CYP52M1 enzyme mutant and application thereof in efficient production of sophorolipid

Abstract

The invention belongs to the field of enzymes, and particularly relates to a CYP52M1 enzyme mutant and application of the CYP52M1 enzyme mutant in sophorolipid production. The CYP52M1 enzyme mutant is a CYP52M1 enzyme mutant which is obtained by mutating at least one site of amino acids at the 141 site, the 148 site and the 476 site of an amino acid sequence as shown in SEQ ID NO.1, and the CYP52M1 enzyme mutant is a CYP52M1 enzyme mutant, according to the mutation, phenylalanine at the 141 site of the amino acid sequence shown in SEQ ID NO.1 is mutated into alanine, tryptophan at the 148 site is mutated into alanine, or proline at the 476 site is mutated into alanine. The mutant enhances the substrate binding orientation and omega-hydroxylation ability, improves the fatty acid conversion efficiency, reduces the cost, and promotes the efficient synthesis of sophorolipid. The invention provides a new technical approach for improving the yield of the biosurfactant through an enzyme engineering means.

Description

Technical Field

[0001] This invention belongs to the field of enzymes, specifically relating to a CYP52M1 enzyme mutant and its application in the efficient production of sophorolipids. Background Technology

[0002] Sophorolipids are an important class of glycolipid biosurfactants with advantages such as low toxicity, environmental friendliness, renewability, and biodegradability, and are widely used in detergents, cosmetics, food, pharmaceuticals, and environmental remediation. In the biosynthetic pathway of sophorolipids, fatty acids first undergo ω-terminal or ω-1 hydroxylation via CYP52M1 (cytochrome P450 monooxygenase) to generate hydroxy fatty acids, which then proceed to glycosyl transfer and further modification steps. Therefore, CYP52M1 is the rate-limiting key enzyme in the synthesis of sophorolipids. However, wild-type CYP52M1 enzymes have limited catalytic efficiency and poor stability under high oil concentrations or fermentation conditions, resulting in limited overall sophorolipid yields. Existing research has focused primarily on process optimization and substrate improvement, with no reports on targeted modification of CYP52M1. Therefore, a modified CYP52M1 enzyme is urgently needed to overcome the yield bottleneck of sophorolipids. Summary of the Invention

[0003] The purpose of this invention is to provide a CYP52M1 enzyme mutant and its application in the production of sophorolipids.

[0004] To achieve the above objectives, the first aspect of the present invention adopts the following technical solution: a CYP52M1 enzyme mutant, wherein the CYP52M1 enzyme mutant is either 1) or 2) below.

[0005] 1) A CYP52M1 enzyme mutant obtained by mutating at least one of the amino acid positions 141, 148, and 476 of the amino acid sequence shown in SEQ ID NO.1; wherein the mutation is to mutate phenylalanine at position 141 to alanine, tryptophan at position 148 to alanine, or proline at position 476 to alanine;

[0006] 2) CYP52M1 enzyme mutants with P450 enzyme activity that have amino acid sequence homology greater than 99%, 95%, 90%, 85%, or 80% with the CYP52M1 enzyme mutant in 1).

[0007] Preferably, the CYP52M1 enzyme mutant is obtained by mutating two or three sites in the amino acid sequence shown in SEQ ID NO.1, specifically at positions 141, 148, and 476.

[0008] Furthermore, the amino acid sequence of the CYP52M1 enzyme mutant is shown in SEQ ID NO.2.

[0009] A second aspect of the present invention is to disclose the coding gene encoding the aforementioned mutant.

[0010] A third aspect of the invention is to provide an expression vector comprising the aforementioned coding gene.

[0011] A fourth aspect of the invention is to disclose a host cell comprising the aforementioned expression vector or the aforementioned coding gene.

[0012] Preferably, the coding gene is integrated or not integrated into the host cell's genome. The host cell is a genetically engineered bacterium.

[0013] A fifth aspect of the present invention is to provide a catalyst comprising the aforementioned mutant, the aforementioned coding gene, the aforementioned expression vector, or the aforementioned host cell.

[0014] The sixth aspect of the present invention discloses the use of the aforementioned mutant, the aforementioned coding gene, the aforementioned expression vector, the aforementioned host cell, or the aforementioned catalyst in improving the efficiency of fatty acid ω-hydroxylation or the yield of sophorolipid.

