Recombinant algal strain for expressing glycolate oxidase, catalase and oxalate oxidase as well as construction method and application of recombinant algal strain

Abstract

The invention provides a recombinant algal strain for expressing glycollic acid oxidase, catalase and oxalate oxidase 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 and oxalate oxidase 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, glucose, cane sugar, 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 glycolate oxidase, catalase, and oxalate oxidase, and a construction method and application thereof. Background Art

[0002] With the development of human society, the advancement of the industrialization process and the increase in population, the emissions of CO2 are 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 carbon fixation methods. 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] To further improve the carbon fixation efficiency of microalgae, the prior art mainly improves the microalgae culture conditions, screens and domesticates excellent algal strains, and uses genetic engineering means to transform microalgae cells at the molecular level. 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, currently, 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, and oxalate oxidase. A new photorespiration bypass GCO pathway is introduced into the recombinant microalgae strain, which can achieve high-efficiency carbon fixation and has a high microalgae cell growth rate.

[0005] 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, and oxalate oxidase 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: 3;

[0009] The amino acid sequence of the oxalate oxidase is as shown in SEQ ID NO: 5.

[0010] Preferably, the microalgae include marine microalgae and / or freshwater microalgae;

[0011] Preferably, the freshwater microalgae include Chlamydomonas reinhardtii.

[0012] The present invention provides a method for constructing the recombinant microalgae strain, comprising the following steps:

[0013] The glycolate oxidase gene, catalase gene, and oxalate oxidase gene are respectively or simultaneously fused and expressed with the PsaD promoter and chloroplast transit peptide gene in microalgae.

[0014] Preferably, one, two, or three of the glycolate oxidase gene, catalase gene, and oxalate oxidase gene are transformed into microalgae in the form of a recombinant vector together with the PsaD promoter and chloroplast transit peptide gene.

[0015] Preferably, the recombinant vector includes a recombinant vector expressing glycolate oxidase and catalase and a recombinant vector expressing oxalate oxidase.

[0016] The present invention provides the use of the recombinant microalgae strain or the recombinant microalgae strain constructed by the construction method in CO2 fixation.

[0017] The present invention provides the use 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.

[0018] The present invention provides a method for improving the carbon fixation efficiency of microalgae, comprising the following steps:

[0019] Express glycolate oxidase, catalase, and oxalate oxidase in the chloroplast of microalgae.

[0020] Preferably, the amino acid sequence of the glycolate oxidase is as shown in SEQ ID NO: 1;

[0021] The amino acid sequence of the catalase is as shown in SEQ ID NO: 3;

[0022] The amino acid sequence of the oxalate oxidase is as shown in SEQ ID NO: 5.

[0023] The present invention has the following advantages compared with the prior art:

[0024] The present invention provides a recombinant microalgae strain, which is a microalgae strain that expresses glycolate oxidase, catalase, and oxalate oxidase in the chloroplast. In the recombinant microalgae strain of the present invention, glycolate oxidase catalyzes the oxidative decomposition of glycolate to form glyoxylate and oxalic acid, oxalate oxidase completely decomposes oxalic acid to form carbon dioxide, increasing the CO2 concentration in the chloroplast, and catalase decomposes excessive H2O2 to assist in maintaining the cell homeostasis. The recombinant microalgae strain can decompose the photorespiratory by-product glycolate into carbon dioxide, increasing the CO2 concentration in the chloroplast, effectively improving the carbon fixation efficiency of microalgae, and contributing to the fixation of CO2 into biomass as well as metabolic products and bioactive substances such as glucose, sucrose, starch, and oils. It has broad application prospects in biological carbon fixation, synthesis of microalgae metabolic products, and preparation of microalgae bioactive substances.

