Method for improving synthesis of sakuranetin by using yarrowia lipolytica based on transcriptome analysis and application of method

Abstract

The invention discloses a method for improving synthesis of sakuranetin from yarrowia lipolytica based on transcriptome analysis and application of the method, and belongs to the technical field of gene engineering. Target genes are analyzed and screened through transcriptome sequencing so as to find a method capable of improving sakuranetin synthesis, and the method specifically comprises the steps that an Illumina HiseqTM platform is used for conducting comprehensive transcriptome analysis on an original strain in the logarithmic phase by applying an RNA-seq technology, then transcriptome data are deeply analyzed through KEGG pathway analysis, and the sakuranetin synthesis can be improved. Therefore, the gene showing significant differential expression in the key path of sakuranetin synthesis is identified. And finally, carrying out free expression and integrated expression on the screened key target gene, and verifying the influence on the sakuranetin yield. Finally, a recombinant yarrowia lipolytica strain which is integrated with the YALI0C01859g gene and has high sakuranetin yield is obtained, and the titer of sakuranetin is increased to 344.0 mg / L.

Description

Technical Field

[0001] The invention relates to a method for improving the synthesis of sakurain by Yarrowia lipolytica based on transcriptome analysis and application thereof, belonging to the technical field of genetic engineering. Background Art

[0002] Sakurain (4,5-dihydroxy-7-methoxyflavanone) is a complex natural product belonging to the flavonoid family and is synthesized by some plants (such as shrubs and rice). Sakurain has been found to have a variety of biological activities, including antibacterial, anti-inflammatory, anti-mutagenic, anti-Helicobacter pylori, anti-leishmaniasis and anti-trypanosomal. Since sakurain has potential nutritional and pharmaceutical market value, the development of efficient synthesis methods is of great research significance. At present, traditional sakurain synthesis methods, such as chemical synthesis and plant extraction, have problems such as low synthesis efficiency and high pollution in the synthesis process, and it is difficult to meet the needs of low carbon and high yield. In recent years, emerging microbial synthesis methods have synthesized many natural products such as lipids, terpenoids, and flavonoids, providing a new solution for the sustainable production of sakurain. With the help of synthetic biology and metabolic engineering strategies, the introduction of heterologous synthesis pathways existing in plants into microorganisms is an effective means of green synthesis of sakurain.

[0003] With the advent of the post-genomic era, the development of transcriptomics technology has provided scientists with new means to explore the relationship between gene expression and product yield. Based on the characteristics of transcriptomes that vary over time and space, functional enrichment analysis and pathway analysis have been widely used to explore the biological significance of target genes, providing a basis for subsequent experimental verification. By regulating the expression of these key target genes, gene expression can be optimized, thereby affecting the biological functions and metabolic pathways of cells. However, there is currently no method for screening target genes for transcriptional sequencing analysis of the sakura-like biosynthesis pathway of Yarrowia lipolytica. Summary of the invention

[0004] To solve the above problems, the present invention analyzes the key target genes of the sakura glycoside synthesis pathway based on transcriptional sequencing, in order to provide a feasible way to improve the expression of sakura glycosides. Specifically, by mining the target genes with significant differences in the metabolic pathway of synthesizing sakura glycosides from glucose, 14 significantly improved genes were integrated and expressed, and the effects of expressing genes with different differential expression amounts on sakura glycoside synthesis were verified. It was found that the sakura glycoside production increased the most after overexpressing the YALI0C01859g gene. Therefore, the present invention also constructed a recombinant Yarrowia lipolytica with high sakura glycoside production, which will provide important support for the efficient production of sakura glycosides.

[0005] The first object of the present invention is to provide a recombinant Yarrowia lipolytica with high production of sakura chlorophyll. The recombinant Yarrowia lipolytica comprises the following modifications: overexpression of one or more of the genes YALI0F02607g, YALI0C01859g, and YALI0F05038g.