[0015] A nucleic acid molecule encoding the CYP52M1 enzyme mutant is inserted into an expression vector to obtain a recombinant vector. This recombinant vector is then introduced into a host cell, or the nucleic acid molecule is directly integrated into the host cell's genome to obtain a genetically engineered bacterium. After culturing and purification, the CYP52M1 enzyme mutant can be obtained. The resulting genetically engineered bacterium can be fermented to produce sophorolipids; the substrates used for fermentation are rapeseed oil and glucose.

[0016] In some implementations, the fermentation conditions are as follows: dissolved oxygen (DO) is maintained above 30% under agitation and aeration control, the temperature is 28-32°C, for example 28°C, 29°C, 30°C, 31°C, 32°C, and the pH is controlled within the range of 2.0-7.0.

[0017] For example, in the initial stage, the stirring speed is set to 500 r / min, the aeration rate to 1.0 vvm, the temperature to 30℃, and the pH to 6.5. As the fermentation progresses, the pH in the system naturally decreases to approximately 2.5. Due to the increase in cell biomass leading to a gradual decrease in dissolved oxygen, the stirring speed and aeration rate are automatically adjusted during this period to maintain DO above 30%. The aeration rate can reach up to 3.0 vvm, and the stirring speed can reach up to 900 r / min to ensure sufficient oxygen supply and promote the CYP52M1-catalyzed fatty acid ω-hydroxylation reaction.

[0018] In some implementations, the culture medium used for fermentation consists of: 2–10 g / L yeast extract, 50–150 g / L glucose (sterilized separately), 50–150 g / L rapeseed oil, 1–3 g / L KH₂PO₄, 1–3 g / L Na₂HPO₄, 0.5–2 g / L MgSO₄, and 0.05–0.2 g / L CaCl₂. For example, a culture medium formulation of 3 g / L yeast extract, 120 g / L glucose, 120 g / L rapeseed oil, 2 g / L KH₂PO₄, 2 g / L Na₂HPO₄, 1 g / L MgSO₄, and 0.1 g / L CaCl₂ can be used, and sterilized at 121°C for 20 min.

[0019] In some implementations, the seed culture is prepared using YPD medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose) and cultured at 30°C and 220 r / min for 24–36 h, with an inoculum size of 2–10% (v / v) of the total fermentation volume.

[0020] In some implementation schemes, the cell optical density (OD) at the end of fermentation is... 600 It can reach 180–220.

[0021] The advantages of this invention lie in the following: Through structural analysis, rational design, and site-directed mutagenesis, amino acids at positions 128, 141, 148, 340, 403, 404, and 476 of the CYP52M1 enzyme sequence shown in SEQ ID NO:1 were modified to construct various CYP52M1 enzyme mutants. Functional verification revealed superior mutants with significantly enhanced catalytic performance. These mutants enhance substrate binding orientation and ω-hydroxylation ability, improve fatty acid conversion efficiency, reduce costs, and promote the efficient synthesis of sophorolipids. This invention provides a new technical approach for improving the yield of biosurfactants through enzyme engineering. Attached Figure Description

[0022] Figure 1 The three-dimensional structure of CYP52M1;

[0023] Figure 2 The yield of sophorolipids obtained by fermentation of recombinant bacteria of wild type and various CYP52M1 mutants is shown in the figure.

[0024] Figure 3 Conversion rate of sophorolipids obtained by fermentation of recombinant bacteria of wild type and various CYP52M1 mutants;

[0025] Figure 4Liquid chromatography-mass spectra of samples obtained by fermentation using recombinant strains containing wild-type CYP52M1 enzyme or F141A / W148A / P476A mutants. Detailed Implementation

[0026] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0027] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0028] Example 1: This example describes the construction of a recombinant Candida globosa that produces sophorolipids.

[0029] In this embodiment, a CYP52M1 enzyme mutant with improved catalytic performance was constructed to enhance the efficiency of fatty acid ω-hydroxylation, thereby promoting the efficient synthesis of sophorolipids.

