin resistance and the pDh-124 plasmid has hygromycin resistance. Positive algal strains successfully transformed can be screened by bleomycin and hygromycin. The specific steps for constructing recombinant Chlamydomonas reinhardtii strains are as follows.

[0074] The recombinant vector pDb124-cre-AGAT-2A-cre-BHAA and the recombinant vector pDh124-cre-BHAD-2A-cre-ISR were co-transformed into the Chlamydomonas reinhardtii strain CC-849 by the glass bead transformation method. Specifically, the Chlamydomonas reinhardtii cells CC-849 cultured to the logarithmic growth phase were centrifuged at 5000 rpm at room temperature to collect the cells, and the cell concentration was adjusted to 1×10 8 cell / mL as competent cells. Pipette 500 μL of Chlamydomonas reinhardtii competent cells into an EP tube containing 0.3 g of glass beads, and pipette 1.5 μg of the recombinant vector pDb124-cre-AGAT-2A-cre-BHAA linearized with NotI and 1.5 μg of the recombinant vector pDh124-cre-BHAD-2A-cre-ISR linearized with NotI into the EP tube; place the EP tube on a high-speed vortex mixer and vortex at high speed for 25 s; add 10 mL of antibiotic-free TAP liquid medium to the EP tube in a laminar flow hood, and resuscitate at 22 °C and 110 rpm for 20 h; centrifuge at 3000 rpm for 5 min to remove all the supernatant medium;

[0075] Resuspend the precipitated algal cells with 200 μL of fresh TAP liquid medium, and spread them on a TAP plate containing bleomycin (concentration 10 mg / L), hygromycin (concentration 10 mg / L), and ampicillin (concentration 100 mg / L), and culture at 22 °C for 10 - 14 d;

[0076] Pick monoclonal colonies on the plate and transfer them to a new TAP solid plate containing bleomycin (concentration 10 mg / L), hygromycin (concentration 10 mg / L), and ampicillin (concentration 100 mg / L), and culture overnight at 22 °C and 110 rpm in TAP liquid medium;

[0077] Pipette 1 mL of the algal solution and inoculate it into a triangular flask containing 50 mL of liquid TAP medium, and shake the algae at 22 °C and 110 rpm until the logarithmic growth phase to obtain the recombinant Chlamydomonas reinhardtii strain.

[0078] 4) DNA-level PCR screening of the recombinant Chlamydomonas reinhardtii strain

[0079] According to the sequence information of AGAT-BHAA on the provided vector, the forward primer AR-F and the reverse primer AR-R were designed. According to the sequence information of BHAD-ISR, the forward primer BI-F and the reverse primer BI-R were designed. The sequence list of the specific primers is shown in Table 1.

[0080] Table 1 Nucleotide sequences of the primers

[0081] Name Nucleotide sequence AR-F 5’-CGGAGTCCGTGCTGTTCTGA-3’(SEQ ID NO: 12) AR-R 5’-GGCACCAGCCGCAGTTTAT-3’(SEQ ID NO: 13) BI-F 5’-TGTGAGCGACTACCTGAACGAGC-3’(SEQ ID NO: 14) BI-R 5’-ATCTCCGTGCCTGCACCAG-3’(SEQ ID NO: 15)

[0082] The detection method is as follows: Pick monoclonal algal cells and culture them in liquid TAP medium for expansion, and then centrifuge to collect the algal cells; Use differential centrifugation to collect Chlamydomonas reinhardtii cell samples; Use a kit to extract the DNA sample of Chlamydomonas reinhardtii cells; Use the designed primer pairs to perform PCR amplification on the DNA sample.

[0083] The results of PCR amplification showed that there was no foreign gene in CC-849, AGAT-BHAA and BHAD-ISR could not be amplified, and the recombinant Chlamydomonas reinhardtii strains amplified the bands of AGAT-BHAA and BHAD-ISR, indicating that the genes of AGAT, BHAA, BHAD and ISR were inserted into the nuclear genes of Chlamydomonas reinhardtii.

[0084] 3) Transcriptional level PCR screening of the engineered Chlamydomonas reinhardtii strain BHAC

[0085] Transcriptional level PCR screening of the recombinant Chlamydomonas reinhardtii strains

[0086] The synthesis of cDNA was first carried out in reverse transcription buffer, and then 1 / 10 of the reaction product was taken out for PCR. By comparing the brightness of the PCR bands, the strength of the gene-expressed mRNA can be known, and the actin PCR amplification product was set as the internal reference control.

