Schizochytrium limacinum RNA (Ribonucleic Acid) interference system and application thereof in increasing grease yield

Abstract

The invention discloses a schizochytrium limacinum RNA (Ribonucleic Acid) interference system and application thereof. According to the system, RNA interference pathways are complemented in schizochytrium limacinum, exogenous RNA-dependent RNA polymerase RdRP is introduced, and shRNA expression driven by an RNA polymerase III type promoter is combined, so that the specific silencing of a target gene after transcription is realized. An efficient RNAi regulation and control platform is constructed by screening and preferably selecting a tRNAGly promoter to drive the expression of shRNA (short hairpin ribonucleic acid). When the system is used for silencing key genes of a fatty acid degradation pathway, the degradation of fatty acid can be remarkably inhibited, and the accumulation level of grease and DHA is improved. The RNA interference system has the advantages of accurate targeting, high regulation and control efficiency, good hereditary stability and the like, is not only suitable for increasing the yield of grease and polyunsaturated fatty acid, but also can be widely applied to schizochytrium limacinum gene function research, metabolic pathway regulation and control and multi-gene synergistic silencing. A universal and efficient genetic regulation tool is provided for metabolic engineering modification of industrial microorganisms and production of high-added-value products.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to an RNA interference system involving Schizochytrium sp. and its application in increasing oil yield. Background Technology

[0002] Schizochytrium sp. is a typical marine oil-producing microorganism with advantages such as rapid growth, short fermentation cycle, and shear resistance, enabling the industrial production of polyunsaturated fatty acids such as docosahexaenoic acid (DHA) and other oil products. However, with continuous optimization of strain selection, culture medium formulation, and fermentation operations, the effect of fermentation regulation on increasing oil and unsaturated fatty acid yields has reached a bottleneck. Currently, genetic modification of Schizochytrium mainly includes random mutagenesis, homologous recombination knockout, and CRISPR / Cas9 gene editing. Traditional homologous recombination is inefficient in Schizochytrium: for example, when introducing exogenous DNA via electroporation, homologous recombination efficiency is very low, and knocking out target genes often requires multiple rounds of operations. Previous studies have reported that knocking out the acyl-CoA oxidase (Acox) gene involved in fatty acid β-oxidation in Schizochytrium through homologous recombination can significantly increase the total fatty acid content. However, the multi-gene knockout process is cumbersome and time-consuming, which is not conducive to the breeding of industrial strains. Furthermore, Schizochytrium contains multiple chromosomal copies or alleles, and complete knockout often requires repeated transformations. Therefore, developing an efficient and convenient method to achieve targeted silencing of Schizochytrium genes is of great significance for accelerating functional gene discovery and further improving lipid and DHA production.

[0003] RNA interference (RNAi) technology can efficiently and programmatically downregulate gene expression at the post-transcriptional level, achieving simultaneous silencing of multiple genes in plants, animals, and some fungi. However, a highly efficient RNAi platform suitable for *Schizochytridum* has not yet been established. It has been demonstrated that microorganisms like *Saccharomyces cerevisiae*, which naturally lack RNAi systems, can also construct RNAi systems by introducing the exogenous proteins Dicer and Argonaute, indicating that target gene silencing can also be achieved in microorganisms lacking natural RNAi systems. Based on this, this invention identified key RNAi components (Dicer and Argonaute proteins) naturally present in *Schizochytridum* and additionally introduced the missing gene (RNA-dependent RNA polymerase, RdRP). A suitable RNA polymerase type III promoter was further screened to drive shRNA, constructing an RNAi system suitable for *Schizochytridum*. This system was successfully used to inhibit the expression of key genes for lipid degradation, thereby significantly increasing lipid yield. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a highly efficient regulatory system based on RNAi for inhibiting gene expression in Schizochytrium. This effectively solves the problem of precise gene silencing in this important oil-producing microorganism and can be applied to increase oil yield.

[0005] The present invention achieves the above objectives through the following technical solutions:

[0006] A highly efficient RNAi gene regulation system for Schizochytrium aims to address the lack of effective gene silencing tools and the limited lipid metabolism regulation methods in this type of industrial oil-producing microorganism, thereby increasing the fermentation yield of oils and high-value-added products such as DHA.

[0007] In a first aspect, the present invention provides a Schizochytrium RNA interference system, wherein the RNA interference system is constructed in Schizochytrium to achieve specific silencing of target genes at the post-transcriptional level; the RNA interference system includes the endogenous Dicer ribonuclease and Argonaute protein of Schizochytrium, and the exogenously introduced RNA-dependent RNA polymerase RdRP expressed in Schizochytrium.

