Genetically modified bacteria with high yield of sophorolipids, construction method and application thereof

Abstract

The present application relates to the technical field of genetic engineering, and in particular to a high-yield sophorolipid gene knockout engineering bacterium, a construction method thereof and an application, the engineering bacterium taking Bombus bornensis Candida as a starting strain, and using a gF-Hph-gR nucleotide sequence to replace a gene sequence seq2 in a sophorolipid synthesis path for regulating a lactonization reaction ， The gF-Hph-gR nucleotide sequence is SEQ ID No. 1, and the seq2 gene sequence is SEQ ID No. 2. The method provided by the present application uses fusion PCR to construct a seq2 gene knockout box with a resistance screening marker, and the shortest time required is 1 day, which provides a new way for efficiently constructing a sophorolipid production engineering strain. The high-yield sophorolipid gene knockout strain provided by the present application lays a foundation for large-scale industrial production of sophorolipid and meets market demand, and has a wide application prospect, economic value and social benefit.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to a gene knockout engineered bacterium that produces high levels of sophorolipids, its construction method, and its applications. Background Technology

[0002] Sophorolipids synthesized by microorganisms are a mixture of sophorolipid molecules, primarily produced by yeasts. These sophorolipid molecules consist of both hydrophilic and lipophilic groups. The hydrophilic group is a sophorose, formed by two glucose molecules linked by a β-1,2 glycosidic bond, while the lipophilic group is a saturated / unsaturated long-chain ω- or ω-1 hydroxy fatty acid, with the two groups linked by a glycosidic bond. Sophorolipids possess both hydrophilic and hydrophobic groups, exhibiting general surfactant properties such as surface tension reduction, foaming, and emulsifying properties. They also possess the characteristic of being usable and degradable by microorganisms, making them a type of green biosurfactant that does not pollute the environment. In recent years, the application of sophorolipids as biosurfactants has received increasing attention. The non-toxicity, high-temperature resistance, and excellent surface activity even in high-salt solutions of sophorolipids make them highly valuable for use in detergents such as dishwashing liquid and laundry detergents. Sophorolipids are also used as penetration enhancers in cosmetics due to their strong skin affinity.

[0003] There are many microorganisms that produce sophorolipids, with *Starmerella bombicola* being the most commonly used yeast. Microbially synthesized sophorolipids have two molecular structures: lactone and acid. Sophorolipids with different molecular structures exhibit different properties in terms of antibacterial activity and surface activity.

[0004] The use of sophorolipids as a cleaning agent or cosmetic ingredient inevitably leads to competition with currently used, relatively inexpensive chemically synthesized surfactants. However, the low yield and high purification cost of wild-type *Candida bumblebee* strains result in high costs for large-scale production of sophorolipids, which is a major obstacle to its industrial production and widespread application. Therefore, there is an urgent need for an engineered strain that produces high levels of sophorolipids as one of the main means to reduce the production cost of sophorolipids. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a gene knockout engineered bacterium that produces high levels of sophorolipids and can inhibit genes that regulate lactone formation in the sophorolipid synthesis pathway.

[0006] To solve the above-mentioned technical problems, the purpose of this invention is achieved through the following technical solution: providing a gene knockout engineered bacterium that produces high levels of sophorolipids, wherein the engineered bacterium is based on *Candida bacillus* as the starting strain, and the gene sequence seq2 that regulates the lactone reaction in the sophorolipid synthesis pathway is replaced by the gF-Hph-gR nucleotide sequence.

[0007] The gF-Hph-gR nucleotide sequence is SEQ ID No. 1, and the seq2 gene sequence is SEQ ID No. 2.

[0008] In addition, the technical problem to be solved by the present invention is to provide a method for constructing a gene knockout engineered bacterium that produces high levels of sophorolipids.

[0009] To solve the above-mentioned technical problems, the objective of this invention is achieved through the following technical solution: a method for constructing a gene knockout engineered bacterium that produces high levels of sophorolipids, characterized by comprising the following steps:

[0010] S1. Construct the knockout box;

[0011] S2. Preparation of wild-type Candida albicans competent cells.

[0012] Preferably, step S1 includes the following steps:

[0013] A1. Optimize the hygromycin resistance gene gF-Hph-gR with strong promoters and terminators;

[0014] A2. Insert the gF-Hph-gR nucleotide sequence into the pUC57 plasmid to construct the hygromycin selection marker plasmid pUHP.