[0025] The present invention provides a method for constructing the recombinant microalgae strain, comprising the following steps: separately or simultaneously fusing the glycolate oxidase gene, catalase gene, and oxalate oxidase gene with the PsaD promoter and the chloroplast transit peptide gene for expression in microalgae. The method of the present invention can optimize the photorespiratory bypass of microalgae, establish the GCO pathway in the chloroplast, increase the carbon dioxide concentration, and promote the growth, development, 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 sugars, organic acids, and alcohols of the recombinant microalgae strain are significantly improved; upon detecting the dry weight, when cultured only with acetate as the carbon source, the recombinant Chlamydomonas reinhardtii strain is increased by 88.4%; when cultured only with carbon dioxide as the carbon source, the recombinant Chlamydomonas reinhardtii strain is increased by 18.2%; when using both acetate and carbon dioxide simultaneously, the recombinant Chlamydomonas reinhardtii strain is increased by 29.2%. Description of the Drawings

[0026] Figure 1 It is the plasmid map of the pDb124-cre-GLO-2A-cre-CAT recombinant vector;

[0027] Figure 2 It is the plasmid map of the pDh124-cre-OXO recombinant vector;

[0028] 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 1-4 lanes are the PCR products of the internal reference actin, GLO, CAT, and OXO respectively; B is the detection result of the transcriptional level of the recombinant Chlamydomonas reinhardtii strain: the M lane is the RNA Marker; the 1-4 lanes are the PCR products of the internal reference actin, GLO, CAT, and OXO respectively;

[0029] Figure 4 Growth curve of the recombinant Chlamydomonas reinhardtii strain under different culture conditions;

[0030] Figure 5 Detection result diagram of the phenotypic characteristics of the recombinant Chlamydomonas reinhardtii strain under different culture conditions. Detailed implementation manners

[0031] The present invention provides a recombinant microalgae strain, which is a microalgae strain expressing glycolate oxidase, catalase and oxalate oxidase in the chloroplast.

[0032] In the present invention, the microalgae include marine microalgae and / or freshwater microalgae. The freshwater microalgae preferably include Chlamydomonas reinhardtii. In the embodiments 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.

[0033] 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: 3; the oxalate oxidase is derived from rice, GeneBank accession number: BAS85865.1, and the amino acid sequence is preferably as shown in SEQ ID NO: 5.

[0034] The nucleotide sequence length of the glycolate oxidase gene is 1107 bases, containing 369 amino acids, and the molecular weight is 40.4 kDa; the nucleotide sequence length of the catalase gene is 2259 bases, containing 753 amino acids, and the molecular weight is 84.16 kDa; the molecular weight of the oxalate oxidase gene is 24 kDa.

[0035] Photorespiration is an energy-consuming side reaction of photosynthesis, which occurs in all photosynthetic cells under light and high-oxygen and low-carbon dioxide conditions, and glycolate is formed as a by-product. During the decomposition of glycolate, CO2 is released, but energy ATP cannot be produced, and nearly 20% of the energy generated by photosynthesis is lost. In the recombinant microalgae strain described in the present invention, glycolate oxidase catalyzes the oxidative decomposition of glycolate to form glyoxylate and oxalic acid, oxalate oxidase completely decomposes oxalic acid to form carbon dioxide, increasing the CO2 concentration in the chloroplast, and catalase decomposes excessive H2O2 to assist in maintaining the cell's homeostasis. The GCO pathway constructed by the three enzymes significantly improves the carbon fixation efficiency of the recombinant microalgae strain described in the present invention compared to the wild-type strain. The recombinant microalgae strain described in the present invention expresses the glycolate oxidase gene (GLO), catalase gene (CAT), and oxalate oxidase gene (OXO) in the chloroplast, establishing a new photorespiration bypass GCO pathway in the chloroplast, which can decompose the photorespiration by-product glycolate into carbon dioxide, increase the carbon dioxide concentration in the chloroplast, effectively improve the carbon fixation efficiency of Chlamydomonas, and contribute to the fixation of CO2 into biomass and metabolites and bioactive substances such as glucose, sucrose, starch, and oil. It has broad application prospects in biological carbon fixation, synthesis of microalgae metabolites, and preparation of microalgae bioactive substances.

[0036] The present invention provides a method for constructing the recombinant microalgae strain, comprising the following steps:

[0037] Fusion expression of the glycolate oxidase gene, catalase gene, and oxalate oxidase gene separately or simultaneously with the PsaD promoter and the chloroplast transit peptide gene in microalgae.

