Recombinant algal strain for expressing glycolate oxidase, catalase, glyoxylate polyaldolase and hydroxymalonate semialdehyde reductase as well as construction method and application of recombinant algal strain

Abstract

The invention provides a recombinant algal strain for expressing glycollate oxidase, catalase, glyoxylate polyaldolase and hydroxymalonate semialdehyde reductase as well as a construction method and application of the recombinant algal strain, and belongs to the technical field of genetic engineering. The invention provides a recombinant microalgae strain, which is a microalgae strain for expressing glycollic acid oxidase, catalase, glyoxylic acid polyaldolase and hydroxymalonic semialdehyde reductase in chloroplast. 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 specifically relates to a recombinant algal strain expressing glycolate oxidase, catalase, glyoxylate polyaldehyde enzyme and hydroxymalonic semialdehyde reductase, 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 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. 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 genetically modifies microalgae cells at the molecular level by applying genetic engineering means. Among them, constructing genetically 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 microalgae cell growth rate. Summary of the Invention

[0004] In view of this, the present invention provides a recombinant microalgae strain expressing glycolate oxidase, catalase, glyoxylate polyaldehyde enzyme and hydroxymalonic semialdehyde reductase. The new photorespiratory bypass GCGT pathway is introduced into the recombinant microalgae strain, which can achieve high-efficiency carbon fixation and has a high microalgae cell growth rate.

[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 glycolate oxidase, catalase, glyoxylate polyaldehyde enzyme and hydroxymalonic semialdehyde reductase in chloroplasts.

[0007] Preferably, the amino acid sequence of the glycolate oxidase is as shown in SEQ ID NO: 1;

[0008] The amino acid sequence of the catalase is as shown in SEQ ID NO: 2;

[0009] The amino acid sequence of the glyoxylate polyaldehyde enzyme is as shown in SEQ ID NO: 3;

[0010] The amino acid sequence of the hydroxymalonic semialdehyde 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] Fusion expression of the glycolate oxidase gene, catalase gene, glyoxylate polyaldehyde enzyme gene and hydroxymalonic semialdehyde reductase gene respectively or simultaneously with the PsaD promoter and the chloroplast transit peptide gene in microalgae.

[0015] Preferably, one, two, three or four genes of the glycolate oxidase gene, catalase gene, glyoxylate polyaldehyde enzyme gene and hydroxymalonic semialdehyde reductase gene are transformed into microalgae in the form of a recombinant vector together with the PsaD promoter and the chloroplast transit peptide gene.

[0016] Preferably, the recombinant vector includes a recombinant vector expressing glycolate oxidase and catalase and a recombinant vector expressing glyoxylate polyaldehyde enzyme and hydroxymalonic semialdehyde reductase.

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

[0018] The present invention provides an 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 substance.

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

[0020] Express glycolate oxidase, catalase, glyoxylate polyaldehyde enzyme and hydroxymalonic semialdehyde reductase in the chloroplast of microalgae.

[0021] Preferably, the amino acid sequence of the glycolate oxidase is as shown in SEQ ID NO: 1;

[0022] The amino acid sequence of the catalase is as shown in SEQ ID NO: 2;

[0023] The amino acid sequence of the glyoxylate polyaldehyde enzyme is as shown in SEQ ID NO: 3;

[0024] The amino acid sequence of the hydroxymalonic semialdehyde 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 that expresses glycolate oxidase, catalase, glyoxylate polyaldehyde enzyme and hydroxymalonic semialdehyde reductase in chloroplasts. In the recombinant microalgae strain of the present invention, glycolate oxidase catalyzes the oxidative decomposition of glycolate to form glyoxylate and oxalic acid, and the highly efficient glyoxylate polyaldehyde enzyme and hydroxymalonic semialdehyde reductase catalyze the formation of fatty acids and CO2, increasing the CO2 concentration in chloroplasts while reducing carbon loss. Catalase decomposes excessive H2O2 to assist in maintaining cell homeostasis. The recombinant microalgae strain can decompose the photorespiratory by-product glycolate into carbon dioxide, increase the carbon dioxide concentration in chloroplasts, 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 oils. 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, which includes the following steps: fusing and expressing the glycolate oxidase gene, catalase gene, glyoxylate polyaldehyde enzyme gene and hydroxymalonic semialdehyde reductase gene separately or simultaneously with the PsaD promoter and the chloroplast transit peptide gene in microalgae. The method of the present invention can optimize the photorespiratory bypass of microalgae, establish the GCGT pathway in chloroplasts, transfer the CO2 originally released in mitochondria to be released in chloroplasts, reduce the loss caused by photorespiration and form a CO2 concentration mechanism similar to that of C4 plants, thereby increasing the carbon dioxide concentration and promoting the growth and development of microalgae and the photosynthetic carbon fixation efficiency. 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 all significantly improved; after detecting OD750, when cultured only with acetate as the carbon source, the dry cell weight of the recombinant Chlamydomonas reinhardtii strain is increased by 42.1%; when cultured only with carbon dioxide as the carbon source, the dry cell weight of the recombinant Chlamydomonas reinhardtii strain is increased by 39.5%; when using acetate and carbon dioxide simultaneously, the dry cell weight of the recombinant Chlamydomonas reinhardtii strain is increased by 27.6%. Description of the Drawings

