A method for increasing the pentadecanoic acid content in Schizochytrium

Abstract

This invention relates to the fields of genetic engineering and microbial lipid metabolism regulation, specifically to a method for increasing the pentadecanoic acid content in *Schizochytrium*. The method includes constructing a gene knockout strain, wherein the ScAMPKα1, ScAMPKα2, or ScAMPKα3 genes are individually knocked out in *Schizochytrium* using homologous recombination technology. The amino acid sequence of the protein encoded by the ScAMPKα1 gene is shown in SEQ ID NO.1, the amino acid sequence of the protein encoded by the ScAMPKα2 gene is shown in SEQ ID NO.2, and the amino acid sequence of the protein encoded by the ScAMPKα3 gene is shown in SEQ ID NO.3. This invention is the first to knock out the adenosine monophosphate-activated protein kinase AMPKα subunit gene, effectively increasing the accumulation level of pentadecanoic acid in *Schizochytrium*.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and microbial lipid metabolism regulation, specifically to a method for increasing the pentadecanoic acid content in Schizochytrium. Background Technology

[0002] Pentadecanoic acid (C15:0), an odd-chain saturated fatty acid, has attracted widespread attention in recent years due to its unique physiological activities. Studies have shown that pentadecanoic acid not only participates in the structural composition of cell membranes, affecting membrane fluidity and stability, but also plays an important role in improving insulin sensitivity, inhibiting inflammatory responses, and reducing the risk of cardiovascular disease by regulating key metabolic signaling pathways such as AMP-activated protein kinase (AMPK). Compared with traditional sources of animal and vegetable oils, the microbial fermentation method for producing pentadecanoic acid has advantages such as short cycle time, high controllability, and no seasonal or geographical limitations, making it a research hotspot in this field. Among them, *Schizochytrium* sp., a high-oil-producing marine microorganism, has been widely used in the industrial production of polyunsaturated fatty acids such as docosahexaenoic acid (DHA) due to its strong fatty acid synthesis capacity, and has also been regarded as a potential excellent host for odd-chain fatty acid synthesis in recent years.

[0003] Currently, the synthesis of pentadecanoic acid (PCA) using microorganisms mainly relies on enhancing fatty acid synthesis pathways through metabolic engineering or fermentation condition optimization. However, several bottlenecks remain in existing technologies. On the one hand, the biosynthesis of odd-chain fatty acids typically depends on propionyl-CoA (PCOA) as the starting unit. However, under conventional fermentation conditions, the supply of PCOA within microbial cells is limited, resulting in a generally low proportion of PCA in total fatty acids, making efficient accumulation difficult. On the other hand, the expression intensity of key enzymes in the fatty acid synthesis pathway, the supply balance of reducing power (NADPH), and the distribution of intermediate metabolites all significantly affect the final concentration and production intensity of the target product. Traditional single-factor optimization strategies are insufficient to systematically address the synergistic regulation issues within these metabolic networks.

[0004] To address the aforementioned shortcomings, existing research has largely focused on increasing pentadecanoic acid (PCA) production through the exogenous addition of precursors (such as propionate and methylmalonic acid) or overexpression of key enzymes in the propionyl-CoA synthesis pathway. While this has improved PCA production to some extent, it suffers from high costs, heavy metabolic burden, and poor strain stability. In recent years, strategies to reshape cellular metabolic flux distribution by regulating global metabolic nodes have gained increasing attention. AMPK, a highly conserved core regulator of energy metabolism in eukaryotes, plays a crucial role in sensing cellular energy status and coordinating the balance between catabolism and anabolism. In oil-producing microorganisms such as Schizochytrium, the activity state of AMPK directly affects the phosphorylation level of key lipid synthases such as acetyl-CoA carboxylase (ACC), thereby regulating the flux of fatty acid synthesis pathways. However, there are currently no reports, either domestically or internationally, on the targeted increase of PCA production by knocking out the AMPK gene in Schizochytrium. In existing technologies, the regulation of AMPK is mostly focused on the addition of small molecule agonists (such as metformin and AICAR). However, such exogenous regulation methods have problems such as high cost, short duration of action and possible interference with other physiological processes in cells, making it difficult to achieve stable and efficient metabolic remodeling.

