Method for increasing yield of sophorolipid and recombinant bacteria used by method

Abstract

The invention discloses a method for increasing the yield of sophorolipid and recombinant bacteria used by the method. The recombinant strain is an engineering improved strain 2 * CYP which is obtained by over-expressing CYP52M1 protein with an amino acid sequence shown as SEQ ID NO: 2 in candida bumblebee ATCC 22214 capable of producing sophorolipid. Experiments prove that compared with the candida bumblebee ATCC 22214, the sophorolipid content in the fermentation liquor obtained by fermenting the engineering improved strain 2 * CYP is increased by 22.67% and reaches a remarkable level. According to the method, the CYP52M1 protein is expressed by adopting free plasmids in the bumblebee candida ATCC 22214 for the first time, so that the yield of sophorolipid is remarkably increased, and the construction operation difficulty and the experiment cost of recombinant bacteria are simplified. The method has an important application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for increasing the yield of sophorolipids and the recombinant bacteria used therein. Background Technology

[0002] Sophorolipids (SLs) are a class of glycolipid biosurfactants produced by yeast metabolism. They have good antibacterial, anti-inflammatory, antitumor, and even anti-HIV activities, and have important theoretical research value and application development potential in the pharmaceutical field.

[0003] Currently, sophorolipids are produced via fermentation, but the yield is low. Therefore, increasing the fermentation yield of sophorolipids has become a hot topic in the production and application of sophorolipids. The copy number of the gene encoding the rate-limiting enzyme in the sophorolipid synthesis gene cluster is a factor affecting sophorolipid yield, but the effect of gene copy number on expression level remains unpredictable. In most cases, the expression level of exogenous proteins increases with increasing copy number. Currently, the common approach is to insert multiple copies of genes related to sophorolipid synthesis into the genome to increase the gene dose. This can be achieved by constructing plasmids containing the sophorolipid synthesis gene cluster via RecET recombination, or by linking the target gene expression cassette together with a homosupase to construct a recombinant expression plasmid containing multiple copies of the expression cassette and integrating it into the host strain genome, thereby obtaining transformants containing high-copy exogenous genes. Previous reports suggested that integration into chromosomes in *Candida bumblebee* resulted in more stable expression than expression in free vectors, but recent studies have found that the integration region may undergo meaningless single crossovers with genes in the original sophorolipid synthesis gene cluster in *Candida bumblebee*. Furthermore, the integration of exogenous genes into the yeast genome can adversely affect the cell's own physiological metabolism, resulting in reduced growth rate and cell viability in high-copy recombinant strains integrated into the genome. In contrast, free vectors can amplify within the cell independently of the host cell's replication cycle, thus significantly increasing the copy number of genes in the sophorolipid synthesis gene cluster. Simultaneously, the expression mode of free vectors has minimal impact on the bacterial genome. Currently, all plasmids used for *Candida albicans* are integrative plasmids; no examples have been found of using free expression plasmids to increase the expression level of rate-limiting enzyme proteins in the sophorolipid synthesis gene cluster. Summary of the Invention

[0004] The purpose of this invention is to increase the yield of sophorolipids.

[0005] This invention first protects and improves CYP 52 The expression level and / or activity of M1 protein can be determined by at least one of A1)-A4): A1) Constructing a recombinant strain for producing sophorolipids; A2) Production of sophorolipids; A3) Increase the yield of sophorolipids; A4) Prepare products for the production of sophorolipids.

[0006] In the above applications, the CYP 52 The M1 protein can be (a1), (a2), (a3), or (a4): (a1) A protein with the amino acid sequence shown in SEQ ID NO:2; (a2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein described in (a1); (a3) The protein shown in (a1) or (a2) is a protein that has undergone substitution and / or deletion and / or addition of one or more amino acid residues and is related to the production of sophorolipids; (a4) is a protein derived from yeast and has more than 98% identity with (a1) or (a2) and is related to the production of sophorolipids.

[0007] The proteins mentioned in (a2) above are labeled as shown in Table 1.