[0038] In the present invention, one, two, or three of the glycolate oxidase gene, catalase gene, and oxalate oxidase gene are transformed into microalgae in the form of a recombinant vector together with the PsaD promoter and the chloroplast transit peptide gene.

[0039] In the present invention, the glycolate oxidase gene (cre-GLO), catalase gene (cre-CAT), and oxalate oxidase gene (cre-OXO) are preferably obtained by optimizing sequences according to the codon preference of Chlamydomonas reinhardtii. The nucleotide sequence of the glycolate oxidase gene is shown in SEQ ID NO: 2; the nucleotide sequence of the catalase gene is shown in SEQ ID NO: 4; the nucleotide sequence of the oxalate oxidase gene is shown in SEQ ID NO: 6.The nucleotide sequence of the PsaD promoter gene is as shown in SEQ ID NO: 11 (GATCCCACACACCTGCCCGTCTGCCTGACAGGAAGTGAACGCATGTCGAGGGA GGCCTCACCAATCGTCACACGAGCCCTCGTCAGAAACACGTCTCCGCCACGCTCTCCCTCTCACGGCCGACCCCGCAGCCCTTTTGCCCTTTCCTAGGCCACCGACAGGACCCAGGCGCTCTCAGCATGCCTCAACAACCCGTACTCGTGCCAGCGGTGCCCTTGTGCTGGTGATCGCTTGGAAGCGCATGCGAAGACGAAGGGGCGGAGCAGGCGGCCTGGCTGTTCGAAGGGCTCGCCGCCAGTTCGGGTGCCTTTCTCCACGCGCGCCTCCACACCTACCGATGCGTGAAGGCAGGCAAATGCTCATGTTTGCCCGAACTCGGAGTCCTTAAAAAGCCGCTTCTTGTCGTCGTTCCGAGACATGTTAGCAGATCGCAGTGCCACCTTTCCTGACGCGCTCGGCCCCATATTCGGACGCAATTGTCATTTGTAGCACAATTGGAGCAAATCTGGCGAGGCAGTAGGCTTTTAAGTTGCAAGGCGAGAGAGCAAAGTGGGACGCGGCGTGATTATTGGTATTTACGCGACGGCCCGGCGCGTTAGCGGCCCTTCCCCCAGGCCAGGGACGATTATGTATCAATATTGTTGCGTTCGGGCACTCGTGCGAGGGCTCCTGCGGGCTGGGGAGGGGGATCTGGGAATTGGAGGTACGACCGAGATGGCTTGCTCGGGGGGAGGTTTCCTCGCCGAGCAAGCCAGGGTTAGGTGTTGCGCTCTTGACTCGTTGTGCATTCTAGGACCCCACTGCTACTCACAACAAGCCAAA); the nucleotide sequence of the chloroplast transit peptide gene is preferably as shown in SEQ ID NO: 7. The adjacent two genes are connected by a 2A peptide gene, and the nucleotide sequence of the 2A peptide gene is preferably as shown in SEQ ID NO: 9.

[0040] In the present invention, when one or two genes of the glycolic acid oxidase gene, the catalase gene, and the oxalate oxidase gene are constructed into a recombinant vector with the PsaD promoter and the 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 to construct a recombinant vector expressing oxalate oxidase with the oxalate oxidase gene. 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 embodiment of the present invention, the chloroplast transit peptide gene, the glycolic acid oxidase gene, the 2A peptide gene, and the 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 PmlI 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 and the oxalate oxidase gene are sequentially ligated to obtain the fusion gene CTP-cre-OXO, and the fusion gene CTP-cre-OXO is cloned into the Pml I and Nhe I multiple cloning sites of the backbone vector pDh124 to obtain a recombinant vector expressing oxalate oxidase (pDh124-cre-OXO). The present invention does not specifically limit the construction method of the fusion gene, and the conventional fusion gene construction method in the art can be used. In the embodiment of the present invention, Nanjing GenScript Biotech Corporation was commissioned to synthesize the fusion gene CTP-cre-GLO-2A-cre-CAT and the fusion gene CTP-cre-OXO, and PmlI and Nhe I restriction sites were added during the synthesis.