[0005] Therefore, there is an urgent need in this field to develop a method that can systematically enhance the pentadecanoic acid synthesis capacity of Schizochytrium. Summary of the Invention

[0006] This invention addresses the technical bottleneck of low pentadecanoic acid synthesis efficiency in Schizochytrium fungi by providing a method to increase pentadecanoic acid production by knocking out the gene encoding the AMPKα subunit.

[0007] The technical principle of this invention is to target and knock out the gene encoding the AMPKα subunit of adenosine monophosphate-activated protein kinase in Schizochytrium, thereby relieving its negative regulation of the lipid synthesis pathway and synergistically enhancing the supply of propionyl-CoA precursor and the flux of fatty acid synthesis, thus breaking through the production bottleneck of existing technologies and providing an efficient and feasible technical solution for the green biomanufacturing of pentadecanoic acid.

[0008] This invention first identifies candidate genes for the AMPKα subunit from the whole genome of Schizochytrium using bioinformatics methods, and analyzes their conserved domains and phylogenetic characteristics. Based on this, knockout vectors for the three AMPKα subunit genes are constructed using a homologous recombination strategy. Stable single-gene knockout strains are obtained by electroporation. Under standard culture conditions, the biomass, oil yield, and fatty acid composition of wild-type and knockout strains are systematically compared to evaluate the functional contribution of each subunit in metabolic regulation.

[0009] This invention is the first to target the AMPKα subunit of Schizochytrium as a metabolic engineering target. By knocking out the gene, the negative regulation of lipid synthesis by AMPK is relieved, and precursor supply and metabolic flux are synergistically enhanced. Experiments show that the engineered strain exhibits significantly increased pentadecanoic acid production with genetic stability, providing a new technical pathway for the green and efficient production of odd-chain fatty acids, and possessing promising prospects for industrial application.

[0010] The technical solution adopted by this invention to solve the technical problem is:

[0011] A method for increasing the pentadecanoic acid content in *Schizochytrium* includes constructing a gene knockout strain. The constructed gene knockout strain is obtained by individually knocking out the ScAMPKα1 gene, ScAMPKα2 gene, or ScAMPKα3 gene in *Schizochytrium* using homologous recombination technology. The amino acid sequence of the protein encoded by the ScAMPKα1 gene is shown in SEQ ID NO.1, the amino acid sequence of the protein encoded by the ScAMPKα2 gene is shown in SEQ ID NO.2, and the amino acid sequence of the protein encoded by the ScAMPKα3 gene is shown in SEQ ID NO.3.

[0012] The mutant strain in this experiment was obtained by knocking out three homologous genes ScAMPKα1, ScAMPKα2 and ScAMPKα3 of the AMPKα subunit on the genome of Schizochytrium ATCC20888, respectively. Their amino acid sequences are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, and their encoding gene nucleotide sequences are shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.

[0013] Furthermore, in the homologous recombination technology, the homologous recombination vector includes the upstream homologous arm shown in SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9, the downstream homologous arm shown in SEQ ID NO.10, SEQ ID NO.11 or SEQ ID NO.12, and the intermediate resistance selection marker gene.

[0014] Furthermore, the Schizochytrium is *Schizochytrium* sp. ATCC20888.

[0015] Furthermore, the resistance selection marker gene is the norsyl trichosin resistance gene, the nucleotide sequence of which is shown in SEQ ID NO. 13.

[0016] This invention provides a method for constructing a mutant strain of Schizochytrium, comprising the following steps:

[0017] (1) Homologous arm gene acquisition: The ATCC20888 strain was sequenced to obtain the upstream and downstream homologous arm sequences of the three homologous genes of the AMPKα subunit (shown in SEQ ID NO.7–12), and each DNA fragment was amplified by PCR;

[0018] (2) Vector construction and verification: The homologous arms were inserted into the pMD19T-NTC vector using seamless cloning to construct the knockout recombinant plasmid, which was transformed into E. coli DH5α. After being verified by sequencing, it was stored at -80 ℃.

[0019] (3) Electroconversion: Take 15 μL (about 5 μg) of linearized recombinant plasmid and introduce it into the host protoplast. The electroconversion parameters are 1.8 kV, 200 Ω, and 25 μF.

