A method for weakening RNA helicase to improve the growth and environmental tolerance of glycerol-producing Candida

Abstract

The present invention discloses a method for weakening RNA helicase to improve the growth and environmental tolerance of Candida glycerolgensis, belonging to the field of biotechnology. The method first uses reverse PCR cloning to obtain an antisense sequence containing DBP7 and part of its 5' non-coding region, and designates it as Cgantidbp7a or Cgantidbp7b. The DBP7 homologous gene of Candida glycerolgensis is designated as CgDBP7. By integrating and expressing Cgantidbp7a or Cgantidbp7b into a uracil-deficient strain of Candida glycerolgensis, the RNA helicase DBP7 is weakened, thereby improving the high salt tolerance of Candida glycerolgensis.

Description

Technical Field

[0001] The invention relates to a method for weakening RNA helicase to improve the growth and environmental tolerance of glycerol-producing Candida albicans, and belongs to the field of biotechnology. Background Art

[0002] Microorganisms face multiple environmental pressures during industrial fermentation, such as high temperature, osmotic pressure, high sugar, acetic acid, and hydrogen peroxide. Glycerol-producing Candida albicans is an industrial stress-resistant strain with high glycerol production screened from nature. It is resistant to osmotic pressure, high temperature, organic acids, oxidative pressure, etc., and is a potential excellent industrial biotechnology host.

[0003] Candida glyceroliferans, a non-traditional yeast, possesses superior hyperosmotic tolerance compared to the model strain Saccharomyces cerevisiae. Previous studies have shown that under conditions of high osmotic pressure, cells balance osmotic pressure by altering the accumulation of intracellular metabolites with osmotic regulatory functions, such as sugars (sucrose and fructose), sugar alcohols (mannose and methylated inositol), and complex sugars (trehalose, raffinose, and fructans). At the same time, hyperosmotic conditions induce the accumulation of calcium ions in the cytoplasm, triggering the calcineurin pathway and thereby regulating intracellular ion homeostasis. Furthermore, high salt conditions alter the fluidity and permeability of the cell membrane, thereby attenuating the cytotoxicity caused by sodium ion influx.

[0004] RNA helicases, a class of ATP-dependent helicases, unwind RNA duplexes and participate in processes such as pre-mRNA splicing, ribosome biogenesis, nucleocytoplasmic transport, translation, RNA degradation, and structural gene expression. RNA helicases also contribute to plant responses to environmental stress. For example, in barley, the RNA helicase HVD1 participates in the response to salt stress. Similarly, in fungi, RNA helicases are involved in responses to environmental stress. Summary of the Invention

[0005] The present invention first clones antisense sequences of DBP7 and part of its 5' non-coding region through reverse PCR and names them Cgantidbp7a or Cgantidbp7b. The DBP7 homologous gene of Candida glycerolgensis is designated CgDBP7. By integrating and expressing Cgantidbp7a or Cgantidbp7b into a uracil-deficient strain of Candida glycerolgensis, the RNA helicase DBP7 is attenuated, improving the high-salt tolerance of Candida glycerolgensis.

[0006] The present invention provides a recombinant glycerol-producing Candida. The recombinant glycerol-producing Candida integrates and expresses an antisense sequence of RNA helicase CgDBP7 in its genome. The antisense sequence of RNA helicase CgDBP7 is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.5.

[0007] In one embodiment of the present invention, the nucleotide sequence encoding the RNA helicase CgDBP7 is shown as SEQ ID NO.3.

[0008] The amino acid sequence of the RNA helicase CgDBP7 is shown in SEQ ID NO.4.

[0009] The integration site of the antisense sequence is the 5.8S site on the genome of Candida glycerologenes.

[0010] In one embodiment of the present invention, the recombinant Candida glycerologen is based on Candida glycerologen CCTCC M93018 as a chassis cell.

[0011] In one embodiment of the present invention, pURGAPU plasmid is used as an integration expression vector.