[0008] In some embodiments, the Schizochytrium is an RNAi+ type Schizochytrium engineered strain containing endogenous Dicer as shown in SEQ ID NO:1 and Argonaute as shown in SEQ ID NO:2, and exogenously introduced and expressed RdRP as shown in SEQ ID NO:3; and expressing shRNA targeting the target gene driven by an RNA polymerase III promoter in the engineered strain.

[0009] In some implementations, the RdRP gene is derived from Schizochytrium accharomyces pombe and optimized for Schizochytrium codon preferences.

[0010] In some embodiments, the RNA polymerase type III promoter is selected from one or more of the following: the 5S rRNA promoter from Schizochytrium, the U6 small nuclear RNA promoter, the tRNAGly promoter, and the tRNAMet promoter.

[0011] In some embodiments, the nucleotide sequence of the 5S rRNA promoter is shown in SEQ ID NO:4 or SEQ ID NO:5, the nucleotide sequence of the U6 small nuclear RNA promoter is shown in SEQ ID NO:6, the nucleotide sequence of the tRNAGly promoter is shown in SEQ ID NO:7, and the nucleotide sequence of the tRNAMet promoter is shown in SEQ ID NO:8.

[0012] In some embodiments, the RNA polymerase type III promoter is the tRNAGly promoter, which can achieve significant silencing of the reporter gene eGFP, reducing the eGFP fluorescence intensity to approximately 15.2% of the control group.

[0013] In some embodiments, the shRNA targets one or more key genes in the fatty acid degradation pathway, the key genes being selected from CPT1, FadA, FadD, FadE, Lipase, and PEX10, and their corresponding shRNA sequences are shown in SEQ ID NO:11 to SEQ ID NO:16.

[0014] In some implementations, multi-target synergistic RNA interference includes the simultaneous expression of shRNA targeting FadD and shRNA targeting CPT1;

[0015] The shRNA sequence targeting FadD is shown in SEQ ID NO:13, and the shRNA sequence targeting CPT1 is shown in SEQ ID NO:11.

[0016] In some embodiments, the shRNA can also target DGAT, a key enzyme in lipid synthesis, and its shRNA sequence is shown in SEQ ID NO:17;

[0017] Among them, the oil yield of the engineered strain targeting DGAT decreased to 30.2 g / L.

[0018] In a second aspect, the present invention provides the application of the Schizochytrium RNA interference system described in any of the first aspects in improving oil yield, by constructing single-target RNAi engineered strains or dual-target RNAi engineered strains to improve oil yield; wherein, when the single-target RNAi engineered strain inhibits FadD or CPT1, the oil yield reaches 78.6 g / L and 75.4 g / L, respectively; and the oil yield of the dual-target RNAi engineered strain reaches 87.3 g / L.

[0019] Thirdly, the present invention provides the application of any of the Schizochytrium RNA interference systems described in the first aspect in the study of Schizochytrium gene function, metabolic pathway regulation, or multi-gene synergistic silencing.

[0020] In some implementations, the dual-target RNAi engineered strain achieved an oil yield of 91.4 g / L under 5 L fermentation conditions, while maintaining a DHA content of 51.7% and a DHA yield of 47.2 g / L; in contrast, the wild-type Schizochytrium strain without RNA interference regulation had an oil yield of 61.2 g / L and a DHA yield of 30.5 g / L.

[0021] In some embodiments, in the Schizochytrium RNA interference system described in any of the first aspects or in any of the applications described in the second and third aspects, the Schizochytrium is Schizochytrium sp. HX-308, with accession number CCTCC No. M209059.

[0022] In some embodiments, in any of the applications described in the second and third aspects, *Schizochytrium* is inoculated into a seed culture medium to activate and obtain a fermentation strain, which is then inoculated into a fermentation culture medium for fermentation. After fermentation, the cells are collected to extract oil; wherein the OD of the fermentation strain is... 600 The inoculum size is 8–10, and the inoculum volume is 8–15% of the fermentation medium volume; the shake flask fermentation conditions are 25–32℃, 150–200 rpm, and culture for 96–144 h.

[0023] In some implementations, the inoculum size is 10% of the fermentation medium volume; the shake-flask fermentation conditions are 28°C, 170 rpm, and 120 h.

[0024] In some embodiments, the seed culture medium comprises: 50 g / L glucose, 2.3 g / L yeast extract, 20 g / L monosodium glutamate, 3 g / L MgCl2·7H2O, 16 g / L Na2SO4, 1 g / L KCl, 1 g / L NaCl, and 3 g / L KH2PO4; and the fermentation culture medium comprises: 100 g / L glucose, 4 g / L yeast extract, 25 g / L monosodium glutamate, 5 g / L MgCl2·7H2O, 20 g / L Na2SO4, 1 g / L KCl, 1 g / L NaCl, 4 g / L KH2PO4, and 4 g / L (NH4)2SO4.