[0015] A3. Using a high-fidelity enzyme, the genomic DNA of *Candida bumblebee* was used as a template, and SBLEdl F1 / SBLEdlR1 was used as a template to amplify the seq2 gene sequence; then, using the seq2 gene sequence as a template, the upstream and downstream homologous arm sequences 5'flanking and 3'flanking of the seq2 gene were amplified using primers SBLEdlF2 / SBLEdlR2 and SBLEdlF4 / SBLEdlR4, respectively.

[0016] A4. Using the hygromycin selection marker plasmid pUHP as a template, and primers SBLEdlF3 / SBLEdlR3, a high-efficiency hygromycin expression cassette gF-Hph-gR with 20bp homologous sequences upstream and downstream of the seq2 gene at both ends was amplified.

[0017] A5. The 5' flanking and 3' flanking arms of the seq2 gene obtained by gel extraction and purification, gel extraction and high-efficiency expression cassette gF-Hph-gR with homologous sequences;

[0018] A6. Fusion PCR: The three purified genes are fused to obtain a linear seq2 gene knockout cassette. Preferably, in step A2, the sequences of primers SBLEdlF1 / SBLEdlR1 are shown in SEQ ID No. 5 / SEQ ID No. 6, the sequences of primers SBLEdlF2 / SBLEdlR2 are shown in SEQ ID No. 7 / SEQ ID No. 8, the sequences of primers SBLEdlF4 / SBLEdlR4 are shown in SEQ ID No. 9 / SEQ ID No. 10, and the sequences of primers SBLEdlF3 / SBLEdlR3 are shown in SEQ ID No. 11 / SEQ ID No. 12.

[0019] Preferably, the 5' and 3' homologous sequences of the hygromycin gene linear expression cassette with homologous sequences are shown in SEQ ID No. 3-1 and SEQ ID No. 3-2, respectively.

[0020] Preferably, in step S2, the linear seq2 gene knockout cassette is transformed into competent cells, and strains that grow normally on YPD plates containing hygromycin are selected for YPD liquid culture to extract genomic DNA; using the extracted genomic DNA as a template, TaKaRaPremixTaq is used. TM The enzyme was used to amplify the DNA fragment by PCR using SBLEdl F2 / SBLEdlR4 as primers.

[0021] Furthermore, this invention also provides an application of a gene knockout engineered bacterium that produces high levels of sophorolipids, particularly in the fermentation production of sophorolipids.

[0022] Preferably, the fermentation process for producing sophorolipids is as follows:

[0023] B1. Inoculate the engineered bacteria into a test tube containing 5mLYPD seed culture medium and culture it.

[0024] B2. Inoculate the seed culture at an inoculation rate of 2% (v / v) into a shake flask containing 50 ml of LYPD medium for primary shake flask fermentation.

[0025] B3, pending OD 600 =1.0, and then inoculate at a rate of 5% (v / v) into a 500mL shake flask containing 100mL of fermentation medium and incubate for 5-10 days.

[0026] Preferably, the YPD culture medium consists of 1% (w / v) yeast extract, 2% (w / v) peptone and 2% (w / v) glucose.

[0027] Preferably, the fermentation medium consists of: 1% (w / v) yeast extract, 4% (w / v) linoleic acid, 8% (w / v) glucose, 0.1% KH2PO4, 0.1% Na2HPO4·12H2O, and 0.05% MgSO4·7H2O.

[0028] Preferably, the fermentation culture time is 5 days.