[0012] The present invention provides a method for increasing the biomass of glycerol-producing Candida albicans. The method comprises integrating and expressing an antisense sequence of RNA helicase CgDBP7 into the genome of the glycerol-producing Candida albicans. The antisense sequence of RNA helicase CgDBP7 is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.5.

[0013] The integration site of the antisense sequence is the 5.8S site on the genome of Candida glycerologenes.

[0014] In one embodiment of the present invention, the glycerogenic Candida is glycerogenic Candida CCTCCM93018.

[0015] In one embodiment of the present invention, the pURGAPU plasmid is used as the integration expression vector.

[0016] The present invention provides a method for improving the tolerance of recombinant Candida glycerologen under a pressure stress environment. The method comprises integrating and expressing an antisense sequence of RNA helicase CgDBP7 in the genome of Candida glycerologen, wherein the antisense sequence of RNA helicase CgDBP7 is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.5; the pressure stress includes but is not limited to temperature stress, osmotic stress, glucose stress, organic acid stress, and oxidant stress, and the integration site of the antisense sequence is the 5.8S site on the Candida glycerologen genome.

[0017] In one embodiment of the present invention, the pressure stress environment is a temperature of 44° C. or 120 mmol / L acetic acid or 3 g / L furfural or 400 g / L sorbitol or 1 mol / L NaCl or 350 g / L glucose.

[0018] In one embodiment of the present invention, the glycerogenic Candida is glycerogenic Candida CCTCCM93018.

[0019] In one embodiment of the present invention, the pURGAPU plasmid is used as the integration expression vector.

[0020] The present invention also provides a method for constructing the above-mentioned recombinant glycerol-producing Candida, which comprises:

[0021] (1) Using the genome of Candida glyceroliferans as a template, the upstream and downstream primers Cgantidbp7aF and Cgantidbp7aR; Cgantidbp7bF and Cgantidbp7bR were used to amplify Cgantidbp7a and Cgantidbp7b, i.e., DBP7 and its partial 5' non-coding region antisense sequence, by PCR. The nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2; the primer sequences (5'-3') are as follows:

[0022] Cgantidbp7aF:acacaattacaaaaaggatccCTTTTATGCTTGATTTCTTGTCTTTTT;

[0023] Cgantidbp7aR:gtcgccgttttcgagggtaccATACAGCATTCAAGAGAACCGTTACG;

[0024] Cgantidbp7bF:acacaattacaaaaaggatccAAGGATGATACAATTTGGCCATTT;

[0025] Cgantidbp7bR:gtcgccgttttcgagggtaccCTGTTCAACGTGCCTTGGCG;

[0026] Among them, the lowercase part is the homology arm sequence;

[0027] (2) An integration expression vector containing Cgantidbp7a and Cgantidbp7b was constructed, and the vector was linearized with HindIII and then transformed into the uracil-deficient strain of Candida glycerinogenes CCTCC M93018;

[0028] (3) verifying the recombinant C.glycerinogenes obtained in step (2) using a colony PCR detection method;

[0029] (4) The growth of recombinant C.glycerinogenes and its tolerance to various environmental stress conditions were further analyzed and identified through spot plate experiments and shake flask culture.

[0030] The present invention also provides a method for improving the NaCl tolerance of Candida glycerologen. The method comprises integrating and expressing the reverse sequence Cgantidbp7a derived from Candida glycerologen as shown in SEQ ID NO.1.

[0031] In one embodiment of the present invention, the glycerogenic Candida is Candida glycerinogenes CCTCC M93018.

[0032] Beneficial effects

[0033] (1) The present invention utilizes the gene Cgantidbp7a derived from Candida glycerinogenes CCTCC M93018 to improve the growth ability of glycerol-producing Candida. By using the method provided by the present invention, the CgDBP7 gene is antisense inhibited in Candida glycerinogenes CCTCC M93018, thereby improving the growth ability of Candida glycerinogenes CCTCC M93018.

[0034] (2) Using the method provided by the present invention, after antisense inhibition of CgDBP7 in Candida glycerinogenes CCTCC M93018, the growth ability of the recombinant strain was improved under various environmental stress conditions, including but not limited to temperature stress, osmotic stress, and glucose stress.