[0025] The advantages and positive effects of this invention are as follows:

[0026] 1. This invention is the first to reconstruct the RNA interference pathway in Schizochytrium, introduce an exogenous RdRP completion module, and establish a sustainable RNAi system, solving the technical problem of the long-standing lack of programmable gene silencing methods in this type of microalga.

[0027] 2. This invention screened and verified a variety of RNA polymerase type III promoters suitable for Schizochytrium fungi, and selected tRNA as the preferred promoter. Gly As a highly efficient shRNA driver element, it improves RNAi expression efficiency and enhances gene silencing effects.

[0028] 3. This invention constructs shRNA expression modules targeting key genes in the fatty acid degradation pathway and verifies that they can significantly enhance lipid accumulation, with silencing FadD and CPT1 showing the most significant effects; synergistic silencing of the two genes further amplifies the lipid gain.

[0029] 4. The RNAi system maintains stable expression in a 5L fermenter. The target engineered strain can achieve a double increase in oil and DHA production without changing the DHA ratio, which has good prospects for industrial application.

[0030] In summary, the Schizochytrium RNA interference system constructed in this invention has the advantages of simple construction, precise targeting, strong adaptability, and significant metabolic regulation effect, providing a new and effective genetic tool for lipid product production and metabolic engineering breeding. Attached Figure Description

[0031] Figure 1 Schematic diagram of the pNeoR expression vector;

[0032] Figure 2 Schematic diagram of the pBleR expression vector;

[0033] Figure 3 Schematic diagram of the pNatR expression vector;

[0034] Figure 4 Evaluation of the silencing efficiency of Pol III promoter-driven shRNAs using fluorescence screening;

[0035] Figure 5 Comparative example of 5 L fermentation tank (oil yield vs. DHA yield). Detailed Implementation

[0036] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0037] The various conventional molecular cloning, gene synthesis, PCR amplification, agarose gel electrophoresis, fluorescence detection, oil extraction and gas chromatography detection involved in the embodiments of this invention are all conventional techniques known to those skilled in the art; unless otherwise specified, they can be implemented with reference to the literature, instructions or experimental manuals (such as "Molecular Cloning: A Laboratory Manual") published before the filing date of this invention.

[0038] I. General Approach

[0039] This invention provides a method for increasing lipid and DHA production by regulating the fatty acid degradation pathway of Schizochytrium using an RNA interference system. The basic idea is as follows: complete the RNAi pathway in Schizochytrium, introduce exogenous RdRP, and construct an RNAi+ engineered strain; screen suitable RNA polymerase type III promoters under the RNAi+ background to drive shRNA expression; construct an shRNA expression module targeting the genes encoding key fatty acid degradation enzymes using the optimized promoter; further construct a multi-target synergistic silencing strain, and verify its fermentation performance under fermenter conditions.

[0040] Preferably, the Schizochytrium sp. HX-308 is the accession number CCTCC No. M209059.

[0041] Preferably, the RNA interference system includes the Dicer and Argonaute proteins of Schizochytrium itself, and exogenously introduced and expressed RdRP, wherein the coding sequences of Dicer and Argonaute are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0042] Preferably, the RdRP gene is derived from fissile yeast and optimized for the codon preference of fissile chytrid fungus. The optimized nucleotide sequence is shown in SEQ ID NO:3.

[0043] Preferably, the optimal promoter for the subsequent expression of the shRNA expression module is tRNA, which is the type III RNA polymerase promoter. Gly .

[0044] II. Culture medium and fermentation conditions (applicable to all examples)

[0045] The culture medium components used in each embodiment are as follows, but are not limited to these:

[0046] Preferably, the seed culture medium can be formulated as follows: 50 g / L glucose, 2.3 g / L yeast extract, 20 g / L sodium glutamate, 3 g / L MgCl2·7H2O, 16 g / L Na2SO4, 1 g / L KCl, 1 g / L NaCl, and 3 g / L KH2PO4, with water as the solvent, and autoclaved at 121°C for 15 min before use.

[0047] Preferably, the fermentation medium can be formulated as follows: 100 g / L glucose, 4 g / L yeast extract, 25 g / L sodium glutamate, 5 g / L MgCl2·7H2O, 20 g / L Na2SO4, 1 g / L KCl, 1 g / L NaCl, 4 g / L KH2PO4 and 4 g / L (NH4)2SO4, with water as solvent and autoclaved at 121°C for 15 min before use.