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

[0029] Figure 2 It is the plasmid map of the pDh124-cre-GCL*cre-TSR recombinant vector;

[0030] Figure 3 Figure for detecting the transcriptional level of the recombinant Chlamydomonas reinhardtii strain; A shows the detection result of the transcriptional level of the Chlamydomonas reinhardtii strain CC-849: Lane M is the RNA Marker; Lanes 1-5 are the PCR products of the internal reference actin, GLO, CAT, GCL, and TSR respectively; B shows the detection result of the transcriptional level of the recombinant Chlamydomonas reinhardtii strain: Lane M is the RNA Marker; Lanes 1-5 are the PCR products of the internal reference actin, GLO, CAT, GCL, and TSR respectively;

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

[0032] Figure 5 Figure for the detection result 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 glycolate oxidase, catalase, glyoxylate polyaldehyde enzyme, and hydroxymalonic semialdehyde reductase in the chloroplast.

[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 advantage of the carbon fixation efficiency of the recombinant microalgae strain.

[0035] In the present invention, the glycolate oxidase is derived from rice, GeneBank accession number: NP_001389210.1, and the amino acid sequence is preferably as shown in SEQ ID NO: 1; the catalase is derived from Escherichia coli, GeneBank accession number: WP_000077872.1, and the amino acid sequence is preferably as shown in SEQ ID NO: 2; the glyoxylate polyaldehyde enzyme is derived from Escherichia coli, GeneBank accession number: WP_061352092.1, and the amino acid sequence is as shown in SEQ ID NO: 3; the hydroxymalonic semialdehyde reductase is derived from Escherichia coli, GeneBank accession number: WP_021571777.1, and the amino acid sequence is preferably as shown in SEQ ID NO: 4.

[0036] In the present invention, the nucleotide sequence of the glycolic acid oxidase gene is 1107 bases in length, encoding 369 amino acids with a molecular weight of 40.4 kDa; the nucleotide sequence of the catalase gene is 2259 bases in length, encoding 753 amino acids with a molecular weight of 84.16 kDa; the nucleotide sequence of the glyoxylate polyaldehyde enzyme gene is 1779 bases in length, encoding 593 amino acids with a molecular weight of 64.75 kDa. The nucleotide sequence of the hydroxymalonic semialdehyde reductase gene is 879 bases in length, encoding 293 amino acids with a molecular weight of 30.99 kDa.

[0037] Photorespiration is a side reaction of photosynthesis that consumes 20 - 30% of the energy. It occurs in all photosynthetic cells under light and high-oxygen, low-carbon dioxide conditions, where glycolic acid is formed as a by-product. During its decomposition, CO2 is released, but no energy ATP is produced, consuming nearly 20% of the energy generated by photosynthesis. In the recombinant microalgae strain of the present invention, glycolic acid oxidase, catalase, glyoxylate polyaldehyde enzyme, and hydroxymalonic semialdehyde reductase are four key enzymes in the GCGT pathway. Glycolic acid oxidase catalyzes the oxidative decomposition of glycolic acid to form glyoxylate and oxalic acid. Efficient glyoxylate polyaldehyde enzyme and hydroxymalonic semialdehyde reductase catalyze the formation of fatty acids and CO2, increasing the CO2 concentration in the chloroplast while reducing carbon loss. Catalase decomposes excess H2O2 to assist in maintaining cell homeostasis. The GCGT pathway constructed by these 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 glycolic acid oxidase gene (GLO), catalase gene (CAT), glyoxylate polyaldehyde enzyme (GCL), and hydroxymalonic semialdehyde reductase (TSR) in the chloroplast to establish a new photorespiratory bypass GCGT pathway in the chloroplast, which can decompose the photorespiratory by-product glycolic acid 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, organic acids, starch, and oils. 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] Fusion expression of the glycolic acid oxidase gene, catalase gene, glyoxylate polyaldehyde enzyme gene, and hydroxymalonic semialdehyde reductase gene separately or simultaneously with the PsaD promoter and the chloroplast transit peptide gene in microalgae.

[0040] In the present invention, one, two, three or four genes of the glycollate oxidase gene, catalase gene, glyoxylate polyaldehyde enzyme gene, hydroxymalonic semialdehyde reductase gene are transformed into microalgae in the form of a recombinant vector together with the PsaD promoter and the chloroplast transit peptide gene.