[0020] (4) Screening and verification: The transformation product was plated on GPY medium containing 50 μg / mL NTC and cultured for 7 days to obtain resistant transformants; the gene knockout was confirmed to be successful by genomic PCR and fatty acid composition analysis after fermentation, and the mutant was stored at -80 ℃.

[0021] (5) Comparison between mutant and wild type: The mutant and wild type (WT) were cultured in GPY medium for 24-48 h to obtain seed liquid, which was then transferred to fermentation medium. After 72 h of culture, the cells were collected for biomass determination and fatty acid content and composition analysis.

[0022] In some implementations, the GPY culture medium is formulated as follows: 3% glucose, 1% peptone, 0.5% yeast extract, and 2% sea salt.

[0023] In some embodiments, the 1L fermentation medium contains the following components: 100 g glucose, 5 g yeast extract, 1.43 g MgSO4, 1 g KH2PO4, 0.5 g (NH4)2SO4, 0.264 g KCl, 0.04 g CaCl2, 0.001 g vitamin B1, and 0.001 g vitamin B2. 12 .

[0024] This invention utilizes homologous recombination technology to individually knock out the ScAMPKα1, ScAMPKα2, or ScAMPKα3 genes, resulting in mutant strains of *Schizochytridactylate* ΔScAMPKα1, ΔScAMPKα2, and ΔScAMPKα3. Compared to the wild type, the proportion of the odd-carbon saturated fatty acid pentadecanoic acid was significantly increased in all knockout strains. In particular, ΔScAMPKα2 showed a 132.4% increase in pentadecanoic acid proportion compared to the wild type.

[0025] The beneficial effects achieved by this invention are: 1) This invention is the first to knock out the AMPKα subunit gene of adenosine monophosphate-activated protein kinase, which effectively increases the accumulation level of pentadecanoic acid in Schizochytrium; 2) Under standard fermentation conditions, the modified Schizochytrium engineered strain has significantly increased the yield of pentadecanoic acid and its proportion of total fatty acids compared with the wild-type strain. Moreover, the engineered strain has good genetic stability and does not require the addition of exogenous precursors or inducers, which greatly reduces the production cost. This invention provides a new technical path for the green and efficient biomanufacturing of pentadecanoic acid. Attached Figure Description

[0026] Figure 1 Phylogenetic tree analysis of the AMPKα subunit of Schizochytrium;

[0027] Figure 2 Plasmid map of the ScAMPKα1 knockout vector;

[0028] Figure 3 Plasmid map of ScAMPKα2 knockout vector;

[0029] Figure 4 Plasmid map of ScAMPKα3 knockout vector;

[0030] Figure 5 Agarose gel electrophoresis image to verify the knockout of three AMPKα subunit genes in Schizochytrium ATCC 20888;

[0031] Figure 6 Colony morphology of wild-type and ScAMPKα knockout strains;

[0032] Figure 7 Cell morphology and lipid droplet distribution of wild-type and ScAMPKα knockout strains;

[0033] Figure 8 Phenotypic analysis of wild-type and ScAMPKα knockout strains under standard culture conditions;

[0034] Figure 9 This is a graph showing the fatty acid analysis of wild-type and ScAMPKα knockout strains under standard culture conditions. Detailed Implementation

[0035] Example 1

[0036] Evolutionary analysis of AMPK homologs in Schizochytrium

[0037] Using the *Saccharomyces cerevisiae* Snf1 (AMPK) protein kinase as the query sequence, a local alignment of the *Schizochytrium* genome database yielded five significantly similar sequences. Three of these genes were identified as potential AMPKα subunit encoding genes, exhibiting a multi-gene pattern similar to *Arabidopsis thaliana*, unlike the single-gene pattern of *Saccharomyces cerevisiae*, suggesting a potentially more complex regulatory network. Structural analysis revealed that only ScAMPKα1 possesses the complete binary structure of a typical AMPKα subunit, with a complete serine / threonine protein kinase catalytic domain at the N-terminus and a characteristic regulatory domain at the C-terminus, indicating that ScAMPKα1 may serve as the catalytic subunit of the *Schizochytrium* AMPK signaling pathway. In contrast, ScAMPKα2 and ScAMPKα3 contain only a single C-terminal regulatory domain and an N-terminal kinase domain, respectively, suggesting they are atypical members of this kinase family that evolved within *Schizochytrium*. Open reading frame predictions indicate that all three contain complete coding sequences with lengths of 1959 bp, 1449 bp, and 1134 bp, respectively, encoding 653, 483, and 378 amino acids.