[0048] The specific fermentation process for each embodiment can be carried out as follows:

[0049] First, wild-type strains of Schizochytrium or engineered strains (such as RNAi+-CPT1) were inoculated into seed culture medium and cultured at 28°C with shaking for about 24 h to obtain primary seeds.

[0050] The primary seeds were transferred to fresh seed culture medium at an inoculation rate of 10% (v / v) and cultured for another 24 h at the same temperature to obtain secondary seeds.

[0051] Then, transfer the culture medium to the next round of seed culture medium at the same inoculation ratio, and incubate at 28℃ for 24 h to complete cell activation and obtain the fermentation strain.

[0052] Preferably, the OD of the fermentation strain is... 600 The value is 8–10; the inoculation amount of the fermentation strain is 8–15% of the fermentation medium volume, more preferably 10%.

[0053] Preferably, the fermentation strain is inoculated into the fermentation medium under shake flask conditions and cultured at 25–32°C and 150–200 rpm for 96–144 h, more preferably at 28°C and 170 rpm for about 120 h.

[0054] In scale-up cultivation, if a 5 L fermenter is used, the same inoculation ratio as in shake flasks can be applied. Preferably, a suitable dissolved oxygen level is maintained by adjusting the stirring speed or aeration rate; more preferably, the fermentation parameters are 28°C, 170 rpm, and 120 h of cultivation.

[0055] Oil and fat testing can be performed as follows (the following steps are merely examples, and those skilled in the art can substitute or optimize them according to equipment and requirements):

[0056] After fermentation, a certain volume of fermentation broth was taken, and the pH of the system was adjusted to approximately 11 using NaOH. A cell wall lysing enzyme was added at 0.3% (by weight) of the fermentation broth, and enzymatic hydrolysis was performed at 50°C for approximately 4 hours. Subsequently, hexane was added for extraction, and the mixture was allowed to stand and separate into layers. The upper yellow organic phase was collected, and the extraction was repeated until the upper layer became lighter in color or nearly colorless. The organic phases obtained from multiple extractions were combined, and the solvent was evaporated to dryness to obtain the crude oil.

[0057] The obtained oil was mixed with 15% NaOH aqueous solution and 70% methanol solution, and subjected to methyl esterification reaction in a water bath at 65°C for about 1.5 h. After cooling, chromatographic grade n-hexane was added to extract fatty acid methyl esters. The upper organic phase was filtered through a microporous membrane to remove impurities, and then analyzed by gas chromatography to determine the fatty acid composition.

[0058] Example 1: Construction of RNAi+ type Schizochytrium engineered strain

[0059] This embodiment aims to illustrate how to complete the naturally incomplete RNA interference pathway in Schizochytrium and construct an RNAi+ engineered strain that can stably express exogenous RdRP, serving as the base strain for subsequent shRNA expression and functional verification.

[0060] First, the whole genome sequence of *Schizochytridactylum* HX-308 was analyzed, confirming that it contains some key factors in the RNA interference pathway, including the coding sequences for Dicer ribonuclease and Argonaute protein (SEQ ID NO:1 and SEQ ID NO:2). Further comparison revealed that the typical RdRP gene was not found in its genome, indicating that *Schizochytridactylum* naturally lacks a complete small RNA amplification mechanism; therefore, exogenous RdRP supplementation is needed to construct a more efficient RNAi system.