[0041] In the present invention, it is preferred to optimize the sequences according to the codon preference of Chlamydomonas reinhardtii to obtain the glycolate oxidase gene (cre-GLO), catalase gene (cre-CAT), glyoxylate polyaldehyde enzyme gene (cre-GCL), and hydroxymalonic semialdehyde reductase gene (cre-TSR). The nucleotide sequence of the glycolate oxidase gene is preferably as shown in SEQ ID NO: 5; the nucleotide sequence of the catalase gene is preferably as shown in SEQ ID NO: 6; the nucleotide sequence of the glyoxylate polyaldehyde enzyme gene is preferably as shown in SEQ ID NO: 7; the nucleotide sequence of the hydroxymalonic semialdehyde reductase gene is preferably as shown in SEQ ID NO: 8.The nucleotide sequence of the PsaD promoter gene is as shown in SEQ ID NO: 13 (GATCCCACACACCTGCCCGTCTGCCTGACAGGAAGTGAACGCATGTCGAGGGAGGCCTCACCAATCGTCACACGAGCCCTCGTCAGAAACACGTCTCCGCCACGCTCTCCCTCTCACGGCCGACCCCGCAGCCCTTTTGCCCTTTCCTAGGCCACCGACAGGACCCAGGCGCTCTCAGCATGCCTCAACAACCCGTACTCGTGCCAGCGGTGCCCTTGTGCTGGTGATCGCTTGGAAGCGCATGCGAAGACGAAGGGGCGGAGCAGGCGGCCTGGCTGTTCGAAGGGCTCGCCGCCAGTTCGGGTGCCTTTCTCCACGCGCGCCTCCACACCTACCGATGCGTGAAGGCAGGCAAATGCTCATGTTTGCCCGAACTCGGAGTCCTTAAAAAGCCGCTTCTTGTCGTCGTTCCGAGACATGTTAGCAGATCGCAGTGCCACCTTTCCTGACGCGCTCGGCCCCATATTCGGACGCAATTGTCATTTGTAGCACAATTGGAGCAAATCTGGCGAGGCAGTAGGCTTTTAAGTTGCAAGGCGAGAGAGCAAAGTGGGACGCGGCGTGATTATTGGTATTTACGCGACGGCCCGGCGCGTTAGCGGCCCTTCCCCCAGGCCAGGGACGATTATGTATCAATATTGTTGCGTTCGGGCACTCGTGCGAGGGCTCCTGCGGGCTGGGGAGGGGGATCTGGGAATTGGAGGTACGACCGAGATGGCTTGCTCGGGGGGAGGTTTCCTCGCCGAGCAAGCCAGGGTTAGGTGTTGCGCTCTTGACTCGTTGTGCATTCTAGGACCCCACTGCTACTCACAACAAGCCAAA); the nucleotide sequence of the chloroplast transit peptide gene is preferably as shown in SEQ ID NO: 9. Two adjacent genes are connected by a 2A peptide gene or (Gly4Ser)3. The nucleotide sequence of the 2A peptide gene is preferably as shown in SEQ ID NO: 11; the nucleotide sequence of (Gly4Ser)3 is preferably as shown in SEQ ID NO: 14.

[0042] In the present invention, when two genes among the glycolic acid oxidase gene, catalase gene, glyoxylate polyaldehyde enzyme gene, and hydroxymalonic semialdehyde reductase gene are constructed into a recombinant vector with the PsaD promoter and chloroplast transit peptide gene, it is preferred to ligate the glycolic acid oxidase gene and the catalase gene to construct a recombinant vector expressing glycolic acid oxidase and catalase, and ligate the glyoxylate polyaldehyde enzyme gene and the hydroxymalonic semialdehyde reductase gene to construct a recombinant vector expressing glyoxylate polyaldehyde enzyme and hydroxymalonic semialdehyde 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 transit peptide gene, glycolic acid oxidase gene, 2A peptide gene, and catalase gene are sequentially ligated to obtain the fusion gene CTP-cre-GLO-2A-cre-CAT, and the fusion gene CTP-cre-GLO-2A-cre-CAT is cloned into the Pml I and Nhe I multiple cloning sites of the backbone vector pDb124 to obtain a recombinant vector expressing glycolic acid oxidase and catalase (pDb124-cre-GLO-2A-cre-CAT); the chloroplast transit peptide gene, glyoxylate polyaldehyde enzyme gene, (Gly4Ser)3, and hydroxymalonic semialdehyde reductase gene are sequentially ligated to obtain the fusion gene CTP-cre-GCL*cre-TSR, and the fusion gene CTP-cre-GCL*cre-TSR is cloned into the PmlI and Nhe I multiple cloning sites of the backbone vector pDh124 to obtain a recombinant vector expressing glyoxylate polyaldehyde enzyme and hydroxymalonic semialdehyde reductase (pDh124-cre-GCL*cre-TSR). 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 examples of the present invention, Nanjing Genscript Biotech Co., Ltd. was entrusted to synthesize the fusion gene CTP-cre-GLO-2A-cre-CAT and the fusion gene CTP-cre-GCL*cre-TSR, and PmlI and Nhe I restriction sites were added during the synthesis.

[0043] In the present invention, the fusion expression in microalgae preferably includes transforming microalgae and screening microalgae. The transformation of microalgae preferably involves transforming the constructed pDb124-cre-GLO-2A-cre-CAT recombinant vector and pDh124-cre-GCL*cre-TSR recombinant vector 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 method or electroporation method. 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. After the transformation, microalgae screening is carried out. The microalgae screening is preferably by plate screening, and the culture medium used for 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 plate screening, the glycolate oxidase gene, catalase gene, glyoxylate polyaldehyde enzyme gene, and hydroxymalonic semialdehyde reductase gene in the recombinant microalgae strain are preferably identified by molecular methods. The primers for identifying the glycolate oxidase gene and catalase gene include a forward primer with a nucleotide sequence as shown in SEQ ID NO: 15 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 16; the primers for identifying the glyoxylate polyaldehyde enzyme gene include a forward primer with a nucleotide sequence as shown in SEQ ID NO: 17 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 18; the primers for identifying the hydroxymalonic semialdehyde reductase gene include a forward primer with a nucleotide sequence as shown in SEQ ID NO: 19 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 20. After molecular identification, a successfully transformed recombinant microalgae strain is obtained.