[0038] Phylogenetic analysis ( Figure 1 The results indicate that ScAMPKα1 clusters with typical AMPKα / Snf1 catalytic subunits in diatoms, plants, animals, and fungi, forming the mainstream evolutionary clade and confirming its typical identity as a conserved catalytic core. ScAMPKα2 and ScAMPKα3, however, are independent of this typical clade, forming independent evolutionary clusters with atypical homologous sequences from various green algae. Within these clusters, they cluster with sequences from different algae, suggesting further differentiation within them. These phylogenetic relationships are consistent with the results of conserved domain analysis: ScAMPKα1 has a complete structure, while ScAMPKα2 and ScAMPKα3 exhibit simplified domain characteristics and are widely associated with atypical algal homologs, suggesting that these genotypes may have independent functional significance in some algal lineages.

[0039] Example 2

[0040] Schizochytrium AMPKα subunit gene knockout experiment

[0041] The method for constructing a mutant strain of Schizochytrium includes the following steps:

[0042] (1) Construction of recombinant plasmid pMD19T-ScAMPKα-NTC

[0043] Using the genome of *Schizochytrium ATCC* 20888 as a template, PCR amplification was performed using primer pairs AMPKα1-up-F / R, AMPKα1-do-F / R, AMPKα2-up-F / R, AMPKα2-do-F / R, AMPKα3-up-F / R, and AMPKα3-do-F / R. The upstream and downstream homologous arm DNA fragments of the AMPKα subunit gene were ultimately obtained. The primer sequences are shown in Table 1 below.

[0044] Table 1 Primer sequences for amplifying the upstream and downstream homologous arms of the scAMPKα subunit gene.

[0045]

[0046] The upstream and downstream homologous arms of ScAMPKα1, ScAMPKα2, and ScAMPKα3 obtained from the above amplification were ligated to plasmid pMD19T-NTC using a seamless cloning method and incubated in a 50 ℃ water bath for 30 min. The vector maps of the three knockout plasmids after final construction are shown below. Figure 2 , Figure 3 and Figure 4 As shown.

[0047] The seamless cloning described above was performed using the following steps: the ligation system was ligated at 50 °C for 30 min, and the ligation system was transformed into competent E. coli DH5α cells; E. coli transformation was performed, and the cells were cultured at 200 rpm at 37 °C for 1 h, then centrifuged at 4000 rpm for 2 min, and finally plated on LB solid medium containing 100 μg / mL ampicillin and incubated upside down overnight at 37 °C for 12-16 h until transformants appeared;

[0048] (2) Construction of Schizochytrium mutant strains ΔScAMPKα1, ΔScAMPKα2 and ΔScAMPKα3

[0049] The three recombinant plasmids pMD19T-ScAMPKα1-NTC, pMD19T-ScAMPKα2-NTC, or pMD19T-ScAMPKα3-NTC were double-digested with the restriction enzymes AflII and NdeI, respectively. The linearized vectors were obtained by gel extraction and used for gene knockout experiments in Schizochytrium. The linearized vectors were stored at -20 °C.

[0050] Preparation of competent cells of Schizochytrium

[0051] The preparation of competent *Schizochytrium* cells was carried out according to the following steps: Single colonies were picked from a plate and inoculated into 50 mL of *Schizochytrium* seed culture medium. The culture was incubated in a shaker at 28 °C and 180 rpm for 24 h to obtain the seed culture. After confirming the absence of microbial contamination under a microscope, 50 mL of the bacterial culture was collected by centrifugation at 4000 rpm for 5 min, and the supernatant was discarded. Subsequently, the bacterial cells were resuspended in 30 mL of 1 M sorbitol solution and centrifuged at 4 °C and 4000 rpm for 5 min, and the supernatant was discarded again; this step was for washing the cells. 5 mL of transformation treatment solution was added to the bacterial cells for resuspending, and 50 μL of 1 M DTT solution was added (to achieve a final concentration of 10 mM). The mixture was gently pipetted and incubated at room temperature for 30 min. After incubation, the cells were centrifuged at 4 °C and 4000 rpm for 5 min, and the supernatant was discarded to collect the treated bacterial cells. The bacterial cells were washed twice with 30 mL of pre-chilled 1 M sorbitol solution (centrifuged at 4 °C, 4000 rpm for 5 min each time, and the supernatant was discarded). After the final wash, the bacterial cells were resuspended in 1 mL of pre-chilled 1 M sorbitol solution and aliquoted into pre-chilled 1.5 mL EP tubes and kept on ice. This completes the preparation of *Schizochytrium* competent cells.