[0041] In the present invention, the fusion expression in microalgae preferably includes transforming microalgae and screening the microalgae. The transformation of microalgae preferably involves transforming the constructed pDb124-cre-GLO-2A-cre-CAT recombinant vector and pDh124-cre-OXO 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, the microalgae are screened. The screening of the microalgae is preferably carried out by 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 glycolate oxidase gene, catalase gene, and oxalate oxidase 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: 12 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 13; the primers for identifying the oxalate oxidase 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, a successfully transformed recombinant microalgae strain is obtained.

[0042] In the present invention, the glycolate oxidase gene, catalase gene, and oxalate oxidase 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 GCO pathway in the chloroplast, increase the carbon dioxide concentration, and promote the growth and development of microalgae and the photosynthetic carbon fixation efficiency. The GCO 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 glycolate generated by photorespiration completely into CO2 in the chloroplast, directly increasing the CO2 concentration in the chloroplast, thus forming a photosynthetic CO2 concentrating mechanism similar to the C4 pathway, improving the carbon fixation efficiency. Modifying the chloroplast helps to improve the carbon fixation efficiency and helps to fix 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, contents of sugars, organic acids, and alcohols 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 in enhancing the carbon fixation efficiency of microalgae.

[0043] The present invention provides an application of the recombinant microalgae strain or the recombinant microalgae strain constructed by the construction method in CO2 fixation.

[0044] 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 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.

[0045] 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, glucose content, starch content, and glycerol content of Chlamydomonas. It has been measured that the biomass of the recombinant microalgae strain of the present invention is significantly increased, and the 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 CO2 fixation and the cultivation for increasing the biomass and starch content of Chlamydomonas.

[0046] 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.

[0047] The present invention provides a method for improving the carbon fixation efficiency of microalgae, comprising the following steps:

[0048] Express glycolate oxidase, catalase, and oxalate oxidase in the chloroplast of microalgae.

[0049] In the present invention, the amino acid sequence of the glycolate oxidase preferably is as shown in SEQ ID NO: 1; the amino acid sequence of the catalase preferably is as shown in SEQ ID NO: 3; the amino acid sequence of the oxalate oxidase preferably is as shown in SEQ ID NO: 5. The method for expressing the glycolate oxidase, catalase, and oxalate oxidase in the chloroplast of 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.

[0050] The expression of glycolate oxidase, catalase and oxalate oxidase in microalgae chloroplasts in the present invention 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 strains 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 strains were significantly increased. 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 dry weight of the recombinant microalgae strain increased by 88.4%; when cultured with carbon dioxide as the carbon source only (cultured in TP medium + 5% CO2), the dry weight of the recombinant microalgae strain increased by 29.2%; when using acetate and carbon dioxide simultaneously (cultured in TAP medium + 5% CO2), the recombinant microalgae strain increased by 18.2%. It shows that the expression of glycolate oxidase, catalase and oxalate oxidase in microalgae chloroplasts can effectively improve the carbon fixation efficiency of microalgae.

[0051] To further illustrate the present invention, a recombinant algal strain expressing glycolate oxidase, catalase and oxalate oxidase, its construction method and application provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0052] In this example, the methods used were all operated according to conventional methods unless otherwise specified, and the reagents used were all conventional reagents or reagents prepared according to conventional methods unless otherwise specified.

[0053] Example 1

[0054] The cre-GLO gene, cre-CAT gene and cre-OXO gene were obtained by screening through database comparison

[0055]

[0056]

[0057] The GeneBank accession number of the reported protein sequence of rice oxalate oxidase: BAS85865.1, and the amino acid sequence is as shown in SEQ ID NO: 5 (MEYGFKAAGLVFVVLLLQQAPVLIRATDADPLQDFCVADLNSEVTVNGHACKPASAAGDEFLFSSKIATGGDVNANPNGSNVTELDVAEWPGVNTLGVSMNRVDFAPGGTNPPHVHPRATEVGIVLRGELLVGIIGTLDTGNRYYSKVVRAGETFVIPRGLMHFQFNVGKTEATMVVSFNSQNPGIVFVPLTLFGSNPPIPTPVLVKALRVDAGVVELLKSKFTGGY). The coding region length of the oxalate oxidase gene is 681 bp. Using the online analysis of conserved domains Conserved domains