[0029] Common methods for obtaining high-yielding sophorolipid-producing strains mainly involve mutagenesis screening or multiple gene knockout and overexpression, which are characterized by randomness and operational complexity. Furthermore, the methods for constructing gene knockout and overexpression cassettes often involve ligating a pre-modified gene fragment with a linearized vector to obtain a recombinant plasmid, transforming competent cells with the recombinant plasmid, plating the transformed competent cells, selecting single colonies, screening successfully transformed single colonies using ColonyPCR, culturing the single colony, extracting the recombinant plasmid from the cells, and finally amplifying the linear gene knockout / overexpression cassette using the recombinant plasmid as a template, which takes 3 days or more. The method for obtaining high-yielding sophorolipid-producing strains provided by this invention uses a fusion PCR method to construct a seq2 gene knockout cassette with an resistance selection marker, which can be obtained in as little as 1 day, improving gene modification efficiency and providing a new approach for efficiently constructing genetically engineered strains that produce sophorolipids. The genetically engineered strain constructed in this invention increased the total sophorolipid yield by 39.8% compared to the wild strain, significantly reducing the production cost of sophorolipids. In particular, the yield of acidic sophorolipids increased by 258.9% compared to the wild strain. Furthermore, acidic sophorolipids, due to their high solubility, good foaming and emulsifying properties, can be widely used in the detergent, oil extraction, skin care, food and pharmaceutical industries. Therefore, this gene knockout strain lays the foundation for large-scale industrial production of sophorolipids and meets market demand, with broad application prospects, economic value and social benefits. Attached Figure Description

[0030] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This invention employs a fusion PCR method to construct the seq2 knockout strain Δseq2 through homologous recombination. (Schematic diagram)

[0032] Figure 2 The agarose gel electrophoresis results show the three gene fragments used to construct the seq2 gene knockout cassette and the seq2 gene knockout cassette obtained by fusion PCR amplification.

[0033] Figure 3 The results of agarose gel electrophoresis of DNA fragments amplified by PCR using wild-type strain and Δseq2 strain genomic DNA as templates and SBLEdlF2 / SBLEdlR4 as primers are shown.

[0034] Figure 4 The above figures show the fermentation results of sophorolipids in the fermentation broths of wild-type strain and Δseq2 strain in this invention.

[0035] Figure 5 This is a comparison chart showing the differences between the crude fermentation broth of the wild-type strain and the Δseq2 strain in an embodiment of the present invention. Detailed Implementation

[0036] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

[0038] It should also be understood that the terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.

[0039] It should also be further understood that the term "and / or" as used in this specification and the appended claims refers to any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.

[0040] Furthermore, terms like "roughly" and "basically" are used to indicate that the content does not require absolute precision, but rather allows for a certain degree of deviation. For example, "roughly equal" does not simply mean absolute equality; in actual production and operation, achieving absolute "equality" is difficult, and a certain degree of deviation is generally present. Therefore, besides absolute equality, "roughly equal to" also includes the aforementioned situation where a certain degree of deviation exists. Using this as an example, in other cases, unless otherwise specified, terms like "roughly" and "basically" have similar meanings.

[0041] This invention provides a gene knockout engineered strain that produces high levels of sophorolipids. The high-yield sophorolipid gene knockout engineered strain is based on *S. bombicola*, with the gF-Hph-gR nucleotide sequence replacing the seq2 gene sequence that regulates the lactone reaction in the sophorolipid synthesis pathway, thereby inhibiting the lactone reaction. The gF-Hph-gR nucleotide sequence is shown in SEQ ID No. 1, and the seq2 gene sequence is shown in SEQ ID No. 2. The amino acid sequence encoded by the seq2 gene sequence is shown in SEQ ID No. 4. Compared with the wild-type strain, the total sophorolipid yield is increased by more than 35%, and the yield of acidic sophorolipids is increased by more than 200%, thus qualifying as a high-yield strain.

[0042] The gene knockout mutant strain of high-yield sophorolipid produced by this invention was deposited on August 25, 2022, at the Guangdong Provincial Microbial Culture Collection Center (5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, Institute of Microbiology, Guangdong Academy of Sciences), with accession number GDMCC No:62738 and taxonomic name: Starmerellabombicola.

[0043] The construction method of the above-mentioned gene knockout engineered bacteria that produces high levels of sophorolipids includes the following steps:

[0044] S1. Construction of the knockout cassette: The hygromycin resistance gene gF-Hph-gR with strong promoters and terminators was optimized to enable its efficient expression in *Candida albicans*. The gF-Hph-gR nucleotide sequence was inserted into the pUC57 plasmid to construct the hygromycin selection marker plasmid pUHP. The high-fidelity enzyme Prime was used... MaxDNAPolymerase was used to amplify the seq gene sequence using *Candida bumblebee* genomic DNA as a template and SBLEdlF1 / SBLEdlR1 as a template. Then, using the obtained seq gene sequence as a template, the upstream and downstream homologous arm sequences of the seq2 gene were amplified using primers SBLEdlF2 / SBLEdlR2 and SBLEdlF4 / SBLEdlR4, respectively. Using the hygromycin selection marker plasmid pUHP as a template, primers SBLEdlF3 / SBLEdlR3 were used to amplify a high-efficiency hygromycin expression cassette with 20bp homologous sequences upstream and downstream of the seq2 gene at both ends. The upstream and downstream homologous arms of the seq2 gene and the high-efficiency hygromycin expression cassette with homologous sequences were purified using the TaKaRaMiniBESTAgaroseGelDNAExtractionKit. The three purified genes were fused using fusion PCR to obtain a linear seq2 gene knockout cassette. The nucleotide sequence of the seq2 gene is shown in SEQ ID. As shown in No. 2, the 5' and 3' homologous sequences of the linear expression cassette of the hygromycin gene with homologous sequences are shown in SEQ ID No. 3-1 and SEQ ID No. 3-2, respectively.

[0045] S2. Wild-type *S. bombicola* competent cells were prepared using the lithium acetate method. A linear seq2 gene knockout cassette was transformed into the competent cells. Strains that grew normally on YPD plates containing hygromycin were selected for YPD liquid culture, and genomic DNA was extracted. Using the extracted genomic DNA as a template, TaKaRaPremixTaq was used. TM The enzyme, using SBLEdlF2 / SBLEdlR4 as primers, amplified the DNA fragment by PCR. DNA sequencing confirmed that the amplified fragment was the seq2 gene knockout cassette sequence, indicating that the seq2 gene knockout was successful.

[0046] In addition, the gene knockout engineered bacteria for high-yield sophorolipid production provided by this invention are applied in the fermentation production of sophorolipid.

[0047] The method for producing sophorolipids by fermentation is as follows: Gene-knockout engineered bacteria are inoculated into test tubes containing 5 ml of LYPD seed culture medium and cultured at 30°C and 200-250 rpm for 24-36 hours. The seed culture is then inoculated at a rate of 2% (v / v) into shake flasks containing 50 ml of LYPD medium for primary shake-flask fermentation at 25-30°C and 200-300 rpm for 24-48 hours until OD (oxidative stress) is achieved. 600=1.0, and then inoculate 5% (v / v) into a 500mL shake flask containing 100mL of fermentation medium, and culture at 25-30℃ and 200-250rpm for 5-10 days; the total sophorolipid and lactone-type sophorolipid content in the fermentation broth is determined by the anthrone method. The total sophorolipid content minus the lactone-type sophorolipid content is the acid sophorolipid content. The glucose residue in the fermentation broth is determined by a biosensor analyzer SBA-40E to evaluate the engineered bacteria and its fermentation production effect.

[0048] The YPD medium consists of 1% (w / v) yeast extract, 2% (w / v) peptone and 2% (w / v) glucose.

[0049] The fermentation medium consists of: 1% (w / v) yeast extract, 4% (w / v) linoleic acid, 8% (w / v) glucose, 0.1% KH2PO4, 0.1% Na2HPO4·12H2O, and 0.05% MgSO4·7H2O.

[0050] The preferred culture conditions for the above seed culture are: 30℃, rotation speed of 200-250rpm, and culture time of 24-30h.

[0051] The preferred conditions for the first-stage shake flask fermentation are as follows: the seed culture is inoculated into 50 mL of culture medium at a rate of 2-4% (v / v), and the culture conditions are: 30℃, 200-300 rpm, and culture for 36-48 h.

[0052] The preferred conditions for the shake flask fermentation are as follows: inoculate the primary shake flask fermentation broth at 5% (v / v) into 100 mL of culture medium and incubate at 30°C and 200-250 rpm for 120 hours.

[0053] Analysis of fermentation products of the high-yield sophorolipid engineered strain and wild-type strain used in this invention:

[0054] (1) Wild-type strains and engineered strains were fermented in a fermentation medium. After fermentation, samples of the fermentation broth were taken to determine and analyze the residual glucose content, total sophorolipid yield, and lactone and acidic sophorolipids.

[0055] (2) For the determination of residual sugar content: the glucose concentration in the culture medium was determined using a biosensor analyzer SBA-40E.