[0008]

[0009] The protein in (a3) ​​above, wherein the substitution and / or deletion and / or addition of one or more amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0010] The proteins mentioned in (a3) ​​above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0011] The gene encoding the protein in (a3) ​​above can be obtained by deleting one or more amino acid residues from the DNA sequence shown in SEQ ID NO:1 from position 601 to 2217 from the 5' end, and / or by performing a missense mutation of one or more base pairs, and / or by attaching the coding sequence of the tag shown in Table 1 to its 5' end and / or 3' end.

[0012] This invention also protects CYPs containing any of the codes described above. 52 Nucleic acid molecules of the M1 protein and / or any of the above-mentioned methods to increase CYP 52 The expression level of M1 protein and / or the application of expression cassettes, recombinant vectors, or recombinant bacteria for substances that enhance M1 protein expression and / or activity can be at least one of A1)-A4): A1) Constructing a recombinant strain for producing sophorolipids; A2) Production of sophorolipids; A3) Increase the yield of sophorolipids; A4) Prepare products for the production of sophorolipids.

[0013] The above-described encoding of any of the above-described CYPs52 The nucleic acid molecule of the M1 protein can be a DNA molecule as shown in (b1), (b2), (b3), or (b4) below: (b1) A DNA molecule with a coding region as shown in SEQ ID NO:1, positions 601-2217 from the 5' end; (b2) A DNA molecule with a nucleotide sequence as shown in SEQ ID NO:1 from position 601 to 2217 starting from the 5' end; (b3) Hybridizes under stringent conditions to a DNA molecule defined in (b1) or (b2) and encodes any of the CYPs described above. 52 The DNA molecule of the M1 protein; (b4) Derived from yeast and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the DNA molecule defined in (b1) or (b2) and encoding any of the CYPs described above. 52 The DNA molecule of the M1 protein.

[0014] The stringent conditions were: hybridization in a 2×SSC, 0.1% SDS solution at 68°C with two washes of 5 min each, followed by hybridization in a 0.5×SSC, 0.1% SDS solution at 68°C with two washes of 15 min each.

[0015] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0016] Any of the above-mentioned methods for improving CYP 52 The substance that expresses and / or activates the M1 protein may be the DNA fragment shown in SEQ ID NO:1.

[0017] Any of the recombinant vectors mentioned above can be the overexpression plasmid pFA6a-5FLAG-hphMX6-CYP mentioned in the examples. 52 M1.

[0018] Any of the recombinant bacteria mentioned above may be the engineered improved strain 2×CYP mentioned in the examples.

[0019] This invention also protects a recombinant strain, characterized by overexpression of any of the above-described CYPs in the starting yeast. 52 M1 protein, resulting in recombinant bacteria; the starting yeast can produce sophorolipids.

[0020] In the above-mentioned recombinant strains, the overexpression can be achieved by introducing the encoding of any of the aforementioned CYPs into the starting yeast strain. 52 The nucleic acid molecule of the M1 protein was realized.

[0021] In the above-mentioned recombinant bacteria, the overexpression can be achieved by introducing the overexpression plasmid pFA6a-5FLAG-hphMX6-CYP mentioned in the examples into the starting yeast. 52 M1 implementation.

[0022] In the above-mentioned recombinant strains, the starting yeast can be *Candida bumblebee*. The *Candida bumblebee* can be *Candida bumblebee* ATCC 22214.

[0023] The recombinant bacteria may specifically be the engineered improved strain 2×CYP mentioned in the examples.

[0024] This invention also protects a method for increasing the yield of sophorolipids, which may include the following steps: increasing the yield of any of the above-described CYPs in the starting yeast. 52 The expression level and / or activity of M1 protein were used to obtain recombinant bacteria; the yield of sophorolipids produced by the fermentation recombinant bacteria was higher than that of the original yeast.

[0025] In the above method, the enhancement of any of the CYPs in the starting yeast is described above. 52 The expression level and / or activity of the M1 protein can be determined by introducing a CYP encoding any of the above-described proteins into the starting yeast. 52 The nucleic acid molecule of the M1 protein was realized.

[0026] In the above method, the enhancement of any of the CYPs in the starting yeast is described above. 52 The expression level and / or activity of the M1 protein can be determined by introducing the overexpression plasmid pFA6a-5FLAG-hphMX6-CYP mentioned in the examples into the starting yeast. 52 M1 implementation.

