A method for constructing a carbon-fixing Thraustochytrium strain

Abstract

The present invention discloses a method for constructing a carbon-fixing Thraustochytrium strain, including obtaining the Thraustochytrium genomic sequence and functional annotation; obtaining the amino acid sequences of the enzymes in the endogenous reductive glycine carbon fixation pathway of known model organisms; performing a multiple sequence similarity alignment between the amino acid sequences of the enzymes in the endogenous reductive glycine carbon fixation pathway of known model organisms and the Thraustochytrium amino acid sequences; analyzing the conserved domains of the amino acid sequences of the enzymes related to the reductive glycine carbon fixation pathway in Thraustochytrium; identifying the function of the reductive glycine carbon fixation pathway in Thraustochytrium; expressing the genes related to acetyl-CoA synthesis in Thraustochytrium; performing laboratory adaptive evolution on Thraustochytrium; 13 performing 13C isotope tracer detection. This method can quickly identify the carbon fixation potential of Thraustochytrium strains and, through gene editing combined with an adaptive evolution strategy, enable Thraustochytrium to utilize carbon atoms from formic acid and CO2 to participate in the biosynthesis of high-value secondary metabolites.

Description

Technical Field

[0001] The present invention belongs to the field of microbial metabolic engineering, and specifically relates to the identification of an endogenous formate-reducing glycine carbon fixation pathway in Thraustochytrium and the construction of a carbon-fixing Thraustochytrium strain. Background Art

[0002] Thraustochytrium is an important group of eukaryotic heterotrophic protists that widely exist in marine environments. Thraustochytrium includes a total of 9 genera, namely Aurantiochytrium, Thraustochytrium, Botryochytrium, Parietichytrium, Sicyoidochytrium, Japonochytrium, Ulkenia, Schizochytrium, and Monorhizochytrium. Thraustochytrium can grow rapidly by heterotrophically utilizing organic substrates and can synthesize various high-value products such as DHA, EPA and other long-chain polyunsaturated fatty acids, and has important application prospects in the field of industrial microorganisms.

[0003] One-carbon compounds such as CO2, formic acid, etc. have been proposed as cheap and sustainable microbial raw materials. Global warming is becoming increasingly serious, and one of the main reasons is the continuous increase in the concentration of CO2 in the atmosphere. Since the Industrial Revolution, the large-scale use of fossil fuels has led to a sharp increase in CO2 emissions, far exceeding the environmental capacity. Therefore, using microorganisms to fix CO2 is one of the important technical means to slow down climate warming. In addition, formic acid is a one-carbon compound and is mainly used as a source of synthetic materials in industry. As an economic and sustainable carbon source, microorganisms fixing one-carbon compounds such as CO2 and formic acid into cell components will further reduce the medium cost and fix CO2 in the environment, which will contribute to the realization of "green bio-manufacturing" while implementing the country's "dual carbon" strategy. The formate-reducing glycine carbon fixation pathway can effectively utilize formic acid and CO2 as carbon sources to synthesize glycine, and ultimately be incorporated into protein synthesis and enter central carbon metabolism.

[0004] Due to the rapid growth of Thraustochytrium and its strong ability to synthesize high-value secondary metabolites, if it has the ability to fix CO2 and formic acid, enabling it to fix cheap and sustainable one-carbon compounds such as CO2 and formic acid while synthesizing high-value secondary metabolites such as docosahexaenoic acid and squalene, further reducing the fermentation cost and achieving carbon negativity, will significantly enhance the fermentation production potential of Thraustochytrium, promote the expansion of the application range of Thraustochytrium, and provide new ideas for slowing down global warming. Summary of the Invention

[0005] The present invention aims to overcome the deficiencies of the prior art and provides a simple and rapid method for identifying the glycine reduction pathway of Thraustochytrium, constructing a carbon-fixing Thraustochytrium strain, and enabling Thraustochytrium to utilize carbon atoms from formic acid and CO2 to participate in the biosynthesis of high-value secondary metabolites.