[0058] (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi?INPUT_TYPE=live&SEQUENCE=BAS85865.1) Protein domain analysis was performed on the predicted amino acid sequence of the oxalate oxidase gene, and it was found that this protein contains an oxalate oxidase domain (cupin domain), which is consistent with the characteristics of the oxalate oxidase gene. The ORF region sequence of the oxalate oxidase gene was optimized according to the codon preference of Chlamydomonas reinhardtii to obtain the optimized OXO gene (cre-OXO gene), and the nucleotide sequence is as shown in SEQ ID NO: 6 (ATGGAGTACGGCTTCAAGGCCGCGGGGCTGGTGTTTGTTGTTCTGCTGCTGCAG CAGGCTCCAGTGCTCATCCGCGCAACCGACGCGGACCCGCTGCAAGACTTCTGCGTGGCAGACCTGAACTCAGAGGTGACAGTCAACGGGCACGCCTGCAAGCCCGCCTCTGCGGCCGGCGACGAGTTCCTGTTCTCGAGCAAGATCGCCACGGGTGGGGATGTAAACGCCAACCCGAATGGCTCCAACGTGACTGAACTGGATGTGGCGGAGTGGCCTGGGGTGAACACCCTGGGCGTCAGCATGAACCGGGTCGACTTTGCGCCCGGCGGCACCAACCCGCCCCATGTGCACCCGCGAGCCACTGAGGTGGGCATTGTGCTGCGGGGCGAGCTTCTGGTCGGCATCATAGGTACGCTGGACACAGGCAACCGCTATTACTCGAAAGTCGTGCGTGCGGGCGAGACGTTCGTGATTCCACGCGGGCTCATGCACTTCCAGTTCAATGTGGGCAAGACCGAGGCGACCATGGTGGTGTCCTTTAACAGCCAGAACCCCGGAATCGTGTTCGTGCCGCTCACGTTGTTCGGCAGCAACCCTCCCATCCCCACGCCGGTGCTGGTCAAGGCGCTGCGCGTTGACGCTGGCGTGGTGGAGCTGCTAAAGTCGAAGTTCACCGGTGGCTAC).

[0059] Example 2

[0060] Construction method of recombinant Chlamydomonas reinhardtii strain

[0061] The Chlamydomonas reinhardtii strain used in this example is CC-849, and the Chlamydomonas reinhardtii strain CC-849 used is provided by the Aquatic Biology Laboratory of the 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 used are provided by the Aquatic Biology Laboratory of the College of Life and Ocean Sciences, Shenzhen University; the construction of the 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..

[0062] The nucleotide sequence of the chloroplast transit peptide gene is as shown in SEQ ID NO: 7 (atggccgtcatgatgcgcacccaggcgcccgctgccactcgcgcttcatcgcgcgtcgctgttgccgctcgcccggctgctcgccgcgccgtggtggtccgcgcc); the amino acid sequence of the chloroplast transit peptide is as shown in SEQ ID NO: 8 (MAVMMRTQAPAATRASSRVAVAARPAARRAVVVRA).

[0063] The nucleotide sequence of the 2A peptide gene is as shown in SEQ ID NO: 9 (gtgaagcagaccctgaacttcgacctgctgaagctggcgggcgacgtggagagcaaccccggcccc); the amino acid sequence of the 2A peptide is as shown in SEQ ID NO: 10 (VKQTLNFDLLKLAGDVESNPGP).

[0064] 1) Construction of the pDb124-cre-GLO-2A-cre-CAT recombinant vector

[0065] Entrust Nanjing Genscript Biotech Co., Ltd. to synthesize the fusion gene CTP-cre-GLO-2A-cre-CAT of chloroplast transit peptide gene, glycolate oxidase gene, 2A peptide gene and catalase gene with Pml I and Nhe I restriction sites, and ligate them into pDb124 at the Figure 1 BglII, SpeI sites, KspAI and PmeI sites shown as follows.

