Genetically engineered bacterium of galactosylceramide as well as construction method and application of genetically engineered bacterium

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a genetically engineered bacterium of galactosylceramide as well as a construction method and application of the genetically engineered bacterium of galactosylceramide. On the basis of an existing delta pyrG aspergillus oryzae auxotroph strain, the metabolic flux of neutral glycosphingolipid is enhanced through overexpression of an AoCerS gene; meanwhile, the expression of a neutral sphingolipid glucosylceramide (GlcCers) synthetase gene AoUGCG is inhibited, so that the common intermediate metabolite flux of the GlcCers and galactosylceramide (GalCers) flows to the synthesis of the GalCers. The GalCers relative content of the constructed aspergillus oryzae engineering strain is increased by more than two times compared with that of a control strain, and is increased by about 70% compared with that of the control strain.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to a genetic engineering bacterium of galactosylceramide and a construction method and application thereof. Background Art

[0002] Neutral glycosphingolipids, as important intracellular bioactive substances, have great application value in the fields of pharmaceuticals, cosmetics, and food. Ceramide is a key molecule for maintaining and forming the epidermal permeability barrier. In keratinocytes, neutral glycosphingolipid glucosylceramide (GlcCers) can be hydrolyzed by β-glucosylceramide hydrolase to release ceramide and store it in the stratum corneum to improve dry skin and atopic dermatitis. Oral administration of GlcCers can also protect the colon surface from the toxicity of various drugs, play a role in preventing or repairing intestinal damage, and regulate intestinal microbial flora to improve the intestinal microenvironment. Studies have shown that whether in skin stratum corneum cells or intestinal cells, GlcCers must release ceramide under the action of β-glucosylceramide hydrolase to improve atopic dermatitis or play a role in repairing intestinal damage. Neutral glycosphingolipid galactosylceramide (GalCers) can activate β-glucosylceramide hydrolase, promote the hydrolysis of GlcCers and increase the content of ceramide. Neutral glycosphingolipids GlcCers currently used in cosmetics or health products are mainly derived from plants, but plant neutral glycosphingolipids do not have GalCers. GalCers are mainly extracted from animal brain tissue, which has problems such as high cost, limited sources, and zoonotic diseases. Chemical synthesis of neutral glycosphingolipids is not suitable for large-scale production due to its complicated steps, high cost, low yield and many by-products. Therefore, it is urgent to explore new safe biological resources that can synthesize GlcCers and GalCers.

[0003] Among fungi, there are many food-grade Aspergillus that can synthesize two types of neutral glycosphingolipids, GalCers and GlcCers, at the same time, which makes up for the defect that plants only contain GlcCers, and has great development and application prospects. In particular, the food-grade strain Aspergillus oryzae will be an ideal biological resource for the development of neutral glycosphingolipids. However, the content of GalCers fermented by Aspergillus directly isolated from nature is not high, and the strain needs to be genetically engineered to obtain a high-yield strain. At present, the regulatory mechanism of neutral glycosphingolipid synthesis at home and abroad has not been clarified, which directly limits the development and application of Aspergillus neutral glycosphingolipids. Therefore, it is still a major challenge to construct a strain that produces a higher yield of neutral glycosphingolipid galactosylceramide. Summary of the invention

[0004] The purpose of the present invention is to inhibit the expression of AoUGCG gene by overexpressing AoCerS gene and RNAi technology, to provide a method for constructing a genetically engineered strain with high yield of GalCers, and the application of the genetically engineered strain in preparing GalCers by microbial fermentation.

[0005] In order to achieve the above object, the present invention provides the following technical solutions: A galactosylceramide genetically engineered bacterium is obtained by firstly enhancing the metabolic flow of neutral glycosphingolipids by overexpressing the AoCerS gene, and then inhibiting the expression of the neutral glycosphingolipid glucosylceramide synthase gene AoUGCG. The recipient bacterium overexpressing the AoCers gene is a ΔpyrG Aspergillus oryzae auxotrophic strain.

[0006] A method for preparing a galactosylceramide-producing genetically engineered bacterium comprises the following steps: Overexpressing the AoCerS gene, inserting the AoCerS gene into an overexpression vector, constructing an AoCerS overexpression vector, transforming the AoCerS overexpression vector into a ΔpyrG Aspergillus oryzae auxotrophic strain, inducing the promoter, and obtaining an overexpression strain: Inhibit the expression of AoUGCG gene, insert the reverse sequence and the forward sequence of AoUGCG gene into the inhibition vector respectively to obtain the AoUGCG inhibition gene vector, and transfer the AoUGCG inhibition gene vector into the overexpression strain to obtain the genetic engineering bacteria of galactosylceramide.