[0052] Electroporation transformation of Schizochytrium competent cells

[0053] Take 80 μL of the prepared competent cells and gently pipette-mix them with 15 μL (approximately 5 μg) of purified linearized DNA fragments. Transfer the mixture to a 2 mm electroporation cuvette and incubate on ice for 20 min. Wipe the outer wall of the cuvette clean with lint-free paper and place it in an electroporator for electroporation. The electroporation parameters are set as follows: voltage 1800 V, resistance 200 Ω, and capacitance 25 μF. To improve transformation efficiency, this electroporation process is repeated twice. Immediately after electroporation, add 1 mL of pre-chilled 1 M sorbitol solution to the cuvette and gently pipette-mix to resuspend the cells. Then transfer the suspension to a sterile 1.5 mL EP tube. Place the EP tube in a shaker at 28 ℃ and 180 rpm for 4 h of recovery culture. After recovery culture, take 100 μL of bacterial culture and spread it evenly on a selection plate containing 50 μg / mL NTC antibiotic. The plates were inverted and incubated in a 28 ℃ constant temperature incubator for 7 days, awaiting subsequent screening and validation.

[0054] PCR verification of transformants

[0055] After clearly visible single colonies grew on the plates, transformants were picked using a sterile pipette tip and cultured in shake flasks in liquid medium. Finally, the successfully cultured strains were added to glycerol and stored in a laboratory -80 °C cryogenic freezer. Each transformant strain was inoculated into *Schizochytrium* seed liquid medium and cultured in a shaker at 28 °C and 180 rpm for 24 h. The bacterial cells were collected, and their genomic DNA was extracted. Using the genomic DNA of the wild-type strain ATCC20888 as a negative control template, PCR amplification was performed using specific primers designed for the target gene knockout site. The amplification products were analyzed by agarose gel electrophoresis. By comparing the size and presence of bands in the transformants and wild-type controls, the complete knockout of the target gene was verified. The agarose gel electrophoresis results for verifying the knockout of the three AMPKα subunit genes in *Schizochytrium* ATCC20888 were as follows: Figure 5 As shown.

[0056] Example 3: Basic phenotypic analysis of Schizochytrium mutant strains

[0057] Colony and cell morphology were compared between the wild type and the three knockout strains. Figure 6 As shown, on the GPY solid plate of Schizochytrium, the colony morphology, size, color and edge characteristics of the knockout strain were not significantly different from those of the wild type.

[0058] To further investigate the effect of knockout on lipid droplet distribution within *Schizochytrium* cells, cells were observed using laser confocal microscopy. Figure 7 As shown, Figure 7 In the diagram, A represents dark field, B represents superposition, and C represents bright field. Scale bar = 10 μm. Both wild-type and knockout strains exhibited typical spherical cells with similar size distribution and intracellular lipid droplet sizes. These results indicate that, compared to the wild-type strain, knockout of the three AMPKα subunits did not induce any noticeable significant changes at the cellular and colony morphology level or at the intracellular lipid droplet level.

[0059] Example 4: Fermentation Culture Analysis of Schizochytrium Mutant Strains

[0060] Microscopic observation was used to subculture vigorous and uncontaminated wild-type and mutant strains of the fungus to ensure its healthy growth. Fermentation culture was then conducted using seed cultures of vigorous and uncontaminated wild-type and mutant strains. 1 L of fermentation medium contained the following components: 100 g glucose, 5 g yeast extract, 1.43 g MgSO4, 1 g KH2PO4, 0.5 g (NH4)2SO4, 0.264 g KCl, 0.04 g CaCl2, 0.001 g vitamin B1, and 0.001 g vitamin B2. 12Add the seed culture to the fermentation medium, and inoculate with the seed culture at a volume of 2.5% of the medium volume.