[0006] In the present invention:

[0007] Firstly, a method for discovering target genes for sakura biosynthesis based on transcriptome sequencing analysis is provided: Firstly, using Illumina Hiseq TM The platform uses RNA-seq technology to conduct a comprehensive transcriptome analysis of the starting strain that can synthesize sakura novo in the logarithmic growth phase, efficiently capturing the expression levels of all transcripts in the cell. Next, through KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis, the transcriptome data is deeply analyzed, and the two key enriched pathways for sakura nodosum synthesis (aromatic amino acid metabolism pathway and carbon metabolism pathway) are found through the enriched pathways of functional enrichment analysis, and then the genes with significant differential expression in the sakura nodosum synthesis pathway are found, providing an important direction for exploring gene regulation of sakura nodosum synthesis. Subsequently, the sakura nodosum production of the recombinant strain is verified by overexpressing the genes that are significantly upregulated in the sakura nodosum synthesis pathway in the starting strain.

[0008] Furthermore, the overexpression is integrated expression and / or episomal expression.

[0009] Furthermore, the integration site on the genome includes but is not limited to the YALI0E30965g site.

[0010] Furthermore, the gene sequence of YALI0E30965g is shown in SEQ ID NO.16.

[0011] Furthermore, the episomal expression or integrated gene is driven by the promoter P TEF Start expression.

[0012] Furthermore, the gene sequence of YALI0F02607g is shown in SEQ ID NO.2, the gene sequence of YALI0C01859g is shown in SEQ ID NO.3, and the gene sequence of YALI0F05038g is shown in SEQ ID NO.10.

[0013] Furthermore, the transformation is the transformation of the recombinant Yarrowia lipolytica relative to the starting strain, and the starting strain includes any transformed or untransformed Yarrowia lipolytica, wherein the transformed Yarrowia lipolytica includes the following transformations:

[0014] The ku70 gene on the Yarrowia lipolytica host strain genome is knocked out and the NOMT encoding gene for expressing naringenin methyltransferase is integrated at the site, and the adenosyl homocysteine ​​enzyme encoding gene YALI0F11759p, the 5-methyltetrahydropteroyltriglutamate-homocysteine ​​methyltransferase encoding gene YALI0E12683p and the S-adenosylmethionine synthetase encoding gene YALI0B14509p are overexpressed. The selection of the Yarrowia lipolytica host strain includes but is not limited to Yarrowia lipolytica Po1f, such as Yarrowia lipolytica Po1fΔku70-NOMT+pYL-P HIS5 -YALI0F11759p-P TEF -YALI0E12683p-P POT1 -YALI0B14509p.

[0015] A second object of the present invention is to provide a method for constructing the recombinant Yarrowia lipolytica, comprising the following steps:

[0016] One or more of the genes YALI0F02607g, YALI0C01859g, and YALI0F05038g are overexpressed in a starting strain of Yarrowia lipolytica.

[0017] Furthermore, the starting strain includes but is not limited to Yarrowia lipolytica Po1fΔku70-NOMT+pYL-P HIS5 -YALI0F11759p-P TEF -YALI0E12683p-P POT1 -YALI0B14509p.

[0018] The third object of the present invention is to provide a method for producing sakura chlorophyll, comprising the following steps: using the recombinant Yarrowia lipolytica to carry out fermentation production.

[0019] Furthermore, production is carried out using glucose as a substrate.

[0020] Furthermore, the fermentation includes shake flask fermentation or fed-batch fermentation.

[0021] Furthermore, the fermentation comprises: preparing a seed solution, inoculating the seed solution into a fermentation medium for culturing;

[0022] The culturing comprises culturing at 28-35° C. and 220-280 rpm (shaking flask);

[0023] Or the culturing includes culturing at 28-35°C, 400-700rpm, and a ventilation volume of 1-2vvm, and controlling the glucose concentration to be 20-30g / L and the pH to be 4.0-7.0 (fermentation tank) during the fermentation process.

[0024] Furthermore, the fermentation process includes: when the glucose concentration is lower than 20 g / L, adding feed medium to control the glucose concentration to 20-30 g / L; the feed medium contains 500-700 g / L glucose.

[0025] Furthermore, the shake flask fermentation seed culture medium contains: 15-25 g / L glucose, 1-3 g / L YNB, 4-6 g / L (NH4)2SO4, and 0.5-1.5 g / L CSM-leu.

[0026] Furthermore, the shake flask fermentation medium contains: 30-50 g / L glucose, 1-3 g / L YNB, 4-6 g / L (NH4)2SO4, 0.5-1.5 g / L CSM-leu, and 4-6 g / L L-tyrosine.

