Sucrose non-fermentation 1 related protein kinase gene RkSnf1 and application thereof

Abstract

The invention discloses a sucrose non-fermentation 1 related protein kinase gene RkSnf1. The nucleotide sequence of the sucrose non-fermentation 1 related protein kinase gene RkSnf1 is as shown in SEQ ID NO: 1; according to the invention, a knockout gene RkSnf1 plasmid is constructed according to the gene and is transferred into rhodosporidium rhodosporicola YM25235, and an experimental result shows that the total carotenoid synthesis amount of an obtained RkSnf1 gene knockout strain is remarkably increased compared with that of wild rhodosporidium rhodosporicola YM25235, and the result shows that a gene knockout engineering bacterium constructed based on the gene provided by the invention can be used for synthesis of carotenoid; the method has important theoretical significance and potential economic value for research on regulation and control of carotenoid synthesis.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a sucrose non-fermenting protein kinase 1 gene RkSnf1 and its application in promoting the production of carotenoids by Rhodosporidium kratochvilovae. Background Art

[0002] Carotenoids are a general term for a class of important natural pigments, belonging to a class of terpenoids, having multiple conjugated double bonds, and generally showing yellow, orange or red colors. The structure of carotenoids is based on isoprene as the basic unit. Most typical carotenoid chemical structures contain 40 carbon atoms and are polymerized by 8 isoprenes. Carotenoids have a variety of biological functions. Carotenoids are precursors of vitamin A in the human body, but humans and animals cannot synthesize them by themselves. Intaking carotenoids can improve human immunity. In addition, carotenoids also have coloring, antioxidant, anti-apoptotic and anti-cancer effects. Microbial cells sense and transduce some external stimuli through signal transduction pathways and synthesize metabolites such as unsaturated fatty acids and carotenoids that can help microorganisms resist adverse environments.

[0003] In yeast, the sucrose non-fermenting 1 (SNF1)-related protein kinase (SnRK) is homologous to the mammalian AMPK protein complex. It is a trimer composed of a Snf1 catalytic subunit, a Snf4 regulatory subunit, and one of the Gal83 / Sip1 / Sip2 protein families. In the absence of glucose, it promotes microbial respiratory metabolism, glycogen accumulation and gluconeogenesis by inhibiting the transcription factor Mig1 or by stimulating the transcription activators Cat8 and Sip4. In addition to participating in the de-repression of glucose-controlled genes, the SNF1-related protein kinase is also involved in a variety of physiological processes, including meiosis and sporulation, aging, autophagy and biofilm formation. The SNF1-related protein kinase is also involved in lipid metabolism in yeast. It can activate transcription factors and enhance the expression of genes related to fatty acid oxidation, thereby enhancing the β-oxidation of fatty acids and playing an important role in the regulation of intracellular acetyl-CoA homeostasis and global histone acetylation. Studies have shown that the SNF1-related protein kinase is also involved in the response of yeast to environmental stresses such as heat shock, alkaline pH and salt stress.

[0004] The Snf1 subunit is the catalytic core of SNF1-related protein kinases, which has protein kinase activity and plays a key role in the energy metabolism, stress response, and carbon source utilization of yeast cells. It contains a typical serine / threonine protein kinase domain and helps cells maintain homeostasis and viability by regulating carbon metabolism, gene expression, and activating stress response pathways. The Snf1 subunit plays an important role in carbon metabolism. Under low glucose conditions, the Snf1 subunit phosphorylates various metabolic enzymes, regulates the expression of transcription factors and carbon metabolic pathways. The Snf1 subunit phosphorylates glycolysis-related enzymes to inhibit the glycolysis pathway and reduce glucose consumption. It can also activate key enzymes in the gluconeogenesis pathway to promote the conversion of non-carbohydrate substances into glucose. Moreover, the Snf1 subunit also regulates fatty acid metabolism, inhibits fatty acid synthesis, and promotes fatty acid oxidation to provide energy for cells. Summary of the Invention

[0005] The present invention provides a sucrose non-fermenting 1-related protein kinase gene RkSnf1, which is isolated from Rhodosporidium kratochvilovae YM25235; the nucleotide sequence of this gene is as shown in SEQ ID NO:1 or a nucleotide sequence complementary to SEQ ID NO:1. The gene sequence is 2872 bp in length, and the gene encodes a polypeptide with the amino acid sequence as shown in SEQ ID NO:2. Knocking out the gene RkSnf1 in Rhodosporidium kratochvilovae promotes the synthesis of carotenoids in Rhodosporidium kratochvilovae.