[0027] The starting yeast mentioned in the above method can be *Candida bumblebee*. The *Candida bumblebee* can be *Candida bumblebee* ATCC 22214.

[0028] In the above method, the recombinant bacteria may specifically be the engineered improved strain 2×CYP mentioned in the examples.

[0029] This invention also protects a method for producing sophorolipids, comprising the following steps: fermenting and culturing any of the recombinant bacteria described above, collecting the fermentation products, and obtaining sophorolipids therefrom.

[0030] This invention also protects the application of any of the recombinant bacteria described above, which may be at least one of A2)-A4): A2) Production of sophorolipids; A3) Increase the yield of sophorolipids; A4) Prepare products for the production of sophorolipids.

[0031] Experiments showed that the sophorolipid content in the fermentation broth obtained from *Candida albicans* ATCC 22214 was 220.68 mg / L; while the content of sophorolipids obtained by introducing the overexpression plasmid pFA6a-5FLAG-hphMX6-CYP into *Candida albicans* ATCC 22214 was... 52 The sophorolipid content in the fermentation broth of the engineered strain 2×CYP obtained from M1 was 270.39 mg / L. Compared with *Candida albicans* ATCC 22214, the sophorolipid content in the fermentation broth of the engineered strain 2×CYP was increased by 22.67%, reaching a significant level. This invention is the first to express CYP in *Candida albicans* ATCC 22214 using a free plasmid. 52 The M1 protein not only significantly increased the yield of sophorolipids but also simplified the construction of recombinant bacteria and reduced experimental costs. This invention has significant application value. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the structure of plasmid pFA6a-5FLAG-hphMX6.

[0033] Figure 2 The result is the agarose gel electrophoresis detection result of step 1 in Example 1.

[0034] Figure 3 The result is the agarose gel electrophoresis detection result in step 2 of Example 1.

[0035] Figure 4 The plasmid is pFA6a-5FLAG-hphMX6-CYP 52 Structural diagram of M1.

[0036] Figure 5 This is the glucose standard curve plotted in step 2 of Example 2. Detailed Implementation

[0037] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0039] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0040] Bumblebee Candida ATCC 22214 is a product of Beijing Bio-Biobio Biotechnology Co., Ltd., with product catalog number Bio-53347. Bumblebee Candida ATCC 22214 will be referred to as Bumblebee Candida ATCC 22214 in the following text.

[0041] A schematic diagram of the structure of plasmid pFA6a-5FLAG-hphMX6 is shown below. Figure 1 It is a product of Wuhan Miaoling Biotechnology Co., Ltd., with product catalog number P34591 (see http: / / www.miaolingbio.com / search.html?keywords=P34591).

[0042] Example 1: Obtaining the engineered improved strain 2×CYP I. Overexpression plasmid pFA6a-5FLAG-hphMX6-CYP 52 M1 Construction 1. Extraction of genomic DNA from *Candida albicans* (bumblebee) (1) Place 800 μL of *Candida bacillus* bacterial culture into a 1.5 mL centrifuge tube, centrifuge at 4 °C and 9000 rpm for 1 min, and collect the bacterial cells.

[0043] (2) Add 500 μL of extraction buffer (the solute and its concentration are Tris 24.23 g / L, NaCl 14.61 g / L, EDTA 9.31 g / L and SDS 2.0 g / L, the solvent is water, and the pH value is 8.0) and an appropriate amount of quartz sand to the bacterial cells collected in step (1). Vortex for 5 min, then place in a water bath and heat at 65°C for 10 min to obtain the bacterial suspension.

[0044] (3) Add 250 μL of 7.5 M ammonium acetate solution to the bacterial suspension obtained in step (2), mix by inverting, then place on ice for 8 min, then centrifuge at 4 °C and 12000 rpm for 10 min, and collect the supernatant.

[0045] (4) Take a new 1.5 mL centrifuge tube, add 600 μL of the supernatant collected in step (3) and 300 μL of pre-cooled isopropanol, mix by inverting, and let stand at -20℃ for more than 30 min; then centrifuge at 4℃ and 12000 rpm for 15 min, discard the supernatant, add 1 mL of 75% (v / v) ethanol aqueous solution to the tube, centrifuge at 4℃ and 12000 rpm for 4 min, discard the supernatant; open the cap and let stand at room temperature for 20 min, after the ethanol has evaporated completely, add 30 μL of ddH2O to dissolve, and obtain the genomic DNA of Candida bacillus.