[0066] The fusion gene CTP-cre-GLO-2A-cre-CAT and the plasmid pDb124 were digested with PmlI and Nhe I and then ligated to obtain the recombinant vector pDb124-cre-GLO-2A-cre-CAT.

[0067] 2) Construction of the pDh124-cre-OXO recombinant vector

[0068] Entrust Nanjing Genscript Biotech Co., Ltd. to synthesize the fusion gene CTP-cre-OXO of chloroplast transit peptide gene and oxalate oxidase gene with Pml I and Nhe I restriction sites, and ligate them into pDh124 at the Figure 2 SpeI and NheI sites shown as follows.

[0069] The fusion gene CTP-cre-OXO and the plasmid pDh124 were digested with Pml I and Nhe I and then ligated to obtain the recombinant vector pDh124-cre-OXO.

[0070] 3) Construction of recombinant Chlamydomonas reinhardtii strains

[0071] Glycolate oxidase, catalase and oxalate oxidase are three key enzymes in the GCO pathway. The recombinant vectors pDb124-cre-GLO-2A-cre-CAT and pDh124-cre-OXO contain three key essential genes in the GCO pathway.

[0072] The constructed recombinant vectors pDb124-cre-GLO-2A-cre-CAT and pDh124-cre-OXO 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, and the successfully transformed positive algal strains can be screened by bleomycin and hygromycin. The specific steps for the construction of recombinant Chlamydomonas reinhardtii strains are as follows.

[0073] The recombinant vector pDb124-cre-GLO-2A-cre-CAT and the recombinant vector pDh124-cre-OXO were co-transformed into the Chlamydomonas reinhardtii strain CC-849 by the glass bead transformation method. Specifically, the Chlamydomonas reinhardtii strain CC-849 cultured to the logarithmic growth phase was centrifuged at 5000 rpm at room temperature to collect 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-OsGLO-2A-OsCAT linearized with NotI and 1.5 μg of the recombinant vector pDh124-OsOXO linearized with 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 a 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 to remove all the supernatant medium;

[0074] 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;

[0075] 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;

[0076] 1 mL of the algal solution 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 strain.

[0077] 4) DNA-level PCR screening of the recombinant Chlamydomonas reinhardtii strain

[0078] Forward primer CAT-F and reverse primer CAT-R were designed according to the sequence information of GLO and CAT on the provided vector, and forward primer OXO-F and reverse primer OXO-R were designed according to the sequence information of OXO. The sequence list of the specific primers is shown in Table 1.

[0079] Table 1 Nucleotide sequences of the primers

[0080] Name Nucleotide sequence CAT-F 5’-GGGCATCCGCTTCTTCC-3’(SEQ ID NO: 12) CAT-R 5’-TGCTGTTGGGCTCGTAGTT-3’(SEQ ID NO: 13) OXO-F 5’-GGGGCTGGTGTTTGTTGT-3’(SEQ ID NO: 14) OXO-R 5’-ATGGTCGCCTCGGTCTT-3’(SEQ ID NO: 15)

[0081] The detection method is as follows: Pick monoclonal algal cells and culture them in liquid TAP medium for expansion, and then 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.

[0082] The results of PCR amplification showed that Chlamydomonas reinhardtii strain CC-849 could not be amplified, indicating the absence of exogenous genes CAT and OXO. The recombinant Chlamydomonas reinhardtii strain amplified the bands of GLO and CAT and the band of OXO, indicating that the genes GLO, CAT, and OXO were inserted into the nuclear genes of Chlamydomonas reinhardtii.

[0083] 5) Transcriptional level PCR screening of recombinant Chlamydomonas reinhardtii strains

[0084] 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 mRNA expression of this gene can be known, and the actin PCR amplification product was set as the internal reference control.