[0087] The detection method is as follows: Culture the algal cells and centrifuge to collect the algal cells; Use the TransZol method to extract the total RNA of the sample; Reverse transcribe the RNA sample with a reverse transcription kit to obtain cDNA; Use specific primers to amplify ATAG, BHAA, BHAD, ISR and actin, and the sequence list of the specific primers is shown in Table 2; Use a PCR instrument for amplification; Observe the brightness of the PCR product bands in the gel imaging system.

[0088] The results are shown in Figure 3 . According to Figure 3 It can be known that the genes of AGAT, BHAA, BHAD and ISR were not amplified in the Chlamydomonas reinhardtii strain CC-849, and the recombinant Chlamydomonas reinhardtii strains amplified the genes of AGAT, BHAA, BHAD and ISR, indicating that the construction of the recombinant Chlamydomonas reinhardtii strains was successful.

[0089] Table 2 Nucleotide sequences of the primers

[0090]

[0091] Example 3

[0092] Detection of phenotypic characteristics of the recombinant Chlamydomonas reinhardtii strains (TAP)

[0093] 1) Detection of growth indexes of the Chlamydomonas reinhardtii strains

[0094] The recombinant Chlamydomonas reinhardtii strain or Chlamydomonas reinhardtii strain CC-849 was inoculated into antibiotic-free TAP liquid medium and cultured with shaking at 22 °C and 110 rpm until the stationary phase;

[0095] During the culture, 200 μL of the algal solution was taken every 24 h and placed in a 96-well plate with three replicates. It was detected by an enzyme-linked immunosorbent assay (ELISA) reader, shaken three times, and the OD 750 was recorded, and the OD 750 absorbance value was used to plot the growth curve;

[0096] After culturing to the stationary phase, 50 mL of the algal solution was collected, centrifuged at 5000 rpm for 5 min at 4 °C to collect the algal cells. After washing the cells twice with distilled water, they were placed in a freeze dryer and freeze-dried to a constant weight, and the dry weight was weighed.

[0097] Upon analysis, CO2 was efficiently converted into biomass in the recombinant Chlamydomonas reinhardtii strain engineering strain. According to the analysis of the plotted growth curve, its growth rate was significantly increased by 1.15 times. According to the analysis of the weighed dry weight, its dry weight was 0.044 g.

[0098] 2) Detection of Chlamydomonas reinhardtii products

[0099] The recombinant Chlamydomonas reinhardtii strain or Chlamydomonas reinhardtii strain CC-849 was inoculated into antibiotic-free TAP liquid medium and cultured with shaking at 22 °C and 110 rpm until the logarithmic growth phase, and then the algal cells were collected. The contents of starch, pyruvate, glucose, sucrose, and glycerol in the algal strain were measured respectively.

[0100] The starch content was detected using a starch detection kit (Beijing Solarbio Science & Technology Co., Ltd.), and the specific detection is as follows.

[0101] 50 mL of the algal solution was taken, centrifuged at 5000 rpm for 5 min at 4 °C to collect the algal cells. After washing the cells twice with distilled water, they were placed in a freeze dryer and freeze-dried overnight. 0.1 g of the dried bacterial powder was weighed and placed in a 2.0 mL centrifuge tube, 1 mL of reagent one was added and homogenized thoroughly, and extracted in a water bath at 80 °C for 30 min; at room temperature, centrifuged at 8000 rpm for 5 min to collect the algal cells; 0.5 mL of distilled water was added to resuspend, gelatinized in a boiling water bath for 15 min, cooled, 0.35 mL of reagent two was added, and extracted at room temperature for 15 min, shaken 3 - 5 times; 0.85 mL of double-distilled water was added, mixed well, centrifuged at 3000 g at room temperature for 10 min; 0.2 mL of the supernatant and 1 mL of reagent three working solution were taken into an EP tube, incubated in a water bath at 95 °C for 10 min, cooled naturally to room temperature, and the absorbance value A was recorded at a wavelength of 620 nm.

[0102] Upon analysis, CO2 was efficiently fixed as starch in the recombinant Chlamydomonas reinhardtii strain. Calculated using the standard curve, its starch content was 6.19 g / 100 g of algal cell dry weight.

[0103] The pyruvate content in the algal strain was detected by using the Pyruvate Assay Kit (Nanjing Jiancheng Bioengineering Institute, A081-1-1, China) to extract and determining the pyruvate content by ultraviolet spectrophotometry; the glycerol content in the algal strain was detected by using the Glycerol Assay Kit (Nanjing Jiancheng Bioengineering Institute, F005-2-1, China) to extract and determining the glycerol content by ultraviolet spectrophotometry; the glucose content in the algal strain was detected by using the Glucose Kit (Nanjing Jiancheng Bioengineering Institute, A154-2-1, China) to extract and determining the glucose content of the algal powder by ultraviolet spectrophotometry. The sucrose content in the algal strain was detected by using the Sucrose Measurement Kit (Nanjing Jiancheng Bioengineering Institute, A099-1-1, China) to extract and determining the sucrose content of the algal powder by ultraviolet spectrophotometry.