[0061] Based on the amino acid sequence information of RdRP in *Schizochytrium*, its coding sequence was codon optimized to suit the codon usage preferences of *Schizochytrium*, resulting in an optimized RdRP gene fragment (SEQ ID NO:3). To facilitate subsequent cloning, primer pairs specifically designed for amplifying the optimized RdRP fragment were designed, denoted as P1 (SEQ ID NO:18) and P2 (SEQ ID NO:19), with an overlap region at their 5' ends that overlaps with the vector backbone. The RdRP gene fragment was cloned by PCR, with the PCR amplification program being: 98℃ for 30 s, 57℃ for 30 s, 72℃ for 2 min, for 34 cycles. After obtaining the RdRP PCR fragment, it was coupled to the *Schizochytrium* expression vector pNeoR (containing the P2902 promoter (SEQ ID NO:20), the CYC1 terminator (SEQ ID NO:21), and the neomycin resistance gene NeoR (SEQ ID NO:22). Figure 1The pNeoR gene was first linearized, and primers P3 (SEQ ID NO:23) and P4 (SEQ ID NO:24) were designed based on the vector sequence. A linear backbone was obtained by PCR and purified via gel extraction. The optimized RdRP fragment (SEQ ID NO:3) was then ligated to the linearized pNeoR backbone using the Gibson method. A typical system contains 3–5 μL of RdRP fragment, 1–2 μL of vector backbone, and 5 μL of Gibson enzyme. The assembly product was obtained by incubation at 50°C for 15–30 min. The reaction system was transformed into E. coli DH5α competent cells for amplification. After screening with antibiotic plates and confirming the correct insertion direction by colony PCR and sequencing of positive clones, the recombinant plasmid pNeoR-RdRP containing the complete P2902-RdRP-CYC1 expression cassette was obtained. The recombinant plasmid was linearized with HindIII and used to transform *Schizochytrium* competent cells. 10–20 μL of the linearized vector was added to 100 μL of competent cells, mixed thoroughly, and incubated on ice. Electroporation was then performed at 0.75 kV, 200 Ω, and 50 μF. Following this, 1 mL of seed culture medium was added, and the cells were incubated at 28°C and 150–200 rpm for 1 h. The incubated bacterial culture was plated onto selection plates containing neomycin (400 μg / mL) and cultured at 28°C in the dark for 3–4 days. Resistant colonies were picked, and genomic PCR was used to identify whether NeoR and RdRP were integrated. After 3–4 generations of continuous subculturing to verify stable heritability, an RNAi+ engineered strain capable of continuously expressing exogenous RdRP in *Schizochytrium* was obtained. This strain can synergize with its endogenous Dicer and Argonaute to form a functional RNA interference pathway, serving as the basic host for subsequent construction of shRNA modules, screening of Pol III promoters, and research on fatty acid degradation gene silencing.

[0062] Example 2: Screening and Silencing Efficiency Verification of shRNA Driven by RNA Polymerase III Promoter

[0063] To quantitatively evaluate the gene silencing efficiency of shRNA driven by different RNA polymerase III promoters, this embodiment first constructs a reporter system stably expressing eGFP in an RNAi+ type Schizochytrium engineered strain. Specifically, the eGFP coding sequence was optimized using Schizochytrium codons, and the optimized nucleotide sequence is shown in SEQ ID NO:9. Using the optimized eGFP gene as a template, specific amplification primer pairs P5 (SEQ ID NO:25) and P6 (SEQ ID NO:26) were designed, each containing an overlap region at its 5′ end that can overlap with the vector backbone. The eGFP gene fragment was obtained by PCR cloning, with the PCR amplification program being: 98℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min, for 34 cycles. After obtaining the eGFP PCR fragment, it was coupled with a primer containing the strong Schizochytrium promoter P... 2902 The Schizochytrium expression vector pBleR (containing the CYC1 terminator and the bleomycin resistance gene bleR (SEQ ID NO:27)) Figure 2The pBleR vector was first linearized. Primers P7 (SEQ ID NO:28) and P8 (SEQ ID NO:29) were designed based on the vector sequence. The linearized vector backbone was obtained by PCR amplification and purified by agarose gel electrophoresis. The obtained eGFP fragment (SEQ ID NO:9) was then ligated to the linearized pBleR backbone using the Gibson assembly method. A typical reaction system included 3–5 μL of eGFP fragment, 1–2 μL of linearized vector backbone, and 5 μL of Gibson assembly enzyme. The reaction was carried out at 50°C for 15–30 min to obtain the assembly product. The reaction system was transformed into *E. coli* DH5α competent cells for amplification. After screening on kanamycin-containing resistant plates, positive clones were verified by colony PCR and sequencing to confirm the sequence correctness and orientation of the eGFP insert, yielding the recombinant plasmid pBleR-P2902-eGFP containing the complete P2902-eGFP-CYC1 expression cassette. The recombinant plasmid was linearized with HindIII and used to transform RNAi+ *Schizochytridum* competent cells. Specifically, 10–20 μL of the linearized vector was added to 100 μL of *Schizochytridum* competent cells, mixed, and incubated on ice. Electroporation was then performed at 0.75 kV, 200 Ω, and 50 μF. Immediately afterwards, 1 mL of seed culture medium was added, and the cells were incubated at 28°C and 150–200 rpm for 1 h. After resuscitation, the bacterial culture was plated onto selection plates containing G418 (400 μg / mL) and incubated at 28°C in the dark for 3–4 days. Resistant colonies were picked and genomic PCR was used to identify successful integration of the KanR and eGFP expression cassettes. After 3–4 generations of subculturing to verify genetic stability, an RNAi+-eGFP reporter strain capable of stably expressing eGFP was obtained. Fluorescence detection confirmed that this strain could continuously and stably produce fluorescent signals even without shRNA introduction, making it suitable for subsequent screening of Pol III promoter-driven shRNAs and evaluation of gene silencing efficiency. Figure 4 ).