[0007] Furthermore, overexpressing the AoCerS gene comprises the following steps: Primers OEAoCerS-F and OEAoCerS-R were designed based on the AoCerS gene sequence. The AoCerS gene was obtained by PCR amplification using the cDNA of Aspergillus oryzae 3.042 as a template. The gene was then connected to the PmlⅠ site of the pEX2B vector by homologous recombination. The vector successfully connected with the AoCerS gene was named pEX2B-AoCerS. The pEX2B-AoCerS vector was transformed into the genome of the ΔpyrG Aspergillus oryzae auxotrophic strain by PEG-mediated protoplast transformation. AoCerS was successfully overexpressed by PCR and qRT-PCR to obtain the AoCerS overexpression strain OEAoCerS. The primers for PCR were pEX2B-F and pEX2B-R, and the primers for qRT-PCR were qAoCerS-F, qAoCerS-R, Ref-HistoneH4-F and Ref-HistoneH4-R.

[0008] Further, inhibiting the expression of AoUGCG gene comprises the following steps: Using the cDNA of Aspergillus oryzae 3.042 as a template and AoUGCG-senseF and AoUGCG-senseR as primers, PCR amplification was performed to obtain the AoUGCG gene fragment, which was then ligated to the XholⅠ site on the pEX1 vector by homologous recombination to obtain the AoUGCG-sense sequence; using AoUGCG-Anti-senseF and AoUGCG-Anti-senseR as primers, PCR amplification was performed to obtain the AoUGCG gene fragment, which was then ligated to BamHⅠ on the pEX1 vector by homologous recombination to obtain the AoUGCG-Anti-sense sequence. The vector in which the AoUGCG-sense sequence and the AoUGCG-Anti-sense sequence were successfully connected was named RNAi-AoUGCG; the RNAi-AoUGCG vector was transferred into the above-mentioned OEAoCerS strain by the PEG-mediated protoplast transformation method to inhibit the expression of the AoUGCG gene; PCR (pEX1-F and pEX1-R) and qRT-PCR (primers qAoUGCG-F and qAoUGCG-R, Ref-HistoneH4-F and Ref-HistoneH4-R) were used to determine that the expression of the AoUGCG gene was successfully inhibited, and the engineered strain OEAoCerS-RNAiAoUGCG was obtained.

[0009] A galactosylceramide genetically engineered bacterium obtained by the preparation method of the galactosylceramide genetically engineered bacterium.

[0010] The above-mentioned galactosylceramide genetic engineering is used for producing galactosylceramide.

[0011] The invention discloses an application of a galactosylceramide-producing genetically engineered bacterium, comprising the following steps: inoculating the galactosylceramide-producing genetically engineered bacterium into a CD fermentation medium, carrying out fermentation culture at 30°C and 180 rpm for 96 h, collecting the fermented bacterial bodies after the fermentation is completed, and using the fermented bacterial bodies to produce galactosylceramide.

[0012] Furthermore, the CD fermentation medium is composed of 2% maltose, 0.2% NaNO 3 , 0.1%KH 2 PO 4 , 0.05%MgSO 4 , 0.05%KCl, 0.05%NaCl, 0.002%FeSO 4 , the solvent is deionized water, pH 5.8-7.0.

[0013] The present invention also provides a method for constructing a genetically engineered strain of galactosylceramide (GalCers), the method comprising: (1) constructing the AoCerS gene onto an overexpression vector pEX2B using conventional molecular biological principles and techniques to obtain a pEX2B-AoCerS vector; transforming the pEX2B-AoCerS vector into the genome of a ΔpyrG Aspergillus oryzae auxotrophic strain by a PEG-mediated protoplast transformation method to increase the expression of the AoCerS gene; controlling the overexpression of the AoCerS gene using a maltose-inducible promoter amyB on the pEX2B vector to obtain an AoCerS overexpression strain (OEAoCerS); (2) inhibiting the expression of the AoUGCG gene using RNAi technology; and According to biological principles and techniques, the forward sequence and reverse sequence of the AoUGCG gene are respectively constructed into the RNAi vector pEX1 to obtain the RNAi-AoUGCG vector; the RNAi-AoUGCG vector is transferred into the OEAoCerS strain obtained in the above (1) by a PEG-mediated protoplast transformation method to inhibit the expression of the AoUGCG gene and obtain the engineered strain OEAoCer-RNAiAoUGCG; (3) the vector pEX2B without the AoCerS gene is transferred into the ΔpyrG Aspergillus oryzae auxotrophic strain to obtain the empty vector pEX2B strain; the pEX1 without the AoUGCG gene is transferred into the strain containing the empty vector pEX2B to obtain the control strain of the present invention, and the control strain is named Control.