[0104] Example 4

[0105] Phenotypic characterization of the recombinant Chlamydomonas reinhardtii algal strain cultured with 5% CO2 (TP + 5% CO2)

[0106] 1) Detection of the growth index of the Chlamydomonas reinhardtii algal strain

[0107] The recombinant Chlamydomonas reinhardtii algal strain or the Chlamydomonas reinhardtii algal strain CC-849 was cultured in an antibiotic-free TP liquid medium with 5% CO2 bubbled in at 22 °C and 110 rpm until the stationary phase;

[0108] During the culture period, 200 μL of the algal solution was taken every 24 h and placed in a 96-well plate with three replicates, and then detected by a microplate reader with shaking three times to detect the OD 750 , and the OD 750 absorbance value was recorded to plot the growth curve;

[0109] After culturing to the stationary phase, 50 mL of the algal solution was collected, and the algal cells were collected by centrifugation at 5000 rpm for 5 min at 4 °C. After washing the cells twice with distilled water, they were placed in a freeze dryer and freeze-dried to constant weight, and then the dry weight was weighed.

[0110] After analysis, CO2 in the recombinant Chlamydomonas reinhardtii algal strain was efficiently converted into biomass. According to the analysis of the plotted growth curve, there was no significant difference in the growth rate. According to the analysis of the weighed dry weight, its dry weight was 0.044 g.

[0111] 2) Detection of Chlamydomonas reinhardtii products

[0112] The recombinant Chlamydomonas reinhardtii strain or Chlamydomonas reinhardtii strain CC-849 was inoculated into an antibiotic-free TP liquid medium, and 5% CO2 was introduced. The culture was shaken at 22 °C and 110 rpm until the logarithmic growth phase; the contents of starch, pyruvate, glucose, sucrose, and glycerol were detected according to the method in Example 3.

[0113] After analysis, CO2 in the recombinant Chlamydomonas reinhardtii strain was efficiently fixed as starch. Calculation was performed using the standard curve, and its starch content was 11.14 g / 100 g dry weight of algal cells.

[0114] Example 5

[0115] Detection of phenotypic characteristics of the recombinant Chlamydomonas reinhardtii strain using acetate and 5% CO2 (TAP + 5% CO2)

[0116] 1) Detection of growth indicators of Chlamydomonas reinhardtii strain

[0117] The recombinant Chlamydomonas reinhardtii strain or Chlamydomonas reinhardtii strain CC-849 was cultured in an antibiotic-free TAP liquid medium with 5% CO2 introduced, and shaken at 22 °C and 110 rpm until the plateau phase;

[0118] During the culture, 200 μL of the algal solution was aspirated every 24 h and placed in a 96-well plate with three replicates. It was detected using a microplate reader, shaken three times, and the OD 750 was recorded, and the OD 750 absorbance value was recorded to plot the growth curve;

[0119] After culturing to the plateau phase, 50 mL of the algal solution was collected, centrifuged at 5000 rpm for 5 min at 4 °C to collect the algal cells. The cells were washed twice with distilled water and then freeze-dried in a freeze dryer until constant weight, and the dry weight was weighed.

[0120] After analysis, CO2 in the recombinant Chlamydomonas reinhardtii strain was efficiently converted into biomass. According to the analysis of the plotted growth curve, its growth rate was significantly increased by 2.78 times. According to the analysis of the weighed dry weight, its dry weight was 0.11 g.

[0121] 2) Detection of Chlamydomonas reinhardtii products

[0122] The recombinant Chlamydomonas reinhardtii strain or Chlamydomonas reinhardtii strain CC-849 was inoculated into an antibiotic-free TAP liquid medium, and 5% CO2 was introduced. The culture was shaken at 22 °C and 110 rpm until the logarithmic growth phase; the contents of starch, pyruvate, glucose, sucrose, and glycerol were detected according to the method in Example 3.

[0123] After analysis, CO2 in the recombinant Chlamydomonas reinhardtii strain was efficiently fixed as starch. Calculation was performed using the standard curve, and its starch content was 11.77 g / 100 g dry weight of algal cells.