[0085] The detection method is as follows: Culture algal cells and centrifuge to collect algal cells; Extract total RNA of the sample using TransZol method; Reverse transcribe the RNA sample using a reverse transcription kit to obtain cDNA; Use specific primers to amplify GLO, CAT, OXO, and actin. The sequence list of 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, and the results are shown in Figure 3 . According to Figure 3 it can be known that Chlamydomonas reinhardtii strain CC-849 did not amplify the GLO gene, the CAT PCR gene, and the OXO gene, and the recombinant Chlamydomonas reinhardtii strain amplified the GLO gene, the CAT PCR gene, and the OXO gene, indicating that the construction of the recombinant Chlamydomonas reinhardtii strain was successful.

[0086] Table 2 Nucleotide sequences of primers

[0087]

[0088]

[0089] Example 3

[0090] Detection of phenotypic characteristics of recombinant Chlamydomonas reinhardtii strains cultured (TAP)

[0091] 1) Detection of growth indexes of Chlamydomonas reinhardtii strains

[0092] Inoculate the recombinant Chlamydomonas reinhardtii strain or Chlamydomonas reinhardtii strain CC-849 into antibiotic-free TAP liquid medium and culture it with shaking at 22°C and 110 rpm until the plateau phase;

[0093] During the cultivation period, 200 μL of algal solution was aspirated every 24 h and placed in a 96-well plate with three replicates. It was detected by a microplate reader, shaken three times, and the OD was measured. 750 , and the OD 750 absorbance value was recorded, and a growth curve was plotted.

[0094] After culturing to the plateau phase, 50 mL of algal solution was collected, centrifuged at 5000 rpm for 5 min at 4 °C to collect the algal bodies. After washing the cells twice with distilled water, they were placed in a freeze dryer and freeze-dried to constant weight, and the dry weight was weighed.

[0095] After analysis, the recombinant Chlamydomonas reinhardtii strain containing the GCO pathway efficiently converted CO2 into biomass. According to the analysis of the plotted growth curve, its growth rate was significantly increased by 1.99 times. According to the analysis of the weighed dry weight, its dry weight was 0.063 g.

[0096] 2) Detection of Chlamydomonas reinhardtii products

[0097] 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. Then the algal cells were collected, and the contents of starch, pyruvate, glucose, sucrose, and glycerol in the algal strains were measured respectively.

[0098] The starch content was detected using a starch detection kit (Beijing Solarbio Science & Technology Co., Ltd.). The specific detection method is as follows.

[0099] 50 mL of algal solution was aspirated, centrifuged at 5000 rpm for 5 min at 4 °C to collect the algal bodies. 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 algal powder was weighed and placed in a 2.0 mL centrifuge tube, and 1 mL of reagent one was added and homogenized thoroughly. It was extracted in a water bath at 80 °C for 30 min; at room temperature, it was centrifuged at 8000 rpm for 5 min to collect the algal bodies; 0.5 mL of distilled water was added to resuspend, and it was gelatinized in a boiling water bath for 15 min. After cooling, 0.35 mL of reagent two was added, and it was 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 the reagent three working solution were taken into an EP tube, incubated in a water bath at 95 °C for 10 min, and naturally cooled to room temperature. The absorbance value A was measured at a wavelength of 620 nm.

[0100] After analysis, the recombinant Chlamydomonas reinhardtii strain containing the GCO pathway efficiently fixed CO2 as starch. Using the standard curve for calculation, its starch content was 1.33 g / 100 g of algal body dry weight.

[0101] The pyruvate content in the algal strain was detected using the Pyruvate Assay Kit (Nanjing Jiancheng Bioengineering Institute, A081-1-1, China), and the pyruvate content was extracted and determined by ultraviolet spectrophotometry; the glycerol content in the algal strain was detected using the Glycerol Assay Kit (Nanjing Jiancheng Bioengineering Institute, F005-2-1, China), and the glycerol content was extracted and determined by ultraviolet spectrophotometry; the glucose content in the algal strain was detected using the Glucose Kit (Nanjing Jiancheng Bioengineering Institute, A154-2-1, China), and the glucose content of the algal powder was extracted and determined by ultraviolet spectrophotometry. The sucrose content in the algal strain was detected using the Sucrose Measurement Kit (Nanjing Jiancheng Bioengineering Institute, A099-1-1, China), and the sucrose content of the algal powder was extracted and determined by ultraviolet spectrophotometry.