[0006] To achieve the above invention objectives, the present invention adopts the following technical solutions. A method for constructing a carbon-fixing Thraustochytrium strain mainly comprises the following steps:

[0007] S1 Obtain the genomic sequence, genes, and amino acid sequences of Thraustochytrium and perform functional annotation

[0008] (1) After obtaining the raw genomic sequencing (Raw Data) of Thraustochytrium, use the fastp (version: 0.21.0) software to remove the low-quality sequences and adapter sequences therein, and finally obtain the available filtered sequences (Clean Data). Use two software, GenomeScope (version: 1.0) and jellyfish (version: 2.2.10), to perform genomic survey analysis to estimate the genome size, heterozygosity, and other conditions;

[0009] (2) For the Clean Data, use the NECAT (version: 20200119) software to perform preliminary assembly on it, and then use the Racon (version: 1.4.13) software to perform two rounds of error correction on the preliminary assembled sequences, and finally obtain the final assembly result;

[0010] (3) For gene structure prediction, a method combining homology prediction, de novo prediction, and transcript prediction is used. Among them, homolog homology prediction (select at least two to three closely related species; software: Exonerate (version: v2.4.0)), de novo prediction (software: Augustus (version: 3.3.2), Genscan (version: 1.0), GlimmerHMM (version: 3.0.4), etc.), and the transcripts reconstructed from RNA-seq data through the stringtie (version: 2.1.4) software. Use the TransDecoder (version: v5.1.0) software to predict the coding frame. Use the MAKER (version: 2.31.10) software to integrate the gene sets predicted by various methods. Through filtering, a non-redundant and more complete gene set and amino acid set are formed;

[0011] (4) Gene function annotation mainly includes the following two methods. First, the sequence similarity search method is used to align the protein sequences encoded by genes in the gene set with the existing protein databases Uniprot, NR, and the metabolic pathway database KEGG using the diamond blastp software (version: 2.0.11.149) to obtain the functional information of the sequences and the information on the metabolic pathways that the proteins may participate in. Among them, KEGG annotation is associated with KEGG ORTHOLOGY and PATHWAY using the KOBAS (version: 3.0) software. The Uniprot database records the correspondence between each protein family and the functional nodes in Gene Ontology, and the biological functions performed by the protein sequences encoded by genes are predicted through this system. Subsequently, the Motif similarity search method is used to align with the CDD, Gene3D, Hamap, Panther, Pfam, Phobius, Pirsf, Pirsr, Prints, Prosite, Sfld, Smart, Superfamily, Tigrfam, and Tmhmm databases in the InterPro sub-database of the secondary database using InterProScan (version: 5.52-86.0) to obtain the conserved sequences, motifs, and domains of the proteins. In addition, the software hmmscan (version: 3.3.2) is also used for domain prediction to obtain the conserved sequences, motifs, and domains of the proteins.

[0012] S2 Obtaining Amino Acid Sequences of Enzymes in the Reductive Glycine Pathway of Known Model Organisms

[0013] (1) The amino acid sequences of endogenous reductive glycine carbon fixation pathway enzymes are retrieved by searching in the Genbank database.

[0014] (2) Endogenous reductive glycine carbon fixation pathway enzymes include: formate-THF ligase, methenyI-THF-cyclohydrolase, methylene-THF-dehydrogenase, aminomethyltransferase (glycine decarboxylase subunit T), glycine decarboxylase (glycine decarboxylase subunit P), dihydrolipoyl dehydrogenase, and glycine decarboxylase subunit H.

[0015] S3 Multiple sequence similarity alignment of the amino acid sequences of enzymes in the reductive glycine pathway of known model organisms and those of Thraustochytrium. (1) A library was constructed for all Thraustochytrium amino acid sequences using the software Blast+ (version: 2.14.0).

[0016] (2) Subsequently, the blastp function in Blast+ was used to score and align the amino acid sequences of enzymes in the reductive glycine pathway of known model organisms as target sequences against the Thraustochytrium amino acid sequences in turn. According to the alignment results, it was determined whether the corresponding genes were present in Thraustochytrium. (The Thraustochytrium amino acid sequences with a score greater than 200 and an E value less than 1e-40 were selected as candidate genes)

[0017] S4 Analysis of the conserved domains of the amino acid sequences of enzymes related to the endogenous reductive glycine carbon fixation pathway in Thraustochytrium. (1) The software NCBI conserve domains search (version: V3.20) was used to analyze the conserved domains of the predicted amino acid sequences of Thraustochytrium enzymes in the reductive glycine pathway and compare them with the conserved domains of the amino acid sequences of enzymes in the reductive glycine pathway of known model organisms, in order to further determine the similarity of the predicted amino acid sequences of Thraustochytrium enzymes in the reductive glycine pathway to those of known model organisms in terms of protein function.