[0124] Figure 4 and Figure 5 are the growth curve graphs of the recombinant Chlamydomonas reinhardtii strains under different culture conditions and the detection result graphs of dry weight, pyruvate content, glucose content, sucrose content, starch content, and lipid content. Among them, WT represents the Chlamydomonas reinhardtii strain CC-849, and BHAC38 represents the recombinant Chlamydomonas reinhardtii strain. According to Figure 4 and Figure 5 , it can be seen that compared with the Chlamydomonas reinhardtii strain CC-849, the growth rate, biomass, and content of organic acids of the recombinant microalgae strain have all increased significantly, the sugar content has decreased significantly, and under the culture condition of using 5% CO2, the glycerol content of the recombinant microalgae strain has increased significantly.

[0125] In summary, through the Chlamydomonas reinhardtii eukaryotic expression system, the present invention verifies its function and discovers that aspartate-glyoxylate aminotransferase, β-hydroxyaspartate aldolase, β-hydroxyaspartate dehydratase, and iminosuccinic acid reductase can efficiently fix and convert CO2 into biomass and starch. The aspartate-glyoxylate aminotransferase gene, β-hydroxyaspartate aldolase gene, β-hydroxyaspartate dehydratase gene, and iminosuccinic acid reductase gene in the recombinant Chlamydomonas reinhardtii strain of the present invention participate in the synthetic metabolism of CO2, significantly improving the CO2 conversion efficiency in the chloroplast of Chlamydomonas reinhardtii and promoting the synthesis of starch, and having a wide application prospect in improving photosynthetic efficiency and carbon fixation.

[0126] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. Other embodiments can also be obtained based on this embodiment without creative efforts, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A recombinant microalgae strain, characterized in that, Microalgae strains expressing aspartate-glyoxylate aminotransferase, β-hydroxyaspartate aldolase, β-hydroxyaspartate dehydratase, and iminosuccinic acid reductase.

2. The recombinant microalgae strain according to claim 1, characterized in that, The amino acid sequence of the aspartate-glyoxylate aminotransferase is shown in SEQ ID NO: 1; The amino acid sequence of the β-hydroxyaspartate aldolase is shown in SEQ ID NO: 2; The amino acid sequence of the β-hydroxyaspartate dehydratase is shown in SEQ ID NO: 3; The amino acid sequence of the iminosuccinic acid reductase is shown in SEQ ID NO:

4.

3. The recombinant microalgae strain according to claim 1 or 2, characterized in that, The microalgae include marine microalgae and / or freshwater microalgae; Preferably, the freshwater microalgae include Chlamydomonas reinhardtii.

4. A method for constructing the recombinant microalgae strain according to any one of claims 1 to 3, characterized in that, Comprising the following steps: Fusion expression of the aspartate-glyoxylate aminotransferase gene, β-hydroxyaspartate aldolase gene, β-hydroxyaspartate dehydratase gene, and iminosuccinic acid reductase gene separately or simultaneously with the PsaD promoter in microalgae.

5. The construction method according to claim 4, wherein One, two, three, or four genes of the aspartate-glyoxylate aminotransferase gene, β-hydroxyaspartate aldolase gene, β-hydroxyaspartate dehydratase gene, and iminosuccinic acid reductase gene are transformed into microalgae in the form of a recombinant vector with the PsaD promoter.

6. The construction method according to claim 5, characterized in that, The recombinant vector includes a recombinant vector expressing aspartate-glyoxylate aminotransferase and β-hydroxyaspartate aldolase and a recombinant vector of β-hydroxyaspartate dehydratase and iminosuccinic acid reductase.

7. Use of the recombinant microalgae strain according to any one of claims 1 to 3 or the recombinant microalgae strain constructed by the construction method according to any one of claims 4 to 6 in CO2 fixation.

8. Use of the recombinant microalgae strain according to any one of claims 1 to 3 or the recombinant microalgae strain constructed by the construction method according to any one of claims 4 to 6 in the preparation of at least one of the following products: feed, fertilizer, food, cosmetics, biofuel, and bioactive substances.

9. A method for improving the carbon fixation efficiency of microalgae, characterized in that, Comprising the following steps: Expressing aspartate-glyoxylate aminotransferase, β-hydroxyaspartate aldolase, β-hydroxyaspartate dehydratase, and iminosuccinic acid reductase in microalgae.

10. The method according to claim 9, wherein The amino acid sequence of the aspartate-glyoxylate aminotransferase is shown in SEQ ID NO: 1; The amino acid sequence of the β-hydroxyaspartate aldolase is shown in SEQ ID NO: 2; The amino acid sequence of the β-hydroxyaspartate dehydratase is shown in SEQ ID NO: 3; The amino acid sequence of the iminosuccinic acid reductase is shown in SEQ ID NO: 4.

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