[0030] like Figure 1 As shown, the CYP52M1 enzyme is a cytochrome P450 monooxygenase derived from *Starmerella bombicola*, capable of catalyzing the terminal hydroxylation of long-chain fatty acids, a key step in the synthesis of sophorolipids. Structural analysis reveals that the orientation of the substrate within the enzyme's hydrophobic channels significantly influences the hydroxylation site. Therefore, optimizing key amino acid residues in the substrate binding pocket through site-directed mutagenesis can enhance the spatial proximity of the terminal carbon atom to the catalytic oxygen, thereby improving ω-hydroxylation activity.

[0031] First, the endogenous CYP52M1 gene in the host bacterium *Starmerella bombicola* was deleted using genome editing methods to avoid background expression interference with subsequent functional evaluation of the mutant. The knockout was performed using a self-knockout system based on hygromycin resistance markers to construct a knockout cassette. The construction process of this knockout cassette is as follows:

[0032] (1) Using the genome of Candida bombicola ATCC 22214 as a template, and CYP52M1-F / CYP52M1-R as primers, a 1.6 kb CYP52M1 gene fragment was amplified by PCR. The fragment was ligated to the vector pMD19-T (Simple) using SolutionI ligase and then transformed into Escherichia coli JM 109 to obtain the recombinant plasmid Ts-CYP52M1.

[0033] (2) Using recombinant plasmid Ts-CYP52M1 as a template and RCYP52M1-F / RCYP52M1-R as primers, reverse PCR was performed under the action of Extaq enzyme to obtain the vector CYP52M1-Ts-CYP52M1 with a length of 3.3 kb.

[0034] (3) A 3.1 kb hphex fragment (SEQ ID NO.17) was synthesized by Hangzhou Qingke Biotechnology Co., Ltd. This fragment is a hygromycin resistance expression cassette, which is surrounded at both ends by self-excisable Rec-Six recombination recognition sequences.

[0035] (4) CYP52M1-Ts-CYP52M1 and the fragment hphex were digested with enzymes Mlu I and Kpn I at the same time.

[0036] (5) CYP52M1-Ts-CYP52M1 and the fragment hphex are ligated under the action of enzyme Solution I to form the recombinant plasmid Ts-CYP52M1-hphex-CYP52M1, which is the CYP52M1 gene knockout mutant cassette.

[0037] CYP52M1 gene knockout mutation cassette quilt Transformed into competent *S. bombicola* ATCC 22214 cells, positive clones were screened in hygromycin-resistant medium. Subsequently, a self-knockout system was induced under antibiotic-free culture conditions, causing the hphex element to be excised spontaneously, retaining only the downstream complete genome repair sequence, resulting in the ΔCYP52M1 strain with the CYP52M1 gene knocked out. PCR and sequencing confirmed the successful knockout.

[0038] The gene encoding the wild-type CYP52M1 enzyme shown in SEQ ID NO:1 is derived from the genome of Starmerellabombicola. Genomic DNA was first extracted from the laboratory-preserved S. bombicola ATCC 22214 (S. bombicola is Starmerellabombicola) strain (purchased from Guangdong Institute of Microbiology). The genomic DNA was extracted using a commercial DNA extraction kit (TIANGEN Yeast Genomic DNA Kit). Specific primer pairs (SEQ ID NO.18-19) were designed to amplify the genomic DNA by PCR. The recovered products were sent to Hangzhou Qingke Sequencing. Using the recombinant plasmid Ts-CYP52M1 with accurate sequencing as a template, single mutant plasmids of CYP52M1 were obtained by site-directed mutagenesis PCR amplification and DpnI enzyme digestion (specific conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 90 s, 35 cycles; 72℃ final extension for 10 min; using the corresponding primers F and R of the mutants in Table 1, 50 μl PCR system was prepared according to the instructions of the 2×Hieff® PCR Master Mix kit).

[0039] Single and combined mutations were constructed, with mutation sites including R128A, F141A, W148A, T340Y, V403F, L404A, and P476A.