[0027] Furthermore, the fermentation tank seed culture medium contains: 5-15 g / L yeast powder, 15-25 g / L peptone, and 15-25 g / L anhydrous glucose.

[0028] Furthermore, the fermentation medium in the fermentation tank contains: 5-15 g / L yeast powder, 15-25 g / L peptone, and 75-85 g / L anhydrous glucose.

[0029] The fourth object of the present invention is to provide a method for improving the synthesis of sakuramin by Yarrowia lipolytica, comprising the step of overexpressing at least one of the genes YALI0F02607g, YALI0C01859g, and YALI0F05038g in a starting strain of Yarrowia lipolytica.

[0030] Furthermore, the overexpression is free expression and / or integrated expression, and the manner and means are as above.

[0031] Beneficial effects of the present invention:

[0032] The present invention explores the effect of genes with different differential expression on the synthesis of sakura by exploring the target genes with significantly increased differences in the metabolic pathway of synthesizing sakura from glucose, and expresses 14 significantly increased genes in a free manner. It is found that some of the genes have a positive promoting effect on the synthesis of sakura, and the recombinant strain overexpressing the YALI0C01859g gene has the most efficient sakura synthesis pathway. Subsequently, the YALI0C01859g gene is integrated and expressed, and it is found that the sakura titer of the integrated strain in the metabolic pathway of synthesizing sakura from glucose is increased to 344.0 mg / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the volcano map in Example 2 of the present invention;

[0034] Figure 2 is the KEGG enrichment pathway diagram in Example 2 of the present invention;

[0035] Figure 3 is a map of the plasmid pUrloxp of the present invention;

[0036] Figure 4-17 The plasmids of the present invention are pUrloxp-YALI0F22121g, pUrloxp-YALI0F02607g, pUrloxp-YALI0C01859g, pUrloxp-YALI0D23815g, pUrloxp-YALI0B22550g, pUrloxp-YALI0D08690g, and pUrloxp-YALI0D0126 5g, pUrloxp-YALI0B14619g, pUrloxp-YALI0E12573g, pUrloxp-YALI0F05038g, pUrloxp-YALI0B22902g, pUrloxp-YALI0B21846g, pUrloxp-YALI0E20977g, and pUrloxp-YALI0D06930;

[0037] Fig.18 is the fermentation yield of sakurain of the free expression recombinant strain YliSaK-G14 of the present invention and the starting strain;

[0038] Fig.19 It is the fermentation yield of sakura glycosides of the integrated expression recombinant strain of the present invention. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0040] 1. The genes and sequences involved in the present invention are as follows:

[0041] YALI0F22121g gene: SEQ ID NO.1

[0042] YALI0F02607g gene: SEQ ID NO.2

[0043] YALI0C01859g gene: SEQ ID NO.3

[0044] YALI0D23815g gene: SEQ ID NO.4

[0045] YALI0B22550g gene: SEQ ID NO.5

[0046] YALI0D08690g gene: SEQ ID NO.6

[0047] YALI0D01265g gene: SEQ ID NO.7

[0048] YALI0B14619g gene: SEQ ID NO.8

[0049] YALI0E12573g gene: SEQ ID NO.9

[0050] YALI0F05038g gene: SEQ ID NO.10

[0051] YALI0B22902g gene: SEQ ID NO.11

[0052] YALI0B21846g gene: SEQ ID NO.12

[0053] YALI0E20977g gene: SEQ ID NO.13

[0054] YALI0D06930g gene: SEQ ID NO.14

[0055] Plasmid pUrloxp: SEQ ID NO.15

[0056] Integration site YALI0E30965g: SEQ ID NO.16

[0057] 2. The materials and methods of the present invention are as follows:

[0058] (I) Starting strain

[0059] The starting strain used in the following examples is described in the Chinese patent publication number CN116478844A, which is the recombinant strain Yarrowia lipolytica Po1fΔku70-NOMT+pYL-P constructed in Example 2. HIS5 -YALI0F11759p-P TEF -YALI0E12683p-P POT1 -YALI0B14509p. Specifically involves the following modifications:

[0060] The gene encoding naringenin methyltransferase NOMT was integrated into the ku70 locus of the host yeast Po1f and expressed episomally under the control of the promoter P HIS5 The adenosine homocysteinease encoding gene YALI0F11759p regulated by the promoter P TEFThe 5-methyltetrahydropteroyltriglutamate-homocysteine ​​methyltransferase encoding gene YALI0E12683p and the promoter P POT1 Regulated S-adenosylmethionine synthetase encoding gene YALI0B14509p three-gene expression cassette.