[0014] The present invention uses a ΔpyrG Aspergillus oryzae auxotrophic strain as the AoCerS gene receptor strain. The strain is obtained by knocking out the pyrG gene by using CRISPR-Cas9 technology in the wild-type Aspergillus oryzae 3.042 strain (CICC40092). The construction method of the ΔpyrG Aspergillus oryzae auxotrophic strain is referred to Fan J, Zhang Z, Long C, et al. Identification and functional characterization of glycerol dehydrogenase reveal the role of inkojic acid synthesis in Aspergillus oryzae. World J Microbiol Biotechnol 36,136(2020). https: / / doi.org / 10.1007 / s11274-020-02912-4. The method for overexpressing the AoCerS gene is as follows: using the AoCerS gene sequence to design primers OEAoCerS-F and OEAoCerS-R through Primer5 software, using the cDNA of Aspergillus oryzae 3.042 as a template, obtaining the AoCerS gene through PCR amplification, connecting to the PmlⅠ site of the pEX2B vector through a homologous recombination method, and naming the vector successfully connected with the AoCerS gene as pEX2B-AoCerS; transforming the pEX2B-AoCerS vector into the genome of the ΔpyrG Aspergillus oryzae auxotrophic strain through a PEG-mediated protoplast transformation method, confirming that AoCerS is successfully overexpressed through PCR (primers pEX2B-F and pEX2B-R) and qRT-PCR (primers qAoCerS-F and qAoCerS-R, Ref-HistoneH4-F and Ref-HistoneH4-R), and obtaining the AoCerS overexpression strain, which is named OEAoCerS.

[0015] The method for inhibiting the expression of the AoUGCG gene is as follows: (1) using the cDNA of Aspergillus oryzae 3.042 as a template, AoUGCG-senseF and AoUGCG-senseR as primers, PCR amplification to obtain an AoUGCG gene fragment, and connecting it to the XholⅠ site on the pEX1 vector through a homologous recombination method to obtain an AoUGCG-sense sequence; using AoUGCG-Anti-senseF and AoUGCG-Anti-senseR as primers, PCR amplification to obtain an AoUGCG gene fragment, and connecting it to BamHⅠ on the pEX1 vector through a homologous recombination method to obtain an AoUGCG-Anti-sense sequence. The vector containing the AoUGCG-sense sequence and the AoUGCG-Anti-sense sequence was successfully connected and named RNAi-AoUGCG; (2) the RNAi-AoUGCG vector was transferred into the above-mentioned OEAoCerS strain by PEG-mediated protoplast transformation to inhibit the expression of the AoUGCG gene; (3) PCR (pEX1-F and pEX1-R) and qRT-PCR (primers qAoUGCG-F and qAoUGCG-R, Ref-HistoneH4-F and Ref-HistoneH4-R) were used to confirm that the expression of the AoUGCG gene was successfully inhibited. The resulting engineered strain was named OEAoCerS-RNAiAoUGCG; The construction method of the control strain is as follows: (1) by using a PEG-mediated protoplast transformation method, the pEX2B vector (without the AoCerS gene) is directly transferred into the ΔpyrG Aspergillus oryzae auxotrophic strain to obtain a pEX2B empty vector strain, named pEX2B-CK; (2) by using a PEG-mediated protoplast transformation method, pEX1 (without the AoUGCG gene) is directly transferred into the above (1) pEX2B-CK strain to obtain a strain carrying the pEX2B vector and the pEX1 vector, named the control strain (Control).

[0016] The present invention also provides the above-mentioned genetically engineered Aspergillus oryzae strain of galactosylceramide (GalCers). A genetically engineered Aspergillus oryzae strain with high production of GalCers was constructed through the above invention, and the relative content of GalCers in the strain under CD solid culture conditions was more than 2 times higher than that of the control strain, and was increased by about 70% compared with the control strain.

[0017] CD medium: 2% maltose, 0.2% NaNO 3 , 0.1%KH 2 PO 4 , 0.05%MgSO 4 , 0.05%KCl, 0.05%NaCl, 0.002%FeSO 4 , 1.8% agar, solvent is deionized water, pH 5.8-7.0.

[0018] The present invention also provides the use of the genetically engineered Aspergillus oryzae strain producing galactosylceramide (GalCers) in microbial fermentation. The application method comprises: inoculating the genetically engineered Aspergillus oryzae strain producing high-yield GalCers into CD fermentation medium, and fermenting and culturing the medium at 30°C and 180 rpm for 96 h. After the fermentation, the collected bacterial cells can be used to extract neutral glycosphingolipids.

[0019] The liquid fermentation medium consists of 2% maltose, 0.2% NaNO 3 , 0.1%KH 2 PO 4 , 0.05%MgSO 4 , 0.05%KCl, 0.05%NaCl, 0.002%FeSO 4 , the solvent is deionized water, pH 5.8-7.0.