[0018] S5 Functional identification of the endogenous reductive glycine carbon fixation pathway in Thraustochytrium

[0019] (1) Thraustochytrium cells were grown in the acclimated M4 fermentation medium containing 13-C labeled formic acid (250 mM) or sodium bicarbonate (100 mM) for 3 days (using unlabeled formic acid and sodium bicarbonate as the control groups; using 13 C-labeled formic acid and unlabeled sodium bicarbonate, unlabeled formic acid and 13 C-labeled sodium bicarbonate, and 13 C-labeled formic acid and containing 13 C-labeled sodium bicarbonate as 3 experimental groups). The cells were harvested by centrifuging 9 mL of the culture at 10,000 rpm for 5 minutes. After washing the cells with distilled water, the cells were lysed using 6M HCl and hydrolyzed at 95°C for 24 hours, and then the samples were completely dried at 95°C. The hydrolyzed samples were resuspended in 1 mL of distilled water and prepared for liquid chromatography-mass spectrometry (LC-MS) detection of the 13 C-isotope situation. LCMS was used to determine whether the amino acids such as glycine in the cell lysate contained carbon atoms from formic acid or sodium bicarbonate, so as to indicate the existence and function of the endogenous reductive glycine carbon fixation pathway in Thraustochytrium.

[0020] (2) The LC-MS parameters were as follows: for liquid chromatography, a C18 reverse-phase column was used, and the mobile phases were (A) water + 0.1% formic acid and (B) acetonitrile + 0.1% formic acid; the flow rate was 0.4 mL / min; the gradient was 0–1 min: 99% A; 1–5 min: from 99% A to 82% A (linear gradient); 5–6 min: from 82% A to 1% A (linear gradient); 6–8 min: 1% A; 8–8.5 min: from 1% A to 99% A (linear gradient); 8.5–11 min: 99% A; for the mass spectrometer part, the positive ionization mode was used, the scanning range was 50.0–300.0 m / z, spectra were recorded within the first 5 minutes of the LC gradient, and finally the precursor ion signal abundance was determined based on the target molecular weight. By comparing the results of different experimental groups, it was determined whether the carbon atoms of glycine in Thraustochytrium cells were derived from CO2 and formic acid to determine its CO2 fixation and formic acid assimilation capabilities.

[0021] Expression of genes related to acetyl-CoA synthesis in Thraustochytrium

[0022] (1) The target gene sequences were obtained from the GenBank database, including serine hydroxymethyltransferase gene, serine deaminase gene, glycine reductase gene, phosphotransacetylase gene, acetate kinase, and acetyl-CoA synthetase.

[0023] (2) Expression vectors of the target genes in Thraustochytrium (containing bleomycin resistance tags) were constructed, namely the serine hydroxymethyltransferase gene-serine deaminase gene expression vector, glycine reductase gene expression vector, and phosphotransacetylase gene-acetate kinase-acetyl-CoA synthetase expression vector.

[0024] (3) After mixing the above 3 linearized expression vectors with Thraustochytrium competent cells, using the electroporation method, after electroporation at 2000 v voltage, they were spread on M4 fermentation medium containing 1.5 mg / mL bleomycin and cultured at 28 °C for 5 days. Preparation of M4 fermentation medium: glucose (60 g / L), yeast extract (15 g / L), potassium dihydrogen phosphate (0.25 g / L), artificial seawater (33 g / L), pH 7.0.

[0025] Laboratory adaptive evolution of Thraustochytrium

[0026] (1) First, determine the tolerance of Thraustochytrium to formic acid at different concentrations. Inoculate the Thraustochytrium seed liquid into M4 medium (glucose: 20 g / L, artificial seawater: 33 g / L, potassium dihydrogen phosphate: 0.25 g / L, yeast extract: 1 g / L, bacteriological peptone: 1.5 g / L) containing sodium formate at concentrations of 1 mM, 3 mM, 30 mM, 50 mM, 250 mM, 500 mM, and 800 mM and 100 mM sodium bicarbonate, and culture at 28 °C and 170 rpm. Subsequently, measure the OD600 value of the bacterial liquid every day for 6 days to make a growth curve graph.

[0027] (2) According to the tolerance of Thraustochytrium to formic acid at different concentrations, select M4 medium containing 500 mM formic acid and 100 mM sodium bicarbonate as the domestication medium. Inoculate the Thraustochytrium seed liquid into the domestication medium and continuously subculture at 28 °C and 170 rpm for 180 days.