[0061] (II) Culture medium

[0062] Seed culture medium CSM: glucose 20.0 g / L, YNB 1.7 g / L, (NH4)2SO4 5.0 g / L, CSM-leu 0.74 g / L.

[0063] Fermentation medium CSM: glucose 40.0 g / L, YNB 1.7 g / L, (NH4)2SO4 5.0 g / L, CSM-leu 0.74 g / L, L-tyrosine 5 g / L.

[0064] YPD seed medium: 10g / L yeast powder, 20g / L peptone, 20g / L anhydrous glucose.

[0065] YPD fermentation medium: 10g / L yeast powder, 20g / L peptone, 80g / L anhydrous glucose.

[0066] (III) Determination method

[0067] OD600 detection: The absorbance value detected by UV spectrophotometer at 600nm represents the biomass (OD600).

[0068] Sakura glycoside detection: The sample needs to be processed before the determination. In order to quantify sakura glycoside, first mix 0.1 mL of fermentation culture with 900 mL of anhydrous methanol, rotate thoroughly, centrifuge at 12000 rpm for 10 min, take 1 mL of supernatant, and then pass it through a water filter membrane (pore size of 0.22 μm) to remove impurities and perform liquid phase detection. High performance liquid chromatography (HPLC) detection parameters, chromatographic column, and detection conditions are: Agilent 1260, UV-VIS detector, ZORBAX Eclipse Plus C18 chromatographic column (4.6×100 mm, 3.5 μm, Agilent); mobile phase is 70% methanol (v / v); flow rate is 0.7 mL / min; UV detector, detection wavelength is 225 nm; injection volume is set to 10 μL; column temperature is set to 35°C. The sakura glycoside standard is configured with 5 gradient concentrations, and the corresponding response values ​​are detected to obtain a standard curve of concentration and absorbance value, and the correlation coefficient R 2 The value should be greater than 0.99, and then the content of sakurain in the sample is determined based on the standard curve.

[0069] Example 1 Method for screening key target genes in the synthesis of sakuramin in Yarrowia lipolytica strains

[0070] (1) Total RNA extraction and transcriptome sequencing: The starting strain Yarrowialipolytica Po1fΔku70-NOMT+pYL-P was selected to synthesize sakuramin from glucose HIS5 -YALI0F11759p-P TEF -YALI0E12683p-P POT1 -YALI0B14509p was used as the experimental object. The total RNA of the samples was extracted by the Trizol method. The integrity of RNA (RIN) was detected using the Agilent 2100 bioanalyzer system, and RIN>8 indicated good integrity. The purity and concentration of RNA were detected using the NanoDrop 2000 ultra-micro spectrophotometer, among which the purity (OD260 / OD280) of 1.9 to 2.1 indicated high purity. Each group of RNA samples was quantified to 500 ng / μL by RNasefreeddH2O and used for subsequent library construction. Subsequently, cDNA was synthesized using the TruSeqTM RNA sample preparation kit, and 12 sequencing libraries with a fragment size of 300±50bp were obtained by PCR amplification. In the Illumina Hiseq TMRNA-seq technology was used on the platform to analyze the transcription level of the logarithmic phase cells of the starting strain and the changes in gene expression of the engineered strain. The raw data obtained by sequencing was processed using Trimmomatic, and the steps were as follows: remove sequences with N bases; remove adapter sequences in reads; remove low-quality bases (Q value < 20) from the 3' to 5' direction of reads; remove low-quality bases (Q value < 20) from the 5' to 3' direction of reads; use the sliding window method to remove bases with a quality value of less than 20 at the tail of reads (window size is 5bp); remove reads themselves and their paired reads with a length of less than 35nt. The reference genome was used as the reference sequence, and the quality-controlled sequencing sequence was aligned with the reference genome using HISAT2, and the alignment results were statistically compared using RSeQC. TPM (Transcripts PerMillion) was used to estimate gene expression levels. DESeq was used to perform differential analysis of gene expression. The results of differential expression analysis were screened according to the differential expression fold (Foldchange, FC) and significant q-value of each gene. The screening threshold was set as: expression difference fold |log2FoldChange|>1, significant q-value<0.05. Genes that met the screening criteria were confirmed as differentially expressed genes. To further explore the effect of up-regulated differentially expressed genes on the production of sakura in Yarrowia lipolytica, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis was used to find out the pathways with significant enrichment of up-regulated differentially expressed genes (p<0.05).