[0064] Based on this, several RNA polymerase III promoters were selected to drive the expression of shRNA targeting eGFP, including two 5S rRNA promoters (SEQ ID NO:4 and SEQ ID NO:5) from Schizochytrium, one U6 small nuclear RNA promoter (SEQ ID NO:6), and two tRNA promoters. Gly and tRNA Phe(SEQ ID NO:7 and SEQ ID NO:8). A uniform shRNA sequence was designed targeting the eGFP gene, with its sense strand, hairpin loop, and antisense strand as its overall structure (SEQ ID NO:10), and was similarly assembled via Gibson into the vector pNatR containing noseminated intradermal resistance NatR (SEQ ID NO:30). Figure 3 The shRNA-eGFP was ligated downstream of different Pol III promoters to construct multiple promoter-shRNA expression modules. These modules were then introduced into RNAi+-eGFP reporter strains via electroporation. After resistance screening and PCR identification, engineered strains with different promoter-driven shRNAs were obtained.

[0065] The engineered strains with different promoters-shRNA obtained above and the RNAi+-eGFP strain without shRNA were used as controls. After being cultured to the logarithmic growth phase under the same culture medium and conditions, the unit cell fluorescence intensity of each strain was detected using a fluorescence microplate reader. The unit fluorescence intensity of the control strain was used as a baseline value to calculate the relative fluorescence intensity changes under different promoter-driven shRNA conditions, thus evaluating the gene silencing effect. The experimental results showed that different RNA polymerase III promoters had significantly different abilities to drive shRNA expression in Schizochytrium. Figure 4 Among them, shRNA driven by the tRNAGly promoter showed the most significant inhibitory effect on eGFP expression, with a unit fluorescence intensity of 15.2% of the control group; tRNA Phe The promoter also showed strong inhibitory ability, with a fluorescence intensity per unit of 34.5% of the control group; while the silencing effects of the 5S rRNA promoter and U6 promoter were relatively weak, with fluorescence intensities per unit of 79.8%, 75.2%, and 85.6% of the control group, respectively. Based on these results, the tRNA was determined. Gly The promoter can efficiently and stably drive shRNA expression and achieve a significant gene silencing effect in Schizochytrium. Therefore, tRNA is preferred in subsequent examples. Gly The promoter serves as the driving element of shRNA.

[0066] Example 3: Validation of RNA interference targeting key genes in the fatty acid degradation pathway and its effect on increasing lipid yield.

[0067] The preferred RNA polymerase type III promoter tRNA identified in Example 2 was screened. Gly Building upon this foundation, this embodiment further utilizes the constructed RNAi system to target and silence key genes in the fatty acid degradation pathway of Schizochytrium, and verifies its impact on lipid accumulation.

[0068] First, shRNA expression modules were designed targeting several key enzyme-encoding genes in the fatty acid degradation pathway of *Schizochytrium*. These target genes include carnitine palmitoyltransferase 1 (CPT1), thiolase FadA, fatty acid kinase FadD, fatty acid dehydrogenase FadE, triglyceride lipase Lipase, and peroxisome forming factor PEX10. The shRNA nucleotide sequences corresponding to each target gene are shown in SEQ ID NO:11 to SEQ ID NO:16. Each shRNA sequence was driven by the tRNAGly promoter validated in Example 2. The shRNA sequences were ligated into the *Schizochytrium* expression vector pNatR containing the noseminatedin resistance gene NatR (SEQ ID NO:30) via Gibson assembly. After linearization, the vector was transformed into RNAi+ strains via electroporation. Resistant colonies were selected, and genomic PCR was used to identify successful integration of NatR and the corresponding shRNA expression cassette. After 3–4 generations of continuous subculturing to verify genetic stability, several single-target RNAi engineered strains were obtained.