[0035] (3) Using the method provided by the present invention, after antisense inhibition of CgDBP7 in Candida glycerinogenes CCTCC M93018, the biomass of the recombinant strain increased by 10.91% compared with the control strain under 1 mol / L NaCl stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 : Agarose gel electrophoresis image of CgDBP7.

[0037] Figure 2 : Agarose gel electrophoresis images of Cgantidbp7a and Cgantidbp7b.

[0038] Figure 3 : Growth of recombinant strains at 30°C; C.gUra5 was complemented as the control strain.

[0039] Figure 4 : Growth of recombinant strains under stress conditions; among them, C.gUra5 was complemented as the control strain.

[0040] Figure 5 : Growth of different RNA helicases under stress conditions; among them, C.gUra5 was complemented as the control strain. DETAILED DESCRIPTION

[0041] The pURGAPU plasmid involved in the following examples is described in the Chinese invention patent publication number CN112553098B.

[0042] The culture medium involved in the following examples is as follows:

[0043] YPD medium (g / L): 20g / L glucose, 20g / L peptone, 10g / L yeast powder. If preparing solid culture medium, add 20g / L agar powder.

[0044] MM medium (g / L): YNB 6.7, glucose 20, pH 6.2-6.5. If preparing solid culture medium, add 20g / L agar powder to the liquid culture medium.

[0045] LB medium (g / L): yeast powder 5, peptone 10, NaCl 10. If preparing solid culture medium, add 20g / L agar powder to the liquid culture medium.

[0046] The detection methods involved in the following embodiments are as follows:

[0047] Tolerance performance test:

[0048] (1) Dot plate experiment:

[0049] The recombinant strain of glycerol-producing Candida was transferred to 10 mL MM liquid medium, cultured at 30°C, 200 rpm / min in a shake flask for 12 h, and then transferred to a new 10 mL MM medium at a 2% (v / v) inoculation rate and cultured at 30°C, 200 rpm / min in a shake flask for 4-8 h as seed liquid. The seed liquid OD 600 Adjust to 1 and serially dilute to 10 -1 , 10 -2 , 10 -3 , 10 -4 Used for spot-plate experiments. After spotting, the culture dishes were placed in a 30°C incubator for 24-60 hours. The culture media used in the experiment contained different stress conditions: a final concentration of 1 mol / L NaCl, 120 mmol / L acetic acid, 3 g / L furfural, 400 g / L sorbitol, and 350 g / L glucose. The temperature was set at 44°C.

[0050] (2) Shake flask experiment:

[0051] Different stress conditions were added to the culture medium, and the absorbance at a wavelength of 600 nm was measured every 8 hours at 30°C and 100 rpm / min using an ultraviolet spectrophotometer.

[0052] The transformation methods of the competent cells involved in the following examples are as follows:

[0053] The conversion method adopts lithium acetate conversion method, and the steps are as follows:

[0054] Candida glycerinogenes CCTCC M93018 uracil-deficient strain was inoculated into YPD liquid medium and cultured in a shaking flask at 30°C and 200 rpm / min. 600 After the value reaches 3-4, the cells were collected by centrifugation at 6000 rpm for 2 minutes and washed twice with sterile double-distilled water. The cells were resuspended in a transformation system consisting of 240 μL of 50% PEG 3350, 36 μL of 1 mol / L LiAc, 20 μL of 5 mg / mL fish sperm DNA (boiled in a water bath for 10 minutes before use), 15 μL of linearized plasmid, and the volume was brought to 360 μL with double-distilled water. After thorough mixing, the cells were heat-shocked at 42°C for 60 minutes, centrifuged at 6000 rpm for 2 minutes, and resuspended in 1 mL of YPD liquid medium. The cells were cultured for 3 hours (30°C, 200 rpm / min). The cells were collected by centrifugation, washed three times with sterile double-distilled water, and 100 μL of double-distilled water was added. After mixing thoroughly, the cells were spread on MM solid medium and cultured at 30°C for 24-48 hours to obtain recombinant Candida glycerinogenes CCTCC M93018.