[0020] The present invention has the following beneficial effects: The present invention uses conventional molecular biology to overexpress the AoCerS gene on the basis of the existing ΔpyrG Aspergillus oryzae auxotrophic strain to enhance the metabolic flux of neutral glycosphingolipids and improve the biosynthesis and accumulation of neutral glycosphingolipids in the strain; at the same time, RNAi technology is used to inhibit the expression of the AoUGCG gene, so that the intermediate product flux in the neutral glycosphingolipid metabolic pathway flows to the synthesis of galactosylceramide (GalCers). The relative content of GalCers in the strain thus constructed under solid culture conditions is more than 2 times higher than that of the control strain, and is about 70% higher than that of the control strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 It is a diagram of the metabolic synthesis pathway and modification sites of galactosylceramide in a specific embodiment of the present invention; Figure 2 This is a schematic diagram of the construction of the AoCerS gene and AoUGCG gene vectors in a specific embodiment of the present invention; Figure 3 This is a diagram of the construction of the strain overexpressing the AoCerS gene and the mRNA expression level of AoCerS in Example 1 in a specific embodiment of the present invention. Figure 3 A in the middle is the construction diagram of the strain overexpressing the AoCerS gene. Figure 3 Middle B is the graph of mRNA expression level of AoCerS; Figure 4 This is a diagram of the strain construction for inhibiting the expression of the AoUGCG gene in the OEAoCerS strain in Example 2 of the specific embodiment of the present invention and a diagram of the mRNA expression level of AoUGCG, Figure 4 A in the middle is the strain construction diagram for inhibiting the expression of AoUGCG gene in OEAoCerS strain. Figure 4 Middle B is the graph of mRNA expression level of AoUGCG; Figure 5 This is a graph showing the relative content of galactosylceramide in the OEAoCerS-RNAiAoUGCG engineered bacteria that overexpress the AoCerS gene and inhibit the expression of the AoUGCG gene in Example 3 of a specific embodiment of the present invention. DETAILED DESCRIPTION

[0023] The following describes the embodiments of the present invention by specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied by other different specific embodiments, and the details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, in the absence of conflict, the features in the following embodiments and embodiments can be combined with each other. In the embodiments of the present invention, the methods used are all conventional methods unless otherwise specified, and the reagents used can be obtained from commercial sources.

[0024] In the following examples, the final concentration of kanamycin in the culture medium is 50 mg / L, and the final concentration of pyrithiamine in the culture medium is 50 mg / L.

[0025] The Aspergillus oryzae 3.042 strain of the present invention is from the China Industrial Microbiological Culture Collection Administration Center, with a deposit date of December 20, 2005 and a strain deposit number of CICC40092. The Aspergillus oryzae 3.042 strain was obtained by knocking out the pyrG gene using CRISPR-Cas9 technology to obtain a ΔpyrG Aspergillus oryzae auxotrophic strain. The pyrG knockout method is specifically referred to Fan J, Zhang Z, Long C, et al. Identification and functional characterization of glyceroldehydrogenase reveal the role in kojic acid synthesis in Aspergillus oryzae. World J Microbiol Biotechnol Biotechnol 36, 136 (2020). https: / / doi.org / 10.1007 / s11274-020-02912-4. The pEX2B plasmid and pEX1 were transformed from pEX2 (V012516) and pEX1 (V012517). The specific transformation methods refer to (1) Nguyen KT, Ho QN, Do LTBX. et al. A new and efficient approach for construction of uridine / uracil auxotrophic mutants in the filamentous fungus Aspergillus oryzae using Agrobacterium tumefaciens-mediated transformation. World J Microbiol Biotechnol 33, 107 (2017). https: / / doi.org / 10.1007 / s11274-017-2275-9. (2) Nguyen KT, Ho QN, Pham TH. et al. The construction and use of versatile binary vectors carrying pyrG auxotrophic marker and fluorescent reporter genes for Agrobacterium-mediated transformation of Aspergillusoryzae. World J Microbiol Biotechnol 32, 204 (2016). https: / / doi.org / 10.1007 / s11274-016-2168-3. Galactosylceramide content determination method: qualitative and quantitative analysis of lipid group metabolism. The relative content of galactosylceramide in the present invention is determined by Biomarker Biotechnology Co., Ltd. through lipid group metabolism based on the LC-QTOF platform.

[0026] See also Figure 1 , Figure 1 A flow chart of the fungal glycosphingolipid metabolic pathway is described, with an emphasis on the regulation of the metabolic pathway by genetic engineering methods (such as overexpression of the AoCerS gene and RNAi inhibition of the AoUGCG gene) to increase the production of the target product galactosylceramide (GalCers).

[0027] Main contents of the picture: 1.I. Precursor synthesis stage L-Serine and Palmitoyl-CoA are enzymatically catalyzed to produce 3-keto dihydrosphinganine (3-ketoDhSph).

[0028] It is subsequently converted to dihydrosphingosine (DhSph).

[0029] 2.II. Neutral glycosphingolipid metabolic pathway (target product GalCers) Over expression AoCerS: Overexpression of AoCerS promotes the production of OH-dihydroceramide (DhCer) and OH-ceramide (Cer).

[0030] The final product is OH-Δ4-Δ8-9-Me-ceramide, which can be further converted into: Glucosylceramides (GlcCers): regulated by the AoUGCG gene, inhibition of this gene (RNAi AoUGCG) reduces the production of GlcCers.

[0031] Galactosylceramides (GalCers): the target product.

[0032] 3.III. Acidic sphingolipid metabolic pathway Dihydrosphingosine (DhSph) is converted into phytosphingosine (PhSph), which further generates phytoceramide (PhCer), inositol-phosphoceramide (IPC), and finally forms mannosylinositolphosphorylceramide (MIPC) and mannosyldiinositol phosphorylceramide [M(IP)2C].