[0028] 13C isotope tracer detection of high-value secondary metabolites of Thraustochytrium 13 13C isotope tracer detection

[0029] (1) After growing Thraustochytrium cells in the domesticated M4 fermentation medium containing 13-C-labeled formic acid (250 mM) or sodium bicarbonate (100 mM) for 3 days (using unlabeled formic acid and sodium bicarbonate as the control group; using 13 13C-labeled formic acid and unlabeled sodium bicarbonate, unlabeled formic acid and 13 13C-labeled sodium bicarbonate, and 13 13C-labeled formic acid and containing 13 13C-labeled sodium bicarbonate as 3 experimental groups), centrifuge 9 mL of the culture at 10,000 rpm for 5 minutes to harvest the cells. After washing the cells with distilled water, lyse the cells using 6 M HCl and hydrolyze at 95 °C for 24 hours, and then completely dry the sample at 95 °C. Resuspend the hydrolyzed sample in 1 mL of distilled water to prepare for liquid chromatography-mass spectrometry (LC-MS) detection 13 of the 13C-isotope situation.

[0030] (2) LC-MS detection parameters of DHA: For liquid chromatography, a C18 reverse-phase column is used, and the mobile phase is a formic acid-acetonitrile system to form a non-polar gradient with a flow rate of 0.2 mL / min; for the mass spectrometer part, the negative ionization mode is used, the scanning range is 100.0–1000.0 m / z, and the multiple reaction monitoring mode is used. Finally, the precursor ion signal abundance is determined according to the molecular weight of DHA. LC-MS detection parameters of squalene: For liquid chromatography, a C18 reverse-phase column is used, and the mobile phase is a methanol-water system to form a non-polar gradient with a flow rate of 0.3 mL / min; for the mass spectrometer part, the positive ionization mode is used, the scanning range is 50.0–500.0 m / z, and the multiple reaction monitoring mode is used. Finally, the precursor ion signal abundance is determined according to the molecular weight of squalene.

[0031] The beneficial effects of the present invention are as follows: First, the rapid identification of the carbon fixation potential of Thraustochytrium helps to quickly identify the carbon fixation potential of Thraustochytrium, which is conducive to screening out Thraustochytrium chassis cells with high carbon fixation potential from nature. The acquisition of Thraustochytrium with high carbon fixation ability: The Thraustochytrium with carbon fixation obtained by this method has strong carbon fixation ability and high metabolic flux of the intracellular reductive glycine pathway, realizing the fixation of one-carbon compounds such as CO2 and formic acid while fermenting and producing DHA or squalene by Thraustochytrium, responding to the national "dual carbon" strategy, and further improving the sustainability of the fermentation synthesis of natural active products by Thraustochytrium. Brief Description of the Drawings

[0032] Figure 1 Functional verification result diagram of the reductive glycine pathway of Thraustochytrium TWZ-97.

[0033] Figure 2 Growth curve diagram of Thraustochytrium TWZ-97 under different concentrations of formic acid addition. Detailed Embodiments

[0034] The present invention will be further described below in conjunction with specific embodiments.

[0035] In the embodiments of the present application, Thraustochytrium TWZ-97 is taken as an example, but the present invention is not limited in any way, and other wild Thraustochytrium can also be used in the present invention:

[0036] S1 Acquisition of the genomic sequence, gene, and amino acid sequence of Thraustochytrium

[0037] (1) Filtering and assembly of the downloaded data:

[0038] After genome sequencing of Thraustochytrium sp. TWZ-97, the sequencing data was obtained. The low-quality sequences and adapter sequences were removed using the fastp (version: 0.21.0) software, and finally the available filtered sequences (CleanData) were obtained. Two software, GenomeScope (version: 1.0) and jellyfish (version: 2.2.10), were used for genome survey analysis to estimate the genome size, heterozygosity rate, etc. The results showed that the genome size was 62,086,562 bp and the heterozygosity rate was 0.98%. For the Clean Data, the software NECAT (version: 20200119) was used for preliminary assembly, and then the software Racon (version: 1.4.13) was used to correct the preliminary assembled sequences in two rounds. Finally, the final assembly result was obtained, and the specific assembly results are shown in the following table.

[0039] Assembly length (bp) 62,493,101.00 Number of contigs 26.00 GC content 45.01 N50 (bp) 2,583,946.00 N90 (bp) 1,678,149.00 Average contig length 2,403,580.81 Median contig length 2,372,063.00 Minimum contig length 1,119,645.00 Maximum contig length 4,079,669.00

[0040] Judging from the results, the continuity of the assembly is good, and the contig N50 is 2,583,946 bp; the genome integrity is good, and the proportion of Complete and single-copy BUSCOs is 90%.