[0071] (2) Screening of key target genes: The starting strain was compared with the genome information of the model strain Yarrowia lipolytica CLIB122 as a reference. A total of 36,098,976 clean data reads were processed, with an average Q20 value of 98.4% and a Q30 value of 95.3%. The GC content in the clean data was determined to be 54.7%. Figure 1 As shown in the figure, the analysis results showed that there were 730 differentially expressed genes, of which 35 differentially expressed genes were up-regulated and 695 differentially expressed genes were down-regulated. The results of KEGG pathway enrichment analysis were as follows Figure 2The results showed that the up-regulated differentially expressed genes were mainly enriched in the following pathways: Phenylalanine, tyrosine and tryptophan biosynthesis, Pyruvate metabolism, Phenylalanine metabolism, Tyrosine metabolism, Glycolysis / Gluconeogenesis, Tryptophan metabolism, Valine, Leucine and isoleucine degradation. These pathways are closely related to aromatic amino acid metabolism and carbon pathways.

[0072] Among the 35 up-regulated differentially expressed genes, 8 genes involved in aromatic amino acid metabolism and 18 genes involved in carbon metabolism were identified. It is worth noting that YALI0B21846g, YALI0E20977g, YALI0E17787g and YALI0A16379g among the up-regulated differentially expressed genes were significantly up-regulated in the aromatic amino acid and carbon metabolism-related pathways. In order to determine the key factors affecting the synthesis of sakura chlorophyll, we selected 14 up-regulated genes with the highest differential expression fold for further verification (see Table 1).

[0073] Table 1 Key target gene information

[0074]

[0075] Example 2 Construction of episomal expression recombinant strain

[0076] (1) The plasmid pUrloxp (sequence shown in SEQ ID NO. 15, map shown in Figure 3 lipolytica genome as a template, F22121g-F / F22121g-R as primers, amplification to obtain the YALI0F22121g fragment with homology arms; then using The target gene fragment was inserted into the linearized vector using rapid cloning technology to obtain the recombinant plasmid Purloxp-YALI0F22121g.

[0077] (2) According to the method of step (1), the homologous arms of the key target genes YALI0F22121g-F, YALI0F22121g-R, YALI0F02607g-F, YALI0F02607g-R, YALI0C01859g-F, YALI0C01859g-R, YALI0D23815g-F, YALI0D23815g-R, YALI0B22550g-F, YALI0B22550g-R, YALI0D08690g-F, YALI0D08690g-R, YALI0D01265g-R -F, YALI0D01265g-R, YALI0B14619g-F, YALI0B14619g-R, YALI0E12573g-F, YALI0E12573g-R, YALI0F05038g-F, YALI0F05038g-R, YALI0B22902g-F, YALI0B22902g-R, YALI0B21846g-F, YALI0B21846g-R, YALI0E20977g-F, YALI0E20977g-R, YALI0D06930g-F, and YALI0D06930g-R fragments; using AvrII and Sal The plasmid pUrloxp stored in the laboratory was linearized into a vector using restriction endonuclease I. Rapid cloning technology is used to insert the target gene fragment into the linearized vector to obtain a recombinant plasmid

[0078] Purloxp-YALI0F22121g, Purloxp-YALI0F02607g, Purloxp-YALI0C01859g, Purloxp-YALI0D23815g, Purloxp-YALI0B22550g, Purloxp-YALI0D08690g, Purloxp-YALI0D01265g, Purloxp-YALI0B14619g, Purloxp-YALI0E12573g, Purloxp-YALI0F05038g, Purloxp-YALI0B22902g, Purloxp-YALI0B21846g, Purloxp-YALI0E20977g, Purloxp-YALI0D06930g (see the map) Figure 4-17 ).