[0044] In the present invention, the glycolic acid oxidase gene, catalase gene, glyoxylate polyaldehyde enzyme gene, and hydroxymalonic semialdehyde reductase gene are respectively or simultaneously fused and expressed with the PsaD promoter and the chloroplast transit peptide gene in microalgae, which can optimize the photorespiratory bypass, establish the GCGT pathway in chloroplasts, increase the carbon dioxide concentration, and promote the growth, development, and photosynthetic carbon fixation efficiency of microalgae. The GCGT 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 chloroplasts, directly increasing the CO2 concentration in chloroplasts, thereby forming a photosynthetic CO2 concentration mechanism similar to the C4 pathway, 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 method of the present invention has broad application prospects in improving the biomass of Chlamydomonas and the synthesis of its metabolites and 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 preferably to culture the recombinant microalgae strain in an environment containing a carbon source. The carbon source for culturing the recombinant microalgae strain preferably includes organic carbon and / or inorganic carbon. 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 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 chloroplasts, improve the photosynthetic efficiency, promote the conversion of carbon dioxide into more biomass, and increase the growth rate of Chlamydomonas, the glucose content, the starch content, and the glycerol content. It is measured that the biomass of the recombinant microalgae strain of the present invention is significantly increased, and the pyruvate content, the starch content, and the number of starch granules are significantly increased. Therefore, the recombinant microalgae strain of the present invention has broad application prospects in CO2 fixation and the culture for increasing the biomass, pyruvate content, and starch content of Chlamydomonas.

[0048] Based on the fact that the recombinant microalgae strain contains various biomasses, the present invention provides an application of 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.

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

[0050] Express glycolate oxidase, catalase, glyoxylate polyaldehyde enzyme and hydroxymalonic semialdehyde reductase in microalgae chloroplasts.

[0051] In the present invention, the amino acid sequence of the glycolate oxidase is preferably as shown in SEQ ID NO: 1; the amino acid sequence of the catalase is preferably as shown in SEQ ID NO: 2; the amino acid sequence of the glyoxylate polyaldehyde enzyme is as shown in SEQ ID NO: 3; the amino acid sequence of the hydroxymalonic semialdehyde reductase is preferably as shown in SEQ ID NO: 4. The method for expressing the glycolate oxidase, catalase, glyoxylate polyaldehyde enzyme and hydroxymalonic semialdehyde reductase in microalgae chloroplasts in the present invention is the same as the method for constructing the recombinant microalgae strain in the above technical solution, and will not be elaborated here.

[0052] The present invention expresses glycolate oxidase, catalase, glyoxylate polyaldehyde enzyme and hydroxymalonic semialdehyde reductase in microalgae chloroplasts, which can more efficiently increase the carbon dioxide concentration in microalgae chloroplasts, improve photosynthetic efficiency, and promote the conversion of carbon dioxide into more biomass. 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 algal cell density, compared with the wild type strain, when cultured only with acetate as the carbon source (cultured in TAP medium), the recombinant microalgae strain increased by 42.1%; when cultured only with carbon dioxide as the carbon source (cultured in TP medium + 5% CO2), the recombinant microalgae strain increased by 39.5%; when using acetate and carbon dioxide simultaneously (cultured in TAP medium + 5% CO2), the recombinant microalgae strain increased by 27.6%. It shows that expressing glycolate oxidase, catalase, glyoxylate polyaldehyde enzyme and hydroxymalonic semialdehyde reductase in microalgae chloroplasts can effectively improve the carbon fixation efficiency of microalgae.

[0053] To further illustrate the present invention, a recombinant algal strain expressing glycolate oxidase, catalase, glyoxylate polyaldehyde enzyme and hydroxymalonic semialdehyde reductase, and its construction method and application provided by the present invention will be described in detail below with reference to the drawings and examples, but they cannot be understood as limiting the protection scope of the present invention.

[0054] In this example, 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-GLO gene, cre-CAT gene, cre-GCL gene and cre-TSR gene were obtained by screening through database comparison

[0057]

[0058]

[0059]