[0061] Wild-type and mutant strains were fermented for 72 h, and cell counts were collected for biomass determination and lipid extraction.

[0062] Biomass determination method: Take 1 mL of Schizochytrium fermentation broth, centrifuge at 8000 rpm for 5 min, discard the supernatant, wash the obtained cell precipitate twice with distilled water, centrifuge again at 8000 rpm for 5 min, discard the supernatant, dry in an oven at 80 ℃ for 3 days, and weigh.

[0063] Oil extraction method: Collect 35 ml of fermentation broth from the fermentation medium into a centrifuge tube, centrifuge at 8000 ×g for 5 min, discard the supernatant, add 10 ml of 50% (v / v) HCl to the centrifuge tube, and acid hydrolyze at 80 ℃ for 4 h. Then add 8 ml of extraction solution (methanol:chloroform = 1:1), invert and mix well to ensure complete extraction. Transfer to a shaker, incubate for 20 min, add 50% (v / v) of 0.1 M NaCl solution, vortex and centrifuge at 8000 ×g for 5 min, transfer the lower layer to a flask, and evaporate to dryness using a rotary evaporator at 80 ℃. The weight of the produced oil can then be determined.

[0064] like Figure 8 As shown, the biomass of each knockout strain did not show a significant difference compared to the wild-type strain. The biomass of the wild-type strain was 16.32 g / L, while the biomass of the knockout strains ΔScAMPKα1, ΔScAMPKα2, and ΔScAMPKα3 were 16.6 g / L, 16.58 g / L, and 16.04 g / L, respectively. This indicates that the absence of any single ScAMPKα subunit under standard culture conditions did not significantly affect the basal growth capacity of Schizochytrium cells. Regarding oil dry weight, the oil dry weight of the wild-type strain was 6.15 g / L, while the oil dry weights of the knockout strains ΔScAMPKα1, ΔScAMPKα2, and ΔScAMPKα3 were 7.15 g / L, 6.76 g / L, and 6.39 g / L, respectively. Compared to the wild-type, the oil dry weight of ΔScAMPKα1, ΔScAMPKα2, and ΔScAMPKα3 increased by 16.26%, 9.92%, and 3.90%, respectively. This difference suggests that ΔScAMPKα1 may be the key catalytic subunit regulating lipid synthesis in the Schizochytrium AMPK complex, while ΔScAMPKα2 and ΔScAMPKα3 may play only minor regulatory roles in oil production due to insufficient integrity of the kinase domain or poor assembly efficiency with the β / γ subunit.

[0065] Example 5: Fatty acid composition analysis of Schizochytrium mutant strains

[0066] Then, the oil extracted in step 4 was methylated and analyzed for fatty acid composition using gas chromatography-mass spectrometry (GC-MS) with a Thermo Scientific™ TRACE™ ISQ QD300 instrument.

[0067] like Figure 9 As shown, knockout altered the fatty acid composition, exhibiting a trend of decreasing proportions of major saturated fatty acids and increasing proportions of multiple unsaturated fatty acids. The proportions of palmitic acid (C16:0), the major saturated fatty acid, were 32.00%, 31.27%, and 32.25% in the three knockout strains, respectively, a decrease of approximately 10.00% compared to the wild type on average; the proportion of myristic acid (C14:0) also decreased from 6.13% to 4.65%, 5.47%, and 5.23%, respectively. Simultaneously, the proportions of multiple unsaturated fatty acids increased, with EPA showing the most significant increase, more than doubling compared to the wild type; the proportion of DHA increased from 36.44% in the wild type to 38.42%, 37.01%, and 37.13%, respectively. This change in fatty acid composition, with a decrease in the proportion of saturated fatty acids and an increase in the proportion of unsaturated fatty acids such as EPA and DHA, suggests that AMPK may regulate fatty acid desaturation pathways, but its specific molecular mechanisms require further elucidation. Notably, the proportion of the odd-chain saturated fatty acid pentadecanoic acid (C15:0) was significantly increased in all knockout strains. Compared to the wild type, the increases in the three knockout types were: ΔScAMPKα1 (14.8%), ΔScAMPKα2 (132.4%), and ΔScAMPKα3 (32.4%). Knockout type 2 showed the most significant increase in C15:0 content, indicating a substantial enhancement in the synthesis flux of odd-chain saturated fatty acids after gene knockout, demonstrating significant potential in regulating fatty acid composition. While knockout types 1 and 3 also showed increases, the magnitude was smaller. Previous studies have shown that odd-chain saturated fatty acid C15:0 is an activator of AMPK and can exert physiological effects through the AMPK pathway. Therefore, it is speculated that after AMPKα subunit knockout leads to inactivation of this pathway, cells may upregulate C15:0 synthesis or accumulation through feedback regulation as a potential metabolic compensation mechanism to attempt to restore or replace the lost AMPK function.