[0056] Centrifuge the fermentation broth and collect the supernatant. Filter the supernatant through a 0.45 μm filter membrane. Dilute the filtrate to an appropriate ratio and inject 25 μL. The residual glucose content of the fermentation broth (g / L) = n × m / 100 (n is the instrument reading, m is the dilution ratio).

[0057] (3) The content of sophorolipids in the fermentation broth was determined by the anthrone-sulfuric acid method.

[0058] Take 500 μL of fermentation broth, add 1 mL of ethanol, shake to mix, and incubate at 12,000 rpm. -1 Centrifuge for 10 min. Take 20 μL of the supernatant and add it to an 8 mL EP tube, then add 980 μL of distilled water (diluted 50 times). Determine the total sugar content using the anthrone-sulfuric acid method. The total sophorolipid content is calculated by removing residual glucose from the fermentation broth from the total sugar content. The sophorolipid content is determined based on the ratio between the molecular weights of sophorolipids and glucose, i.e., 1.91 g of sophorolipids is equivalent to 1 g of glucose.

[0059] Take 500 μL of fermentation broth, add 1 mL of ethyl acetate, and vortex to extract thoroughly at 12,000 rpm. 1 Centrifuge for 10 min. Add 20 μL of the supernatant to an 8 mL EP tube, and add 380 μL of distilled water (diluted 20 times). Measure the OD value of the sophorolipid sample at 620 nm using the anthrone-sulfuric acid method. Calculate the glucose content from the glucose standard curve. The lactone-type sophorolipid content can be obtained by applying a ratio of 1:1.91. Acidic sophorolipid content = Total sophorolipid content - Lactone-type sophorolipid content.

[0060] Experimental results show that the high-yield sophorolipid engineered strain Δseq2 constructed by the method described in this invention achieved a total sophorolipid yield of 64.3 g / L after 5 days of shake-flask fermentation, which is 39.8% higher than that of the wild-type strain. The yield of acidic sophorolipid reached 50.6 g / L, which is 258.9% higher than that of the wild-type strain. Due to the high solubility and good foaming and emulsifying properties of acidic sophorolipid, it can be widely used in the food, pharmaceutical, cosmetic, and cleaning industries. This makes the Δseq2 engineered strain a superior strain for the production of sophorolipids, especially acidic sophorolipids.

[0061] The following describes the specific implementation details with reference to the embodiments.

[0062] Example 1: Construction of a gene knockout cassette with gF-Hph-gR resistance selection marker

[0063] (1) Using genomic DNA of *S. bombicola* as a template, the nucleotide sequence fragment shown in SEQ ID NO:2 was obtained by PCR amplification using primers SBLEdl F1 and SBLEdlR1. Using this gene fragment as a template, the upstream region fragment 5'flanking was amplified by PCR using primers SBLEdlF2 and SBLEdlR2; similarly, the downstream region fragment 3'flanking was obtained by PCR amplification using primers SBLEdlF4 and SBLEdlR4. Using pUHP plasmid as a template, the gF-Hph-gR fragment was obtained by PCR amplification using primers SBLEdlF3 and SBLEdlR3.

[0064] (2) Finally, using SBLEdlF2 and SBLEdlR4 as primers, the three fragments 5'flanking, 3'flanking, and gF-Hph-gR were ligated via fusion PCR, thus obtaining a linear knockout cassette for the gene fragment shown in SEQ ID NO:2. The fusion PCR results are as follows: Figure 2 As shown in the image.

[0065] Gene knockout cassettes with resistance selection markers can be constructed using the above method in as little as one day.

[0066] Example 2 Production of gene knockout strains

[0067] A single colony of *S. bombicola* was inoculated into a 250 mL shake flask containing 25 mL of LYPD medium and cultured at 30 °C and 300 rpm for 18 hours.

[0068] The culture medium from the previous step was seeded at a rate of 2% (v / v) into a 250 mL shake flask containing 50 mL of LYPD medium, and the cells were cultured at 30 °C and 120 rpm until OD (outcome limit) was reached. 600 The value is between 1 and 2.

[0069] Transfer the bacterial culture into a 50mL centrifuge tube, centrifuge at 3000g and 4℃ for 5 minutes to collect the bacterial cells, then resuspend the precipitate in 50mL of sterile water cooled on ice, centrifuge at 3000g and 4℃ for 5 minutes, and discard the supernatant; resuspend the precipitate in 50mL of sterile water cooled on ice and centrifuge again.