[0055] Example 1: Preparation of recombinant Candida glycerologenous yeast with antisense inhibition of RNA helicase CgDBP7

[0056] The specific steps are as follows:

[0057] (1) Using the genome of Candida glycerinogenes CCTCC M93018 as a template, the upstream and downstream primers Cgantidbp7aF, Cgantidbp7aR, Cgantidbp7bF, and Cgantidbp7bR were used to obtain the Cgantidbp7a and Cgantidbp7b gene fragments by PCR. The PCR reaction system was as follows (50 μL): 25 μL Prime STAR Max Premix (2×), 15 pmol primers, 150 ng template, and double-distilled water to 50 μL (primers were synthesized by Shanghai Shenggong Biotechnology Co., Ltd., and the rest were purchased from TaKaRa). The amplification reaction conditions were as follows: 98°C, 10 s; 60°C, 15 s; 72°C, 1 kb / min, 35 cycles. After the electrophoresis of the amplified product was completed (such as Figure 2 The gel was excised and recovered according to the kit instructions (the column recovery kit was purchased from Shanghai Shenggong Bioengineering Co., Ltd.) to obtain the antisense sequences Cgantidbp7a and Cgantidbp7b (nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2).

[0058] The primer sequences (5'3') are as follows:

[0059] Cgantidbp7aF:acacaattacaaaaaggatccCTTTTATGCTTGATTTCTTGTCTTTTT;

[0060] Cgantidbp7aR:gtcgccgttttcgagggtaccATACAGCATTCAAGAGAACCGTTACG.

[0061] Cgantidbp7bF:acacaattacaaaaaggatccAAGGATGATACAATTTGGCCATTT;

[0062] Cgantidbp7bR:gtcgccgttttcgagggtaccCTGTTCAACGTGCCTTGGCG;

[0063] Among them, the lowercase part is the homology arm sequence;

[0064] (2) connecting the Cgantidbp7a and Cgantidbp7b products obtained in step (1) with the pURGAPU expression vector (the integration site of the plasmid is 5.8S) by homologous recombination;

[0065] The recombinant vectors pURGAPU-Cgantidbp7a and pURGAPU-Cgantidbp7b were prepared respectively;

[0066] The ligation products were transformed into Escherichia coli JM109, and cells were transferred from the plates to LB liquid medium supplemented with ampicillin and cultured at 37°C, 200 rpm, for 10–12 h. Plasmids were then extracted (plasmid extraction kit purchased from Shanghai Sangon Biotechnology Co., Ltd.). The extracted plasmids were digested with HindIII Quickcut enzyme using the following digestion system (100 μL): 5 μL HindIII Quickcut enzyme, 10 μL Quickcut buffer, and a final template concentration of 200–300 ng / μL. Double-distilled water was added to 100 μL (Quickcut enzyme and accompanying Quickcut buffer purchased from TaKaRa). The reaction was incubated at 37°C in a water bath for 2 h. The linearized plasmid was then recovered by column extraction according to the kit instructions (column extraction kit purchased from Shanghai Sangon Biotechnology Co., Ltd.).

[0067] (3) The recovered linearized plasmids were transformed into the uracil-deficient strain of Candida glycerinogenes CCTCC M93018 to prepare recombinant Candida glycerinogenes CCTCC M93018 / pURGAPU-Cgantidbp7a and C.glycerinogenes CCTCC M93018 / pURGAPU-Cgantidbp7b.

[0068] Control strain:

[0069] According to the above method, the linearized empty plasmid pURGAPU was transformed into the uracil-deficient strain of Candida glycerinogenes CCTCC M93018 to prepare the control strain: recombinant Candida glycerinogenes CCTCC M93018 / pURGAPU.