[0068] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0069] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still be protected by the present invention.

Claims

1. A method for increasing the pentadecanoic acid content of Schizochytrium, characterized in that: The invention includes constructing gene knockout strains, wherein the constructed gene knockout strains are obtained by knocking out the ScAMPKα1 gene, ScAMPKα2 gene, or ScAMPKα3 gene separately in Schizochytrium using homologous recombination technology. The amino acid sequence of the protein encoded by the ScAMPKα1 gene is shown in SEQ ID NO.1, the amino acid sequence of the protein encoded by the ScAMPKα2 gene is shown in SEQ ID NO.2, and the amino acid sequence of the protein encoded by the ScAMPKα3 gene is shown in SEQ ID NO.

3. The Schizochytrium is *Schizochytrium* sp. ATCC20888.

2. The method for increasing the pentadecanoic acid content of Schizochytrium according to claim 1, characterized in that: The nucleotide sequence encoding ScAMPKα1 is shown in SEQ ID NO.4, the nucleotide sequence encoding ScAMPKα2 is shown in SEQ ID NO.5, and the nucleotide sequence encoding ScAMPKα3 is shown in SEQ ID NO.

6.

3. The method for increasing the pentadecanoic acid content of Schizochytrium according to claim 1, characterized in that: In the homologous recombination technology, the homologous recombination vector includes the upstream homologous arm shown in SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9, the downstream homologous arm shown in SEQ ID NO.10, SEQ ID NO.11 or SEQ ID NO.12, and the intermediate resistance selection marker gene.

4. The method for increasing the pentadecanoic acid content of Schizochytrium according to claim 3, characterized in that: The resistance selection marker gene is the norsyl styridine resistance gene, and its nucleotide sequence is shown in SEQ ID NO.

13.

5. A method for constructing a mutant strain of Schizochytrium, characterized in that: Includes the following steps, (1) Obtaining homologous arm genes: The upstream and downstream homologous arm sequences of the three homologous genes of the AMPKα subunit were obtained by sequencing of Schizochytrium sp. ATCC20888, as shown in SEQ ID NO.7-12. Each DNA fragment was amplified by PCR. (2) Vector construction and verification: The homologous arms were inserted into the pMD19T-NTC vector by seamless cloning to construct the knockout recombinant plasmid, which was transformed into Escherichia coli DH5α. After being verified by sequencing, it was stored at -80 ℃. (3) Electroconversion: Take 15 μL of linearized recombinant plasmid and introduce it into the host protoplast. The electroconversion parameters are 1.8 kV, 200 Ω, and 25 μF. (4) Screening and verification: The transformation product was plated on GPY medium containing 50 μg / mL NTC and cultured for 7 days to obtain resistant transformants; the gene knockout was confirmed to be successful by genomic PCR and fatty acid composition analysis after fermentation, and the mutant was stored at -80 ℃. (5) Comparison between mutant and wild type: The mutant and wild type WT were cultured in GPY medium for 24-48 h to obtain seed liquid, which was then transferred to fermentation medium. After 72 h of culture, the cells were collected for biomass determination and fatty acid content and composition analysis.

6. The method for constructing a Schizochytrium mutant strain as described in claim 5, characterized in that: The GPY culture medium formula is: 3% glucose, 1% peptone, 0.5% yeast extract, and 2% sea salt.

7. The method for constructing a Schizochytrium mutant strain as described in claim 5, characterized in that: The fermentation medium, 1L, contains the following components: 100 g glucose, 5 g yeast extract, 1.43 g MgSO4, 1 g KH2PO4, 0.5 g (NH4)2SO4, 0.264 g KCl, 0.04 g CaCl2, 0.001 g vitamin B1, and 0.001 g vitamin B2. 12 .

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