[0069] The different single-target RNAi engineered strains constructed above and the RNAi+ control strain without shRNA were cultured in shake flasks under the same culture medium and fermentation conditions. After 120 h of fermentation, the oil yield of each strain was measured according to the aforementioned oil extraction and gas chromatography analysis methods. The results showed that, compared with the control strain, the RNAi engineered strains targeting key genes in the fatty acid degradation pathway all showed varying degrees of increased oil accumulation. Among them, the engineered strains inhibiting fatty acid kinase FadD and carnitine palmitoyltransferase CPT1 showed the most significant increase in oil yield, reaching approximately 78.6 g / L and 75.4 g / L, respectively (Table 1). The fatty acid ratio of each engineered strain did not change significantly from the control group (Table 1). As a control experiment, a shRNA engineered strain targeting the key enzyme in oil synthesis, diacylglycerol acyltransferase DGAT, was further constructed. The corresponding shRNA sequence is shown in SEQ ID NO:17. The engineered strain was fermented under the same construction and culture conditions as above, and the oil yield was measured. The results showed that its oil yield was significantly reduced, only about 30.2 g / L. The above results indicate that the RNA interference system constructed in this invention can achieve specific silencing of target genes in Schizochytrium, and the changes in lipid production caused by gene silencing are consistent with the biological function of the target gene in lipid metabolism.

[0070] Table 1

[0071]

[0072] Example 4: Validation of multi-target synergistic RNA interference in lipid production

[0073] Based on the experimental results of Example 3, two fatty acid degradation-related target genes, FadD and CPT1, which showed the most significant effect on increasing lipid accumulation, were selected, and a dual shRNA co-expression system was designed and constructed. Specifically, corresponding shRNA sequences were designed for FadD and CPT1, respectively, and their nucleotide sequences are shown in SEQ ID NO:13 and SEQ ID NO:11, respectively. Each shRNA expression cassette used tRNAs selected in Example 2. Gly The promoter was used to drive the expression of the *Schizochytrium* strain containing the noseminated resistance gene NatR (SEQ ID NO:30) via Gibson assembly, forming a dual shRNA co-expression plasmid. The dual-target RNAi engineered strain, the single-target RNAi engineered strain constructed in Example 3, and the RNAi+ control strain without shRNA were cultured under the same shake-flask fermentation conditions, and their lipid yields were measured. The results showed that the lipid yield of the dual-target RNAi engineered strain was further increased, reaching approximately 87.3 g / L, significantly higher than the control group (Table 2), indicating that multi-target synergistic silencing can produce an additive or synergistically enhanced lipid gain effect without affecting the fatty acid ratio (Table 2).

[0074] Table 2

[0075]

[0076] Comparative analysis of oil yield optimization in *Schizochytrium* using multi-target synergistic RNA interference and analysis of oil and fatty acid composition in *Schizochytrium* without RNA interference regulation under 5L fermenter conditions.

[0077] To compare and verify the effects of a multi-target synergistic RNA interference strategy on lipid accumulation and fatty acid composition in *Schizochytrium* under scaled-up fermentation conditions, the engineered strain with multi-target synergistic RNA interference described in Example 4 and the control strain of *Schizochytrium* without RNA interference regulation were fermented under the same 5 L fermenter conditions, and their lipid yield and fatty acid composition were analyzed. Specifically, the dual-target RNAi engineered strain constructed in Example 4 and the control strain of *Schizochytrium* without any shRNA expression module were inoculated into 5 L fermenters. Both fermentations used the same culture method, as detailed in the culture conditions section. The results showed that, under 5 L fermenter conditions, the engineered strain of *Schizochytrium* regulated by multi-target synergistic RNA interference exhibited significantly improved lipid accumulation capacity, with a lipid yield of approximately 91.4 g / L. Fatty acid composition analysis showed that while the total lipid content was significantly increased, the proportion of DHA in the total fatty acids remained stable at approximately 51.7%, with a final DHA yield of 47.2 g / L. In contrast, the wild-type control strain of Schizochytrium without RNA interference regulation had an oil yield of only 61.2 g / L and a DHA yield of only 30.5 g / L, significantly lower than the multi-target RNAi engineered strain. Figure 3 ).

[0078] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A Schizochytrium RNA interference system, characterized in that: An RNA interference system was constructed in Schizochytrium to achieve specific post-transcriptional silencing of target genes; the RNA interference system includes the endogenous Dicer ribonuclease and Argonaute protein of Schizochytrium, and the exogenously introduced RNA-dependent RNA polymerase RdRP expressed in Schizochytrium.

2. The Schizochytrium RNA interference system according to claim 1, characterized in that: The Schizochytrium is an RNAi+ type Schizochytrium engineered strain containing the endogenous Dicer as shown in SEQ ID NO:1 and Argonaute as shown in SEQ ID NO:2, and exogenously introduced and expressed RdRP as shown in SEQ ID NO:3; and expressing shRNA driven by the RNA polymerase III promoter and targeting the target gene in the engineered strain.

3. The Schizochytrium RNA interference system according to claim 2, characterized in that: The RdRP gene is derived from Schizochytrium pombe and optimized for codon preferences specific to Schizochytrium.