[0040] Among them, F141A, W148A, L404A, R128A, and P476A increase the efficiency of substrate entry into the catalytic active site by expanding the substrate binding pocket, thereby improving catalytic activity; T340Y introduces a large aromatic ring with a π-electron cloud, making the substrate binding pocket locally tighter and more rigid, and enabling it to form π-interactions with the substrate; V403F can form new π-interactions by introducing aromatic side chains or by repositioning the substrate through aromatic volume, bringing the substrate closer to or further away from the heme center, thus affecting catalytic efficiency and selectivity. The construction method of the single mutant expression cassette is as follows:

[0041] Using the genome of *S. bombicola* ATCC 22214 (also known as *Candida bombicola*) as a template, expression cassettes were constructed and engineered strains were prepared according to the following steps: First, PCR amplification was performed separately: ① A 1000 bp upstream fragment of the CYP52M1 gene in the *S. bombicola* genome was amplified as an endogenous promoter using Pcyp-F and Pcyp-R primers, with the *S. bombicola* genome as the template; ② Using plasmids of each CYP52M1 single-point mutant as templates, PCR amplification was performed using CYP52M1-F / CYP52M1-R primers to obtain the CDS sequences (i.e., coding sequences) of each single-point CYP52M1 mutant; ③ A 200 bp downstream fragment of the CYP52M1 gene in the *S. bombicola* genome was amplified as an endogenous terminator using Tcyp-F and Tcyp-R primers, with the *S. bombicola* genome as the template; ④ A 3.1 kb fragment was artificially synthesized by Hangzhou Qingke Biotechnology Co., Ltd. The hphex fragment (SEQ ID NO.17) was spliced ​​together by fusion PCR to form a "promoter-CYP52M1 mutant-terminator-hphex resistance gene" expression cassette.

[0042] Take 50 μL of ΔCYP52M1 bacterial suspension and 10 μL of the expression cassette fragment to be transformed, mix them in a 1.5 mL EP tube, and incubate on ice for 5 min. Transfer to a pre-chilled 1 mm electroporation cuvette, place on ice for 3-5 min, gently wipe the cuvette dry, and then electroporate using an electroporator (1.5 kV, 200 Ω, 25 μF). Immediately after removing the cuvette, add 1 mL of pre-chilled sorbitol, pipette, and transfer to a 1.5 mL EP tube. Incubate in a 30°C water bath for 1 h. Immediately remove the bacterial suspension, take 200 μL, spread it on a hygromycin-resistant YPD plate, invert the plate, and incubate at 30°C for 3 days until a single colony grows on the plate. Pick the hygromycin-resistant colonies, and verify by colony PCR and sequencing to confirm that the CYP52M1 mutant expression cassette is correctly integrated into the ΔCYP52M1 strain genome target site, thus obtaining the target recombinant strain. In addition to single mutants, after screening for high-performing single-point mutation sites, their mutant plasmids were used as templates for subsequent mutations. Combined mutants were constructed by progressively stacking mutation sites (see the single mutant plasmid construction process for PCR system and conditions), thereby obtaining corresponding double-mutant and triple-mutant expression lines, including but not limited to: F141A / W148A and F141A / W148A / P476A (SEQ ID NO.2), to systematically evaluate the synergistic effect of different site combinations on hydroxylation activity. The construction process of the F141A / W148A mutant was as follows: using the F141A mutant plasmid as a template, PCR amplification and DpnI digestion were performed using primers for W148A to obtain the F141A / W148A mutant plasmid. The CDS sequence of the F141A / W148A mutant was obtained by PCR amplification using primers CYP52M1-F / CYP52M1-R. Construction of the F141A / W148A / P476A mutant: Using the F141A / W148A mutant plasmid as a template, PCR amplification and DpnI digestion were performed using primers for P476A to obtain the F141A / W148A / P476A mutant plasmid. The CDS sequence of the F141A / W148A / P476A mutant was obtained by PCR amplification using primers CYP52M1-F / CYP52M1-R. Following the aforementioned method for constructing single-mutant expression cassettes, a combined mutant expression cassette was constructed, and engineered strains were prepared. Colony PCR and sequencing verification confirmed that the corresponding CYP52M1 mutant expression cassette was correctly integrated into the ΔCYP52M1 strain genomic target site.

[0043] The amino acid sequences of the wild-type CYP52M1 enzyme and the F141A / W148A / P476A mutant are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The gene for the wild-type CYP52M1 enzyme is shown in SEQ ID NO.26. The listed mutation sites and primers used are shown in Table 1.