[0070] Example 2: Growth assay of recombinant glycerol-producing Candida albicans

[0071] (1) The recombinant glycerogenic Candida albicans C.glycerinogenes CCTCCM93018 / pURGAPU, C.glycerinogenes CCTCC M93018 / pURGAPU-Cgantidbp7a, and C.glycerinogenes CCTCC M93018 / pURGAPU-Cgantidbp7b prepared in Example 1 were inoculated into MM liquid culture medium, and cultured at 30°C and 200 rpm for 12 hours to prepare seed solutions;

[0072] (2) inoculating the prepared seed solution into MM liquid culture medium at a 2% (v / v) inoculation rate, and culturing at 30°C and 200 rpm to obtain a secondary seed solution;

[0073] (3) The obtained secondary seed solution was plated on MM solid medium and cultured in a 30°C incubator for 24 to 48 hours. The plate results were as follows: Figure 3 shown.

[0074] The secondary seed solution cultured in step (2) was inoculated into MM medium and cultured in a shake flask at 30°C and 100 rpm. The growth of C. glycerinogenes was measured every 8 hours. The results are shown in Table 1.

[0075] C.glycerinogenes CCTCC M93018 / pURGAPU was cultured as described above, and the growth of C.glycerinogenes was measured as a control. The results are shown in Table 1.

[0076] Table 1: Growth of strains at 30°C (OD 600 express)

[0077]

[0078] The experimental results show that under no stress conditions at 30°C, the antisense inhibition recombinant strain constructed with the antisense inhibition sequence has a significant growth advantage. The spot plate results show that the growth advantage of the antisense inhibition recombinant strain a is more obvious. At 56 hours, the biomass of the antisense inhibition strain a increased by 16.93% compared with the control strain.

[0079] The above results indicate that antisense inhibition of RNA helicase DBP7 has a significant positive effect on the growth of glycerol-producing Candida albicans.

[0080] Example 3: Growth determination of recombinant glycerol-producing Candida albicans under stress conditions

[0081] (1) The recombinant glycerol-producing Candida albicans (C.glycerinogenes CCTCCM93018 / pURGAPU), C.glycerinogenes CCTCC M93018 / pURGAPU-Cgantidbp7a, and C.glycerinogenes CCTCC M93018 / pURGAPU-Cgantidbp7b prepared in Example 1 were inoculated into MM liquid culture medium and cultured at 30°C and 200 rpm for 12 hours to prepare seed solution;

[0082] (2) inoculating the prepared seed solution into MM liquid culture medium at a rate of 2% (v / v) and culturing at 30°C and 200 rpm to obtain a secondary seed solution;

[0083] (3) The obtained secondary seed solution was plated on MM solid medium with different pressure elements and cultured in a 30°C incubator for 24 to 60 hours;

[0084] The pressure elements are: 1 mol / L NaCl solution; acetic acid with a final concentration of 120 mmol / L; furfural with a final concentration of 3 g / L; sorbitol with a final concentration of 400 g / L; glucose with a final concentration of 350 g / L; a high temperature environment of 44°C, and the spot plate results are as follows Figure 4 shown.

[0085] (4) inoculating the secondary seed solution obtained in step (2) into MM liquid culture medium supplemented with pressure elements and performing shake flask culture at 30° C. and 100 r / min;

[0086] The stress element is 1 mol / L NaCl solution stress or acetic acid stress with a final concentration of 120 mmol / L;

[0087] The growth of C.glycerinogenes was measured every 8 h. C.glycerinogenes CCTCC M93018 / pURGAPU was cultured as described above as a control. The results are shown in Tables 2 and 3. Figure 4 As shown;

[0088] Table 2: Tolerance of recombinant glycerol-producing Candida albicans strains to NaCl (OD 600 express)

[0089]

[0090] Table 3: Tolerance of recombinant glycerol-producing Candida strains to acetic acid (OD 600 express)

[0091]

[0092] The experimental results show that under the condition of 1 mol / L NaCl, the antisense inhibition recombinant strain has a significant growth advantage, and the biomass of the antisense inhibition recombinant strain increased by 10.91% compared with the control strain.

[0093] From the above results, it can be seen that weakening RNA helicase DBP7 can effectively improve the tolerance of glycerol-producing Candida albicans under high salt stress conditions.