[0006] In order to achieve the above object of the present invention, the technical solution of the present invention is as follows:

[0007] 1. Extract the genomic DNA of Rhodosporidium kratochvilovae YM25235 strain by CTAB method. Using the genomic DNA as a template, and primers RkSnf1-F and RkSnf1-R, the sucrose non-fermenting protein kinase 1 gene RkSnf1 sequence is obtained by PCR amplification. After designing gRNA using the CRISPR online website and comprehensively evaluating through the online website, the gRNA1 and gRNA2 sequences are obtained;

[0008] 2. According to the gRNA1 and gRNA2 sequences, primers RkSnf1-sgRNA1-F, RkSnf1-sgRNA1-R, RkSnf1-sgRNA2-F, and RkSnf1-sgRNA2-R were synthesized by Sangon Biotech Co., Ltd. Using plasmid pRU2034 as a template, the sgRNA1 fragment 1 was amplified with pRGEcoRI-F and RkSnf1-sgRNA1-R; the sgRNA1 fragment 2 was amplified with RkSnf1-sgRNA1-F and pRUGPD1-EcoRI-R. The sgRNA1 fragment 1 and fragment 2 were ligated to the digested pRU2034 vector to obtain the RkSnf1 gene knockout plasmid pRU2034 / RkSnf1-sgRNA1.

[0009] Using pRU2034 as a template, the sgRNA2 fragment 1 was amplified with pRGEcoRI-F and RkSnf1-sgRNA2-R; the sgRNA2 fragment 2 was amplified with RkSnf1-sgRNA2-F and pRUGPD1-EcoRI-R. The sgRNA2 fragment 1 and fragment 2 were ligated to the digested pRU2034 vector to obtain the RkSnf1 gene knockout plasmid pRU2034 / RkSnf1-sgRNA2.

[0010] 3. The RkSnf1 gene knockout plasmids pRU2034 / RkSnf1-sgRNA1 and pRU2034 / RkSnf1-sgRNA2 were transformed into the strain YM25235 / RkURA3Δ by PEG-mediated protoplast transformation method. The transformants were screened to obtain the RkSnf1 gene knockout strain YM25235 / RkSnf1Δ. The knockout strain was cultured, and the carotenoid content was extracted and measured.

[0011] Advantages and technical effects of the present invention:

[0012] The present invention provides a new sucrose non-fermenting 1-related protein kinase gene RkSnf1. According to this gene, a knockout gene plasmid was constructed and transferred into Rhodosporidium toruloides YM25235. The experimental results showed that compared with the wild Rhodosporidium toruloides YM25235, the total carotenoid synthesis amount of the RkSnf1 gene knockout strain was significantly increased. The results indicate that the gene knockout engineering bacteria constructed based on the gene provided by the present invention can be used for the synthesis of carotenoids. The present invention has important theoretical significance and potential economic value for the research on regulating carotenoid synthesis. Description of the Drawings

[0013] Figure 1Schematic diagram of the PCR amplification result of the RkSnf1 gene. In the figure: Lane 1 is the DNA marker; Lane 2 is the negative control; Lane 3 is the PCR amplification product of the RKSnf1 gene;

[0014] Figure 2 Schematic diagram of the plasmid map of the gene knockout plasmid pRU2034 / RkSnf1-sgRNA1;

[0015] Figure 3 Schematic diagram of the plasmid map of the gene knockout plasmid pRU2034 / RkSnf1-sgRNA2;

[0016] Figure 4 Schematic diagram of the colony PCR verification results of the knockout plasmid pRU2034 / RkSnf1-sgRNA1 and the knockout plasmid pRU2034 / RkSnf1-sgRNA2; In the figure: Lane 1 is the DNA marker; Lanes 2-7 are the colony PCR products of the 1-6th transformants of the pRU2034 / RkSnf1-sgRNA1 knockout plasmid; Lanes 8-13 are the colony PCR products of the 1-6th transformants of the pRU2034 / RkSnf1-sgRNA2 knockout plasmid;

[0017] Figure 5 PCR verification of the transformation of the gene knockout plasmid into Rhodosporidium toruloides; In the figure: Lane 1, DNA marker; Lane 2, blank control group (template is ddH2O); Lane 3, PCR product with the YM25235 genome as the template (containing introns); Lane 4, transformant YM25235 / RkSnf1Δ-741;