[0046] The genomic DNA extracted from *Candida bumblebee* was detected by agarose gel electrophoresis.

[0047] Test results are shown Figure 2 (The first lane is for DNA markers, with molecular weights of 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, and 100bp from top to bottom. The other lanes contain genomic DNA extracted from bumblebee Candida albicans.)

[0048] 2. Obtaining the target fragment (1) Using the genomic DNA of *Candida bumblebee* extracted in step 1 as a template, primer P was used. cyp52m1 For: 5'-CC TTAATTAA GCTTCGTATCCGTCGTCCGTAA-3' (underlined is the recognition site of restriction endonuclease PacⅠ) and primer P cyp52m1 Rev:5'-AT CGTACG The primer pair consisting of TAACATCAGGCACCGCAATCG-3' (underlined is the recognition site of restriction endonuclease BsiWⅠ) was used for PCR amplification to obtain the PCR amplification product.

[0049] (2) The PCR amplification products obtained in step (1) are detected by agarose electrophoresis.

[0050] Test results are shown Figure 3 (The first lane is for DNA markers. The molecular weights of the DNA markers from top to bottom are 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, and 100bp. The arrows indicate the target fragments.)

[0051] (3) After completing step (2), cut the gel and recover the DNA fragment with a size of 3526bp, which is the target fragment.

[0052] 3. Overexpression plasmid pFA6a-5FLAG-hphMX6-CYP 52 Construction and identification of M1 (1) The plasmid pFA6a-5FLAG-hphMX6 was digested with restriction endonucleases PacⅠ and BsiWⅠ, and the 4497bp linear vector backbone was recovered.

[0053] (2) The target fragment was digested with restriction endonucleases PacⅠ and BsiWI, and the 3518bp CYP was recovered. 52 M1 segment.

[0054] (3) The carrier skeleton recovered in step (1) and the CYP recovered in step (2) 52 The M1 fragment was ligated using T4 ligase to obtain the overexpression plasmid pFA6a-5FLAG-hphMX6-CYP. 52 M1.

[0055] (4) The overexpression plasmid pFA6a-5FLAG-hphMX6-CYP obtained in step (3) 52 M1 is the template, and primer P is used. cyp52m1 For and primer P cyp52m1 PCR amplification was performed using primers composed of Rev, yielding a PCR product of 3526 bp. This indicates that the overexpression plasmid pFA6a-5FLAG-hphMX6-CYP... 52 M1 was successfully built.

[0056] (5) The overexpression plasmid pFA6a-5FLAG-hphMX6-CYP 52 M1 was sequenced. Sequencing results showed that the overexpression plasmid pFA6a-5FLAG-hphMX6-CYP... 52 M1 is a recombinant plasmid obtained by replacing the small DNA fragment between the restriction endonuclease PacⅠ and BsiWI recognition sites of plasmid pFA6a-5FLAG-hphMX6 with the DNA fragment shown in SEQ ID NO:1. Positions 601-2217 of SEQ ID NO:1 are CYP sequences from the 5' end. 52 M1 gene. Overexpression plasmid pFA6a-5FLAG-hphMX6-CYP 52 M1 expresses the CYP shown in SEQ ID NO:2. 52 M1 protein.

[0057] Recombinant plasmid pFA6a-5FLAG-hphMX6-CYP 52 A structural diagram of M1 is shown below. Figure 4 .

[0058] II. Obtaining and Identifying the Engineered Strain Breed 2×CYP 1M LiAc: Add 5.1g LiAc to 50ml of sterile water, dissolve completely, adjust the pH to 7.5, and sterilize at 121℃ for 15min.

[0059] 50% PEG3350: Add 25g of PEG3350 to 30mL of sterile water, dissolve it thoroughly with a magnetic stirrer (50℃ to aid dissolution), then bring the volume to 50mL with sterile water and sterilize at 121℃ for 15min.