[0094] Example 4: Preparation of recombinant strains that inhibit RNA helicase with antisense inhibition

[0095] The specific steps are as follows:

[0096] (1) Using the genome of Candida glycerinogenes CCTCC M93018 as a template, the upstream and downstream primers Cgantidbp7F, Cgantidbp7R, Cgantided1F, and Cgantided1R were used to obtain the Cgantidbp7 and Cgantided1 gene fragments by PCR. The PCR reaction system was as follows (50 μL): 25 μL Prime STAR Max Premix (2×), 15 pmol primers, 150 ng template, and double-distilled water to 50 μL (primers were synthesized by Shanghai Shenggong Biotechnology Co., Ltd., and the rest were purchased from TaKaRa). The amplification reaction conditions were as follows: 98°C, 10 s; 60°C, 15 s; 72°C, 1 kb / min, 35 cycles. After the electrophoresis of the amplified product was completed (such as Figure 1 The gel was excised and recovered according to the kit instructions (column recovery kit was purchased from Shanghai Shenggong Bioengineering Co., Ltd.) to obtain the antisense sequences of Dbp7 and Ded1 genes (nucleotide sequences are shown in SEQ ID NO.5 and SEQ ID NO.6).

[0097] The primer sequences (5'3') are as follows:

[0098] Cgantidbp7F:acacaattacaaaaaggatccAGCAAAGTTAAACTCTTCGTTTTGC;

[0099] Cgantidbp7R:gtcgccgttttcgagggtaccATGGCTGACAATGATGATGGG;

[0100] Cgantided1F:acacaattacaaaaaggatccCCACCATTGAGAAGATGAGTTGTT;

[0101] Cgantided1R:gtcgccgttttcgagggtaccATGGCATACGTTCCTCCACACA;

[0102] (2) connecting the Cgantidbp7 and Cgantided1 products obtained in step (1) with the pURGAPU expression vector by homologous recombination;

[0103] The recombinant vectors pURGAPU-Cgantidbp7 and pURGAPU-Cgantided1 were prepared respectively.

[0104] The ligation products were transformed into Escherichia coli JM109, and cells were transferred from the plates to LB liquid medium supplemented with ampicillin and cultured at 37°C, 200 rpm / min, for 10–12 h. Plasmids were then extracted (plasmid extraction kit purchased from Shanghai Sangon Biotechnology Co., Ltd.). The extracted plasmids were digested with HindIII Quickcut enzyme using the following digestion system (100 μL): 5 μL HindIII Quickcut enzyme, 10 μL Quickcut buffer, and a final template concentration of 100–200 ng / μL. Double-distilled water was added to 100 μL (Quickcut enzyme and accompanying Quickcut buffer purchased from TaKaRa). The reaction was incubated at 37°C in a water bath for 2 h. The linearized plasmid was then recovered by column extraction according to the kit instructions (column extraction kit purchased from Shanghai Sangon Biotechnology Co., Ltd.).

[0105] (3) The recovered linearized plasmids were transformed into the uracil-deficient strain of Candida glycerinogenes CCTCC M93018 to prepare recombinant Candida glycerinogenes CCTCC M93018 / pURGAPU-Cgantidbp7 and C.glycerinogenes CCTCC M93018 / pURGAPU-Cgantided1.

[0106] Control strain:

[0107] According to the above method, the linearized empty plasmid was transformed into the uracil-deficient strain of Candida glycerinogenes CCTCC M93018 to prepare the control strain: recombinant Candida glycerinogenes CCTCC M93018 / pURGAPU.

[0108] Example 5: Analysis of stress resistance of recombinant strains inhibiting RNA helicase with antisense inhibition

[0109] (1) The recombinant glycerol-producing Candida albicans C. glycerinogenes CCTCCM93018 / pURGAPU-Cgantidbp7 and C. glycerinogenes CCTCC M93018 / pURGAPU-Cgantided1 prepared in Example 4 were inoculated into MM liquid culture medium and cultured at 30° C. and 200 rpm for 12 hours to prepare a seed solution;

[0110] (2) inoculating the prepared seed solution into MM liquid culture medium at a rate of 2% (v / v) and culturing at 30°C and 200 rpm to obtain a secondary seed solution;

[0111] (3) The obtained secondary seed liquid was spotted on MM solid culture medium with different pressures and cultured in a 30°C incubator for 24 to 60 hours.