4. The Schizochytrium RNA interference system according to claim 3, characterized in that: The RNA polymerase type III promoter is selected from one or more of the following: 5S rRNA promoter, U6 small nuclear RNA promoter, tRNAGly promoter, and tRNAMet promoter derived from Schizochytrium.

5. The Schizochytrium RNA interference system according to claim 4, characterized in that: The nucleotide sequence of the 5S rRNA promoter is shown in SEQ ID NO:4 or SEQ ID NO:5, the nucleotide sequence of the U6 small nuclear RNA promoter is shown in SEQ ID NO:6, the nucleotide sequence of the tRNAGly promoter is shown in SEQ ID NO:7, and the nucleotide sequence of the tRNAMet promoter is shown in SEQ ID NO:

8.

6. The Schizochytrium RNA interference system according to claim 4, characterized in that: The RNA polymerase type III promoter is the tRNAGly promoter, which can significantly silence the reporter gene eGFP, reducing the eGFP fluorescence intensity to about 15.2% of the control group.

7. The Schizochytrium RNA interference system according to any one of claims 2 to 6, characterized in that: The shRNA targets one or more key genes in the fatty acid degradation pathway, the key genes being selected from CPT1, FadA, FadD, FadE, Lipase, and PEX10, and their corresponding shRNA sequences are shown in SEQ ID NO:11 to SEQ ID NO:

16.

8. The Schizochytrium RNA interference system according to claim 7, characterized in that: Multi-target synergistic RNA interference includes the simultaneous expression of shRNA targeting FadD and shRNA targeting CPT1; wherein the sequence of the shRNA targeting FadD is shown in SEQ ID NO:13, and the sequence of the shRNA targeting CPT1 is shown in SEQ ID NO:

11.

9. The application of the Schizochytrium RNA interference system according to any one of claims 1 to 8 in increasing oil yield, characterized in that: Oil yield was increased by constructing single-target RNAi engineered strains or dual-target RNAi engineered strains. Among them, the oil yield of the single-target RNAi engineered strain reached 78.6 g / L and 75.4 g / L when FadD or CPT1 was inhibited, respectively; the oil yield of the dual-target RNAi engineered strain reached 87.3 g / L.

10. The application of the Schizochytrium RNA interference system according to any one of claims 1 to 8 in the study of Schizochytrium gene function, metabolic pathway regulation, or multi-gene synergistic silencing.

11. The application according to claim 9, characterized in that: The dual-target RNAi engineered strain achieved an oil yield of 91.4 g / L under 5 L fermentation conditions, with a DHA content of 51.7% and a DHA yield of 47.2 g / L. In contrast, the wild-type Schizochytrium strain without RNA interference regulation had an oil yield of 61.2 g / L and a DHA yield of 30.5 g / L.

12. The Schizochytrium RNA interference system according to any one of claims 1 to 8, characterized in that: The shRNA can also target DGAT, a key enzyme in lipid synthesis, and its shRNA sequence is shown in SEQ ID NO:17; among them, the lipid yield of the engineered strain targeting DGAT decreased to 30.2 g / L.

13. The Schizochytrium RNA interference system according to any one of claims 1 to 8, 12, or the application according to any one of claims 9 to 11, characterized in that: The Schizochytrium sp. HX-308 was identified as having the accession number CCTCC No. M209059.

14. The application according to any one of claims 9 to 11, characterized in that: Schizochytrium fungi was inoculated into a seed culture medium to activate and obtain a fermentation strain. This fermentation strain was then inoculated into a fermentation medium for fermentation. After fermentation, the cell bodies were collected to extract the oil. The OD value of the fermentation strain was... 600 The inoculum size is 8–10, and the inoculum volume is 8–15% of the fermentation medium volume; the shake flask fermentation conditions are 25–32℃, 150–200 rpm, and culture for 96–144 h.

15. The application according to claim 14, characterized in that: The inoculum size was 10% of the fermentation medium volume; the shake-flask fermentation conditions were 28℃, 170 rpm, and 120 h.

16. The application according to claim 14, characterized in that: The seed culture medium consists of: 50 g / L glucose, 2.3 g / L yeast extract, 20 g / L sodium glutamate, 3 g / L MgCl2·7H2O, 16 g / L Na2SO4, 1 g / L KCl, 1 g / L NaCl, and 3 g / L KH2PO4; the fermentation culture medium consists of: 100 g / L glucose, 4 g / L yeast extract, 25 g / L sodium glutamate, 5 g / L MgCl2·7H2O, 20 g / L Na2SO4, 1 g / L KCl, 1 g / L NaCl, 4 g / L KH2PO4, and 4 g / L (NH4)2SO4.

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