[0060] 10×TE buffer: The solute and its concentration is 10mM EDTA, the solvent is 100mM Tris-HCl buffer, and the pH value is 7.5.

[0061] Salmon sperm DNA (ssDNA): Add 100 mg of salmon sperm DNA to 10 mL of sterile 10×TE buffer, mix well, incubate overnight at 4°C, aliquot into centrifuge tubes, and store at -20°C.

[0062] 2×YPDA liquid culture medium: Dissolve 5 g of yeast extract, 10 g of peptone, 10 g of glucose and 25 mg of adenine hemisulphate in ddH2O, then bring the volume to 200 ml with ddH2O; sterilize at 115℃ for 20 min.

[0063] 1. Inoculate a single colony of *Candida bumblebee* into 20 mL of 2×YPDA liquid medium and culture at 30℃ and 200 rpm for 16-18 h with shaking to obtain culture solution 1.

[0064] 2. Take a 1.5 mL sterile centrifuge tube, add 1 mL of culture solution 1, centrifuge at 12000 rpm for 30 s at room temperature, and collect the precipitate.

[0065] 3. After completing step 2, add 1 mL of ddH2O to the centrifuge tube for resuspending, centrifuge at 12000 rpm for 30 s at room temperature, and discard the supernatant.

[0066] 4. After completing step 3, add 100µL of 1.1×TE / LiAC to the centrifuge tube and resuspend at 4℃ to obtain competent cells of Candida bacillus.

[0067] Each 5 mL 1.1×TE / LiAC consists of 550 µL 10×TE buffer, 550 µL 1M LiAc, and 3.9 mL ddH2O.

[0068] 5. After completing step 4, add competent cells of *Candida baccata* to the conversion mixture, incubate in a 30°C water bath for 45 minutes, inverting and mixing every 5-10 minutes during the process to prevent precipitation from affecting the conversion efficiency.

[0069] The transformation mixture consisted of 600 µL of PEG3350 mixture, 10 µL of denatured salmon sperm DNA, and 10 µL (0.1–1 µg) of overexpression plasmid pFA6a-5FLAG-hphMX6-CYP. 52 Composed of M1.

[0070] The salmon sperm DNA (ssDNA) was denatured in a boiling water bath for 10 minutes, and then quickly placed on ice to cool before use, thus obtaining denatured salmon sperm DNA.

[0071] The PEG3350 mixture consists of 8 mL of 50% PEG3350, 1 mL of 1M LiAc, and 1 mL of 10×TE buffer.

[0072] 6. After completing step 5, add 70µL DMSO, incubate in a 42℃ water bath for 20min, inverting and mixing every 5-10min; then centrifuge at 700g for 5min and collect the precipitate; add 1mL 2×YPDA liquid culture medium to the precipitate, and revive at 30℃ and 200rpm for 1h to obtain the transformed yeast culture.

[0073] 7. After completing step 6, spread the transformed yeast culture onto a hygromycin screening plate and incubate at 28°C for 96 hours to obtain several yeast single colonies.

[0074] Hygromycin screening plates: Dissolve 10g of yeast extract, 20g of peptone, 20g of glucose and 10g of agar in distilled water, then bring the volume to 1000ml with distilled water and sterilize at 121℃ for 15min; when the temperature drops to about 50℃, add 2ml of 50mg / ml hygromycin solution, mix well and pour into petri dishes, then allow to cool naturally.

[0075] 8. After completing step 7, perform colony PCR verification on each yeast single colony. Specifically: using each yeast single colony as a template, and primer P... hyg F: 5'-CAGCGAGAGCCTGACCTATTG-3' and primer P hyg PCR amplification was performed using a primer pair consisting of R: 5'-GCGACCAGCATTCACATACGAT-3'. The PCR amplification products were then subjected to agarose gel electrophoresis. The following determination was made: if the PCR amplification product of a single yeast colony contained a DNA fragment of 974 bp, then the yeast colony was considered a positive yeast colony, i.e., it was overexpressed by the plasmid pFA6a-5FLAG-hphMX6-CYP.52 M1 was successfully transformed into Candida bumblebee; if the PCR amplification product of a single yeast colony does not contain a DNA fragment of size 974 bp, then the single yeast colony is not a positive yeast colony.