[0112] The pressure elements are: 1 mol / L NaCl solution; acetic acid with a final concentration of 120 mmol / L; furfural with a final concentration of 3 g / L; sorbitol with a final concentration of 400 g / L; glucose with a final concentration of 350 g / L; hydrogen peroxide with a final concentration of 10 mmol / L; temperature environments of 30°C and 44°C. The spot plate results are as follows: Figure 5 shown.

[0113] (4) C.glycerinogenes CCTCC M93018 / pURGAPU-Cgantidbp7 and C.glycerinogenes CCTCC M93018 / pURGAPU-Cgantided1 were inoculated into YPD medium with different stresses (1 mol / L NaCl solution; acetic acid with a final concentration of 120 mmol / L; furfural with a final concentration of 3 g / L; sorbitol with a final concentration of 400 g / L; glucose with a final concentration of 350 g / L, hydrogen peroxide with a final concentration of 10 mmol / L; temperature environment of 30°C and 44°C) according to the above method. The culture was shaken at 30°C and 200 r / min. The growth of C.glycerinogenes was measured every 6 hours. C.glycerinogenes CCTCC M93018 / pURGAPU was cultured according to the above method as a control. The results are shown in Table 4 and Figure 5 As shown;

[0114] Table 4: Growth of recombinant strains under different stress conditions at 42 h

[0115]

[0116] In the table, the control strain is: C.glycerinogenes CCTCC M93018 / pURGAPU, Cg-antidbp7 is: C.glycerinogenes CCTCC M93018 / pURGAPU-Cgantidbp7, and Cg-antided1 is: C.glycerinogenes CCTCC M93018 / pURGAPU-Cgantided1.

[0117] The results showed that compared with weakening the expression of other RNA helicases, weakening the RNA helicase DBP7 could significantly improve the growth of glycerol-producing Candida under stress conditions.

[0118] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A recombinant glycerol-producing Candida, characterized in that: The recombinant Candida glycerologenis has an antisense sequence of RNA helicase CgDBP7 integrated and expressed in its genome, and the antisense sequence of RNA helicase CgDBP7 is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.5; The recombinant glycerol-producing Candida albicans uses glycerol-producing Candida albicans CCTCC M93018 as a chassis cell.

2. The recombinant glycerogenic Candida according to claim 1, wherein The pURGAPU plasmid was used as the integration expression vector.

3. A method for increasing the biomass of glycerol-producing Candida albicans, characterized in that: The method comprises integrating and expressing an antisense sequence of RNA helicase CgDBP7 in the genome of Candida glycerologenes, wherein the antisense sequence of RNA helicase CgDBP7 is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.5; The glycerol-producing Candida is glycerol-producing Candida CCTCC M93018.

4. The method according to claim 3, wherein The pURGAPU plasmid was used as the integration expression vector.

5. A method for improving the tolerance of Candida glycerologen under stress environment, characterized in that: The method comprises the following steps: integrating and expressing an antisense sequence of RNA helicase CgDBP7 into the genome of glycerol-producing Candida albicans, wherein the antisense sequence of RNA helicase CgDBP7 is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.5; the pressure stress is NaCl stress with a final concentration of 1 mol / L, acetic acid stress with a final concentration of 120 mmol / L, furfural stress with a final concentration of 3 g / L, sorbitol stress with a final concentration of 400 g / L, glucose stress with a final concentration of 350 g / L, hydrogen peroxide stress with a final concentration of 10 mmol / L, or temperature stress of 30°C or 44°C; and the glycerol-producing Candida albicans is glycerol-producing Candida albicans CCTCCM93018.

6. The method according to claim 5, wherein The pURGAPU plasmid was used as the integration expression vector.

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