[0044] SEQ ID NO.1 CYP52M1 WT amino acid sequence MLIKDIILTPMSLSAVAGLLPLLFVAFLVLHEPIWLLWYRYAARRHKCSMPRFIEKSFPLGIQRTMDMIKTAKSYTLLEVQYDRVFNKFKARTYLRQAPLQYQIFTIEPENIKTILATKFNDFGLGARFHTVGK VFGQGIFTLSGNGWKQSRSMLRPQFTKDQVCRIDQISSHAAELIKEMNRAMKVDQFIDVQHYFHKLTLDTATEFLFGESCESSLNPENQSCIVARDGSEITAEQFVESYNFLLNYAFKRTLSSKVYWLFNSKEFRD HKKRAQSYIDYYVDKALYATSFAAENSIAEKDAAAESSGIYVFSLEMAKVTRDPVTIRDQIFNILIAGRDTTAATLSFAIHFLARNPDVFNKLREEVLDHFGTKEEQRPLSFELLKQAPYLKQVINEVLRLAPV LPLNFRTAVRDTTLPIGGGPEQKDPIFVPKGTAVYYSIYMVHRDIKYWGPDAHEFNPNRWENLKLDNVWAFLPFNGGPRICLGQQFALTELSLTLVRLLQEYSKIEMGPDFPESPRFSTTLTAQHAPPGVVVRFS

[0045] SEQ NO.26 CYP52M1 WT encoding sequence

[0046] SEQ ID NO.2 CYP52M1 F141A / W148A / P476A mutant MLIKDIILTPMSLSAVAGLLPLLFVAFLVLHEPIWLLWYRYAARRHKCSMPRFIEKSFPLGIQRTMDMIKTAKSYTLLEVQYDRVFNKFKARTYLRQAPLQYQIFTIEPENIKTILATKFNDFGLGARFHTVGKVFGQGIATLSGNGAKQSRSMLRPQFTKDQVCRIDQISSHAAELIKEMNRAMKVDQFIDVQHYFHKLTLDTATEFLFGESCESLNPENQSCIVARDGSEITAEQFVESYNFLLNYAFKRTLSSKVYWLFNSKEFRDHKKRAQSYIDYYVDKALYATSFAAENSIAEKDAAAESSGIYVFSLEMAKVTRDPVTIRDQIFNILIAGRDTTAATLSFAIHFLARNPDVFNKLREEVLDHFGTKEEQRPLSFELLKQAPYLKQVINEVLRLAPVLPLNFRTAVRDTTLPIGGGPEQKDPIFVPKGTAVYYSIYMVHRDIKYWGPDAHEFNPNRWENLKLDNVWAFLAFNGGPRICLGQQFALTELSLTLVRLLQEYSKIEMGPDFPESPRFSTTLTAQHAPPGVVVRFS

[0047] Nucleic acid sequence of SEQ ID NO.17 Hphex expression cassette (hphex fragment):

[0048] Table 1. Mutation sites and primers used

[0049] Example 2: This example describes the fermentation production of recombinant Candida albicans that produces sophorolipids.

[0050] The recombinant Candida albicans expressing the CYP52M1 mutant constructed in Example 1 was fermented to verify its ability to produce sophorolipids. Wild-type Candida albicans (S. bombicola ATCC 22214, without CYP52M1 enzyme gene knockout) was used for fermentation as a wild-type control.

[0051] The principle of sophorolipid preparation: rapeseed oil is hydrolyzed into fatty acids (such as oleic acid) by extracellular lipases secreted by yeast. The fatty acids are absorbed by the cells, and under the action of wild-type or mutant CYP52M1, the ω-1 position of the fatty acids is hydroxylated. The hydroxylated fatty acids are then combined with glucose by the yeast's own endogenous glycosyltransferases UGTA1 / UGTB1 to generate sophorolipids.

[0052] Recombinant *Candida globosa* expressing the CYP52M1 enzyme mutant or wild-type *Candida globosa* was inoculated into 50 mL of YPD liquid medium and cultured at 30°C and 220 r / min for 30 h as a seed culture. Subsequently, 5% (v / v) of the seed culture was inoculated into a 5 L fermenter containing fermentation medium, resulting in a total fermentation volume of 2.5 L. The main components of the fermentation medium included 3 g / L yeast extract, 120 g / L glucose (sterilized separately), 120 g / L rapeseed oil, 2 g / L KH₂PO₄, 2 g / L Na₂HPO₄, 1 g / L MgSO₄, and 0.1 g / L CaCl₂.