[0070] Resuspend the bacterial pellet in 4 mL of ice-cold 1 M sterile sorbitol solution, centrifuge at 3000 g and 4 °C for 5 minutes, and discard the supernatant.

[0071] The precipitate was suspended in 4 mL of freshly prepared 0.1 M lithium acetate solution (3500 μL water, 400 μL lithium acetate, 100 μL DTT), and left at room temperature for 15 minutes. Then, it was centrifuged at 3000 g and 4 °C for 5 minutes and the supernatant was discarded.

[0072] Resuspend the bacterial pellet in 4 mL of ice-cold 1 M sterile sorbitol solution, centrifuge at 3000 g and 4 °C for 5 minutes, and discard the supernatant.

[0073] Suspend the bacterial cells in 1M sorbitol solution, place on ice, and use as soon as possible.

[0074] Pipette 50 μg of the yeast suspension into a centrifuge tube, add 2.53 μg of transformation DNA solution (i.e., the gene knockout fragment with the nucleotide sequence shown in SEQ ID NO:2) to the yeast suspension, mix well, and pre-cool on ice for 5 minutes.

[0075] The mixture was then transferred to an electroporation cup with a 0.2 cm gap and placed on ice for 5 minutes. Subsequently, a 5 ms pulse with a 2.5 kV pulse was applied to the mixture using a MicroPulser (Bio-Rad).

[0076] Remove the electroporation vessel and immediately add ice-cold 1M sorbitol. Gently mix and transfer to a 1.5 mL centrifuge tube. Incubate the mixture at 30°C for 1 hour. Spread 100 μL of the mixture onto a selective medium and incubate at 30°C for approximately 1 week. For the selective medium, use agar medium containing 1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose, and 500 ppm hygromycin.

[0077] Genomic DNA was extracted from single colonies after liquid amplification culture. Using both wild-type strain and selected single-colony genomic DNA as templates, PCR amplification was performed using primers SBLEdlF2 / SBLEdlR4. The amplified DNA fragments were analyzed; the DNA fragment length amplified using single-colony genomic DNA as a template matched the constructed knockout cassette, thus confirming the successful acquisition of the target gene knockout engineered strain (Δseq2 strain). The results are as follows: Figure 3 As shown.

[0078] Example 3: Sophorolipid fermentation production from Δseq2 strain and wild-type strain

[0079] Culture of strains:

[0080] Single colonies of the Δseq2 strain and its wild-type strain obtained in Example 2 were inoculated into test tubes containing 5m LYPD seed culture medium and cultured at 30°C and 200-250 rpm for 24-36 hours. The seed culture was then inoculated at a rate of 2% (v / v) into shake flasks containing 50m LYPD medium for primary shake-flask fermentation at 25-30°C and 200-300 rpm for 24-48 hours until OD (Organic Demand) was reached. 600 =1.0, and then inoculate 5% (v / v) into a 500mL shake flask containing 100mL of fermentation medium, and incubate at 25-30℃ and 200-250rpm for 5-10 days.

[0081] The preferred culture conditions for the above seed culture are: 30℃, rotation speed of 200-250rpm, and culture time of 24-30h.

[0082] The preferred conditions for the first-stage shake flask fermentation are as follows: the seed culture is inoculated into 50 mL of culture medium at a rate of 2-4% (v / v), and the culture conditions are: 30℃, 200-300 rpm, and culture for 36-48 h.

[0083] The preferred conditions for the shake flask fermentation are as follows: inoculate the primary shake flask fermentation broth at 5% (v / v) into 100 mL of culture medium and incubate at 30°C and 200-250 rpm for 120 hours.

[0084] Example 4: Detection and Evaluation of Sophorolipid Production

[0085] After cultivation, the residual sugar content and sophorolipid content in the culture medium were measured. Total sophorolipid yield was classified into three levels: high-yielding, medium-yielding, and low-yielding strains, based on yields of ≥60 g / L, 40–60 g / L, and <40 g / L. Acidic sophorolipid yield was also classified into three levels: high-yielding, medium-yielding, and low-yielding, based on yields of ≥45 g / L, 30–45 g / L, and <30 g / L with a proportion greater than 80% of total sophorolipids.