[0102] Example 4

[0103] Phenotypic characterization of the recombinant Chlamydomonas reinhardtii algal strain cultured with 5% CO2 (TP + 5% CO2)

[0104] 1) Detection of growth indicators of the Chlamydomonas reinhardtii algal strain

[0105] 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 at 22 °C and 110 rpm until the stationary phase;

[0106] During the culture, 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. After shaking 3 times, the OD 750 was measured, and the OD 750 absorbance value was recorded, and a growth curve was plotted;

[0107] 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 freeze-dried in a freeze dryer until constant weight, and the dry weight was weighed.

[0108] After analysis, the recombinant Chlamydomonas reinhardtii algal strain containing the GCO pathway efficiently utilized CO2 and converted CO2 into biomass. According to the analysis of the plotted growth curve, its growth rate was significantly increased by 31.6%. According to the analysis of the weighed dry weight, its dry weight was 0.048 g.

[0109] 2) Detection of Chlamydomonas reinhardtii products

[0110] The engineered 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.

[0111] After analysis, CO2 in the recombinant Chlamydomonas reinhardtii strain containing the GCO pathway was efficiently fixed into starch. Calculated using the standard curve, the starch content was 2.25 g / 100 g dry weight of algal cells.

[0112] Example 5

[0113] Detection of phenotypic characteristics of the recombinant Chlamydomonas reinhardtii strain using acetate and 5% CO2 (TAP + 5% CO2)

[0114] 1) Detection of growth indexes of Chlamydomonas reinhardtii strain

[0115] 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;

[0116] During the culture, 200 μL of the algal solution was taken every 24 h, placed in a 96-well plate with three replicates, and detected using a microplate reader. After shaking 3 times, OD 750 was measured, and the absorbance value of OD 750 was recorded to plot the growth curve;

[0117] 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 to a constant weight in a freeze dryer and weighed for the dry weight.

[0118] After analysis, the recombinant Chlamydomonas reinhardtii strain containing the GCO pathway efficiently converted CO2 into biomass. According to the analysis of the plotted growth curve, its growth rate was significantly increased by 0.85 times. According to the analysis of the weighed dry weight, its dry weight was 0.109 g.

[0119] 2) Detection of Chlamydomonas reinhardtii products

[0120] The engineered 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.

[0121] After analysis, CO2 in the recombinant Chlamydomonas reinhardtii strain containing the GCO pathway was efficiently fixed into starch. Using the standard curve for calculation, its starch content was 0.69 g / 100 g of algal dry weight.

[0122] 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 lipid content, where WT represents the Chlamydomonas reinhardtii strain CC-849 and GCO2 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, contents of carbohydrates, organic acids and alcohols of the recombinant microalgae strain are all significantly improved.

[0123] In summary, through the Chlamydomonas reinhardtii eukaryotic expression system, the present invention verified its function and found that glycolate oxidase, catalase and oxalate oxidase can efficiently fix CO2 into biomass and starch. Glycolate oxidase, catalase and oxalate oxidase 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 being able to participate in the synthesis of starch, and having broad application prospects in improving photosynthetic efficiency and carbon fixation.

[0124] 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 these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A recombinant microalgae strain, characterized in that, Microalgae strains that express glycolate oxidase, catalase, and oxalate oxidase 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: 3; The amino acid sequence of the oxalate oxidase is shown in SEQ ID NO:

5.

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 glycolate oxidase gene, catalase gene, and oxalate oxidase 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, or three of the glycolate oxidase gene, catalase gene, and oxalate oxidase gene are transformed into microalgae in the form of a recombinant vector together 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 oxalate oxidase.

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 glycolate oxidase, catalase, and oxalate oxidase 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 ID NO: 1; The amino acid sequence of the catalase is shown in SEQ ID NO: 3; The amino acid sequence of the oxalate oxidase is shown in SEQ ID NO: 5.

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