[0053] The temperature was maintained at 30°C during fermentation. In the initial stage of fermentation, the aeration rate was controlled at 1 vvm, the stirring speed at 600 rpm, and the pH at 6.5. DO was maintained above 30% throughout fermentation. The cells entered a stable growth phase after 72 hours, and the pH of the fermentation broth gradually decreased to approximately 2.5, while OD... 600 The value reached approximately 200. Fermentation lasted for a total of 192 hours before ending.

[0054] Example 3: Product Analysis

[0055] This embodiment analyzes the fermentation products obtained in Example 2 to verify the effect of the CYP52M1 mutant on the composition of sophorolipid products.

[0056] Mix 0.5 mL of fermentation broth with 1 mL of anhydrous ethanol and shake vigorously for 5 min to fully dissolve extracellular products. Then centrifuge at 15000 rpm for 5 min to remove cells and impurities. Filter the supernatant through a 0.22 μm PTFE membrane. Dilute the filtrate appropriately with 50% ethanol and use directly for high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis. Detection was performed using a high-performance liquid chromatography system coupled with mass spectrometry (HPLC-MS, Micromass Quattro LC; Shimadzu LC system), and separation was performed at 30°C using an EC-C18 (100 mm × 4.6 mm, Merck KGaA) column. The mobile phase consisted of an aqueous solution (A) containing 0.1% formic acid and methanol (B). The gradient elution program was as follows: initial 10% B, linearly increasing to 60% within 10 min and holding for 30 min; then increasing to 100% within 80 min and holding for 20 min; finally decreasing to 10% and equilibrating within the last 4 min. The flow rate was 1.0 mL / min, and the total analysis time was 150 min. The mass spectrometry detection mode was positive ion scan, and the mass scan range was set to 215–1100 g / mol.

[0057] Glucose measurement: After centrifuging the fermentation broth, take the supernatant, dilute it to the range of 0-1 g / L, and measure it using a biosensor SBA-40D.

[0058] The content of sophorolipids was determined using the sulfuric acid-anthrone colorimetric method. The specific steps are as follows: Take 0.5 mL of fermentation broth, add 1.0 mL of anhydrous ethanol, mix well, centrifuge at 15000 r / min for 5 min, and collect the supernatant. Extract the supernatant twice with an equal volume of ethyl acetate, combine the organic phases, dry under a nitrogen stream, and reconstitute the residue with 1.0 mL of deionized water. Take 0.5 mL of the reconstituted solution, add an equal volume of 4 mol / L hydrochloric acid, and heat in a 100°C water bath for 1 h for hydrolysis. After cooling, neutralize with 4 mol / L NaOH to pH 6–7, and bring the volume to 1.0 mL with deionized water to obtain the test solution. The anthrone reagent was prepared at a mass fraction of 0.2% (w / v): slowly dissolve 0.2 g of anthrone in 100 mL of concentrated sulfuric acid under ice bath conditions, and use immediately after preparation. Take 0.5 mL of the test solution into a colorimetric tube, add 4.5 mL of anthrone-sulfuric acid reagent, mix well, and react in a 95°C water bath for 10 min. Remove and cool to room temperature. Measure the absorbance at 620 nm using a UV-Vis spectrophotometer. Prepare a series of standard solutions ranging from 0 to 100 μg / mL using glucose as a standard, and perform the colorimetric reaction according to the same procedure to plot a standard curve. Calculate the glucose equivalent content from the standard curve based on the absorbance value of the sample, and express the sophorolipid concentration as glucose equivalents. If conversion to sophorolipid mass is required, molar mass conversion can be performed based on the molecular composition of the target product.

[0059] In the description of this invention, "sophorolipid yield" refers to the percentage of sophorolipid mass produced by each mutant strain relative to the sophorolipid yield of the wild-type strain (WT) under the same fermentation conditions, with WT yield defined as 100% as the relative benchmark.

[0060] The “substrate conversion rate” mentioned in this invention refers to the efficiency of converting a unit mass of rapeseed oil into sophorolipids, expressed as the ratio of the mass of sophorolipids to the mass of oil consumed (g sophorolipids / g rapeseed oil), which is used to directly reflect the ability of substrate carbon to flow into the target product.