[0086] (1) For the determination of residual sugar content: the glucose concentration in the culture medium was determined using a biosensor analyzer SBA-40E.

[0087] Centrifuge the fermentation broth and collect the supernatant. Pass the supernatant through a 0.45 cm centrifuge and collect the filter membrane. Dilute the filtrate to an appropriate factor and inject 25 μL. The residual glucose content of the fermentation broth (g / L) = n × m / 100 (n is the instrument reading, and m is the dilution factor).

[0088] (2) For the determination of sophorolipid content: the total sugar content in the culture medium was determined by the anthrone method. Then, based on the ratio between the molecular weights of sophorolipid and glucose, i.e., 1.91g of sophorolipid is equivalent to 1g of glucose, the sophorolipid content can be calculated.

[0089] Total sophorolipid content: The total sugar concentration in the supernatant was determined by ethanol extraction. The glucose content in the sophorolipid was obtained by subtracting the residual sugar in the supernatant. The total sophorolipid content can then be calculated.

[0090] (3) Content of lactone-type sophorolipids: The glucose content of lactone-type sophorolipids in the supernatant of the ethyl acetate layer was determined by extraction with ethyl acetate, and the content of lactone-type sophorolipids could be obtained by conversion.

[0091] Comparative Example 1

[0092] A yeast strain producing only acidic sophorolipids was constructed by knocking out the lactonease-encoding gene SBLE and the peroxisome membrane transporter-encoding gene PXA1, and by overexpressing the glucosyltransferase gene UGTB. In a fermentation broth containing 132.0 g / L glucose, 4.0 g / L yeast extract, 4.0 g / L sodium citrate, 1.0 g / L KH₂PO₄, 0.16 g / L K₂HPO₄·12H₂O, 0.7 g / L MgSO₄·7H₂O, 37.5 g / L rapeseed oil, 0.5 g / L NaCl, and 0.27 g / L CaCl₂·2H₂O, after 9 days of cultivation, the total yield of acidic sophorolipids was 44 g / L. (Source: Zhang Jiangrui et al. Acta Microbiologica Sinica, 2019)

[0093] In Comparative Example 2, a high-yield sophorolipid-producing engineered strain was obtained by knocking out the pH-responsive protein rlp gene, the leu3 transcription factor which regulates branched-chain amino acid synthesis, and the zinc finger transcription factor ztf1 in yeast. After 7 days of cultivation in a fermentation broth containing 80.0 g / L glucose, 3.0 g / L yeast extract, 1.0 g / L KH2PO4, 1.0 g / L Na2HPO4·12H2O, 0.5 g / L MgSO4·7H2O, and 80 ml / L rapeseed oil, the total sophorolipid yield reached 102 g / L, but the proportion of acidic sophorolipids was relatively low, approximately 53%.

[0094] The total sophorolipid and acidic sophorolipid yields of strain Δseq2 were 64.3 g / L and 50.6 g / L, respectively, representing increases of 39.8% and 258.9% compared to the wild-type strain's 46.0 g / L and 14.1 g / L. The results are as follows... Figure 4 and Figure 5 As shown in Table 1, the statistical results are shown in Table 2, and the yield evaluation is shown in Table 2.

[0095] Table 1: Test Results

[0096]

[0097] Table 2: Yield Evaluation

[0098]

[0099] In summary, this invention successfully constructed an engineered strain that produces high-yield sophorolipids, particularly acidic sophorolipids. Using a fusion PCR method, a gene knockout cassette was efficiently constructed, resulting in an engineered strain (a *Candida bumblebee* strain with genotype Δseq2) that significantly increased both the total and acidic sophorolipid yields. This provides an excellent, stable, and high-yield strain for the industrial production of sophorolipids and also has significant theoretical research value. Acidic sophorolipids exhibit excellent solubility, foaming, emulsification, and cleaning properties, showing broad application prospects in industries such as household cleaning, personal care, food, pharmaceuticals, and oil extraction, demonstrating considerable practical application value.

[0100] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A genetically knockout engineered bacterium with high yield of sophorolipids, characterized in that: The engineered bacteria is *Candida albicans* (bumblebee-borne yeast). Starmerella bombicola The starting strain was seq2, which replaced the gene sequence regulating lactone formation in the sophorolipid synthesis pathway with the gF-Hph-gR nucleotide sequence. The gF-Hph-gR nucleotide sequence is SEQ ID No. 1, and the seq2 gene sequence is SEQ ID No.