[0033] AoCerS gene overexpression: enhanced the synthesis of target products in the neutral glycosphingolipid metabolic pathway.

[0034] RNAi inhibition of AoUGCG gene: reduced the production of competitive byproduct GlcCers and further increased the production of GalCers.

[0035] Example 1 Construction of strains overexpressing the AoCerS gene Taking the ΔpyrG Aspergillus oryzae auxotrophic strain as the starting strain, the AoCerS gene overexpression vector was constructed using conventional molecular biological techniques. The AoCerS gene regulated by the amyB promoter (nucleotide sequence as shown in SEQ ID No. 1) was integrated into the genome of the ΔpyrG Aspergillus oryzae auxotrophic strain through PEG-mediated protoplast transformation to enhance the expression intensity of the AoCerS gene.

[0036] (1) Construction of pEX2B-AoCerS vector: pEX2B plasmid was digested with PmlⅠ (Thermo Scientific Eco72I), and the digested product was purified by agarose gel electrophoresis and then recovered by DNA purification kit (DP214) to obtain the pEX2B digested fragment. The cDNA of Aspergillus oryzae 3.042 was used as a template and OEAoCerS-F and OEAoCerS-R were used as primers for PCR amplification. The PCR product was recovered by DNA purification kit (DP214) to obtain the AoCerS gene fragment. The pEX2B digested fragment and the AoCerS gene fragment were ligated according to the instructions of homologous recombination ligase (Vazyme C112-01). The ligated product was transformed into E. coli DH5a, screened on kanamycin LB plates, and sequenced to verify the correct pEX2B-AoCerS plasmid vector, which was used for subsequent transformation of ΔpyrG Aspergillus oryzae auxotrophic strain.

[0037] (2) pEX2B-AoCerS plasmid vector was used to transform the ΔpyrG Aspergillus oryzae auxotrophic strain: a. The ΔpyrG Aspergillus oryzae auxotrophic strain was inoculated into 100 mL DPY liquid culture medium containing 0.2% uridine and 0.2% uracil. After incubation at 30 °C and 160 rpm for 20 h, the cells were collected by filtration using sterile gauze (filter paper); b. The cells were incubated in 10 mL TF solution I (lysis solution) for 3 h and shaken at 30 °C and 50 rpm to separate the protoplasts from the mycelium; c. The protoplast suspension was diluted with an equal amount of TF solution II, the mycelium was removed by filtration to obtain a protoplast solution, and then the supernatant was removed by centrifugation at 4 °C and 700 g for 8 min. The precipitated protoplasts were resuspended in TF solution II at a concentration of 1×10 7 -5×10 7 / mL; d. Mix 5 µL of pEX2B-AoCerS plasmid with 200 µL of protoplast suspension and incubate on ice for 30 min; e. Add TF solution III to the protoplast mixture in three steps of 250, 250, and 850 µL, mix well in sequence, and place at room temperature in the dark for 20 min; f. Dilute the protoplast suspension in step e with 5-10 mL of TF solution II, centrifuge at 2000 rpm at 4 °C for 8 min, and remove the supernatant; g. Gently resuspend the protoplasts with 500 µL of TF solution II, add 4 mL of MM medium (containing 1.2 M sorbitol and 0.8% agar) to the protoplast suspension, and then pour the mixture into MM agar medium (containing 1.2 M sorbitol and 1.5% agar); h. Culture at 30 °C for 3-4 After 14 days, transformants were visible on the agar medium and inoculated onto CD medium (screening) without uridine and uracil. i. Mycelium was picked and verified by PCR using primers pEX2B-F and pEX2B-R to obtain the A. oryzae transformed strain with overexpression of AoCerS gene, which was named OEAoCerS.

[0038] (3) Obtaining the pEX2B-CK control strain: Using the pEX2B plasmid vector that does not contain the AoCerS gene, the pEX2B plasmid was transformed into the ΔpyrG Aspergillus oryzae auxotrophic strain according to the operation method of (2) above. The strain was used as the control strain for the OEAoCerS Aspergillus oryzae transformed strain and was named pEX2B-CK.

[0039] (4) qRT-PCR analysis of the AoCerS gene expression level in the OEAoCerS strain to determine the successful overexpression of the AoCerS gene: OEAoCerS and pEX2B-CK strains were inoculated on CD solid medium (expansion culture) and cultured at 30 °C for 96 h. The fungi were collected and RNA was extracted according to the instructions of the fungal RNA extraction kit (R6840-01, OMEGA). The extracted RNA was reverse transcribed to obtain cDNA according to the instructions of the reverse transcription kit (RR037Q, TAKARA). Using cDNA as a template, AoCerS gene primers (qAoCerS-F and qAoCerS-R) and internal reference gene HistoneH4 primers (RefH4-F and RefH4-R) were used, respectively, according to TBGreen® Premix Ex Taq™II FAST The qRT-PCR reaction system and reaction program were configured according to the qPCR (CN830A, TAKARA) instruction manual, and amplification was performed on a CFX96 (Bio-Rad) fluorescent PCR instrument to obtain the mRNA expression level of the AoCerS gene in the OEAoCerS strain.