[0076] The positive yeast single colony was named the engineered improved strain 2×CYP.

[0077] Example 2: Production of sophorolipids by fermentation using the engineered improved strain 2×CYP obtained in Example 1. The experiment was repeated 6 times in parallel, and the average value was calculated. The steps for each repetition are as follows: 1. The engineered improved strain 2×CYP or *Candida bumblebee* obtained in Example 1 was inoculated into 250 ml of fermentation medium (the solutes and their concentrations were glucose 90 g / L, corn oil 90 g / L, peptone 0.7 g / L, sodium citrate 5 g / L, yeast extract 3 g / L, potassium dihydrogen phosphate 1 g / L, ammonium sulfate 2 g / L, magnesium sulfate heptahydrate 4 g / L, calcium chloride dihydrate 0.1 g / L and sodium chloride 0.1 g / L, the solvent was water, and the pH value was 6.0), and fermented at 28℃ and 200 rpm for 7 days to obtain the fermentation broth.

[0078] 2. Detect the sophorolipid content in the fermentation broth. The content of sophorolipids in fermentation broth was determined using the sulfuric acid-anthrone colorimetric method. The principle of the sulfuric acid-anthrone colorimetric method for determining total sugars is as follows: This method is specifically designed for sugar determination and can be used to determine hexoses, pentoses, and hexuronic acids. Whether in a free or polymerized state in polysaccharides, they react with anthrone reagent to produce a blue-green color, exhibiting maximum light absorption at 620 nm. The principle for determining sophorolipid content is as follows: Since sophorolipids consist of both sophorose and lipid components, the sulfuric acid-anthrone colorimetric method can measure the OD value of sophorose at 620 nm. Further, a glucose standard curve can be plotted based on the data to determine the glucose content. The molecular weight of sophorolipids is constant compared to glucose; 1g of glucose is equivalent to 1.91g of sophorolipids. Therefore, the sophorolipid content in the sample can be determined using the sulfuric acid-anthrone colorimetric method.

[0079] The specific steps are as follows: (1) Weigh 0.1g of anthrone accurately, then add 100mL of concentrated sulfuric acid and mix well to obtain the anthrone reagent.

[0080] (2) Accurately weigh 0.1 g of analytical grade anhydrous glucose that has been dried to constant weight in a drying oven at 105℃, then add it to a 100 mL volumetric flask, dissolve it completely with distilled water, and dilute to 100 mL with distilled water to obtain a glucose mother solution with a glucose concentration of 1000 mg / L. Add 0.5 mL, 1 mL, 2 mL, 3 mL and 4 mL of the glucose mother solution to a 100 mL volumetric flask respectively, and dilute to 100 mL with distilled water to obtain glucose solution 1 with a concentration of 5 mg / L, glucose solution 2 with a concentration of 10 mg / L, glucose solution 3 with a concentration of 20 mg / L, glucose solution 4 with a concentration of 30 mg / L and glucose solution 5 with a concentration of 40 mg / L.

[0081] (3) Place 4 mL of anthrone reagent and 1 mL of the test solution (distilled water (as blank control), glucose solution 1, glucose solution 2, glucose solution 3, glucose solution 4 or glucose solution 5) in a stoppered test tube, cool quickly in an ice bath, then heat in a boiling water bath for 10 min and continue cooling in an ice bath. After cooling is complete, detect the OD value at 620 nm.

[0082] A glucose standard curve was plotted with the concentration of the glucose solution on the x-axis and the OD value at 620 nm on the y-axis.

[0083] The glucose standard curve is shown below. Figure 5 Where Y = 0.0073X + 0.00056 (R) 2 =0.99928), Y is the OD value at 620nm, and X is the concentration of the glucose solution.

[0084] (4) Take 1 mL of the fermentation broth obtained in step 1, add 1 mL of ethyl acetate, mix well and let stand for 5 min; then centrifuge at 5000 r / min for 5 min, collect 10 µL of the upper ethyl acetate and add it to a stoppered test tube, evaporate in a water bath; finally wash with n-hexane and evaporate to dryness, add 1 mL of water and 4 mL of anthrone reagent in sequence, cool quickly in an ice bath, then heat in a boiling water bath for 10 min and continue to cool in an ice bath. After cooling is complete, detect the OD value at 620 nm.