[0018] Figure 6 Deletion analysis result of the RkSnf1 gene;

[0019] Figure 7 Analysis result of the total carotenoid content of the knockout strain YM25235 / RkSnf1Δ-741 and the control strain YM25235. Detailed implementation manners

[0020] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. However, the protection scope of the present invention is not limited to the described content. The reagents and methods used in the embodiments, unless otherwise specified, are all conventional reagents and conventional methods;

[0021] The plasmid pRU2034 and the strain YM25235 / RkURA3Δ in the following embodiments were prepared according to the method in Xiong Chao's "Research on the Relationship between RkACS2 and the Synthesis of Carotenoids and Oils in Rhodosporidium toruloides under Glucose Starvation Stress [D]. Kunming University of Science and Technology, 2024."

[0022] Solid medium formula for SCD(UΔ) (1 L): 20 g of glucose, 100 mL of 10× mother liquor of amino acids (UΔ), 5 g of ammonium sulfate, 1.7 g of YNB, 18 g of agar powder, add 900 mL of ddH2O;

[0023] 10× mother liquor of amino acids (UΔ) (100 mL): 0.1 g of cysteine, 0.1 g of arginine, 0.1 g of lysine, 0.1 g of threonine, 0.05 g of aspartic acid, 0.05 g of isoleucine, 0.05 g of phenylalanine, 0.05 g of proline, 0.05 g of serine, 0.05 g of tyrosine, 0.05 g of valine, 0.05 g of methionine, 0.1 g of tryptophan, 0.05 g of histidine, 0.1 g of leucine, 0.1 g of adenine;

[0024] Citric acid buffer: 0.58 g of citric acid, 10.97 g of mannitol, 0.58 g of sodium citrate, make up the volume to 200 mL, pH 5.4, filter with a 0.22 μm sterile membrane;

[0025] PTC buffer: 0.156 g of Tris-HCl, 1.108 g of anhydrous calcium chloride, 50 g of polyethylene glycol 3350, make up the volume to 100 mL, filter with a 0.22 μm sterile membrane;

[0026] STC buffer: 1.108 g of anhydrous calcium chloride, 21.86 g of sorbitol, 0.156 g of Tris-HCl, make up the volume to 100 mL, sterilize at 115 °C for 20 min;

[0027] Enzyme mixture: 0.03 g of lywallzyme (Guangdong Institute of Microbiology), 0.125 g of snailase (Solarbio), make up the volume to 4 mL with sodium citrate buffer, filter with a 0.22 μm sterile membrane;

[0028] CTAB genomic extraction solution: 4 g of CTAB, 3.152 g of Tris-HCl, 1.169 g of EDTA (anhydrous), 16.3632 g of NaCl, make up the volume to 200 mL, pH 8.0.

[0029] Example 1: Obtaining of sucrose non-fermenting protein kinase 1 gene RkSnf1

[0030] 1. Extract the genome of Rhodosporidium toruloides YM25235 strain by CTAB method. Using this genome as a template, with primers RkSnf1-F: 5’-ATGAGCAACCGCGTCCACC-3’ and RkSnf1-R: 5’-TCATGCCGCCGAGGGAG-3’, obtain the gene fragment by PCR amplification. The PCR amplification system is as follows:

[0031]

[0032] The PCR amplification conditions were pre-denaturation at 95°C for 5 min, then denaturation at 95°C for 30 s, annealing at 62°C for 30 s, extension at 72°C for 3 min, for a total of 30 cycles, and finally extension at 72°C for 10 min.

[0033] Through agarose gel electrophoresis analysis ( Figure 1 ), sequencing was performed to obtain the gene RkSn1 with a fragment size of 2872 bp.