[0079] (3) The recombinant plasmids Purloxp-YALI0F22121g, Purloxp-YALI0F02607g, Purloxp-YALI0C01859g, Purloxp-YALI0D23815g, Purloxp-YALI0B22550g, Purloxp-YALI0D08690g, Purloxp-YALI0D01265 g, Purloxp-YALI0B14619g, Purloxp-YALI0E12573g, Purloxp-YALI0F05038g, Purloxp-YALI0B22902g, Purloxp-YALI0B21846g, Purloxp-YALI0E20977g, and Purloxp-YALI0D06930g were introduced into the starting strains and named

[0080] YliSaK-G14-1~YliSaK-G14-14 (see Table 3).

[0081] Table 2 Primer sequences

[0082]

[0083] Table 3 Key target genes and corresponding recombinant strains

[0084]

[0085]

[0086] Example 3 Synthesis of sakurain by free expression recombinant strain

[0087] To verify the sakura glycoside synthesis of the YliSaK-G14 engineered strain, we fermented 14 strains of bacteria. The specific steps are as follows:

[0088] In shake flask culture, a 14 ml shake tube was used to prepare the seed culture solution (using seed culture medium CSM), and cultured at 250 rpm and 30°C for 32-48 h. The seed culture solution (0.8 ml) was inoculated into the fermentation medium CSM (30 ml in a 250 ml shake flask), and cultured at 30°C and 250 rpm for 120 h. During the shake flask culture, 1 ml of the cell suspension was taken every 24 h.

[0089] Results Fig.18 (Strain No. 0 is the starting strain), and it was found that the yield of engineered strains such as YliSaK-G14-2, YliSaK-G14-3, and YliSaK-G14-10 was significantly improved.

[0090] Example 4 Construction of integrated expression recombinant strain

[0091] (1) Take Yarrowia lipolytica Po1fΔku70-NOMT+pYL-P HIS5 -YALI0F11759p-P TEF -YALI0E12683p-P POT1 -YALI0B14509p was used as the starting strain, and the strain was cultured in 2 mL of YPD medium to the exponential growth phase (16-24 h), and po1fk bacteria in 1 mL of the fermentation broth were collected. After centrifugation and discarding the supernatant, 90 μL of 50% volume PEG4000 solution, 5 μL of lithium acetate (25 M), 5 μL of single-stranded DNA (salmon sperm) and 5 μL of the key target gene knockout frame (obtained by AvrII digestion, the recombinant plasmid Purloxp-YALI0C01859g obtained in Example 3) were added, mixed and incubated at 37°C for 1 h, and then coated on a uracil-deficient plate (CSM-URA), integrated into the YALI0E30965g site of Yarrowia lipolytica, and screened to obtain the recombinant engineered bacteria with the integrated YALI0C01859g gene.

[0092] Example 5 Fermentation production of sakura glycosides by integrating expression of recombinant strains

[0093] (1) picking up the recombinant Yarrowia lipolytica obtained in Example 4, inoculating it into a seed culture medium, and culturing it at a temperature of 30° C. and a rotation speed of 220 rpm for 48 h to obtain a recombinant Yarrowia lipolytica seed solution;

[0094] (2) The seed culture was expanded in a 250 mL shake flask containing 30 mL YPD seed medium, shaken at 250 rpm, and maintained at 30°C for 48 hours.

[0095] (3) 60 mL of the seed culture was transferred to 3-L YPD fermentation medium in a 5-L fermentor (T&J Biotechnology Co., Ltd.). During the entire fermentation process, 2 M NaOH and 2 M HCl were used to maintain the pH at 6.8 and the temperature was kept constant at 30°C. The aeration rate was 1.5 vvm and the stirring speed was 600 rpm. In addition, 600 g / L of glucose was added during the fermentation process to ensure a consistent glucose concentration. Specifically, when the glucose concentration was lower than 20 g / L, sugar supplementation was started, with 50 ml (600 g / L) added each time.

[0096] (4) Obtain fermentation broth according to the above method, and after fermentation, detect the content of sakurain in the fermentation broth of the integrated recombinant strain by the sakurain determination method. Fig.19As shown, the titer of sakura vinifera increased to 344.0 mg / L after further integration and expression of the key target gene for sakura vinifera synthesis.

[0097] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A recombinant Yarrowia lipolytica with high production of sakura sakura The recombinant Yarrowia lipolytica comprises the following modifications: overexpression of one or more of the genes YALI0F02607g, YALI0C01859g, and YALI0F05038g.