[0060] The reported protein sequence GeneBank accession number of Escherichia coli hydroxymalonic semialdehyde reductase: WP_021571777.1, and the amino acid sequence is as shown in SEQ ID NO: 4 (MKLGFIGLGIMGTPMAINLARAGHQLHVTTIGPVADELLSLGAVSVETARQVTEASDIIFI MVPDTPQVEEVLFGENGCTKASLKGKTIVDMSSISPIETKRFARQVNELGGDYLDAPVSGGEIGAREGTLSIMVGGDEAVFERVKPLFELLGKNITLVGGNGDGQTCKVANQIIVALNIEAVSEALLFASKAGADPVRVRQALMGGFASSRILEVHGERMIKRTFNPGFKIALHQKDLNLALQSAKALALNLPNTATCQELFNTCAANGGSQLDHSALVQALELMANHKLAW). The coding region length of the cre-TSR gene is 879 bp. Using the online analysis of conserved domains Conserved domains (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi?INPUT_TYPE=live&SEQUENCE=WP_021571777.1) to analyze the protein domain of the deduced amino acid sequence of the hydroxymalonic semialdehyde reductase gene, it was found that this protein contains a hydroxymalonic semialdehyde reductase domain of 293 amino acids, which is consistent with the characteristics of the hydroxymalonic semialdehyde reductase gene.The ORF region sequence of the hydroxymalonic semialdehyde reductase gene was optimized according to the codon preference of Chlamydomonas reinhardtii to obtain the optimized TSR gene (cre-TSR gene), and the nucleotide sequence is as shown in SEQ ID NO: 8 (ATGAAGCTGGGCTTCATTGGGCTTGGAATCATGGGCACCCCCATGGCCATTAACCTG GCCAGGGCGGGCCACCAGCTGCATGTGACAACCATCGGCCCCGTGGCTGACGAGCTCCTGTCCCTAGGGGCAGTGTCCGTGGAGACGGCGCGGCAGGTGACAGAGGCCTCAGACATCATCTTCATCATGGTCCCAGACACGCCGCAGGTGGAGGAGGTCCTGTTTGGGGAGAACGGCTGCACCAAGGCGAGCCTCAAGGGCAAGACCATAGTGGATATGTCCAGCATCTCGCCAATCGAGACCAAGCGCTTCGCCCGGCAGGTCAACGAGTTGGGCGGCGACTACCTGGATGCCCCTGTGTCGGGGGGTGAGATCGGGGCGCGCGAGGGGACTCTCTCCATCATGGTGGGCGGCGATGAGGCGGTCTTCGAGCGTGTAAAACCCCTTTTCGAGCTGTTGGGTAAGAACATCACGCTGGTGGGAGGCAACGGCGACGGGCAGACCTGCAAGGTGGCGAATCAGATTATCGTCGCACTCAACATCGAAGCTGTGAGCGAGGCGCTGCTGTTCGCCAGCAAGGCCGGCGCGGACCCAGTGCGGGTGCGCCAGGCGCTGATGGGCGGCTTCGCGAGCTCCCGCATACTAGAAGTGCACGGCGAGCGCATGATCAAGCGCACCTTCAACCCCGGCTTCAAGATCGCCCTGCACCAGAAAGATCTCAACCTGGCGCTCCAGAGCGCCAAGGCGCTGGCGTTGAATCTGCCCAACACCGCCACGTGCCAGGAGCTGTTCAACACGTGTGCCGCCAATGGTGGCTCACAGCTGGATCACAGCGCGCTGGTGCAGGCACTGGAGCTGATGGCCAACCACAAGCTAGCCTGG).

[0061] Example 2

[0062] Method for constructing 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, and 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 chloroplast transit peptide gene is shown in SEQ ID NO: 9 (atggccgtcatgatgcgcacccaggcgcccgctgccactcgcgcttcatcgcgcgtcgctgttgccgctcgcccggctgctcgccgcgccgtggtggtccgcgcc); The amino acid sequence of the chloroplast transit peptide is shown in SEQ ID NO: 10 (MAVMMRTQAPAATRASSRVAVAARPAARRAVVVRA).

[0065] 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: 12 (VKQTLNFDLLKLAGDVESNPGP).

[0066] (Gly4Ser)3 nucleotide sequence is preferably shown as SEQ ID NO: 14 (GGCGGGGGGGGCAGCGGTGGAGGTGGATCTGGCGGGGGCGGGTC).

[0067] 1) Construction of pDb124-cre-GLO-2A-cre-CAT recombinant vector

[0068] Nanjing Genscript Biotechnology Co., Ltd. was commissioned to synthesize the fusion gene CTP-cre-GLO-2A-cre-CAT of the chloroplast transit peptide gene, glycolate oxidase gene, 2A peptide gene and catalase gene with Pml I and Nhe I restriction sites, and ligated them into pDb124 at the BglII, SpeI sites, KspAI and PmeI sites as shown in Figure 1 the figure.

[0069] The fusion gene CTP-cre-GLO-2A-cre-CAT and the plasmid pDb124 were digested with Pml I and Nhe I and then ligated to obtain the pDb124-cre-GLO-2A-cre-CAT recombinant vector.

[0070] 2) Construction of pDh124-cre-GCL*cre-TSR recombinant vector

[0071] Nanjing Genscript Biotechnology Co., Ltd. was commissioned to synthesize the fusion gene CTP-cre-GCL*cre-TSR of the chloroplast transit peptide gene, glyoxylate polyaldehyde enzyme gene, (Gly4Ser)3 and hydroxymalonic semialdehyde reductase gene with Pml I and Nhe I restriction sites, and ligated them into pDh124 at the SpeI and NheI sites as shown in Figure 2 the figure.

[0072] The fusion gene CTP-cre-GCL*cre-TSR and the plasmid pDh124 were digested with Pml I and Nhe I and then ligated to obtain the pDh124-cre-GCL*cre-TSR recombinant vector.