[0034] Example 2: Construction of the RkSnf1 gene knockout plasmid

[0035] 1. For the gene RkSn1 sequence obtained in Example 1, gRNAs were designed using the CRISPR online website. After designing the gRNAs on the website and comprehensively evaluating them through the online website, the gRNA1 and gRNA2 sequences were obtained, and the sequences are as follows:

[0036] gRNA1: 5’-GAAGAGGTTGCAGATCTGTG(TGG)-3’, sense strand 1371 - 1390 bp;

[0037] gRNA2: 5’-GTGCAGTGGTGTCAGCACGG(CGG)-3’, antisense strand 2337 - 2356 bp;

[0038] According to the gRNA1 and gRNA2 sequences, primers RkSnf1-sgRNA1-F, RkSnf1-sgRNA1-R, RkSnf1-sgRNA2-F, and RkSnf1-sgRNA2-R were synthesized by Sangon Biotech Co., Ltd., and the primer sequences are as follows:

[0039] pRGEcoR I-F: 5’-TACTGAATTAACGCCGAATTG-3’;

[0040] pRUGPD1-EcoR I-R: 5’-AGAGAACAAGAATTCCGAAGTTATATTAAGGGTTGTC-3’;

[0041] RkSnf1-sgRNA1-F: 5’-GAGATCGTCGATGACCTCTGGTTTTAGAGCTAGAAATAGC-3’; RkSnf1-sgRNA1-R: 5’-CAGAGGTCATCGACGATCTCAGGAGCTCGCCTGTATC-3’;

[0042] RkSnf1-sgRNA2-F: 5’-GTGCAGTGGTGTCAGCACGGGTTTTAGAGCTAGAAATAGC-3’; RkSnf1-sgRNA2-R: 5’-CCGTGCTGACACCACTGCACAGGAGCTCGCCTGTATC-3’;

[0043] 2. Using plasmid pRU2034 as a template, amplify the sgRNA1 fragment 1 (SEQ ID NO:3) with pRGEcoR I-F and RkSnf1-sgRNA1-R; amplify the sgRNA1 fragment 2 (SEQ ID NO:4) with RkSnf1-sgRNA1-F and pRUGPD1-EcoRI-R. The amplification system is as follows:

[0044] Amplification system for sgRNA1 fragment 1:

[0045]

[0046]

[0047] The PCR amplification conditions are pre-denaturation at 95°C for 5 min, then denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, for a total of 30 cycles, and finally extension at 72°C for 10 min.

[0048] Amplification system for sgRNA1 fragment 2:

[0049]

[0050] The PCR amplification conditions are pre-denaturation at 95°C for 5 min, then denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, for a total of 30 cycles, and finally extension at 72°C for 10 min.

[0051] 3. Digest the plasmid pRU2034 with the restriction enzyme EcoR I, recover it, and ligate the sgRNA1 fragment 1 and fragment 2 with the digested pRU2034 vector by one-step cloning to obtain the RkSnf1 gene knockout plasmid pRU2034 / RkSnf1-sgRNA1 ( Figure 2 );

[0052] The digestion system is as follows:

[0053]

[0054] Gently mix the prepared mixture, briefly centrifuge, and then place it at 37°C for static reaction for 5 hours;

[0055] The ligation system is as follows:

[0056]

[0057] React at 37 °C for 30 minutes.

[0058] 4. Referring to the methods in Steps 2 and 3, using pRU2034 as a template, amplify to obtain sgRNA2 fragment 1 (SEQ ID NO:5) with pRGEcoR I-F and RkSnf1-sgRNA2-R; amplify to obtain sgRNA2 fragment 2 (SEQ ID NO:6) with RkSnf1-sgRNA2-F and pRUGPD1-EcoRI-R; ligate sgRNA2 fragment 1 and fragment 2 with the digested pRU2034 vector to obtain the RkSnf1 gene knockout plasmid pRU2034 / RkSnf1-sgRNA2( Figure 3 );

[0059] 5. Transform the RkSnf1 gene knockout plasmid into Escherichia coli and verify

[0060] (1) Take out the competent Escherichia coli DH5α stored at -80 °C, place it on ice to thaw, and take 50 μL for standby;

[0061] (2) Take 5 μL of each of the RkSnf1 gene knockout plasmids pRU2034 / RkSnf1-sgRNA1 and pRU2034 / RkSnf1-sgRNA2 and add them to the competent DH5α bacterial suspension respectively. After gently flicking and mixing, let it stand on ice for 30 minutes;

[0062] (3) Heat shock the mixture in a 42 °C water bath for 45 s, and then immediately place it on ice to cool for 2 - 3 minutes;

[0063] (4) Add 900 μL of pre-cooled LB liquid medium to the above mixture, and incubate it in a shaker at 37 °C and 100 rpm for 1 hour;