[0040] AoCerS gene vector construction diagram can be found in Figure 2 Middle A.

[0041] Depend on Figure 3 It can be seen that the AoCerS gene was successfully overexpressed. Figure 3 In Figure A, the colony growth of pEX2B-CK (empty control strain) was significantly greater than that of the OEAoCerS series strains, and the colony growth of the OEAoCerS series strains was inhibited, indicating that the overexpression of AoCerS may have a certain negative impact on the growth of the strain; see Figure 3 In B, the expression level of AoCerS gene in pEX2B-CK (empty control strain) was extremely low. In the OEAoCerS series of strains, the expression level of AoCerS was significantly increased, and the expression level of OEAoCerS-1 was the highest, reaching about 60. The expression levels of the remaining strains (OEAoCerS-2 to OEAoCerS-4) were similar, but lower than that of OEAoCerS-1.

[0042] Example 2 Construction of strains inhibiting AoUGCG gene expression Taking the OEAoCerS obtained in Example 1 as the starting strain, RNAi technology was used to inhibit the expression of the AoUGCG gene (nucleotide sequence such as SEQ ID No. 2) in the OEAoCerS strain, and the expression of the original AoUGCG gene in the genome of the OEAoCerS strain was inhibited to weaken the metabolic flow of glucosylceramide (GlcCers), thereby further enhancing the biosynthesis of galactosylceramide.

[0043] (1) Construction of RNAi-AoUGCG vector: a. Using the cDNA of Aspergillus oryzae 3.042 as a template and AoUGCG-senseF and AoUGCG-senseR as primers, PCR amplification was performed. The PCR product was purified by agarose gel electrophoresis and then recovered using a DNA purification recovery kit (DP214) to obtain the AoUGCG-sense gene fragment; b. Using the same method, AoUGCG-Anti-senseF and AoUGCG-Anti-senseR were used to amplify the AoUGCG-sense gene fragment. GCG-Anti-sense gene fragment; c. pEX1 plasmid was digested with XholⅠ (1094S, TAKARA) according to the instructions, and the digestion product was recovered by agarose gel electrophoresis and DNA purification recovery kit (DP214) to obtain pEX1 plasmid XholⅠ digestion fragment; d. pEX1 plasmid XholⅠ digestion fragment and AoUGCG-sense gene fragment were connected according to the instructions of homologous recombination ligase (VazymeC112-01), and the ligation product was transformed into E.coli DH5a, kanamycin LB plate screening, sequencing verification to obtain the correct pEX1-AoUGCG-sense plasmid; e. pEX1-AoUGCG-sense plasmid was digested according to the instructions of BamHⅠ (1010S, TAKARA), and the digestion product was recovered by agarose gel electrophoresis and DNA purification recovery kit (DP214) to obtain the pEX1-AoUGCG-sense plasmid BamHⅠ digestion fragment; f. pEX1-AoUGCG-sense plasmid BamHⅠ digestion fragment and AoUGCG-Anti-sense gene fragment were ligated according to the instructions of homologous recombination ligase (VazymeC112-01), and the ligation product was transformed into E.coli DH5a, kanamycin LB plate screening, sequencing verification to obtain the correct RNAi-AoUGCG plasmid vector.

[0044] (2) Transformation of OEAoCerS strain with RNAi-AoUGCG plasmid vector: a. The OEAoCerS strain was inoculated into DPY liquid medium, cultured at 30 °C, 160 rpm for 20 h, and then filtered using sterile gauze (filter paper) to collect the bacteria; the remaining transformation methods bg were the same as step (2) in Example 1; h. After culture at 30 °C for 3-4 d, transformants were visible on the agar medium, and they were inoculated into CD medium (screening) containing 50 mg / L final concentration of pyrithiamine; i. Mycelium was picked and verified by PCR using primers pEX1-F and pEX1-R to obtain the Aspergillus oryzae transformed strain that inhibited the expression of the AoUGCG gene, which was named OEAoCerS-RNAiAoUGCG.

[0045] (3) Obtaining the control strain Control: Using the pEX2B-CK obtained in Example 1 as the base strain and the pEX1 plasmid vector without the AoUGCG gene, the pEX1 plasmid was transformed into the pEX2B-CK strain according to the operation method of Example 1 (2) above. The pEX1 plasmid was used as the control strain for the OEAoCerS-RNAiAoUGCG Aspergillus oryzae transformed strain and was named Control.