2. The recombinant Yarrowia lipolytica according to claim 1, characterized in that The overexpression is integrated expression and / or episomal expression.

3. The recombinant Yarrowia lipolytica according to claim 2, characterized in that The integration site includes the YALI0E30965g site; the gene sequence of YALI0E30965g is shown in SEQ ID NO.

16.

4. The recombinant Yarrowia lipolytica according to claim 1, characterized in that The transformation is the transformation of the recombinant Yarrowia lipolytica relative to the starting strain, and the starting strain includes any transformed or untransformed Yarrowia lipolytica; Preferably, the starting strain is a modified Yarrowia lipolytica, comprising the following modifications: The ku70 gene on the Yarrowia lipolytica host bacteria genome is knocked out and the NOMT encoding gene for expressing naringenin methyltransferase is integrated at the site, and the adenosyl homocysteine ​​enzyme encoding gene YALI0F11759p, the 5-methyltetrahydropteroyltriglutamate-homocysteine ​​methyltransferase encoding gene YALI0E12683p and the S-adenosylmethionine synthetase encoding gene YALI0B14509p are overexpressed; preferably, the Yarrowia lipolytica host bacteria includes Yarrowia lipolytica Po1f.

5. The recombinant Yarrowia lipolytica according to claim 1 or 4, characterized in that The gene sequence of YALI0F02607g is shown in SEQ ID NO.2, the gene sequence of YALI0C01859g is shown in SEQ ID NO.3, and the gene sequence of YALI0F05038g is shown in SEQ ID NO.

10.

6. A method for constructing a recombinant Yarrowia lipolytica, characterized in that: The following steps are involved: Overexpressing one or more of the genes YALI0F02607g, YALI0C01859g, and YALI0F05038g in a starting strain of Yarrowia lipolytica; Optionally, the starting strain of Yarrowia lipolytica is a modified or unmodified Yarrowia lipolytica; Modified Yarrowia lipolytica Includes the following modifications: The ku70 gene on the Yarrowia lipolytica host bacteria genome is knocked out and the NOMT encoding gene for expressing naringenin methyltransferase is integrated at the site, and the adenosyl homocysteine ​​enzyme encoding gene YALI0F11759p, the 5-methyltetrahydropteroyltriglutamate-homocysteine ​​methyltransferase encoding gene YALI0E12683p and the S-adenosylmethionine synthetase encoding gene YALI0B14509p are overexpressed; preferably, the Yarrowia lipolytica host bacteria includes Yarrowia lipolytica Po1f.

7. A method for producing sakura chlorophyll, characterized in that: The method comprises the following steps: using the recombinant Yarrowia lipolytica according to any one of claims 1 to 5 or the recombinant Yarrowia lipolytica according to claim 6 for fermentation production.

8. The method according to claim 7, characterized in that Contains at least one of the following characteristics: (1) Production using glucose as substrate; (2) The fermentation includes shake flask fermentation or fed-batch fermentation; (3) The seed culture medium for shake flask fermentation contains: 15-25 g / L glucose, 1-3 g / L YNB, 4-6 g / L (NH4)2SO4, and 0.5-1.5 g / L CSM-leu; (4) The shake flask fermentation medium contains: 30-50 g / L glucose, 1-3 g / L YNB, 4-6 g / L (NH4)2SO4, 0.5-1.5 g / L CSM-leu, 4-6 g / L L-tyrosine (5) The fermentation tank seed culture medium contains: 5-15 g / L yeast powder, 15-25 g / L peptone, and 15-25 g / L anhydrous glucose; (6) The fermentation medium in the fermentation tank contains: 5-15 g / L yeast powder, 15-25 g / L peptone, and 75-85 g / L anhydrous glucose.

9. The method according to claim 7 or 8, characterized in that: The fermentation comprises the step of culturing in a fermentation medium; The culturing comprises culturing at 28-35° C. and 220-280 rpm; Or the culturing includes culturing at 28-35°C, 400-700rpm, and a ventilation volume of 1-2vvm, and controlling the glucose concentration to be 20-30g / L and the pH to be 4.0-7.0 during the fermentation process.

10. A method for improving the synthesis of sakuramin by Yarrowia lipolytica, characterized in that: The method comprises the step of overexpressing at least one of the genes YALI0F02607g, YALI0C01859g and YALI0F05038g in a starting strain of Yarrowia lipolytica.

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