[0073] 3) Construction of recombinant Chlamydomonas reinhardtii strains

[0074] Glycolate oxidase, catalase, glyoxylate polyaldehyde enzyme and hydroxymalonic semialdehyde reductase are four key enzymes in the GCGT pathway. The recombinant vectors pDb124-cre-GLO-2A-cre-CAT and pDh124-cre-GCL * *cre-TSR contain four key essential genes in the GCGT pathway.

[0075] The constructed recombinant vectors pDb124-cre-GLO-2A-cre-CAT and pDh124-cre-GCL*cre-TSR 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 the recombinant *Chlamydomonas reinhardtii* algal strains are as follows.

[0076] The recombinant vectors pDb124-cre-GLO-2A-cre-CAT and pDh124-cre-GCL*cre-TSR 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. 500 μL of *Chlamydomonas reinhardtii* competent cells were pipetted into an EP tube containing 0.3 g of glass beads, and 1.5 μg of the recombinant vector pDb124-cre-GLO-2A-cre-CAT linearized by NotI and 1.5 μg of the recombinant vector pDh124-cre-GCL*cre-TSR linearized by NotI were pipetted into the EP tube; the EP tube was placed on a high-speed vortex mixer and vortexed at high speed for 25 s; in the laminar flow hood, 10 mL of antibiotic-free TAP liquid medium was added to the EP tube, and the cells were resuscitated at 22 °C and 110 rpm for 20 h; centrifuged at 3000 rpm for 5 min, and the supernatant medium was completely removed;

[0077] The precipitated algal cells were resuspended with 200 μL of fresh TAP liquid medium and spread on a TAP plate containing bleomycin (concentration 10 mg / L), hygromycin (concentration 10 mg / L), and ampicillin (concentration 100 mg / L), and cultured at 22 °C for 10 - 14 d;

[0078] Single colonies were picked from the plate and transferred to a new TAP solid plate containing bleomycin (concentration 10 mg / L), hygromycin (concentration 10 mg / L), and ampicillin (concentration 100 mg / L), and cultured overnight at 22 °C and 110 rpm in TAP liquid medium;

[0079] 1 mL of the algal liquid was pipetted and inoculated into a triangular flask containing 50 mL of liquid TAP medium, and the algae were shaken at 22 °C and 110 rpm until the logarithmic growth phase to obtain the recombinant *Chlamydomonas reinhardtii* algal strain.

[0080] 4) PCR screening of the recombinant *Chlamydomonas reinhardtii* algal strain at the DNA level

[0081] Forward primer CAT-F and reverse primer CAT-R were designed according to the sequence information of GLO and CAT on the provided vector. Forward primer GCL-F, reverse primer GCL-R, forward primer TSR-F and reverse primer TSR-R were designed according to the sequence information of GCL and TSR. The sequence list of the specific primers is shown in Table 1.

[0082] Table 1 Nucleotide sequences of the primers

[0083] Name Nucleotide sequence CAT-F 5’-GGGCATCCGCTTCTTCC-3’(SEQ ID NO: 15) CAT-R 5’-TGCTGTTGGGCTCGTAGTT-3’(SEQ ID NO: 16) GCL-F 5’-CATCGCCAAACCCGTCTCCAAG-3’(SEQ ID NO: 17) GCL-R 5’-TCCACGCCATAGCCGTTCACCT-3’(SEQ ID NO: 18) TSR-F 5’-GGGCTTCATTGGGCTTGGA-3’(SEQ ID NO: 19) TSR-R 5’-GTGGCGGTGTTGGGCAGAT-3’(SEQ ID NO: 20)

[0084] The detection method is as follows: Pick monoclonal algal cells into liquid TAP medium for expanded culture, and centrifuge to collect algal cells; Use differential centrifugation to collect Chlamydomonas reinhardtii cell samples; Extract Chlamydomonas reinhardtii cell DNA samples using a kit; Use the designed primer pairs to perform PCR amplification on the DNA samples.

[0085] The PCR amplification results showed that the Chlamydomonas reinhardtii strain CC-849 had no foreign genes, and CAT, GCL, and TSR could not be amplified. Bands of GLO and CAT, a band of GCL, and a band of TSR were amplified from the recombinant Chlamydomonas reinhardtii strain. It indicated that the genes GLO, CAT, GCL, and TSR were inserted into the Chlamydomonas reinhardtii nuclear gene.

[0086] Transcriptional level PCR screening of the recombinant Chlamydomonas reinhardtii strain

[0087] 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 mRNA expression of the gene could be known. The actin PCR amplification product was set as an internal reference control.

[0088] The detection method is as follows: Culture algal cells and centrifuge to collect algal cells; Extract the total RNA of the sample using the TransZol method; Reverse transcribe the RNA sample using a reverse transcription kit to obtain cDNA; Amplify GLO, CAT, GCL, TSR, and actin using specific primers. 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 a gel imaging system. The results are shown in Figure 3 . According to Figure 3 It can be seen that the Chlamydomonas reinhardtii strain CC-849 did not amplify GLO, CAT, GCL, and TSR, and the recombinant Chlamydomonas reinhardtii strain amplified GLO, CAT, GCL, and TSR, indicating that the recombinant Chlamydomonas reinhardtii strain was successfully constructed.