[0064] (5) Centrifuge at 5000 rpm for 5 minutes, discard 900 μL of the supernatant, gently pipette and mix the bacterial cell pellet, and evenly spread the mixed bacterial liquid on the LB solid medium containing Spe antibiotic, and culture it upside down at 37 °C overnight;

[0065] (6) Pick a single colony from the LB plate, add it to 10 μL of sterile ddH2O, pipette and mix well, place it in a PCR instrument, heat it at 98 °C for 10 minutes. After the reaction is completed, centrifuge at 10000 rpm for 5 minutes, and take the supernatant as the template for colony PCR;

[0066] (7) Using the supernatant as a template, perform PCR amplification with the primers pRGEcoR I-F and pRUGPD1-EcoR I-R. The amplification system is as follows:

[0067]

[0068] The PCR amplification conditions were as follows: pre-denaturation at 95°C for 5 min, then denaturation at 95°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 1 min, for a total of 30 cycles, and finally extension at 72°C for 10 min.

[0069] The amplified products were analyzed and verified by agarose gel electrophoresis, and the results were as Figure 4 shown. The fragment size was consistent with the expectation, preliminarily indicating that the RkSnf1 gene knockout plasmids pRU2034 / RkSnf1-sgRNA1 and pRU2034 / RkSnf1-sgRNA2 were successfully constructed;

[0070] The single colonies verified correctly by colony PCR were inoculated into LB liquid medium containing Spe antibiotic (100 μg / mL) and cultured overnight with shaking at 37°C and 160 rpm. Subsequently, 5 mL of the bacterial solution was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The primers used for sequencing were pRGEcoR I-F and pRUGPD1-EcoR I-R; the sequencing results were consistent with the expectation, indicating that the RkSnf1 gene knockout plasmids pRU2034 / RkSnf1-sgRNA1 and pRU2034 / RkSnf1-sgRNA2 were successfully constructed.

[0071] Example 3: Obtaining of RkSnf1 gene knockout strains

[0072] 1. The RkSnf1 gene knockout plasmid was transformed into the auxotrophic strain YM25235 / RkURA3Δ by the protoplast transformation method

[0073] (1) A single colony of the auxotrophic strain YM25235 / RkURA3Δ activated by streaking was picked and inoculated into 5 mL of YPD liquid medium and cultured at 30°C and 160 rpm for 12 hours;

[0074] (2) An appropriate amount of the above bacterial solution was inoculated into 50 mL of YPD liquid medium and cultured at 30°C and 160 rpm until the OD600 value was approximately 0.58;

[0075] (3) Centrifugation was carried out at 5000 rpm and 4°C, the supernatant was discarded, and the bacterial cells were collected; the bacterial cells were resuspended with 10 mL of pre-cooled sodium citrate buffer, centrifuged again to discard the supernatant, and after repeating once, the bacterial cells were resuspended with 1 mL of pre-cooled sodium citrate buffer;

[0076] (4) 4 mL of the enzyme mixture was added to the bacterial suspension and enzymolyzed at 30°C and 90 rpm for 2.5 hours to break the cell wall to obtain protoplasts;

[0077] (5) After the enzymatic hydrolysis is completed, centrifuge at 1300 r and 4 °C for 11 minutes to collect the bacterial cells, resuspend the bacterial cells with 10 mL of pre-cooled STC buffer, centrifuge again and discard the supernatant. After repeating once, resuspend the bacterial cells with 1 mL of STC buffer; Prepare pre-cooled 5 mL sterile centrifuge tubes, and dispense 100 μL of the bacterial suspension into each tube;

[0078] (6) Add 3 μg of the equal mixture of plasmid pRU2034 / RkSnf1-sgRNA1 and plasmid pRU2034 / RkSnf1-sgRNA2, let it stand on ice for 10 minutes, then add 200 μL of pre-cooled PTC buffer to each tube, place it on ice for 10 minutes. After repeating once, add 800 μL of pre-cooled PTC buffer, gently pipette and mix evenly, and place it in a 42 °C water bath for 30 minutes;

[0079] (7) Add 2 mL of YPD medium containing 0.4 M sucrose, and incubate at 30 °C and 90 rpm for 24 hours;

[0080] (8) Then add 2 mL of 0.4 M sucrose YPD medium, and incubate at 28 °C and 90 rpm for 24 h;