[0046] (4) qRT-PCR analysis of the AoUGCG gene expression level in the OEAoCerS-RNAiAoUGCG strain to determine that the expression of the AoUGCG gene was successfully inhibited: the OEAoCerS-RNAiAoUGCG and Control strains were inoculated on CD solid medium (expansion culture) and cultured at 30 °C for 96 h. The fungi were collected and RNA was extracted according to the instructions of the fungal RNA extraction kit (R6840-01, OMEGA). The extracted RNA was reverse transcribed to obtain cDNA according to the instructions of the reverse transcription kit (RR037Q, TAKARA). Using cDNA as a template, AoUGCG gene primers (qAoUGCG-F and qAoUGCG-R) and internal reference gene Histone H4 primers (RefH4-F and RefH4-R) were used, respectively, according to TB Green® Premix Ex Taq™ II FAST The qRT-PCR reaction system and reaction program were configured according to the qPCR (CN830A, TAKARA) instruction manual, and amplification was performed on a CFX96 (Bio-Rad) fluorescent PCR instrument to obtain the mRNA expression level of the AoUGCG gene in the OEAoCerS-RNAiAoUGCG strain.

[0047] AoUGCG gene vector construction diagram can be found in Figure 2 Middle B.

[0048] See also Figure 4 . RNAi knockdown of AoUGCG gene was successful.

[0049] AoUGCG gene knockdown affects strain growth: Figure 4 Figure A shows that the growth of the strain was inhibited after the AoUGCG gene was knocked down compared with the control strain, which indicates that the AoUGCG gene plays an important role in the normal metabolism and growth of the strain.

[0050] Figure 4 Center B shows that RNAi operation effectively inhibited the mRNA expression level of AoUGCG gene, indicating that the RNAi system was successfully constructed and functioned.

[0051] Knockdown level consistency: The knockdown effects and colony growth of the two engineered strains, RNAiAoUGCG-1 and RNAiAoUGCG-2, were similar, indicating that the constructed RNAi system was stable and reproducible.

[0052] Example 3 Determination of the content of galactosylceramide in OEAoCerS-RNAiAoUGCG engineered bacteria The OEAoCerS-RNAiAoUGCG engineered strain and the control strain Control obtained in Example 2 were used as starting strains, and the content of galactosylceramide (GalCers) in the engineered strains overexpressing the AoCerS gene and inhibiting the expression of the AoUGCG gene was measured.

[0053] (1) The OEAoCerS-RNAiAoUGCG engineered strain and the control strain Control were inoculated on CD solid medium (expansion culture) and cultured at 30 °C for 96 h. The cells were collected and immediately quick-frozen in liquid nitrogen. The samples were then freeze-dried. Each sample weighed at least 5 g.

[0054] (2) The samples processed in (1) were sent to Biomarker Biotechnology Co., Ltd. for lipidome metabolic analysis to obtain the relative content of GalCers in the OEAoCerS-RNAiAoUGCG engineered strain and the control strain Control.

[0055] (3) By Figure 5 It can be seen that the level of AoCerS gene overexpression in the genome and the inhibition of AoUGCG gene expression, the strain thus constructed had a relative content of GalCers under solid culture conditions that was more than 2 times higher than that of the control strain Control, and was about 70% higher than that of the control strain Control.

[0056] The culture medium or related solutions involved in Examples 1-3 are specifically as follows: TF solution I: 50 mM maleic acid (pH 5.5), 1% Yatalase (lyase), 0.6 M (NH 4 ) 2 SO 4 , prepare and filter sterilize before use.

[0057] TF solution II: 1.2 M sorbitan, 50 mM CaCl 2 ·2H 2 O, 35 mM NaCl, 10 mM Tris-HCl (pH 7.5), prepared with deionized water and sterilized by autoclaving at 115 °C for 15 min.

[0058] TF Solution III: 60% PEG 4000, 50 mM CaCl 2 ·2H 2 O, 10 mM Tris-HCl (pH 7.5), prepared with deionized water, and sterilized by autoclaving at 115 °C for 15 min.

[0059] LB medium: 10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, solvent is deionized water, pH 6.5-7.0.

[0060] DPY medium: 2% glucose, 1% tryptone, 0.5% yeast extract, 0.5% KH 2 PO 4 , 0.05%MgSO 4 , 0.1% uridine, 0.1% uracil, the solvent is deionized water, and 1.6% agar powder is added to the solid culture medium, with a pH value of 5.5-6.0.

[0061] M+Met medium (MM): 0.2% NH 4 Cl, 0.1% (NH 4 ) 2 SO 4 , 0.05%KCl, 0.05%NaCl, 0.1%KH 2 PO 4 , 0.05%MgSO 4 ⋅7H 2 O, 0.002%FeSO 4 ⋅7H 2 O, 2% glucose, 0.15% methionine, prepared with deionized water, pH 5.5, and autoclaved at 115 °C for 15 min.

[0062] CD medium (screening): 2% glucose, 0.2% NaNO 3 , 0.1%KH 2 PO 4 , 0.05%MgSO 4 , 0.05%KCl, 0.05%NaCl, 0.002%FeSO 4 The solvent is deionized water, pH 5.8-7.0, and 1.8% agar is added to the solid culture medium.