[0041] (2) Gene structure prediction:

[0042] The method of combining homology prediction, de novo prediction and transcript prediction was used for gene structure prediction. Among them, homolog homology prediction (at least two to three closely related species were selected; software: Exonerate (version: v2.4.0)), de novo prediction (software: Augustus (version: 3.3.2), Genscan (version: 1.0), GlimmerHMM (version: 3.0.4), etc.), the transcripts reconstructed from RNA-seq data by the software stringtie (version: 2.1.4), and the software TransDecoder (version: v5.1.0) was used to predict the coding frame. The software MAKER (version: 2.31.10) was used to integrate the gene sets predicted by various methods. Through filtering, a non-redundant and more complete gene set and amino acid set were formed, and the total number of predicted genes was 11,858.

[0043] Obtaining the amino acid sequences of enzymes in the reductive glycine pathway of Saccharomyces cerevisiae S2

[0044] (1) The amino acid sequences of endogenous reductive glycine carbon fixation pathway enzymes were retrieved by searching in the Genbank database.

[0045] (2) The enzymes of the endogenous reductive glycine carbon fixation pathway include: formate-THF ligase, methenyl-THF-cyclohydrolase, methylene-THF dehydrogenase, aminomethyltransferase (glycine decarboxylase subunit T), glycine decarboxylase (glycine decarboxylase subunit P), dihydrolipoyl dehydrogenase, and glycine decarboxylase subunit H.

[0046] S3 Multiple sequence similarity alignment of the amino acid sequences of the enzymes in the reductive glycine pathway of Saccharomyces cerevisiae and Thraustochytrium (1) A database was constructed for all the amino acid sequences of Thraustochytrium TWZ-97 using the software Blast+ (version: 2.14.0). (2) Subsequently, the blastp function in Blast+ was used to score and align the amino acid sequences of the enzymes in the reductive glycine pathway of Saccharomyces cerevisiae as target sequences against the amino acid sequences of Thraustochytrium. The results showed that each amino acid sequence of the enzymes in the reductive glycine pathway of Saccharomyces cerevisiae had at least one corresponding amino acid sequence of Thraustochytrium TWZ-97 with a very high score and a very low expectation value.

[0047] S4 Analysis of the conserved domains of the amino acid sequences of the enzymes related to the endogenous reductive glycine carbon fixation pathway in Thraustochytrium

[0048] (1) The conserved domain analysis of the predicted amino acid sequences of the enzymes in the reductive glycine pathway of Thraustochytrium was performed using the software NCBI conserve domains search (version: V3.20), and compared with the conserved domains of the amino acid sequences of the enzymes in the reductive glycine pathway of Saccharomyces cerevisiae to further determine the similarity of the predicted amino acid sequences of the enzymes in the reductive glycine pathway of Thraustochytrium to known model organisms in terms of protein function. The results showed that the conserved domains contained in each predicted amino acid sequence of the enzymes in the reductive glycine pathway of Thraustochytrium were highly similar to the corresponding amino acid sequences of Saccharomyces cerevisiae.

[0049] S5 Functional identification of the endogenous reductive glycine carbon fixation pathway in Thraustochytrium

[0050] (1) After growing Thraustochytrium cells in the acclimated M4 fermentation medium containing 13-C labeled formic acid (250 mM) or sodium bicarbonate (100 mM) for 3 days, centrifuge 9 mL of the culture at 10,000 rpm for 5 minutes to harvest the cells. The grouping information is as follows: a. M4 medium with 250 mM formic acid and 100 mM sodium bicarbonate; b. M4 medium with 250 mM 13C-formic acid and 100 mM sodium bicarbonate; c. M4 medium with 250 mM formic acid and 100 mM 13C-sodium bicarbonate; d. M4 medium with 250 mM 13C-formic acid and 100 mM 13C-sodium bicarbonate. After washing the cells with distilled water, lyse the cells using 6 M HCl and hydrolyze at 95 °C for 24 hours, followed by completely drying the sample at 95 °C. Resuspend the hydrolyzed sample in 1 mL of distilled water and prepare for liquid chromatography-mass spectrometry (LC-MS) to detect the 13C-isotope situation, as Figure 1 shown.