2. The preservation number of the high-yield sophorolipid gene knockout engineering bacteria is GDMCC No: 62738.

2. The method for constructing a genetically knockout engineered bacterium with high yield of sophorolipids according to claim 1, characterized in that, The method comprises the following steps: S1, constructing a knockout cassette; comprising the following steps: A1, optimizing the hygromycin resistance gene gF-Hph-gR with a strong promoter and terminator; A2, inserting the gF-Hph-gR nucleotide sequence into the pUC57 plasmid to construct a hygromycin selection marker plasmid pUHP; A3, using high-fidelity enzyme, taking the Bombicola bombicola genome DNA as a template, and taking SBLEdl F1 and SBLEdl R1 as primers to amplify the seq2 gene sequence; then taking the seq2 gene sequence as a template, and using primers SBLEdl F2 and SBLEdl R2, SBLEdl F4 and SBLEdl R4 to amplify the upper and lower homologous arm sequences 5' flanking and 3' flanking of the seq2 gene; A4, taking the hygromycin selection marker plasmid pUHP as a template, and using primers SBLEdl F3 and SBLEdl R3 to amplify the hygromycin high-efficiency expression cassette gF-Hph-gR with the upper and lower 20 bp homologous sequences of the seq2 gene at both ends; A5, gel purification to obtain the upper and lower homologous arm sequences 5' flanking and 3' flanking of the seq2 gene, and gel purification of the hygromycin high-efficiency expression cassette gF-Hph-gR with the homologous sequence; A6, fusion pcr: fusing the three genes obtained by purification to obtain a linear seq2 gene knockout cassette; S2, preparing a wild-type Bombicola bombicola competent cell and verifying the transformation result; The sequences of the primers SBLEdl F1 and SBLEdl R1 are shown in SEQ ID No. 5 and SEQ ID No. 6, the sequences of the primers SBLEdl F2 and SBLEdl R2 are shown in SEQ ID No. 7 and SEQ ID No. 8, the sequences of the primers SBLEdl F4 and SBLEdl R4 are shown in SEQ ID No. 9 and SEQ ID No. 10, and the sequences of the primers SBLEdl F3 and SBLEdl R3 are shown in SEQ ID No. 11 and SEQ ID No.

12.

3. The method for constructing a genetically knockout strain of high-yield sophorolipid according to claim 2, characterized in that: The 5' end and 3' end homologous sequences of the hygromycin gene linear expression cassette with the homologous sequence are ggaactctta actctcccagacatc and tggcactacg ctgtagttgg caag respectively.

4. The method for constructing a genetically knockout engineered bacterium with high production of sophorolipids according to claim 2, characterized in that: In the step S2, the linear seq2 gene knockout cassette is transformed into the competent cells, the transformed competent cells are coated on the YPD plate containing hygromycin for culture, the normally grown colonies on the plate are selected for YPD liquid culture, and the genomic DNA of the colonies is extracted; the extracted genomic DNA is used as a template, Premix Taq enzyme is used, SBLEdl F2 and SBLEdl R4 are used as primers, and the length of the DNA fragment obtained by PCR amplification is used to judge the transformation result.

5. Application of the high-yield sophorolipid gene knockout engineering bacteria according to claim 1 in the fermentation production of sophorolipids. The fermentation production of sophorolipids is carried out as follows: B1, inoculating the engineering bacteria into a test tube containing 5 mL of YPD seed culture medium for culture; B2, inoculating the seed liquid into a shake flask containing 50 mL of YPD culture medium at an inoculation amount of 2% v / v for primary shake flask fermentation; B3, OD 600 = 1.0, and inoculated into 500 mL shake flasks containing 100 mL of fermentation medium at an inoculum of 5% v / v and incubated for 5-10 days.

6. Use according to claim 5, characterized in that, The composition of the fermentation culture medium is as follows: 1% w / v yeast extract, 4% w / v linoleic acid, 8% w / v glucose, 0.1% KH2PO4, 0.1% Na2HPO4·12H2O, and 0.05% MgSO4·7H2O.

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