[0089] Table 2 Nucleotide sequences of the primers

[0090]

[0091]

[0092] Example 3

[0093] Phenotypic Characterization of Cultured Recombinant Chlamydomonas reinhardtii Strains (TAP)

[0094] 1) Detection of Growth Indexes of Chlamydomonas reinhardtii Strains

[0095] The recombinant Chlamydomonas reinhardtii strain or the Chlamydomonas reinhardtii strain CC-849 was inoculated into an antibiotic-free TAP liquid medium and cultured with shaking at 22 °C and 110 rpm until the stationary phase;

[0096] During the culture period, 200 μL of the algal solution was aspirated every 24 h and placed in a 96-well plate with three replicates. It was detected by a microplate reader and shaken three times to detect OD 750 , and the OD 750 absorbance value was recorded to plot the growth curve;

[0097] 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 a constant weight, and the dry weight was weighed.

[0098] After analysis, CO2 was efficiently converted into biomass in Chlamydomonas reinhardtii containing the GCGT pathway. According to the analysis of the plotted growth curve, its growth rate was significantly increased by 2.02 times. According to the analysis of the weighed dry weight, its dry weight was 0.048 g.

[0099] 2) Detection of Products of Chlamydomonas reinhardtii

[0100] The recombinant Chlamydomonas reinhardtii strain or the Chlamydomonas reinhardtii strain CC-849 was inoculated into an 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 strains were measured respectively.

[0101] The starch content was detected using a starch detection kit (Beijing Solarbio Science & Technology Co., Ltd.). The specific detection is as follows.

[0102] Absorb 50 mL of algal solution, centrifuge at 4°C and 5000 rpm for 5 min to collect the algal bodies. After washing the algal bodies twice with distilled water, place them in a freeze dryer and freeze-dry overnight. Weigh 0.1 g of the dried algal powder and put it into a 2.0 mL centrifuge tube, add 1 mL of Reagent 1 and homogenize thoroughly, extract in a water bath at 80°C for 30 min; at room temperature, centrifuge at 8000 rpm for 5 min to collect the algal bodies; add 0.5 mL of distilled water to resuspend, gelatinize in a boiling water bath for 15 min, after cooling, add 0.35 mL of Reagent 2, extract at room temperature for 15 min, and shake 3 - 5 times; add 0.85 mL of double-distilled water, mix well, centrifuge at 3000 g at room temperature for 10 min; take 0.2 mL of the supernatant and 1 mL of the working solution of Reagent 3 into an EP tube, heat in a water bath at 95°C for 10 min, cool naturally to room temperature, and record the absorbance value A at a wavelength of 620 nm.

[0103] After analysis, CO2 is efficiently fixed as starch in Chlamydomonas reinhardtii containing the GCGT pathway. Using the standard curve for calculation, the starch content is 12.84 g / 100 g of dry algal body weight.

[0104] The detection of pyruvate content in the algal strain is carried out by using the Pyruvate Assay Kit (Nanjing Jiancheng Bioengineering Institute, A081 - 1 - 1, China) for extraction and determining the pyruvate content by ultraviolet spectrophotometry; the detection of glycerol content in the algal strain is carried out by using the Glycerol Assay Kit (Nanjing Jiancheng Bioengineering Institute, F005 - 2 - 1, China) for extraction and determining the glycerol content by ultraviolet spectrophotometry; the detection of glucose content in the algal strain is carried out by using the Glucose Kit (Nanjing Jiancheng Bioengineering Institute, A154 - 2 - 1, China) for extraction and determining the glucose content of the algal powder by ultraviolet spectrophotometry. The detection of sucrose content in the algal strain is carried out by using the Sucrose Measurement Kit (Nanjing Jiancheng Bioengineering Institute, A099 - 1 - 1, China) for extraction and determining the sucrose content of the algal powder by ultraviolet spectrophotometry.

[0105] Example 4

[0106] Phenotypic characteristic detection of the cultivation of recombinant Chlamydomonas reinhardtii strains using 5% CO2 (TP + 5% CO2)

[0107] 1) Detection of the growth index of Chlamydomonas reinhardtii strains

[0108] The recombinant Chlamydomonas reinhardtii strain or Chlamydomonas reinhardtii 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 was reached with shaking.

[0109] During the cultivation, 200 μL of the algal solution was taken every 24 h and placed in a 96-well plate with three replicates. It was then detected using a microplate reader and shaken three times to measure the OD 750 and the OD 750 absorbance value was recorded to plot the growth curve.

[0110] 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. The cells were washed twice with distilled water and then freeze-dried in a freeze dryer until a constant weight was reached, and the dry weight was weighed.

[0111] Upon analysis, CO2 in Chlamydomonas reinhardtii containing the GCGT pathway was efficiently converted into biomass. According to the analysis of the plotted growth curve, its growth rate was significantly increased by 0.88 times. According to the analysis of the weighed dry weight, its dry weight was 0.0548 g.

[0112] 2) Detection of products of Chlamydomonas reinhardtii

[0113] The recombinant Chlamydomonas reinhardtii strain or Chlamydomonas reinhardtii strain CC-849 was inoculated into an antibiotic-free TP liquid medium with 5% CO2 bubbled in at 22 °C and 110 rpm until the logarithmic growth phase was reached with shaking. The contents of starch, pyruvate, glucose, sucrose, and glycerol were detected according to the method in Example 3.