[0051] (2) Use LCMS to determine whether amino acids such as glycine in the cell lysate contain carbon atoms from formic acid or sodium bicarbonate, so as to indicate the existence and function of the endogenous reductive glycine carbon fixation pathway in Thraustochytrium. The LC-MS parameters are as follows: for liquid chromatography, use a C18 reverse-phase column, and the mobile phases are (A) water + 0.1% formic acid and (B) acetonitrile + 0.1% formic acid; the flow rate is 0.4 mL / min; the gradient is 0–1 min: 99% A; 1-5 min: from 99% A to 82% A (linear gradient); 5–6 min: from 82% A to 1% A (linear gradient); 6-8 min: 1% A; 8-8.5 min: from 1% A to 99% A (linear gradient); 8.5–11 min: 99% A; for the mass spectrometer part, use the positive ionization mode, the scanning range is 50.0–300.0 m / z, record the spectrum within the first 5 minutes of the LC gradient, and finally determine the abundance of the parent ion signal according to the target molecular weight.

[0052] Expression of genes related to acetyl-CoA synthesis in Thraustochytrium

[0053] (1) Obtain the target gene sequences in the GenBank database, including serine hydroxymethyltransferase gene, serine deaminase gene, glycine reductase gene, phosphoacetyltransferase gene, acetate kinase, and acetyl-CoA synthetase.

[0054] (2) Construct the expression vectors of the target genes in Thraustochytrium (containing bleomycin resistance tags), namely the serine hydroxymethyltransferase gene-serine deaminase gene expression vector, glycine reductase gene expression vector, and phosphoacetyltransferase gene-acetate kinase-acetyl-CoA synthetase expression vector.

[0055] (3) After mixing the above three linearized expression vectors with the competent cells of Thraustochytrium, using the electroporation method, after electroporation at a voltage of 2000 v, coat them on the M4 fermentation medium containing 1.5 mg / mL bleomycin, and culture at 28 °C for 5 days. Preparation of M4 fermentation medium: glucose (60 g / L), yeast extract (15 g / L), potassium dihydrogen phosphate (0.25 g / L), artificial sea salt (33 g / L), pH 7.0.

[0056] S7 Laboratory Adaptive Evolution of Thraustochytrium

[0057] (1) First, determine the tolerance of Thraustochytrium to different concentrations of formic acid. Inoculate the Thraustochytrium seed liquid into the M4 medium (glucose: 20 g / L, artificial sea salt: 33 g / L, potassium dihydrogen phosphate: 0.25 g / L, yeast extract: 1 g / L, bacterial peptone: 1.5 g / L) containing 1 mM, 3 mM, 30 mM, 50 mM, 250 mM, 500 mM and 800 mM sodium formate and 100 mM sodium bicarbonate respectively, and culture at 28 °C, 170 rpm. Subsequently, measure the OD600 value of the bacterial liquid every day for 6 days, make a growth curve, as Figure 2 shown.

[0058] (2) According to the tolerance of Thraustochytrium to different concentrations of formic acid, select the M4 medium containing 500 mM formic acid and 100 mM sodium bicarbonate as the domestication medium, inoculate the Thraustochytrium seed liquid into the domestication medium, and continuously subculture at 28 °C, 170 rpm for 180 days.

[0059] S8 13 13C Isotope Tracing Detection of High-Value Secondary Metabolites of Thraustochytrium

[0060] (1) Grow Thraustochytrium cells in the domesticated M4 fermentation medium containing 13-C labeled formic acid (250 mM) or sodium bicarbonate (100 mM) for 3 days (using unlabeled formic acid and sodium bicarbonate as the control group; using 13 13C labeled formic acid and unlabeled sodium bicarbonate, unlabeled formic acid and 13 13C labeled sodium bicarbonate and 13 13C labeled formic acid and containing 13 13C labeled sodium bicarbonate as 3 experimental groups), centrifuge 9 mL of the culture at 10000 rpm for 5 minutes to harvest the cells. After washing the cells with distilled water, use 6M HCl to lyse the cells, hydrolyze at 95 °C for 24 hours, and then completely dry the sample at 95 °C. Resuspend the hydrolyzed sample in 1 mL of distilled water, and prepare for liquid chromatography-mass spectrometry (LC-MS) detection 13 of 13C-isotope situation.

[0061] (2) LC-MS detection parameters for DHA: The liquid chromatography uses a C18 reverse-phase column, the mobile phase is a formic acid-acetonitrile system to form a non-polar gradient, and the flow rate is 0.2 mL / min; for the mass spectrometer part, the negative ionization mode is used, the scanning range is 100.0–1000.0 m / z, the multiple reaction monitoring mode is used, and finally the parent ion signal abundance is determined according to the molecular weight of DHA. LC-MS detection parameters for squalene: The liquid chromatography uses a C18 reverse-phase column, the mobile phase is a methanol-water system to form a non-polar gradient, and the flow rate is 0.3 mL / min; for the mass spectrometer part, the positive ionization mode is used, the scanning range is 50.0–500.0 m / z, the multiple reaction monitoring mode is used, and finally the parent ion signal abundance is determined according to the molecular weight of squalene.