[0081] (9) After the incubation is completed, centrifuge at 1300 r and 4 °C for 11 min to collect the bacterial cells, discard most of the supernatant, resuspend the bacterial cells with the remaining supernatant, and evenly spread them on the SCD(UΔ) solid medium for screening using uracil deficiency;

[0082] 2. Screening of RkSnf1 gene knockout strains

[0083] (1) Transfer the colonies that can grow normally on the SCD(UΔ) solid medium to a new SCD(UΔ) solid medium and number them. Then inoculate them into 5 mL of YPD liquid medium respectively, and culture at 28 °C and 160 rpm for 12 hours;

[0084] (2) Use the CTAB method to extract the genomic DNA of the strains;

[0085] (3) Using the extracted genomic DNA as a template, perform PCR amplification using primers RkSnf1-F and RkSnf1-R. The PCR reaction system is as follows:

[0086]

[0087] The PCR amplification program is: pre-denaturation at 95 °C for 5 min, then denaturation at 95 °C for 30 s, annealing at 62 °C for 30 s, extension at 72 °C for 3 min, a total of 30 cycles, and finally extension at 72 °C for 10 min.

[0088] After PCR amplification, the PCR products were separated by agarose gel electrophoresis; using the YM25235 strain as a control, the results were as Figure 5 shown. The size of the amplified band of the YM25235 strain was normal, while the amplified band of the transformant was significantly smaller than that of the control. The size of the deleted fragment was similar to the distance between the two sgRNAs, suggesting that the RkSnf1 gene had been successfully knocked out.

[0089] Further verification was carried out by genomic sequencing. The PCR products were sent to Shanghai Bioengineering Co., Ltd. for sequencing, and the primers were RkSnf1-F and RkSnf1-R; the sequencing results were as Figure 6 shown. The sequencing results showed that both RkSnf1sgRNA1 and RkSnf1sgRNA2 successfully guided the Cas protein to specifically recognize their corresponding sites on the RkSnf1 gene, cleavage occurred at 3 bp upstream of the sgRNA1 PAM sequence and near 4 bp downstream of the sgRNA2 PAM sequence, causing the DNA to break, resulting in a 953-bp fragment deletion, and the gene RkSnf1 was successfully knocked out. The strain with the RkSnf1 gene knocked out was named YM25235 / RkSnf1Δ-741.

[0090] Example 4: Analysis of the carotenoid content of the RkSnf1 gene knockout strain YM25235 / RkSnf1Δ-741

[0091] The positive transformant YM25235 / RkSnf1Δ-741 was inoculated into 50 mL of YPD liquid medium and fermented at 28 °C and 160 rpm for 168 h. Then, the cells were collected by centrifugation at 4500 rpm for 6 min in a 50 mL centrifuge tube, washed twice with pre-cooled ddH2O, and finally centrifuged at 4500 rpm for 8 min to completely remove the supernatant. The tube wall was gently tapped to make the cells evenly adhere to the inner wall of the tube, and then dried in an oven at 55 °C and ground into powder. 0.4 g of the cell powder was used to extract total carotenoids with an acetone-methanol mixture (Vacetone: Vmethanol = 4:1). Referring to the method of "Improvement of the method for determining total carotenoids in seabuckthorn oil by ultraviolet spectrophotometry [J]. Journal of Minzu University of China (Natural Sciences Edition), 2009, 18(03): 5-8." by Yang Wanzheng et al., using a UV-visible spectrophotometer, with the wild-type Rhodosporidium toruloides YM25235 strain as a control, the absorbance was measured at 450 nm and the content of total carotenoids was calculated; among them, the total carotenoid synthesis amount of the wild-type Rhodosporidium toruloides YM25235 strain was 3.63 ± 0.15 mg / g DCW, while the carotenoid synthesis amount of the knockout strain YM25235RkSnf1Δ-741 was 6.04 ± 0.64 mg / g DCW, which was significantly higher than that of the wild-type Rhodosporidium toruloides YM25235 strain. As Figure 7As shown, the results show that knocking out the RkSnf1 gene can cause an increase in the total carotenoid content in the Rhodosporidium toruloides YM25235 strain.

Claims

1. A sucrose non-fermenting 1-related protein kinase gene RkSnf1, whose nucleotide sequence is shown in SEQ ID NO:

1.

2. Use of the sucrose non-fermenting protein kinase 1 gene RkSnf1 according to claim 1 in promoting the production of carotenoids by Rhodosporidium kratochvilovae.

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