[0114] Upon analysis, CO2 in Chlamydomonas reinhardtii containing the GCGT pathway was efficiently fixed as starch. Using the standard curve for calculation, its starch content was 14.30 g / 100 g of algal cell dry weight.

[0115] Example 5

[0116] Detection of phenotypic characteristics of the recombinant Chlamydomonas reinhardtii strain cultured with acetate and 5% CO2 (TAP + 5% CO2)

[0117] 1) Detection of growth indicators of Chlamydomonas reinhardtii strain

[0118] The recombinant Chlamydomonas reinhardtii strain or Chlamydomonas reinhardtii strain CC-849 was cultured in an antibiotic-free TAP liquid medium with 5% CO2 bubbled in at 22 °C and 110 rpm until the stationary phase was reached with shaking.

[0119] During the cultivation, 200 μL of the algal solution was taken every 24 h and placed in a 96-well plate with three replicates. It was then detected using a microplate reader and shaken three times to measure the OD 750 and the OD 750 absorbance value was recorded to plot the growth curve.

[0120] After culturing to the plateau phase, 50 mL of algal solution was collected, and the algal bodies 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 a constant weight, and the dry weight was weighed.

[0121] After analysis, CO2 was efficiently converted into biomass in Chlamydomonas reinhardtii containing the GCGT pathway. According to the growth curve analysis, its growth rate was significantly increased by 1.01 times. According to the analysis of the weighed dry weight, its dry weight was 0.108 g.

[0122] 2) Detection of Chlamydomonas reinhardtii products

[0123] The recombinant Chlamydomonas reinhardtii strain or the Chlamydomonas reinhardtii strain CC-849 was inoculated into an antibiotic-free TAP liquid medium, and 5% CO2 was introduced, and the cells were cultured with shaking 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.

[0124] After analysis, CO2 was efficiently fixed as starch in Chlamydomonas reinhardtii containing the GCGT pathway. Using the standard curve for calculation, its starch content was 12.69 g / 100 g algal body dry weight.

[0125] Figure 4 and Figure 5 are the growth curve graph of the recombinant Chlamydomonas reinhardtii strain under different culture conditions and the detection result graphs of dry weight, pyruvate content, glucose content, sucrose content, starch content, and oil content, where WT represents the Chlamydomonas reinhardtii strain CC-849, and GCGT3 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 organic acid content of the recombinant microalgae strain were all significantly increased, and the sugar and alcohol contents were significantly decreased.

[0126] In summary, through the eukaryotic expression system of Chlamydomonas reinhardtii, the present invention verified its function and found that glycolate oxidase, catalase, glyoxylate polyaldehyde enzyme, and hydroxymalonic semialdehyde reductase can efficiently fix and convert CO2 into biomass and starch. The glycolate oxidase gene, catalase gene, glyoxylate polyaldehyde enzyme gene, and hydroxymalonic semialdehyde reductase gene in the recombinant Chlamydomonas reinhardtii strain of the present invention are involved 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 broad application prospects in improving photosynthetic efficiency and carbon fixation.

[0127] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. Other embodiments can also be obtained according to these embodiments 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 that express glycolate oxidase, catalase, glyoxylate polyaldehyde enzyme, and hydroxymalonic semialdehyde reductase in chloroplasts.

2. The recombinant microalgae strain according to claim 1, characterized in that, The amino acid sequence of the glycolate oxidase is shown in SEQ ID NO: 1; The amino acid sequence of the catalase is shown in SEQ ID NO: 2; The amino acid sequence of the glyoxylate polyaldehyde enzyme is shown in SEQ ID NO: 3; The amino acid sequence of the hydroxymalonic semialdehyde 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, Including the following steps: Fusion expression of the glycolate oxidase gene, catalase gene, glyoxylate polyaldehyde enzyme gene, and hydroxymalonic semialdehyde reductase gene separately or simultaneously with the PsaD promoter and the chloroplast transit peptide gene in microalgae.

5. The construction method according to claim 4, characterized in that, One, two, three, or four genes of the glycolate oxidase gene, catalase gene, glyoxylate polyaldehyde enzyme gene, and hydroxymalonic semialdehyde reductase gene are transformed into microalgae in the form of a recombinant vector with the PsaD promoter and the chloroplast transit peptide gene.

6. The construction method according to claim 5, characterized in that The recombinant vector includes a recombinant vector expressing glycolate oxidase and catalase and a recombinant vector expressing glyoxylate polyaldehyde enzyme and hydroxymalonic semialdehyde 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, Including the following steps: Express glycolate oxidase, catalase, glyoxylate polyaldehyde enzyme, and hydroxymalonic semialdehyde reductase in the chloroplasts of microalgae.

10. The method according to claim 9, wherein The amino acid sequence of the glycolate oxidase is shown in SEQ IDNO: 1; The amino acid sequence of the catalase is shown in SEQ ID NO: 2; The amino acid sequence of the glyoxylate polyaldehyde enzyme is shown in SEQ ID NO: 3; The amino acid sequence of the hydroxymalonic semialdehyde reductase is shown in SEQ ID NO: 4.

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