[0062] References:

[0063] 1. Yishai, O., Bouzon, M., V., & Bar-Even, A. (2018). In Vivo Assimilation of One-Carbon via a Synthetic Reductive Glycine Pathway in Escherichia coli. ACS synthetic biology, 7(9), 2023–2028.

[0064] 2. Gonzalez de la Cruz, J., Machens, F., Messerschmidt, K., & Bar-Even, A. (2019). Core Catalysis of the Reductive Glycine Pathway Demonstrated in Yeast. ACS synthetic biology, 8(5), 911–917.

[0065] 3. Sánchez-Andrea, I., Guedes, I.A., Hornung, B., Boeren, S., Lawson, C.E., Sousa, D.Z., Bar-Even, A., Claassens, N.J., & Stams, A.J.M. (2020). The reductive glycine pathway allows autotrophic growth of Desulfovibrio desulfuricans. Nature communications, 11(1), 5090.

Claims

1. A method for constructing a carbon-fixing Thraustochytrium strain, characterized in that, It includes the following steps: (1) Obtain the genomic sequence, coding genes, and amino acid sequences of Thraustochytrium and perform functional annotation: Obtain its genomic sequence, all coding genes, amino acid sequences, and annotation information by performing adapter removal, data filtering, genomic survey analysis, genomic assembly, and annotation on the initial sequencing data obtained from genomic sequencing of Thraustochytrium; (2) Obtain the amino acid sequences of enzymes in the endogenous glycine reduction pathway of known model organisms: Retrieve the amino acid sequences of enzymes in the endogenous glycine carbon fixation pathway through the Genbank database search; (3) Perform multiple sequence similarity alignment between the amino acid sequences of enzymes in the endogenous glycine reduction pathway of known model organisms and the amino acid sequences of Thraustochytrium: Use the BLAST software to perform multiple sequence alignment analysis of the amino acid sequences of enzymes in the endogenous glycine carbon fixation pathway of each known model organism against all amino acid sequences of Thraustochytrium; Among them, the known model organism is Saccharomyces cerevisiae; (4) Analyze the conserved domains of the amino acid sequences of enzymes related to the endogenous glycine carbon fixation pathway in Thraustochytrium: Use the NCBI conserved domain search software to perform domain function analysis on the predicted amino acid sequences of enzymes related to the endogenous glycine pathway in Thraustochytrium; (5) Identify the function of the endogenous glycine carbon fixation pathway in Thraustochytrium: Add 13 C-labeled formic acid and sodium bicarbonate to the Thraustochytrium medium, lyse the cells after culturing for 2 generations, and take the supernatant for liquid chromatography-mass spectrometry to detect whether glycine in the cells carries 13 C-labeled formic acid and sodium bicarbonate carbon atoms; (6) Express the genes related to acetyl-CoA synthesis in Thraustochytrium: Overexpress the serine hydroxymethyltransferase gene, serine deaminase gene, glycine reductase gene, phosphoacetyltransferase gene, acetate kinase, and acetyl-CoA synthase gene in Thraustochytrium TWZ-97 respectively to obtain engineering strains; (7) Laboratory adaptive evolution of Thraustochytrium: Continuously subculture the Thraustochytrium engineering strains obtained in the above steps in the evolutionary M4 fermentation medium. The formula of the evolutionary M4 fermentation medium is as follows: glucose 60 g / L, yeast extract 15 g / L, potassium dihydrogen phosphate 0.25 g / L, artificial sea salt 33 g / L, sodium formate 500 mM, sodium bicarbonate 100 mM, and pH 7.0; (8) High-value secondary metabolites of Thraustochytrium 13 C isotope tracer detection: Add 13 formic acid labeled with 13 C and sodium bicarbonate to the Thraustochytrium medium. After culturing for 2 generations, lyse the cells, take the supernatant and perform liquid chromatography-mass spectrometry to detect whether docosahexaenoic acid and squalene in the cells carry 13 carbon atoms of formic acid labeled with 13 C and sodium bicarbonate.