[0061] The yield of sophorolipids and the conversion rate of rapeseed oil in fermentation broth samples from wild-type and mutant strains were determined, and the results are as follows: Figure 2 , 3As shown, with the wild-type strain as the control, the sophorolipid yield was defined as 100%, corresponding to a conversion rate of approximately 0.457 g / g. Most single-point mutations showed varying degrees of performance changes. The catalytic efficiency of R128A, T304Y, and L404A was lower than that of the wild type. Among them, the sophorolipid yield and conversion rates of the F141A, W148A, P476A, and V403F mutants were all higher than those of the wild type, indicating that these sites are involved in substrate binding or catalytic efficiency regulation. For example, the sophorolipid yield of W148A reached 164%, and the conversion rate was approximately 0.750 g / g, which was about 64% higher than that of the wild type, indicating that the mutation at this site can significantly enhance the fatty acid ω-hydroxylation ability. Compared with single-point mutations, the combined mutants showed a more significant yield increase effect. Among them, the F141A / W148A double mutant strain showed superior yield compared to all single mutant strains, with a sophorolipid yield of approximately 170% and a corresponding conversion rate of approximately 0.777 g / g, suggesting a synergistic enhancement effect between the mutant sites. Among all tested strains, the triple mutant F141A / W148A / P476A performed best, with a sophorolipid yield of 210% and a corresponding conversion rate of 0.960 g / g, which is approximately 2.10 times that of the wild type. These results indicate that multi-site mutation guided by structural analysis can effectively improve the substrate recognition ability and catalytic efficiency of CYP52M1 and further enhance the sophorolipid synthesis capacity.

[0062] Finally, the sophorolipid products of the wild-type and triple mutant strains F141A / W148A / P476A were analyzed by HPLC-MS, and the results are as follows: Figure 4 The acidic sophorolipid (Mw=706) had its main elution time at 9.6 min, while the lactone-type sophorolipid (Mw=688) had its main elution times around 11.8 min and 17.8 min, confirming the production of sophorolipids. The product spectrum of the recombinant strain CYP52M1 F141A / W148A / P476A mutant showed significant changes compared to the wild type, with enhanced signal peaks for hydroxylated fatty acid derivatives, indicating that the mutant possesses higher catalytic activity for the ω-hydroxylation reaction of fatty acids, thereby increasing the overall yield of sophorolipids.

[0063] The above embodiments are only used to illustrate the technical solutions of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way. All technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A mutant of CYP52M1 enzyme, characterized in that, The CYP52M1 enzyme mutant is the following 1) or 2): 1) a CYP52M1 enzyme mutant obtained by mutating at least one of the amino acids at positions 141, 148, and 476 of the amino acid sequence shown in SEQ ID NO. 1; the mutation is mutating the phenylalanine at position 141, the tryptophan at position 148, or the proline at position 476 of the amino acid sequence shown in SEQ ID NO. 1 to alanine; 2) a CYP52M1 enzyme mutant having P450 enzyme activity and having more than 99%, more than 95%, more than 90%, more than 85%, or more than 80% homology with the amino acid sequence of the CYP52M1 enzyme mutant in 1).

2. The mutant CYP52M1 enzyme according to claim 1, wherein The CYP52M1 enzyme mutant is obtained by mutating two or three of the amino acids at positions 141, 148, and 476 of the amino acid sequence shown in SEQ ID NO.

1.

3. The mutant CYP52M1 enzyme according to claim 1, wherein The amino acid sequence of the CYP52M1 enzyme mutant is shown in SEQ ID NO.

2.

4. A coding gene encoding the mutant of any one of claims 1-3.

5. An expression vector comprising the coding gene of claim 4.

6. A host cell comprising the expression vector of claim 5 or the coding gene of claim 4.

7. The host cell of claim 6, wherein, The coding gene is integrated or not integrated in the genome of the host cell.

8. The host cell of claim 6, wherein, The host cell is a genetically engineered bacterium.

9. A catalyst characterized by, It comprises the mutant of any one of claims 1-3, the coding gene of claim 4, the expression vector of claim 5, or the host cell of claim 6.

10. Use of the mutant of any one of claims 1-3, the coding gene of claim 4, the expression vector of claim 5, the host cell of claim 6, or the catalyst of claim 9 in improving the efficiency of fatty acid ω-hydroxylation or the yield of sophorolipids.

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