[0063] CD medium (for expansion): 2% maltose, 0.2% NaNO 3 , 0.1%KH 2 PO 4 , 0.05%MgSO 4, 0.05%KCl, 0.05%NaCl, 0.002%FeSO 4 The solvent is deionized water, pH 5.8-7.0, and 1.8% agar is added to the solid culture medium.

[0064] The primer sequence information involved in Examples 1-3 is shown in Table 1, and the nucleotide sequences are shown in SEQ ID No. 3 to SEQ ID No. 18.

[0065] Table 1. Primer sequences The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope of the present invention.

Claims

1. A genetically engineered bacterium producing galactosylceramide, characterized in that: The genetically engineered bacteria are obtained by first enhancing the metabolic flow of neutral glycosphingolipids by overexpressing the AoCerS gene, and then inhibiting the expression of the neutral glycosphingolipid glucosylceramide synthase gene AoUGCG. The recipient bacteria that overexpress the AoCers gene are ΔpyrG Aspergillus oryzae auxotrophic strains.

2. A method for preparing the genetically engineered bacteria for preparing the galactosylceramide according to claim 1, characterized in that: The following steps are involved: Overexpressing the AoCerS gene, inserting the AoCerS gene into an overexpression vector, constructing an AoCerS overexpression vector, transforming the AoCerS overexpression vector into a ΔpyrG Aspergillus oryzae auxotrophic strain, inducing a promoter, and obtaining an overexpression strain; Inhibit the expression of AoUGCG gene, insert the reverse sequence and the forward sequence of AoUGCG gene into the inhibition vector respectively to obtain the AoUGCG inhibition gene vector, and transfer the AoUGCG inhibition gene vector into the overexpression strain to obtain the genetic engineering bacteria of galactosylceramide.

3. The preparation method according to claim 2, characterized in that: The ΔpyrG Aspergillus oryzae auxotrophic strain is obtained by knocking out the pyrG gene of the wild-type Aspergillus oryzae 3.042 strain.

4. The preparation method according to claim 2, characterized in that: RNAi technology was used to inhibit AoUGCG gene expression.

5. The preparation method according to claim 3, characterized in that: Overexpression of the AoCerS gene includes the following steps: Primers OEAoCerS-F and OEAoCerS-R were designed based on the AoCerS gene sequence. The AoCerS gene was obtained by PCR amplification using the cDNA of Aspergillus oryzae 3.042 as a template. The gene was then connected to the PmlⅠ site of the pEX2B vector by homologous recombination. The vector successfully connected with the AoCerS gene was named pEX2B-AoCerS. The pEX2B-AoCerS vector was transformed into the genome of the ΔpyrG Aspergillus oryzae auxotrophic strain by PEG-mediated protoplast transformation. PCR and qRT-PCR were used to determine that AoCerS was successfully overexpressed, and the AoCerS overexpression strain OEAoCerS was obtained.

6. The preparation method according to claim 3, characterized in that: Inhibition of AoUGCG gene expression includes the following steps: Using the cDNA of Aspergillus oryzae 3.042 as a template and AoUGCG-senseF and AoUGCG-senseR as primers, the AoUGCG gene fragment was amplified by PCR and connected to the XholⅠ site on the pEX1 vector by homologous recombination to obtain the AoUGCG-sense sequence; using AoUGCG-Anti-senseF and AoUGCG-Anti-senseR as primers, the AoUGCG gene fragment was amplified by PCR and connected to the BamHⅠ on the pEX1 vector by homologous recombination to obtain the AoUGCG-sense sequence. UGCG-Anti-sense sequence; the vector of AoUGCG-sense sequence and AoUGCG-Anti-sense sequence was successfully connected and named RNAi-AoUGCG; the RNAi-AoUGCG vector was transferred into the above-mentioned OEAoCerS strain by PEG-mediated protoplast transformation method to inhibit the expression of AoUGCG gene; PCR and qRT-PCR were used to determine that the expression of AoUGCG gene was successfully inhibited, and the obtained engineered strain was named OEAoCerS-RNAiAoUGCG.

7. A genetically engineered bacterium producing galactosylceramide obtained by the method for preparing the genetically engineered bacterium producing galactosylceramide according to any one of claims 2 to 6.

8. Use of the genetically engineered bacteria producing galactosylceramide according to claim 1 or 7 in producing galactosylceramide.

9. Use of the genetically engineered bacteria of galactosylceramide according to claim 8 in producing galactosylceramide, characterized in that: The method comprises the following steps: inoculating the genetically engineered bacteria of galactosylceramide into CD fermentation medium, carrying out fermentation culture at 30°C and 180 rpm for 96 hours, collecting the fermented bacteria after the fermentation is completed, and using the fermented bacteria to produce galactosylceramide.

10. Use of the genetically engineered bacteria of galactosylceramide according to claim 9 in the production of galactosylceramide, wherein the CD fermentation medium is composed of: 2% maltose, 0.2% NaNO3, 0.1% KH2PO4, 0.05% MgSO4, 0.05% KCl, 0.05% NaCl, 0.002% FeSO4, the solvent is deionized water, and the pH of the CD fermentation medium is 5.8-7.0.

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