[0085] The results are as follows: The OD values ​​obtained from six parallel replicate experiments of the fermentation broth obtained by fermentation of the engineered strain 2×CYP were 0.914, 0.944, 0.924, 0.885, 1.197 and 1.338, respectively, with an average OD value of 1.034.

[0086] The OD values ​​obtained from six parallel replicate experiments of the fermentation broth obtained by fermentation of Candida bumblebee were 0.783, 0.741, 0.833, 0.846, 1.016 and 0.843, respectively, with an average OD value of 0.844.

[0087] Based on the above results, the sophorolipid content in the fermentation broth obtained from the engineered strain 2×CYP was 270.39 mg / L, while the sophorolipid content in the fermentation broth obtained from *Candida bumblebee* was 220.68 mg / L. Compared with *Candida bumblebee*, the sophorolipid content in the fermentation broth of the engineered strain 2×CYP increased by 22.67%. Therefore, it is evident that the engineered strain 2×CYP significantly improves the sophorolipid content produced by fermentation compared to *Candida bumblebee*.

[0088] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Increase CYP 52 The application of substances that increase the expression level and / or activity of M1 protein, and are at least one of A1)-A4): A1) Constructing a recombinant strain for producing sophorolipids; A2) Production of sophorolipids; A3) Increase the yield of sophorolipids; A4) Prepare products for the production of sophorolipids; The CYP 52 The M1 protein is (a1) or (a2) or (a3) ​​or (a4): (a1) A protein with the amino acid sequence shown in SEQ ID NO:2; (a2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein described in (a1); (a3) The protein shown in (a1) or (a2) is a protein that has undergone substitution and / or deletion and / or addition of one or more amino acid residues and is related to the production of sophorolipids; (a4) is a protein derived from yeast and has more than 98% identity with (a1) or (a2) and is related to the production of sophorolipids.

2. Contains the CYP encoding described in claim 1 52 The nucleic acid molecule of M1 protein and / or the enhancement of CYP as described in claim 1 52 The application of expression cassettes, recombinant vectors, or recombinant bacteria for substances that express and / or activate M1 protein, and are at least one of A1)-A4): A1) Constructing a recombinant strain for producing sophorolipids; A2) Production of sophorolipids; A3) Increase the yield of sophorolipids; A4) Prepare products for the production of sophorolipids.

3. Recombinant bacteria, characterized in that: Overexpression of the CYP described in claim 1 in the starting yeast 52 M1 protein, yielding recombinant bacteria; The starting yeast can produce sophorolipids.

4. The recombinant bacteria according to claim 3, characterized in that: The overexpression is achieved by introducing the CYP encoding the CYP described in claim 1 into the starting yeast. 52 The nucleic acid molecule of the M1 protein was realized.

5. The recombinant bacteria according to claim 3, characterized in that: The starting yeast is *Candida bumblebee*.

6. A method for increasing the yield of sophorolipids, comprising the following steps: increasing the CYP content of the starting yeast as described in claim 1. 52 The expression level and / or activity of M1 protein were used to obtain recombinant bacteria; The yield of sophorolipids produced by the recombinant fermentation strain was higher than that produced by the original yeast strain.

7. The method according to claim 6, characterized in that: The improved starting yeast strain contains the CYP as described in claim 1. 52 The expression level and / or activity of the M1 protein are determined by introducing the CYP protein encoding the one described in claim 1 into the starting yeast. 52 The nucleic acid molecule of the M1 protein was realized.

8. The method according to claim 6, characterized in that: The starting yeast is *Candida bumblebee*.

9. A method for producing sophorolipids, comprising the following steps: fermenting and culturing the recombinant bacteria as described in any one of claims 3 to 5, collecting the fermentation products, and obtaining sophorolipids therefrom.

10. The application of the recombinant bacteria according to any one of claims 3 to 5, comprising at least one of A2) to A4): A2) Production of sophorolipids; A3) Increase the yield of sophorolipids; A4) Prepare products for the production of sophorolipids.

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