2. The construction method of a carbon-fixing Thraustochytrium strain according to claim 1, characterized in that, For the step (1) of obtaining the genomic sequence, coding genes, and amino acid sequences of Thraustochytrium and performing functional annotation, the following software is used: (1) fastp, GenomeScope, jellyfish: for genomic survey analysis; (2) NECAT, Racon: for genome third-generation sequencing sequence assembly and error correction; (3) Exonerate, Augustus, Genscan, TransDecoder, stringtie, and Maker: for performing genome structure prediction, including homology prediction, de novo prediction, open reading frame prediction, and evidence integration, thereby obtaining the genomic genes and amino acid sequences of Thraustochytrium; (4) InterProScan, Uniprot, hmmscan, Pfam, NR, KOBAS: for performing genome function and domain annotation, thereby obtaining the gene function information of Thraustochytrium.

3. The construction method of a carbon-fixing Thraustochytrium strain according to claim 1, characterized in that, The multiple sequence similarity alignment of the amino acid sequences of the endogenous reductive glycine pathway enzymes of the known model organism and the Thraustochytrium amino acid sequences in step (3) is performed using BLAST.

4. The construction method of a carbon-fixing Thraustochytrium strain according to claim 1, characterized in that, The conserved domain analysis of the amino acid sequences of the endogenous reductive glycine carbon fixation pathway-related enzymes of Thraustochytrium in step (4) is performed using the NCBI conserved domain search software.

5. The construction method of a carbon-fixing Thraustochytrium strain according to claim 1, characterized in that, Functional identification of the endogenous reductive glycine carbon fixation pathway of Thraustochytrium, with unlabeled formic acid at 250 mM and sodium bicarbonate at 100 mM as the control group; respectively with 13 250 mM of 13C-labeled formic acid and 100 mM of unlabeled sodium bicarbonate, 250 mM of unlabeled formic acid and 13 100 mM of 13C-labeled sodium bicarbonate, and 13 250 mM of 13C-labeled formic acid and 100 mM of sodium bicarbonate containing 13 13C as three experimental groups; in addition, the LC-MS parameters use a C18 reverse-phase column for liquid chromatography, with the mobile phase being A: water + 0.1% formic acid, B: acetonitrile + 0.1% formic acid; the flow rate is 0.4 mL / min; the gradient is: 0–1 min, 99% A; 1-5 min, 99% A to 82% A; 5–6 min, 82% A to 1% A; 6-8 min, 1% A; 8-8.5 min, from 1% A to 99% A; 8.5–11 min, 99% A; the mass spectrometer uses the positive ionization mode, and the scanning range is 50.0–300.0 m / z.

6. The construction method of a carbon-fixing Thraustochytrium strain according to claim 1, characterized in that, The expression of the genes related to the synthesis of acetyl-CoA in Thraustochytrium in step (6): The overexpression of the serine hydroxymethyltransferase gene and the serine deaminase gene converts glycine containing formate carbon atoms and CO2 carbon atoms into pyruvate; the overexpression of the glycine reductase gene and the phosphotransacetylase gene or acetate kinase and acetyl-CoA synthetase converts glycine containing formate carbon atoms and CO2 carbon atoms into pyruvate acetyl-CoA.

7. The construction method of a carbon-fixing Thraustochytrium strain according to claim 1, characterized in that, The 13 C isotope tracer detection of the high-value secondary metabolites of Thraustochytrium includes: using unlabeled formic acid at 250 mM and sodium bicarbonate at 100 mM as the control group; respectively using 13 250 mM of 13 C-labeled formic acid and 100 mM of unlabeled sodium bicarbonate, 250 mM of unlabeled formic acid and 13 100 mM of 13 C-labeled sodium bicarbonate, and 250 mM of C-labeled formic acid and 100 mM of C-labeled sodium bicarbonate as three experimental groups; in addition, the LC-MS detection parameters for DHA are: for liquid chromatography, using a C18 reversed-phase column, with a mobile phase of formic acid-acetonitrile system, forming a non-polar gradient, and a flow rate of 0.2 mL / min; for the mass spectrometer part, using the negative ionization mode, with a scanning range of 100.0–1000.0 m / z, using the multiple reaction monitoring mode, and finally determining the precursor ion signal abundance according to the molecular weight of DHA; the LC-MS detection parameters for squalene are: for liquid chromatography, using a C18 reversed-phase column, with a mobile phase of methanol-water system, forming a non-polar gradient, and a flow rate of 0.3 mL / min; for the mass spectrometer part, using the positive ionization mode, with a scanning range of 50.0–500.0 m / z, using the multiple reaction monitoring mode, and finally determining the precursor ion signal abundance according to the molecular weight of squalene.

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